N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids as inhibitors of ureases

Article abstract of DOI:10.1007/s00726-011-0920-4

Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based

Full text of DOI:10.1007/s00726-011-0920-4

Amino Acids (2012) 42:1937–1945
DOI 10.1007/s00726-011-0920-4
ORIGINAL ARTICLE
N-substituted aminomethanephosphonic
and aminomethane-P-methylphosphinic
acids as inhibitors of ureases
•
Łukasz Berlicki Marta Bochno
•
•
Agnieszka Grabowiecka Arkadiusz Białas
•
•
Paulina Kosikowska Paweł Kafarski
Received: 30 December 2010 / Accepted: 16 April 2011 / Published online: 11 May 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Small unextended molecules based on the
diamidophosphate structure with a covalent carbon-to-
phosphorus bond to improve hydrolytic stability were
developed as a novel group of inhibitors to control microbial
urea decomposition. Applying a structure-based inhibitor
design approach using available crystal structures of bacte-
rial urease, N-substituted derivatives of aminomethylphos-
phonic and P-methyl-aminomethylphosphinic acids were
designed and synthesized. In inhibition studies using urease
from Bacillus pasteurii and Canavalia ensiformis, the N,N-
dimethyl derivatives of both lead structures were most
effective with dissociation constants in the low micromolar
range (Ki = 13 0.8 and 0.62 0.09 lM, respectively).
Whole-cell studies on a ureolytic strain of Proteus mirabilis
showed the high efficiency of N,N-dimethyl and N-methyl
derivatives of aminomethane-P-methylphosphinic acids for
urease inhibition in pathogenic bacteria. The high hydrolytic
stability of selected inhibitors was confirmed over a period of
30 days using NMR technique.
et al. 1997; Krajewska 2009). Subsequently, carbamate
decomposes spontaneously to carbon dioxide and a second
molecule of ammonia (Fig. 1).
Ureolysis is a major virulence factor that enables Heli-
cobacter pylori to colonize the highly acidic stomach
environment due to the increase in pH caused by ammonia
production (Burne and Chen 2000; Mobley et al. 1995;
Mobley and Hausinger 1989). High ammonia concentra-
tion impairs hydrogen ion transport through mucosal tissue,
which leads to ulcers and possibly cancer (Goodwin et al.
1986; Chen et al. 1986). Ureolytic bacteria infections of the
urinary tract by Proteus species and Ureaplasma urealyti-
cum with non-physiological alkalization of the urine induce
stone formation, lead to chronic inflammatory disease
combined with nephro- and ureolithiasis and cause a pre-
disposition to opportunistic infections (Griffith 1979;
Soriano and Tauch 2008; Worcester and Coe 2008).
The nitrogen cycle contributes to the ecosystem balance
and includes nitrogen fixation, mineralization, nitrification
and denitrification. Soil microorganisms play a crucial role
in those mechanisms and maintaining balance is strongly
dependent upon available nitrogen. Therefore, excessive
urea fertilization and microbial enzymatic decomposition,
which lead to uncontrolled ammonia release, are concern-
ing (Mobley and Hausinger 1989). The use of urea in
agriculture constitutes more that 50% of global N-fertilizer
usage in addition to its growing application as an animal
feed additive (Sahrawat 1980). Ammonia serves as the
primary substrate in the two-step nitrification process that
is conducted by autotrophic nitrifying bacteria. Enhanced
ureolysis and nitrification in urea-fertilized soils results in
N-losses due to ammonia volatilization and nitrate leach-
ing. The local increase in pH due to high urease activity
can damage plants in addition to the toxic effects of
accumulating nitrate on seeds and germinating seedlings.
