An improved method for the quaternization of nicotinamide and antifungal activities of its derivatives

Article abstract of DOI:10.3390/molecules24061001

The quaternization reactions of nicotinamide, with different electrophiles: methyl iodide and substituted 2-bromoacetophenones (4-Cl, 4-Br, 4-H, 4-CH₃, 4-F, 4-OCH₃, 4-Ph, 2-OCH₃, 4-NO₂) are reported. The preparations were carried out by conventional synthesis and under microwave irradiation in absolute ethanol and acetone. The synthesis performed by microwave dielectric heating significantly improved yield, up to 8 times, and shortened down the reaction time from ca. one day in conventional, to 10–20 min. The structures of the synthesized compounds were confirmed by IR, ¹H- and ¹³C-NMR spectroscopy, mass spectrometry and elemental analysis. The compounds have been screened for antifungal activities against Fusarium oxysporum, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum at concentrations of 10 μg/mL and 100 μg/mL. Six compounds showed the strong inhibition of mycelium growth at a concentration of 10 μg/mL. All tested compounds revealed the great inhibitory activities against S. sclerotiorum at a concentration of 100 μg/mL.

Full text of DOI:10.3390/molecules24061001

molecules
Article
An Improved Method for the Quaternization of
Nicotinamide and Antifungal Activities of
Its Derivatives
2
3,4
Tamara Siber 1, Valentina Bušic´ , Dora Zobundžija 2, Suncˇica Roca 3, Dražen Vikic´-Topic´
,
1
2,
Karolina Vrandecˇic´ and Dajana Gašo-Sokacˇ
*
1
Faculty of Agrobiotechnical Sciences, Josip Juraj Strossmayer University of Osijek, Vladimira Preloga 1,
HR–31000 Osijek, Croatia; tamara.siber@pfos.hr (T.S.); kvrandecic@pfos.hr (K.V.)
Faculty of Food Technology, Josip Juraj Strossmayer University of Osijek, Kuhacˇeva 20, HR–31000 Osijek,
Croatia; vbusic@ptfos.hr (V.B.); dzobundzija@ptfos.hr (D.Z.)
NMR Centre, Ruđer Boškovic´ Institute, Bijenicˇka cesta 54, HR–10000 Zagreb, Croatia; sroca@irb.hr (S.R.);
vikic@irb.hr (D.V.-T.)
2
3
4
Department of Natural and Health Sciences, Juraj Dobrila University of Pula, Zagrebacˇka 30,
HR–52100 Pula, Croatia
*
Correspondence: dajana.gaso@ptfos.hr; Tel.: +385-31-224327
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 February 2019; Accepted: 9 March 2019; Published: 13 March 2019
Abstract: The quaternization reactions of nicotinamide, with different electrophiles: methyl iodide
and substituted 2-bromoacetophenones (4-Cl, 4-Br, 4-H, 4-CH3, 4-F, 4-OCH3, 4-Ph, 2-OCH3, 4-NO2)
are reported. The preparations were carried out by conventional synthesis and under microwave
irradiation in absolute ethanol and acetone. The synthesis performed by microwave dielectric heating
significantly improved yield, up to 8 times, and shortened down the reaction time from ca. one day
in conventional, to 10–20 min. The structures of the synthesized compounds were confirmed by
IR, 1H- and 13C-NMR spectroscopy, mass spectrometry and elemental analysis. The compounds
have been screened for antifungal activities against Fusarium oxysporum, Fusarium culmorum,
Macrophomina phaseolina and Sclerotinia sclerotiorum at concentrations of 10
µg/mL and 100
µg/mL.
Six compounds showed the strong inhibition of mycelium growth at a concentration of 10
µg/mL.
All tested compounds revealed the great inhibitory activities against S. sclerotiorum at a concentration
of 100 µg/mL.
Keywords: quaternization; nicotinamide; substituted 2-bromoacetophenones; microwave synthesis;
antifungal activity
1. Introduction
Nicotinamide is an important heterocyclic compound, which together with nicotinic acid, belongs
to vitamin B3. Vitamin B3 is biosynthetically converted to nicotinamide adenine dinucleotide
(NAD+), a versatile acceptor of hydride equivalents, to form the reduced dinucleotide, NADH.
Nicotinamide is a water-soluble B complex vitamin which is naturally present in animal products,
whole cereals and legumes [
of their antimicrobial [ ], fungicidal [
plant growth regulator activity [17], cytotoxic properties [18
1
]. Derivatives of nicotinamide have attracted much attention because
11], insecticidal [12 13] and herbicidal activity [14 16],
19], and antiangiogenesis activity [20].
2–
6
7–
,
–
,
Some nicotinamide derivatives, like boscalid, are used as commercial antifungicides (Figure 1). All
fungicide synthesis studies revealed changes in the sub-structural unit of the amide backbone group
of nicotinamide.
Molecules 2019, 24, 1001; doi:10.3390/molecules24061001
www.mdpi.com/journal/molecules

