Antimicrobial activities of new analogues of benzalkonium chloride

Article abstract of DOI:10.1016/S0223-5234(99)00216-0

(Alkoxymethyl)dimethyl{2-hydroxy-5-[(4-X-phenyl)azo]benzyl}ammonium chlorides were prepared in high yield. All these chlorides, new analogues of benzalkonium chloride, showed antimicrobial activity. Activity depends on the length and kind of substituent a

Full text of DOI:10.1016/S0223-5234(99)00216-0

Eur. J. Med. Chem. 34 (1999) 765−771
© 1999 Editions scientifiques et médicales Elsevier SAS. All rights reserved
765
´
Laboratory note
Antimicrobial activities of new analogues of benzalkonium chloride
J. Pernaka*, I. Mirskab, R. Kmiecika
aPoznan University of Technology, Sklodowskiej-Curie 2, 60-965 Poznan, Poland
bK. Marcinkowski University of Medical Sciences, Sieroca 10, 61-771 Poznan, Poland
(Received 29 December 1998; revised 15 March 1999; accepted 18 March 1999)
Abstract – (Alkoxymethyl)dimethyl{2-hydroxy-5-[(4-X-phenyl)azo]benzyl}ammonium chlorides were prepared in high yield. All these
chlorides, new analogues of benzalkonium chloride, showed antimicrobial activity. Activity depends on the length and kind of substituent at
´
the quaternary nitrogen atom. © 1999 Editions scientifiques et médicales Elsevier SAS
analogue of benzalkonium chloride / 4-hydroxyazobenzenes / Mannich bases / chloromethylalkyl ether / antimicrobial activity
1. Introduction
2. Chemistry
Benzalkonium chloride (BAC) is the product of a
nucleophilic substitution reaction of alkyldimethylamine
with benzyl chloride [1]. Chemically, it is monoalkyldi-
methylammonium chloride with one long-chain alkyl
group representing a mixture of the alkyls from C8H17 to
C18H37. Following Domagk’s publication in 1935 [2], a
large number of application areas were developed for
BAC. It is used as a pharmaceutical aid (preservative),
cationic surface active agent, germicide, antiseptic (topi-
cal), antiseptic for skin preoperatively or for wounds,
burns, etc. BAC is often present as a preservative or
stabilising agent in nebulizer solutions used to treat
asthma and chronic obstructive pulmonary disease [3].
Also it is widely used as an antimicrobial agent in the
treatment of common infections of the mouth and throat.
Mannich reaction (or aminomethylation) of variously
substituted phenols is a well known process and compre-
hensive reviews have been published [4–7]. A few Man-
nich bases of phenolic azobenzenes have demonstrated
cytotoxicity towards murine and human cancers [8].
Mannich bases methiodides have a promising cytotoxic
activity in a wide variety of tumours [9].
The new analogues of benzalkonium chloride (3–10)
were prepared by the reaction of 2-[(dimethylamino)-
methyl]-4-[(4-chlorophenyl)azo]phenol (2a) or 2-[(dime-
thylamino)methyl]-4-[(4-methylphenyl)azo]phenol (2b)
with chloromethylalkyl or chloromethylcycloalkyl ethers
giving yields between 80–98%. In this case, Mannich
base is a compound which is easily transformed into a
quaternary ammonium salt.
We now report the synthesis and antimicrobial activi-
ties of new quaternary ammonium compounds, the ana-
logues of benzalkonium chloride. We plan to find com-
pounds with antimicrobial activity which are diluted in
water giving a coloured solution. Commercial products
with these compounds will not have to contain any dye.
Chloromethylalkyl and chloromethylcycloalkyl ethers
were synthesised from the corresponding alcohols. Man-
nich bases (2a–b) were prepared by treatment of the
4-hydroxyazobenzenes with equimolar quantities of
formaldehyde and dimethylamine in 75% yield.
The 4-hydroxyazobenzenes (azo dyes) were prepared
by several authors in the 19th century [10]. At the present
time, only one compound, 4-hydroxyazobenzene (Sol-
vent Yellow 7), is commercially available.
