Synthesis and biological evaluation of 4-hydroxy-5-oxo-2,5-dihydro-1: H -pyrrole-3-carboxamides and their zinc(II) complexes as candidate antidiabetic agents

Article abstract of DOI:10.1039/c7nj00970d

The newly designed 1-(arylmethyl)-2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole-3-carboxamides (1a-e) were synthesized by using N-tritylated acrylamide as a starting material, and their zinc(ii) complexes (10a-e) were readily prepared by simply mixing 1a-e and ZnSO₄ in the presence of LiOH. The aldose reductase (AR) inhibitory activity of 1a-e was evaluated to reveal important structural features for 2,5-dihydro-5-oxo-1H-pyrrole derivatives to exhibit high AR inhibitory activity. The in vitro insulin-mimetic activity of these complexes was evaluated from 50% inhibitory concentrations (IC₅₀) on free fatty acid (FFA) release from isolated rat adipocytes treated with epinephrine. Four out of the five newly synthesized complexes exhibited higher in vitro insulin-mimetic activities than ZnSO₄, a positive control. This study proved that the newly synthesized N₂O₂-coordination-type zinc(ii) complexes based on pyrrole-3-carboxamide derivatives (1a-d) are potential hypoglycemic agents.

Full text of DOI:10.1039/c7nj00970d

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Tamura, S. Kawano, Y. Yoshikawa, A. Kato, K. SASAKI and H. Yasui, New J. Chem., 2017, DOI:
10.1039/C7NJ00970D.
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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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DOI: 10.1039/C7NJ00970D
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ARTICLE
Synthesis and biological evaluation of 4-hydroxy-5-oxo-2,5-
dihydro-1H-pyrrole-3-carboxamides and their zinc(II) complexes
as candidate antidiabetic agents
Received 00th January 20xx,
Accepted 00th January 20xx
Ryota Saito,*a,b Moe Tamura,a Saya Kawano,a Yutaka Yoshikawa,c Akihiro Kato,a Kaname Sasaki,a
and Hiroyuki Yasuid
DOI: 10.1039/x0xx00000x
www.rsc.org/
The newly designed 1-(arylmethyl)-2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole-3-carboxamides (1a–e) were synthesized by
using N-tritylated acrylamide as a starting material, and their zinc(II) complexes (10a–e) were readily prepared by simply
mixing 1a–e and ZnSO4 in the presence of LiOH. Aldose reductase (AR) inhibitory activity of 1a–e were evaluated to reveal
important structural features for 2,5-dihydro-5-oxo-1H-pyrrole derivatives to exhibit high AR inhibitory activity. The in vitro
insulin–mimetic activity of these complexes was evaluated from 50% inhibitory concentrations (IC50) on free fatty acid (FFA)
release from isolated rat adipocytes treated with epinephrine. Four out of the five newly synthesized complexes exhibited
higher in vitro insulin-mimetic activities than ZnSO4, a positive control. This study proved that the newly synthesized N2O2-
coordination-type zinc(II) complexes based on pyrrole-3-carboxamide derivatives (1a–d) are potential hypoglycemic agents.
