Synthesis and antifungal activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols

Article abstract of DOI:10.1002/ardp.200900136

Mono-Mannich bases, 1-aryl-3-phenethylamino-1-propanone hydrochlorides, 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, and semi-cyclic mono-Mannich bases, 3-aroyl-4-aryl-1-phenethyl-4-piperidinols, 1b, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, were synthesized by a non-classi

Full text of DOI:10.1002/ardp.200900136

Arch. Pharm. Chem. Life Sci. 2010, 343, 291–300
E. Mete et al.
291
Full Paper
Synthesis and Antifungal Activity of 1-Aryl-3-phenethylamino-
1-propanone Hydrochlorides and 3-Aroyl-4-aryl-1-phenethyl-4-
piperidinols
Ebru Mete1, Canan Ozelgul2, Cavit Kazaz1, Dilsad Yurdakul3, Fikrettin Sahin3, and Halise Inci Gul2
1 Department of Chemistry, Faculty of Sciences, Ataturk University, Erzurum, Turkey
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
3 Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University,
Istanbul, Turkey
Mono-Mannich bases, 1-aryl-3-phenethylamino-1-propanone hydrochlorides, 1a, 2a, 3a, 4a, 5a,
6a, 7a, 8a, 9a, and semi-cyclic mono-Mannich bases, 3-aroyl-4-aryl-1-phenethyl-4-piperidinols, 1b,
2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, were synthesized by a non-classical Mannich reaction. The aryl part
was: C6H5 for 1a, 1b; 4-CH3C6H4 for 2a, 2b; 4-CH3OC6H4 for 3a, 3b; 4-ClC6H4 for 4a, 4b; 4-FC6H4 for
5a, 5b; 4-BrC6H4 for 6a, 6b; 2,4-(Cl)2C6H3 for 7a, 7b; 4-NO2C6H4 for 8a, 8b; and C4H3S(2-yl) i.e., 2-
thienyl for 9a, 9b. Piperidinol compounds 2b, 3b, 4b, 5b, 7b, 8b, and 9b are reported here for the
first time. The synthesized compounds were tested against seven types of plant pathogenic fungi
and three types of human pathogenic fungi using the agar dilution assay. Itraconazole was
tested against Candida parapsilosis as the reference compound, while Nystatin was tested as the
reference compound against the other fungi. Compounds 1a, 1b, 2a, 4a, 4b, 5a, 5b, 6a, 7a, 8a, 9a,
and 9b can be selected as model compounds to develop new antifungal agents against the
human pathogen Microsporum canis. Compounds 8a and 8b, which had a similar antifungal
activity compared with the reference compound Nystatin against the plant pathogen Aspergillus
flavus, can serve as model compounds to develop new antifungal agents to solve agricultural
problems.
Keywords: Antifungal activity / Mono-Mannich bases / Piperidinol / Synthesis /
Received: June 17, 2009; Accepted: October 14, 2009
DOI 10.1002/ardp.200900136
reaction [1]. Mannich bases have several biological activ-
ities such as antimicrobial [2–11], cytotoxic [4, 12–26],
anticancer [1, 27–30], analgesic [31], anti-inflammatory
[4, 32–35], diuretic [36], and anticonvulsant activities
[37–40]. It has been reported that they have an inhibiting
effect on DNA topoisomerase I [12, 13] and II [41].
Primary and opportunistic fungal infections in
humans continue to increase rapidly because of the
increased number of immunocompromised persons
such as patients with AIDS, cancer, and transplants [42].
In addition, the development of resistance to current
antifungal therapeutics continues to drive the search for
more effective new drugs.
Introduction
Mannich bases are generally formed by the reaction
between a compound containing a reactive hydrogen
atom, formaldehyde, and a secondary amine. On occa-
sion, aldehydes other than formaldehyde may be
employed, and the secondary amine may be replaced by
ammonia and primary amines. The process whereby
these compounds are formed is known as the Mannich
Correspondence: Professor H. Inci Gul, PhD., Department of Pharma-
ceutical Chemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzu-
rum, Turkey.
E-mail: incigul1967@yahoo.com
Fax: +90 442 236-0962
Yet, fungal infections are also existent in plants; these
pathogens cause paleness, leaf burns, and decay of
plants, decrease the quality and yield of crops in agricul-
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Table 1. 1H-NMR and 13C-NMR spectra of the synthesized compounds.
Compound 1H-NMR (DMSO)
13C-NMR (DMSO)
1b
d: 1.56 (br d, 1H, J = 13.2 Hz), 2.00–2.07 (m, 1H), 2.58–2.93
(m, 8H), 4.43 (dd, 1H, J = 11.4, 3.7 Hz), 4.93 (d, OH, J = 1.8 Hz), 128.5, 128.902, 128.947, 129.3, 129.4, 134.4, 136.8, 141.1,
7.02–7.58 (m, 13H), 7.82 (dd, 2H, J = 8.4, 1.1 Hz). 148.3, 204.3.
d: 33.5, 39.9, 49.1, 50.6, 52.7, 60.2, 73.3, 125.5, 126.5, 127.1,
2b
d: 1.52 (br d, 1H, J = 13.6 Hz), 1.91–1.97 (m, 1H), 2.14 (s, 3H), d: 21.1, 21.8, 33.5, 40.1, 49.2, 50.1, 52.8, 60.0, 73.2, 125.3,
2.32 (s, 3H), 2.59–2.91 (m, 8H), 4.39 (dd, 1H, J = 11.4, 3.7 Hz), 126.5, 128.9, 129.1, 129.2, 129.4, 130.1, 134.1, 136.0, 141.1,
4.95 (d, OH, J = 2.2 Hz), 6.97 (d, 2H, J = 8.4 Hz), 7.14–7.27 (m, 145.2, 145.5, 204.2.
