Urease inhibition and anti-leishmanial assay of substituted benzoylguanidines and their copper(ii) complexes

Article abstract of DOI:10.1039/c1dt10464k

A series of N,N′,N′′-trisubstituted guanidines (1-6) and their copper(ii) complexes, [κ²(O,N)-C₆H ₅CONHC(NHC₆H₄Cl)NR]₂Cu(ii) (R = iso-propyl (1a), n-butyl (2a), sec-butyl (3a), tert-butyl (

Full text of DOI:10.1039/c1dt10464k

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PAPER
Urease inhibition and anti-leishmanial assay of substituted benzoylguanidines
and their copper(II) complexes†
Ghulam Murtaza,a Amin Badshah,*a Muhammad Said,a Hizbullah Khan,a Ajmal Khan,b Samreen Khan,b
Sadia Siddiq,b M. Iqbal Choudhary,b Jose´e Boudreauc and Fre´de´ric-Georges Fontainec
Received 18th March 2011, Accepted 21st June 2011
DOI: 10.1039/c1dt10464k
A series of N,N¢,N¢¢-trisubstituted guanidines (1–6) and their copper(II) complexes,
2
[k (O,N)-C6H5CONHC(NHC6H4Cl)NR]2Cu(II) (R = iso-propyl (1a), n-butyl (2a), sec-butyl (3a),
tert-butyl (4a), benzyl (5a), and para-tolyl (6a)) were synthesized and characterized using elemental
analysis, FTIR and NMR spectroscopy. DFT studies were used to assess the location of the protons in
the free ligands. However, calculations have shown that, in all cases, hydrogen bonding from either N–H
group gives conformations that are very similar in energy. Single crystal XRD studies were used to
characterize ligands 1 and 4 and the related complexes 1a and 4a. The structures reveal that these
complexes are mononuclear in the solid state and that copper adopts a regular square planar geometry.
In both metallic species, the N, N¢, N¢¢-trisubstituted guanidine ligands chelate the Cu(II) atom using
the oxygen and one nitrogen. The synthesized compounds were investigated for urease inhibition using
thiourea as a standard drug. Most complexes exhibit a better activity than the respective guanidines
and compound 1a was found to be the most active with IC50 = 9.83 0.07 mM (the IC50 for thiourea is
21.0 0.1 mM). The species were also screened for their anti-leishmanial activity. However, all of the
compounds were devoid of any significant activity.
Recently, a number of compounds have been proposed as
Introduction
urease inhibitors,5 which are considered interesting new targets
Urease is a nickel-containing metalloenzyme that catalyzes the
for anti-ulcer drugs and for the treatment of infections caused
by urease producing bacteria.6 Inhibitors of urease can be
broadly classified into two categories: (i) organic compounds,
such as acetohydroxamic acid, humic acid, triazoles and 1,4-
benzoquinone,7 and (ii) transition metal ions, such as Cu2+, Zn2+,
Pd2+ and Cd2+.8 Metal complexes have also been reported to
act as urease inhibitors; those of Sn(IV), V(IV), Bi(III), Cu(II)
and Cd(II) have interesting urease inhibitory activities.9 Bismuth
complexes are amongst the most widely used urease inhibitors for
the treatment of peptic ulcers and Helicobacter pylori infections.10
However, they exhibit many side effects, such as darkening of
the tongue, vomiting, diarrhoea and dizziness. Cu(II) complexes
are of interest since they exhibit greater activity when inhibiting
jack bean urease than complexes of Ni(II), Co(II) and Zn(II),
and might provide fewer side effects than the bismuth containing
drugs.11
Leishmaniasis is one of the deadliest diseases after malaria,
which is caused by protozoan parasites of genus Leishmania and
is transmitted by the bite of sand flies. The various manifestations
of Leishmania parasites include cutaneous Leishmaniasis (CL),
mucocutaneous Leishmaniasis and visceral Leishmaniasis (VL).12
Human leishmaniasis is distributed worldwide, but mainly occurs
in the tropics and sub-tropics, where there is a prevalence of 12
million cases, with approximately 0.5 million cases of VL and
1.5 million cases of CL.13 Classical antiparasitic drugs are highly
hydrolysis of urea to form ammonia and carbamate, which further
decomposes to yield a second equivalent of ammonia and carbon
dioxide.1 Bacterial ureases have been reported as an important
virulence factor in the development of many harmful clinical
conditions for human and animal health. Urease is directly
involved in the formation of infectious stones and contributes
to pathogenesis.2 It is the major cause of pathogenesis induced
by Helicobacter pylori, which plays an important role in peptic
ulcers and may lead to stomach cancer.3 Urease also affects the
efficiency of soil nitrogen fertilization and ammonia volatilization
and damages roots.4
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad, 45320,
Pakistan. E-mail: aminbadshah@yahoo.com
bH. E. J. Research Institute of Chemistry, International Center for Chemical
and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
cDe´partement de Chimie, Centre de recherche sur les proprie´te´s des interfaces
et de la catalyse, Universite´ Laval, 1045 Avenue de la Me´decine, Que´bec,
Que´bec, G1V 0A6, Canada
† DFT data are available. Crystallographic data have been deposited
with CCDC. CCDC 816344 for 1, 816342 for 4, 816341 for 1a and
816343 for 4a. These data can be obtained upon request from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK, e-mail: deposit@ccdc.cam.ac.uk, or via the internet at
www.ccdc.cam.ac.uk. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1dt10464k
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Table 1 Characteristic benzoylguanidine resonances by NMR
spectroscopy
toxic and most are no longer effective due to the emergence of
drug resistance in the parasites.
Guanidines are well-known physiologically active substances
with a broad spectrum of biological activity that includes anti-
hypertensive, anti-glaucoma and cardiotonic and anti-histaminic
activities.14 Substituted guanidines are also reported to exhibit
anti-leishmanial activity.15 In this paper, we wish to report the
synthesis and characterization of substituted benzoylguanidines
(1–6) and their Cu(II) complexes (1a–6a) using a large array of
spectroscopic techniques, including X-ray diffraction analysis for
1, 4, 1a and 4a. All of the species were tested for their activity in
urease and leishmanial inhibition in order to look at the effect of
peripheral segments on the biological activity and to investigate
the activity of the organic molecules compared to their metal
complexes. To our knowledge, it is the first report demonstrating
significant urease inhibition by guanidine complexes.