Keywords Urease inhibition Á Transition state
analogues Á Diamidophosphate Á Aminophosphonic acid Á
Aminophosphinic acid
Introduction
Urease (urea amidohydrolase, E.C. 3.5.1.5) catalyzes the
hydrolysis of urea to ammonia and carbamate (Karplus
Ł. Berlicki (&) Á M. Bochno Á A. Grabowiecka Á A. Białas Á
P. Kosikowska Á P. Kafarski
Department of Bioorganic Chemistry, Faculty of Chemistry,
Wrocław University of Technology,
´
Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
e-mail: lukasz.berlicki@pwr.wroc.pl
_
123

1938
Ł. Berlicki et al.
were recorded in D2O (99.8% D) with NaOD on Brucker
Avance 300 or 600 MHz spectrometers. High-resolution
mass spectra were measured on an LCT Premier XE
(Waters) apparatus with electrospray ionization (positive
ions). Preparative HPLC purifications were performed with
Varian ProStar equipped with a C18 (21.4 mm 9 250 mm)
column (solvent A: 0.1% TFA in water, solvent B: 0.1%
TFA in acetonitrile). The elute was monitored at both 210
and 254 nm. Gradient A (t [min], %B; flow rate: 10 mL/
min): 0 min, 0%; 25 min, 18%; 35 min, 65%.
O
O
H2O,
NH3 +
H2N
NH2
NH2
HO
NH2
urease
O
NH3 + CO2
HO
Fig. 1 Enzymatic hydrolysis of urea
Nitrogen losses resulting from these processes can amount
to 50% of the fertilizer used (Gioacchini et al. 2002).
New strategies to regulate microbial urease activity both
for therapeutic and agronomical purposes are currently being
developed. Structurally diverse classes of urease inhibitors
have been successfully characterized (Amtul et al. 2002).
The most potent inactivators are phosphordiamides, which
are classical transition state analogues (Faraci et al. 1995;
Dominguez et al. 2008). Hydroxamates (Kobashi et al. 1962,
1971, 1975; Odake et al. 1992, 1994), imidazoles (Nagata
et al. 1993; Kuehler et al. 1995), benzoquinones (Zaborska
et al. 2002; Ashiralieva and Kleiner 2003), thiols (Ambrose
et al. 1950; Kot et al. 2000), thioureas and selenoureas
constitute other classes (Sivapriya et al. 2007). However, the
most effective structures (particularly phosphordiamidates)
lack stability in aqueous environments. A new class of
compounds containing a hydrolytically stable C–P bond is
one strategy for creating inhibitors with the desirable char-
acteristics (Vassiliou et al. 2008, 2010). In our previous
work, we used available crystal structures of bacterial urease
for molecular modeling and refined chemical synthesis of
peptidic derivatives of P-methyl- and P-hydroxymethyl-
aminomethanephosphinic acids. We demonstrated the
potential of this approach for the construction of active
urease inhibitors.
Chemistry
Compound 1 is commercially available (Aldrich). Com-
pounds 2 (Rohovec et al. 1996), 5, 8, 10 (Tyka and Hagele
1984), 7 and 12 (Kudzin et al. 2005) were obtained based
on literature protocols.
N,N-dimethylaminomethanephosphonic acid (4)
A mixture of N,N-dimethylamine (0.63 g, 10 mmol), die-
thyl phosphite (1.29 mL, 10 mmol) and paraformaldehyde
(0.30 g, 10 mmol) was heated for 3 h at 80°C. The solution
was diluted with diethylether (50 mL) and dried over
magnesium sulfate. After evaporation of the solvent, the
crude compound was purified using a preparative HPLC
method. Pure diethyl N,N-dimethylaminomethanepho-
sphonate was added to concentrated HCl (10 mL) and
refluxed for 6 h. Evaporation of the reaction mixture in
vacuo yielded crude product, which was dissolved in eth-
anol. Propylene oxide was added to the solution (to achieve
pH 7) and pure compound 4 was precipitated. The com-
pound was filtered and washed with acetone. Yield 0.97 g
(70%). 1H NMR (D2O, ppm): d 2.93 (s, 6H, 29CH3,
NCH3), 3.26 (d, 2H, J = 12.6 Hz, CH2P), 31P NMR (D2O,
ppm): d 7.35. HR-ESI MS (M ? 1), expected 140.0477,
found 140.0472.
In this paper, we describe the chemistry and biological
activity of N-substituted derivatives of aminomethylphos-
phonic and P-methyl-aminomethylphosphinic acids towards
plant and bacterial ureases. In comparison with previously
studied compounds, the structure of these derivatives is less
extended and less susceptible to hydrolysis. Molecular
modeling studies helped to elucidate their mode of binding
and to interpret kinetic data obtained in vitro.
