Design, synthesis, and evaluation of novel organophosphorus inhibitors of bacterial ureases

Article abstract of DOI:10.1021/jm800570q

A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in vitro against urease from Bacillus pasteurii and Proteus vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with K _i = 170 nM (against B. pasteurii enzyme) for the most active N-(N′-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid.

Full text of DOI:10.1021/jm800570q

5736
J. Med. Chem. 2008, 51, 5736–5744
Design, Synthesis, and Evaluation of Novel Organophosphorus Inhibitors of Bacterial Ureases
Stamatia Vassiliou,‡ Agnieszka Grabowiecka,† Paulina Kosikowska,† Athanasios Yiotakis,‡ Paweł Kafarski,† and
Łukasz Berlicki†,*
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw UniVersity of Technology, Wyb. Wyspian´skiego 27,
50-370 Wrocław, Poland, Laboratory of Organic Chemistry, Department of Chemistry, UniVersity of Athens, Panepistimopolis, Zografou,
15771 Athens, Greece
ReceiVed May 15, 2008
A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using
the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-
methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in Vitro against urease
from Bacillus pasteurii and Proteus Vulgaris. The studied compounds constitute a group of competitive,
reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures
exhibited kinetic characteristics of potent, slow binding urease inhibitors, with Ki ) 170 nM (against B.
pasteurii enzyme) for the most active N-(N′-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic
acid.
Introduction
transition state analogue.10,15 However, hydroxamic acids are
the best recognized urease inhibitors.13,19-22 The simplest
analogue in this group is acetohydroxamic acid, which exhibited
a competitive slow-binding inhibition with Ki ) 2.6 µM against
Klebsiella aerogenes enzyme. Various hydroxamate analogues,
several aminoalkanehydroxamic acids, their N-substituted de-
rivatives, as well as dipeptides, were examined (N-glycylgly-
cinehydroxamic acid exhibited IC50 ) 0.79 µM against Heli-
cobacter pylori urease).23,24 Interestingly, simple organosulfur
compounds represented, for example, by ꢀ-mercaptoethanol, also
exhibited considerable inhibitory activity.25 This phenomenon
was explained by the analysis of the crystal structure of
ꢀ-mercaptoethanol-urease complex, in which the sulfur atom
was bridged between the two nickel ions present in the active
site.6
Urease (urea amidohydrolase, E.C. 3.5.1.5) is an enzyme that
catalyzes hydrolysis of urea to ammonia and carbamate, which
is the final step of nitrogen metabolism in living organisms.1,2
Carbamate decomposes rapidly and spontaneously, yielding a
second molecule of ammonia. These reactions cause significant
increase of solution pH.
Bacterial ureases are large heteropolymeric metalloproteins
with nickel(II) ions present in their active sites.3-6 A significant
amino acid sequence similarity was observed between all ureases
of a bacterial origin.7,8 The mechanism of enzymatic reaction
has been studied extensively by several research groups for
many years.2,5,9-11 On the basis of several crystal structures of
complexes of Bacillus pasteurii urease, Ciurli and co-workers
proposed the most reliable enzymatic reaction mechanism.10 The
active site of the native enzyme binds three water molecules
and a hydroxide ion bridged between two nickel ions. Urea
replaces these three water molecules and is bound by a network
of hydrogen bonds as well as by the nickel ions.12 An activated
carbon atom of urea is attacked by the Ni-bridging hydroxide
ion, forming a tetrahedral transition state. Subsequently, am-
monia is released from the active site followed by the negatively
charged carbamate.
Several classes of compounds are known to show considerable
inhibitory activity against this enzyme with phosphoramidates
being the most active.13-17 It was shown that phenyl phosphor-
diamidate (PPDa) and its 4-substituted derivatives exhibit
inhibitory properties in nanomolar range (PPD was a competitive
slow-binding inhibitor with Ki* ) 0.6 nM against B. pasteurii
enzyme).16,18 Its mode of action relays on hydrolysis in the
active site and release of phosphorodiamidic acid (1), which is
the actual enzyme inhibitor and represents enzymatic reaction
The studies on novel urease inhibitors are essential not only
for the basic research on urease biochemistry but also for the
possible development of a highly needed therapy for urease
mediated bacterial infections.1,8,26,27 Ammonia released in the
urease catalyzed reaction is either a direct cause of clinical
conditions or a crucial factor to the pathogen survival and a
host colonization. Ureolytic activity of several microorganisms,
i.e., Proteus mirabilis, Proteus Vulgaris, and Ureaplasma
urealyticum, is involved in the formation of urinary tract stones,
which may lead to the chronic inflammation of kidney and its
pelvis.28-31 Additionally, urinary catheter obstruction in patients
is caused by its colonization by urease-producing microorgan-
isms, mainly P. mirabilis.32,33 Moreover, the overproduction of
ammonia by infectious microorganisms may contribute to
ammonia encephalopathy or hepatic coma.34-36Acetohydroxamic
acid, a potent urease inhibitor, was shown to be an efficient
drug against diseases caused by ureolytic bacteria.37-39 Another
mechanism of urease involvement in pathogenic bacteria
infection relies on the creation of a microenvironment suitable
for the pathogen existence. Helicobacter pylori infection of
stomach is possible only after local neutralization of gastric acid
by released ammonia.27,40,41 Furthermore, high concentration
of ammonia disturbs mucosal permeability, in particular hy-
drogen ions passage through mucosal surface, and causes
formation of peptic ulcers.42,43 The immense significance of this
medical problem was emphasized by the 2005 Nobel Prize in
* To whom correspondence should be addressed. Phone: +48 71 320
40 80. Fax: +48 71 328 40 64. E-mail: lukasz.berlicki@pwr.wroc.pl.
