Synthesis and in vivo anti- or pro-inflammatory activity of new bisphosphonates and vinylphosphonates

Article abstract of DOI:10.1007/s00706-018-2328-2

Abstract: We herein report the synthesis and in vivo anti-inflammatory activity of a series of new bisphosphonate and vinylphosphonate derivatives of pyrrolidine and piperidine through a short route of synthesis. The C-alkylation of tetraethylmethylene diphosphonate with N-(bromoacetyl)pyrrolidine or N-(bromoacetyl)piperidine, respectively, yielded the corresponding α-substituted bisphosphonates in excellent yields (82–89%). Next, the Horner–Wadsworth–Emmons reaction of these bisphosphonates with aromatic aldehydes afforded final vinylphosphonates in moderate yields (26–36%). Synthesized bisphosphonates and vinylphosphonates were tested by two models of acute inflammation in male BalB/c mice, founding excellent edema inhibition by topical TPA (12-O-tetradecanoylphorbol-13-acetate) model (67.53–72.10% in comparison with indomethacin = 64.89%). However, remarkably pro-inflammatory effect by systematic carrageenan model (??9.78 to ??36.18) was observed, probably due to biotransformation. In conclusion, the new vinylphosphonates emerged as attractive topical anti-inflammatory compounds that “twist” its pharmacological activity to route of administration. Further research is needed to understand the dual effect.

Full text of DOI:10.1007/s00706-018-2328-2

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-018-2328-2
ORIGINAL PAPER
Synthesis and in vivo anti‑ or pro‑infammatory activity of new
bisphosphonates and vinylphosphonates
Oscar Abelardo Ramírez‑Marroquín1 · María Adelina Jiménez‑Arellanes2 · Abimelek Cortés‑Pacheco1 ·
Oscar R. Zambrano‑Vásquez2 · Adolfo López‑Torres1
Received: 28 August 2018 / Accepted: 25 October 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
We herein report the synthesis and in vivo anti-infammatory activity of a series of new bisphosphonate and vinylphospho-
nate derivatives of pyrrolidine and piperidine through a short route of synthesis. The C-alkylation of tetraethylmethylene
diphosphonate with N-(bromoacetyl)pyrrolidine or N-(bromoacetyl)piperidine, respectively, yielded the corresponding
α-substituted bisphosphonates in excellent yields (82–89%). Next, the Horner–Wadsworth–Emmons reaction of these bis-
phosphonates with aromatic aldehydes aforded fnal vinylphosphonates in moderate yields (26–36%). Synthesized bis-
phosphonates and vinylphosphonates were tested by two models of acute infammation in male BalB/c mice, founding
excellent edema inhibition by topical TPA (12-O-tetradecanoylphorbol-13-acetate) model (67.53–72.10% in comparison
with indomethacin=64.89%). However, remarkably pro-infammatory efect by systematic carrageenan model (− 9.78 to
− 36.18) was observed, probably due to biotransformation. In conclusion, the new vinylphosphonates emerged as attractive
topical anti-infammatory compounds that “twist” its pharmacological activity to route of administration. Further research
is needed to understand the dual efect.
Graphical abstract
Keywords Phosphorus compounds · Alkenes · Wittig reaction · Infammation
Introduction
Electronic supplementary material The online version of this
In recent years, vinylphosphonates have been gaining atten-
tion in need of their interesting and underexplored phar-
macological activities. For example, many vinylphospho-
nates have applications as antimicrobials [1–3], enzymatic
inhibitors [4, 5], anticancer agents [6, 7] and have also been
used in siRNA gene silencing technology [8–11] and drug
delivery systems [12]. In this context, it is worth mention-
ing dehydrophos [1], a natural broad spectrum antibiotic,
tamiphosphor [2], a synthetic bioisostere of the antiviral
article (https://doi.org/10.1007/s00706-018-2328-2) contains
supplementary material, which is available to authorized users.
* Oscar Abelardo Ramírez-Marroquín
oramirez@unpa.edu.mx
1
Instituto de Química Aplicada, Universidad del Papaloapan,
Tuxtepec, Mexico
2
Unidad de Investigación Médica en Farmacología, Hospital
de Especialidades, CMN Siglo XXI, Instituto Mexicano del
Seguro Social, Mexico City, Mexico
Vol.:(0123456789)
1 3

O. A. Ramírez-Marroquín et al.
on vinylphosphonates have just received modest attention
[26–28]. An interesting example of in vitro anti-infamma-
tory activity of vinylphosphonates was reported by Al Qun-
tar et al. [28]. Compounds 1–3 (Fig. 2a) modulated the pro-
duction of reactive oxygen species, nitric oxide, and tumoral
necrosis factor (TNF-α) (all of them involved in the devel-
opment of infammation) in cultured murine macrophages.
