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Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant

Phenolic Aldehydes

Article  in  Journal of the Brazilian Chemical Society · November 2016

DOI: 10.21577/0103-5053.20160208

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J. Braz. Chem. Soc., Vol. 00, No. 00, 1-8, 2016.

Article
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0103 - 5053 $6.00+0.00

Urease Inhibitors of Agricultural Interest Inspired by Structures of

Plant Phenolic Aldehydes

Lívia P. Horta,a Yane C. C. Mota,a Gisele Maria Barbosa,a Taniris C. Braga,b

Ivanildo E. Marriel,c Ângelo de Fátima*,b and Luzia V. Modolo*,a

Grupo de Estudos em Bioquímica de Plantas, GEBioPlan, Departamento de Botânica, ICB and

a

b
Grupo de Estudos em Química Orgânica e Biológica, GEQOB, Departamento de Química, ICEx,
Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil
c
Empresa Brasileira de Pesquisa Agropecuária, Centro Nacional de Pesquisas de Milho e Sorgo,
EMBRAPA/CNPMS, Sete Lagoas-MG, Brazil

The plant phenolic natural products (PNPs) protocatechuic aldehyde, syringaldehyde and
vanillin were used as platforms for obtaining four urease inhibitors. Urea (urease substrate) or
thiourea (urease inhibitor) core was added to the structure of newly synthesized compounds
to provide inhibitors up to 230-fold more active than the PNPs they originated from. The PNP
derivatives are mixed inhibitors with higher affinity to urease active site. Two compounds were as
efficient as N-(butyl)thiophosphoric triamide (NBPT) toward soil. Overall, PNPs derivatives are
promising urease inhibitors for use as additive in urea-based fertilizers formulations.

Keywords: urea, urease inhibitor, natural product, phenolic aldehyde, fertilizer additive

Introduction the other hand, soil ureases can also be detrimental for
crop production specially when using the technique of
Nitrogen (N) is a key nutrient absorbed by plants covering fertilization. In this case, the urea applied on soil
mostly as nitrate (NO 3−) and/or ammonium (NH 4+). surface is rapidly hydrolyzed by soil ureases releasing
Despite the great abundance of N in nature, less than NH3 far away from rhizosphere allowing for N losses
2% is bioavailable to plants. Biological N fixation, soil to the atmosphere due to the volatile nature of NH3. In
organic matter mineralization and lightening are natural fact, depending on climate and soil physicochemical
processes known to increase the input of absorbing N in properties, more than 50% of the N-urea applied to soil
soil.1,2 However, these sources of bioavailable N are not surface can be lost mainly by NH3 volatilization.4,8 Besides
enough to guarantee food supply for the growing world’s negatively affecting plant N nutrition, excessive N losses to
population predicted to reach over 9.5 billion people atmosphere as NH3 remarkably impact natural ecosystems
within the next 35 years.3 Then, N fertilizers have been by contributing either directly or indirectly to acid rain,
widely used to improve crop productivity, in particular lakes and rivers eutrophication and formation of nitrous
urea, due to its high N content (46%), low price per N oxide, an atmospheric pollutant.8
unit and easy management.4 One of the strategies that have been adopted to minimize
Urease (EC [Link]; urea amidohydrolase) catalyzes N losses as NH 3 is the use of urea-based fertilizers
the hydrolysis of urea furnishing the gaseous products supplemented with urease inhibitors to slow down urea
ammonia (NH3) and carbon dioxide (CO2).5 It occurs in hydrolysis on soil surface and further increase the possibility
a variety of organisms including plants, fungi, bacteria of urea incorporation to soil by rain and irrigation.9
and some vertebrates. Soil ureases play essential roles The potential of several classes of substances to
in N global cycle, also contributing to agriculture with inhibit the ureolytic activity of soil ureases have been
respect to the availability of N as NH4+ to plant growth investigated. Recently, our group has described that
in soils supplemented with urea-based fertilizers.6,7 On phosphoramidate, benzothiazole and benzoylthiourea
compounds are also promising urease inhibitors.10-12 Up to
*e-mail: adefatima@[Link], lvmodolo@[Link] now, phosphorodiamide and phosphorotriamide derivatives
2 Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant Phenolic Aldehydes J. Braz. Chem. Soc.