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Figure 1. The structure of boscalid.
Related to our previous research [21 22] in which we carried out the quaternization of vitamin B6
,
derivatives with substituted 2-bromoacetophenones, in this study we investigated whether vitamin B3
is a good-suited nucleophile for the microwave (MW) assisted quaternization reaction with the same
electrophiles. For this purpose, alkyl halides (methyl iodide) and substituted 2-bromoacetophenone
have been used as electrophiles. New potential fungicidal molecules were prepared by using a simple
and easily repeatable microwave assisted method. We investigated whether the derivatives with the
quaternary nitrogen atom and the phenacyl unit are effective fungicides comparing to commercial
agricultural ones. Fungicides are mainly toxic substances, so the goal of this work was to produce
environmentally friendly fungicides, the derivatives of vitamin B3, which will be less toxic than
commercial ones. In this research, fungicides from the group strobilurin and dithiocarbamates that
have been approved for a specific culture of fungi in Croatia have been used as the standard (Figure 2).
(Zn)y
Azoxystrobin
Mancozeb
Cyproconazol
Figure 2. Some fungicides used in commercial products.
2. Results
2.1. Chemistry
Here we report the synthesis of ten (1–10) nicotinamide derivatives (Schemes 1 and 2).
(A) EtOH, reflux, 17 h, rt
CH3I
(B) EtOH or acetone,
MW, 440 W, 20 min
I−
(1)
Scheme 1.
Conventional (A) and microwave irradiation (B) synthesis of 3-carbamoyl-1-
methylpyridinium iodide (1).

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(A) Acetone, reflux,
1 h at 60 oC and 24 h at rt
Br−
(B) EtOH or acetone,
MW, 440 W, 10 min
(2−10)
=
(2)
(3)
(4)
(5)
(9)
(6)
(10)
(7)
(8)
Scheme 2. Conventional (A) and microwave irradiation (B) synthetic routes to nicotinamide derivatives
with 2-bromoacetophenones (2–10).
Optimum conditions for performing microwave-assisted reactions were ascertained by carrying
out reaction of nicotinamide and 2-bromoacetophenone (4). The results, summarized in Table 1,
showed that the maximum yield of product (93%) was obtained by heating the sample at 440 W for
10 min.
Table 1. Screening of reaction times in the quaternization of nicotinamide and 2-bromoacetophenone
(4) at 440 W.
Time (min)
2
4
6
8
10
12
14
16
Yield (%)
22
28
55
71
93
85
89
90
The main disadvantages of the conventional method are a long reaction time and lower yields.
Conventional synthesis of nicotinamide and methyl iodide (1) took place up to 17 h, and with the
substituted 2-bromoacetophenone (3) up to 25 h.
Differences between results obtained by conventional synthesis of quaternary salts of nicotinamide
and by microwave-assisted method are shown in Table 2. The highest yield (78%) in the
conventional method was achieved by quaternization of nicotinamide and methyl iodide (1). Among
the investigated substituted 2-bromoacetophenones, the highest yield (51%) was obtained with
2-brom-4-bromoacetophenone (
) (18%). All other compounds (
synthesized nicotinamide salts ( ) and (
3
). The lowest yield was found for compound (
10) have yields between 31–39%. Zhuravlev et al. have
) by conventional heating in anhydrous alcohol for 1 h, which
7) (11%) and for
(5
2, 6, 8–
4
7
gave rise to yield of 44% for compound (4), and 21% for compound (7) [23]. The same reactions were
performed in our study by conventional method in acetone, giving yields of 25% and 11%, respectively.

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Table 2. Experimental times and yields of products (1–10) obtained by conventional method and by
microwave-assisted method, in ethanol and acetone.
Conventional Method (A)
Compound
Microwave-Assisted Method (B)
EtOH
Acetone
Time (h)
Yield (%)
Time (min)
Solvent
Yield (%)
Yield (%)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
EtOH
17 (rt)
78
32
51
25
18
37
11
31
36
39
20
95
67
64
93
88
71
90
44
41
43
92
65
56
73
76
46
75
80
36
42
◦
1 (60 C) +
Acetone
10
24 (rt)
The nucleophilic substitution of halogen in the α-haloketones (3) is considerably easy because of
the greater electrophilicity of the carbocation adjacent to the acceptor carbonyl group. In nicotinamide
the substituent decreases the nucleophilicity of nitrogen due to its negative inductive –I effect
[σm(CONH2) = +0.28]. Consequently, the presence in the substrate molecule of a highly acceptor
carbonyl group, as in the case of phenacyl bromide, leads to redistribution of electron density in the
molecule, and the weakening of C–Br bond resulted in easier removal of the leaving group.
Significantly reducing reaction times from ca. 1 day in the conventional method to 10–20 min in
the MW procedure is the big advantage of using microwave synthesis. Generally, all obtained yields
in microwave synthesis are higher in ethanol than in acetone, except for (
most probably due to the influence of the phenyl group, and for ( ) and (10) where obtained yields
are almost equal in both solvents. The highest yields in ethanol and acetone were achieved for (
8) where it is the opposite,
2
1
)
(>92%). We can assume that methyl iodide is the most reactive since the iodides are better leaving
groups in nucleophilic substitution than bromides. Among substituted 2-bromoacetophenones, the
highest yield in ethanol was obtained for (
were found for ( ) (71%), ( ) (67%) and ( ) (64%), while the lowest yields were for (
between 41% and 44%. The highest yield in acetone was obtained for ( ) (80%), and only a little
less was yielded for ( ), ( ) and ( ) (73–76%). The yield for ( ) was 65%, for ( ) was 56%, and for
) was 46%. The lowest yields in ethanol and acetone were obtained for ( ), amounting 41% and
36%, respectively. This corresponds with steric hindrance of –OCH3 group in ( ), in ortho position
of 2-bromoacetophenone. The yield differences in used solvents can be attributed to the nature of
the molecules. The highest yields in ethanol can be explained with the tangent value, which is less
= 0.941), while
4
) (93%), (7) (90%) and (
5) (88%). Slightly lower yields
6
2
3
8–10), which are
8
4
5
7
2
3
(6
9
9
δ
for ethanol than for acetone [24]. Ethanol is a high medium absorbing solvent (tan
δ
acetone is a low medium one (tan δ = 0.054).
The chemical structures of investigated compounds were determined on the basis of spectral data
analysis of IR, 1H- and 13C-NMR spectra in solution, elemental analysis and mass spectral analysis.
All compounds show the absorption bands for
3400, 3350, 3180, 1690–1650 and 1620 cm–1. The absorption bands of aromatic rings,
1400 cm–1 and C–HAr 3030, 3010 cm–1, were also observed. The molecular ion peak of cationic form
with intensity of 100% was detected in the mass spectra of all investigated compounds.
In the 1H-NMR spectra of all synthesized compounds (
10) only one set of signals is present.
ν
N–H,
ν
NH2,
ν
NH2,
ν
C=O and
δ
N–C–C groups at
ν
C–CAr 1650, 1580,
ν
1–
The number, multiplicity and integrals of signals correspond to expected molecular structures. The
enumeration scheme used for the assignment of the NMR spectra is presented in Figure 3. The aromatic
area of 1H-NMR spectra contains four signals from pyridine moiety (from H-2 to H-6), two signals
from the amide group (NH2) and two doublets, corresponding to the phenyl protons (H-10/14 and
H-11/13, (2–8, 8) also contains three signals from
10)). The aromatic area of the 1H-NMR spectrum of (