N-Benzyl-N-methylaminomethanephosphonic acid (6)
Diethyl N-(benzyl-N-methyl)aminomethanophosphonate
(2.1 g, 8 mmol) (Karp 1999) was hydrolyzed with con-
centrated HClaq using the above-described procedure for
diethyl N,N-dimethylaminomethanephosphonate. Yield
1.46 g (85%). 1H NMR (D2O, ppm): d 2.75 (s, 3H, NCH3),
3.13 (bs, 2H, CH2P), 4.39 and 4.17 (2*bs, 2H, CH2Ph),
7.34 (m, 5H, Ar), 31P NMR (D2O, ppm): d 8.0. HR-ESI MS
(M ? 1), expected 216.0790, found 216.0790.
Materials and methods
General
N-methylaminomethanephosphonic acid (3)
All reagents obtained from commercial suppliers (Aldrich,
Fluka, Sigma, Merck, POCh) were of analytical grade and
were used without further purification. All NMR spectra
N-benzyl-N-methylaminomethanephosphonic acid (6)
(2.75 g, 12.69 mmol) was dissolved in acetic acid
123

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids
1939
(N-benzyl-N-methylamino)methane-P-methylphosphinic
acid (11)
(150 mL) and palladium catalyst (10% Pd/C) was added.
Hydrogenation at 1 atm was performed for 12 h. After
filtration of the catalyst, the solution was evaporated in
vacuo. The residue was dissolved in water and evaporated
again. Then, the residue was dissolved in HCl (20%,
80 mL) and evaporated. The crude hydrochloride product
was dissolved in ethanol (100 mL) and propylene oxide
was added to achieve a pH of 7. Precipitated compound 3
was filtered off and washed with acetone. Yield 0.93 g
A 100 mL flask equipped with a condenser was filled with
benzylmethylamine (33 mmol, 4.25 mL) in 30 mL of water
and heated on a steam bath. Next, 37% formalin (49 mmol
1.48 g) was added to the hot mixture. After 1 h, another
portion of formalin was added followed by heating for an
additional 2 h. Then the reaction mixture was cooled down to
room temperature and the volatiles were removed in vacuo.
The residue was dissolved in chloroform, washed with brine
and dried over anhydrous sodium sulfate. The desiccant was
filtered off and chloroform was removed in vacuo to yield
crude N-hydroxymethyl-N-methylbenzylamine.
1
(44%). H NMR (D2O, ppm): d 2.62 (s, 3H, CH3N), 2.96
(d, 2H, J = 12 Hz, CH2P), 31P NMR (D2O, ppm): d 9.5.
HR-ESI MS (M ? 1), expected 126.0320, found 126.0309.
N,N-dimethylaminomethane-P-methylphosphinic
acid (9)
A portion of the crude N-hydroxymethyl-N-methylben-
zylamine (12.6 mmol, 1.90 g) was dissolved in 30 mL of
glacial acetic acid and added dropwise to dichloromethyl-
phosphine (11.5 mmol, 1.0 mL). The mixture was mag-
netically stirred and cooled in an ice-water bath under inert
atmosphere. After 15 min, the cooling bath was removed,
and the mixture was heated under reflux for 40 min. When
boiling ceased, 20% hydrochloric acid (40 mL) was added
carefully to the reaction through the condenser. Hydrolysis
was performed for an additional 30 min under reflux. The
mixture was cooled, and the volatile products were
removed in vacuo. The remaining residue was dissolved in
35 mL of ethanol and treated with propylene oxide until
formation of precipitate could be observed. The precipi-
tated white solid was filtered off, washed with acetone and
dried. The product was separated with HPLC chromatog-
raphy (gradient A) to yield 0.85 g (35% yield) of 4.
1H NMR (D2O, ppm): d 1.25 (d, J = 14.4 Hz, 3H, PCH3);
2.85 (s, 3H, NCH3); 3.19 (bs, 2H, PCH2) 4.35 (s, 1H,
PhCH2), 7.43 (s, 5H, Ph). 31P NMR (D2O, ppm): d 28.80.
HR-ESI MS (M ? 1), expected 214.0997, found 214.0981.
