Synthesis, antiinflammatory activity, and molecular docking studies of bisphosphonic esters as potential MMP-8 and MMP-9 inhibitors

Article abstract of DOI:10.3762/bjoc.16.108

Bisphosphonic acids (or bisphosphonates) have been successfully used in the clinic treatment of bone diseases for over decades. Additionally, the antiinflammatory activity of these compounds has been gaining attention. In our previous work, we synthesized and in vivo evaluated the bisphosphonic esters 1 and 2, finding a moderate edema inhibition upon oral and topical administration on BALB/c mice. Thus, in this work, the bioisosteric replacement of an amide functional group for an ester afforded the new bisphosphonates 3–6, which had a moderate oral edema inhibition (25 mg/kg dose) and a significant topical antiinflammatory activity (2 mg/ear) on BALB/c mice, with 6 being the most active hit (55.9% edema inhibition), comparable to the positive control (55.5% edema inhibition) on a TPA topical model. Next, to assess the acute toxicity of the synthesized derivatives, test animals were administered with 50–100 mg/kg of 3–6, respectively, by an oral route, and after 14 days, neither lethality nor a significative weight loss were observed. Finally, a structure–activity relationship (SAR) and a molecular docking analysis of 3–6 helped us to explain the trend observed in biological tests. Considering all these aspects, we propose the inhibition of MMP-8 and MMP-9 as a possible action mechanism of the synthesized derivatives.

Full text of DOI:10.3762/bjoc.16.108

Synthesis, antiinflammatory activity, and molecular docking
studies of bisphosphonic esters as potential MMP-8
and MMP-9 inhibitors
Abimelek Cortes-Pacheco1,2, María Adelina Jiménez-Arellanes2,
Francisco José Palacios-Can3, José Antonio Valcarcel-Gamiño3,
Rodrigo Said Razo-Hernández3, María del Carmen Juárez-Vázquez2,
Adolfo López-Torres1 and Oscar Abelardo Ramírez-Marroquín*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 1277–1287.
1Instituto de Química Aplicada, Universidad del Papaloapan.
Tuxtepec, 68301, Mexico, 2Unidad de Investigación Médica (UIM) en
Farmacología, UMAE Hospital de Especialidades, Centro Médico
Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social
(IMSS). Av. Cuauhtémoc 330, Col. Doctores 06720, Ciudad de
México (CdMx), Mexico and 3Centro de Investigación en Dinámica
Celular, Universidad Autónoma del Estado de Morelos Avenida
Universidad 1001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico
doi:10.3762/bjoc.16.108
Received: 12 March 2020
Accepted: 25 May 2020
Published: 08 June 2020
Associate Editor: I. Baxendale
© 2020 Cortes-Pacheco et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Oscar Abelardo Ramírez-Marroquín* - oramirez@unpa.edu.mx
* Corresponding author
Keywords:
inflammation; molecular docking; organophosphorus compounds
Abstract
Bisphosphonic acids (or bisphosphonates) have been successfully used in the clinic treatment of bone diseases for over decades.
Additionally, the antiinflammatory activity of these compounds has been gaining attention. In our previous work, we synthesized
and in vivo evaluated the bisphosphonic esters 1 and 2, finding a moderate edema inhibition upon oral and topical administration on
BALB/c mice. Thus, in this work, the bioisosteric replacement of an amide functional group for an ester afforded the new bisphos-
phonates 3–6, which had a moderate oral edema inhibition (25 mg/kg dose) and a significant topical antiinflammatory activity
(2 mg/ear) on BALB/c mice, with 6 being the most active hit (55.9% edema inhibition), comparable to the positive control (55.5%
edema inhibition) on a TPA topical model. Next, to assess the acute toxicity of the synthesized derivatives, test animals were
administered with 50–100 mg/kg of 3–6, respectively, by an oral route, and after 14 days, neither lethality nor a significative weight
loss were observed. Finally, a structure–activity relationship (SAR) and a molecular docking analysis of 3–6 helped us to explain
the trend observed in biological tests. Considering all these aspects, we propose the inhibition of MMP-8 and MMP-9 as a possible
action mechanism of the synthesized derivatives.