Molecules 2019, 24, 1001
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the substituted phenyl ring (H-16/20, H-17/19, H-18). The aromatic area of the 1H-NMR spectrum
of ( ) contains four different signals because of the –OCH3 group substituted in position C-14. In the
1H-NMR spectra only signals of H-4 and H-6 atoms were overlapping and they were distinguished
9
by correlation signals in COSY spectra of (
2
5
) and (
8). Signal of H-7 atom was observed at 6.55 ppm,
and signals of –CH3 and –OCH3 group (
1
,
,
7,
9
) were found from 2.44 to 4.42 ppm. In the 13C APT
spectra, the most dshielded signals were of C-8 (189.6 ppm) and CONH2 (162.7 ppm) nuclei.
4
17
20
3
16
15
5
6
18
19
11
2
12
10
9
Br−
7
8
13
14
(8)
1
Figure 3. Structure and H-13C HMBC spectrum of (
8) in DMSO-d6. Red arrows and circles mark
the key correlation signals which proved the structure of obtained compound. The one-dimensional
600 MHz 1H-NMR spectrum is shown at the top edge and the 150 MHz 13C-NMR spectrum at the
left-hand edge.
All aromatic 13C nuclei were found in the range 150–120 ppm, so each signal was assigned
to its atom in the molecule by using C–H correlations over three bonds in HMBC spectra. The key
correlation signals were found between atoms H-7 and C-2/6, H-10/14 and C-8, and in the spectra of (8)
between H-16/20 and C-12 and H-11/13 and C-15 (Figure 3). These correlation signals unambiguously
confirmed the structures of obtained compounds. The C-7 atom signal is at 66.3 ppm, while C-signals
of methyl at 48.2 ppm (1) and at 21.2 ppm (5) and of methoxy groups at 55.8 ppm (7) and at 56.3 ppm
(9). NMR spectra of synthesized compounds can be found in Supplementary files.

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2.2. Antifungal Activity
Different concentration of compounds (
1
–10) were evaluated for their antifungal activities in vitro
against four fungal pathogens (Fusarium oxysporum, Fusarium culmorum, Macrophomina phaseolina,
Sclerotinia sclerotiorum), Table 3.
The newly synthesized compound (
fungal pathogens at concentrations of 10 and 100
F. oxysporum, great inhibitory activities at both concentrations were detected for compound (
against F. culmorum, ( ) showed the highest inhibitory effects. The compound ( ) showed the lowest
activity at concentration of 10 g/mL against M. phaseolina, while at concentration of 100 g/mL
the lowest activity was observed for compound ( ). The compounds ( ) and (10) indicate the lowest
3
) and (
4
) showed very good inhibitory activities against all
g/mL. More precisely, against M. phaseolina and
), while
µ
3
4
5
µ
µ
8
9
activity on mycelial growth of F. culmorum. All compounds tested here showed the great inhibitory
activities against S. sclerotiorum at a concentration of 100 µg/mL.
Compounds (1), (5), (7
), (9) and (10) also showed the strong inhibition of mycelium growth at
a concentration of 10
µ
g/mL, although between them and compounds (
2–
4
) and ( ), there was no
8
statistically significant difference. For S. sclerotiorum the same trend of mycelial growth inhibition is
continued over the next 72 h and 96 h. Comparing fungicides and the prepared and tested compounds
at both concentrations revealed no statistically significant difference between either, but the compounds
will be further tested with intention of deepening their mechanism of action.
Literature data suggest that the mechanism of action of the fungicide derived from the strobilurin
group proceeds through inhibition of mitochondrial respiration of fungi. The fungicides derived from
the dithiocarbamates group are preventively and curatively fungicides, not systemic ones characterized
by multiple-action.
The used fungicides are known as being highly efficient against Fusarium [25,26],
S. sclerotiorum [27] and M. phaseolina fungal pathogens [28]. It was confirmed in other investigations that
the pyridine carboxamide group of pesticides have a common target receptor, succinate dehydrogenase
(SDH, EC 1.3.5.1.). They act as SDH inhibitors (SDHIs) and disrupt the mitochondrial tricarboxylic
acid cycle and respiration chain [29].