(N-benzylamino)methane-P-methylphosphinic acid 10
(15 mmol, 3.0 g) and sodium carbonate (45 mmol, 4.8 g)
were dissolved with stirring in 30 mL of water. The mix-
ture was cooled in an ice-water bath. Next, iodomethane
(39 mmol, 2.43 mL) in 30 mL of dioxane was added drop
wise, and the mixture was stirred overnight. Precipitated
solid (NaI) was filtered off and volatiles were removed in
vacuo. Excess methyl iodide was removed by extraction
with ether, and the residue was cautiously acidified with
2.0 M HCl until evolution of CO2 ceased. After evapora-
tion of water, the solid residue was dissolved in methanol,
and insoluble inorganic salts were filtered off. Methanol
was then removed in vacuo to obtain 3.75 g (95%) of
(N-benzyl-N,N-dimethylamino)methane-P-methylphosphinic
acid hydrochloride.
Potassium carbonate (7.9 g) and 10% palladium on
a carbon catalyst (0.9 g) were added to the stirred
solution of crude (N-benzyl-N,N-dimethylamino)methane-
P-methylphosphinic acid hydrochloride. Hydrogen was
passed through the mixture under atmospheric pressure.
After 3 h, NMR analysis of the mixture showed com-
plete conversion of the substrate. The solids were filtered
off and the solvent was removed in vacuo. The residue
was dissolved in water and acidified with 2.0 M HCl
(pH \ 2). The resulting yellow–brown mixture was
washed several times with chloroform to remove chlo-
roform-soluble color contaminants. Water was then
removed in vacuo and the residue was dissolved in
acetonitrile. Insoluble inorganic salts were filtered off
and the solvent of the filtrate was removed in vacuo.
Finally, 1.9 g (80%) of 8 hydrochloride was obtained.
1H NMR (D2O, ppm): d 1.43 (d, J = 14.4 Hz, 3H,
PCH3); 3.00 (s, 6H, N(CH3)2); 3.37 (d, J = 8.6 Hz, 2H,
PCH2). 31P NMR (D2O, ppm): d 34.12. HR-ESI MS
(M ? 1), expected 138.0684, found 138.0661.
Enzyme purification
Bacillus pasteurii CCM 2056T was grown in a nutrient
media containing 20 g urea, 20 g/L of yeast extract with
addition of 1 mM NiCl2, pH 8 at 30°C. The cultures were
incubated for 48 h, yielding about 4.7 g/L of wet cells. The
collected cells were resuspended in lysis buffer containing
50 mM phosphate, pH 7.5, 1 mM b-mercaptoetanol, and
1 mM EDTA and sonicated. Unbroken cells and cell debris
were removed by centrifugation. The supernatant was
clarified using a 0.22 lm filter (RotilaboÒ, ROTH) and
then desalted on a BioGel column (Bio-Rad). The obtained
fractions were used as the starting material for the urease
purification. The enzyme preparation procedure con-
sisted of three steps: anion-exchange (Q Sepharose, GE
123

1940
Ł. Berlicki et al.
Healthcare), hydrophobic (Phenyl Sepharose, GE Health-
care) and affinity chromatography (Cellufine Sulphate,
Chisso Corporation). Initially the sample was loaded onto a
Q Sepharose column equilibrated with 50 mM phosphate
buffer at pH 7.5. Urease-containing fractions were eluted
with a linear gradient of NaCl (0–1.5 M). The ionic
strength of the obtained fractions was increased to 1 M
(NH4)2SO4 and then applied onto a Phenyl Sepharose
column. Urease was developed with a descending gradient
of (NH4)2SO4 in 50 mM phosphate buffer, pH 7.5. The
collected fractions were dialyzed against 20 mM phosphate
buffer, pH 6.5 and then loaded onto a Cellufine Sulphate
column, which had been equilibrated with the same buffer.
Enzyme elution was performed with 20 mM phosphate
buffer containing 0.5 M NaCl, pH 7.5. All of the purifi-
cation steps were performed at 10°C (cold room) using an
AKTA Prime system (Amersham Biosciences).
containing 0.2 mM phenol red and 100 mM urea in 10 mM
phosphate buffer, pH 7.0 after incubation at 37°C.