effects, including hypoglycemia, nausea, and convulsions.4 In
addition, continuous repression of the blood glucose levels
using pharmacotherapy can be misinterpreted by the body that
it no longer needs to synthesize insulin and thus, it quits
synthesizing the peptide, eventually forcing the patients to rely
on exogenous insulin injections.4
Introduction
Diabetes is characterized as a state of chronic hyperglycemia
that is caused by a decrease in the level and/or function of
insulin, which is a peptide hormone secreted by the
β cells of
the pancreatic islets of Langerhans to regulate the blood
glucose level.1 Diabetes is generally categorized into type 1 and
type 2,2 where type 2 diabetes patients comprise more than
90% of all diabetes patients.3
Type 1 diabetes results from an absolute shortage of insulin,
caused by destruction of the pancreatic
insulitis, or other related conditions, and hence it is not a
lifestyle-related disease.2 Type 1 diabetes develops in childhood
or early adulthood and, to date, the only available treatment
involves direct administration of insulin as insulin injections,
which generally lead to physical and emotional distress in
diabetes patients. In contrast, type 2 diabetes is a lifestyle-
related disease, caused by obesity, stress, insufficient exercise,
and aging.2 These factors can result in insufficient secretion and
impaired action of insulin. Primary treatments of type 2
diabetes involve exercise and diet management without
pharmacotherapeutic intervention. When these methods
become ineffective, administration of synthetic hypoglycemic
agents, such as sulfonylureas, meglitinides, and biguanides, is
initiated.4 However, some oral antidiabetic agents exhibit side
In addition, diabetes can lead to serious complications,
including neuropathy, retinopathy and nephropathy.2 The onset
of the complications is the most critical aspect for diabetics, and
is strongly related to the polyol metabolic pathway. Under the
circumstances of hyperglycemia, the amount of glucose taken
up into the cells increases and glucose overflows from the
normal glycolytic pathway into the polyol metabolic pathway to
consume the excess glucose.5, 6 During the first step of the
polyol metabolic pathway, glucose is reduced to sorbitol by
aldose reductase (AR) using NADPH as a coenzyme and then,
the resulting sorbitol is oxidized to fructose (Fig. 1).5, 6 Because
the second step is slower than the first one, sorbitol abnormally
accumulates in the cells, accompanied by excessive
consumption of NADPH. This state increases the intracellular
osmolality, inhibits the uptake of important molecules and ions
into the cell, and eventually causes macular edema. In addition,
excessive consumption of NADPH hinders the other pathways
that utilize NADPH as a coenzyme, leading to increased
oxidative stress that results in various complications.5, 7 Thus,
inhibition of AR, involved in the first step of the pathway, is
considered essential to prevent the onset or progression of
diabetes complications.8, 9 Based on these findings, it is highly
desirable to develop new antidiabetic agents, which not only
replace the subcutaneous insulin injections and the oral
hypoglycemic drugs but also simultaneously inhibit AR to
decrease the risk of diabetes complications.
β cells, autoimmune
a.Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-
8510 Japan. E-mail: saito@chem.sci.toho-u.ac.jp
b.Research Center for Materials with Integrated Properties, Toho University, 2-2-1
Miyama, Funabashi, Chiba 274-8510, Japan.
c. Department of Health, Sports and Nutrition, Kobe Women’s University, 4-7-2
Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan
d.Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical
University, Kyoto 607-8414, Japan.
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Fig.
2 Structure of ethyl 1-benzyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
carboxylate (EBPC).
View Article Online
DOI: 10.1039/C7NJ00970D
Results and Discussion
Synthesis
Fig. 1 Schematic presentation of the polyol pathway of glucose metabolism.
First, we synthesized 1a–e, the corresponding conjugate acids
of the ligands in the target zinc(II) complexes, using the same
reaction scheme as that used for synthesizing EBPC and its
derivatives.22 Briefly, conjugate addition of the benzyl amines,
Zinc(II) ion has received considerable attention by medicinal
chemists as a new diabetic agent since the insulin-mimetic
activity of zinc(II) ion was discovered by Coulston et al. in
1980.10 Numerous studies have been conducted during the last
two decades to develop new oral antidiabetic drugs based on
zinc(II) ion. For instance, oral administration of ZnCl2 to
streptozotocin-induced diabetic rats could decrease the high
blood glucose levels,11, 12 and zinc oxide nanoparticles improved
insulin levels in diabetic rats.13 Despite the promising potency,
the inorganic form of zinc(II) is poorly absorbed; this feature
necessitates higher dosages, which subsequently increase the
adverse drug reactions.10, 13 Thus, organic zinc(II) compounds
were used, which, in addition, exhibited enhanced therapeutic
effects.10 Thereafter, many zinc(II) complexes have been
developed and showed insulin-mimetic activity both in vitro and
in vivo.10, 14-21
4a
conjugate adducts, 5a
conjugate adducts, 5a
–
e
, toward acrylamide was conducted and the corresponding
, were obtained in good yield. The
, were sequentially reacted with diethyl
–
e
e
–
oxalate in the presence of sodium ethoxide (Scheme 1). When
5a was subjected to the reaction, the target amide, 1a, was
obtained accompanied by the formation of the ester, 6a. The
ratio of 1a
spectroscopy. Similar amide-ester transformations occurred
with 5b . Although determination of the product ratio and
purification of the esters were not fulfilled, it is obvious that this
side reaction decreased the yields of the desired amides, 1b
:
6a was determined as 1.3:1 by 1H-NMR
–
e
–e.