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Table 3. The effect of different compounds on mycelial growth (mm) at 10 µg/mL and 100 µg/mL concentrations.
Mycelial Growth (mm)
M. phaseolina
F. culmorum
F. oxysporum
S. sclerotiorum
Compounds
10 µg/mL
100 µg/mL
10 µg/mL
100 µg/mL
10 µg/mL
100 µg/mL
10 µg/mL
100 µg/mL
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
21.5 ± 5.8
18.75 ± 4.6
16.25 ± 4.5
20.5 ± 5.2
23.75 ± 3.9
21 ± 6.2
16.75 ± 3.2
18.75 ± 4.3
15 ± 4.8
1.9 ± 4.5
23 ± 3.4
21 ± 5.2
20 ± 8.1
24.2 ± 3.9
22 ± 2.9
19 ± 3.6
25.25 ± 3
23.75 ± 1.5
24 ± 0
19.25 ± 0.9
20.25 ± 1.7
19.5 ± 0.6
18.5 ± 0.6
26 ± 1.8
15 ± 1.4
15 ± 2.8
15 ± 1.4
15.5 ± 0.6
16.5 ± 1
15 ± 1.6
13.75 ± 1.2
14.5 ± 1
16 ± 0.8
16.25 ± 0.5
4 ± 0
12.75 ± 1.2
14 ± 1.6
4 ± 0
4.5 ± 1
4.25 ± 0.5
4.75 ± 0.9
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
12.25 ± 0.9
13.5 ± 2.4
12.5 ± 1.3
12.5 ± 0.6
12.25 ± 0.9
16.25 ± 0.9
14.25 ± 0.9
15 ± 0.8
22.5 ± 1.9
25.75 ± 1.5
26.5 ± 2.6
26.5 ± 3.1
26.5 ± 2.2
29.5 ± 1.3
29 ± 1.1
26 ± 2.4
4 ± 0
20.5 ± 4.2
16.5 ± 6.4
20.5 ± 5.8
22.25 ± 2.2
25 ± 2.4
4 ± 0
31.75 ± 1.2
29.75 ± 0.9
31.75 ± 0.5
4 ± 0
4.25 ± 0.5
4.5 ± 1
4 ± 0
(10)
Strobilurin a
Dithio-carbamates a
LSD test
4 ± 0
4 ± 0
4 ± 0
4 ± 0
4 ± 0
6.91
6.34
2.79
2.06
1.91
1.74
0.80
0
a
Mean
±
SD determinated from Fhiser’s test, when these means were compared with controls, mean values were considered significantly different when p
≤
0.05 [30]. The commercial
fungicide strobilurin and dithiocarbamates were used for comparison.

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3. Materials and Methods
3.1. General Experimental Procedures
All reactions were performed in a controllable single-mode microwave reactor by Microwave
Synthesis Labstation Start S (Milestone, Shelton, CT, USA). The reactor is equipped with a magnetic
stirrer as well as with temperature and power controls (220 V/50–60 Hz, 2.4 kW).
Solvents and reagents were purchased from Merck (Darmstadt, Germany) and used without
further purification. To identify a compound by an investigation of its infrared spectrum Cary 630
FTIR, Agilent Technologies (Santa Clara, CA, USA) was used with DTGS (Deuterated triglycine sulfate)
detector. NMR spectra were recorded on Bruker AV600 spectrometer (Bruker, Rheinstetten, Germany),
operating at 600.135 MHz for the 1H nucleus and 150.903 MHz for the 13C nucleus. All data were
recorded in DMSO-d6 at 298 K in 5 mm NMR tubes. Chemical shifts in 1H and 13C spectra (
δ/ppm)
were referenced to the methyl signal of tetramethylsilane (TMS). Proton spectra with spectral width
of 12019 Hz and a digital resolution of 0.37 Hz per point were measured with 32 scans. 13C APT
spectra with spectral width of 39,370 Hz and a digital resolution of 0.60 Hz per point, respectively,
were collected with
≈
300 scans. Assignments of the 1H- and 13C-NMR signals were performed using
gradient-selected two-dimensional homo- and heteronuclear correlation experiments (1H-1H COSY,
1H-13C HMQC and H-13C HMBC). Melting points were determined with a Stuart melting point
apparatus SMP3 (Mettler Toledo, Croatia).
1
3.2. Synthesis of 3-Carbamoyl-1-methylpyridinium Iodide (1)
Conventional method (A): Nicotinamide (0.244 g, 2 mmol) were dissolved in 10 mL of absolute
ethanol and added to methyl iodide (0.187 mL, 3 mmol), which had also been previously dissolved in
10 mL of absolute ethanol. The reaction mixture was yellow after 17 h of reflux. Slow cooling of the
reaction mixture promoted the formation of pure yellowish pearlescent needles crystals. They were
washed with diethyl ether and recrystallized from methanol
MW synthesis in ethanol (B): To nicotinamide (0.244 g, 2 mmol) dissolved in 10 mL of absolute
ethanol, 0.187 mL of methyl iodide (3 mmol) was added. The reaction mixture was subjected to MW
irradiation (20 min at 440 W) until the product was visible on TLC. Slow cooling of the reaction mixture
promoted formation of pure yellowish pearlescent needles crystals. They were washed with diethyl
ether and recrystallized from methanol.
MW synthesis in acetone (B): To nicotinamide (0.244 g, 2 mmol) dissolved in 5 mL of acetone,
0.187 mL of methyl iodide (3 mmol) was added. The reaction mixture was subjected to MW irradiation
(20 min at 440 W) until the product was visible on TLC. Slow cooling of the reaction mixture promoted
formation of pure yellowish pearlescent needles crystals. They were washed with diethyl ether and
recrystallized from methanol.
3-carbamoyl-1-methylpyridinium iodide (
1
). Yield: conventional method, 411 mg, MW from ethanol
◦
501 mg, MW from acetone 487 mg; m.p. 203–204 C; IR (KBr) νmax 3324.8, 3153.3, 3011.7, 2780.6, 1848.8,
1677.3, 1440.2, 1388.6, 1118.2, 775.3 cm–1; 1H-NMR (DMSO-d6, 600.135 MHz):
(1H, d, J = 6.2 Hz, H-6), 8.91 (1H, td, J = 8.2, 1.59 Hz, H-4), 8.50 (1H, br s, NHa), 8.26 (1H, dd, J = 8.1,6.1
Hz, H-5), 8.13 (1H, br s, NHb), 4.42 (3H, s, CH3) ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
162.8 (C,
δ 9.41 (1H, s, H-2), 9.12
δ
CONH2) 147.1 (CH, C-6), 145.6 (CH, C-2), 142.8 (CH, C-4), 133.2 (C, C-3), 127.3 (CH, C-5), 48.2 (CH3,
CH3) ppm; MS m/z 137 [M+] (100); anal. Calcd. For C7H9N2OI: C 31.84, H 3.44, I 48.06, N 10.61, O
6.06%. Found: C 31.40, H 3.27, N 10.48%.
3.3. Synthesis of Nicotinamide Derivatives with Substituted 2-Bromoacetophenones (2–10)
Conventional method (A): Nicotinamide (0.244 g, 2 mmol) and substituted 2-bromoacetophenone
(2 mmol) were dissolved in 100 mL of acetone. The reaction mixture was mixed on a magnetic stirrer
◦
and heated for 1 h at 60 C, and for another 24 h at room temperature. The crude product was filtered

