Synthesis and Antimicrobial Evaluation of Bis-morpholine Triazine Quaternary Ammonium Salts

Article abstract of DOI:10.1002/cmdc.202100409

Efficient, environmentally and economically sustainable, and nontoxic antibacterial products are of global relevance in the fight against microorganism contamination. In this work, an easy and straightforward method for the synthesis of bis-morpholino triazine quaternary ammonium salts (bis-mTQAS) is reported, starting from 2,4,6-trichloro-1,3,5-triazine or 2,4-dichloro-6-methoxy-1,3,5-triazine and various N-alkylmorpholines. Bis-mTQAS were tested as antimicrobials against Gram-negative and Gram-positive bacterial strains. The best-performing bis-mTQAS were found to achieve total disinfection against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 at 50 and 400 μg/mL, respectively. Distinctively, bis-mTQAS with the highest antimicrobial efficiency had lowest cytotoxicity.

Full text of DOI:10.1002/cmdc.202100409

Communications
doi.org/10.1002/cmdc.202100409
ChemMedChem
Synthesis and Antimicrobial Evaluation of Bis-morpholine
Triazine Quaternary Ammonium Salts
Andrea Morandini,[a] Benedetta Leonetti,[b] Pietro Riello,[a, c] Roberto Sole,[a, d]
Vanessa Gatto,[a, e] Isabella Caligiuri,[f] Flavio Rizzolio,[a, f] and Valentina Beghetto*[a, e]
Since their first introduction by Domagk in 1935,[3] quater-
Efficient, environmentally and economically sustainable, and
nary ammonium salts (QAS) have become among the most
nontoxic antibacterial products are of global relevance in the
fight against microorganism contamination. In this work, an
easy and straightforward method for the synthesis of bis-
morpholino triazine quaternary ammonium salts (bis-mTQAS) is
reported, starting from 2,4,6-trichloro-1,3,5-triazine or 2,4-
dichloro-6-methoxy-1,3,5-triazine and various N-alkylmorpho-
lines. Bis-mTQAS were tested as antimicrobials against Gram-
negative and Gram-positive bacterial strains. The best-perform-
ing bis-mTQAS were found to achieve total disinfection against
Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC
25922 at 50 and 400 μg/mL, respectively. Distinctively, bis-
mTQAS with the highest antimicrobial efficiency had lowest
cytotoxicity.
widespread and used disinfectants worldwide.[4]
Generally, QAS contain four alkyl, aryl or heterocyclic groups
bonded to a nitrogen atom (see Figure 1) and are subdivided
into mono-, bis- and multi-QAS according to the overall charge
present on the compound. The number of charges and the
nature of the functional groups bonded to the nitrogen atom
define both the physicochemical properties of QAS and their
antimicrobial activity and toxicity.[5] Commercially available
mono-QAS such as benzalkonium chloride, cetylpyridinium
chloride, cetyltrimethylammonium chloride, have been widely
used for different applications (Figure 1).[6]
As far as their mechanism is concerned, it is generally
accepted that positively charged QAS electrostatically interact
with the surface of the bacterial membrane (negatively
charged) and penetrate the cell wall; this phenomenon is
promoted by the affinity of the long lipophilic chain within the
cell membrane (Figure 2) gradually causing the cell death.[7]
Thus, antimicrobial efficiency of QAS depends on the
presence of a hydrophobic chain (tail), which is similar in
structure to the bilayer of the membrane, and one or more
polar “heads” interacting with the phospholipid acids. One of
the disadvantages of QAS is their high toxicity together with
Today, the fight against infectious diseases still poses a serious
challenge for healthcare worldwide.[1] According to statistical
data reported in the literature, bacteria are becoming quickly
resistant to commercially available antibiotics and antiseptics.[2]
[a] Dr. A. Morandini, Prof. P. Riello, Dr. R. Sole, V. Gatto, Prof. F. Rizzolio,
Prof. V. Beghetto
Dipartimento di Scienze Molecolari e Nanosistemi
Università Ca’ Foscari di Venezia
Via Torino 155, 30172, Venezia Mestre, (Italy)
E-mail: beghetto@unive.it
[b] B. Leonetti
Brenta S.r.l. – Nine trees group
Viale Milano, 26, 36075 Montecchio Maggiore Vicenza (Italy)
[c] Prof. P. Riello
European Centre for Living Technology (ECLT)
Ca’ Bottacin, Dorsoduro 3911, 30123 Venice (Italy)
[d] Dr. R. Sole
Figure 1. Examples of commercially available mono-QAS. 1) benzalkonium
chloride, 2) cetylpyridinium chloride, 3) cetyltrimethylammonium chloride.