1H
13C
Species
R
N–H
N–H
CN3
C
O
1
2
3
4
5
6
iso-propyl
n-butyl
sec-butyl
tert-butyl
benzyl
4.53
4.82
4.50
4.78
5.18
8.43
12.27
12.25
12.30
12.26
12.36
11.93
157.3
158.4
158.3
157.4
158.4
156.5
177.8
177.9
178.2
177.4
178.0
178.5
para-tolyl
One interesting feature of these molecules is the presence of
two different N–H groups that can form intramolecular hydrogen
bonds with the C O of the amide moiety. They can be observed
using FT-IR, where they appear as two peaks, one broad and
one sharp, in the 3205–3385 cm-1 range, and as two resonances
in the 1H NMR spectra. All of the species have one N–H proton
Results and discussion
1
in the H NMR spectra that is observed in the 12.25–12.36 ppm
range and another that is observed between 4.50–5.18 ppm (with
the exception of 6, for which the latter signal appears at d 8.43).
The downfield signal is attributed to the N–H fragment that is
hydrogen bonded with the carbonyl oxygen, while the other N–H
is typical for secondary amines in the absence of hydrogen bonding
(Table 1).
Single crystals of 1 and 4 suitable for X-ray crystallographic
studies were obtained from a recrystallization in ethanol. The
ORTEP representations are illustrated in Figs 1 and 2. These
results confirm that intramolecular hydrogen bonding is occurring
between the N–H from the aniline moiety and the carbonyl
in both molecules. The –C(O)N C(NH–)(NH–) core in both
species exhibit a large amount of electron delocalization due to
the Y-aromaticity,19 as can be observed by the C–N bond lengths
Synthesis and characterization of polysubstituted guanidines
The guanidine ligands 1–6 were synthesized using
a
mercury-promoted guanylation reaction from N-benzoyl-N¢-(2-
chlorophenyl)thiourea,16 which was synthesized by an extensively
used methodology (Scheme 1).17 For all of the syntheses, the
products were isolated in good yields (72–81%) and the purity
was confirmed by elemental analysis.
The IR spectra were taken for ligands 1– 6. The C N stretching
frequencies for all of the ligands were observed in the range 1544–
1595 cm-1, which indicates some degree of conjugation between
all three of the nitrogen atoms extending to the carbonyl group,
the stretching frequency of which appears in the 1601–1727 cm-1
1
range. Using 13C{ H} NMR spectroscopy, it was possible to locate
˚
˚
the C O carbonyl moieties in the range of 177.4–178.5 ppm
for all of the compounds, while the CN3 resonate in the 156.5–
158.4 ppm range, which is the specific region for the guanidine
moiety (Table 1).18 All of the other aromatic and aliphatic carbons
that vary between 1.328–1.362 A and 1.333–1.355 A in 1 and 4,
respectively. Furthermore, the O(1)–C(7)–N(1)–C(8), N(2)–C(8)–
N(1)–C(7) and N(3)–C(8)–N(1)–C(7) torsion angles only slightly
deviate from the expected 0◦ (or 180◦) values for a fully planar
core (average values of 12.7 and 6.5◦ for 1 and 4, respectively).
While only the intramolecular hydrogen bond could be observed
1
1
appeared in the normal regions, for both H and 13C{ H} NMR
spectroscopy.
Scheme 1 Synthesis of guanidines 1–6 with carbon numbering
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Fig. 1 ORTEP diagram of 1. Thermal ellipsoids are presented at
50% probability. Some hydrogen atoms were omitted for clarity. The
˚
dashed lines represent hydrogen bonding. Selected bond lengths (A) and
angles (◦): C(7)–O(1) 1.251(4); C(7)–N(1) 1.345(4); C(8)–N(1) 1.332(4);
C(8)–N(2) 1.362(4); C(8)–N(3) 1.328(4); O(1)–C(7)–N(1) 126.5(3);
C(7)–N(1)–C(8) 121.4(3); N(1)–C(8)–N(2) 122.9(3); N(1)–C(8)–N(3)
118.6(3); N(2)–C(8)–N(3) 118.4(3); O(1)–C(7)–N(1)–C(8) -14.1(5);
N(2)–C(8)–N(1)–C(7) -13.3(4); N(3)–C(8)–N(1)–C(7) 169.2(3).
Fig.
2
ORTEP diagram of 4. Thermal ellipsoids are presented
at 50% probability. The dashed lines repr◦esent hydrogen bond-
˚
ing. Selected bond lengths (A) and angles ( ): C(7)–O(1) 1.246(2);
in the solid state structure of species 4, a more extended hydrogen
bond network exists in the solid state structure of 1. In addition to
C(7)–N(1) 1.349(2); C(8)–N(1) 1.335(2); C(8)–N(2) 1.355(2); C(8)–N(3)
1.333(2); O(1)–C(7)–N(1) 127.75(17); C(7)–N(1)–C(8) 120.63(16);
N(1)–C(8)–N(2) 122.50(16); N(1)–C(8)–N(3) 119.23(16); N(2)–C(8)–N(3)
118.27(16); O(1)–C(7)–N(1)–C(8) -7.8(3); N(2)–C(8)–N(1)–C(7) -6.3(3);
N(3)–C(8)–N(1)–C(7) 174.66(17).