Molecules 2019, 24, 1001
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and washed with acetone. The reaction course was monitored by TLC chromatography with a mixture
of chloroform: methanol (6:1). Recrystallization from appropriate solvent gave pure product.
MW synthesis (B): To a solution of nicotinamide (0.244 g, 2 mmol) in 10 mL of solvent (abs. ethanol
or acetone), substituted 2-bromoacetophenones (2 mmol) were added and the reaction mixture was
irradiated at 440 W for a 10 min. The crystals were removed from the solution by filtration, washed
with diethyl ether and acetone and recrystallized from methanol.
3-Carbamoyl-1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium bromide (2). Yield: conventional method
227 mg, MW from abs. ethanol 480 mg, MW from acetone 462 mg; m.p. 252–254 ◦C, IR (KBr) νmax
1
3486.8, 3198.1, 1689.8, 1650, 1590, 1394, 1341, 984, 902, 708.2, 620.2, 574 cm–1; H-NMR (DMSO-d6,
600.135 MHz):
s, NHa), 8.41 (1H, dd, J = 8.2,6.1 Hz, H-5), 8.18 (1H, br s, NHb), 8.08 (2H, d, J = 8.5 Hz, H-10, H-14),
7.76 (2H, d, J = 8.5 Hz, H-11, H-13), 6.56 (2H, s, H-7) ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
189.7
δ 9.51 (1H, s, H-2), 9.15 (1H, d, J = 6.1 Hz, H-6), 9.12 (1H, d, J = 8.2 Hz, H-4), 8.64 (1H, br
δ
(C, C-8), 162.7 (C, CONH2), 147.7 (CH, C-6), 146.6 (CH, C-2), 144.0 (CH, C-4), 139.5 (C, C-12), 133.5 (C,
C-3), 132.2 (C, C-9), 130.1 (CH, C-10, C-14), 129.2 (CH, C-11, C-13), 127.4 (CH, C-5), 66.3 (CH2, C-7)
ppm; MS m/z 273.20 [M+](100); anal. Calcd. for C14H12BrClN2O2: C 47.29, H 3.40, Br 22.47, Cl 9.97, N
7.88, O 9.00. Found: C47.21, H 3.19, N 7.74%.
1-(2-(4-Bromophenyl)-2-oxoethyl)-3-carbamoylpyridin-1-ium bromide (
3). Yield: conventional method
410 mg, MW from abs. ethanol 510 mg, MW from acetone 450 mg; m.p. 256–258 ◦C, IR (KBr)
ν
max
3414.2, 3220.4, 2907.3, 1893.5, 1689.8, 1650, 1590, 1334.4, 1394, 1133.1, 984.0, 626.2, 566.6 cm–1; 1H-NMR
(DMSO-d6, 600.135 MHz): 9.50 (1H, s, H-2), 9.13 (1H, dt, J = 6.2, 1.2 Hz, H-6), 9.11 (1H, dt, J = 8.2,1.2
δ
Hz, H-4), 8.63 (1H, br s, NHa), 8.40 (1H, dd, J = 8.2, 6.2 Hz, H-5), 8.18 (1H, br s, NHb), 7.98 (2H, d,
J = 8.8 Hz, H-10, H-14), 7.90 (2H, d, J = 8.6 Hz, H-11, H-13), 6.53 (2H, s, H-7) ppm. 13C-NMR (DMSO-d6,
150.903 MHz):
δ 189.9 (C, C-8), 162.7 (C, CONH2), 147.8 (CH, C-6), 146.7 (CH, C-2), 144.0 (CH, C-4),
133.5 (C, C-3), 132.6 (C, C-9), 132.2 (CH, C-11, C-13), 130.2 (CH, C-10, C-14), 128.8 (C, C-12), 127.5 (CH,
C-5), 66.3 (CH2, C-7) ppm; MS m/z 321.10 [M+](100); anal. Calcd. For C14H12Br2N2O2: C 42.03, H 3.02,
Br 39.95, N 7.00, O 8.00. Found: C 42.10, H 2.76, N 6.74%.
3-Carbamoyl-1-(2-oxo-2-phenylethyl)pyridin-1-ium bromide (4). Yield: conventional method 160 mg, MW
from abs. ethanol 600 mg, MW from acetone 470 mg; m.p. 236.5–238 ◦C; IR (KBr) νmax 3333.2, 3265.1,
3175.7, 2899.9, 2825.3, 2109.8, 1915.9, 1984.8, 1617.7, 1493.4, 1341.8, 1207.7, 888.9, 767.8 cm–1; 1H-NMR
(DMSO-d6, 600.135 MHz):
δ
9.57 (1H, s, H-2), 9.21 (1H, d, J = 6.0 Hz, H-6), 9.15 (1H, d, J = 8.3 Hz
,
H-4), 8.67 (1H, br s, NHa), 8.42 (1H, dd, J = 8.0, 6.1 Hz, H-5), 8.21 (1H, br s, NHb), 8.08 (2H, dd, J = 8.2
,
1.1 Hz, H-10, H-14), 7.80 (1H, t, J = 7.4 Hz, H-12), 7.68 (2H, t, J = 8.2 Hz, H-11, H-13), 6.63 (2H, s, H-7)
ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
190.5 (C, C-8), 162.7 (C, CONH2), 147.8 (CH, C-6), 146.6
δ
(CH, C-2), 144.0 (CH, C-4), 134.7 (CH, C-12), 133.5 (C, C-9), 133.4 (C, C-3), 129.1 (CH, C-11, C-13),
128.2 (CH, C-10, C-14), 127.5 (CH, C-5), 66.3 (CH2, C-7) ppm; MS m/z 241 [M+](100); anal. Calcd. for
C14H13N2O2Br: C 52.36, H 4.08, N 8.72. Found: C 52.07, H 3.78, N 8.67%.
3-Carbamoyl-1-(2-oxo-2-(p-tolyl)ethyl)pyridin-1-ium bromide (
from abs. ethanol 590 mg, MW from acetone 510 mg; m.p. 248.3–248.8 C; IR (KBr)
5
). Yield: conventional method 120 mg, MW
◦
ν
3578.2, 3332.2,
max
3235.3, 3168.2, 3056.4, 2937.1, 2117.1, 1878.6, 1684.8, 1610.2, 1463.7, 1401.5, 1334.4, 1215.1, 118.2, 991.5,
986, 909.5, 805.1 cm−1; 1H-NMR (DMSO-d6, 600.135 MHz):
δ
9.55 (1H, s, H-2), 9.19 (1H, dt, J = 6.2
,
0.9 Hz, H-6), 9.13 (1H, dt, J = 8.1, 1.3 Hz, H-4), 8.66 (1H, br s, NHa), 8.41 (1H, dd, J = 8.1,6.2 Hz, H-5),
8.19 (1H, br s, NHb), 7.97 (2H, d, J = 8.04 Hz,H-10, H-14), 7.47 (2H, d, J = 8.04 Hz, H-11, H-13), 6.58
(2H, s, H-7), 2.44 (3H, s, CH3) ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
δ 189.9 (C, C-8), 162.7 (C,
CONH2), 147.7 (CH, C-6), 146.5 (CH, C-2), 145.3 (C, C-12), 143.9 (CH, C-4), 133.4 (C, C-3), 130.9 (C,
C-9), 129.6 (CH, C-11, C-13), 128.3 (CH, C-10, C-14), 127.4 (CH, C-5), 66.2 (CH2, C-7), 21.2 (CH3, C-15)
ppm; MS m/z 255 [M+] (100); anal. Calcd. mass for C15H15BrN2O2:C 53.75, H 4.51, Br 23.84, N8.36, O
9.55. Found: C 52.62, H 4.49, N 8.35%.