are recognized as the most efficient urease inhibitors for health promoters include protocatechuic aldehyde (PA),
crop production purposes.13-15 syringaldehyde (SA) and vanillin (VA).
Indeed, the N-(butyl)thiophosphoric triamide (NBPT) The aim of the study herein presented was to use the
was found to become a very effective inhibitor when natural products PA, SA and VA as building blocks for
transformed to its corresponding oxo-derivative the development of four urease inhibitors of agricultural
(oxo‑NBPT) by soil microorganisms. 16 The NBPT interest (Figure 1). Urea (urease substrate) or thiourea
effectiveness is directly related to soil properties in which (urease inhibitor) core was also introduced to the structure
low concentration of this urease proinhibitor is needed of phenolic aldehyde derivatives synthesized (Figure 1).
to achieve the desired result in temperate soils while Then, in vitro assays were performed with pure jack bean
greater concentrations are required for tropical soils.17-20 urease to check the potential of synthesized compounds as
Additionally, NBPT is more efficient in neutral soils with inhibitors of ureolytic activity and disclose the mechanism
limited organic matter.17,21 Tropical soils exhibit organic of action of promising molecules. The effect of such
matter and microbial biomass dynamics different from phenolic aldehyde derivatives on soil ureases was addressed
temperate soils.22 The amendment of soil with organic to confirm the potential of synthesized compounds for use
matter demanded from 2- to 4-fold NBPT to alleviate N as additives in urea-based fertilizers.
volatilization by 20% in comparison to soils devoid of
crop residues.23 Although other works investigated the Experimental
NBPT effectiveness in tropical soils,24-26 further research
is needed for the development of novel and cost-effective Preparation of phenolic aldehyde derivatives
urease inhibitors with improved efficiency in tropical soils
and different environmental conditions. An ethanolic mixture containing protocatechuic
Nature is undoubtedly a source of metabolites with aldehyde (PA), syringaldehyde (SA) or vanillin (VA)
potential to interfere with the activity of ureases as individually (1 mmol; Sigma-Aldrich), ethyl acetoacetate
determined by in vitro assays with pure enzymes from (1.5 mmol) and urea or thiourea (1.5 mmol), here referred to
Helicobacter pylori or Canavalia ensiformis (jack bean).27 as (thio)urea, and p-sulfonic acid calix[4]arene (0.5 mol%)
Among natural products produced by plants, phenolic was maintained under reflux and vigorous stirring for
compounds, such as flavonoids, methyl gallate and 8 h. After then, the mixture was filtered and the phenolic
stilbenoids have been shown to inhibit ureases.28-30 aldehyde derivative formed was recrystallized using
Although there is no report on the ability of natural ethanol. The phenolic aldehyde derivatives synthesized
phenolic aldehydes to inhibit ureases, it is likely that such based on urea (2A7) or thiourea (2A9, 2B10 and 2D2)
secondary metabolites may work on ureases or be good structure were characterized by 1H and 13C nuclear
prototypes for the design of urease inhibitors. Examples magnetic resonance (NMR), infrared, melting point and
of natural phenolic aldehydes that have been explored as elemental analysis and the data compared to those reported

Figure 1. Design of the urease inhibitors of agricultural interest 2A7, 2A9, 2B10 and 2D2 based on protocatechuic aldehyde (PA), vanilin (VN) or
syringaldehyde (SA) structures combined with (thio)urea cores.
Vol. 00, No. 00, 2016 Horta et al. 3