7H), 7.39 (d, 2H, J = 8.1 Hz), 7.78 (d, 2H, J = 8.4 Hz).
3b
4b
5b
6b
d: 1.51 (br d, 1H, J = 13.6 Hz), 1.91–1.97 (m, 1H), 2.58–2.89
(m, 8H), 3.62 (s, 3H), 3.80 (s, 3H), 4.37 (dd, 1H, J = 11.4, 3.3
Hz), 5.04 (d, OH, J = 1.8 Hz), 6.72 (d, 2H, J = 8.8 Hz), 6.97 (d,
2H, J = 8.8 Hz), 7.14–7.28 (m, 5H), 7.43 (d, 2H, J = 8.8 Hz),
7.87 (d, 2H, J = 8.8 Hz).
d: 33.5, 40.2, 49.2, 49.7, 53.0, 55.5, 56.3, 60.1, 73.1, 113.9,
114.8, 126.5, 126.6, 128.9, 129.3, 129.4, 131.5, 140.6, 141.1,
158.4, 164.5, 203.2.
d: 1.55 (br d, 1H, J = 13.6 Hz), 2.04–2.11 (m, 1H), 2.56–2.91
(m, 8H), 4.30 (dd, 1H, J = 11.0, 3.7 Hz), 4.96 (d, OH, J = 1.1
Hz), 7.15 (quasi d, 2H, J = 8.8 Hz), 7.21–7.28 (m, 5H), 7.43
(quasi d, 2H, J = 8.8 Hz), 7.50 (quasi d, 2H, J = 8.4 Hz), 7.74
(quasi d, 2H, J = 8.8 Hz).
d: 33.5, 39.6, 48.9, 51.3, 52.3, 60.2, 73.1, 126.5, 127.7, 128.3,
128.9, 129.3, 129.4, 130.7, 131.8, 136.0, 138.9, 141.1, 147.2,
202.5.
d: 1.55 (br d, 1H, J = 13.6 Hz), 2.04–2.11 (m, 1H), 2.57–2.90
(m, 8H), 4.35 (dd, 1H, J = 11.0, 3.7 Hz), 4.96 (d, OH, J = 1.1
Hz), 6.94 (br t, 2H, J = 8.9 Hz), 7.14–7.28 (m, 7H), 7.52 (dd,
2H, J = 8.8, 5.5 Hz), 7.84 (dd, 2H, J = 8.9, 5.1
Hz).
d: 33.5, 39.8, 49.0, 51.1, 52.5, 60.2, 73.1, 115.5 (d,2JC-F = 124
Hz), 115.8 (d,2JC-F = 126 Hz), 126.5, 127.7 (d,3JC-F = 8 Hz), 128.9,
129.4, 131.9 (d, 3JC-F = 9 Hz), 133.9 (d, 4JC-F = 2 Hz), 141.1, 144.3
(d,4JC-F = 3 Hz), 161.5 (d,1JC-F = 243 Hz), 165.7 (d,1JC-F = 252 Hz),
202.3.
d: 1.55 (br d, 1H, J = 13.6 Hz), 2.02–2.09 (m, 1H), 2.55–2.92
(m, 8H), 4.28 (dd, 1H, J = 11.2, 3.5 Hz), 4.96 (d, OH, J = 1.5
Hz), 7.14–7.26 (m, 5H), 7.30 (d, 2H, J = 8.4 Hz), 7.43 (d, 2H,
J = 8.4 Hz), 7.56 (d, 2H, J = 8.4 Hz), 7.64 (d,
d: 33.5, 39.6, 48.9, 51.3, 52.3, 60.2, 73.1, 120.4, 126.5, 128.1,
128.2, 128.9, 129.4, 130.8, 131.3, 132.3, 136.3, 141.1, 147.6,
202.6.
2H, J = 8.8 Hz).
7b
8b
d: 1.04 (t, 1H, J = 6.9 Hz), 1.38–1.41 (m, 1H), 2.41–2.97 (m,
8H), 4.55 (dd, 1H, J = 11.0, 2.9 Hz), 5.59 (d, OH, J = 1.5 Hz),
7.01 (d, 1H, J = 8.4 Hz), 7.11 (dd, 1H, J = 8.4, 1.8 Hz), 7.15–
7.29 (m, 8H), 7.63 (d, 1H, J = 8.8 Hz).
d: 1.92 (br d, 1H, J = 14.3 Hz), 2.98–3.05 (m, 1H), 3.15–3.77
(m, 8H), 4.86 (dd, 1H, J = 11.3, 3.3 Hz), 6.14 (s, OH), 7.24–
d: 33.7, 35.7, 48.7, 51.0, 52.4, 60.4, 73.7, 126.5, 127.2, 127.4,
128.9, 129.4, 129.5, 130.2, 130.4, 130.9, 131.0, 131.5, 132.9,
135.5, 139.2, 141.2, 142.7, 202.4.
d: 30.3, 36.3, 48.5, 49.8, 51.5, 57.3, 72.1, 123.67, 123.69,
127.51, 127.55, 129.3, 129.4, 129.8, 137.7, 143.4, 147.0,
7.40 (m, 5H), 7.46 (quasi d, 2H, J = 8.8 Hz), 7.56 (quasi d, 2H, 149.5, 153.2, 198.2.
J = 8.8 Hz), 7.89 (quasi d, 2H, J = 8.8 Hz), 7.94 (quasi d, 2H,
J = 8.8 Hz).