Whole-cell urease activity assay
Proteus mirabilis CCM 1944 was routinely grown at 37°C
on solidified medium containing 2% yeast extract, 2% urea
and 1 mM NiCl2 (Benini et al. 1996). Christensen urea agar
was used to confirm the ureolytic activity of the strain
(Vuye and Pijck 1973).
Cells harvested in early log phase (20 h of culture) were
suspended in saline at OD650 = 0.1 (Cfu = 1.2 9 105
mL-1). Urease was assayed in Eppendorf polypropylene
microplates (50–350 lL well) in a 100 lL mixture con-
taining modified Christensen liquid medium depleted of
phosphate and urea, adjusted to pH 5.5 with citric acid. For
IC50 estimation, 10 lL of the cell suspension were prein-
cubated in the presence of a defined inhibitor concentration
at 37°C for 15 min and the reactions were started with the
addition of 5 mM urea. The IC50 values were calculated as
described elsewhere (Vassiliou et al. 2008). The micro-
plates were incubated in ELMI SkyLine thermostatic
incubator at 37°C with gentle stirring (120 rpm). The
reactions were stopped and ammonia quantified by sub-
sequent addition of phenol/nitroprusside and NaOH/hypo-
chlorite solutions (100 lL each) (Vassiliou et al. 2008).
The plates were read at 650 nm using a TECAN Sunrise
microplate reader. The progress curves were initiated by
the addition of 20 lL of a cell suspension into 200 lL
reactions in Eppendorf tubes in the presence of 10 mM
urea at 37°C. Reactions were stopped by the addition of
phenol and hypochlorite solutions (500 lL each) and then
read at 650 nm.
Partially purified urease from Bacillus pasteurii CCM
2056T exhibited Michaelis–Menten saturation kinetics with
a KM of 17.6 0.9 mM, Vmax and a specific activity of
approximately 1,530.3 U/mg.
Urease activity was tested during purification by two
different methods: a qualitative phenol red urease test based
on the color change of the phenol red pH indicator as a result
of urea hydrolysis (during the hydrophobic column chro-
matography step) and a quantitative indophenol assay mea-
suring the amount of released ammonia (all other steps).
Inhibition test
The inhibitory properties of the analyzed compounds were
evaluated using a colorimetric indophenol assay. Aliquots
of the enzyme solution (10 lL) were incubated with
commercially available jack bean urease (Sigma) with an
estimated KM of 2.7 0.6 mM or partially purified urease
from Bacillus pasteurii CCM 2056T (KM 17.6 0.9 mM)
in 10 mM phosphate buffer, pH 7.0 in the presence of
various inhibitor concentrations (0.001–1 mM) in 96-well
plates. The IC50 determination was performed with 50 mM
urea for bacterial urease and 20 mM for plant urease. For
the Ki measurements, the urea concentration varied from 10
to 100 mM and from 0.5 to 15 mM for the abovementioned
enzymes, respectively. After 20 min of incubation at 37°C,
the reaction was stopped by adding phenol-hypochlorite
reagents and the absorbance of the formed indophenol blue
complexes was measured at 650 nm with a Tecan Sunrise
absorbance reader. The IC50 and Ki parameters of the
analyzed inhibitors were calculated with GraphPad Prism 5
software. All inhibitory tests were run in triplicate.
Molecular modeling
The crystal structure of the phosphate-urease (Bacillus
˚
pasteurii) complex refined to 1.85 A obtained from the
Protein Data Bank (1IE7) was used as the starting point
(Benini et al. 2001). All calculations were done with the
use of the Insight 2000 (Accelrys) molecular modeling
program package. Hydrogen atoms were added and the
protonation states for all side chains were set for pH 7.0.
The structure of the ligand was appropriately modified with
the Builder module and the inhibitor-enzyme complex was
optimized using the Discover program and cff97 force
field.