In spite of all the above mentioned and rheumatoid arthri-
tis as an emerging problem of public health [29], we herein
report the synthesis of new potential anti-inflammatory
agents. These vinylphosphonates are structurally related to
parent compounds 1–3. The new vinylphosphonates 4–6 con-
serve the α,β-unsaturated phosphonate in 1–3, an aromatic
ring and also a nitrogen atom at four-bond distance from phos-
phonate (Fig. 2b). Hence, the synthesis of target compounds
4–6 was achieved through HWE reaction of bisphosphonates
9–10 with aromatic aldehydes as the key step. Furthermore,
an in vivo evaluation of 4–6 anti-infammatory agents was
carried out to develop new promising antiarthritic compounds.
Fig. 1 Structure of some biologically active vinylphosphonates
oseltamivir and (S)-FTY720, a potential anticancer agent
[6] (Fig. 1). On the other hand, bisphosphonates have been
used as the primary therapeutic agents in the treatment of
bone diseases such as osteoporosis, metastatic and osteo-
lytic bone disease, Paget’s disease of bone and osteopenia.
For instance, zoledronate, alendronate, pamidronate, and
etidronate (all commercial drugs) inhibit bone resorption
interfering with the action of the bone-resorbing osteoclasts
by diferent molecular modes of action [13–15].
Results and discussion
Synthesis
In addition to the structural relationship between target com-
In other instances, the synthesis of vinylphosphonates
has been carried out through Horner–Wadsworth–Emmons
(HWE) reaction [16, 17], hydrophosphorylation of alkynes
[18–21], cross coupling of dialkylphosphites and dibro-
moalkenes [22, 23], and Heck reaction starting from sim-
ple vinylphosphonates and aryl halides [24, 25], mainly.
Despite the importance and potential of these compounds
to medicinal chemistry, anti-infammatory activity studies
pounds 4–6 and anti-infammatory vinylphosphonates 1–3,
Scheme 1
(a)
(b)
Fig. 2 a Previously reported in vitro anti-infammatory vinylphosphonates [28]; b newly proposed vinylphosphonates as anti-infammatory
agents
1 3

Synthesis and in vivo anti‑ or pro‑infammatory activity of new bisphosphonates and…
we used the Prediction of Activity Spectra for Substances
(PASSOnline) database, getting probability of activity as
antiarthritic agents>0.9 (in scale from 0 to 1) in 4–6. PAS-
Sonline predicts more than 7000 types of biological activity
with an average accuracy>95% [30] and has been used suc-
cessfully to predict in vitro and in vivo diverse pharmaco-
logical activities of new compounds (and possible adverse
efects) [31–34]. Moreover, proposed vinylphosphonates 4–6
agreed with Lipinski’s rules [35] as a clear indicator of its
drug-likeness.
As a fnal step in the synthesis, using bisphosphonates
9–10, respectively, we carried out the HWE reaction with
aromatic aldehydes, under inert conditions. The reaction
of 9 with p-anisaldehyde using NaHMDS as base, aforded
product 4 as trace. A 31P NMR analysis of crude reaction
mixture showed an E/Z ratio of 65:35. Next, the use of LDA
as a hard base improved yield to 12%; the crude reaction’s
NMR analysis showed an E/Z ratio of 13:87. Subsequent
chromatographic purifcation aforded 8% of (Z)-4 and 4%
of (E)-4. Finally, the use of the hardest base n-BuLi pro-
duced compound 4 in 36% yield, with an E/Z ratio of 17:83.
This time again it was possible to separate diastereomers
(Z)-4 in 16% and (E)-4 in 20% yields. Employing n-BuLi in
subsequent experiments aforded vinylphosphonates 5 and
6 in moderate yields (26–32%). It is worth to mentioning
that for compounds 5 and 6, just E isomer was detected and
isolated (Scheme 3).
For the synthesis of target compounds 4–6 our strategy
started with amidation of pyrrolidine or piperidine with bro-
moacetyl bromide, respectively, under previously established
conditions [36], obtaining excellent yields without purifca-
tion (Scheme 1).
Then we tried to prepare the key bisphosphonate interme-
diates 9 and 10 through C-alkylation reaction of tetraethyl-
methylene diphosphonate with alkyl halides 7 and 8 under
anhydrous conditions. At the frst attempt, using NaHMDS
as base and alkyl halide 7, product 9 was detected by NMR
analysis. Due to the instability of 9 in silica gel column
chromatography, the product could not be obtained under
these conditions. Therefore, the use of neutral alumina as
stationary phase during purifcation of 9 or 10 under identi-
cal reaction conditions allowed obtention of desired products
in excellent yields (Scheme 2).
