A new bactericidal lead structure for the protection of materials

Article abstract of DOI:10.1016/j.bmcl.2004.11.036

Our search for a new broad spectrum bactericide for preserving materials lead to the discovery of a highly active bicyclic amine (1) and a number of derivatives. The synthesis and biological evaluation as well as a first toxicological assessment of these compounds are described. Compound 1 shows strong bactericidal activity down to levels of below 100 ppm but unfortunately increases the number of mutations in Ames tests.

Full text of DOI:10.1016/j.bmcl.2004.11.036

Bioorganic & Medicinal Chemistry Letters 15 (2005) 625–629
A new bactericidal lead structure for the protection of materials
Erasmus Vogl,a,* Rainer Bruns,a Oliver Kretschik,b Hermann Uhr,a Johannes Kaulen,a
Martin Kugler,b Peter Wachtler,b Wolfgang Kreissc and Gunther Eberzd
¨
aBayer Chemicals AG, Material Protection Products, BCH-R&D, Geb. I1, 51368 Leverkusen, Germany
bBayer Chemicals AG, Material Protection Products, BCH-MPP-TM, Geb. R54, 47829 Uerdingen, Germany
cBayer Industry Services, SUA-BM, Geb. Q 21, 51368 Leverkusen, Germany
dBayer AG, Corporate Center, BAG-GPA, Geb. 9115, 51368 Leverkusen, Germany
Received 28 October 2004; accepted 15 November 2004
Available online 16 December 2004
Abstract—Our search for a new broad spectrum bactericide for preserving materials lead to the discovery of a highly active bicyclic
amine (1) and a number of derivatives. The synthesis and biological evaluation as well as a first toxicological assessment of these
compounds are described. Compound 1 shows strong bactericidal activity down to levels of below 100 ppm but unfortunately
increases the number of mutations in Ames tests.
Ó 2004 Elsevier Ltd. All rights reserved.
Table 1. Minimum inhibitory concentrations (MICs) of 1, determined
in vitro (Agar)
Microbicides are chemicals, which, at small concentra-
tions, provide effective protection against the microbio-
logical spoilage of a wide variety of products, including
foodstuffs, paints, polymer emulsions, carbonate slur-
ries, metal working fluids, detergents, cosmetics, adhe-
sives, or plastics. The relevant market for biocides/
microbicides is projected to stand at approximately
€ 3 billion in 2005.1 New chemical classes of such micro-
bicides have become extremely rare.2 The challenge with
new microbicides lies in finding long lasting broad spec-
trum activity against a variety of extremely different
organisms while toxicity toward mammals and persis-
tency should be minimal. In addition, the biocide
industry is also affected by regulatory issues and the
increasing cost of registration. As existing biocides are
relatively inexpensive chemicals, the cost of new actives
must not be above approximately € 100 per kg.
NH2
O
N
1
Bacterium
MIC (ppm)
Bacillus subtilis
5
Pseudomonas aeruginosa ATCC 15442
P. aeruginosa NCIB 6749
P. aeruginosa NCIB 12201
Alcaligenes faecalis
5
<1
7.5
<1
<1
Corynebacterium
We subsequently initiated a program on testing other
analogues of 1. By focusing on the bicyclic ring structure
we thought to track down essential parts in respect to
activity. However, much to our surprise, the results at
this stage were disappointing. For example, none of
the analogues displayed in Figure 1 showed any signifi-
cant activity against the bacteria tested. The aliphatic
five-membered ring structure as well as the isoxazoline
part both seemed indispensable.
Here we want to report on a new lead structure for a
bactericide and the course of our discovery process.
Our attention was first caught while microbiologically
screening libraries of small organic molecules. In a mod-
ified sensor assay3 and classical screening, a compound
1, containing an isoxazole moiety, exhibited excellent
in vitro activity against a variety of bacteria (Table 1).
That finding was also significant as being in perspicuous
contrast to the series of isothiazolone biocides, estab-
lished on the market (Fig. 2). Changes on the ring sys-
tem are obviously tolerated here, without activity
Keywords: Biocide; Lead structure.
*
Corresponding author. Tel.: +49 214 30 52731; fax: +49 214 30
67966; e-mail: erasmus.vogl.ev@bayerchemicals.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.036