*Correspondence and reprints

766
3. Antimicrobial activity
substituent (5 and 8); and group 4, chlorides with
CH2O(CH2)nC6H11 substituent (6 and 10). The calculated
average MIC values for cocci, rods and fungi are shown
in figures 1 and 2. As shown by the results in these tables
and figures, all the chlorides studied are very active
against cocci and active against rods and fungi. The
microbial activity depends on the length and kind of
substituent at the quaternary nitrogen atom. Figures 1 and
2 reveal a decrease in the MIC value to the optimum
value and these values increase for chlorides from groups
1, 2 and 3. The same correlation is observed for the MBC
values. Generally the MBC values are slightly higher
than MIC values. To the most active compounds in group
1 belong chlorides which have butoxymethyl, hexyl-
oxymethyl and octyloxymethyl substituent, in group 2
chloride with heptyloxymethyl chain, and in group 3
chlorides which have cyclopentyloxymethyl, cyclohexyl-
oxymethyl and cycloheptyloxymethyl substituent. To the
worst compounds belong chlorides with dodecyloxy-
methyl and cyclododecyloxymethyl chain. Chlorides
from group 4 have comparable values of MIC. In this
group the microbial activity does not depend on the
substituent – CH2O(CH2)nC6H11 where n = 1 or 2.
All synthesised quaternary ammonium chlorides
(3–10) were tested for antimicrobial activity against
cocci, rods and fungi.
4. Results and discussion
4-Hydroxyazobenzenes react readily with formalde-
hyde and secondary amines to give Mannich bases. The
structures of two prepared mono Mannich bases (2a–b)
were characterized by their microanalysis CHN and by
1
their H and 13C NMR spectroscopy.
Quaternization of 2a and 2b with chloromethylalkyl or
chloromethylcycloalkyl ethers produced red crystalline
quaternary ammonium chlorides 3–10 (table I) diluted in
water. The water solution of these chlorides is stable and
1
orange in colour. H and 13C NMR-spectral analysis of
prepared chlorides allowed easy elucidation of their
structure.
The minimal inhibitory concentration (MIC) and the
minimal bactericidal concentration (MBC) values deter-
mined for all forty chlorides are given in tables II and III.
The chlorides studied were divided into four groups with
respect to the kind of substituent: group 1, chlorides with
an alkoxymethyl substituent with an even number of
carbon atoms (3 and 7); group 2, chlorides with the same
substituent but with an odd number of carbon atoms (4
and 8); group 3, chlorides with a cycloalkoxymethyl
The most active chlorides against microorganisms
were: {5-[(4-chlorophenyl)azo]-2-hydroxybenzyl}dimethyl-
(cyclohexyloxymethyl)ammonium (5b), {5-[(4-chloro-
phenyl)azo]-2-hydroxybenzyl}dimethyl(cyclohexyl-
methyloxymethyl)ammonium (6a), {2-hydroxy-5-[(4-
methylphenyl)azo]benzyl}dimethyl(hexyloxymethyl)-

767
Table I. (Alkoxymethyl)dimethyl{5-[(4-chlorophenyl)azo]-2-hydroxybenzyl}ammonium chlorides (3–6) and (alkoxymethyl)dimethyl{2-
hydroxy-5-[(4-methylphenyl)azo]benzyl}ammonium (7–10) chlorides.
Chloride
R1
R2
Yield (%) Chloride
R1
R2
Yield (%)
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
C2H5
C4H9
C6H13
C8H17
C10H21
C12H25
C3H7
C5H11
C7H15
C9H19
C11H23
C5H9
C6H11
C7H13
C8H15
C12H23
C6H10CH3
CH2C6H11
CH2CH2C6H11
CH2CH2CH2C6H11
80
96
98
90
85
86
90
80
80
90
96
80
80
90
80
80
80
90
86
85
7a
7b
7c
7d
7e
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C4H9
C6H13
C8H17
C10H21
C12H25
C3H7
C5H11
C7H15
C9H19
C11H23
C5H9
C6H11
C7H13
C8H15
C12H23
C6H10CH3
CH2C6H11
CH2CH2C6H11
CH2CH2CH2C6H11
80
86
90
80
80
96
80
87
80
80
90
88
84
92
80
86
84
80
80
80
a
a
a
a
a
a
a
a
a
a
7f
a
a
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
a
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
b
b
b
9f
b
b
6a
6b
6c
10a
10b
10c
b
b
b
b
a
b
linear alkyl, alicyclic.
ammonium (7c), {2-hydroxy-5-[(4-methylphenyl)azo]ben-
zyl}dimethyl(cyclopentyloxymethyl)ammonium (9a).