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3-Carbamoyl-1-(2-(4-fluorophenyl)-2-oxoethyl)pyridin-1-ium bromide (
6
). Yield: conventional method
250 mg, MW from abs. ethanol 480 mg, MW from acetone 309 mg; m.p. 246–249 ◦C; IR (KBr)
ν
max
3518.5, 3332.2, 3235.3, 3175.7, 3078.8, 2937.1, 2117.1, 1900.9, 1684.8, 1595.3, 1401.5, 1326.9, 1230.0, 1103.3,
909.5, 752.9, 834.9, 685.8, 566.6 cm–1; 1H-NMR (DMSO-d6, 600.135 MHz):
δ
9.52 (1H, s, H-2), 9.16 (1H,
d, J = 6.1 Hz, H-6), 9.13(1H, dt, J = 8.2, 1.3 Hz, H-4), 8.64 (1H, br s, NHa), 8.41 (1H, dd, J = 8.0, 6.0 Hz,
H-5), 8.19 (1H, br s, NHb), 8.16 (2H, m,H-10, H-14), 7.52 (2H, t, J = 8.8 Hz, H-11, H-13), 6.57 (2H, s, H-7)
ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
δ 189.1 (C, C-8), 165.7 (C, JC,F = 253 Hz, C-12), 162.7 (C,
CONH2), 147.7 (CH, C-6), 146.6 (CH, C-2), 144.0 (CH, C-4), 133.5 (C, C-3), 131.4 (CH, JC,F = 10 Hz, C-10,
C-14), 130.3 (C, JC,F = 3 Hz, C-9), 127.4 (CH, C-5), 116.2 (CH, JC,F = 22 Hz, C-11, C-13), 66.2 (CH2, C-7)
ppm; MS m/z 259 [M+] (100); anal. Calcd for C14H12BrFN2O2:C 49.58, H3.57, N 8.26, O9.43. Found: C
48.48, H3.47, N 8.05%.
3-Carbamoyl-1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridin-1-ium bromide (
7
). Yield: conventional method
◦
80 mg, MW from abs. ethanol 630 mg, MW from acetone 530 mg; m.p. 238–240 C; IR (KBr)
ν
max 3257.7,
3123.5, 2952.1, 2947.7, 2117.1, 1900.9, 1677.3, 1602.8, 1401.5, 1244.9, 1125.7, 641.1, 605.1, 574.0, 395 cm–1;
1H-NMR (DMSO-d6, 600.135 MHz):
δ
9.54 (1H, s, H-2), 9.19 (1H, dt, J = 6.1, 1.0 Hz,H-6), 9.13 (1H, dt,
J = 8.1, 1.4 Hz, H-4), 8.65 (1H, br s, NHa), 8.40 (1H, dd, J = 8.2,6.0 Hz, H-5), 8.19 (1H, br s, NHb), 8.05
(2H, d, J = 8.8 Hz,H-10, H-14), 7.19 (2H, d, J = 8.8 Hz, H-11, H-13), 6.56 (2H, s, H-7), 3.91 (3H, s, H-15)
ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
δ 188.7 (C, C-8), 163.3 (C, C-12), 162.8 (C, CONH2), 147.8
(CH, C-6), 146.6 (CH, C-2), 143.9 (CH, C-4), 133.4 (C, C-3), 130.7 (CH, C-10, C-14), 127.4 (CH, C-5), 126.3
(C, C-9), 114.4 (CH, C-11, C-13), 66.7 (CH2, C-7), 55.8 (CH3, C-15) ppm; MS m/z 271 [M+] (100); anal.
Calcd. for C15H15BrN2O3: C 51.30, H 4.31, Br 22.75, N 7.98, O 13.67. Found: C51.21, H 4.05, N 7.73%.
1-(2-([1,10-Biphenyl]-4-yl)-2-oxoethyl)-3-carbamoylpyridin-1-ium bromide (8). Yield: conventional method
250 mg, MW from abs. ethanol 350 mg, MW from acetone 630 mg; m.p. 252.5–261 ◦C, IR (KBr)
ν
max
3362.1, 3287.5, 3123.5, 2877.5, 2117.1, 1886, 1677.3, 1620, 1453.7, 1386.6, 1244.9, 1148, 998.9, 634.9, 775.3,