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Introduction
Bisphosphonic acids (or bisphosphonates) are organophos-
phorus compounds characterized by a P–C–P moiety. These
organic compounds are valuable drugs for the treatment of bone
diseases as osteoporosis, Paget’s disease, and malignant hyper-
calcemia [1-3]. Specifically, bisphosphonates act as osteoclast
resorption inhibitors, augmenting the bone density and
preventing osteoporosis [4]. Moreover, some bisphosphonates
have gained attention as potential antiinflammatory agents by in
vitro and in vivo assays [5-8]. Additionally, bisphosphonates
have been reported as inhibition and downregulation matrix
metalloproteinase (MMP) agents [9-11]. In this regard, MMPs
are a family of extracellular proteinases (24 isoenzymes in
human) involved in tissue regeneration and are closely related
to physiologic and physiopathological processes, such as
inflammation, angiogenesis, and metastasis in cancer [12-15],
pointing at bisphosphonates as potential treatments for cancer
and other inflammation-related diseases. In this respect, MMP
inhibition by phosphonates or bisphosphonates has been previ-
ously studied through computational or X-ray diffraction
analyses to describe the enzyme inhibitor site binding modes
[16-18].
Figure 1: Previously reported antiinflammatory bisphosphonates 1 and
2. edema inhibition (in %, carrageenan model, 50 mg/kg) for 1: 7.0; for
2: 22.2.
Furthermore, in the search of more potent and low-toxicity de-
rivatives, in this work, we have focussed our attention on the
molecular modification of the derivatives 1 and 2 through the
bioisosteric replacement [20] of the amide functional group by
an aliphatic or aromatic ester. The potential antiinflammatory
activity of the new bisphosphonates was predicted using the
Prediction of Activity Spectra for Substances (PASSOnline)
database, which compares the molecular structure of test com-
pounds vs a large training set of experimental bioactive or inac-
tive compounds [21]. The results of the prediction are summa-
rized as probability of activity (Pa) and probability of inactivity
(Pi) values, both ranging from 0 to 1 (Figure 2), where a higher
Pa value is desired. Thus, a Pa value of new bisphosphonates ≥
the Pa value of 1 and 2 was the applied inclusion criterion in
this study. As can be seen in Figure 2, the Pa is greater for the
new derivatives 3–6 (0.63–0.77) compared to the previous bio-
active compounds 1 and 2 (0.51). In order to explore the SAR in
the proposed compounds 3–6, we evaluated the effect of the
volume of the ester group by the inclusion of ethyl or tert-butyl
substituents in the aliphatic derivatives 3 and 4. On the other
hand, the replacement of the aliphatic chains by an aromatic
portion led us to the derivatives 5 and 6 where the effect of
benzyl or 4-methoxybenzyl substituents was assessed
(Figure 2).
Thus, in this work, the bisphosphonates 3–6 were synthesized
by a two-step method and then evaluated through two in vivo
acute inflammation models in BALB/c mice. Furthermore, the
acute toxicity was determined for these derivatives, and molec-
ular docking studies were performed to account for a possible
action mechanism as MMP-8 and MMP-9 inhibitors.
Results and Discussion
Chemistry
As part of our ongoing interest in the discovery of new antiin-
flammatory agents, our research group have previously
addressed the synthesis and in vivo antiinflammatory activity
evaluation of the bisphosphonic esters 1 and 2, observing activi-
ty by oral (carrageenan model) and topical administration (TPA Furthermore, the bisphosphonic esters 3–6 passed the Lipinski’s
model) in BALB/c mice (Figure 1) [19].
rules [22] as criteria for drugs for an oral administration as we
Figure 2: Designed bisphosphonic esters as antiinflammatory agents.