˚
the intramolecular N–H ◊ ◊ ◊ O bond (H–O of 2.01(3) A), another
intramolecular interaction exists between the chloride and the
˚
same N–H (H–Cl of 2.49(3) A) and an intermolecular interaction
between the N–H of iso-propyl amine and the carbonyl of an
˚
adjacent molecule (H–O of 2.11(3) A).
chloroaniline moiety and the carbonyl group, which is expected to
be favored, and the other between the functionalized secondary
alkyl- or aryl- amine and the carbonyl group. Minimum energy
structures were localized for both the NHR (A) and NH(o-Cl–
Ph) (B) interactions (Table 2). Fig. 3 shows that the optimized
geometry for 4B reproduces the solid state structure of 4. However,
In order to get more information on the stability of the hydrogen
bonds in species 1–6, numerical analyses were carried out using
density functional theory with the hybrid functional B3LYP and
the 6-31g(d,p) double zeta basis set. Two conformations were
looked at; one with an internal hydrogen bond between the
Table 2 Optimized distances for the hydrogen bonding obtained by DFT for species 1–6 (corresponding crystallographic values in bold)
Angle (◦)
DGA→B (kcal mol-1)
˚
R
Bond distances (A)
N–H
NH ◊ ◊ ◊ O
1.745
1.723
2.01(3)
1.749
1.728
1.745
1.722
1.725
1.706
1.92(2)
1.741
1.720
1.715
1.726
N ◊ ◊ ◊ O
2.596
2.581
2.663(3)
2.591
2.584
2.596
2.581
2.600
2.571
2.596(2)
2.591
2.579
2.579
2.582
NHO
137.2
137.5
132(3)
136.3
137.2
137.5
137.5
140.1
138.2
139(2)
137.2
137.5
138.3
137.3
1A
1B
iPr
iPr
1.029
1.034
0.87 (4)
1.028
1.033
1.029
1.034
1.030
1.035
0.82(2)
1.027
1.034
1.033
1.033
-0.97
2A
2B
3A
3B
4A
4B
nBu
nBu
sBu
sBu
tBu
tBu
0.29
-0.62
-1.2
5A
5B
6A
6B
Bn
Bn
pTol
pTol
-0.24
1.6
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stable conformation in this particular study, especially as it does
not account for the solvation effects that will be important in this
particular case.
Synthesis and characterization of polysubstituted guanidine Cu(II)
complexes
The entire series of polysubstituted guanidine Cu(II) complexes
1a–6a were synthesized in good yield using Cu(II) acetate in
methanol, as illustrated in Scheme 2. For all of the complexes, the
products were isolated in good yields (72–82%) and the elemental
analysis confirms that two guanidine ligands are coordinated to
each metal ion. Since all of the complexes are paramagnetic,
no NMR studies were carried out. However, the magnetic
susceptibility for all of the complexes is between 1.40–1.74 BM,
which is characteristic for the 3d9 electronic configuration of Cu(II)
complexes.
Fig. 3 A representation of the minimized structure of 4 using DFT
(B3LYP 6-31g(d,p)).
The stretching frequencies of the carbonyl groups in all metallic
species are shifted to smaller wavenumbers compared to the free
ligands, confirming the coordination of the guanidines by the
carbonyl moiety. The systematic comparison is, however, difficult,
since the C O band merges with the C N band in many
complexes. The stretching frequencies for the C N vibrations
did show only nominal changes, since this fragment does not
coordinate the metal center. The disappearance of a weak N–
H stretch is also observed, which provides supporting information
for the complexation of the ligand that occurs through the
coordination of an amido moiety. One sharp band associated
with the N–H stretching frequency appears in the 3205–3405 cm-1
region, which indicates a shift towards higher values compared to
the free ligand. For all of the complexes, bands are observed in
the normal region for Cu–N and Cu–O bonds (274–276 cm-1 and
430–490 cm-1, respectively).20
for the fragments involved in the N–H ◊ ◊ ◊ O interaction, a longer
N–H bond (1.04 A) and a shorter O–H bond (1.71 A) are obtained
˚
˚
˚
by calculations, but a very similar N ◊ ◊ ◊ O distance (2.57 A) and
N–H ◊ ◊ ◊ O angle (138◦) are observed (crystallographic values for
1 and 4 are available in Table 2 and the optimized geometry
coordinates and figures for all of the representations are available
in the supplementary information†). As can be seen in Table 2, the
free energy differences between conformations A and B (DGA→B
)
for compounds 1–6 are less than 2.0 kcal mol-1. Nevertheless,
one can suggest that in the case of species 6, conformation A is
more readily available (DGA→B = 1.6 kcal mol-1) and might be in
equilibrium with conformation B, which could explain the upfield
shift for the downfield N–H resonance and the downfield shift
for the upfield N–H resonance (at d 11.93 and 8.43 compared to
average values of d 12.3 and 4.8, respectively). However, it should
be noted that the DG values are less than the precision of the
method and do not allow for a definitive identification of the most
Single crystals of 1a and 4a were isolated from a 1 : 1 CHCl3/n-
hexane solution, thus confirming the connectivity and the pseudo
Scheme 2 Synthesis of guanidine complexes
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square planar geometry around the metal center (Figs 4 and
5). Species 1a crystallizes in the monoclinic P21/c space group
with Z = 6. One of the two independent molecules has an
inversion center on the copper center, making both of the ligands
crystallographically identical with the summation of the angles
around the copper being 360◦. The other independent molecule
shows some degree of disorder, with the major model (60%) with
both chlorines on the aniline moiety on opposite sides of the square
planar plane, whereas the minor model (40%) has the chlorines on
the same side of the plane. As a consequence, there are important
distortions around the copper atom, with the summation of the
angles around the Cu being 363.7(4)◦. The tert-butyl analogue
¯
4a crystallizes in the R3 rhombohedral space group with Z =
18 and also exhibits some degree of disorder where the chloride
atoms alternate on both sides of the plane in a 80 : 20 ratio. The
summation of the angles around the metal center in this solid state
structure is 360.1(3)◦. In both complexes, the Cu–N bonds are
˚
longer than the Cu–O bonds (1.938(2)–1.962(2) A vs. 1.892(2)–
˚
1.913(2) A, respectively) and are in the expected range for Cu(II)
Fig. 5 An ORTEP diagram of 4a. The major model is presented (80%).
guanidine complexes.21
The thermal ellipsoids are at 50% probability. Most of the hydrog◦en
˚
atoms are omitted for clarity. Selected bond lengths (A) and angles ( ):
Cu(1)–O(1) 1.9022(16); Cu(1)–O(51) 1.9031(15); Cu(1)–N(2) 1.9543(19);
Cu(1)–N(52) 1.9543(19); O(1)–Cu(1)–N(2) 89.56(7); O(51)–Cu(1)–N(52)
89.91(7); O(51)–Cu(1)–N(2) 90.43(7); O(1)–Cu(1)–N(52) 90.23(7);
N(2)–Cu(1)–N(52) 177.99(9); O(1)–Cu(1)–O(51) 176.07(8).