9b
d: 1.77 (br d, 1H, J = 13.6 Hz), 2.00–2.07 (m, 1H), 2.52–2.94
(m, 8H), 4.15 (dd, 1H, J = 11.4, 3.7 Hz), 5.42 (d, OH, J = 1.1
Hz), 6.82 (dd, 1H, J = 4.9, 3.5 Hz), 7.05 (dd, 1H, J = 3.5, 1.3
Hz), 7.14–7.30 (m, 7H), 7.94–7.97 (m, 2H, overlapped 2H
of thiophene).
d: 33.5, 41.0, 48.9, 52.8, 53.2, 60.0, 72.7, 122.8, 124.5, 126.5,
127.7, 128.9, 129.4, 129.7, 135.3, 137.1, 141.1, 144.4, 154.4,
196.1.
ture, and increase the cost to produce crops, which reported findings directed us to synthesize mono-Man-
means that plant pathogens are harmful for the agricul- nich bases, 1-aryl-3-phenethylamino-1-propanone hydro-
tural economy [43].
chlorides, and semi-cyclic mono-Mannich bases, 3-aroyl-
It has been reported that Mannich bases of conjugated 4-aryl-1-phenethyl-4-piperidinols, and to evaluate their
styryl ketones [10], isatin N-Mannich bases [44], acetophe- antifungal activity.
none derived mono- and bis-Mannich bases, piperidinols
It has also been possible to see the alterations in biolog-
and azine derivatives of mono-Mannich bases [2, 3, 5, 6, ical activity of the compounds depending on their chem-
10] have antifungal activity. Further, Mannich ketones ical structures, which allowed us to find the most suit-
possess bioactivity which may be due to the alkylating able compounds for further studies to develop new effec-
ability of a,b-unsaturated ketones that are liberated in tive antifungal compounds against pathogens in humans
vivo following deamination [15, 17, 45–49]. These and/or plants.
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Antifungal Activity of Mannich Bases
293
Table 2. Results of UV, IR, and MS spectra of the synthesized compounds.
UV§
kmax
MS (m/z)
IR (KBr, cm–1)
C=O stretching OH stretching
IR (KBr, cm–1)
Compound Aryl
Exact
Mass
LogE
C (10–5 M)
[M+]
1b
2b
3b
4b
5b
C6H5
385.2
413.2
445.2
453.1
421.2
252
261
251
260
211
250
263
205
257
265
203
235
265
294
4.07
4.31
4.31
4.30
4.24
3.88
4.30
4.57
3.93
4.43
4.14
3.95
3.94
3.91
5.20
4.83
4.48
4.40
11.86
385.5
413.8
445.7
453.8
421.4
1679
1671
1664
1677
1676
3204
3326
3324
3167
3181
4-CH3C6H4
4-CH3OC6H4
4-ClC6H4
4-FC6H4
6b
7b
4-BrC6H4
2,4-(Cl)2C6H3
541.0
521.1
3.68
9.50
541.4
521.7
1676
1665
3433
3462
8b
9b
4-NO2C6H4
C4H3S(2-yl)
475.2
397.1
3.15
8.80
475.8
397.5
1684
1642
3390
3387
§ UV spectra of the compounds were taken in ethanol except for compound 7b, which was taken in methanol.
Table 3. Results of the elemental analyses of the synthesized compounds.
Compound Formula
Aryl
Elemental Analyses
Calculated (%)
N
Found (%)
N
C
H
S
C
H
S
1b
2b
3b
4b
5b
6b
7b
8b
9b
C26H27NO2
C28H31NO2
C28H31NO4
C26H25Cl2NO2
C26H25F2NO2
C26H25Br2NO2
C26H23Cl4NO2
C26H26ClN3O6
C22H23NO2S2
C6H5
81.01
81.32
75.48
68.72
74.09
57.48
59.68
61.00
66.47
7.06
7.56
7.01
5.55
5.98
4.64
4.43
5.12
5.83
3.63
3.39
3.14
3.08
3.32
2.58
2.68
8.21
3.52
81.41
81.24
75.66
68.76
74.28
57.16
59.30
61.13
66.36
7.03
7.59
7.04
5.43
5.87
4.64
4.50
5.15
5.82
3.66
3.69
3.29
3.27
2.94
2.61
2.56
8.18
3.68
4-CH3C6H4
4-CH3OC6H4
4-ClC6H4
4-FC6H4
4-BrC6H4
2,4-(Cl)2C6H3
4-NO2C6H4
C4H3S(2-yl)
16.13
16.28
None of the compounds were found to be effective
against Fusarium oxysporum CE1, Botrytis cinerea MFD3,
and Candida albicans EA07 at the concentration range
studied. On the other hand, Nystatin was also ineffective
against Microsporum canis AO5 at the concentration range
studied. The antifungal-activity results of the compounds
are shown in Table 4.
Results and discussion
Of the compounds synthesized, piperidinol compounds,
2b, 3b, 4b, 5b, 7b, 8b, and 9b, will be reported for the first
time in this study. The chemial structures of the com-
pounds were confirmed by 1H-NMR, 13C-NMR, UV, IR, and
MS spectra and the purity level of the compounds was
determined by elemental analyses. The spectral data of
the mono-Mannich bases were reported in our previous
Synthesis of the compounds
study [50] and the spectral data for the piperidinol com- The mono-Mannich bases 1a–9a reported in this study
pounds are shown in Tables 1 and 2. The results of the ele- were synthesized as described in our previous study by
the classical Mannich reaction in an acidic solution of
mental analyses for the piperidinols, which are reported
here for the first time, are shown in Table 3. The elemen- ethanol under reflux conditions [50]. The optimal reac-
tal-analyses (C, H, N, S) results of the compounds were tion conditions for the synthesis of these compounds, 1-
within € 0.4% of the calculated values.
aryl-3-phenethylamino-1-propanone
hydrochlorides,
were investigated by changing the molar ratios of reac-
Itraconazole was tested against Candida parapsilosis
EA08 as the reference compound, while Nystatin was tants and solvent, and the acidity level of the reaction
tested as the reference compound against other fungi. medium using compound 1a and 9a as representative
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Table 4. Antifungal activities of the synthesized compounds as minimal inhibitory concentration (MIC in lg/mL).