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Beilstein J. Org. Chem. 2020, 16, 1277–1287.
wanted to test these derivatives through in vivo acute inflamma- canoylphorbol-13-acetate (TPA) through topical administration
tion models [23]. Then, the above-named derivatives were syn- [25] and b) a carrageenan model, through oral administration
thesized in a first stage by the esterification of bromoacetyl bro- [25]. When the bisphosphonates 3–6 (2 mg/ear) were assayed
mide and the corresponding alcohol. The reaction of ethyl or with a TPA model, the derivatives 5 (40.7% edema inhibition)
tert-butyl alcohol and bromoacetyl bromide in the presence of and 6 (55.9% edema inhibition) were the most active ones, with
triethylamine in CH2Cl2 yielded the bromoaceto esters 7 and 8 6 having a comparable edema inhibition to the positive control
in 42 and 71% yield (Scheme 1b, method A). Nonetheless, (indomethacin, 55.5% edema inhibition, Table 1). Nevertheless,
when benzyl or 4-methoxybenzyl alcohol were used under the the derivatives 3 and 4 exhibited a moderate antiinflammatory
same reaction conditions, 9 and 10 were obtained in poor yields. activity (25.5% and 23.9% edema inhibition, respectively).
Thus, the subsequent use of NaHCO3 as a base in CH3CN [24] Thus, the inclusion of an aromatic ring in the derivatives 5 and
afforded the bromoaceto esters 9 and 10 in 71 and 91% yield 6 clearly potentiated the desired pharmacological effect on
(Scheme 1b, method B). It is important to note that 4-methoxy- mice. The rationale behind this could be the difference in
benzyl alcohol was prepared by the reduction of 4-methoxy- lipophilicity between the aliphatic esters 3 and 4, which were
benzaldehyde (Scheme 1a).
less lipophilic (by means of the clogP value of 0.58 and 1.39,
respectively) compared to the aromatic derivatives 5 and 6
Next, the treatment of tetraethyl methylenediphosphonate with (clogP = 1.80 and 1.85, respectively), with the last being the
NaHMDS under an anhydrous atmosphere, followed by the ad- most permeable one through mice skin (Table 1). Nevertheless,
dition of 7–10, respectively, afforded the final products 3–6, re- it is important to note that the aliphatic derivatives 3 and 4
spectively, in 41–73% yield (Scheme 2).
considerably differed in the clogP value between each other
(0.58 and 1.39) but had a comparable edema inhibition, indicat-
ing that for this study, the volume of the ester group had little
Pharmacological activity
With the target compounds on hand, we proceed to evaluate importance for the pharmacological activity. More important
them with two acute inflammation models: a) 12-O-tetrade- was the replacement of the aliphatic for an aromatic residue in
Scheme 1: Synthesis of the intermediate bromoaceto esters 7–10.
Scheme 2: Synthesis of the bisphosphonates 3–6.
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Table 1: Antiinflammatory activity of 3–6 using a TPA topical model. 2 mg/ear of the test compounds was used.a
treatment
auricular edema (mg)
% inhibition
clogPb
DE50 (mg/ear)
TPA
8.80 mg ± 0.46
3.92 mg ± 0.37c
6.56 mg ± 0.19c,d
6.70 mg ± 0.39c,d
5.22 mg ± 0.37c,d
3.88 mg ± 0.21c
–
–
–
indomethacin (2 mg/ear)
55.5
25.5
23.9
40.7
55.9
–
–
3
4
5
6
0.58
1.39
1.80
1.85
n.d.
n.d.
1.4
0.9
aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is given in respect to the TPA group. Statistical
analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bCalculated using the Molinspiration property engine v2018.10b [31]. cVs TPA control. dVs
indomethacin; n = 5.
