Synthesis of novel 4,6-di(substituted)amino- 1,2-dihydro-1,3,5-triazine derivatives as topical antiseptic agents

Article abstract of DOI:10.1021/jm8014712

A series of novel 4,6-di(substituted)arnino-1,2-dihydro-1,3,5- triazine derivatives designed to have ClogP of 5.1-7.5 was synthesized and evaluated for their antiseptic properties by MIC and MBC tests against Gram-positive and Gram-negative bacteria, incl

Full text of DOI:10.1021/jm8014712

J. Med. Chem. 2009, 52, 597–600
597
Synthesis of Novel 4,6-Di(substituted)amino-
1,2-dihydro-1,3,5-triazine Derivatives as
Topical Antiseptic Agents
Shirou Maeda,* Toshiko Kita, and Kanji Meguro
CT Laboratory, Hamari Chemicals, Ltd., 1-4-29 Kunijima,
Higashiyodogawa-ku, Osaka 533-0024, Japan
ReceiVed NoVember 22, 2008
Abstract: A series of novel 4,6-di(substituted)amino-1,2-dihydro-1,3,5-
triazine derivatives designed to have ClogP of 5.1-7.5 was synthesized
and evaluated for their antiseptic properties by MIC and MBC tests
against Gram-positive and Gram-negative bacteria, including MRSA,
VRE, and P. aeruginosa. Among these compounds, 4-alkyl-6-aralkyl
derivatives having ClogP of 6.6-7.1 and 4-alkyl-6-aryl or 4,6-dialkyl
derivatives with ClogP of 6.0-6.4 showed pronounced antibacterial
activities in both tests.
Figure 1. Biguanide-type antiseptics and synthetic target (A).
A large number of antibacterial drugs, such as antibiotics and
synthetic or semisynthetic antibacterial agents, have been
developed and used clinically to combat various infectious
diseases, and these drugs have contributed dramatically to human
health. On the other hand, bacteria that have gained resistance
against these drugs, including so-called MRSAa and VRE, are
recently becoming more prevalent and causing serious social
problems, such as nosocomial infections, opportunistic infec-
tions, and so on.
Although one of the useful ways to reduce these risks is to
prevent infections beforehand by applying appropriate, fast-
acting antiseptics or disinfectants topically on the human body,
such as on the hands or skin, or in the medical environment,
no new compound has been developed as an effective topical
antiseptic or disinfectant for more than 50 years. Thus, since
the discovery of benzalkonium chloride (1935), benzethonium
chloride (1943), alkyldiaminoethylglycine hydrochloride (1953),
CHG (Figure 1, 1954), and povidone-iodine (1956), these agents
have continued to be widely used despite their unsatisfactory
antiseptic potential and spectrum. Furthermore, many bacteria
are now said to be becoming resistant1 even to these agents.
Therefore, there is an urgent need to develop new compounds
having highly potent antiseptic property against wide variety
of bacteria, including various drug-resistant organisms.
In one such attempt, Tsubouchi et al.2 reported in 1997 that
they had succeeded in modifying the antiseptic potential of CHG
by chemical modification focused on its biguanide structure and
identified the N1,N5-disubstituted biguanide derivative 1 (OPB-
2045,2 Figure 1) as a candidate for clinical testing, but as far as
we are aware, it has not yet been put to practical use.
We now report on our approach to develop a new antiseptic
agent for topical use, and the synthesis and antiseptic properties
of novel 4,6-di(substituted)amino-dihydrotriazine derivatives are
presented.
Figure 2. Structures of proguanil, cycloguanil, trimethoprim, and
compound B.
4,6-Di(substituted)amino-dihydrotriazine structure (A, Figure
1) is thought to be one of the cyclized forms of biguanide
antiseptic agents such as CHG or 1, which stimulated our interest
to study the antiseptic potential of this skeleton. Syntheses of a
large number of compounds possessing this skeleton have been
reported so far, especially since the discovery of cycloguanil
in 1946 as an active metabolite of a biguanide-type antimalarial
agent proguanil3 (Figure 2), and various biological activities
such as antimalarial, antibacterial, antitumor, antitoxoplasmic,
anthelmintic, anticoccidium activities, and so on have been
described.4 However, it is well-known that these activities
mainly depend on their inhibition of dihydrofolate reductase.
Therefore, it is anticipated that even if many such compounds
have antibacterial activity, this mode of action should not be
bactericidal but rather bacteriostatic because of their inhibition
of folic acid metabolism. To our knowledge, no folate inhibitor
has been reported to have acute bactericidal properties and
therefore has not been applied as antiseptic drug for topical use.
According to our own experiments, for example, it was shown
that trimethoprim, a representative antifolate antibacterial agent
with a diaminopyrimidine structure (Figure 2), had antibacterial
activity only in the MIC test but not in the MBC test, which is
often used2 to evaluate acute bactericidal properties (data not
shown). A simple 4-amino-6-benzylamino-dihydrotriazine com-
pound (B)5 (Figure 2) was prepared as the acetate to examine
its antibacterial property, but this was inactive even in our MIC
test.
* To whom correspondence should be addressed. Phone: 06-6328-8740.
Fax: 06-6326-6744. E-mail: shirou-maeda@hamari.co.jp.
a Abbreviations: MIC, minimal inhibitory concentration; MBC, minimal
bactericidal concentration; MRSA, methicillin-resistant Staphylococcus
aureus; VRE, vancomycin-resistant enterococcus; ClogP, calculated log P;
CHG, chlorhexidine gluconate; S. aur, Staphylococcus aureus; E. coli,
Escherichia coli; P. aerug, Pseudomonas aeruginosa.
10.1021/jm8014712 CCC: $40.75
2009 American Chemical Society
Published on Web 12/31/2008