Compound Aryl
Rhizoctonia Sclerotinia Aspergillus Alternaria Macro-
Microspo-
rum
canis AO5
Candida
parapsilosis
EA08
solani 2001 sclerotio-
rum FD3
flavus FD7
alternata
FS2002
phamina
phaseoli
CE4
1a
1b
2a
2b
3a
3b
4a
4b
C6H5
C6H5
50
25
25
4-CH3C6H4
4-CH3C6H4
4-CH3OC6H4
4-CH3OC6H4
4-ClC6H4
200
200
200
25
4-ClC6H4
200
5a
4-FC6H4
25
100
5b
6a
6b
7a
7b
8a
8b
9a
4-FC6H4
12.5
25
4-BrC6H4
4-BrC6H4
2,4-(Cl)2C6H3
2,4-(Cl)2C6H3
4-NO2C6H4
4-NO2C6H4
C4H3S(2-yl)
C4H3S(2-yl)
100
25
50
100
12.5
12.5
100
100
100
100
50
200
200
25
25
9b
Nystatin
Itraconazole
12.5
25
200
50
12.5
compounds from our previous paper [50]. It has been the ß-aminopropiophenones compared with the dihydro-
reported that the most suitable mol ratio of the reactants quinolones [31].
was 1:1.2:1, compared with 2:2:1 suitable for ketone,
Compound 4a was synthesized by heating the ketone,
paraformaldehyde, and phenethylamine hydrochloride. paraformaldehyde, and amine (1:1.7:1 equivalent ratio)
The most suitable reaction medium was ethanol with in acidic isopropanol with a yield of 35% (as given in [51]).
added concentrated hydrochloric acid (compared to the This compound had a muscle-relaxant activity at 300
medium without solvent and only ethanol) [50]. As mg/kg [51]. As described in detail above, the reported
reported in the literature, compound 3a, is a mono-Man- compounds 3a and 4a were synthesized according to the
nich base with methoxy substitution [31], and compound classical Mannich reaction. There is no bioactivity data
4a, is a mono-Mannich base with a chloride substitution related to mono-Mannich bases, except for compounds
[51].
3a and 4a. The mono-Mannich bases presented in this
Here, a brief description of the synthesis of 3a [31]: 0.1 study were synthesized by an experimental procedure
mol of suitable ketone and 0.12 mol amine hydrochlor- different from that in our previous study [50].
ide were heated in ethanol for a while; 0.12 mol para-
Of the semi-cyclic mono-Mannich bases or piperidi-
formaldehyde was added and the heating continued for nols, the non-substituted phenyl derivative 1b and the 4-
7 h. More paraformaldehyde (0.05 mol) was added and bromophenyl derivative 6b are reported in the literature
1
the heating continued for another 2 h. Then, the solvent [52–54]: Upton et al. reported the potent H-antagonistic
was evaporated under low pressure, water was added to activity of indeno[2,2-c]pyridines and their 4-arylpiperidi-
1
the reaction medium, and the mixture was washed with nol precursors [52]. They reported to have an H-antago-
diethyl ether. The mixture was alkalized by 50% NaOH, nistic activity of 26 and 23 nM, respectively, at the 1H-hist-
washed and dried. Compound 3a was obtained by passing amine receptor in guinea-pig ileum with the compounds
HCl gas through the ether solution of the substance. In having a piperidine structure; the aryl part in this case
the above-mentioned reference [31], similar compounds was phenyl, and the alkyl residues on nitrogen were
i.e., b-aminopropiophenones, were designed as open- methyl and ethyl. The activity level was comparable with
chain analogues of the dihydroquinolones and their that for the clinically-established phenindamine, which
analgesic effects were tested. b-Aminopropiophenones has an IC50 value of 36 nM. These data made Upton et al.
had better analgesic activity than the other compounds. screen eleven piperidine compounds in vivo, including
This can be explained by a better receptor suitability of compound 1b of this study, using the 48/80 challenge
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Arch. Pharm. Chem. Life Sci. 2010, 343, 291–300
Antifungal Activity of Mannich Bases
295
test. None of the compounds was capable of protecting
mice against a lethal dose of 48/80. The calculated lipo-
philicity for most of the piperidines including compound
1b was between 10 to 100 times that of the analogue,
with the methyl substitution on the nitrogen of the
piperidine structure. Researchers attributed this situa-
tion to the affected pharmacokinetics of the drug with
respect to its site of action [52].
Compound 1b was synthesized according to [53] by
heating of 0.1 mol of phenethylamine dissolved in 20 mL
of ethanol together with a solution of 0.2 mol of b-dime-
thylamino propiophenone hydrochloride in 50 mL water
on a water bath for 2 h. The oily base, which was pro-
duced, was taken up in diethyl ether and converted to
the hydrochloride salt with isopropanolic hydrochloride.
The hydrochloride salt of compound 1b was obtained in
34% yield (13 g). The compound was recrystallized from
ethanol. The reported melting point was 204–2058C [55];
in [53], the melting point of compound 1b is given as
202–2038C together with UV and IR spectral data. In both
cases, compound 1b was obtained in form of the hydro-
chloride salt, while, in this study, it is in base form. Yet,
the synthetic methods used in the literature differed
from ours used in this study. Phenethylamine and a
mono-Mannich base different from its corresponding
one were used for the synthesis of the piperidinol com-
pound 1b in the literature.