Table 3 IC50 values for the urease inhibition of the synthesized
compounds
Compound
% inhibition
IC50 S.E.M (mM)
1
6
0
3.1
5.7
27
—
—
—
—
—
—
2
3
4
5
6
0
1a
95.1
97.6
39.7
0
86.4
0
9.83 0.07
11.50 0.05
—
2a
3a
4a
—
5a
107.1 3.5
—
21.00 0.01
Fig. 4 An ORTEP diagram of 1a. One of the two independent molecules is
6a
presented with thermal ellipsoids at 50% probability. The hyd◦rogen atoms
Thiourea
98.2
˚
are omitted for clarity. Selected bond lengths (A) and angles ( ): Molecule
1: Cu(1)–O(1) 1.913(2); Cu(1)–O(21) 1.903(2); Cu(1)–N(2) 1.945(3);
Cu(1)–N(22) 1.938(3); O(1)–Cu(1)–N(2) 90.38(11); O(21)–Cu(1)–N(22)
90.15(10); O(21)–Cu(1)–N(2) 92.05(11); O(1)–Cu(1)–N(22) 91.09(10);
N(2)–Cu(1)–N(22) 167.99(12); O(1)–Cu(1)–O(21) 162.36(11). Molecule 2
(an inversion center is present on Cu(2)): Cu(2)–O(41) 1.892(2); Cu(2)–
N(42) 1.962(3); O(41)–Cu(2)–N(42) 90.45(10); O(41)–Cu(2)–N(42)*
89.55(10).
than thiourea (IC50 = 21.00 0.01) and comparable to literature
precedents for copper complexes.22 From the observed data, it
can be suggested that the steric hindrance of the R group plays a
significant effect on the urease inhibition properties (% inhibition
n-butyl (2a) > iso-propyl (1a) > sec-butyl (3a) > tert-butyl (4a)).
Also, the presence of an aromatic ring as the R group in both 6 and
6a seems to prevent significant inhibition properties, since both
species did not show any activity. Finally, the guanidine species
are all more active as metal complexes than the neutral ligands.
The various synthesized compounds were also tested for
leishmanicidal activity and compared with Amphotericin B and
Pentamidine (Table 4). It is indeed known that the combina-
tion of organic ligands with various transition metals can be
used to enhance drug performance. Indeed, organic drugs, like
chloroquine or clotrimazole, can be used with metal-containing
fragments to enhance their biological activity against malaria and
Chagas diseases.23 Out of the 12 compounds, compound 5a shows
Urease inhibition and anti-leishmanial activities
The synthesized compounds were tested for jack bean urease
inhibition (Table 3). The six benzoylguanidine species (1–6) did
not show any significant inhibition, with the best inhibitor, 5, only
showing 27% urease inhibition. On the other hand, complexes 1a–
6a, with the exception of 4a, demonstrated much better inhibition
properties than the corresponding ligands. Compounds 1a and 2a
have shown excellent inhibition activity with IC50 values of 9.83
0.07 and 11.50 0.05, respectively, which is significantly better
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Table 4 IC50 values of different compounds for anti-leishmanial activities
determined on an Atomic Absorption Spectrophotometer Perkin
Elmer 2380. The magnetic susceptibility of the complexes was
recorded by a Magnetic Susceptibility Balance Auto MSB. The
melting points were determined on a Bio Cote SMP10-UK and
are uncorrected.
Compound
IC50 S.E.M (mg ml-1)
1
>100
>100
84.21 0.05
>100
Not Screened
>100
2
3
4
General synthesis of N,N¢,N¢¢-trisubstituted guanidines 1–6
5
6
The guanidine ligands were synthesized from N-benzoyl-N¢-(2-
chlorophenyl)thiourea using a guanylation reaction method. The
thiourea was mixed with the desired substituted amine in DMF
in an equimolar ratio with two equivalents of triethylamine. The
temperature was maintained below 5 ◦C using an ice bath and
one equivalent of mercuric chloride was added to the reaction
mixture with vigorous stirring. The ice bath was removed after
30 min, while the stirring continued overnight. The progress of the
reaction was monitored using TLC until all of the thiourea was
consumed. 20 ml of chloroform was added to the reaction mixture
and the suspension was filtered through a sintered glass funnel
to remove the HgS residue. The solvents were evaporated under
reduced pressure and the solid residue was dissolved in 20 ml of
CH2Cl2, then washed with water (4 ¥ 30 ml) and the organic phase
was dried over anhydrous MgSO4. The solvent was evaporated
and the residue recrystallized in ethanol to get crystals of the title
compounds (see Scheme 1).
1a
>100
2a
78.0 0.4
80.8 1.0
>100
57.73 0.45
>100
3a
4a
5a
6a
Amphotericin B
Pentamidine
0.29 0.05
5.09 0.04
a moderate activity with an IC50 value of 57.73 0.45, while 2a, 3a
and 3 did exhibit some leishmanicidal activity, albeit very low, with
respective IC50 values of 78.0 0.4, 80.8 1.0 and 84.21 0.05).
Therefore, all of the species tested have very low leishmanicidal
activity against the promastigote stage of the parasite.
Conclusion
A
series of N,N¢,N¢¢-trisubstituted guanidines (1–6) and
their Cu(II) complexes [C6H5CONHC(NHC6H4Cl)NR]2Cu(II)
where –CH(CH3)2 (1a), –CH2CH2CH2CH3 (2a),
N-Isopropyl-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine
(1).
Quantities used were 2.8 ml (20 mmol) of triethylamine, 0.9 ml
(10 mmol) of iso-propylamine, 2.9 g (10 mmol) of N-benzoyl-N¢-
(2-chlorophenyl)thiourea and 2.72 g (10 mmol) mercuric chloride.