598 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Letters
Scheme 1a
4,6-Di(substituted)amino-1,2-dihydrotriazine derivatives (7)
were conveniently synthesized as shown in Scheme 1. The
primary alkylamine hydrochloride (2) was reacted with sodium
dicyanamide to give the cyanoguanidine (3). Reaction of 3 with
another primary amine hydrochloride (4) gave a key intermediate
biguanide (5) as the dihydrochloride. When cyclized with
acetone in the presence of acid (e.g., hydrochloric acid) under
reflux, two types of compounds 6 and 7 were obtained
depending on the substituent R. In the case where R was an
aryl group, the 4-alkylamino-6-amino-1-aryl-1,2-dihydro-1,3,5-
triazin-6-amine (6) was obtained, in which the aryl group was
incorporated in the triazine ring as an N1-substituent. However,
heating 6 with sodium hydroxide in ethanol resulted in isomer-
ization to the desired 4,6-di(substituted)amino-1,2-dihydrotri-
azine (7) with two exocyclic substituted-amino groups. On the
other hand, in the case where R in 5 is an aralkyl or an alkyl
group, cyclization with acetone gave a mixture of 6 (minor)
and 7 (major). Although 7 can be isolated by column chroma-
tography, it was possible to obtain 7 as a single compound by
treating the mixture with sodium hydroxide. Compounds 6 and
7 were able to be isolated as acid salts by treating with suitable
acids. In the case of 7, treatment of the free base with wet
carboxylic esters easily produced the carboxylic acid salts, e.g.,
acetic acid salt from ethyl acetate, by rapid hydrolysis of the
esters due to the strong basicity of 7.
a Reagents and conditions: (a) NaN(CN)2, CH3CN, reflux; (b) xylene,
reflux, concentrated HCl; (c) acetone, MeOH, concentrated HCl, reflux;
(d) 5 M NaOH, EtOH (aq), reflux.
To generate compounds possessing acute bactericidal proper-
ties, we have introduced a concept that a suitable balance
between molecular hydrophilicity and hydrophobicity is neces-
sary for a compound to interact or participate with the bacterial
cell wall to kill bacteria instantly. Thus, we designed hitherto
unknown, novel 4,6-di(substituted)amino-dihydrotriazine deriva-
tives (A) with ClogP 6 of 5-7 as an index because the free
bases of CHG and 1 have ClogP of 6.18 and 6.19, respectively.
Compounds with a suitable combination of aryl, aralkyl, and
alkyl side chains at the two amino groups were prepared, and
their antiseptic activity was evaluated by MIC and MBC tests
for Gram-positive bacteria, S. aur 209PJC and MRSA 97-115,
and for Gram-negative bacteria, E. coli NIHI JC-2 and P. aerug
PAO-1. MIC and MBC values were determined, after 24 h of
incubation and after 1, 3, and 5 min of contact, with the test
organisms, respectively.
According to the above-mentioned general procedure, 8-36
having ClogP between 5.1 and 7.5 were designed and synthe-
sized using a dimethyl structure for R′′ and R′′′ of the target
(A) because it was easy and economical to prepare. It was also
thought to be desirable to avoid the added complexity arising
from the introduction of an asymmetric center at the C2 position.
Most of the dihydrotriazine derivatives reported here were
obtained as the acetates with some exceptions (Table 1). MIC
Table 1. Antibacterial Activity (MIC) of Compounds 7 (8-36) and CHG
MIC (µg/mL)
compda
Rb
alkyl
ClogPc
S. aurd
MRSAe
E. colif
P. aerugg
8
9
Bzl
Bzl
Bzl
Bzl
undecyl
decyl
nonyl
octyl
decyl
nonyl
octyl
heptyl
undecyl
decyl
nonyl
octyl
7.43
7.02
6.60
6.18
7.50
7.08
6.67
6.25
7.31
6.89
6.47
7.10
6.74
6.74
7.15
6.74
6.32
7.03
6.81
6.39
5.97
5.55
7.22
6.38
6.38
5.97
5.55
5.13
6.31
6.18
0.4
0.4
0.8
0.8
0.8
0.8
0.4
0.8
0.4
0.4
0.8
0.4
0.8
0.8
0.8
0.4
0.8
0.4
0.4
0.8
1.6
6.3
0.8
0.8
0.8
1.6
1.6
1.6
0.8
0.2
0.8
0.4
1.6
1.6
0.8
0.8
0.4
1.6
0.8
0.8
1.6
0.8
0.8
0.8
0.8
0.8
1.6
0.8
0.4
0.8
1.6
6.3
1.6
0.8
0.8
1.6
3.1
6.3
1.6
3.1
100
12.5
12.5
12.5
100
25
12.5
25
100
25
25
50
25
25
25
12.5
25
100
25
12.5
25
100
50
12.5
50
100
50
50
25
25
10
11
12
13
14
15
16
17
18h
19
20
21
22
23
24
25i
26
27
28
29
30
31
32
33
34
35
36
4-Me-Bzl
4-Me-Bzl
4-Me-Bzl
4-Me-Bzl
4-MeO-Bzl
4-MeO-Bzl
4-MeO-Bzl
4-CF3-Bzl
2-Cl-Bzl
2-Cl-Bzl
Ph-CH2CH2
Ph-CH2CH2
Ph-CH2CH2
4-MeO-Ph-CH2CH2
Ph
Ph
Ph
Ph
octyl
heptyl
octyl
octyl
octyl
octyl
octyl
25
25
12.5
25
50
decyl
nonyl
octyl
decyl
decyl
nonyl
octyl
heptyl
octyl
heptyl
hexyl
pentyl
butyl
50
12.5
12.5
25
100
12.5
50
100
>100
50
50
25
50
100
50
50
100
12.5
12.5
25
25
octyl
dodecyl
CHG
propyl
ethyl
100
12.5
1.6
50
a Evaluated as acetates. b Bzl ) benzyl; Ph ) phenyl. c Free base. d S. aur 209PJC. e MRSA 97-115. f E. coli NIHJ JC-2. g P. aerug PAO-1. h Mesylate.
i Hydrochloride.