The synthesis of the bromine-containing piperidinol
compound 6b as described in [54]: 0.25 equivalent amine
hydrochloride were added to the solution of one equiva-
lent arylmethyl ketone and one equivalent paraformal-
dehyde in acetonitrile; then, the mixture was refluxed
for 20 h in the presence of hydrochloric acid. After cool-
ing the reaction mixture to room temperature, the sol-
vent was removed under vacuum. The residue was dis-
solved in dichloromethane and washed with bicarbonate
solution, water, and brine, respectively. After the organic
phase was dried, the crude compound was purified by
column chromatography using triethylamine/diethyl
ether as eluent. The bromo compound 6b was obtained
in 74% yield. 1H-NMR and 13C-NMR data of this compound
are reported in reference [54]. In [56], the yield of com-
pound 6b is also reported with 74%, but it was obtained
as a syrup. This differs from our result: in our hands 6b is
solid. This compound is reported to be an inhibitor of the
dopamine transporter for illnesses related to dopamine
transportation. Having a phenethyl substituent on the
nitrogen atom, 6b had shown 18 times lower activity
compared to the compound having an ethyl substituent
on the nitrogen. In a structure-activity relationship study
it was noted that the positions of the substituent on two
phenyl rings play an important role for the binding and
Figure 1. Chemical structure of the synthesized compounds.
re-absorption of this type of compounds [54]. Addition-
ally, with this series of compounds [54], a molecular-mod-
eling study was also realized. As seen, compound 6b was
synthesized by a classical Mannich reaction using aceto-
nitrile as reaction solvent, which distinguishes it from
the method used in this study.
The synthetic pathway used here to synthesize all com-
pounds included taking ketone, aldehyde, and amine
hydrochloride in the mol ratio 2:2:1 and heating them in
a medium without solvent. This is a non-classical
method, differing from the methods described in the
above mentioned literature [50, 52, 54–56]. The advan-
tage of the method used here makes it possible to synthe-
size both compounds simultaneously (the mono-Man-
nich bases and piperidinols) in a single reaction. The syn-
thesized mono-Mannich bases come in the form of HCl
salts, the piperidinol compounds in base form, except for
compound 8b, which comes as HCl salt. The reason for
this was the purification by direct crystallization of com-
pound 8b from the reaction medium, while the other
piperidinol-type compounds were purified by passing
them through a basic aluminum oxide column.
Spectral data of the compounds confirmed their chem-
ical structures (Fig. 1). As an example, and in agreement
1
with the chemical structure, H- and 13C-NMR data of
compound 2b (Fig. 2) are as follows: While the H15 pro-
tons on the aromatic ring neighboring the carbonyl give
doublets (J = 8.4 Hz) at d = 7.78 ppm, H20 protons on the
aromatic ring connected to piperidine give doublets at d
= 7.39 ppm (J = 8.1 Hz), when the 1H-NMR spectrum of the
compound was analyzed. Multiplet signals at d = 7.14–
7.27 ppm belong to the H10, H11, H12, and H16 protons
on the benzene rings. The H21 protons of the 4-methyl-
substituted benzene ring connected directly to the piper-
idine ring and give a doublet at d = 6.97 ppm (J = 8.4 Hz).
The hydroxyl (OH) proton gives a doublet at d = 4.95 ppm
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Arch. Pharm. Chem. Life Sci. 2010, 343, 291–300
8a, and 9a were found to be effective against fungi, which
are pathogenic in human. Mono-Mannich bases, which
were effective against plant pathogenic fungi: the 2,4-
dichloro derivative, compound 7a, exhibited 50% of the
antifungal activity the reference compound Nystatin
against Rhizoctonia solani, while the bromo derivative 6a
was effective against Sclerotinia sclerotiorum (25% of the
reference). Compound 7a was twice as effective against
Aspergillus flavus as the reference compound. Antifungal
activity equal to Nystatin was found in compound 8a,
which is a nitro derivative, against Aspergillus flavus. The
methoxy derivative 3a and the chloro derivative 4a were
effective against Macrophamina phaseoli (25% of the refer-
ence compound).
Mono-Mannich bases effective against human patho-
genic fungi: While reference compound Nystatin was
ineffective against Microsporum canis, the non-substi-
tuted compound 1a was active at 50 lg/mL, the methyl
derivative 2a, the fluoro derivative 5a, the bromo deriv-
ative 6a, and compound 9a, which has thiophene ring,
Figure 2. Representative compound 3-(4-methylbenzoyl)-4-(4-
methylphenyl)-1-phenethyl-4-piperidinol numbered for the NMR
studies.
(J = 2.2 Hz) by long-distance interaction with the H5b pro- were active at 25 lg/mL, the 2,4-dichloro derivative 7a
ton. The H3 proton, which is neighbor to a carbonyl, has and nitro derivative 8a at 12.5 lg/mL, the chloro deriv-
been observed as a doublet of doublets at d = 4.39 ppm (J = ative 4a was found effective at 200 lg/mL. Fluoro deriv-
11.4, 3.7 Hz) by interacting with the H2 protons. Methy- ative 5a, 2,4-dichloro derivative 7a, nitro derivative 8a,
lene protons H2, H6, and H7, which are next to a nitrogen and compound 9a (thiophene ring) had an antifungal
in the piperidine ring, and the benzylic proton H8 proton activity of 12.5% of that of the reference compound Itra-
have been observed as multiplets at d = 2.59–2.91 ppm. conazole.