the ester group, leading to the more active derivatives 5 and 6. were predicted to be more bioactive than 5 and 6 (Table 2). Ad-
The clogP value of 5 and 6 was more similar between them, but ditionally, this tendency was strongly connected to the clogP
a remarked difference in the edema inhibition was observed value, where the edema inhibition was inversely proportional to
(40.7 vs 55.9%), indicating that the introduction of an electron- the clogP value (Table 2). Thus, the higher the predicted Pa and
donating 4-methoxy substituent on the phenyl ring of 6 potenti- the lower the clogP, the higher the observed activity. It is worth
ated the antiinflammatory activity compared to the nonsubsti- to mention that the oral efficacy of the tested compounds was
tuted derivative 5. Next, the DE50 value was assessed for the opposed to that observed with the TPA topical model. This may
more interesting targets 5 (DE50 = 1.4 mg/ear) and 6 (DE50 = be a consequence of the lower lipophilicity of 3 (clogP = 0.58)
0.9 mg/ear), with the methoxy derivative 6 having a higher po- and 4 (clogP = 1.39) compared to 5 (clogP = 1.80) and 6
tency and efficacy (55.9% edema inhibition) in the series (clogP = 1.85), influencing the better dissolution of 3 and 4 in
(Table 1). Our results were in good accordance with the an aqueous medium prior to its absorption through gut mice
previous observation of the antiinflammatory activity of the few (Table 2). Finally, the synthesized bisphosphonates 3 and 4
bisphosphonic esters [26-30].
have proven to be more active (24.6% and 20.9% edema inhibi-
tion, respectively, at a 25 mg/kg dose) by oral administration
Next, the antiinflammatory activity of the bisphosphonates 3–6 than the parent compounds 1 and 2 (7.0% and 22.2% edema
was assayed with a carrageenan model by intragastric adminis- inhibition, respectively, at a 50 mg/kg dose, Figure 1) [19]. In
tration. As can be seen in Table 2, the derivatives 3 and 4 were addition, 3 and 4 had a comparable activity than what was re-
the more active ones this time (24.6% and 20.9% edema inhibi- ported for other bisphosphonic esters [30].
tion, respectively). A remarkable difference was observed for
the derivatives 5 (13.8% edema inhibition) and 6 (9.1% edema Following this, the acute toxicity of 3–6 was determined
inhibition) where a low antiinflammatory activity was observed. through one oral administration of 50 or 100 mg/kg in BALB/c
In this regard, a clear correlation between the experimental and mice, and after 14 days, no significant weight loss or lethality
predicted activity was observed. Thus, the compounds 3 and 4 was observed in the individuals. Additionally, the post-mortem
Table 2: Antiinflammatory activity of 3–6 with a carrageenan oral model. Test compounds: 25 mg/kg.a
treatment
paw edema (mm)b
% inhibition
Pa
clogPc
carrageenan
0.830 ± 0.017d
0.530 ± 0.04d
0.626 ± 0.03d,e
0.657 ± 0.02d,e
0.716 ± 0.03d,e
0.755 ± 0.06e
–
–
–
indomethacin (20 mg/kg)
36.1
24.6
20.9
13.8
9.1
–
–
3
4
5
6
0.77
0.71
0.66
0.63
0.58
1.39
1.80
1.85
aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is in respect to the carrageenan group. Statistical
analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bAt 5 h. cCalculated using Molinspiration property engine v2018.10b [31]. dVs control
carrageenan. eVs indomethacin; n = 5.
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inspection of the kidneys, heart, and bowel of the experimental From Table 3, we observed that a correlation between the anti-
mice did not show any significant weight differences to the inflammatory activity of the compounds and the molecular
control group (Supporting Information File 1, Table 1, and properties can be established. The structural modifications are
Table 2).
directly correlated to the molecular weight (MW) of the com-
pounds, and the MW can be correlated to the molecular volume.