CIRCC,
via C. Ulpiani 27, 70126 Bari, Italy
[e] V. Gatto, Prof. V. Beghetto
Crossing S.r.l.
Viale della Repubblica 193/b, 31100 Treviso (Italy)
[f] I. Caligiuri, Prof. F. Rizzolio
Pathology Unit,
Centro di Riferimento Oncologico (CRO) IRCCS
Via F. Gallini 2, 33081, Aviano (Italy)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100409
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
Non-Commercial NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.
Figure 2. General mechanism of action of QAS where the phospholipid
membranes are indicated in blue and quaternary ammonium salts in red.
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the development of bacterial resistance.[8,9] On the contrary, bis-
QAS are more efficient as antimicrobials, less toxic and induce
lower bacterial resistance compared to mono-QAS.[10]
Additionally, QAS are mostly prepared by direct alkylation
of a tertiary amine in the presence of haloalkanes[11] requiring
high temperatures, long reaction times and often, toxic
reagents.[11b]
Our research group has long been interested in sustain-
ability issues related to reduced consumption of toxic chem-
icals, process simplification and implementation of industrial
sustainability.[12]
In this concern, extensive studies have been carried out on
the synthesis and use of 1,3,5-triazine derived quaternary
ammonium salts, such as 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-
methyl-morpholinium chloride (4, Scheme 2) as dehydro-con-
densation agents for the synthesis of fine chemicals and
polymers.[13] Although 4 is a quaternary ammonium salt, the
possibility to use 4 or similar triazine based QAS as antimicro-
bial agents has never been taken into consideration and
therefore, we deemed it interesting to verify this possibility.
Few features have been taken into consideration to achieve
the proposed objective: 4 and other triazine derived quaternary
ammonium salts prepared according to the literature are very
efficient and reactive dehydro-condensation agents with re-
duced stability in solution.[14] It is generally accepted that the
presence of a methyl substituent on the tertiary amine
improves the reactivity of the condensation agent and con-
sequently reduces its stability in time. According to the
literature 4 and in general 4-(4,6-dialkoxy[1,3,5]triazin-2-yl)-4-
methyl-morpholinium chlorides are subject to demethylation of
the methyl-morpholine moiety leading to the formation of a
neutral unreactive species (see Scheme 1).
Since antimicrobial agents require to be stable in solution
for long times, the stability of the triazine quaternary
ammonium salts had to be improved. According to the
literature substitution of the N-methyl group with an N-ethyl
substituent should improve the stability of the triazine salt.
Further, efficient antimicrobial QAS require a hydrophobic
chain and a hydrophilic head, i.e. one or more nitrogen atoms
bearing a positive charge.[7a–b,10] Thus, we investigated the
possibility to synthesize multi-charged TQAS similar to 4,
starting from 2-chloro-4,6-disubstituted-1,3,5-triazine and mor-
pholino amines bearing hydrocarbon chains of different length
(see Scheme 2a).
First experiments were carried out to study the stability of
different triazine quaternary ammonium salts prepared by
reaction of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and
different tertiary amines, respectively N-methyl, N-ethyl and N-
butyl morpholine (Scheme 2, compounds 4–6), the synthesis of
the first two compounds was already reported by Kunishima
et al.[15] While water solutions of 4 totally decomposed within
48 h, 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-ethyl-morpholynium
chloride (5) was stable in solution for over 60 days. These data
clearly highlight that TQAS stability is strongly dependent on
the nature of the amine, and careful choice of the tertiary
amines allowed preparing very stable TQAS. Nevertheless,
probably due to the absence of a long alkyl chain within the
structure of 5, this TQAS had no antimicrobial activity against
bacterial strains tested.
Therefore, the possibility to prepare N-alkylmorpholine
derived TQAS, bearing a long alkyl chain was tested starting
Scheme 1. Demethylation reaction of 4-(4,6-dialkoxy[1,3,5]triazin-2-yl)-4-
methyl-morpholinium chlorides.
Scheme 2. Synthetic strategy and chemical structure of morpholine TQAS tested.
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from commercially available N-butylmorpholine (6). Unfortu-
nately, in all reaction conditions tested, N-butylmorpholine was
totally unreactive towards CDMT and 6 could not be synthe-
sized. This is probably to be ascribed to steric hindrance
phenomena.