The E and Z confguration in 4–6 was unambiguously
1
assigned through H NMR spectra, specifcally to vinylic
3JH-P coupling, observing a typical J=23.9 Hz for E isomer
and J=47.1 Hz for Z isomer, as has been well documented
elsewhere [37] and it is depicted in Fig. 3.
To the best of our knowledge, both bisphosphonates 9 and
10 and vinylphosphonates 4–6 have not been reported in the
literature, hence constitute new and unexplored bioactive
compounds.
Scheme 2
Scheme 3
1 3

O. A. Ramírez-Marroquín et al.
Fig. 3 NMR 1H spectra of diastereomers (Z)-4 and (E)-4
Table 1 Anti-infammatory activity of 9, 10, and 4–6, 5 h after
Table 2 Anti-infammatory activity of 9, 10, and 4–6, 6 h after
administration, carrageenan model
administration, TPA model
Treatment
Paw edema/mm
% inhibition
Auricular edema/mg
% inhibition
Carrageenan
0.48 0.07
0.38 0.02
–
TPA control
8.31 0.52
2.92 0.41a
–
Indomethacin 20 mg/kga
21.86
Indomethacin 2 mg/eara
64.89
Test compounds 50 mg/kg
Test compounds 2 mg/ear
9b
0.45 0.06
0.38 0.02
0.42 0.09
0.57 0.01
0.53 0.06
0.66 0.08
7.01
22.15
14.35
− 35.78
− 9.78
− 36.18
9b
4.70 0.62b
3.66 0.18b
2.70 0.16a
2.36 0.30a
2.45 0.56a
2.32 0.33a
43.48
55.99
67.53
71.62
70.54
72.10
10b
(E)-4b
(Z)-4b
5b
10b
(E)-4b
(Z)-4b
5b
6b
6b
Each group represents the mean standard error of the mean (SEM).
The percentage of inhibition of edema is with respect to the carra-
geenan group. Two-way analysis of variance (ANOVA) and post hoc
Student–Newman–Keuls (p≤0.05)
Each group represents the mean standard error of the mean (SEM).
The percentage of inhibition of the edema is with respect to the TPA
group. One-way analysis of variance (ANOVA) and post hoc Stu-
dent–Newman–Keuls (p≤0.05)
aVersus carrageenan control
bVersus indomethacin
aVersus carrageenan control
bVersus indomethacin
kg in male BalB/c mice using carrageenan inducing paw
edema as phlogistic agent and indomethacin as positive
control. Astonishingly, just bisphosphonates 9 and 10
exhibited activity with 7.01 and 22.15% edema inhibition,
Anti‑infammatory activity
Anti-infammatory activity evaluation [38] of compounds
9 and 10, and 4–6 was carried out by oral gavage at 50 mg/
1 3

Synthesis and in vivo anti‑ or pro‑infammatory activity of new bisphosphonates and…
Fig. 4 Proposed pro-infammatory metabolites presumably formed by oral administration
respectively (indomethacin showed 21.89% inhibition). In
contrast, vinylphosphonates (Z)-4, 5, and 6 were more pro-
infammatory than carrageenan itself (Table 1). Despite
these previous results, we carried out topical administration
of compounds 9 and 10, and 4–6 at 2 mg per ear in BalB/c
mice using TPA (12-O-tetradecanoylphorbol-13-acetate) as
irritant. In this assay, moderate edema inhibition for bispho-
sphonates 9 and 10 was observed, but signifcant inhibition
for vinylphosphonates 4–6 was achieved (67.53–72.10%),
comparable or superior than indomethacin (64.89%)
(Table 2). In view of the dual behavior of vinylphospho-
nates 4–6 in function of administration route, we propose
a preliminary explanation: the hydrolysis of phosphonate
functional group in 4–6 afords phosphonic acid deriva-
tives 11–13 (Fig. 4) which could have pro-infammatory
activity. In this respect, it is important to mention that com-
pounds 11–13 got probability of infammation as adverse
efect of 0.27–0.35 through PASSonline prediction; but this
prediction was not obtained in bisphosphonates 9 and 10 or
vinylphosphonates 4–6 at all.
Conclusion
The synthesis of new bisphosphonates 9 and 10 and
vinylphosphonates 4–6 provided in vivo anti- or pro-infam-
matory agents, tested by two models of acute infammation.
Interestingly, in function of route of administration, we
found pro-infammatory activity in vinylphosphonates (Z)-4,
5, and 6 with oral dosage (carrageenan model), but promis-
ing anti-infammatory activity with topical administration
(TPA model). A preliminary hypothesis was disclosed for
this dual behavior, but further research is needed to clarify
the dual efect. Lastly, synthesized compounds 9, 10, and
4–6 emerged as promising new topical anti-infammatory
agents which should be the object of future studies.