†
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw
University of Technology.
‡
Laboratory of Organic Chemistry, Department of Organic Chemistry,
University of Athens.
a Abbreviations: PPD, phenyl phosphordiamidate; Cbz, benzyloxycar-
bonyl, TMS, trimethylsilyl; EDC 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide; HOBt, Hydroxybenzotriazol; DIPEA, diisopropylethylamine.
10.1021/jm800570q CCC: $40.75
2008 American Chemical Society
Published on Web 08/22/2008

Organophosphorus Inhibitors of Bacterial Ureases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5737
Scheme 1
Physiology or Medicine, awarded to Warren and Marshall for
the research on the role of H. pylori in gastritis and peptic ulcer
disease.
In this paper, we present the design, synthesis, and evaluation
of novel ogranophosphorus inhibitors of bacterial urease. On
the basis of the crystal structure of Bacillus pasteurii urease,
several P-methyl phosphinic peptidic structures were designed
by using the computer aided techniques. Successful synthesis
of the designed compounds in enantiomerically pure forms
allowed the kinetic evaluation of their potency against ureases
purified from B. pasteurii and P. Vulgaris. Obtained results
showed high activity of the novel structures, with inhibitory
constants in nanomolar range for the most active compounds,
which proves the correctness of the applied strategy.
Figure 1. Computed structure of aminomethyl(P-methyl)phosphinic
acid (2)-urease (B. pasteurii) complex. Hydrogen bonds are indicated
as green dashed lines.
Results and Discussion
Computer-Aided Design. Among the several known urease
inhibitors, phosphordiamidates are the most efficient.15,16,44
A
very high activity of phosphorodiamidic acid (1), the simplest
structure in this group, results from its close similarity to the
transition state of the enzymatic reaction (Scheme 1). The main
disadvantage to the widespread application of this class of
inhibitors is their susceptibility to hydrolysis, particularly at low
pH. To overcome such limitation, we considered the replacement
of unstable P-N bonds with highly inert P-C linkages. Thus,
aminomethyl(P-methyl)phosphinic acid (2) was chosen to be a
lead compound, structurally analogous to phosphorodiamidic
acid (1). It is well established that phosphinic acids act as very
potent inhibitors of metal mediated enzymatic amide bond
hydrolysis.45-47 Moreover, it was shown that this class of
compounds exhibit similar inhibitory properties to phosphora-
mides, as the computed free energy of binding is very
similar.48,49 Additionally, the positively charged aminomethyl
moiety, although extended, is very similar to the transition state
due to the fact that it retains the same charge.50 On the basis of
the crystal structure of 1-B. pasteurii urease complex,10 the
binding mode of 2 in the enzyme active site was modeled
(Figure 1).
Figure 2. Modeled mode of binding of N-glycyl-aminomethyl(P-
methyl)phosphinic acid (3) to bacterial urease active site. Hydrogen
bonds are indicated as green dashed lines.
amide group with ArgR339 and AlaR366, respectively. These
results strongly suggested that compound 3 should exert higher
inhibitory effect toward bacterial urease comparing to 2, which
was further proved by calculation of LUDI scores, which are
the measure of their potential enzyme affinity. The LUDI score
obtained for 3 (SCORE ) 462) was substantially higher than
that calculated for 2 (SCORE ) 338).
The next possible improvement of the inhibitor potency was
the N-terminal amino acid residue structural extension. To
enhance hydrophobic interactions with the enzyme, bulky
substituents were introduced (compounds 4-6). Optimization
of the structure of these inhibitors’ complexes with urease
confirmed the correctness of proposed modifications. All
hydrogen bonds found for 3-urease complex were preserved,
while hydrophobic bulky substituents docked well in the enzyme
cavity. Computed LUDI scores (SCORE ) 575, 593, and 504
for 4, 5, and 6, respectively) also suggested higher possible
efficacy of these inhibitors. Additionally, serine derivative 7 was
chosen for further synthesis, as the molecular modeling showed
that it could exhibit potency similar to 3.
As assumed, the binding pattern of 2 is comparable to that
observed for 1. Both oxygen atoms of 2 form hydrogen bonds
with AspR363 and HisR222 and thus reproduce the same pattern
that has been evidenced for 1. The methyl group of 2 docks in
the place of Ni(2)-bound phosphorodiamidic acid (1) amide
group. Analogously to the distal amide moiety of 1, the
aminomethyl substituent of 2 projects toward the entrance of
the active site and forms hydrogen bonds with HisR323 and
AlaR366
.
Structure 2 offers highly interesting possibilities for further
modifications in order to enhance significantly the affinity
toward the enzyme by derivatization of its amino moiety. Taking
into consideration both the kinetic characteristics of dipeptide
hydroxamic acids23 and the synthetic feasibility, a dipeptidic
analogue of 2 was planned. Thus, the mode of interaction of
the extended, nonsubstituted analogue, N-glycyl-aminomethyl(P-
methyl)phosphinic acid (3) with urease active site, was modeled
(Figure 2). Two novel interactions, in comparison to 2-urease
complex, occurred in the model, namely two hydrogen bonds
formed by the carbonyl oxygen atom and the NH of the inhibitor

5738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Vassiliou et al.
Scheme 2a
a Reagents and conditions: (a) NaH2PO2, CH3OH, HCl, reflux, 30 min.;
(b) Cbz-Cl, K2CO3, rt; (c) TMSCHN2; (d) NaH, CH3I, THF, -15°C to rt.
to catalytic hydrogenolysis followed by treatment with TMSBr,
the serine analogue 7 was obtained (Scheme 4), while reacting
17 only with TMSBr provided the benzyl protected serine
analogue 6. By a similar reaction sequence compound 13 was
transformed to potential inhibitor 18.