626
E. Vogl et al. / Bioorg. Med. Chem. Lett. 15 (2005) 625–629
NH2
O
NH2
O
NH2
O
N
N
N
NH2
O
1
NH2
O
N
NH2
O
N
NH2
NH
NH2
N
NH2
O
O
NH2
S
N
O
NH2
O
O
N
N
O
N
S
N
N
Figure 1. Some analogues of 1. None of them showed significant activity, however.
O
O
O
O
O
O
Cl
Cl
N
N C8H17
N
C8H17
N
NH
N
S
S
S
S
S
S
Cl
MIT
CMIT
OIT
DCOIT
Promexal
BIT
Figure 2. Commercial biocides of the isothiazolone class.
NH2
O
N
N
OH
N
cat. NaOH
H2NOH
NaH
N
toluene
reflux
EtOH
78 °C
PrOH/H2O
60 °C
N
NH2
N
3
4
2
1
60%
20%
80%
Scheme 1.
being abruptly lost. (However, distinct shifts in their
spectrum of activity do occur, e.g., while MIT is only
bactericidal, OIT is only fungicidal and algaecidal.) As-
cribed to the activated nitrogen sulfur bond, a reactive
mechanism of biocidal action has been widely accepted
for this class of microbicides.
ture was distilled for purification. The cyclization to
yield the final product was then initiated by catalytic
amounts of NaOH.6 However, when we tried to utilize
this route to produce 100 g quantities of 1 we encoun-
tered a strongly exothermic decomposition during the
distillation of the hydroxyl amine 4, which previously
had been carried out successfully on a smaller scale.
Hydroxyl amines are not without danger as reactants
in processes and during distillation.7 We thought this dis-
tillation to be essential for the purification of compound
4 as we had old reference samples of 4, which displayed
So for the next step we returned to 1 and focused on
its synthesis. As the H NMR spectrum of 1 seemed
1
relatively unspecific, the structure of 1 was unequivo-
cally confirmed by X-ray analysis.4 Bond lengths are
all in the expected range, the molecule is relatively flat
with the methylene groups being disordered. To produce
larger quantities of the compound required for tests of
more practical relevance, we started from adiponitrile
2 (Scheme 1).
1
perfect H NMR purity, while freshly prepared samples
showed additional signals. During our investigation we
learned, however, that the initially formed 1:2-mixture
of cis–trans-isomers (Scheme 2) will gradually shift to
Compound 2 is available in bulk as it is an intermediate
of Nylon-6,6. Reacting 2 with sodium hydride gave a
cyclized product 3, which after cooling of the reaction
mixture could be filtered off (Thorpe reaction).5 A num-
ber of bases has been suggested for this reaction. Treat-
ing 3 with hydroxyl amine affected a formal ammonia
hydroxyl amine exchange. The resulting product mix-
N
N
OH
N
N
OH
Scheme 2. cis–trans-Isomers of 4.