581.5 cm–1; 1H-NMR (DMSO-d6, 600.135 MHz):
δ
9.58 (1H, s, H-2), 9.21 (1H, d, J = 6.1 Hz, H-6), 9.15
(1H, dt, J = 8.3, 1.4 Hz, H-4), 8.67 (1H, br s, NHa), 8.43 (1H, dd, J = 8.1, 6.1 Hz, H-5), 8.21 (1H, br s, NHb),
8.16 (2H, d, J = 8.7 Hz, H-10, H-14), 7.99 (2H, d, J = 8.5 Hz, H-11, H-13), 7.83 (2H, dd, J = 8.8, 1.4 Hz,
H-16, H-20), 7.55 (2H, t, J = 7.2 Hz, H-17, H-19), 7.48 (1H, t, J = 7.2 Hz, H-18), 6.63 (2H, s, H-7) ppm.
13C-NMR (150.903 MHz, DMSO-d6, 25 ◦C):
δ 190.0 (C, C-8), 162.7 (C, CONH2), 147.7 (CH, C-6), 146.6
(C, C-2), 145.8 (1C, C-12), 144.0 (CH, C-4), 138.4 (C, C-15), 133.5 (C, C-3), 132.3 (C, C-9), 129.1 (CH, C-17,
C-19), 129.0 (CH, C-10, C-14), 128.7 (CH, C-18), 127.4 (CH, C-5), 127.1 (CH, C-16, C-20), 127.0 (CH,
C-11, C-13), 66.3 (CH2, C-7) ppm; MS m/z: 317 [M+] (100); anal. Calcd. for C20H17BrN2O2: C 60.47, H
4.31, Br 20.11, N 7.05, O 8.05. Found: C 60.52, H 4.14, N 6.68%.
3-Carbamoyl-1-(2-(2-methoxyphenyl)-2-oxoethyl)pyridin-1-ium bromide (
9). Yield: conventional method
250 mg, MW from abs. ethanol 287 mg, MW from acetone 253 mg; m.p. 245–247 ◦C; IR (KBr)
ν
max
3339.7, 3235.3, 3135.3, 2952.1, 2117.1, 1886.0, 1684.8, 1595.3, 1461.1, 1386.6, 1289.7, 1207.7, 909.5, 760.4,
678.4, 588.6 cm–1; 1H-NMR (DMSO-d6, 600.135 MHz):
9.54 (1H, s, H-2), 9.17 (1H, d, J = 6.0 Hz, H-6),
9.10 (1H, dt, J = 8.3, 1.2 Hz, H-4), 8.63 (1H, br s, NHa), 8.38 (1H, dd, J = 8.0, 6.2 Hz, H-5), 8.17 (1H, br s,
NHb), 7.89 (1H, dd, J = 7.8, 1.8 Hz, H-10), 7.75 (1H, ddd, J = 7.3, 1.8 Hz, H-12), 7.36 (1H, d, J = 8.4 Hz
H-13), 7.16 (1H, m, H-11), 6.33 (2H, s, H-7), 4.05 (3H, s, OCH3) ppm. 13C-NMR (DMSO-d6, 150.903
MHz): 189.8 (C, C-8), 162.7 (C, CONH2), 160.0 (C, C-14), 147.7 (CH, C-6), 146.4 (CH, C-2), 143.8 (CH,
δ
,
δ
C-4), 136.3 (CH, C-12), 133.3 (C, C-3), 130.3 (CH, C-10), 127.2 (CH, C-5), 122.8 (C, C-9), 120.9 (CH,
C-11), 113.1 (CH, C-13), 70.0 (CH2, C-7), 56.3 (CH3, C-15) ppm; MS m/z 271 [M+] (100); anal. Calcd. for
C15H15BrN2O3: C 51.30, H 4.31, Br 22.75, N 7.98, O 13.67. Found: C 51.50, H 4.39, N 7.77%.
3-Carbamoyl-1-[2-(4-nitrophenyl)-2-oxoethyl]pyridinium bromide (10). Yield: conventional method 252 mg,
MW from abs. ethanol 277 mg, MW from acetone 271 mg; m.p. 262.0–262.5 ◦C; IR (KBr)
ν
1707,
max
1525–1494, 1950, 1558, 1373 cm–1; 1H-NMR (DMSO-d6, 600.135 MHz):
δ 9.57 (1H, s, H-2), 9.21 (1H, d,
J= 6.0 Hz, H-6), 9.17 (1H, d, J= 8.0 Hz, H-4), 8.69 (1H, br s, NHa), 8.48 (2H, d, J = 8.80 Hz, H-11, H-13),