In fact, Kunishima et al. reported that 4 and similar triazine
compounds obtained by reaction of CDMT and a cyclic aliphatic
tertiary amine are formed by interchange of the less abundant
equatorial conformer (E) of the amine which then rearranges to
the most stable axial conformer (A).[15]
A similar behaviour may occur also for the formation of
quaternary ammonium salts by reaction of 2,4-dichloro-1,3,5-
triazine and 2,4,6-trichloro-1,3,5-triazine as reported in
Scheme 3.[15]
In the case of 5, steric hindrance is supposed to derive not
only from the substituents present on the triazine but also from
the butyl-chain of the tertiary amine inhibiting the formation of
the desired product.
To reduce steric hindrance and promote the formation of N-
alkylmorpholine TQAS, an alternative synthetic strategy was
adopted starting from 2,4-dichloro-6-methoxy-1,3,5-triazine or
2,4,6-trichloro-1,3,5-triazine (Scheme 2b), since the covalent
radius of the ClÀ atom is smaller than the one of the À OCH3
group, decreasing steric hindrance around the triazine core.[16]
In this way, 4,4’-(6-methoxy-1,3,5-triazine-2,4-diyl)bis(4-butyl-
morpholin-4-ium)chloride (7) and 4,4’-(6-chloro-1,3,5-triazine-
2,4-diyl)bis(4-butylmorpholin-4-ium)chloride (8) could be syn-
thesized in very mild reaction conditions (see experimental).
Nevertheless, as for 4 and 5, also these two TQAS showed no
antimicrobial activity.
An adequate strategy to introduce longer alkyl chains on
the structure of the triazine was evidently required. Commer-
cially no morpholine with longer N-alkyl chains were available,
so an alternative way to easily prepare a set of tertiary amines
of variable carbon length was devised starting from 4-(3-
aminopropyl)morpholine and different aliphatic carboxylic acids
in the presence of
(Scheme 4).
4 as dehydro-condensation agent
In fact, with this strategy tertiary amines with up to twelve
carbon atoms were synthesized in good yield, from easily
available, cost effective and nontoxic chemicals. According to
the synthetic strategy reported in Scheme 1, 4,4’-(6-methoxy-
1,3,5-triazine-2,4-diyl)bis(4-(3-alkylamidopropyl)morpholin-4-
ium) chlorides (12–14), were prepared by reaction of 2 and
amidoamines 9–11, and analogously 4,4’-(6-chloro-1,3,5-
triazine-2,4-diyl)bis(4-(3-alkylamidopropyl)morpholin-4-ium)
chlorides (15–17), by reaction of 3 and amidoamines 9–11, at
room temperature in 1 h. All compounds were fully character-
ized, and data are reported in the supporting information
section.
Interestingly, according to the ESI-MS analysis only disub-
stituted compounds were isolated even if 2,4-dichloro-6-meth-
oxy-1,3,5-triazine or 2,4,6-trichloro-1,3,5-triazine and amido-
amines 9–11 were used in a molar ratio 1/1. According to the
literature this behaviour may be explained because of the
+
strong electron-withdrawing character of the NR3
substituent[13b,17] promoting nucleophilic substitution in meta
position, despite the presence of very bulky amidoamines 9–11.
In fact, according to the Hammett substituent constants,[18]
+
different NR3 have σm between +0.86 and +1.76, thus highly
more electron-withdrawing than À Cl (σm +0.37) or À OMe (σm
+0.12), justifying the high reactivity of the À Cl atoms of
intermediate mono-TQAS species, promoting the formation of
bis-mTQAS.
Also, for TQAS 15–17, double substitution products pre-
vailed while no trisubstituted products were formed with any of
the amidoamines tested, probably due to the high steric
hindrance of the two substituents and to the low solubility of
15–17 in the reaction solvent, preventing further substitution.
Antimicrobial activity of these morpholinium bis-mTQAS
was evaluated against Gram-positive (Staphylococcus aureus
ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922)
bacterial strains. The lipophilicity of the synthesized bis-mTQAS
was expressed in terms of their partition coefficient values
(LogP) calculated using online platform Chemicalize
(ChemAxon).[21] TQAS 12 and 15 still showed no inhibition
against S. aureus and E. coli, even at concentrations above
400 μg/mL (Table 1). As the length of the alkyl chain present on
the quaternary ammonium salts increased, a gradual improve-
ment of the efficacy of bis-mTQAS was observed. In particular,
best inhibition was achieved with compound 16, giving total
inhibition towards S. aureus at concentrations of 50 μg/mL and
for E. coli at 400 μg/mL. These data are comparable to the
activity of other bis-QAS reported in the literature, giving MIC
values between 8 and 83 mgμg/mL[RS1] for Gram-positive
bacterial strains, while higher concentrations are required for
more resistant Gram-negative bacterial strains.[8,22] Generally,
Scheme 3. Possible mechanism of equatorial, axial interchange of conformer
(E) and conformer (A).