Singlet signals at d = 2.32 ppm and d = 2.14 ppm belong to
methyl protons, which are connected to both aromatic Antifungal activity of semi-cyclic mono-Mannich
rings. The H5b proton belonging to the piperidin ring bases, piperidinols (Table 4)
gives a multiplet between d = 1.91–1.97 ppm, while the Semi-cyclic mono-Mannich base compounds, piperidi-
H5a proton shows a wide doublet at d = 1.52 ppm.
nols, 4b, 7b, and 8b were found to be effective against
In the 13C-NMR spectrum of the compound, carbonyl fungi, pathogenic to plants, while compounds 1b, 4b, 5b,
carbon at d = 204.2 ppm, aromatic carbons at d = 145.5, and 9b were found to be effective against fungi, patho-
145.2, 141.1, 136.0, 134.1, 130.1, 129.4, 129.2, 129.1, genic in humans. Semi-cyclic mono-Mannich bases i.e.,
128.9, 126.5, 125.3 ppm, methylen and methine carbons piperidinols effective against plant fungi can be
at d = 73.2, 60.0, 52.8, 50.1, 49.2, 40.1, 33.5 ppm, methyl described as follows: the 2,4-dichloro derivative 7b had
carbons at d = 21.8, 21.1 ppm have been observed in an antifungal activity of 25% of the reference compound
accordance with the chemical structures. A strong against Rhizoctonia solani and 50% activity of the refer-
absorption peak has been observed in the IR spectrum of ence compound against Alternaria alternata, while the
compound 2b corresponding to a carbonyl at 1671 cm–1 nitro derivative, compound 8b, had antifungal activity
(Table 2) confirming the chemical structure. The maxi- equal with the reference drug against Aspergillus flavus.
mum absorption band in the UV spectrum of compound The chloro derivative 4b had 25% activity of the reference
2b was found at 261 nm in accordance with its structure compound against Alternaria alternata.
(Table 2). The MS spectrum of the same compound taken
The effects of semi-cyclic mono-Mannich bases i.e.,
by EI method provided the m/z peak at 413.8, which corre- piperidinols, which were effective against human patho-
sponds to the base form of compound 2b (Table 2).
genic fungi, can be summarized as follows: Piperidinols
had antifungal activity against Microsporum canis, against
which reference compound Nystatin was uneffective.
Antifungal activity of mono-Mannich bases (Table 4)
Mono-Mannich base compounds 3a, 4a, 6a, 7a, and 8a Non-substituted phenyl 1b, the chloro derivative 4b, and
were found to be effective against fungi, which are patho- compound 9b in which the aromatic part is thiophene,
genic in plants, while compounds 1a, 2a, 4a, 5a, 6a, 7a, showed antifungal activity at a concentration of 25
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Antifungal Activity of Mannich Bases
297
lg/mL, while the fluoro derivative 5b showed activity at
12.5 lg/mL.
The antifungal activity was specific and intense against
Microsporum canis as can be seen in Table 4. Furthermore,
The effect of the replacement of the phenyl ring with a the reference compound Nystatin was not found effective
thiophene ring on the antifungal activity of the com- against this microorganism in the concentration range
pounds could only be observed against Microsporum canis studied. To conclude, compounds 1a, 1b, 2a, 4a, 4b, 5a,
for compounds 1a and 9a, and 1b and 9b. Antifungal 5b, 6a, 7a, 8a, 9a, and 9b can be selected as model com-
activity increased two times when the antifungal activity pounds to develop new antifungal agents against the
of compounds 1a and 9a was compared, while it did not human pathogen Microsporum canis. In addition, com-
change when the antifungal activity of compounds 1b pounds 8a and 8b, which had similar antifungal activity
and 9b was compared. This may suggest that the effect of with the reference compound Nystatin against the plant
ring replacement on the antifungal activity is limited. pathogen Aspergillus flavus, can serve as model com-
Changes in the electronic nature of the compounds did pounds to develop new antifungal agents to solve agricul-
not affect the bioactivity very much.
tural problems.
Yet, the bioactivity was affected when the chemical
structure of the compounds was changed from a mono-
Mannich base to a semi-cyclic mono-Mannich base.
According to this, the antifungal activity against Rhizocto-
nia solani decreased by half in piperidinol when compar-
ing the bioactivities of 7a and its analogue 7b. However,
the antifungal activity was not affected when the bioac-
tivities of 8a and its analogue 8b were compared against
Aspergillus flavus. The antifungal activity against Microspo-
rum canis increased two times in the piperidinol com-
pound when the bioactivities of 1a and its analogue 1b
were compared, while antifungal activity increased eight
times in piperidinol when the bioactivities of compound
4a and its analogue 4b were compared. Antifungal activ-
ity increased two times for the piperidinol compound 5b
compared with 5a against Microsporum canis. However,
the antifungal activity was not affected when the bioac-
tivities of compounds 9a and 9b were compared against
the same microorganism.
Although compounds 5a, 7a, 8a, and 9a, which are
mono bases, were effective at 100 lg/mL against Candida
parapsilosis, their corresponding piperidinols were inef-
fective at the concentration range studied. The mono-
Mannich bases 3a and 4a were effective at 200 lg/mL
against Macrophamina phaseoli, while their correspond-
ing piperidinols were ineffective. Mono-Mannich bases
6a and 7a were effective at 100 lg/mL against Sclerotinia
sclerotiorum and Aspergillus flavus, while their correspond-
ing piperidinols were ineffective at the concentration
range studied. On the other hand, piperidinol com-
pounds 4b and 7b were effective against Alternaria alter-
nata, while their corresponding mono derivatives were
ineffective.