Lastly, in order to acknowledge a potential mechanism of action We can appreciate that 3 and 4 have the lowest volume com-
of the bisphosphonates 3–6, we propose that the tested deriva- pared to 5 and 6. This fact can help us to explain the greater
tives are acting as MMP inhibitors. In this respect, MMP-8 and antiinflammatory activity of 3 and 4 with a carrageenan model;
MMP-9 isoenzymes are related to inflammatory processes in a small molecular volume increases the pharmacokinetic abili-
different tissues [32-35]. Furthermore, for MMP-8 and MMP-9, ties of the compounds. In addition, 3 and 4 have a greater dipole
enzyme–inhibitor interaction modes are well known. For exam- moment compared to 5 and 6. The hydrogen-bond formation
ple, the coordination of the P=O oxygen atom in bisphospho- and the noncovalent interactions are influenced by the dipole
nates with a zinc cation in the catalytic site of the MMPs has moment. This means that an increased dipole moment can
been characterized, both through X-ray diffraction and molecu- improve the binding properties of a molecule. Besides the
lar docking studies [11,36,37]. Consequently, we propose dipole moment, 3 and 4 have the greatest chemical hardness of
MMP-8 and MMP-9 as potential biological targets of 3–6. the bisphosphonates. This descriptor is related to the chemical
susceptibility to an external potential. Therefore, the antiinflam-
Computational and theoretical analysis
Ligands structure–activity relationship
matory activity of these compounds can be related to the size
(volume), solvation (dipole moment), and chemical reactivity
As a first approximation, we studied the structural and physico- (hardness; probably related to a minor metabolic biotransforma-
chemical features of the compounds to explain the antiinflam- tion). These descriptors can be associated with the better phar-
matory activity. The structure geometry used to obtain the mo- macokinetic profile of the derivatives 3 and 4 by oral adminis-
lecular properties of each bisphosphonate represented a tration.
minimum in the potential energy surface since all vibrational
frequency values were positive. In Table 3, all the molecular Molecular docking
properties obtained for the compounds are displayed. The As a second approximation, to study the effect of these struc-
chemical hardness (η) and softness (S) were calculated using tural modification on the pharmacodynamics, we performed a
Equation 1 and Equation 2, which are based in the Koopman’s molecular docking over two MPPs. In Table 4, the interaction
theorem for the determination of the global chemical reactivity energy value (MolDock Score) [38] of each compound with the
descriptors.
two different MMPs obtained from the docking calculation is
displayed. Also, the ligand efficiency (LE) of each bisphospho-
nate is shown; the ligand efficiency stands for the coefficient of
the interaction energy by the number of atoms in the molecule
(excluding hydrogen atoms).
(1)
(2)
By the inspection of the results above, there is a correlation be-
tween the LE parameter in MMP-8 and the experimental activi-
ty of the bisphosphonates, resulting in the highest LE value for
Table 3: Molecular properties of the compounds 3–6.
molecular properties
3
4
5
6
molecular weight (amu)
dipole moment (Debye)
EHOMO (eV)
374.307
3.4
402.361
3.4
436.378
2.43
466.404
2.4
−10.57
0.82
−10.44
0.72
−9.41
0.25
−8.83
0.19
ELUMO (eV)
volume (Å3)
363.78
71.532
5.69
401.16
73.573
5.58
430
455.41
74.198
4.51
PSA (Å2)
70.905
4.83
hardness (η)
softness (S)
0.1756
0.1792
0.207
0.2217
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Table 4: MolDock Score and LE1 values (kcal/mol) for the docking experiments of the molecules 3–6 with MMP-8 and MMP-9 enzymes and the cor-
responding inhibition values.a
ligand
MMP-8
MMP-9
% inhibition
MolDock Score
LE
MolDock Score
LE
3
4
5
6
−146.72
−137.92
−150.42
−168.58
−6.38
−5.52
−5.37
−5.62
−134.16
−147.76
−147.24
−146.50
−5.83
−5.91
−5.26
−4.88
24.57
20.86
13.80
9.06
aIndomethacin was set as the reference (with 36.1272%).
3, with the most potent inhibition observed with the carrageenan In all cases, a distorted tetrahedral coordination geometry was
model (Table 2). The predicted LE value for 4 in MMP-9 was observed for the zinc ion, caused by the chelation with His197,
comparable to 3 but showed less activity than 3. For both dock- Glu198, and His207 residues. His201 was far apart from the
ings, 6 showed some of the highest MolDock Score values, but catalytic site, and the basic nitrogen atom in this residue was
nevertheless, its inhibition activity was the lowest (Table 2). It pointing away from the zinc ion [39]. The calculated distances
is important to note that a clear correlation between the pre- from the Zn2+ ion and the different sites at the ligand and the
dicted interaction energy of 3–6 with MMP-8 and the topical protein are summarized in Table 5.
antiinflammatory activity was observed (TPA model), with the
derivative 6 being the most active one, followed by 5, 3, and 4
Table 5: Calculated distances (Å) for the coordination of the zinc(II) ion
at MMP-8.