Finally, on the basis of the well-established high potency of
organosulfur compounds against urease,6,51 we decided to make
an additional modification by introducing a sulfur atom into
the structure of phosphinic dipeptides. Thus, the oxygen atom
of phosphinic group that bridges nickel ions could be replaced
by a sulfur atom, yielding thiophosphinic dipeptide (8) in order
to obtain a similar pattern of interactions with that observed in
the crystal structure of ꢀ-mercaptoethanol-urease complex.
To obtain Cbz-protected inhibitors starting from 12 and 14,
these compounds were demethylated with excess of aqueous
LiOH in dioxane followed by acidification with diluted HCl
and column chromatography purification to provide pure 19 and
1
20 (Scheme 5) as judged by H NMR.
Chemistry. Although the synthesis of 1-aminomethylphos-
phinic acid 9 (the analogue of glycine) has been intensively
studied,52-54 none of these methods were successful in our hands
either because of multistep character and thus very low total
yield of the process or because of toxic and expensive reagents
required. Therefore, a recently described55 method was at-
tempted and proved to be successful. In this procedure, aqueous
solution of NaH2PO2 and formaldoxime was added to hot
methanolic solution of HCl, and the product was separated by
ion-exchange chromatography (Scheme 2). The main byproduct
of this reaction is the diaddition product, namely bis(aminom-
ethyl)phosphinic acid, which is easily observed in 31P NMR
spectra (14.2 ppm for the monoadduct, 29.2 ppm for the diadduct
in D2O). Benzyloxycarbonyl (Cbz) protection of 9 proceeded
smoothly, and the product 10 was obtained in pure form. NMR
characterization of this compound is possible in D2O/NaOD
solution, otherwise only broad signals are observed, suggesting
that aggregation or micelle formation occurs, most likely as a
result of hydrogen bonding.56 Subsequent O-methylation with
trimethylsilyl diazomethane provided 11 in high yield. Although
no other special precautions were taken, the P-H compound
was immediately used after purification to avoid possibility of
oxidation to the corresponding phosphonate. P-methylation was
achieved by direct alkylation with CH3I using NaH as a base.
By using one equivalent of the base, compound 12 was obtained
while two equivalents of the reagents resulted in both P- and
N-methylation, providing 13 in very good yield after chromato-
graphic purification (Scheme 2).
Further elongation of 12 was achieved by conventional
carbodiimide/benzotriazole procedure. Thus, Cbz-group was
removed by catalytic hydrogenolysis and the free amine was
acylated using several natural amino acids in the presence of
EDC·HCl and HOBt. After column chromatography, com-
pounds 14-17 (Scheme 3) were obtained as mixture of two
enantiomers for the first pseudodipeptide and two diastereoi-
somers in the three other cases. The purified compounds were
finally deprotected in two subsequent synthetic steps. The methyl
ester was removed by using TMSBr and then methanol. The
benzyloxycarbonyl group was removed using HBr in acetic acid.
Compounds 3-5 were purified by means of cation exchange
resin column chromatography by water elution, and the purity
of the final products was confirmed by HPLC using a reverse
phase MZ-Analytical column, Kromasil, C18.
After evaluating the previously synthesized phosphinic inhibi-
tors, we turned our attention to preparation of the thio-analogues
of the most potent ones. The replacement of amide bonds in
physiologically active peptides with thioamide bonds is one of
several backbone modifications used frequently in the search
for more potent and/or selective compounds than the parent
structures. Despite numerous literature examples57 for the
replacement of the oxo group of phosphorus (PdO) with the
thio (PdS), to the best of our knowledge, there is no reference
for thiophosphinic pseudopeptides. Very recently,58 Acher et
al. described the synthesis and agonist activity of a thiophos-
phonate toward group III metabotropic glutamate receptors. To
synthesize the thiophosphinic analogue of 20, Lawesson’s
reagent was chosen as thionating agent because phosphorus
pentasulfide P2S5, commonly used for such transformations in
the past, is associated with low and variable yields, long reaction
times, and large excess of reagent are required.59 Because
Lawesson’s reagent is the reagent of choice for transformations
of functional groups like amides and urethanes, we thought that
compound 12 would be the best substrate for the thionation
because there is no amide bond present. Indeed, taking into
consideration the reactivity of Lawesson’s reagent toward the
carbonyl group60 (Figure 3) and the literature data for the (PdO)
to (PdS) transformation,61 we anticipated that by careful control
of the temperature we could selectively thionate phosphorus
atom without affecting the carbamate function.
Therefore, after detailed experimentation, we found that the
optimal reaction conditions were: heating at 95 °C, for 2 h in
dry toluene. Under these conditions, compound 21 was obtained
in 82% yield after purification (Scheme 6). It can be noted that
the increase of the 31P NMR chemical shift, 54.4 ppm for 12
compared to 96.1 ppm for 21, which is very diagnostic. In the
further step, Cbz group was removed using HBr/AcOH without
affecting the thiophosphinic function. Catalytic hydrogenolysis
was excluded because of the well-known poisoning effect of
sulfur. After liberating the amine from its hydrobromic salt using
DIPEA, conventional carbodiimide method provided protected
thiophosphinic dipeptide 22, using Cbz-Gly-OH as acylating
agent. When we tried to apply the previously described
deprotection sequence (TMSBr then HBr/AcOH), we found out
that after treating 22 with TMSBr, the obtained product was
20, where sulfur-oxygen exchange had occurred quantitatively
as judged by mass spectroscopy (Scheme 7). This prompted us
to search the literature for analogous results. The only similar
example is a study published in 1985,62 describing the high
By careful planning of the deprotection sequence, two
additional inhibitors were obtained. Indeed, by subjecting 17

Organophosphorus Inhibitors of Bacterial Ureases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5739
Scheme 3a
a Reagents and conditions: (a) H2, Pd/C, CH3OH, 1 atm, rt; (b) R1-NH-CH(R2)-COOH, EDC·HCl, HOBt, CH2Cl2, rt, 12 h; (c) TMSBr, CH2Cl2, rt, 2 h,
then CH3OH; (d) 33% HBr/AcOH, rt, 1 h; (e) Dowex AG50 × 4 (H+).