elsewhere.31,32 The phenolic aldehyde derivatives were method as described elsewhere,36 with some modifications.
obtained in 49 to 80% yield. Clayey dystrophic Red Latosol (oxisol) soil was collected
from Brazilian Cerrado (19°28’01.2’’S, 44°10’24.5’’W).
In vitro urease activity assay The physical features of the collected soil were 6, 4, 12 and
78% of coarse sand, fine sand, silt and clay, respectively,
Initially, the phenolic aldehydes and derivatives 2A7, and chemical analyses showed pH 6.3, 10 mg L-3 PMehlich-1,
2A9, 2B10 and 2D2 were screened for the ability to inhibit 129 mg L-3 K, 4.4, 0.9, 0.1 and 2.6 cmolc L-3 of Ca2+, Mg2+,
in vitro the ureolytic activity of purified Canavalia ensiformis Al3+ and H + Al, respectively, sum of bases of 5.6 cmolc L-3,
(jack bean) type III urease (Sigma, St. Louis, Mo, USA). 68% base saturation, organic matter of 2.5 dag kg-1.
Each reaction medium containing 20, 1 and 10 mmol L-1 Sieved soil samples (0.5 g; particles smaller than 2 mm)
of phosphate buffer (pH 7.0), ethylene diamine tetra acetic were incubated with 72 mmol L-1 urea in the absence or
acid (EDTA) and urea, respectively, 12.5 mU urease and 3.2 mmol L-1 of 2A7, 2A9, 2B10, 2D2 or NBPT (used as a
compounds-test at 0 or 1.6 mM was incubated for 10 min reference of soil ureases inhibitor) at 37 oC for 1 h. Ureases
at 25 °C. Reactions were stopped by adding 0.5 volume of activity was stopped by incubating the systems with 5 mL
1% m/v phenol in 5 mg L-1 sodium nitroprusside (SNP) of 1 mol L-1 KCl in 10 mmol L-1 HCl for 30 min at 25 °C.
followed by the addition of 0.7 volume of 0.5% m/v NaOH A supernatant aliquot was taken after soil decantation
in 0.1% v/v NaOCl solution. After samples incubation and added to a solution containing 3.4, 2.5 and 2.5% of
at 50 ºC for 5 min, the absorbance was measured at sodium salicylate, sodium citrate and sodium tartrate,
630 nm to determine the amount of ammonium (NH4+) respectively, and 120 mg L-1 SNP. After 15 min incubation
formed.33 Hydroxyurea (HU) was used as a reference of at 25 °C (under darkness), 0.1 volume of 3.0% NaOH in
urease inhibitor. Urease inhibition was determined in 1.0% sodium hypochlorite was added to each reaction
terms of percentage of NH4+ formed in compounds-test system following incubation under darkness for 1 h at
reactions in relation to total urease activity in reactions 25 °C and 600 rpm. The NH4+ formed was detected by
without compounds. Three independent experiments were spectrophotometric measurements at 660 nm.
performed, each with four replicates. Then, assays using different concentrations (from
0.05 to 3.2 mM) of phenolic aldehyde derivatives or
Effect of phenolic aldehyde derivatives on the kinetic NBPT were performed to determine the concentration of
parameters of jack bean urease compound‑test that causes 50% inhibition of soil ureases
(IC50). Independent experiments were performed, each with
The inhibition profile exhibited by the natural product at least five replicates.
derivatives 2A7, 2A9, 2B10 and 2D2, synthesized in this study,
was determined by incubating inhibitors at concentrations Statistical analysis
necessary to inhibit jack bean urease activity between 30 and
40% (from 0.3 to 1.6 mM) in reaction medium containing Data were subjected to analysis of variance (ANOVA)
20 mmol L-1 phosphate buffer (pH 7.0), 1 mmol L-1 EDTA, by general linear model (GLM) procedure and contrast
urea (ranging from 1 to 32 mmol L-1) and 12.5 mU urease. analysis at 5% significance level using the software R
The stoppage of reactions, NH4+ quantification and urease (Software Foundation, Boston, MA, USA).
inhibition calculation were done as described previously.
Jack bean urease kinetic parameters such as initial velocity Results
(Vo), KM (Michaelian constant) and maximum velocity (Vmax)
were obtained using Hyper32 software.34 The OriginPro8 Inhibition of ureolytic activity of jack bean urease
(Origin Lab, Northamptom, MA) software was used to
pursue Michaelis‑Menten hyperbolas and Lineweaver‑Burk The in vitro assay with purified jack bean type III
plots. The equilibrium dissociation constants for urease- urease showed that, among the natural products tested,
inhibitor complex (K i) and for urease-urea-inhibitor only protocatechuic aldehyde (PA; at 1.6 mM) effectively
complex (K’i) were determined from the α and the α’ values.35 inhibited the enzyme activity (68% inhibition) while
vanillin (VN) and syringaldehyde (SA) marginally reduced
Soil ureases activity assay the production of NH4+ (Figure 2). The derivatives 2A7
and 2B10, originated from PA, were the most potent
The effect of phenolic aldehyde derivatives on the urease inhibitors showing results (94% urease inhibition)
activity of soil ureases was assessed by the salicylate comparable to that observed for the standard inhibitor
 4 Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant Phenolic Aldehydes J. Braz. Chem. Soc.