The qualitative red phenol assay was used during
enzyme purification. The urease activity of the extracted
fractions (10 lL) was monitored spectrophotometrically at
560 nm by measuring the color change of the mixture
Results and discussion
Urease is a large heteropolymeric enzyme with a vari-
able subunit composition depending on the organism
123

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids
1941
O-
O
a
O
P
NH
R
P
OH
N
+
H2N
NH2
R
OEt
H2N
NH3
O-
+ CH2O + HP(O)(OEt)2
OEt
enzymatic reaction
transition state
classical transition
state analogue
O
b
N
P
R
OH
OH
4, (R = CH3),
6, (R = Bzl)
O
O
c
H
O
O
N
P
N
P
OH
OH
OH
OH
H2N
P
H2N
P
OH
OH
OH
3
6
1
2
O
O
d
extended transition state analogues
R
H
N
H
N
R
OH
H
O
P
H
e or f
b
N
R
R'
OH
R
N
O
P
OH
R
N
O
P
OH
5, (R = Bzl, R' = OH),
8, (R = CH3, R' = CH3),
10 (R = Bzl, R' = CH3)
R'
R'
OH
3, (R = CH3, R' = H),
4, (R = CH3, R' = CH3),
5, (R = Bzl, R' = H),
6, (R = Bzl, R' = CH3),
7, (R = CH3CO, R' = H).
8, (R = CH3, R' = H),
9, (R = CH3, R' = CH3),
10, (R = Bzl, R' = H),
11, (R = Bzl, R' = CH3),
12, (R = CH3CO, R' = H).
O
O
g
H
N
P
N+
I-
P
OH
OH
10
O
h
N
P
Fig. 2 Structures of designed compounds
OH
9
(Krajewska 2009; Ham et al. 2001; Benini et al. 2000).
Despite the significant variability in the overall composi-
tion of the enzyme, the structure of its active site is highly
conserved. The active site contains two nickel ions, has a
relatively small volume, and is covered by a flap during the
reaction. The most effective urease inhibitors are diami-
dophosphate (DAP) and its esters, which hydrolyze to the
active molecule (DAP) in the active site (Benini et al.
1999). Diamidophosphate is a classical transition state
analogue. Because the structure of DAP contains hydro-
lyzable (particularly at low pH) P–N bonds, we aimed to
explore extended transition state analogues containing
highly stable P–C bonds, such as aminomethylphosphonic
and aminomethyl-P-methylphosphinic acids (compounds 1
and 2, Fig. 2). Previously, we showed that 2 is a moderate
urease inhibitor and that some of its peptidic derivatives are
very potent. Here, we used N-alkylated derivatives of 2 and
its close analogue 1—structures 3-6 and 8-11. Although the
proposed substituents do not offer the possibility of form-
ing additional hydrogen bonds in contrast to the parent
molecules, they allow for controlling the number of
hydrogen bond donors (methyl moieties) and/or for the
H
N
i, j
O
N
P
OH
11
Scheme 1 Reagents and conditions: a reflux, b HClaq, then popylene
oxide, c H2, 10% Pd/C, d 37% formalin, steam bath, e PCl3, (for
compound 5), f CH3PCl2,(for compounds 8, 10), g CH3I, Na2CO3,
h H2, 10% Pd/C, K2CO3, i CH2Oaq, j CH3PCl2, then HClaq
creation of aromatic interactions (benzyl groups). More-
over, this type of substitution provides highly stable
derivatives.
The parent molecules 1 and 2 are readily available—
either commercially (1) or using standard protocols from the
literature (2) (Rohovec et al. 1996). The use of the parent
molecules in the synthesis of other designed compounds is
not convenient with the exception of N-acetylated structures
7 and 12, which were obtained quantitatively by reaction of
compounds 1 and 2, respectively, with excess acetic anhy-
dride in acetic acid (Kudzin et al. 2005). Disubstituted
phosphonic derivatives 4 and 6 were obtained from a
123

1942
Ł. Berlicki et al.
three-component condensation of the appropriate amine,
formaldehyde and diethylphosphite and by subsequent
acidic hydrolysis (Scheme 1). Compound 6 was also used for
the synthesis of the N-methyl analogue 3 by hydrogenolysis.