Scheme 4a
against urease from Bacillus pasteurii (Sporosarcina pasteurii)
CCM 2056T and Proteus Vulgaris CCM 1956. Affinity purifica-
tion using cellufine sulfate combined with hydroxylapatite ion-
exchange chromatography yielded urease preparations of 3800
nkat mg -1 specific activity (40% recovery) and 2550 nkat mg -1
(27% recovery) for Bacillus and Proteus strains, respectively.
B. pasteurii enzyme was kinetically characterized by Km of 28
mM and Vmax of 0.063 mM s-1, for P. Vulgaris urease Km was
12.4 mM and Vmax 0.035 mM s-1. Urease activity was evaluated
using Berthelot color reaction procedure, as other methods of
ammonia quantification based on pH changes (e.g., phenol red
assay) lead to artificial results due to strong buffering properties
of the studied inhibitors.63 All analyzed compounds exhibited
interesting inhibitory activity against bacterial ureases, however,
their potency varied substantially from Ki 340 µM to 170 nM
a Reagents and conditions: (a) H2, Pd/C, CH3OH, 1 atm, rt; (b) TMSBr,
for 2 and 24, respectively, against B. pasteurii enzyme (Table
1). There were no significant differences in susceptibility of the
CH2Cl2, rt, 2 h, then CH2OH; (c) Dowex AG50 × 4 (H+).
two purified microbial ureases toward presented inhibitors,
Scheme 5a
which is most probably the result of very similar structure of
their active sites.8
As expected, originally designed lead compound 2 exhibited
moderate activity of the competitive, reversible mode (Ki ) 340
µM, B. pasteurii enzyme). Further modification of this structure
caused considerable affinity improvement for all designed
compounds toward chosen enzymes. Noteworthy, a very small
alternation of inhibitor structure by substituting an amine
hydrogen with a methyl group yielded over 1 order of magnitude
decrease of inhibitory constant (Ki ) 18 µM for compound 18).
According to molecular modeling studies, in 2-urease complex,
one of the amine hydrogen atoms of the inhibitor is not engaged
in the hydrogen bonding. Thus, the negative enthaphlic effect
of desolvatation of 2 upon binding to the enzyme is eliminated
by N-methylation.
On the basis of results obtained by Kabashi and co-workers
for hydroxamic acids,23 as well as our molecular modeling
studies, dipeptidyl structures were synthesized and evaluated.
Such extension of the lead compound, represented by structures
3-7, proved successful, with inhibitory constants being in low
micromolar range. Interestingly, the simplest structure in this
group, namely compound 3, appeared to be the most effective
(Ki ) 21 µM for B. pasteurii enzyme).
Combination of sulfur containing compounds with dipeptidyl
structures leading to compound 24 caused exceptional improve-
ment of inhibitory activity, and Ki* ) 170 nM was obtained
for this structure. This value is about 3 orders of magnitude
better than the one of the analogous oxygen compound 20.
Moreover, it was found that kinetic characteristic of 24 was
completely different, showing slow binding mode of action
(Figure 4). Initial rates of the progress curves recorded in the
presence of 24 were hyperbolically dependent on the inhibitor
concentration, suggesting mechanism B of inhibitor binding.64
Thus, the inhibitor most likely undergoes the conformational
change after binding to the enzyme and a slow-dissociating
enzyme-inhibitor complex is formed.
a Reagents and conditions: (a) aq LiOH, dioxane, 48 h, rt; (b) aq HCl.
Figure 3. Reactivity of Lawesson’s reagent toward the carbonyl groups
of protected dipeptides.
acidity catalysis of the sulfur exchange reaction for a very simple
thiophosphinate, namely O-methyl dimethylthiophosphinate.
Furthermore, desulfurization under acidic conditions was re-
ported very recently58 for a thiophosphinate.
Taking this information into consideration, we decided to
perform alkaline hydrolysis of the methyl ester. Indeed, by
treating 22 with LiOH for 48 h, Cbz-protected thiophosphinic
acid 24 (Scheme 6) was obtained after typical workup followed
by column chromatography purification, which was confirmed
by mass spectrometry and 31P NMR comparison with 20 (48.7
ppm for 20 and 88.4 ppm for 24). Analogously, compound 25
was obtained from 21. For the thiophosphinic dipeptide 8, a
similar strategy was pursued, but Fmoc-Gly-OH was used as a
substrate in coupling step in order to receive simultaneous
cleavage of the Fmoc group and the methyl ester upon alkaline
hydrolysis. Dowex purification furnished pure compound 8.
Biological Activity. The inhibitory potential of the synthe-
sized P-methyl phosphinic and tiophosphinic acids was assessed

5740 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Vassiliou et al.
Scheme 6a
a Reagents and conditions: (a) Lawesson’s reagent, 95°C, 2 h; (b) (i) 33% HBr/AcOH; (ii) Cbz-Gly-OH or Fmoc-Gly-OH, DIPEA, EDC ·HCl, HOBt,
CH2Cl2, rt, 12 h; (c) (i) aq LiOH, dioxane, 48 h, rt; (ii) aq HCl 0.5M.