The results presented demonstrate that the new BAC
analogues are very active against cocci. Their activities
are similar to the activity of BAC. The antimicrobial
activities of BAC (Aldrich product, in which R represents
a mixture of alkyls from C8H17 to C18H37) against cocci,
Micrococcus luteus, Staphylococcus epidermidis and Sta-
phylococcus aureus as measured in the same MIC test are
1.5, 3.0 and 1.5 mmol/L, respectively. Tomlinson and
coworkers [11] reported the antibacterial activities of
homologues series (C8–C18) of alkylbenzyldimethyl-
ammonium chlorides against Pseudomonas aeruginosa.
BAC resistance is a potential problem for application, for
example in the food processing industry [12]. We found
compounds with large molecular weights, crystalline,
diluted in water and the orange water solution is stable.
Chloromethylalkyl ethers and chloromethylcycloalkyl
ethers were prepared via the procedures which were
reported earlier [13]. The percentage of ether in a crude
product was determined by an alkalimetric method [14].
2-[(Dimethylamino)methyl]-4-(phenylazo)phenols (2a–b);
general procedure:
Pathway A. To a solution of dimethylamine (30 mmol)
in 95% EtOH (10 mL) paraformaldehyde powder (0.9 g,
30 mmol) was added. The mixture was stirred and heated
when the paraformaldehyde had dissolved, then the
corresponding 4-hydroxyazobenzene (30 mmol) in
100 mL EtOH was added. The reaction mixture was
stirred for 1 h at 60 °C.
Pathway B. To 2-[(dimethylamino)methyl]phenol
(20 mmol) in 50 mL MeOH was added diazonium salt
prepared from dimethylamine (20 mmol). The mixture
was stirred at room temperature for 2 h. The solid
substrate was filtered and then recrystallized from EtOH.
2-[(Dimethylamino)methyl]-4-[(4-chlorophenyl)azo]-
phenol (2a): m.p. 120–122 °C, 1H NMR (CDCl3) δ
ppm = 11.6 (s, OH), 7.83 (dd, J = 6 Hz, 1H), 7.82 (d,
J = 9 Hz, 2H), 7.61 (d, J = 2 Hz, 1H), 7.45 (d, J = 8 Hz,
2H), 6.95 (d, J = 8 Hz, 1H), 3.72 (s, 2H), 2.36 (s, 6H);
13C NMR δ ppm = 161.8, 151.0, 145.5, 135.7, 129.1,
125.2, 123.6, 122.6, 122.1, 116.6, 62.6, 44.4.
5. Experimental protocols
5.1. Chemistry
NMR spectra were recorded on a Varian Model XL 300
1
spectrometer at 300 MHz for H and 75 MHz for 13C at
20 °C with tetramethylsilane as internal reference. Satis-
factory elemental analyses were obtained: C ± 0.32,
H ± 0.29 and N ± 0.24.
2-[(Dimethylamino)methyl]-4-[(4-methylphenyl)azo]-
phenol (2b): m.p. 99–100 °C, lit. 103 °C [10].

768
Table II. The MICa and MBCa values of examined chlorides (3–6).