Yield 2.46 g, 76%. M.p. 94–96 ◦C. FT-IR (KBr, cm-1) 3234, 3064,
2973, 1726, 1586, 1561, 1488, 1374, 1290, 1221, 1128, 926, 744,
R
=
–CH(CH3)(CH2CH3) (3a), –C(CH3)3 (4a), –CH2C6H5 (5a),
and –para-tolyl (6a) were synthesized and characterized. It was
observed, using NMR spectroscopy, crystallographic studies and
DFT analysis, that the guanidines are stabilized by hydrogen
bonds. In the case of 1, an extended network of hydrogen bonds
was observed by crystallographic studies. The structural studies
of the complexes 1a and 4a revealed that the copper atom adopts
a square planner geometry. Urease inhibition studies revealed that
the complexation of the guanidines has a significant effect on their
activity. The steric hindrance of the R group seems also to play a
significant role on the inhibition activity. However, the synthesized
compounds did not show any significant anti-leishmanial activity.
◦
1
718, 699, 673, 598. H NMR (300 MHz, CDCl3, 25 C): d 1.32
3
(d, 6H, J = 7.2 Hz, CH3), 4.45 (m, 1H, CH), 4.53 (s, 1H, NH),
3
7.22–7.52 (m, 7H, Ar–H), 8.28 (d, 2H, J = 6.9 Hz, Ar–H 2 &
◦
6), 12.27 (s, 1H, NH); 13C NMR (75.47 MHz, CDCl3, 25 C): d
22.9 (2C, CH(CH3)3), 43.6 (CH(CH3)3), 125.9, 126.4, 127.9 (2C),
129.0, 129.2 (2C), 130.2, 130.5, 131.3, 134.0, 138.5 (aromatic C),
157.3 (CN3), 177.8 (C O). Anal. calcd for C17N3H18OCl (315.8):
C, 64.66; H, 5.75; N, 13.31; Found: C, 64.13; H, 5.47; N, 13.19%.
N-(n-Butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine
(2).
Quantities used were 2.8 ml (20 mmol) of triethylamine, 1.0 ml
(10 mmol) of n-butylamine, 2.9 g (10 mmol) of N-benzoyl-N¢-(2-
chlorophenyl)thiourea and 2.72 g (10 mmol) of mercuric chloride.
Yield 2.54 g, 77%. Liquid. FT-IR (KBr, cm-1) 3286, 3224, 3065,
2958, 2872, 1591, 1566, 1428, 1445, 1362, ◦1218, 1145, 1061, 894,
751, 710. 1H NMR (300 MHz, CDCl3, 25 C): d 0.99 (t, 3H, 3J =
7.2 Hz, –CH2CH2CH2CH3), 1.42 (m, 2H, –CH2CH2CH2CH3),
1.67 (m, 2H, –CH2CH2CH2CH3), 3.54 (t, 2H, 3J = 6.9 Hz,
–CH2CH2CH2CH3), 4.82 (s, 1H, NH), 7.20–7.57 (m, 7H, Ar–H),
Experimental section
General comments
All experiments were performed under the normal conditions
of temperature and pressure. All reagents were purchased from
Sigma–Aldrich and were used as received without further pu-
rification. All solvents were purified by distillation, dried using
the appropriate drying agents, saturated with nitrogen, stored
˚
over 4 A molecular sieves and degassed before use. NMR
3
8.29 (d, 2H, J = 6.6 Hz, Ar–H 2 & 6), 12.25 (s, 1H, NH); 13C
1
spectra were recorded on a Bruker 300 MHz spectrometer. H
◦
NMR (75.47 MHz, CDCl3, 25 C): d 13.8 (–CH2CH2CH2CH3),
NMR (300.13 MHz): CDCl3 (7.26 ppm from SiMe4); 13C NMR
(75.46 MHz): internal standard TMS. The splitting of proton reso-
nances in the NMR spectra are shown as, s = singlet, d = doublet, t =
triplet and m = multiplet (showing a complex pattern). IR spectra
were recorded as KBr pellets on a Bio-Rad Excalibur FT-IR
Model FTS 3000 MX (400–4000 cm-1) and Bruker FT-IR model,
Tensor 37 (200–400 cm-1). Elemental analyses were performed on
a Fisons EA1108 CHNS analyzer and metal concentrations were
20.2 (-CH2CH2CH2CH3), 31.6 (-CH2CH2CH2CH3), 41.3
(–CH2CH2CH2CH3), 124.8, 127.1, 128.0 (2C), 128.8, 129.0 (2C),
129.9, 131.0, 131.4, 134.0, 138.5, (Aromatic-C), 158.4 (CN3),
177.9 (C O); Anal. calcd for C18N3H20OCl (329.82): C, 65.55;
H, 6.11; N, 12.74; Found: C, 65.26; H, 5.72; N, 12.51%.
N-(sec-Butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine
(3).
Quantities used were 2.8 ml (20 mmol) of triethylamine, 1.0 ml
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(10 mmol) of sec-butylamine, 2.9 g (10 mmol) of N-benzoyl-N¢-(2-
chlorophenyl)thiourea and 2.72 g (10 mmol) of mercuric chloride.
Yield 2.44 g, 74%. M.p. 72–73 ◦C. FT-IR (KBr, cm-1) 3385, 3245,
2966, 2870, 1603, 1589,1565, 1483, 1445, 1368, 1213, 1056, 897,
752, 709, 530, 441. 1H NMR (300 MHz, CDCl3, 25 ◦C): d 1.01 (t,
3H, 3J = 6.9 Hz, –CH(CH3)(CH2CH3)), 1.27 (d, 3H, 3J = 7.2 Hz,
–CH(CH3)(CH2CH3)), 1.64 (m, 2H, –CH(CH3)(CH2CH3)),
4.39 (m, 1H, –CH(CH3)(CH2CH3)), 4.50 (s, 1H, NH), 7.23–
General synthesis of complexes 1a–6a
A solution of (CH3COO)2Cu·H2O in 20 ml of dry methanol was
added into a solution of 2 equivalents of the appropriate guanidine
in 15 ml of methanol at room temperature.24 The reaction mixture
was stirred for 4 h in an inert atmosphere, then filtered and
the residual solid was washed with methanol. The solid was
re-dissolved in CHCl3 and n-hexane (1 : 1) was added to give
the blue crystals of the desired complex which were isolated by
filtration.