in that order (Table 1). Thus, the efficacy of the tested com-
pounds 3–6 was well correlated with the lipophilicity and the
predicted interaction energy with MMP-8 (Table 4). Next, we
searched for other energy interaction features from the docking
calculation.
molecule
3
4
5
6
His207
2.28
2.47
3.84
1.99
3.30
2.46
3.85
1.99
2.36
2.45
4.16
1.99
2.26
2.44
4.13
1.99
His197
Glu198
As expected, 4–6 could bind the Zn2+ ion in a monodentate
fashion through the oxygen atom double bonded to the phos-
phorus atom (P=O) of one of the phosphonate moieties, as re-
oxygen (ligand)
ported in the literature for other structures [38,39]. Only for 3, The zinc chelation in MMP-9 occurred in a different way. The
zinc chelation was observed through the oxygen atom double predicted orientation of the histidine residues at the catalytic site
bonded to the carbon atom (C=O) of the ester group (Figure 3). were not aiming directly at the metal ion. However, it can be
assumed that because of the flexibility of the protein in solution,
the coordination to the metal ion would be possible. The targets
3 and 4 could coordinate the Zn2+ ion through the oxygen atom
double bonded to the phosphorus atom (P=O) of one of the
phosphonate moieties, while 5 and 6 had interactions through
the C=O oxygen atom of the ester group. Because the interac-
tions of the benzyl group of 5 and 6, respectively, with the
Phe110 residue present at the catalytic site through π–π interac-
tions are possible, the orientation of the molecules inside the
catalytic site allowed the coordination through the ester groups
rather than through the phosphonate moieties. The calculated
distances from the Zn2+ ion and the different sites at the ligand
and the protein are summarized in Table 6.
In Figure 4, a schematic representation of the interactions of the
ligands in MMP-8 is shown. As expected, the oxygen atoms
Figure 3: Coordination of the Zn2+ ion by residues and by the carbon-
yl ester oxygen atom of molecule 3. The basic coordinating nitrogen
and oxygen atoms are marked in light blue.
that are coordinating the zinc ion have the greatest contribution.
For example, 3 in MMP-8 has a greater contribution energy
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Table 6: Calculated distances (Å) for the coordination of the zinc(II) ion
at MMP-9.
Table 7: Calculated total (ETotal) and electrostatic energy (EElec) for
the most contributing oxygen atoms to the interaction energy of the
complex. Energies in kcal/mol.
molecule
3
4
5
6
energy
MMP-8
3
4
5
6
His411
2.35
2.72
4.53
2.00
2.67
3.33
4.51
2.00
2.80
3.30
4.12
2.00
2.72
3.17
4.48
2.00
His401
ETotal (O1) −22.03
EElec (O1) −24.20
EElec (Zn) −13.63
−32.22
−39.97
−22.83
−35.880
−38.200
−34.11
−38.71
Glu402
oxygen (ligand)
−20.4092 −19.51
from oxygen O1, with an electrostatic energy (EElec) of Compound 3 displayed two hydrogen bonds. The first one was
−24.20 kcal/mol and a total energy (ETotal) of −22.03 kcal/mol; formed between a hydrogen atom of His162 with an oxygen
the observed energy values are shown in Table 7.
atom from the ester moiety (the oxygen atom bound to the ethyl
Figure 4: 2D schematic representations of the MMP-8 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted
bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions.