Strainsb
Chlorides
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
6a
6b
6c
Cocci
M. luteus
MIC
MBC 21
21
10
10
18
18
17
34
32
32
59
118 78
78
37
37
9
18
33
518 61
61
19
19
36
71
18
18
34
34
479
479
9
9
9
9
8
8
16
16
S. epidermidis
S. aureus
MIC 21
10
18
9
18
8
17
8
16
30
30
156 73
156 146 18
9
16 31
128 31
19
19
18
18
9
9
8
17
8
118
9
9
18
18
8
8
16
64
MBC 21
MIC
260 38
MBC 260 76
217 267 252 238 156 146
217 534 252 477 1256 219 36
9
128 245
518 490 19
9
9
18
9
9
214
214
8
8
18
36
18
36
8
8
16
129
Rods
P. aeuruginosa
MIC
260 304 284 267 504 1908 2512 1750 275 518 980 73
71
553 429 1915 137 137 133 520
MBC 520 304 568 534 504 1908 2515 4695 550 2074 980 146 142 553 429 1915 256 553 268 520
P. vulgaris
MIC
81
76
18
132 252 477 628 219
267 504 954 2512 438 36
9
259 245 19
518 490 73
18
71
9
268 16
9
18
137 66
8
16
129
MBC 161 152 36
137 536 1915 68
K. pneumoniae
E. coli
MIC
260 304 141 132 504 954 1256 875
9
64
490 38
36
36
36
133 239 36
133 958 68
36
36
66
64
MBC 520 304 284 267 504 954 2512 1750 36
1037 980 146 71
133 129
MIC 81 38 141 132 125 477 628 438 136 259 490 38
MBC 326 76 141 267 252 954 628 875 275 1037 980 73
71
142 18
18
66
958 36
18
36
34
66
32
64
133 1915 36
S. marescens
MIC 1302 608 568 534 1008 3816 5025 1750 275 1037 1960 73
142 256 1073 3831 1106 68
133 1041
MBC 1302 1216 568 534 4032 3816 5025 4695 550 2074 1960 146 285 553 2146 3831 256 137 268 1041
Fungi
C. albicans
MIC
325 304 70
267 504 3816 1256 875 136 259 490 146 71
68
268 3831 256 68
34
129
260
MBC 651 608 141 534 1008 3816 2512 1750 1101 4149 980 146 142 256 536 3831 1106 137 66
T. mentagrophytes
Rh. rubra
MIC
161 76
18
66
125 477 1256 438 36
64
122 73
36
36
36
36
268 1915 137 68
286 1915 256 68
34
34
64
129
MBC 161 152 18
132 252 477 1256 875 36
2074 245 73
MIC 161 152 36
MBC 161 304 70
132 252 3816 628 219 36
267 504 3816 1256 219 68
259 490 38
2074 490 73
36
71
36
36
268 1915 256 36
268 3831 256 68
17
34
64
64
a
b
in mmol/L, the number of microorganisms in mL ranged from 104–105.
The quaternary ammonium chlorides 3–10 were pre-
{5-[(4-chlorophenyl)azo]-2-hydroxybenzyl}dimethyl-
(cyclohexyloxymethyl)ammonium chloride (5b): m.p.
120–123 °C, 1H NMR (DMSO-d6) δ ppm = 11.5 (s, OH),
8.07 (d, J = 2 Hz, 1H), 7.96 (dd, J = 7 Hz, 1H), 7.85 (d,
J = 9 Hz, 2H), 7.58 (d, J = 9 Hz, 2H), 7.38 (d, J = 9 Hz,
1H), 4.86 (s, 2H, CH2N), 4.60 (s, 2H, NCH2O), 3.84 (m,
1H), 3.05 (s, 6H), 1.92 (m, 2H), 1.74 (m, 2H), 1.49 (m,
6H); 13C NMR δ ppm = 159.3, 148.7, 143.0, 133.6,
128.3, 127.4, 124.4, 121.8, 115.2, 113.2, 82.5, 78.0
(NCH2O), 56.1 (CH2N), 44.4 [N(CH3)2], 33.4, 27.7,
21.9.
pared by dissolving 2-[(dimethylamino)methyl]-4-(X-
phenylazo)phenol in CH2Cl2 and adding an equimolar
amount of the appropriate chloromethylalkyl or chloro-
methylcycloalkyl ether. The mixture was stirred at room
temperature for 24 h. The solvent was evaporated and the
crude product was extracted three times with hexane.
Finally, the products were crystallized from
CH3COOC2H5/MeOH and dried in vacuum oven.