E. Vogl et al. / Bioorg. Med. Chem. Lett. 15 (2005) 625–629
627
NaOtBu
toluene
N
NH2
O
N
a) H2NOH
b) NaOH
N
or
KOtBu
no solvent
N
NH2
2
3
1
85%[6]
75%[7]
83%
Scheme 3.
one side during drying and storage of the solid sample at
room temperature, affecting the observed simplification
of the NMR spectrum. The initial mixture of isomers
with a proportion of ca. 1:2 can be regenerated by heat-
ing the single isomer to 100 °C for 1 h.
among the strongest broad spectrum bactericides
known, yet structurally simple.
As for the acute toxicity, the LD50 (oral, rat) was deter-
mined to be >100 < 300 mg/kg. Toward alkaline hydro-
lysis, 1 turned out to be relatively stable with T1/2 = 97 h
(pH 9, 50 °C). In addition 1 was only weakly reactive in
enzyme assays with ADH10 and coenzyme A11 but reac-
tive toward glutathione.12 Unfortunately the Ames
test13 for mutagenicity was clearly positive and in addi-
tion, according to the Magnusson–Kligmann test,14
compound 1 was a potential sensitizer. So we could
not proceed with the development of this compound.
After this clarification and some additional experimen-
tation to improve the synthesis employing a phase trans-
fer-catalyst instead of NaH to affect the Thorpe
cyclization we realized that PTC results mainly in the
formation of a dimerized side product.8 But while ini-
tially managing to purify 4 by column chromatography
to avoid decomposition during distillation we found that
crude 4 can favorably be converted to the final product 1
by in situ cyclization after treatment with base. In a
recent study it was shown that 3 can even be produced
under solvent-free conditions.9 We could therefore
arrive at a very short and economically feasible reaction
sequence (Scheme 3).
In our efforts to find other derivatives of comparable
biocidal activity we then synthesized a large number of
different derivatives substituted at the nitrogen atom.
The in vitro screening gave promising MICs with two
N-acylated compounds, 5 and 6.
Once we had larger quantities of 1 at hand we could
continue with the biological study. As shown in Figure
3, isoxazole 1 protects metal working fluids perfectly
against microbiological spoilage. At concentrations of
100 ppm the activity lasts over 10 weeks. In addition
we observed that in a Ôtime-to-killÕ test using Pseudomo-
nas fluorescens the bactericidal effect of 1 was of course
much more rapid than that of the standard bactericide
BIT, both at 20 ppm. This means that compound 1 is
O
O
O
H
N
N
CF3
O
O
N
N
5
6
Even more relevant than the in vitro results, 5 and 6
kept metal working fluids free of microbes for a remark-
able length of time. As shown in Figure 3, activity is par-
ticularly good against yeasts and molds. While both
derivatives are certainly less potent than the parent com-
pound 1, they still do exhibit strong activity, with 5
being a touch better than 6. After analyzing the rates
of hydrolysis of 5 and 6 we found that a simple depro-
tection of 5 or 6 yielding 1 as the real underlying active
compound could not be excluded nor be confirmed
(T1/2 = 105 h for 5 and T1/2 = 6 h for 6, pH 9, 50 °C.
Note that the primary hydrolysis product in case of 6
is not 1.)
O
O
O
H
N
NH2
O
N
N
CF3
O
O
OH
N
N
N
N
1
4
5
6
10
9
8
7
6
5
4
3
2
1
0
bacteria
molds
yeasts
However, our hopes of having identified a development
candidate faded nevertheless when we looked at the
Ames mutation rates: compounds 5 and 6 both clearly
led to increased number of mutations in this test.
We nevertheless tried to find other active derivatives
and started looking at the next generation of backup
candidates, identified during in vitro screening. One
promising class of derivatives were again modified
at the nitrogen atom, resulting in derivatives of
Figure 3. Preservation of a metal working fluid (mineral-oil based) at a
concentration of 100 ppm active ingredient