Monosubstituted derivatives were synthesized using the
known method with N-hydroxymethyl-N-alkylformamides
as the starting material (Tyka and Hagele 1984). The reaction
of an appropriate substrate with phosphorus thrichloride
or dichloromethylphosphine and subsequent hydrolysis
produced compounds 5, 8 and 10. N, N-dimethylaminome-
thane-P-methylphosphinic acid 9 was obtained using
exhaustive methylation of the N-benzylated compound 10
and hydrogenolytic removal of the benzyl group under basic
conditions (Hulme and Rosser 2002). Finally, we synthesized
11 by reaction of benzylmethylamine with formaline
and fosfinylation of the hydroxymethyl intermediate with
CH3PCl2.
binding. The phosphinic acid group of this inhibitor forms
two hydrogen bonds with His222 and Asp363 and interacts
with the two nickel ions (Fig. 3).
In addition to the N-methyl compounds, we also eval-
uated the acetylated and N-benzylated compounds.
Unfortunately, these modifications did not enhance the
inhibitory potency of the compounds. Most likely, the
space available within the active site of urease imposes
steric requirements, which cannot be fulfilled by the
additional aromatic substituents of compounds 5, 6, 10 and
11. The enzyme delivered from plant material was gener-
ally less susceptible to inhibition by the studied compounds
consistent with data published on other urease inhibitors.
Within the analyzed group of inhibitors, analogously to the
bacterial enzyme, structures 4 and 9 were the most potent
with Ki values equal 21 and 3.7 lM, respectively.
We evaluated the hydrolytic stability of the most active
compounds 3, 4, 8 and 9. NMR analysis of aqueous solu-
tions (pH 2) of the compounds showed unchanged spectral
characteristics after 30 days at room temperature. It is
worth to note that, the class of compounds presented herein
is considerably more stable than the widely studied phos-
phoramidates (Pope et al. 1998).
All synthesized compounds were assayed against urease
purified from Bacillus pasteurii CM2056T (Vassiliou et al.
2008) and commercially available enzyme from Canavalia
ensiformis (Table 1). Kinetic data were obtained using the
indophenol blue assay based on the Berthelot reaction
(Chaney and Marbach 1962). Progress curves of the urease
reaction in the presence of the studied compounds revealed
the competitive reversible mode of inhibition within the
micromolar range for most of the compounds. Both parent
molecules (1 and 2) exhibited similar potency against the
bacterial enzyme with inhibitory constants of 314 and
340 lM, respectively. Subsequent substitution of the
nitrogen atom with a methyl group caused a significant
increase in the activity (inhibitors 3 and 8). Introduction of
a second methyl moiety (compounds 4 and 9) further
reduced the inhibition constants, Ki to 13 lM and 620 nM,
respectively. The difference in the free energy of binding
(DDG) between the unsubstituted compound 2 and its
N-methyl analogue 8 as well as between inhibitor 8 and its
N,N-dimethyl counterpart 9 is similar (1.7 and 2.0 kcal/
mol, respectively) and corresponds to the energy of
hydrogen bond breakage. We assume that during the for-
mation of the urease-inhibitor 2 complex, three hydrogen
bonds between bulk water and inhibitor amine group are
broken and replaced with only one interaction with the
enzyme active residues.
For whole-cell urease inhibition studies, we used the
Proteus mirabilis strain, which is a common pathogen of the
human urinary tract (Mobley et al. 1995). The inhibition
studies using whole cells were all performed at pH 5.5, which
corresponds to the acidity of physiological urine. Plate cell
counts proved that the Proteus cells remained viable and
actively multiplied under the assay conditions used.
The effectiveness of urease inhibition against intact cells
was evaluated for compounds 8 and 9, and compared with
well-established acetohydroxamic acid (AHA). In the
presence of 5 mM urea using 1.2 9 103 cells, the IC50
values were 154 6 lM for compound 9, 36 3 lM for
compound 8 and 64.6 3 lM for AHA. Similarly to the
results of the cell-free inhibition studies (Table 1), the
values were in the micromolar range in the whole-cell
assay and the mono-substituted derivative 8 was a more
efficient inhibitor than the N,N-dimethyl compound 9.
Similar to acetohydroxamate, the time course of urease
activity in the presence of compounds 9 and 8 in non-
preincubated system showed a short lag phase of inhibition
(Fig. 4). The non-preincubated system likely requires time
to allow the interaction between the intracellular enzyme
and inhibitor. Because the Proteus urease is located in the
cell cytoplasm, the inhibitory efficiency is strongly
dependent on the compound diffusivity. The obtained
results show that the newly developed small structures
containing the remarkably stable carbon-to-phosphorus
bond are active against urease in intact bacterial cells and
their effectiveness is comparable with acetohydroxamic
acid.