hydroxyurea (HU; Figure 2). Compounds 2A9 and 2D2, derivatives were able to inhibit soil ureases at different
derived from VN and SA, respectively, caused enzyme extents; 2A7 (PA derivative) and 2D2 (SA derivative) were
inhibition that averaged 58.6% (Figure 2). found to be as efficient as NBPT (commercial inhibitor;
40% enzyme inhibition) while the VN-derived 2A9 and
100 a a a
the PA-derived 2B10 inhibited soil ureases by up to 30%
80
b (Figure 4).
Jack bean urease inhibition / %

b b
60 The concentration of 2A7 and 2D2 necessary to cause
40 the inhibition of soil ureases by 50% (IC50) were, in average,
20
3.25 mM. The derivative 2A9 exhibited a maximum
inhibitory activity of 16% when used at 0.05 mM or higher
concentrations. There was not a pattern in the behavior
4
of results observed for the replicates of independent
3
experiments performed with 2B10 and NBPT, which
2
did not allowed for determining the IC50 values for such
c
1
c inhibitors.
0
HU PA 2A7 2B10 VN 2D2 SA 2A9
Thermal stability of natural phenolic-derived urease
Figure 2. Inhibition of jack bean urease by phenolic aldehydes inhibitors
a n d i t s d e r iva t ive s . T h e c o m p o u n d s h y d r o x y u r e a ( H U ) ,
protocatechuic aldehyde (PA), syringaldehyde (SA), vanillin (VA), 2A7
and 2B10 (PA derivatives), 2A9 (SA derivative) and 2D2 (VN derivative) The thermal stability of the natural phenolic aldehyde-
were employed at 1.6 mM in reactions containing 10 mmol L-1 urea derived urease inhibitors, assessed by mass changes of
and 12.5 mU urease. Results are representative of three independent
experiments, each with four replicates. Different letters indicate significant compounds as a function of fast increments in temperature,
difference (p < 0.05 by contrast analysis) among the compounds. revealed that the first event of mass loss (decrease by 5%) for
derivatives 2A7, 2D2, 2A9 and 2B10 occurred at 254, 253,
Mechanism of action of phenolic aldehyde derivatives 244 and 226 ºC, respectively (Figure S1, Supplementary
toward jack bean urease Information section). This same event was observed when
NBPT was subjected to 151 ºC (Figure S1, Supplementary
Urease is categorized as a Michaelian enzyme Information section). The second event, characterized by
since the graph of initial velocity (Vo) versus urea 20% mass loss, took place at 200.5 ºC for NBPT while
concentration exhibits a typical hyperbolic behavior similar percentage of mass loss for the phenolic aldehyde
(Figure 3). The average urea KM (Michaelian constant) derivatives was registered at 241, 269.1, 271.2 and 276.4 ºC
and urease maximum velocity (Vmax) in reactions free for 2B10, 2A7, 2A9 and for 2D2, respectively, (Figure S1,
of urease inhibitor were, respectively, 3.4 ± 0.4 mM and Supplementary Information section).
8.1 ± 0.4 µmol NH4+ min‑1 mg-1 protein. The addition of
2A7, 2A9, 2B10 or 2D2 to the reaction medium caused a Discussion
concentration-dependent increment of urea KM (apparent
Michaelis constant: KM (app)) and decrease of urease Vmax The potential of a series of plant natural products as
(apparent maximum velocity: Vmax (app)) (Figure 3). All the urease inhibitors of clinical and/or agricultural interest has
phenolic aldehyde derivatives tested behaved as mixed been documented.27 Among them, methyl gallate (phenolic
inhibitors, as attested by the lines intersection in the second ester) and its glycosylated derivative isolated from
quadrant of Lineweaver-Burk plots (Figure 3; right column). Paeonia lactiflora roots were shown to be promising with
The derivative 2A7 was the most potent mixed inhibitor respect to the inhibition of H. pylori urease.37 The promising
since the Ki and K’i values for complexes formed with this effect of these phenolic esters prompted us to investigate
compound were the lowest in comparison with the others the potential of phenolic aldehyde derivatives as urease
inhibitors (Table 1). In general, Ki values were lower than inhibitors of agricultural interest. Thus, the plant natural
the K’i values for complexes related to the same inhibitor products protocatechuic aldehyde (PA), vanillin (VN) and
by 2.4- to 15.5-fold. syringaldehyde (SA) were selected as prototypes for the
design of new urease inhibitors based on urea or thiourea
Soil ureases activity assay scaffolds, urease substrate and inhibitor respectively.
In vitro assays revealed that PA per se decreased the
When tested at 3.2 mM, all phenolic aldehyde activity of jack bean type III urease, while VN and SA were
Vol. 00, No. 00, 2016 Horta et al. 5