Experimental
All commercial materials were used as received from
Sigma-Aldrich® without further purification. Flash
chromatography was performed using 230–400-mesh
Silica Flash 60® silica gel. Thin-layer chromatography
was performed with pre-coated TLC sheets of silica
gel (60F254, Merck). NMR spectra were recorded in a
In addition to the above-mentioned hypothesis, the suc-
cessful use of phosphonates as prodrugs, which are hydro-
lyzed to bioactive phosphonic acids by enzymatic processes,
has been well documented [39–41].
1
In spite of all, of the tested compounds (9, 10, and 4–6)
only 9 and 10 were active when administered intragastric;
however, compounds (Z)-4, 5, and 6 induced infammation.
This efect may be a result of biotransformation of phospho-
nates 4–6 to its respective phosphonic acids 11–13, predicted
as potential pro-infammatory metabolites. On the other
hand, all compounds tested (9, 10, and 4–6) by the topical
route were active, so they are candidates for further research
to develop new clinically efective anti-infammatory agents.
Finally, it should be noted that topical and systemic
infammation models carried out in this work are widely
used to explore the anti-infammatory efect of synthetic and
natural products [42–44].
Varian System instrument (400 MHz for H, 100 MHz
for 13C, and 161.9 MHz for 31P), and calibrated with
CDCl3 as the solvent and TMS as the internal standard
signal. Chemical shifts (δ) are reported in parts per mil-
lion. Multiplicities are recorded as follows: s = singlet,
d = doublet, t = triplet, dd = doublet of doublets, td = tri-
plet of doublets, bs = broad singlet, q = quartet, and
m = multiplet. Coupling constants (J) are given in Hz.
High-resolution mass spectra (HRMS) were obtained in
a SYNAPT G2-Si (Waters) spectrometer equipped with
single quadrupole mass filter and time of flight mass
analyzer (Q-TOF).
1 3

O. A. Ramírez-Marroquín et al.
(t, J=135.4 Hz, CHP), 46.08 (CH2N), 46.42 (CH2N), 62.5
(d, J=6.6 Hz, OCH2), 62.90 (d, J=6.4 Hz, OCH2), 167.69
(t, J=7.6 Hz, C=O) ppm; 31P NMR (161.9 MHz, CDCl3):
δ = 23.86 ppm; HRMS [ESI+]: m/z calculated 400.1654,
found 400.1720.
General procedure for the synthesis of alkyl halides
7 and 8
To an ice bath-cooled solution of bromoacetyl bromide
(13.78 mmol) in 20 cm3 of CH2Cl2 was added dropwise
via addition funnel a solution of pyrrolidine or piperidine,
respectively (2 equivalents) in 30 cm3 of CH2Cl2, and the
reaction mixture was stirred overnight. Then the reaction
mixture was quenched by the addition of 20 cm3 of dis-
tilled water and extracted with CH2Cl2 (3 × 20 cm3). Next,
to the combined organic extracts 30 cm3 of HCl 2.0 N was
added. After partition, the fnal organic extract was dried
over Na2SO4, fltered, and evaporated under reduced pres-
sure, yielding desired bromoamides 7 and 8.
Tetraethyl [3‑oxo‑3‑(piperidin‑1‑yl)propane‑1,1‑diyl]‑
bis(phosphonate) (10, C16H33NO7P2) Yield: 780 mg (82%)
1
as a yellow oil. H NMR (400 MHz, CDCl3): δ = 1.33 (t,
J = 7.1 Hz, 12H, CH3), 1.51–1.69 (m, 6H, CH2), 2.85 (td,
J=16.3, 5.5 Hz, 2H, CH2C=O), 3.43–3.65 (m, 5H, CH2N,
CHP), 4.12–4.23 (m, 8H, OCH2) ppm; 13C NMR (100 MHz,
CDCl3): δ=16.33 (d, J=6.4 Hz, CH3), 24.48 (CH2), 25.53
(CH2), 26.34 (CH2), 28.73 (t, J=3.9 Hz, CH2C=O), 31.62
(t, J=135.4 Hz, CHP), 43.55 (CH2N), 46.43 (CH2N), 62.51
(d, J=6.7 Hz, OCH2), 62.91 (d, J=6.5 Hz, OCH2), 167.30
(t, J=7.4 Hz, C=O) ppm; 31P NMR (161.9 MHz, CDCl3):
δ = 23.99 ppm; HRMS [ESI+]: m/z calculated 414.1811,
found 414.1809.