In the case of inhibitor 8, two hydrogen bonds with its
amine group are broken (with bulk water) and one is
formed (with enzyme) upon complexation with urease. The
energy balance is most favorable with inhibitor 9 because
one hydrogen bond is broken and one is formed with its
amine group during the complexation process. The mod-
eled mode of binding of the most active inhibitor 9 shows
that its amine group forms one hydrogen bond with the
carbonyl moiety of Ala366, which is consistent with the
experimental data for differences in the free energy of
123

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids
1943
Table 1 Inhibitory activities of aminomethanephosphonic and aminomethyl-P-methylphosphinic acid derivatives (NI not inhibitory)
No
Compound
Canavalia ensiformis urease
Bacillus pasteurii urease
IC50 [lM] Ki [lM]
IC50 [lM]
Ki [lM]
1
432 37.8
240 64.3
700 23
1100 10.5a
228 15.6
49 1.7
NI
314 8.2
340 22a
70 2,2
13 0.8
O
P
H2N
OH
OH
2
3
4
5
NI
O
P
H2N
OH
678 48
82 26
NI
204 52
O
P
H
N
OH
OH
21 5.7
O
P
N
OH
OH
O
P
H
N
N
OH
OH
6
7
NI
NI
300 6.5
115 7.4
O
P
OH
OH
463 34
148 18.5
O
P
H
N
OH
OH
O
8
884 14
224 39
3.7 1.7
571 119
60.0 0.3a
18.0 0.7a
0.62 0.09
72 3.8
O
H
N
P
OH
9
14.4 4.8
3.8 0.4
O
N
P
OH
10
1 632 226
256 12.8
O
H
N
N
P
OH
11
12
NI
NI
O
P
OH
1 455 189
295 96
258 12.7
95 6.3
O
P
H
N
OH
O
a
Values reported previously (Vassiliou 2008)
123

1944
Ł. Berlicki et al.
Fig. 3 Stereo image of modeled mode of binding of compound 9 to the Bacillus pasteurii urease active site. Hydrogen bonds are marked as thin
solid lines
Conclusions
bacterial urease with a Ki of 0.62 lM, making it one of the
most active reversible urease inhibitors (Kosikowska and
Berlicki 2011; Follmer 2010; Amtul et al. 2002). The most
widely studied and clinically used inhibitor acetohydroxa-
mic acid showed a Ki of 2.6 lM (against Klebsiella aerog-
enes enzyme, Todd 1989) and similar activity against whole
cells. Such characteristics imply that these compounds could
be used in therapeutic and agricultural applications. More-
over, these structures offer the possibility of various modi-
fications, which might provide improved physicochemical
and inhibitory properties.
Herein, we explored the potential of phosphonates and
phosphinates as urease inhibitors. This is the first report on
phosphonic acids designed for use against bacterial urease.
However, the P-methylphosphinates were developed to
enhance the inhibitory activity of previously published
structures (Vassiliou et al. 2008). We investigated chemical
structures based on the C–P bond as an attractive alternative
to known phosphoramidates. Our studies led to the synthesis
of structures with low structural complexity, high hydrolytic
stability and satisfactory biological activity against purified
cytoplasmic urease of pathogenic Proteus species. The most
active compound 9 showed high inhibitory activity against
Acknowledgments This work was supported by a grant from the
Polish Ministry of Science and Higher Education (no. PBZ-MEiN-5/
2/2006 and 681/N-COST/2010/0). The calculations were performed
using hardware and software resources (including the Accelrys pro-
grams) at the Supercomputing and Networking Center in Wrocław.
P. Kafarski, Ł. Berlicki, A. Białas and P. Kosikowska thank the
Foundation for Polish Science for financial support. P. Kosikowska is
grateful for a fellowship that was co-financed by the European Union
and the European Social Fund.
30
25
20
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
control
15
0.1mM 9
0.1 mM AHA
10
0.1mM 8
5
0
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