9.0 1.8
2A7 2A7

7.5 1.5 0.5 mM

V0 / (µmol NH4 min mg prot)

I-free
-1

6.0 1.2
0.3 mM
0.3 mM
-1

1/V
4.5 0.5 mM 0.9
+

3.0 0.6
I-free

1.5 0.3

0.0 0.0
0 5 10 15 20 25 30 35 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Urea / mM -0.3 1/[Urea]

1.2
1.0
2A9 2B10
0.7 mM
1.0
0.8 1.6 mM

0.7 mM 0.8
0.5 mM
0.6 I-free
1/V

0.6 I-free
0.4
1/V 0.4

0.2
0.2

0.0 0.0
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

-0.2 1/[Urea] -0.2 1/[Urea]

1.2
2D2

1.0 1.2 mM

0.8 0.8 mM

0.6 I-free
1/V

0.4

0.2

0.0
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
-0.2 1/[Urea]
Figure 3. Representative Michaelis-Menten hyperbola and Lineweaver-Burk plots for jack bean urease in the presence of phenolic aldehyde derivatives.
The compounds 2A7 and 2B10 (PA derivatives), 2A9 (SA derivative) and 2D2 (VN derivative) were employed at different concentrations (0.3 to 1.6 mM)
in reactions containing urea ranging from 1 to 32 mmol L-1 and 12.5 mU urease. A Vo versus urea concentration plot obtained from data of assays with 2A7
is shown to exemplify the Michaelian behavior of urea catalysis.

found to be inactive as they caused less than 1% enzyme urease.29,30,38,39 Interestingly, structural modifications on PA,
inhibition (Figure 2). Besides methyl gallate and related VN and SA dramatically improved their ability to inhibit
derivatives,37 other plant phenolic compounds, such as (iso) the ureolytic activity of jack bean urease (Figure 2). Indeed,
quercitrin, avicularin, guaijaverin, flavonoid glucosides the conversion of phenolic aldehydes to derivatives bearing
and shoreaphenol, were also reported to inhibit jack bean urea or thiourea core (Figure 1) yielded the VN derivative
 6 Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant Phenolic Aldehydes J. Braz. Chem. Soc.

Table 1. Inhibition constants for phenolic aldehyde derivatives towards phosphate buffer (pH 7.0) was, in average, 3.4 mM and
jack bean type III urease
urease Vmax 8.1 µmol NH4+ min-1 mg-1 prot. Other studies
with jack bean urease, under experimental conditions
Inhibitor Ki / mM K’i / mM
distinct from the one used here, reported urea KM values
2A7 0.23 ± 0.01 1.19 ± 0.07
ranging from 1.0 to 4.0 mM.7,41
2A9 3.83 ± 0.84 9.29 ± 3.78 The kinetic behavior of jack bean urease in the presence
2B10 0.69 ± 0.02 10.71 ± 6.77 of the phenolic aldehyde derivatives is consistent with the
2D2 1.26 ± 0.06 11.22 ± 4.57 one expected for an enzyme in the presence of a mixed
Ki: equilibrium dissociation constant for urease-phenolic aldehyde inhibitor. Mixed inhibitors are known to be capable of
derivative complex; K’i: equilibrium dissociation constant for urease-urea- binding both the free enzyme (forming an enzyme-inhibitor
phenolic aldehyde derivative complex; values are the mean ± standard
deviation of triplicate determinations from a representative experiment.
complex) and the enzyme-substrate complex (forming an
enzyme-inhibitor-substrate complex).42 The values obtained
for the dissociation constants for both urease-inhibitor and
60 a
urease-urea-inhibitor complexes indicate that the phenolic
50
aldehyde derivatives synthesized bind more efficiently
to the urease active site in comparison to allosteric ones
Soil ureases inhibition / %