Experimental
Materials
The following chemicals were used for the synthesis of the com-
pounds in this study: acetophenone, 49-methylacetophenone, 49-
nitroacetophenone, 49-chloroacetophenone, 2-acetylthiophene
(Fluka, Steinheim, Switzerland), 49-methoxyacetophenone, 49-flu-
oroacetophenone, 49-bromoacetophenone, 29,49-dichloroaceto-
phenone (Acros, Geel, Belgium), paraformaldehyde (Merck,
Darmstadt, Germany), methanol, ethyl acetate (Riedel-deHaꢀn,
Seelze, Germany), diethyl ether (Fluka, Steinheim, Switzerland),
and ethanol (J. T. Baker, Deventer, Holland). The 1H- and 13C-NMR
spectra were recorded at 400/100 MHz on a Varian spectrometer
(Varian, Danbury, CT, USA). EI-MS spectra were recorded on a
Thermo-Finnigan mass analyzer (San Jose, CA, USA). UV spectra
of compounds were recorded on a Thermo-Electron Hekios (a)
(UVA 114903) spectrometer (Cambridge, UK). Infrared spectra
were obtained for KBr disks on a Mattson 1000 FT-IR spectropho-
tometer (Cambridge, England). Elemental analyses were carried
out with a Leco CHNS-932 instrument (Leco, St. Joseph, MI, USA).
Melting points were determined on a Bꢁchi 530 (Flawil, Switzer-
land).
Synthesis of mono-Mannich bases and piperidinols
1a–9a and 1b–9b (Fig. 1)
1H-NMR, 13C-NMR, UV, IR, and MS spectral data of semi-cyclic
mono-Mannich bases, piperidinol compounds 1b–9b are shown
in Tables 1 and 2. The purity level of the compounds was deter-
mined by elemental analyses and the results are shown in Table
3. Elemental analyses (C, H, N, S) results of the compounds were
within € 0.4% of the calculated values. Spectral data and elemen-
tal analyses (C, H, N, S) results for mono-Mannich bases were
reported in our previous study [50].
Suitable amounts of ketone, paraformaldehyde, and phenyl-
ethylamine hydrochloride in the mol ratio 2:2:1 were stirred
and heated in an oil bath to synthesize the compounds.
For the compounds 1a, 5a, and 9a, the solid starting com-
pounds began to melt when the temperature reached 88, 90,
and 828C, respectively. The reaction content became clear, trans-
parent mass at 928C, 928C, and 858C, for the compounds 1a, 5a,
and 9a, respectively. Heating was stopped by removing the reac-
tion flask from the oil bath. The temperature inside the balloon
flask spontaneously and suddenly increased to 100, 107, and
1068C, respectively.
Mono-Mannich bases had antifungal activity especially
against Sclerotinia sclerotiorum, Aspergillus flavus, Macro-
phamina phaseoli, Candida parapsilosis, and Rhizoctonia sol-
ani, while the semi-cyclic mono-Mannich bases i. e., piper-
idinols, had antifungal activity especially against Alterna-
ria alternata.
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In the case of compounds 2a, 7a, and 8a, the temperature
started to increase suddenly, without the observation of a melt-
ing or clearing of the starting compounds when the tempera-
ture of the reaction medium reached 87, 93, 808C, respectively.
The reaction flask was quickly removed from the oil bath. The
temperature inside the balloon spontaneously and suddenly
increased to 110, 108, and 1028C, respectively.
When the temperature reached 878C for the compound 3a, all
solid substances in the reaction medium started to melt, the
reaction content turned clear, and the temperature started to
increase spontaneously. Then, the reaction flask was quickly
removed from the oil bath. The temperature of the reaction con-
tent reached 1108C in a short period of time for the compound
3a.
When the temperature reached 928C for the compound 4a,
the solid substance started to melt. When heating continued,
the reaction content solidified again totally. The reaction flask
was quickly removed from the oil bath. The temperature of the
reaction medium spontaneously increased to 1048C.
When the temperature reached 918C for the compound 6a,
the temperature of the reaction content suddenly started to
increase without melting and becoming clear. The reaction flask
was removed from the oil bath. The temperature of the reaction
content reached to 1038C.
In all reactions, following the increase in temperature and
removal of the flask from the oil bath, ethyl acetate (20 mL) was
added to the reaction flask when the temperature had dropped
to 658C. Stirring was continued for 24 h. The formed precipitates
were separated by filtration.
Compound 1a was crystallized from ethyl acetate. Com-
pounds 2a, 6a, 7a, and 8a were crystallized from methanol and
compounds 3a, 4a, and 5a were crystallized from ethanol. After
filtration through the basic Al2O3 column, 9a was crystallized in
ethanol and dried by washing with diethyl ether. The synthetic
yields of the mono-Mannich bases 1a–8a, and 9a were 18%, 24%,
20%, 28%, 33%, 35%, 35%, 30%, and 16%, respectively.
After isolation of compounds 1a, 2a, and 3a from the reaction
medium, the ethyl acetate present in the reaction flask was
removed under low pressure to obtain the piperidine com-
pounds 1b, 2b, and 3b. NaOH (5%) solution was added to the
obtained viscous substances of orange color. The reaction con-
tent was stirred in a water bath at 408C. Reaction content looked
like an emulsion with oil in the beginning; it became viscous
and solidified by time. Stirring was continued for 24 h for the
compound residues of 1a and 2a, while it continued for 120 h
for the residue of 3a, until it was precipitated. The precipitates
were filtered and washed with water. The crude product was
crystallized from methanol. Compounds 1b and 2b obtained
yielded white solid substances with 30 and 21%, respectively. A
viscous substance of yellow color, which was obtained after
removal of the compound 3a, was filtered through the basic
Al2O3 column using ethyl acetate/hexane (60:40) solvent system.