Scheme 7a
are quoted, were chromatographically homogeneous. Reactions were
monitored by thin-layer chromatography (TLC) carried out on 0.25
mm silica gel plates (60F-254) using UV light as visualizing agent
and an aqueous solution of cerium molybdate/H2SO4 (“Blue Stain”)
or 2% (w/v) ninhydrin in ethanol and heat in both cases as
developing agent. Purification of compounds by column chroma-
tography was carried out on silica gel (70-230 mesh). RPHPLC
analyses were carried out on an analytical instrument using a MZ-
Analytical column 250 mm × 4 mm, Kromasil 100 (C18, 5 µm),
at a flow rate of 0.5 mL/min. Solvent A: 10% CH3CN, 90% H2O,
0.1% TFA. Solvent B: 90% CH3CN, 10% H2O, 0.09% TFA. The
following gradients were used: (A) t ) 0 min (0% B), t ) 20 min
(35% B), t ) 25 min (60% B), t ) 32 min (100% B), t ) 34 min
(100% B), t ) 38 min (40% B); (B) t ) 0 min (0% B), t ) 20
min (10% B), t ) 25 min (60% B), t ) 32 min (100% B), t ) 34
min (100% B), t ) 38 min (40% B); (C) t ) 0 min (0% B), t )
20 min (25% B), t ) 25 min (60% B), t ) 32 min (100% B), t )
34 min (100% B), t ) 38 min (40% B); (D) t ) 0 min (0% B),
t ) 10 min (25% B), t ) 45 min (75% B), t ) 50 min (100% B),
t ) 55 min (100% B), t ) 60 min (40% B). Eluted peaks were
detected by a UV detector at 254 or 210 nm. Reported retention
times are counted in minutes. In NMR measurements, CDCl3, D2O
and CD3OD were used as solvents. 1H, 31P, and 13C NMR spectra
were recorded on a 200 MHz spectrometer. Proton and carbon
chemical shifts are referenced to residual solvent. 31P chemical shifts
are reported on δ scale (in ppm) downfield from 85% H3PO4. The
following abbreviations were used to explain the NMR multiplici-
ties: s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet,
br ) broad. Before microanalysis, samples were dried under high
vacuum at 25 °C for 24 h in a dry pistol. All spectroscopy and
analytical data were obtained in the Laboratory of Organic
Chemistry of the University of Athens.
Molecular Modeling. Crystal structure of phosphorodiamidic
acid (1)-Bacillus pasteurii urease complex10 was obtained from
the Protein Data Bank65 (refcode 3UBP) and used as starting point
for all computations. Hydrogen atoms were added using Insight
2000 program from Accelrys, assuming pH 7.0. All molecular
mechanics calculations were done using Discover program with
cff97 force field and conjugate gradient minimizer. Minimizations
were done up to energy change of 0.02 kcal/mol. To obtain designed
inhibitor-enzyme complex, phosphorodiamidic acid were ap-
propriately modified using the Builder module of Insight 2000
program. Then the structures of the obtained complexes were
minimized in two steps. First, positions of all hydrogen atoms were
optimized; second, positions of all atoms of enzyme active site and
inhibitor were minimized. Finally, optimized structures were scored
using LUDI scoring function from Insight 2000 package.66
a Reagents and conditions: (a) TMSBr, CH2Cl2, rt, 2 h, then CH3OH.
A comparison of protected and nonprotected dipeptides 20
and 3 strongly suggests that structure 8 should be more effective
than 24. Although compound 8 was synthesized in pure form,
it was impossible to obtain its reliable kinetic data due to its
high instability in the assay mixture. Nevertheless, incomplete
measurements suggest very promising inhibitory properties (data
not shown). High inhibitory efficiency of sulfur analogues in
comparison to their oxygen counterparts most probably results
from stronger interaction of sulfur atom with nickel ions present
in the urease active site. Similar phenomenon was observed in
the crystal structure of urease complex with mercaptoethanol.6
Conclusions
This paper is the first report on the highly active bacterial
urease inhibitors belonging to the organophosphinate class of
compounds. The computer aided design using crystal structures
of Bacillus pasteurii urease allowed the development of the
novel inhibitors and proved very successful. Combination of
structural features of urease inactivators described so far, namely
phosphoramidates, hydroxamic dipeptides, and sulfur com-
pounds, and computational and experimental verification of this
approach led to structure 24 exhibiting Ki ) 170 nM against B.
pasteurii and 450 nM against P. Vulgaris enzymes. Thus, it ranks
among the most potent small molecular weight inhibitors of
bacterial ureases. Moreover, studied compounds are character-
ized by the presence of a chemically inert C-P bond, assuring
their stability in physiological conditions. Several well-known
examples of successful applications of biologically active
phosphonates and phosphinates (glyphosate, phosphinothricin,
aledronate, phosphomycin) indicate high potential of this group
of compounds. Moreover, proposed scaffold of urease inhibitor
offers the possibility of convenient further modifications and
extensions that could give rise to structures with improved
inhibitory activity or/and selectivity toward enzymes of chosen
origin.
Chemistry. Methyl N-benzyloxycarbonylaminomethyl(P-me-
thyl)phosphinate (12). NaH, 60% dispersion in mineral oil (117
mg, 2.93 mmol), was added to a cooled (-10 °C) solution of 11
(670 mg, 2.93 mmol) in THF (27 mL) under argon. The reaction
mixture was treated with CH3I (0.37 mL, 6.00 mmol) and stirred
at -10 °C for 1 h and at rt for 0.5 h. The reaction was treated with
ethyl acetate, washed with water, dried (Na2SO4), and evaporated.