40 (Table 1). The potency of compounds with respect to the

b
b
binding to urease active site is 2A7 > 2B10 > 2D2 > 2A9.
30 As for the binding to allosteric site(s) the order of potency
is 2A7 >> 2B10 = 2D2 = 2A9.
20 The derivatives 2A7 and 2D2 were the most efficient
b b compounds to inhibit soil ureases, clustering together with
10 the reference inhibitor NPBT (Figure 4). These synthesized
compounds are able to inhibit soil urease activity by 50%
0 when used at 3.25 mM. Notably, the maximum inhibition
2A7 2A9 2B10 2D2 NBPT
of soil ureases exhibited by the SA derivative 2A9 was
Figure 4. Effect of phenolic aldehyde derivatives and NBPT on the activity
of soil ureases. The compounds N-(butyl) thiophosphorictriamide (NBPT), 16% when employed at 0.05 mM, no matter higher
2A7 and 2B10 (PA derivatives), 2A9 (SA derivative) and 2D2 (VN concentrations would be applied on soil. In fact, in the case
derivative) were applied to soil at 3.2 mM in the presence of 72 mmol L-1 of soil ureases, (a)biotic conditions such as temperature,
urea. Results are representative of independent experiments, each with at
least five replicates. Distinct letters indicate significant difference (p < 0.05 pH, moisture and the presence of different types of clays,
by contrast analysis) among the compounds. organic matter and viable microorganisms particularly
are known to affect ureases performance.14 Moreover, soil
2D2 that is 230-fold more potent than VN by itself, the matrix comprises complex physicochemical features and
SA derivative 2A9 of potency 66-fold higher than that of biological processes that may culminate in the chemical
SA by itself and the PA derivatives 2A7 and 2B10 were modifications of xenobiotic substances,43 as is the case of
about 40% more potent in comparison with PA. Under our synthetic urease inhibitors. Such chemical transformations
experimental conditions, the novel urease inhibitors 2A7 triggered by soil microbiota may result in loss or increment
and 2B10 were as potent as hydroxyurea (HU; known of the function of a certain compound.43 Alternatively, the
urease inhibitor), while 2D2 (also novel) and 2A9 was less complex nature of soil matrix may affect the bioavailability
effective than HU (Figure 2). The potential of the derivative of the xenobiotic for interaction with the target enzymes.
2A9 as an inhibitor of one of the jack bean ureases was Taking these into account it is likely that the PA derivative
recently reported,40 although the experimental conditions 2B10 undergoes some structural transformation caused by
were different from the one reported herein. Thus, the soil microbiota as it is known to occur with NBPT.16
outstanding performance of these phenolic derivatives, It is well known that NBPT is sensitive to heat.44
compared to the natural products they originated from, For the most active derivatives on soil (2A7 and 2D2),
might be attributed to the combination of a catechol thermogravimetric analysis shows no decomposition of
skeleton with urea or thiourea core. such compounds up to 170 oC (Figure S1; Supplementary
We performed kinetic experiments varying urea Information section). In addition to the thermal stability, the
concentration in urease-catalyzed reactions containing each derivatives 2A7 and 2D2 are obtained in a single synthesis
inhibitor at fixed concentrations (Figure 3). The urea KM step (78% average yield) after a simple purification
value obtained from reactions carried out in 20 mmol L-1 procedure (recrystallization) that furnishes compounds as
Vol. 00, No. 00, 2016 Horta et al. 7

solid materials. These are desirable features for obtaining 9. Junejo, N.; Khanif, M. Y.; Hanfi, M. M.; Yunus, W. M. Z. W.;
urease inhibitors to be used as additive in urea-based Dharejo, K. A.; Afr. J. Biotechnol. 2011, 10, 3504.
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their ability to inhibit soil ureases, make these compounds, 15. Domínguez, M. J.; Sanmartín, C.; Font, M.; Palop, J. A.; San
especially 2A7 and 2D2, promising candidates for further Francisco, S.; Urrutia, O.; Houdusse, F.; García-Mina, J. M.;
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