N‑(Bromoacetyl)pyrrolidine (7) Yield: 2.42 g (92%) as
1
a white solid that melts at room temperature. H NMR
(400 MHz, CDCl3): δ = 1.87–1.94 (m, 2H, CH2), 1.98–
2.05 (m, 2H, CH2), 3.50 (t, J = 6.9 Hz, 2H, CH2N), 3.54
(t, J = 6.8 Hz, 2H, CH2N), 3.82 (AB system, J= 20.0 Hz,
2H, CH2Br) ppm; 13C NMR (100 MHz, CDCl3): δ=24.31
(CH2), 26.15 (CH2), 27.39 (CH2Br), 46.43 (CH2N), 47.06
(CH2N), 165.07 (C=O) ppm; HRMS [ESI]: m/z calculated
for C6H11BrNO ([M+H]+) 192.0024, found 192.0095.
General procedure for the synthesis
of vinylphosphonates 4–6
A solution of n-BuLi (1.5 equiv) was added dropwise to
a stirred solution of respective bisphosphonate 9 or 10 (1
equiv) in 7 cm3 THF at − 5 °C under nitrogen. Resultant
solution was stirred for 15 min at this temperature and then
10 min at room temperature, followed by the addition of
respective aromatic aldehyde (1.1 equiv) at − 5 °C. Reac-
tion mixture was stirred for 10 min at this temperature and
then for 18 h at − 20 °C. Finally, the reaction mixture was
N‑(Bromoacetyl)piperidine (8) Yield: 2.47 g (87%) as a pale
yellow oil. 1H and 13C NMR spectra were found to be identi-
cal with the ones described in Ref. [45].
General procedure for the synthesis
of bisphosphonates 9 and 10
1
concentrated, and crude product was analyzed by H and
31P NMR and then purifed by column chromatography
supported on silica gel, AcOEt:MeOH (90:10), yielding
vinylphosphonates 4–6.
To a solution of tetraethylmethylene bisphosphonate in
8 cm3 THF was added dropwise NaHMDS (1.5 equiv) at
− 5 °C under nitrogen. The solution was stirred for 15 min
and then 10 min at room temperature. After this time, a solu-
tion of the respective alkyl halide 7 or 8 (2.5 equiv) was
added at − 5 °C. Ten minutes later, reaction mixture was
stirred at − 20 °C for 18 h. Finally, the reaction mixture
(Z)‑Diethyl [1‑(4‑methoxyphenyl)‑4‑oxo‑4‑(pyrrolidin‑1‑yl)‑
but‑1‑en‑2‑yl]phosphonate (4, C19H28NO5P) Yield: 54 mg
(16%) as yellow viscous oil. 1H NMR (400 MHz, CDCl3):
δ=1.12 (t, J=7.1 Hz, 6H, CH3), 1.84–2.00 (m, 4H, CH2),
3.40 (d, J = 13.3 Hz, 2H, CH2C=O), 3.48–3.55 (m, 4H,
CH2N), 3.82 (s, 3H, OCH3), 3.92–4.01 (m, 4H, OCH2),
6.86 (AA′BB′, J=8.8 Hz, 2H, Harom), 7.16 (d, J=47.2 Hz,
1H, CH), 7.56 (AA′BB′, J = 8.9 Hz, 2H, Harom) ppm; 13C
NMR (100 MHz, CDCl3): δ = 15.99 (d, J = 7.0 Hz, CH3),
24.43 (CH2), 26.14 (CH2), 41.81 (d, J=12.0 Hz, CH2C=O),
45.89 (CH2N), 46.76 (CH2N), 55.22 (OCH3), 61.76 (d,
J = 5.7 Hz, OCH2), 113.12, 121.43 (d, J = 178.7 Hz, CP),
128.15 (d, J = 7.5 Hz), 131.14 (d, J = 1.6 Hz), 146.68 (d,
1
was concentrated, and crude product was analyzed by H
and 31P NMR and then purifed by column chromatogra-
phy supported on neutral alumina, CH2Cl2:AcOEt:MeOH
(50:49:01), obtaining corresponding bisphosphonates 9 and
10.
Tetraethyl [3‑oxo‑3‑(pyrrolidin‑1‑yl)propane‑1,1‑diyl]‑
bis(phosphonate) (9, C15H31NO7P2) Yield: 687 mg (89%) as
1
a pale yellow oil. H NMR (400 MHz, CDCl3): δ = 1.31–
1.34 (m, 12H, CH3), 1.83–2.00 (m, 4H, CH2), 2.80 (td,
J = 16.2, 5.8 Hz, 2H, CH2C=O), 3.42–3.57 (m, 5H,
CH2N, CHP), 4.10–4.25 (m, 8H, OCH2) ppm; 13C NMR
(100 MHz, CDCl3): δ = 16.30 (d, J = 6.1 Hz, CH3), 24.37
(CH2), 25.99 (CH2), 30.31 (t, J=4.1 Hz, CH2C=O), 31.59
J=8.8 Hz), 159.85, 168.98 (d, J=4.7 Hz, C=O) ppm; 31
P
NMR (161.9 MHz, CDCl3): δ=18.12 ppm; HRMS [ESI+]:
m/z calculated 382.1783, found 382.1952.