The crude material was purified by column chromatography, eluting
Experimental Section
General. All materials were purchased from commercial sup-
pliers (Aldrich, Sigma, Fluka, Meck) at the highest commercial
quality and used without purification unless otherwise stated. Dry
tetrahydrofuran (THF) and methylene chloride were obtained by
distillation of commercially available predried solvents from NaH.
All of the compounds, for which analytical and spectroscopic data

Organophosphorus Inhibitors of Bacterial Ureases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5741
Table 1. Inhibition of Bacillus pasteurii and Proteus Vulgaris Ureases by Studied Compoundsa
a *Steady state kinetics studies.
with CH2Cl2-CH3OH (9.5:0.5), to give 624 mg of 12 as colorless
removed in vacuo, the reaction mixture was partitioned between
1
oil; (83% yield). H NMR (CDCl3): δ 1.40 (d, J ) 13.9 Hz, 3H),
ethyl acetate and water, the organic phase was successively washed
with 5% NaHCO3, 1% HCl, and water, dried over Na2SO4,
evaporated, and the crude product was purified by column chro-
matography using CH2Cl2-CH3OH (9.5:0.5) as eluent. Compound
14 was obtained in 72% yield (238 mg). 1H NMR (CDCl3): δ 1.46
(d, J ) 13.9 Hz, 3H), 3.48-3.70 (m, 4H), 3.69 (d, J ) 10.3 Hz,
3H), 5.11 (s, 2H), 5.65 (bs, 1H), 7.10 (bs, 1H), 7.33 (s, 5H). 13C
NMR (CDCl3): δ 12.5 (d, JPC ) 93.5 Hz), 38.0 (d, JPC ) 93.5
Hz), 44.7, 51.9 (d, JPC ) 8.0 Hz), 67.4, 128.1, 128.2, 128.5, 137.4,
156.9, 170.0. 31P NMR (CDCl3): δ 53.1. MS (ESI+) m/z 415.1 (M
+ 1).
N-Glycylaminomethyl(P-methyl)phosphinic acid (3). A solu-
tion of phosphinate 14 (31.4 mg, 0.10 mmol) and TMSBr (23 mg,
0.15 mmol) in dry CH2Cl2 (1 mL) was stirred for 2 h at room
temperature. Then the solvent was evaporated and the residue was
treated with ethyl acetate (2 mL) and CH3OH (2 mL). The solution
was stirred for 30 min at room temperature and evaporated in vacuo.
The crude product was triturated with dry diethyl ether to furnish
3.49 (m. 2H), 3.63 (d, J ) 10.3 Hz, 3H), 5.00 (s, 2H), 6.09 (bt,
1H), 7.29 (s, 5H). 13C NMR (CDCl3): δ 11.3 (d, JPC ) 93.3 Hz),
38.1 (d, JPC ) 104.9 Hz), 50.5 (d, JPC ) 7.1 Hz), 67.0, 128.1, 128.2,
128.5, 136.4, 157.0. 31P NMR (CDCl3) δ 54.4. MS (ESI+) m/z
258.1 (M + 1).
Methyl N-(N′-Benzyloxycarbonylglycyl)aminomethyl(P-me-
thyl)phosphinate (14). A mixture of 10% Pd/C (30 mg) and 12
(300 mg, 1.16 mmol) in methanol (30 mL) was stirred at room
temperature under an atmosphere of hydrogen until all the starting
material was consumed as observed by TLC (2.5 h) The catalyst
was filtered off through celite, and the mixture was washed with
methanol and water/methanol (1:9). Solvents were combined and
evaporated in vacuo to give a solid (142 mg) that was dried over
P2O5. Dichloromethane (5 mL) was then added, followed by
addition of Cbz-Gly-OH (206 mg, 1 mmol), EDC ·HCl (190 mg, 1
mmol), HOBt (152 mg, 1 mmol), and the reaction mixture was
stirred at room temperature for 12 h. The organic solvent was then

5742 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Vassiliou et al.
2 M LiOH (0.25 mL) was added and the mixture was vigorously
stirred at room temperature for 48 h. Water was added, the aqueous
solution was washed with ethyl acetate and acidified to pH 1 with
0.5 M HCl, and the aqueous phase was extracted with ethyl acetate
twice. The organic extracts were combined, dried over Na2SO4,
and evaporated in vacuo to give the free hydroxyphosphinyl
compound. The crude product was purified by column chromatog-
raphy using CH2Cl2/CH3OH/AcOH (9:1:0.5) as eluent to give 19
1
(42 mg, 87% yield). H NMR (CDCl3): δ 1.43 (d, J ) 13.9 Hz,
3H), 3.52 (bs, 2H), 5.40 (bs, 1H), 7.31 (s, 5H). 13C NMR (CDCl3):
δ 13.1, (d, JPC ) 97.2 Hz), 38.9 (d, JPC ) 101.2 Hz), 67.6, 127.4,
128.4, 128.5, 128.8, 136.2, 156.3. 31P NMR (CDCl3): δ 52.0. MS
(ESI+) m/z 244 (M + 1). tR ) 16.3 min. HPLC purity > 98%,
gradient D. Anal. (C10H14NO4P) C, H, N.
O-Methyl N-Benzyloxycarbonylaminomethyl(P-methyl)phos-
phinothioate (21). A mixture of 12 (43 mg, 0.17 mmol) and
Lawesson’s reagent (68 mg, 0.17 mmol) was heated at 95 °C in
sodium dried toluene (1 mL) for 2 h. Evaporation in vacuo gave a
pale-yellow oil, which was chromatographed (5% EtOAc-CH2Cl2)
1
to give 38 mg of 21 as viscous oil (82%). H NMR (CDCl3): δ
1.77 (d, J ) 13.9 Hz, 3H), 3.50 (m, 2H), 3.65 (d, J ) 10.4 Hz,
3H), 3.73 (s, 2H), 5.12 (s, 2H), 5.22 (bs, 1H), 7.35 (s, 5H). 13C
NMR (CDCl3): δ 19.7 (d, JPC ) 72.3 Hz), 45.0 (d, JPC ) 84.7
Hz), 51.8 (d, JPC ) 6.8 Hz), 67.6, 128.4, 128.6, 128.8, 136.2, 156.3.