3
7.52 (m, 7H, Ar–H), 8.28 (d, 2H, J = 6.9 Hz, Ar–H 2 & 6),
12.30 (s, 1H, NH); 13C NMR (75.47 MHz, CDCl3, 25 ◦C): d
10.4 (–CH(CH3)(CH2CH3)), 20.5 (–CH(CH3)(CH2CH3)), 29.8
(–CH(CH3)(CH2CH3)), 48.9 (–CH(CH3)(CH2CH3)), 124.7,
127.4, 127.9 (2C), 128.8, 129.2 (2C), 130.0, 130.8, 131.3, 136.4,
138.5, (aromatic C), 158.3 (CN3), 178.2 (C O). Anal. calcd for
C18N3H20OCl (329.82): C, 65.55; H, 6.11; N, 12.74; Found: C,
65.11; H, 5.84; N, 12.27%.
Bis(N-(iso-propyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidina-
to)copper(II) (1a). Quantities used were 1.26 g (4 mmol) of N-
isopropyl-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (1) and 0.4 g
(2 mmol) of (CH3COO)2Cu·H2O. Yield: 1.12 g, 81%. M.p. 225–
◦
226 C. FT-IR (KBr, cm-1): 3415, 3059, 2967, 1591, 1557, 1518,
1446, 1392, 1290, 1175, 1057, 914, 885, 770, 749, 703, 581, 538,
418, 276. Anal. calcd for Cu C34N6H34O2Cl2 (693.12): Cu, 9.17; C,
58.92; H, 4.94; N, 12.12; Found: Cu, 8.98; C, 58.67; H, 4.62; N,
11.96%. m eff, 1.62 BM.
N-(tert-Butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (4).
Quantities used were 2.8 ml (20 mmol) of triethylamine, 1.1 ml
(10 mmol) of tert-butylamine, 2.9 g (10 mmol) of N-benzoyl-
N¢-(2-chlorophenyl)thiourea and 2.72 g (10 mmol) of mercuric
chloride. Yield 2.67 g, 81%. M.p. 110–112 ◦C. FT-IR (KBr, cm-1)
3314, 3170, 3057, 2973, 2927, 1591, 1571, 1486, 1445, 1369, 1220,
1146, 1058, 902, 752, 712, 680, 590. 1H NMR (300 MHz, CDCl3,
25 ◦C): d 1.56 (s, 9H, –C(CH3)3), 4.78 (s, 1H, NH), 7.18–7.53
Bis(N-(n-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidinato)-
copper(II) (2a). Quantities used were 1.32 g (4 mmol) of N-
(n-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (2) and 0.4 g
(2 mmol) of (CH3COO)2Cu·H2O. Yield: 1.13 g, 78%. M.p 155–
◦
3
156 C. FT-IR (KBr, cm-1): 3427, 3063, 2957, 2929, 2869, 1591,
(m, 7H, Ar–H), 8.29 (d, 2H, J = 6.6 Hz, Ar–H 2 & 6), 12.26 (s,
1H, NH); 13C NMR (75.47 MHz, CDCl3, 25 ◦C): d 29.7 (3C,
–C(CH3)3), 52.3 (–C(CH3)3), 124.3, 126.8, 127.9 (2C), 129.0,
129.2 (2C), 131.2, 132.8, 134.2, 138.7, 142.9 (aromatic C), 157.4
(CN3), 177.4 (C O). Anal. calcd for C18N3H20OCl (329.82): C,
65.55; H, 6.11; N, 12.74; Found: C, 65.15; H, 5.89; N, 12.34%.
1556, 1521, 1462, 1390, 1230, 1150, 1058, 939, 752, 711, 573, 525,
449, 274. Anal. Calcd. for CuC36N6H38O2Cl2 (721.18): Cu, 8.81;
C, 59.96; H, 5.31; N, 11.65; Found: Cu, 8.66; C, 59.28; H, 5.29; N,
11.36%. m eff, 1.56 BM.
Bis(N -(sec-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidina-
to)copper(II) (3a). Quantities used were 1.32 g (4 mmol) of
N-(sec-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (3) and
0.4 g (2 mmol) of (CH3COO)2Cu·H2O. Yield: 1.18 g, 82%. M.p.
N-Benzyl-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine
(5).
Quantities used were 2.8 ml (20 mmol) triethylamine, 1.1 ml
(10 mmol) of benzylamine, 2.9 g (10 mmol) of N-benzoyl-N¢-(2-
chlorophenyl)thiourea and 2.72 g (10 mmol) of mercuric chloride.
Yield 2.61 g, 72%. M.p. 90–92 ◦C. FT-IR (KBr, cm-1) 3375, 3063,
3032, 2951, 1728, 1627, 1609, 1589, 1558, 1367, 1219, 879, 752,
701, 680, 680, 588, 555, 495. 1H NMR (300 MHz, CDCl3, 25 ◦C):
d 4.80 (s, 2H, CH2), 5.18 (s, 1H, NH), 7.20–7.54 (m, 12H, Ar–H),
◦
235–237 C. FT-IR (KBr, cm-1): 3408, 3062, 2969, 1590, 1556,
1488, 1447, 1391, 1263, 1160, 1056, 923, 897, 743, 705, 579, 538,
457, 275. Anal. Calcd. for CuC36N6H38O2Cl2 (721.18): Cu, 8.81;
C, 59.96; H, 5.31; N, 11.65; Found: Cu, 8.72; C, 59.51; H, 5.16; N,
11.42%. m eff, 1.71 BM.
3
8.30 (d, 2H, J = 6.6 Hz, Ar–H 2 & 6), 12.36 (s, 1H, NH); 13C
NMR (75.47 MHz, CDCl3, 25 ◦C): d 45.4 (CH2), 125.8, 126.4,
127.3, 127.6 (2C), 127.8, 128.0 (2C), 128.9 (2C), 129.2, 129.3
(2C), 130.8, 131.5, 133.5, 134.6, 138.2 (Aromatic-C), 158.4 (CN3),
178.0 (C O). Anal. calcd for C21N3H18OCl (363.84): C, 69.32;
H, 4.99; N, 11.55; Found: C, 69.14; H, 4.73; N, 11.31%.
Bis(N-(tert-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidina-
to)copper(II) (4a). Quantities used were 1.32 g (4 mmol) of
N-(tert-butyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (4) and
0.4 g (2 mmol) of (CH3COO)2Cu·H2O. Yield: 1.15 g, 80%. M.p.