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 599
MBC (µg/mL)
Table 2. Bactericidal Activity (MBC) of Compounds 7 (9-36) and CHG
MBC (µg/mL)
compd
contact (min)
S. aura
MRSAb
E. colic
P. aerugd
compd
contact (min)
S. aura
MRSAb
E. colic
P. aerugd
9
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
6.3
6.3
6.3
12.5
6.3
6.3
25
12.5
6.3
6.3
3.1
3.1
6.3
3.1
3.1
25
12.5
12.5
12.5
6.3
3.1
6.3
6.3
3.1
12.5
6.3
6.3
3.1
3.1
3.1
12.5
6.3
6.3
50
12.5
6.3
6.3
12.5
6.3
6.3
>50
50
50
25
12.5
12.5
25
25
12.5
50
50
50
25
6.3
25
12.5
6.3
6.3
6.3
3.1
12.5
6.3
6.3
6.3
1.6
1.6
12.5
6.3
6.3
3.1
3.1
1.6
6.3
3.1
3.1
25
12.5
12.5
12.5
3.1
3.1
12.5
6.3
3.1
25
12.5
12.5
12.5
3.1
3.1
25
12.5
12.5
12.5
6.3
6.3
6.3
1.6
1.6
6.3
3.1
23
1mn
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
25
25
12.5
6.3
12.5
3.1
3.1
6.3
3.1
3.1
3.1
1.6
1.6
12.5
6.3
3.1
12.5
6.3
6.3
6.3
3.1
3.1
12.5
6.3
12.5
3.1
1.6
6.3
6.3
3.1
3.1
3.1
1.6
6.3
3.1
3.1
25
12.5
6.3
3.1
1.6
1.6
6.3
3.1
1.6
6.3
3.1
3.1
50
25
12.5
6.3
50
12.5
12.5
50
10
11
13
14
15
17
18
19
20
21
22
24
>100
>100
>100
25
25
12.5
6.3
12.5
6.3
6.3
25
6.3
6.3
6.3
6.3
3.1
12.5
6.3
6.3
3.1
3.1
3.1
25
12.5
6.3
3.1
3.1
1.6
62.5
62.5
62.5
6.3
6.3
25
25
25
25
12.5
12.5
25
27
28
25
31
12.5
12.5
12.5
6.3
6.3
12.5
6.3
3.1
3.1
3.1
3.1
3.1
1.6
1.6
3.1
3.1
1.6
12.5
12.5
12.5
25
12.5
12.5
6.3
6.3
6.3
3.1
3.1
25
12.5
12.5
25
12.5
6.3
1000
250
125
32
6.3
6.3
12.5
6.3
6.3
50
>100
>100
>100
6.3
33
34
6.3
6.3
6.3
6.3
6.3
12.5
6.3
25
12.5
12.5
3.1
12.5
3.1
3.1
36
3.1
3.1
CHG
62.5
31.3
15.6
>500
>500
>500
6.3
1.6
25
12.5
12.5
>50
>50
>50
12.5
12.5
a S. aur 209PJC. b MRSA 97-115. c E. coli NIHJ JC-2. d P. aerug PAO-I.
Table 3. Bactericidal Activity (MBC) of Compounds 14, 32, and CHG
MBC (µg/mL)
and MBC data evaluated for these compounds are shown in
Tables 1 and 2, respectively.
compd contact (min) MRSAa MRSAb
VREc
P. aerugd
Generally speaking, most of the compounds synthesized had
potent antibacterial properties comparable to CHG in the MIC
test (Table 1) and were superior to the CHG in the MBC test
(Table 2), irrespective of the kind of hydrophobic N-substituents
or N-alkyl chain-length. However, it seems that compounds with
a relatively high ClogP (e.g., 8, 12, and 16 in the aralkyl-alkyl
compounds; 26 in phenyl-alkyl compounds; 30 in dialkyl
compounds) and those with a low ClogP value (e.g., 29, 35)
tend to show reduced activity against Gram negative bacteria
despite the fact that they still have potent activity against Gram