31P NMR (CDCl3): δ 96.1. MS (ESI+) m/z 274.2 (M + 1).
O-Methyl N-(N-Benzyloxycarbonylglycyl)aminomethyl(P-me-
thyl)phosphinothioate (22). Cbz-protected compound 21 (270 mg,
1 mmol) was stirred for 1 h at 20 °C in 33% HBr/AcOH (3 mL).
Dry diethyl ether (20 mL) was then added to precipitate a white
solid. The supernatant liquid was decanted, the procedure repeated
twice, and the remained solid was dried over P2O5. DIPEA (170
mL, 1 mmol) was added to a suspension of the resulting hydro-
bromide in CH2Cl2 (5 mL), followed by addition of Cbz-Gly-OH
(206 mg, 1 mmol), EDC ·HCl (190 mg, 1 mmol), and HOBt (152
mg, 1 mmol). The reaction mixture was stirred at room temperature
for 12 h. Then organic solvent was removed in vacuo, the reaction
mixture was partitioned between ethyl acetate and water, the organic
phase was successively washed with 5% NaHCO3, 1% HCl, and
water, dried over Na2SO4, evaporated, and the crude product was
purified by column chromatography using CH2Cl2-CH3OH (9.5:
0.5) as eluent. Compound 22 was obtained in 72% yield (238 mg).
1H NMR (CDCl3): δ 1.73 (d, J ) 13.9 Hz, 3H), 3.65-3.87 (m,
4H), 3.60 (d, J ) 10.3 Hz, 3H), 5.10 (s, 2H), 5.73 (bs, 1H), 6.90
(bs, 1H), 7.33 (s, 5H). 13C NMR (CDCl3): δ 20.0 (d, JPC ) 73.2
Hz), 42.8 (d, JPC ) 81.3 Hz), 44.9, 51.9 (d, JPC ) 6.9 Hz), 67.7,
128.4, 128.6, 128.8, 136.1, 156.8, 169.2. 31P NMR (CDCl3): δ 96.4.
MS (ESI+) m/z 331.2 (M + 1).
N-(N′-Benzyloxycarbonylglycyl)aminomethyl(P-methyl)phos-
phinothioic Acid (24). Following the same procedure described
1
above for 19, compound 24 was obtained (77% yield). H NMR
Figure 4. Time-dependent inhibition of B. pasteurii urease as a
function of 24 concentration in nonincubated (A) and incubated
24-urease systems (B). Steady-state kinetic analysis of the mode of
24 inhibition (C).
(CD3OD): δ 1.49 (d, J ) 13.9 Hz, 3H), 3.40-3.69 (m, 4H), 5.10
(s, 2H), 7.33 (s, 5H). 13C NMR (CD3OD): δ 12.4 (d, JPC ) 92.9
Hz), 38.9 (d, JPC ) 105.1 Hz), 43.7, 66.7, 127.7, 127.8, 128.3,
137.8, 156.1, 171.1. 31P NMR (CD3OD): δ 88.4. MS (ESI+) m/z
316.8 (M + 1). tR ) 30.7 min. HPLC purity > 98% gradient D.
Anal. (C12H17N2O4PS) C, H, N.
the free hydroxyphosphinyl product as solid. To this solid, 33%
HBr/AcOH (1 mL) was added; the reaction mixture was stirred at
room temperature for 1 h. Dry diethyl ether (7 mL) was then added
to precipitate an orange solid. The supernatant was decanted, the
procedure repeated twice, and the remained solid was purified using
a Dowex AG50 × 4 cation exchange resin and water as eluent. The
fractions that gave positive color reaction with ninhydrin were
combined and evaporated under vacuum to give 3 (15 mg, 91% yield).
1H NMR (D2O): δ 1.20 (d, J ) 13.9 Hz, 3H), 3.38 (d, J ) 8.8 Hz,
2H), 3.71 (s, 2H). 13C NMR (D2O): δ 12.8 (d, JPC ) 91.7 Hz), 38.9
(d, JPC ) 104.2 Hz), 40.6, 167.0. 31P NMR (D2O): δ 42.2. MS (ESI+)
m/z 166.9 (M + 1). Analytical HPLC method B: tR ) 12.4 min. HPLC
purity > 97%. Anal. (C4H11N2O3P·0.5H2O) C, H, N.
Urease Purification. Bacillus pasteurii CCM 2056T and Proteus
Vulgaris CCM 1956 were each cultured aerobically for 48 h at 37
°C in 1 L of medium, pH 9.0, containing (per liter) 20 g of yeast
extract, 20 g of urea, and 1.0 mM NiCl2.67 Cell pellet was collected
by centrifugation at 12000g for 15 min at 4 °C, washed twice with
20 mM sodium phosphate, pH 6.5, and disrupted ultrasonically in
20 mM phosphate/1 mM EDTA buffer pH 6.5. Insoluble cell debris
was removed by centrifugation at 15000g for 30 min, and 5 mL of
supernatant was applied to cellufine sulfate step A and B columns
(14 mm × 150 mm) using urease affinity purification protocol
described by Icatlo et al.68 Fractions of 1.5 mL were collected and
assayed for protein content by the method of Bradford69 and urease
activity by the phenol-hypochlorite colorimetric method.63 Step
B column void volume containing partially purified urease was
combined with urease fractionated with 20 mM phosphate pH 7.4/
N-Benzyloxycarbonylaminomethyl(P-methyl)phosphinic acid
(19). rotected phosphinate 12 (51 mg, 0.20 mmol) was dissolved
in dioxane (0.25 mL) and water (0.20 mL). To this solution, aqueous

Organophosphorus Inhibitors of Bacterial Ureases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5743
(3) Dixon, N. E.; Gazzola, C.; Blakeley, R. L.; Zerner, B. Jack bean urease
(EC 3.5.1.5). Metalloenzyme. Simple biological role for nickel. J. Am.