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E. Vogl et al. / Bioorg. Med. Chem. Lett. 15 (2005) 625–629
Table 2. Representative in vitro data for imidodicarbonate derivatives of 1
Bacterium/Agar
MIC (ppm)
P. aeruginosa NCIB 6749 complex medium
P. aeruginosa NCIB 6749 chem. def. medium
10
10
5
10
5
10
10
10
5
10
5
10
Cl
Br
O
Cl
O
N
O
Cl
Cl
O
N
O
O
N
O
O
N
O
O
O
O
O
N
O
O
O
O
O
O
O
N
O
O
O
O
O
O
O
N
O
O
O
N
N
Cl
N
N
N
Cl
Cl
Cl
Br
imidodicarbonates. When trying to synthesize gram
quantities, however, we noticed that these compounds
were thermally not very stable, with strongly exothermic
decompositions taking place around 80 °C. The com-
pounds were perfectly stable at room temperature but
for safety considerations and because the whole class
(we synthesized over 20 derivatives) behaved similarly,
they were abandoned as a whole even though the
in vitro data had been very promising. Table 2 shows
some representative in vitro results.
without mutagenic or sensitizing potential is not feasible
at all. None of the existing broad spectrum bactericides
would comply with the stringent legal requirements, if
they were developed again under current circumstances.
The results of this work clearly underline that the devel-
opment of bactericides being environmentally more
compliant than existing ones is a very challenging goal.
References and notes
1. Biocide Information Service; Kline & Co., 2003.
2. A recent exemption being Bethoxazine introduced by
Janssen in 2003, see: Bosselaers, J.; Blancquaert, P.; Gors,
J.; Heylen, I.; Lauwaerts, A.; Nys, J.; Van der Flaas, M.;
ValckeJanssen, A. Faerg och Lack Scandinavia 2003, 49(1),
5–11.
Finally, and as a last attempt to utilize lead structure 1,
we thought of using a pro-drug approach: when looking
at intermediate 4 we noticed that, as just trace amounts
of base or acid present in the environment or media to
be protected would be sufficient to affect the cyclization
to 1, intermediate 4 itself might be added to materials as
bactericide. The result would be a slow and steady sup-
ply of 1 in low concentrations, possibly sufficient to pro-
vide a bactericidal activity high enough to protect, but
low enough to avoid undesired toxicity issues. The syn-
thesis of larger amounts of 4 was not a trivial undertak-
ing though, due to the aforementioned instability
reasons and the need of purification by column chroma-
tography. But we managed to isolate sufficient amounts
of 4 and our pro-drug idea turned out to be thoroughly
true: Figure 3 shows that only 100 ppm of 4 are suffi-
cient to preserve a metal working fluid for over 10 weeks
against bacteria, molds, and yeasts. We immediately
proceeded with toxicity studies. Unfortunately even 4
had a positive Ames test result and in addition a sensi-
tizing potential according to the Magnusson–Kligmann
test. It is obviously impossible to distinguish at this
point if these unfavorable toxicity data of 4 are due to
a rapid transformation of 4 to 1 or due to inherent sub-
stance characteristics of 4 itself. It would be interesting
(but also tedious) to clarify if 4 is transformed to 1 be-
fore entering the cell or after entering the cell or if 1 is
a product of cell metabolism. But even then the current
Ames test results would not be altered.
3. Eberz, G.; Rast, H.-G.; Burger, K.; Kreiss, W.; Weise-
mann, C. Chromatographia 1996, 43, 5–9.
4. Additional data can be obtained on request.
5. Thorpe reaction of adiponitrile: Francis, J. E.; Benett, D.
A.; Hyun, J. L.; Rovinski, S. L.; Amrick, C. L.; Loo, P. S.;
Murphy, D.; Neale, R. F.; Wilson, D. E. J. Med. Chem.
1991, 34, 2899–2906; Rodriguez-Hahn, L.; Parra, M.;
Martinez, M. Synth. Commun. 1984, 14(10), 967–972;
Schroeder, E.; Rigby, G. W. J. Am. Chem. Soc. 1949, 71,
2205–2207.
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Elnagdi, M. H.; Elgemeie, G. E. H. Monatshefte fu¨r
Chemie 1984, 115, 573–579.
7. Hydroxylamine hydrochloride, MSDS, Acros Organics,
1996: exposure to heat may promote violent decomposi-
tions. See also: Bretherick, L. In Bretherick’s Handbook of
Reactive Chemical Hazards, 4th ed.; Butterworths, ISBN
0-7506-0103-5, 1990; pp 1233–1234.
8. Thompson, Q. E. J. Am. Chem. Soc. 1958, 80, 5483–
5487.
9. Yoshizawa, K.; Toyota, S.; Toda, F. Green Chem. 2002, 4,
68–70.
10. Compound 1 does not readily react with alcohol dehy-
drogenase (ADH). For a related study on the chemical
reactivity of some isothiazolone biocides, see: Collier,
P. J.; Ramsey, A.; Waigh, R. D.; Douglas, K. T.; Austin,
P.; Gilbert, P. J. Appl. Bacteriol. 1990, 69(4), 578–
584.
11. The rate of the reaction of 1 with the thiol groups of
coenzyme A is measured in the presence of coumarinyl-
phenyl-maleimide (CMP), which competes for occupying
the thiol groups. Measurement is done by tracking the
fluorescence of CPM.
In summary, we discovered a remarkably active new
class of biocides.15 Looking at the compounds investi-
gated so far, the activity seems to be confined to a small
window of derivatives of 1, substituted at the nitrogen
atom, if not to 1 exclusively. This narrow confinement
is surprising.
It might be the case that developing a new powerful
broad spectrum bactericide for non-pharma applications
12. The reaction of 1 with glutathione is finished in less than
6 h. For reactivity of fungicides toward thiols, see, for

E. Vogl et al. / Bioorg. Med. Chem. Lett. 15 (2005) 625–629
629
example: Gullner, G.; Cserhati, T.; Mikite, G. Pestic.
Biochem. Physiol. 1991, 39(1), 1–7.
13. Mortelmans, K.; Zeiger, E. Mutat. Res. 2000, 455, 29–
60.
14. Schlede, E.; Eppler, R. Contact Dermatitis 1995, 32(1), 1–4.
15. Bruns, R.; Eberz, G.; Kreiss, W.; Kretschik, O.; Kugler,
M.; Uhr, H.; Vogl, E.; Wachtler, P. (Bayerchemicals AG)
DE 10256186, 2004.