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group of the phosphonate unit), with a calculated distance (Figure 4b). Also, for compound 5, only one hydrogen bond
(O–H) of 1.86 Å (or a 2.60 Å O–N distance) and an energy of interaction was seen. Indeed, the oxygen atom double bonded to
−1.51 kcal/mol. The second interaction, stronger than the first the phosphorus atom of the phosphonate group was strongly
one, was observed from an oxygen atom (P=O) of the phos- bonded to an amide hydrogen atom localized between Ile159
phonate unit to an amide hydrogen atom located between Ile159 and Leu160, with a 3.08 Å O–N distance and an energy of
and Leu160. It possesses an energy of −2.5 kcal/mol and a −2.5 kcal/mol (Figure 4c). Compound 6 showed four hydrogen
1.86 Å distance from the oxygen atom to the amide hydrogen bonds that varied in force. Two were observed between an
atom (or a 3.09 Å O–N distance, Figure 4a). Compound 4 only oxygen atom from an OEt moiety of a phosphonate group to
displayed one hydrogen bond of the double bonded carbonyl amide hydrogen atoms localized between Ile159, Leu160, and
oxygen atom to an amide hydrogen atom located between Ala161 (Figure 4d). These interactions had energy values of
Asn218 and Tyr219. This interaction was regarded as weak, −1.94 and −2.5 kcal/mol, which is regarded as characteristic for
with an energy of −0.47 kcal/mol and a 3.51 Å O–N distance strong hydrogen bonds. The measured O–N distances had
Figure 5: 2D schematic representations of the MMP-9 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted
bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions; green
= CH–π interactions.
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values of 3.15 and 2.67 Å. Another hydrogen-bond interaction
was seen from an OEt moiety (from the phosphonate group
coordinating the zinc ion), with a weak energy of
−0.95 kcal/mol and O–N distance of 3.26 Å. Finally, the me-
thoxy group at the ligand had a weak energy of −1.0 kcal/mol
and a 3.12 Å distance (Figure 4d).
Table 8: Calculated total (ETotal) and electrostatic energy (EElec) for
the most contributing oxygen atoms to the system. Energies in
kcal/mol.
energy
MMP-9
3
4
5
6
ETotal (O1) −38.4103 −39.9732 −23.9506 −23.8484
EElec (O1) −34.1810 −32.8937 −17.2600 −7.5905
EElec (Zn) −20.9699 −19.8742 −20.2706 −19.022
As can be seen in Figure 4, the hydrophobic zones of the cata-
lytic site in MMP-8 were formed by Ile159, Leu160, and
Ala161, as well for Gly158 and Val194. However, for 3, the
ethyl groups were not entirely pointing at this zone. The calcu-
lated C–C distance from a methylene group to a Leu160 methyl actions from an oxygen atom (P=O) of the phosphonate group
group was 3.35 Å. The other methyl groups from the ligand had to amide hydrogen atoms located between Ala189, Leu188, and
interactions with His162 (2.96 Å), His197 (2.98 Å), and His207 Leu187, with energy values of −2.36 and −1.60 kcal/mol
(3.20 Å). Indeed, the rest of the molecules also presented this (Figure 5b). Compound 5 exhibited two weak hydrogen bonds
type of interactions between an ethyl fragment of the ligand from an OEt oxygen atom to an amide hydrogen atom of
with Leu160, and less with His207 and Val194. Weak hydro- Ala189 and Leu188 (Figure 5c). The energy value was
phobic interactions were also observed from Tyr219 and the ar- −0.89 kcal/mol, and the O–N distance was 3.24 Å. The second
omatic ring of 6 as well as π–π interactions to His197. The mol- interaction was very weak, with a value of −0.15 kcal/mol from
ecules were not adequately occupying the cavity of the enzyme a P=O oxygen atom to the His401 amine hydrogen atom.
in MMP-8, as shown in Figure 4. For the compounds 4 and 5, Finally, for 6, the hydrogen bond interactions were lacking.
the tert-butyl and benzyl groups at the ester moiety were From the energy contribution profile seen in Figure 5d, most of
pointing out to the solvent, without any important interactions the interaction energy was due to the coordination of the ligand
with the hydrophobic zone inside the cavity (Figure 4b and to the zinc ion, with the same applying to 5. This could explain
Figure 4c). The only molecule that was occupying the entire why no predicted hydrogen bonding was present in 6 and just
pocket was compound 6 due to the formation of four hydrogen very weak ones in 5.
bonds and a favourable π–π interaction between the methoxy-
benzyl group and His197.
For MMP-9, the hydrophobic zones were formed by Ile159,
Leu160, and Ala161, as was the case for Gly158 and Val194.