1 3

Synthesis and in vivo anti‑ or pro‑infammatory activity of new bisphosphonates and…
(E)‑Diethyl [1‑(4‑methoxyphenyl)‑4‑oxo‑4‑(pyrrolidin‑1‑yl)‑
but‑1‑en‑2‑yl]phosphonate (4, C19H28NO5P) Yield: 69 mg
(20%) as a yellow viscous oil. 1H NMR (400 MHz, CDCl3):
δ=1.33 (t, J=7.1 Hz, 6H, CH3), 1.86–2.02 (m, 4H, CH2),
3.43 (d, J = 18.7 Hz, 2H, CH2C=O), 3.50–3.55 (m, 4H,
CH2N), 3.82 (s, 3H, OCH3), 4.07–4.17 (m, 4H, OCH2), 6.90
(AA′BB′, J=8.8 Hz, 2H, Harom), 7.55 (AA′BB′, J=8.5 Hz,
2H, Harom), 7.66 (d, J=23.9 Hz, 1H, CH) ppm; 13C NMR
(100 MHz, CDCl3): δ = 16.48 (d, J = 6.9 Hz, CH3), 24.67
(CH2), 26.35 (CH2), 33.73 (d, J=10.5 Hz, CH2C=O), 46.20
(CH2N), 47.05 (CH2N), 55.43 (OCH3), 62.08 (d, J=5.1 Hz,
OCH2), 113.99, 121.99 (d, J= 181.2 Hz, CP), 127.88 (d,
J=22.7 Hz), 130.90, 146.61 (d, J=11.7 Hz), 160.25, 168.35
(d, J=1.8 Hz, C=O) ppm; 31P NMR (161.9 MHz, CDCl3):
δ = 22.07 ppm; HRMS [ESI+]: m/z calculated 382.1783,
found 382.1952.
Evaluation of anti‑infammatory activity
Carrageenan-induced edema procedure was performed as
described by Pérez-González et al. [38]. Treated groups
(n=5) received indomethacin (20 mg/kg) by the intragas-
tric route or tested compounds 9, 10, and 4–6 (50 mg/kg)
1 h prior to the injection of carrageenan (20 cm3, 2%). The
samples were solubilized in Tween 80:water (10:90) and the
control received only vehicle. The percentage of inhibition
was calculated by comparing the measurement of the paw
edema at diferent times (1, 3, 5, 7, and 24 h) using a digital
micrometer and the value of time zero (baseline) (E0). The
results were analyzed with the formula described by Pérez-
González et al. [38]:
% Inhibition = (Et − E0) carrageenan − (Et − E0) treated∕
(Et − E0 carrageenan] × 100.
(1)
(E)‑Diethyl [1‑(4‑nitrophenyl)‑4‑oxo‑4‑(pyrrolidin‑1‑yl)‑
but‑1‑en‑2‑yl]phosphonate (5, C18H251N2O6P) Yield: 94 mg
(32%) as viscous pale brown oil. H NMR (400 MHz,
CDCl3): δ = 1.34 (t, J = 7.1 Hz, 6H, CH3), 1.88–2.04 (m,
4H, CH2), 3.36 (d, J = 18.6 Hz, 2H, CH2C=O), 3.49–
3.55 (m, 4H, CH2N), 4.09–4.20 (m, 4H, OCH2), 7.75
(d, J = 23.6 Hz, 1H, CH), 7.80 (AA′BB′, J = 8.9 Hz, 2H,
Harom), 8.22 (AA′BB′, J=8.8 Hz, 2H, Harom) ppm; 13C NMR
(100 MHz, CDCl3): δ = 16.31 (d, J = 6.8 Hz, CH3), 24.49
(CH2), 26.12 (CH2), 33.44 (d, J=10.0 Hz, CH2C=O), 46.12
(CH2N), 46.99 (CH2N), 62.30 (d, J=5.2 Hz, OCH2), 123.63,
128.27 (d, J=179.3 Hz, CP), 129.78 (d, J=1.4 Hz), 141.56
(d, J = 22.3 Hz), 144.05 (d, J = 11.8 Hz), 147.71, 167.36
(d, J=1.6 Hz, C=O) ppm; 31P NMR (161.9 MHz, CDCl3):
δ = 19.21 ppm; HRMS [ESI+]: m/z calculated 397.1528,
found 397.1576.