positive bacteria in the MIC test. Compounds that showed MIC
values less than 100 µg/mL (Table 1) were further evaluated
by the MBC test (Table 2). Although some aralkyl-alkyl
compounds having slightly high or low ClogP values, such as
11, 15, 18, 22, and 24, were less effective against MRSA
compared to other bacteria including ordinary S.aur, it was very
interesting to note that these compounds generally had faster-
acting and more-potent bactericidal activity than CHG, as
indicated by the results of MBC test (Table 2). It is noted that
the difference of substituents on the phenyl ring and the alkyl
chain length does not seem to affect the antimicrobial activities,
as can be seen from 9-36. Therefore, ClogP of the whole
molecule seemed to be a very important determinant factor of
the antiseptic properties of this series. For example, 4-alkyl-6-
aralkyl-dihydrotriazine derivatives having ClogP of 6.6-7.1 and
14
1
3
5
1
3
5
1
3
5
50
50
25
50
25
50
50
25
50
25
25
25
12.5
25
25
6.3
6.3
3.1
3.1
3.1
32
25
25
12.5
1.6
CHG
500
250
250
500
500
500
>1000
>1000
>1000
125
31.3
31.3
a MRSA 97-53. b MRSA KM 97-108. c VRE 49. d P. aerug no. 12.
4-alkyl-6-aryl or 4,6-dialkyl derivatives with ClogP of 6.0-6.4
are expected to have a reasonably good potential as antiseptic
agents.
MBC effects of 14 and 32 against different strains of MRSA
and P. aerug and a VRE strain were further evaluated in
comparison to CHG. As shown in Table 3, these compounds
showed potent bactericidal properties against every strain
including VRE. In contrast, CHG was far less potent than 14
and 32 and appeared to be inactive against the VRE used.
In conclusion, our work has shown that the novel 4,6-
di(substituted)amino-1,2-dihydro-1,3,5-triazine derivatives syn-
thesized had remarkable and acute bactericidal properties and
thus may be promising new antiseptics, which are to be
developed after a 50-year absence of this type of drug. Further
studies on antiseptic properties including mode of action of these
analogues [e.g., 1,2-dihydro-2,2-dimethyl-6-(4-methylben-

600 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Letters
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Supporting Information Available: Details on the synthetic
procedures, compound characterization, and MIC and MBC test
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Synthesis and Structure-Activity Relationships of Novel Antiseptics.
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