{5-[(4-chlorophenyl)azo]-2-hydroxybenzyl}dimethyl-
(dodecyloxymethyl)ammonium chloride (3f): m.p.
124–126 °C, 1H NMR (DMSO-d6) δ ppm = 11.9 (s, OH),
8.06 (d, J = 2 Hz, 1H), 7.96 (dd, J = 7 Hz, 1H), 7.86 (d,
J = 9 Hz, 2H), 7.66 (d, J = 9 Hz, 2H), 7.46 (d, J = 9 Hz,
1H), 4.82 (s, 2H, CH2N), 4.58 (s, 2H, NCH2O), 3.86 (t,
J = 7 Hz, 2H), 3.02 (s, 6H), 2.00 (m, 2H), 1.63 (m, 18H),
0.86 (t, J = 7 Hz, 3H); 13C NMR δ ppm = 161.4, 150.5,
144.6, 135.2, 130.5, 129.5, 125.9, 123.8, 117.1, 114.9,
90.0, 73.0 (NCH2O), 57.9 (CH2N), 46.3 [N(CH3)2], 33.7,
31.3, 29.2, 29.0, 28.8, 28.7, 25.3, 22.1, 13.9.
(Cycloheptyloxymethyl)dimethyl{2-hydroxy-5-[(4-methyl-
phenyl)azo]benzyl}ammonium chloride (9c): m.p.
1
152–154 °C, H NMR (DMSO-d6/CDCl3) δ ppm = 11.3
(OH), 8.03 (d, J = 2 Hz, 1H), 7.92 (dd, J = 6 Hz, 1H),
7.76 (d, J = 9 Hz, 2H), 7.34 (d, J = 9 Hz, 3H), 4.82 (s,
2H, CH2N), 4.59 (s, 2H, NCH2O), 4.02 (m, 1H), 3.05 (s,
6H), 2.42 (s, 3H), 1.99 (m, 2H), 1.81 (m, 4H), 1.58 (m,
4H), 1.54 (m, 2H); 13C NMR δ ppm = 158.7, 148.4,

769
Table III. The MICa and MBCa values of examined chlorides (7–10).
Strainsb
Chlorides
7a 7b 7c
7d 7e
7f
8a 8b 8c
8d 8e
9a 9b 9c
9d 9e
9f
10a 10b 10c BAC
Cocci
M. luteus
MIC
MBC 22 40
22 20
9
9
9
9
65 61 42 20 18
65 61 82 20 18 34 126 40 19 37
9
63 20 10
9
9
9
249
249
9
9
9
9
9
9
17 1.5
17
3
S. epidermidis
S. aureus
MIC 22 10
MBC 22 79
9
9
9
33 248 42 10
9
9
34
67
8
8
20 19
40 38 18 18 16 18
9
9
8
9
9
9
9
9
17
17
3
3
18 33 248 42 10
MIC 11 20 19
MBC 170 40 19
9
9
16 16 42 10
262 16 82 20 36 270 126 154 38 18
9
134 126 77 19
9
9
9
249
997
9
9
37
72
9
9
17 1.5
17
6
Rods
P. aeuruginosa
MIC
344 158 147 138 2103 3972 331 76 143 1083 2043 1239 148 143 139 1994 143 143 280 1088 96
MBC 688 319 298 279 4206 3972 662 308 288 4333 4086 1239 299 289 1122 1994 289 143 561 1088 192
P. vulgaris
MIC 85 79 19 69 262 496 662 308 270 255 20 19 280 16 18 17 12
9
9
9
9
MBC 688 319 298 69 262 993 662 154 143 1083 1021 619 299 18 280 249 289 143 139 561 12
K. pneumoniae
E. coli
MIC 344 79 19 138 262 993 331 39 18 67 63 40 38 18 36 997 18 72 18 67 12
MBC 344 158 38 138 529 1986 331 76 36 1083 255 154 74 71 139 997 37 72 139 134 24
MIC
85 40 19 69 529 1986 82 20 36 270 255 77 19 18 36 498
9
37 36 134
3
6
MBC 85 79 19 138 1051 1986 164 76 143 1083 510 154 38 37 70 997 18 37 69 134
S. marescens
MIC
688 319 147 279 4206 3972 1324 152 143 2166 4086 1239 148 289 1122 1994 289 143 561 2176 12
MBC 1376 638 596 279 4206 3972 1324 308 143 2166 2043 2478 299 289 1122 3988 289 143 561 2176 24
Fungi
C. albicans
MIC
688 319 38 36 1051 1986 331 152 288 541 1021 309 148 37 36 1994 72 143 139 272
3
3
MBC 1376 319 38 69 2103 1986 662 308 576 2166 1021 619 299 143 70 1994 143 289 280 272
T. mentagrophytes
Rh. rubra
MIC
170 40 38
9
262 248 164 39 288 1083 510 154 38 18
9
9
489 37 72 69 272
489 72 143 69 282
3
6
MBC 170 79 74 18 529 248 331 152 288 1083 255 308 148 71
MIC 344 158 19
9
1051 1986 164 20 143 270 255 154 74 37 18 997 72 72 69 272 12
MBC 344 158 38 36 1051 1986 331 39 143 1083 255 154 74 37 36 1994 72 72 131 272 12
a
b
in mmol/L, the number of microorganisms in mL ranged from 104–105.