◦
247–249 C. FT-IR (KBr, cm-1): 3424, 3068, 2961, 2926, 2870,
1590, 1559, 1523, 1460, 1393, 1292, 1150, 1057, 935, 897, 753,
707, 687, 585, 526, 490, 275. Anal. Calcd. for CuC36N6H38O2Cl2
(721.18): Cu, 8.81; C, 59.96; H, 5.31; N, 11.65; Found: Cu, 8.59;
C, 59.43; H, 5.26; N, 11.48%. m eff, 1.74 BM.
N-(4-Methylphenyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine
(6). Quantities used were 2.8 ml (20 mmol) of triethylamine, 1.1 g
(10 mmol) of para-toludine, 2.9 g (10 mmol) of N-benzoyl-N¢-(2-
chlorophenyl)thiourea and 2.72 g (10 mmol) of mercuric chloride.
Yield 2.73 g, 75%. M.p. 87–89 ◦C. FT-IR (KBr, cm-1) 3307, 3110,
3067, 2959, 2930, 1727, 1625, 1586, 1539, 1485, 1350, 1294, 747,
Bis(N -benzyl-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidinato)-
copper(II) (5a). Quantities used were 1.45 g (4 mmol) of N-
benzyl-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (5) and 0.4 g
(2 mmol) of (CH3COO)2Cu·H2O. Yield: 1.17 g, 74%. M.p. 214–
705, 587, 540. H NMR (300 MHz, CDCl3, 25 ◦C): d 2.42 (s,
1
3H, CH3), 8.43 (s, 1H, NH), 7.09–8.43 (m, 13H, Ar–H), 11.93 (s,
◦
◦
1H, NH); 13C NMR (75.47 MHz, CDCl3, 25 C): d 21.1 (CH3),
215 C. FT-IR (KBr, cm-1): 3403, 3053, 3021, 2961, 1590, 1560,
125.0, 125.2, 125.7 (2C), 127.1, 127.3, 128.1 (2C), 129.2, 129.3
(2C), 130.7, 131.6, 132.7, 134.7, 137.5, 138.2 (aromatic C), 156.5
(CN3), 178.5 (C O); Anal. calcd. for C21N3H18OCl (363.84): C,
69.32; H, 4.99; N, 11.55; Found: C, 69.07; H, 4.82; N, 11.46%.
1517, 1462, 1392, 1342, 1240, 1159, 1057, 881, 740, 704, 569, 535,
452, 274. Anal. Calcd. for CuC42N6H34O2Cl2 (789.21): Cu, 8.05;
C, 63.92; H, 4.34; N, 10.65; Found: Cu, 7.83; C, 63.54; H, 4.19; N,
10.38%. m eff, 1.48 BM.
9208 | Dalton Trans., 2011, 40, 9202–9211
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Bis(N-(4-methylphenyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguani-
dinato)copper(II) (6a). Quantities used were 1.45 g (4 mmol) of
N-(paratolyl)-N¢-(2-chlorophenyl)-N¢¢-benzoylguanidine (6) and
0.4 g (2 mmol) of (CH3COO)2Cu·H2O.; Yield: 1.14 g, 72%. M.p.
Vibrational analyses were performed to confirm the optimized
stationary points as true minima on the potential energy surface
and to obtain the zero-point energy and thermodynamic data. The
free Gibbs energies, G, were calculated for T = 298.15 K.
◦
219–220 C. FT-IR (KBr, cm-1): 3397, 3056, 3024, 2919, 1591,
1556, 1524, 1477, 1422, 1367, 1224, 895, 737, 697, 603, 560, 501,
444, 275. Anal. Calcd. For CuC42N6H34O2Cl2 (789.21): Cu, 8.05;
C, 63.92; H, 4.34; N, 10.65; Found: Cu, 7.73; C, 63.49; H, 4.07; N,
10.76%. m eff, 1.45 BM.
Biological assays
The urease inhibition activity studies were carried out according
to a reported protocol.34 Exactly 25 ml of the enzyme (jack
bean urease) solution and 5 ml of the test compounds (0.5 mM
concentration) were incubated in 96-well plates for 15 min at 30 ◦C,
with each well containing 55 ml of buffers containing 100 mM urea.
Ammonia production was measured to probe the urease activity
by the indophenol method. The final volumes were maintained at
200 ml by adding 45 ml of a phenol reagent (1% w/v phenol and
0.005% w/v sodium nitroprusside) and 70 ml of an alkali reagent
(0.5% w/v NaOH and 0.1% of active chloride NaOCl) to each
well. Using a microplate reader (Molecular Devices, CA, USA),
the increase in absorbance was measured at 630 nm after 50 min
at pH 6.8. The results (change in absorbance per minute) were
collected using the SoftMax Pro software (Molecular Devices,
CA, USA). Thiourea was used as the standard inhibitor and the
percentage of inhibition were calculated as follows:
Structural studies
Crystals of 1, 4, 1a, and 2a were grown by slow evaporation
methods. The solvent system for the guanidines was ethanol and
for the complexes was a petroleum ether and chloroform (1 : 1)
mixture. The guanidine crystals were colourless and the complexes
crystals were dark blue. The block crystals were mounted on a
glass fibre using Paratone N hydrocarbon oil. The measurements
were made at 200(2) K on a Bruker APEX II area detector
diffractometer equipped with graphite monochromated Mo-Ka
˚
radiation (0.71073 A). The program used for retrieving the cell
parameters and data collection was APEX 2.25 The data were
integrated using the program SAINT26 and were corrected for
Lorentz and polarization effects. The multiscan absorption correc-
tions were performed using SADABS.27 The structures were solved
and refined using SHELXS-97 and SHELXL-97 (Sheldrick,
1997)28 and all non-H atoms were refined anisotropically, with the
hydrogen atoms placed at idealized positions. The crystallographic
data are given in Table 5.
Percentage inhibition = 100 - (ODtest well/ODcontrol) ¥100
A modified method of S. Habtemariam and T. Mossman
was used to determine the anti-leishmanial activity.35 Serial
dilutions of test samples were made in a 96-well flat bottom
plate (polystyrene, sterile, corning) in a 100 ml volume. Parasites
were diluted with fresh culture medium to a final density of 106
cells ml-1. 100 ml of parasite culture was added to all of the wells.