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8.44 (1H, dd, J= 8.0, 6.0 Hz, H-5), 8.32 (2H, d, J = 8.8 Hz, H-10, H-14), 8.22 (1H,br s, NHb), 6.68 (2H, s,
H-7) ppm. 13C-NMR (DMSO-d6, 150.903 MHz):
189.9 (C, C-8), 162.8 (C, CONH2), 150.6 (C, C-12),
δ
147.8 (CH, C-2), 146.8 (CH, C-6), 144.1 (CH, C-4), 138.2 (C, C-9), 133.5 (C, C-3), 129.7 (CH, C-10, C-14),
127.6 (CH, C-5), 124.1 (CH, C-11, C-13), 66.7 (CH2, C-7) ppm; MS m/z 367 [M+] (100); anal. Calcd. for
C13H11N2O3Br: C 48.32, H 3.43, N 8.67. Found: C 48.74, H 3.36, N 8.77%.
3.4. Antifungal Assay
Antifungal assay was performed on four cultures of phytopathogenic fungi (Fusarium oxysporum,
Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum) provided from the culture
collections of the Chair for phytopathology, Faculty of Agrobiotechnical Sciences, Josip Juraj
Strossmayer University of Osijek, Croatia. The fungicidal activity of ten synthetic compounds at
concentrations of 10 µg/mL and 100 µg/mL were tested.
For antifungal assays sterilized Petri dish (9 cm in diameter) were used. Each Petri dish was
filled with 10 mL of mix of PDA and one of synthetic compounds of specific concentrations. Agar
plugs (4 mm diameter) of one week old culture were picked up with a sterilized needle and placed in
the center of the Petri dishes. For antifungal assays was used according to the method proposed by
Wu et al. [8]. Petri dishes were stored in the chamber at 22
±
1 ◦C, with 12 h light/12 h dark regime
and 70% RH. The fungi colony diameter of each culture was measured 48 h after inoculation.
Each treatment was performed in four replicates. As a control 1% DMSO was used. Commercially
agricultural fungicide was used as positive control. The fungicide Amistar Extra with active ingredient
from the strobilurin group was used for F. culmorum, F. oxysporum and S.sclerotiorum, while the fungicide
Star 80 WP with active ingredient from the dithiocarbamates group was used for M. phaseolina. The
fungicides were mixed with PDA in the recommended concentration.
Statistical analysis of data was performed using factorial analysis of variance ANOVA by grouping
the data depending on coating and concentration applied. Fisher’s LSD test was applied to estimate the
statistical significance of differences between different synthetic compounds and concentrations using
SAS 9.2 Statistical Package (SAS Institute Inc., Cary, NC, USA) [30]. Mean values were considered
significantly different when p ≤ 0.05.
4. Conclusions
The quaternization reactions of nicotinamide with different electrophiles—methyl iodide and
2-bromoacetophenones derivatives were carried out by conventional synthesis and by microwave
irradiation synthesis, in absolute ethanol and acetone. By using microwave dielectric heating we
obtained significantly improved yields, up to 8 times greater, and reduced reaction times down
to 10–20 min. The synthesized compounds have been screened for antifungal activities against
Fusarium oxysporum, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum at
concentrations of 10
µg/mL and 100
µg/mL. Six compounds showed the strong inhibition of mycelium
growth at concentration of 10
µg/mL, while all tested compounds showed the great inhibitory activities
against S. sclerotiorum at a concentration of 100 µg/mL. Such investigations offer an alternative for
more environmentally acceptable fungicides.
Supplementary Materials: The following are available online, copies of 1H- and 13C-NMR spectra, copies of
1H-1H COSY NMR spectra of compounds ( ), copy of 1H-13C HMQC NMR spectrum of compound (
) and ( ),
copies of 1H-13C HMBC NMR spectra of compounds (2), (3) and (9).
2
8
8
Author Contributions: D.G.-S., V.B. and D.Z. designed the experiment and synthesized compounds. D.V.-T. and
S.R. performed NMR experiments and analyzed data, K.V. and T.S. evaluated antifungal activity of compounds.
All authors have written, read and approved the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no competing financial interest.

Molecules 2019, 24, 1001
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