143.3, 139.0, 127.9, 127.8, 124.4, 120.5, 115.2, 112.5,
86.6, 81.1, 56.4, 44.5, 31.9, 26.1, 20.3, 19.4 (CH3).
Microbiology, K. Marcinkowski University of Medical
Sciences, Poznan.
Antimicrobial activity was determined by the tube
dilution method. The method shows, the lowest concen-
tration of a chloride inhibiting cell multiplication (MIC)
or killing them (MBC). Two-fold dilutions of the chlo-
rides were prepared in the Mueller-Hinton broth medium
(bacteria) or in the Sabouraud broth medium (fungi). A
suspension of the standard microorganisms prepared
from 24 h cultures of bacteria in the Mueller-Hinton broth
medium and from 5 and 10 day cultures in the Sabouraud
agar medium for fungi at a concentration of 105 cfu/mL
were added to each dilution in a 1:1 ratio. Growth (or its
lack) of the microorganisms was determined visually
after incubation for 24 h at 37 °C (bacteria) or 5–10 days
at 28–30 °C (fungi). The lowest concentration at which
there was no visible growth (turbidity) was taken as the
MIC.
5.2. Antimicrobial activity
Microorganisms used: eleven standard strains repre-
sentative of cocci; Micrococcus luteus ATCC 9341,
Staphylococcus epidermidis ATCC 12228, Staphylococ-
cus aureus ATCC 6538, rods; Pseudomonas aeruginosa
ATCC 15442, Proteus vulgaris NCTC 4635, Klebsiella
pneumoniae ATCC 4352, Escherichia coli NCTC 8196,
Serratia marcescens ATCC 8100, yeast-like fungi; Can-
dida albicans ATCC 10231, Rhodotorula rubra PhB, and
dermatophytes Trichophyton mentagrophytes var. gyp-
seum ATCC 9533.
Standard strains were supplied by National Collection
of Type Cultures (NCTC), London and American Type
Culture Collection (ATCC). Rhodotorula rubra (PHB)
strain was taken from the Department of Pharmaceutical

770
Figure 2. The MIC mean values for (alkoxymethyl)dimethyl-
{2-hydroxy-5-[(4-methylphenyl)azo]benzyl}ammonium chlo-
rides (7–9).
Figure 1. The MIC mean values for (alkoxymethyl)dimethyl-
{5-[(4-chlorophenyl)azo]-2-hydroxybenzyl}ammonium chlori-
des (3–5).
Then from each tube, one loopful was cultured on an
agar medium with inactivates [14] (0.3% lecithin, 3%
polysorbate 80 and 0.1% cysteine L) and incubated for
48 h at 37 °C (bacteria) or for 5–10 days at 28–30 °C
(fungi). The lowest concentration of the chloride support-
ing no colony formation was defined as the MBC.
Acknowledgements
This investigation received financial support from the
Polish Committee of Scientific Research, Grant KBN 3
T09B 010 15.

771
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