Scheme 4. Synthesis of amidoamines 9–11.
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Table 1. MIC, IC50 and LogP values of TQAS.[a]
Entry TQAS Chain length S. aureus E. coli
IC50. In the case of morpholinium bis-mTQAS there seems to be
an opposite correlation between the chain length and the
toxicity of the product (compare entries 1, 3 or 4, 6, Table 1).
Studies are in progress to further investigate this phenomenon.
In conclusion, in this work the synthesis of a promising new
class of triazine derived antimicrobial agents has been reported.
Distinctively, best MIC values were obtained with less toxic bis-
mTQAS. Moreover, triazine moiety has been verified as an
interesting core structure, highly reactive and versatile which
will allow to prepare a plethora of new antimicrobial agents.
Studies are in progress for the synthesis of different libraries of
mono- and bis-TQAS and their use as antimicrobial agents.
IC50
[μg/mL] [μg/mL][b]
LogP[c]
[μg/mL]
1
2
3
4
5
6
12
13
14
15
16
17
8
>400
300
>400
>400
50
>400
>400
300
>400
400
36
34
62
25
59
67
À 5.13
À 2.07
+1.98
À 4.91
À 1.44
+2.60
12
16
8
12
16
300
200
[a] Minimal inhibitory concentration (MIC) was calculated taking as
reference the negative control as the lowest concentration which inhibits
bacterial growth. All the experiments were carried out both in biological
and technical triplicate. [b] IC50 values were determined on eukaryotic
fibroblasts MRC-5 cells. [c] LogP calculated using online platform Chemic-
alize.
Conflict of Interest
data achieved confirm what Gilbert and Moore reported about
the selective effect of the length of the alkyl chain on Gram-
positive and Gram-negative bacteria.[7c] Very recently
Sapozhnikov[8a] reported the synthesis of mono-QAS containing
aminoamides, prepared from different carboxylic acids and a
dimethyl alkylamine, and highest MIC were observed with lauric
acid residues in the amide fragment, in agreement with data
reported in Table 1 for compound 16.
To gain deeper insight into the behaviour of morpholine
bis-mTQAS, LogP were calculated and are reported in Table 1.
In the case of S. aureus for which morpholine bis-mTQAS are
more efficient, a minimum between MIC values and LogP was
observed, confirming that, as for QAS, lipophilicity and hydro-
phobicity of the compound influence the efficiency of bis-
mTQAS, so that too long or too short alkyl chains adversely
affect the antimicrobial activity of morpholine bis-mTQAS (See
Figure 3).[23]
Interestingly, all IC50 of morpholino bis-mTQAS are rather
high compared to other commercially available QAS indicating
a low cytotoxicity profile of the compounds.[8,23] Distinctively,
bis-mTQAS 16, which gave best MIC, had one of the highest
IC50, consequently the most efficient antimicrobial bis-mTQAS
corresponds also to one of the least cytotoxic. In general, the
toxicity of QAS depends on the length of the alkyl chain and is
correlated to the efficacy as antimicrobial, so that QAS bearing
C12À C14 alkyl chains normally have highest MIC and lowest
The authors declare no conflict of interest.
Keywords: triazine quaternary ammonium salts · antimicrobial
agents · low toxicity antimicrobials · QAS
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Manuscript received: June 7, 2021
Revised manuscript received: July 18, 2021
Accepted manuscript online: July 20, 2021
Version of record online: ■■■, ■■■■
ChemMedChem 2021, 16, 1–6
www.chemmedchem.org
5
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH
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COMMUNICATIONS
Dr. A. Morandini, B. Leonetti, Prof. P.
Riello, Dr. R. Sole, V. Gatto, I.
Caligiuri, Prof. F. Rizzolio, Prof. V.
Beghetto*
1 – 6
Synthesis and Antimicrobial Eval-
uation of Bis-morpholine Triazine
Quaternary Ammonium Salts
An easy and straightforward meth-
odology for the synthesis of bis-mor-
pholino triazine quaternary
ammonium salts (bis-mTQAS) is
reported, starting from 2,4,6-trichloro-
1,3,5-triazine or 2,4-dichloro-6-
methoxy-1,3,5-triazine and various N-
alkylmorpholines. These bis-mTQAS
show significant promise as antimicro-
bial agents against Gram-negative
and Gram-positive bacterial strains.