The solvent was removed under low pressure. The solid sub-
stance obtained was crystallized from methanol. The solid white
compound 3b was obtained with a yield of 24%. The melting
points of the compounds 1b, 2b, and 3b were 139–1418C, 129–
1318C, and 112–1138C, respectively.
and recrystallized from methanol five times. 8b was obtained as
primrose-yellow solid compound with a yield of 22%. The melt-
ing point of the compound was 213–2158C.
After isolating compounds 4a, 5a, 6a, 7a, and 9a from the reac-
tion medium, ethyl acetate present in the reaction flask was
removed under low pressure to obtain the compounds 4b, 5b,
6b, 7b, and 9b. The obtained viscous compounds 4b, 5b, 6b, 7b
and 9b of orange color were purified by column chromatogra-
phy using basic Al2O3 column. The solvent systems used for chro-
matography of the particular compounds were as follows: ethyl
acetate/hexane (10:90) for 4b and 7b, ethyl acetate/hexane (5:95)
for 5b, methanol/ethyl acetate (10:90) for 6b, ethyl acetate/
methanol (10:90) for 9b. The solvent was removed under low
pressure. However, compounds 4b, 5b, 6b, and 9b were recrystal-
lized with methanol, and 7b was recrystallized from ethanol/
diethyl ether. The compounds obtained and the yields were as
follows: 4b solid white with a yield of 18%, 5b solid white with a
yield of 31%, 6b solid white with a yield of 18%, 7b solid white
with a yield of 28%, 9b and solid white with a yield of 28%. The
melting points of the compounds 4b, 5b, 6b, 7b, and 9b were
132–1348C, 145–1468C, 130–1328C, 122–1248C, and 155–1568C,
respectively.
Determination of the antifungal activity
Rhizoctonia solani 2001, Fusarium oxysporum CE1, Sclerotinia scle-
rotiorum FD3, Aspergillus flavus FD7, Alternaria alternata FS2002,
Macrophamina phaseoli CE4, Botrytis cinerea MFD3 as plant patho-
genic fungi and Microsporum canis AO5, Candida albicans EA07,
Candida parapsilosis EA08 as human pathogenic fungi were used
to determine the antifungal activities of the compounds. Micro-
organisms were provided by the Department of Genetics and
Bioengineering, Faculty of Engineering and Architecture, Yedi-
tepe University, Istanbul, Turkey. Antifungal activities of the
compounds were tested using the agar dilution assay in the con-
centration range of 6.25 to 200 lg/mL. Minimal inhibitory con-
centrations (MIC) of the compounds were determined. Nystatin
(Pharmatech, Denver CO, USA) and Itraconazole (Matrix Pharma)
were used as reference compounds.
Microwell dilution assay
The minimal inhibitory concentration (MIC) values were deter-
mined for the yeast isolates, which were sensitive to compounds
in disc diffusion assay. The inocula of the Candida isolates were
prepared from 12-h broth cultures and suspensions were
adjusted to 0.5 McFarland standard turbidity. Compounds dis-
solved in dimethylsulfoxide (DMSO) were first diluted to the
highest concentration (200 lg/mL) to be tested, and then serial
two-fold dilutions were made in order to obtain a concentration
range from 6.25 to 200 lg/mL in 15 mL sterile test tubes contain-
ing Sabouraud dextrose broth (SDB) for yeast. MIC values of com-
pounds against yeast isolates were determined based on a micro-
well dilution method [6]. The 96-well plates were prepared by dis-
pensing 95 lL of nutrient broth and 5 lL of the inoculum into
each well. 100 lL from the stock solutions of compounds pre-
pared at the concentration of 200 lg/mL was added into the sec-
ond wells. Then, 100 lL from their serial dilutions was trans-
ferred into five consecutive wells. The last well on each strip con-
taining 195 lL of nutrient broth without compound and 5 lL of
the inoculum was used as negative control. The final volume in
each well was 200 lL. PSA and Nystatin and Itraconazole at the
concentration range of 200 to 6.25 lg/mL were prepared in
After isolating 8a from the reaction medium, ethyl acetate
present in the reaction flask was removed under low pressure to
obtain compound 8b. Methanol was added to the obtained vis-
cous substance of orange color and the mixture was kept at
room temperature. The crystals formed were taken by filtration
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Arch. Pharm. Chem. Life Sci. 2010, 343, 291–300
Antifungal Activity of Mannich Bases
299
nutrient broth and Sabouraud dextrose broth was used as stand-
ard drug for the positive control. 200 lL of nutrient broth was
transferred into the first wells as positive control. The plate was
covered. The contents of all wells were gently mixed on a plate
shaker at 300 rpm for 20 s and then incubated at appropriate
temperatures for 24 h. Microbial growth in each medium was
determined by reading the respective absorbance at 600 nm
using the ELx 800 universal microplate reader (Biotek Instru-
ment Inc, Highland Park, VT, USA) and confirmed by plating 5
lL samples from clear wells on nutrient agar medium. The MIC
was defined as the lowest concentration of the compounds to
inhibit the growth of microorganisms.
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MIC values of the fungi isolates were studied based on the agar
dilution method, as described previously [6]. Compounds were
added aseptically to sterile molten potato dextrose agar (PDA)
medium containing compounds at the appropriate volume to
produce the concentration range of 6.25 to 200 lg/mL. The
resulting PDA solutions were immediately poured into Petri
plates after vortexing. The plates were spot-inoculated with 5 lL
(104 spore/mL) of each fungal isolate. Nystatin and Itraconazole
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