Two rows were left for negative and positive controls. Negative
controls received medium, while the positive control contained
varying concentrations of standard anti-leishmanial compounds,
e.g. amphotericin B, pentamidine, etc. The plate was incubated at
22–25 ◦C for 72 h. The culture was examined microscopically on an
improved Neubauer counting chamber and the IC50 values of the
compounds possessing anti-leishmanial activity were calculated
Computational details
The density functional theory calculations were carried out with
the restricted B3LYP hybrid functional, as implemented in the G03
program.29 B3LYP is Becke’s three parameter functionals (B3)30
with the non-local correlation provided by the LYP expression31
and VWN functional III for local correlation.32 The 6-31g(d,p)
basis set was used to describe all atoms (a single set of first
polarization functions was added to each atom).33 Geometry op-
timizations were carried out without any geometrical constraints.
Table 5 Crystallographic data
1
4
1a
4a
Formula
FW
Cryst. syst.
C17H18ClN3O
329.82
Monoclinic
C18H20ClN3O
329.82
Monoclinic
P21/c
10.0536(15)
14.736(2)
12.5743(19)
90, 113.060(2), 90
1714.0(4)
C34H34Cl2CuN6O2
693.11
Monoclinic
P21/c
19.020(8)
14.289(6)
18.525(8)
90, 96.193(6), 90
5005(4)
6
1.380
0.855
2154
12034/60387
C36H38Cl2CuN6O2
721.16
Rhombohedral
¯
Space group
P2(1)/n
R3
˚
a/A
13.5978(19)
11.6143(16)
20.150(3)
40.2231(18)
40.2231(18)
11.5607(11)
90, 90, 120
16198.2(19)
18
˚
b/A
˚
c/A
a, b, g (◦)
90, 98.910(2), 90
3143.8(8)
8
1.334
0.248
1328
4938/28021
0.0805
3
˚
V/A
Z
4
Dc/g cm-3
1.278
0.231
696
4170/20438
0.0561
1.331
0.795
6750
8841/67939
0.0688
m/mm-1
F000
No. of unique/total reflns
Rint
0.1093
1.019
R1 = 0.0587, wR2 = 0.1428
R1 = 0.1047, wR2 = 0.1673
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
1.008
1.001
1.040
R1 = 0.0495, wR2 = 0.1075
R1 = 0.1000, wR2 = 0.1339
R1 = 0.0439, wR2 = 0.0831
R1 = 0.0933, wR2 = 0.1005
R1 = 0.0433, wR2 = 0.1015
R1 = 0.0749, wR2 = 0.1155
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©

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by Software Ezfit 5.03 Perella Scientific. All assays were run in
triplicate.
14 (a) R. G. S. Berlinck and M. H. Kossuga, Nat. Prod. Rep., 2005, 22, 516–
550; (b) R. G. S. Berlinck, Nat. Prod. Rep., 2002, 19, 617–649; (c) R. G. S.
Berlinck, Nat. Prod. Rep., 1999, 16, 339–365; (d) R. G. S. Berlinck, Nat.
Prod. Rep., 1996, 13, 377–409; (e) J. V. Greenhill, L. I. Lee, G. P. Ellis
and D. K. Luscombe, in Progress in Medicinal Chemistry, Elsevier
Science, New York, 1993, vol. 30, chapter 5; (f) J. C. Manimala and
E. V. Anslyn, Eur. J. Org. Chem., 2002, 3909–3922.
15 (a) N. Sunduru Nishi, S. Palne, P. M. S. Chauhan and S. Gupta,
Eur. J. Med. Chem., 2009, 44, 2473–2481; (b) S. Singh, A. Jhingran, A.
Sharma, A. R. Simonian, P. Soininen, J. Vepsalainen, A. R. Khomutov
and R. Madhubala, Biochem. Biophys. Res. Commun., 2008, 375, 168–
172.
Acknowledgements
The authors are grateful to HEC (Pakistan) for financial support
and scholarship. F.-G. Fontaine is grateful to NSERC (Canada),
CFI (Canada), FQRNT (Que´bec) and CERPIC (Universite´
Laval) for financial support. J. Boudreau acknowledges NSERC,
FQRNT and Rio-Tinto Alcan for scholarships.
16 (a) D. A. Powell, P. D. Ramsden and R. A. Batey, J. Org. Chem.,
2003, 68, 2300–2309; (b) S. Cunha, M. B. Costa, H. B. Napolitano, C.
Lauriucci and I. Vecanto, Tetrahedron, 2001, 57, 1671–1675.
17 The use of mercury salt is a drawback. However, it is being used for
research purposes since it allows a high yielding and rapid synthetic
pathway: (a) M. K. Rauf, Imtiaz-ud-Din, A. Badshah, M. de Gielen,
M. Ebihara, D. Vos and S. Ahmed, J. Inorg. Biochem., 2009, 103, 1135–
1144; (b) J. Zhang, Y. Shi, P. Stein, K. Atwal and C. Li, Tetrahedron
Lett., 2002, 43, 57–59; (c) Y. Yu, J. M. Ostresh and R. A. Houghten,
J. Org. Chem., 2002, 67, 3138–3141; (d) K. S. Kim and L. Qian,
Tetrahedron Lett., 1993, 34, 6777–7680 The by-product of the synthesis
(HgS) is insoluble in water (Ksp = 2 ¥ 10-53) and is environmentally
benign; (e) H. Y. Son, S. Lee, S. B. Park, M. S. Kim, E. J. Choi, T. S.
Singh, Y. Bae, S. J. Kwack, T. S. Kang, H. I. Shin, M. C. Baek and S. H.
Kim, Immunopharmacol. Immunotoxicol., 2010, 32, 277–283. A greener
option is to use bismuth nitrate, but the reaction time is longer and the
yields are low and it is rarely used; (f) S. Cunha, M. T. Rodrigues, C. de
Silva, H. B. Napolitano, I. Vecanto and C. Lariucci, Tetrahedron, 2005,
61, 10536–10540.
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