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Structure-based rationalization of urease inhibition by phosphate: novel
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778–790.
(12) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Mangani, S.; Ciurli, S.
Molecular Details of Urease Inhibition by Boric Acid: Insights into
the Catalytic Mechanism. J. Am. Chem. Soc. 2004, 126, 3714–3715.
(13) Dixon, N. E.; Gazzola, C.; Watters, J. J.; Blakeley, R. L.; Zerner, B.
Inhibition of jack bean urease (EC 3.5.1.5) by acetohydroxamic acid
and by phosphoramidate. Equivalent weight for urease. J. Am. Chem.
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hydroxamic acids. Biochim. Biophys. Acta 1962, 65, 380–383.
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0.15 M NaCl and applied to hydroxyapatite column (14 mm ×
100 mm) equilibrated with 20 mM phosphate buffer pH 7.4 and
eluted with linear gradient from 20 to 100 mM phosphate (50 mL),
collecting 1.5 mL fractions. Urease-containing fractions eluted at
around 50 mM phosphate were pooled and stored at 4 °C.
Enzyme Assay. Bacterial urease was assayed at 30 °C in 3 mM
phosphate buffer pH 7.0. Separate reactions (200 µL total volume)
were terminated by introduction of 0.5 mL phenol-sodium
nitroprusside solution immediately followed by addition of 0.5 mL
NaOH-sodium hypochlorite mixture.63 The amount of ammonia
liberated was assayed by measuring indophenol blue formed in
Berthelot reaction over 15 min incubation at 30 °C following the
changes at 650 nm. Compounds studied as inhibitors did not affect
indophenol readings when introduced to standard curve assays using
ammonium sulfate.
Kinetic parameters Km and Vmax of noninhibited urease were
determined by measuring initial rates of reactions carried out in
the standard assay mixtures containing 2-100 mM urea.
Inhibition Studies. Progress curves were obtained by initiation
of urease reaction with addition of 50 pkat enzyme into assay
mixtures containing increasing concentrations of inhibitors and urea,
which are in the range of 2 to 100 mM. Each assay was run in
triplicate. Ki values were dertermined from Lineweaver-Burk plots
after testing at least 5 inhibitor concentrations. IC50 values were
obtained from measurements performed in the presence of saturating
urea concentration 100 mM.
Steady-state kinetic measurements were performed in two ways.
The enzyme was preicubated with assayed inhibitor concentration
in separate aliquots of reaction buffer prior to reaction initiation
by incorporation of urea. Amounts of enzyme and inhibitor in each
reaction mixture were analogous to the corresponding progress
curve experiment. In fast dilution assays, reactions were started by
introducing portions of previously preincubated 30 times concen-
trated enzyme-inhibitor mixture into assay buffer samples contain-
ing urea. In each case, the enzyme was allowed to interact with
inhibitor for 30 min before contacting substrate. Linear regression
analysis was used to calculate steady-state inhibition constants Ki*
using equation:64
V
maxS0
Vs )
(1)
I
KM 1 +
+ S0
/
(
)
K
Concentration of inhibitor causing 50% enzyme activity loss
(IC50) was calculated from linear regression of urease activity versus
the logarithm of inhibitor concentration.
Acknowledgment. This work was supported by grant of
Polish Ministry of Science and Higher Education (no. 2 P04B
00729) and Polish-Greek Scientific and Technological Inter-
national Cooperation Joint Project for the Years 2006/2008. The
calculations were carried out using hardware and software
resources (including the Accelrys programs) of the Supercom-
puting and Networking Centre in Wrocław. Paweł Kafarski,
Łukasz Berlicki, and Paulina Kosikowska thank the Foundation
for Polish Science for financial support.
(22) Muri, E. M. F.; Mishra, H.; Avery, M. A.; Williamson, J. S. Design
and synthesis of heterocyclic hydroxamic acid derivatives as inhibitors
of Helicobacter pylori urease. Synth. Commun. 2003, 33, 1977–1995.
(23) Odake, S.; Nakahashi, K.; Morikawa, T.; Takebe, S.; Kobashi, K.
Inhibition of Urease Activity by Dipeptidyl Hydroxamic Acids. Chem.
Pharm. Bull. (Tokyo) 1992, 40, 2764.
Supporting Information Available: elemental analysis and
HPLC purity for all target compounds; details of synthesis and
spectral data for compounds 4-8, 10, 11, 13, 15-18, 20, 23, 25.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(24) Mishra, H.; Parrill, A. L.; Williamson, J. S. Three-Dimensional
Quantitative Structure-Activity Relationship and Comparative Mo-
lecular Field Analysis of Dipeptide Hydroxamic Acid Helicobacter
pylori Urease Inhibitors. Antimicrob. Agents Chemother. 2002, 46,
2613–2618.
(25) Todd, M. J.; Hausinger, R. P. Competitive inhibitors of Klebsiella
aerogenes urease. Mechanisms of interaction with the nickel active
site. J. Biol. Chem. 1989, 264, 15835–15842.
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