TPA-induced ear edema was conducted as described by
Pérez-González et al. [38]. Control was treated with TPA
(2.5 μg) in acetone on the right ear (W’s) and then left ear
received only 25 mm3 of acetone (Wo). The experimental
groups (n=5) received TPA and 30 min later were treated
with test compounds 9, 10, and 4–6 (2 mg/ear) in the right
ear (Ws). Anti-infammatory activity was calculated accord-
ing to the weight diference between the ear sections (6 mm)
at 6 h, compared with the control group, using the following
formula:
% Inhibition = [(W’s − Wo) control − (W’s− Wo) treated∕
(W’s − Wo) control] × 100.
(2)
Acknowledgements The authors thank CONACyT and Secretaría de
Educación Pública of Mexico for fnancial support via project INFRA-
2015-01-252013 and F-PROMEP-39/Rev-04 SEP. ACP also thank Sec-
retaría de Educación Pública of Mexico for undergraduate scholarship.
(E)‑Diethyl [1‑(4‑nitrophenyl)‑4‑oxo‑4‑(piperidin‑1‑yl)‑
but‑1‑en‑2‑yl]phosphonate (6, C19H27N2O6P) Yield: 115 mg
1
(26%) as viscous pale brown oil. H NMR (400 MHz,
CDCl3): δ = 1.35 (t, J = 7.1 Hz, 6H, CH3), 1.55–1.72 (m,
6H, CH2), 3.40 (d, J=18.3 Hz, 2H, CH2C=O), 3.45–3.47
(m, 2H, CH2N), 3.59–3.62 (m, 2H, CH2N), 4.07–4.21 (m,
4H, OCH2), 7.69 (AA′BB′, J=8.5 Hz, 2H, Harom), 7.74 (d,
References
1. Ulrich EC, Bougioukou DJ, van der Donk WA (2018) ACS
Chem Biol 13:537
2. Chen CL, Lin TC, Shie JJ, Tsai KC, Cheng YSE, Jan JT, Lin CJ,
Fang JM, Wong CH (2014) Eur J Med Chem 81:106
3. Harnden MR, Parkin A, Parratt M, Perkins RM (1993) J Med
Chem 36:1343
J=23.8 Hz, 1H, CH), 8.22 (AA′BB′, J=8.8 Hz, 2H, Harom
)
ppm; 13C NMR (100 MHz, CDCl3): δ=16.31 (d, J=6.7 Hz,
CH3), 24.46 (CH2), 25.67 (CH2), 26.49 (CH2), 31.87 (d,
J=9.5 Hz, CH2C=O), 43.35 (CH2N), 47.25 (CH2N), 62.33
(d, J=5.3 Hz, OCH2), 123.62, 128.87 (d, J=180.0 Hz, CP),
129.71 (d, J=1.4 Hz), 141.63 (d, J=22.6 Hz), 143.69 (d,
4. Ettari R, Nizi E, Di Francesco ME, Micale N, Grasso S, Zappalà
M, Vičík R, Schirmeister T (2008) Chem Med Chem 3:1030
5. Al-Quntar AAA, Baum O, Reich R, Srebnik M (2004) Arch
Pharm Pharm Med Chem 337:76
J=11.7 Hz), 147.61, 167.15 (d, J=1.7 Hz, C=O) ppm; 31
P
6. Tonelli F, Alossaimi M, Natarajan V, Gorshkova I, Berdyshev E,
Bittman R, Watson DG, Pyne S, Pyne NJ (2013) Biomolecules
3:316
NMR (161.9 MHz, CDCl3): δ=19.45 ppm; HRMS [ESI+]:
m/z calculated 411.1685, found 411.1690.
1 3