Only for 3 and 4, one of the oxygen atoms of the phosphonate As stated before, π–π interactions from the aromatic ring of the
units was the most contributing atom to the interaction energy. ligands 5 and 6 with the Phe110 residue were displayed
However, for 5 and 6, the electrostatic energy was almost (Figure 5c and 5d). Furthermore, CH–π interactions from the
equally distributed in the three oxygen atoms of the two phos- tert-butyl group with Phe110 were also seen, with a 3.12 Å dis-
phonate and ester groups. For example, in 5, the three above- tance (Figure 5b). All molecules had hydrophobic interactions
mentioned oxygen atoms displayed a total energy-per-atom from an ethyl group of the ligands to a methyl moiety of Val398
value of −17.26 (C=O), −15.62 (P1=O), and −14.07 (P2=O) and with Leu187, with distances below 3.5 Å.
kcal/mol, although the electrostatic energy (EElec) was higher at
the ester oxygen atom (C=O), with a value of −23.95 kcal/mol, For MMP-9, the ligands were even more exposed to the solvent,
while the other two oxygen atoms from the phosphonate units although many more noncovalent interactions were seen be-
had energies of −13.65 and −11.53 kcal/mol, respectively. The tween the catalytic site and the molecules (Figure 5).
same energy trend was observed for 6, which correlated well
with the predicted interactions of the molecule (the C=O and Conclusion
P=O units binding Zn2+) at the catalytic site (Table 8).
In this work, we reported the two-step synthesis of the bisphos-
phonic esters 3–6. For the first time, the antiinflammatory activ-
As before, in Figure 5, the schematic representations of the ity of the compounds was assessed by oral (carrageenan model)
interaction of the ligands in MMP-9 are displayed. Compound 3 and topical administration (TPA model) to mice. Among these,
displayed one hydrogen bond, from the carbonyl oxygen atom the derivative 6 had an excellent edema inhibition, comparable
to an amide hydrogen atom located between Ala189 and to the positive control with the TPA model. On the other hand,
Leu188, with this interaction being regarded as very weak, the bioisosteric replacement of an amide for an ester group in
having an energy of −0.36 kcal/mol and a 3.53 Å O–N distance the parent compounds 1 and 2 afforded the more potent deriva-
(Figure 5a). On the other hand, 4 had two hydrogen bond inter- tives 3 and 4, which had a higher antiinflammatory activity than
1285

Beilstein J. Org. Chem. 2020, 16, 1277–1287.
Oscar Abelardo Ramírez-Marroquín -
the parent bisphosphonates 1 and 2 using a carrageenan model.
Moreover, a lipophilicity–activity relationship was observed for
the two acute inflammation models: the aliphatic hydrophilic
compounds 3 and 4 were the more potent ones by oral adminis-
tration, and the aromatic lipophilic bisphosphonates 5 and 6 had
a better antiinflammatory activity by topical administration. In
addition, the safety of the test compounds 3–6 was evaluated by
an acute toxicity determination where no significant weight loss
or lethality was observed in individuals at a 50 and 100 mg/kg
dose (the two- or four-fold dose as used in the original study).
Finally, a ligand structure–activity relationship and molecular
docking analysis led us to propose MMP-8 and MMP-9 inhibi-
tion as the possible action mechanism of 3–6 due to the good
correlation between the antiinflammatory activity of the bispho-
sphonic esters and the interaction energy with these enzymes
(especially MMP-8). Also, a good correlation between the bio-
logical effects and interaction of the compounds with the Zn2+
cofactor of these enzymes was observed.
https://orcid.org/0000-0003-4354-5468
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The authors thank CONACyT of Mexico for financial support
via project CB-2017-2018 A1-S-10787, INFRA-2015-01-
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CONACyT for the graduate grant 942820. Antonio Valcarcel
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On behalf of all authors, the corresponding author states that
there is no conflict of interest.
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ORCID® iDs
Abimelek Cortes-Pacheco - https://orcid.org/0000-0003-3982-8203
María Adelina Jiménez-Arellanes - https://orcid.org/0000-0002-9379-5601
Rodrigo Said Razo-Hernández - https://orcid.org/0000-0002-9716-9376
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