O. A. Ramírez-Marroquín et al.
7. Lim KG, Tonelli F, Li Z, Lu X, Bittman R, Pyne S, Pyne NJ
(2011) J Biol Chem 286:18633
25. Kabalka GW, Guchhait SK, Naravane A (2004) Tetrahedron Lett
45:4685
8. Parmar RG, Brown CR, Matsuda S, Willoughby JLS, Theile CS,
Charissé K, Foster DJ, Zlatev I, Jadhav V, Maier MA, Egli M,
Manoharan M, Rajeev KG (2018) J Med Chem 61:734
9. Elkayam E, Parmar R, Brown CR, Willoughby JL, Theile CS,
Manoharan M, Joshua-Tor L (2017) Nucleic Acids Res 45:3528
10. Parmar R, Willoughby JLS, Liu J, Foster DJ, Brigham B, Theile
CS, Charisse K, Akinc A, Guidry E, Pei Y, Strapps W, Cancilla
M, Stanton MG, Rajeev KG, Sepp-Lorenzino L, Manoharan
M, Meyers R, Maier MA, Jadhav V (2016) Chem Biol Chem
17:985
26. Barbour M, McNaughton M, Boomkamp SD, MacRitchie N, Jiang
HR, Pyne NG, Pyne S (2017) Br J Pharmacol 174:210
27. Abdou WM, Kamel AA, Shaddy AA (2010) Eur J Med Chem
45:5217
28. Al Quntar AAA, Gallily R, Katzavian G, Srebnik M (2007) Eur J
Pharmacol 556:9
29. Gabriel SE, Michaud K (2009) Arthritis Res Ther 11:3
30. Druzhilovskiy DS, Rudik AV, Filimonov DA, Lagunin AA, Glo-
riozova TA, Poroikov VV (2016) Russ Chem Bull 65:384
31. González-Trujano ME, Uribe-Figueroa G, Hidalgo-Figueroa S,
Martínez AN, Déciga-Campos M, Navarrete-Vázquez G (2018)
Biomed Pharmacother 101:553
11. Prakash TP, Kinberger GA, Murray HM, Chappell A, Riney S,
Graham MJ, Lima WF, Swayze EE, Seth PP (2016) Bioorg Med
Chem Lett 26:2817
32. Thangarasu P, Selvi ST, Manikandan A (2018) Bioorg Chem
81:468
12. Altenbuchner PT, Werz PDL, Schçppner P, Adams F, Kronast
A, Schwarzenböck C, Pçthig A, Jandl C, Haslbeck M, Rieger B
(2016) Chem Eur J 22:14576
33. Razzaghi-Asl N, Mirzayi S, Mahnam K, Sepehri S (2018) J Mol
Graph Model 83:138
13. Russell RGG (2011) Bone 49:2
34. Le TA, Truong HH, Thi TPN, Thi ND, To HT, Thi HP, Soldaten-
kov AT (2015) Mendeleev Commun 25:224
14. Drake MT, Clarke BL, Khosla S (2008) Mayo Clin Proc 83:1032
15. Nancollas GH, Tang R, Phipps RJ, Henneman Z, Gulde S, Wu
W, Mangood A, Russell RGG, Ebetino FH (2006) Bone 38:617
16. Baird LJ, Colomban C, Turner C, Teesdale-Spittle PH, Harvey
JE (2011) Org Biomol Chem 9:4432
35. Lipinski CA, Lombardo F, Dominy BW, Feeney P (1997) Adv
Drug Deliv Rev 23:3
36. Weaver WE, Whaley WM (1947) J Am Chem Soc 69:515
37. Adler P, Fadel A, Rabasso N (2014) Tetrahedron 70:4437
38. Pérez-González M, Gutiérrez-Rebolledo G, Yépez-Mulia L,
Rojas-Tomé I, Luna-Herrera J, Jiménez-Arellanes M (2017)
Biomed Pharmacother 89:89
17. Grison C, Comoy C, Chatenet D, Coutrot P (2002) J Organomet
Chem 662:83
18. Chen T, Zhao CQ, Han LB (2018) J Am Chem Soc 140:3139
19. Evano G, Gaumont AC, Alayrac C, Wrona IE, Giguere JR, Dela-
croix O, Bayle A, Jouvin K, Theunissen C, Gatignol J, Silvanus
AC (2014) Tetrahedron 70:1529
39. Wiemer AJ, Wiemer DF (2015) Top Curr Chem 360:115
40. Pertusati F, Serpi M, McGuigan C (2012) Antivir Chem Chem-
other 22:181
20. Ananikov VP, Khemchyan LL, Beletskaya IP (2009) Synlett 2375
21. Zhao CQ, Han LB, Goto M, Tanaka M (2008) Angew Chem Int
Ed 113:1983
41. Somogyi G, Buchwald P, Bodor N (2004) Pharmazie 59:378
42. Zhang D, Li J, Sun S, Huang C (2018) Toxicon 151:89
43. Gutiérrez-Rebolledo GA, Pérez-González MZ, Zamilpa A, Jimé-
nez-Arellanes MA (2017) Asian Pac J Trop Med 10:141
44. Florentino IF, Silva DPB, Silva DM, Cardoso CS, Moreira ALE,
Borges CL, Soares CMA, Galdino PM, Lião LM, Ghedini PC,
Menegatti R, Costa EA (2017) Nitric Oxide 69:35
45. Leung PSW, Teng Y, Toy PH (2010) Org Lett 12:4996
22. Jouvin K, Coste A, Bayle A, Legrand F, Karthikeyan G, Tadi-
parthi K, Evano G (2012) Organometallics 31:7933
23. Evano G, Tadiparthi K, Couty F (2011) Chem Commun 47:179
24. Lee HS, Pai SH, Liao WT, Yang XJ, Tsai FY (2017) RSC Adv
7:34293
1 3