Recently, we have synthesized zinc(II) complexes using ethyl 1-
benzyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxylate
(EBPC, Fig. 2) and its derivatives as organic ligands. We showed,
for the first time, that they exhibited insulin-mimetic
activities.22 In addition, the ligands themselves have AR
inhibitory activity;23 thereby the complexes are potential
bifunctional chemotherapeutics for both diabetes and its
complications. The coordination mode of the EBPCs toward the
central zinc(II) ion was determined by means of X-ray
crystallographic analysis, which showed that the complex had a
planar trans-conformation, where the zinc(II) ion was
configured with the hydroxyl oxygen at the C3 and the ester
carbonyl oxygen at the C4 of the EBPCs.22 It is known that the
coordination mode of the ligands toward zinc(II) affects their
bioavailability and therapeutic potency.10,24 However, only the
Zn(O4) coordination mode has been investigated regarding the
Zn-EBPC system. Thus, in the present study, which is a part of
our ongoing project on the development of chemotherapeutics
based on the zinc(II) complexes of the EBPC derivatives, we
synthesized several new zinc(II) complexes, with Zn(N2O2)
coordination mode, using the new 4-hydroxy-5-oxo-2,5-
dihydro-1H-pyrrole-3-carboxamide derivatives as ligands. In
addition, we evaluated their insulin-mimetic activities to
investigate whether replacing the oxygen atoms of the EBPCs by
nitrogen atoms affected their insulin-mimetic activity or not.
The in vitro AR inhibitory activity of the new ligands was also
investigated.
Scheme 1 Synthetic route to amides 1a
transformation products.
–e accompanied by undesired amide-ester
This amide-ester transformation reaction was first considered
to occur when the surplus ethoxide attacks the amide carbonyl
carbon of the product. Thus, we carried out the same reaction
using 0.94 equivalent of sodium ethoxide (Scheme 2, eq.1).
However, ester formation was still observed. We also
investigated the effect of the reaction temperature on the
amide-ester transformation reaction. It was found that lowering
the reaction temperature resulted in not only a diminished yield
of the amide but also formation of the ester (Scheme 2, eq.2).
These results suggested that the excess amount of the base and
the reaction temperature did not influence the occurrence of
this side reaction.
OEt
N
O
O
OH
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DOI: 10.1039/C7NJ00970D
O
O
N
NH2
N
H
NH2
H
H
H
O
OEt
EtO
OEt
O
O
EtO
O
Scheme 2 Reaction of amide 5a with diethyl oxalate under various conditions.
H
H
N
NH
O
N
H
N
H
O
O
O
In order to investigate whether the amide groups in the starting
OEt
O
OEt
O
materials of the cyclocondensation reaction and the produced
amides incur the amide-ester transformation reaction with
sodium ethoxide or not, 5a and 1a were sequentially treated
with sodium ethoxide under the same conditions used for the
cyclocondensation reaction in Scheme 1 (Scheme 3). It was
found that the amides remained intact under these conditions,
which indicated that the amide-ester transformation reaction
did not proceed at the initial and final stages of the
cyclocondensation reaction (Scheme 3).
OEt
N
OEt
N
H
NH
H
O
O
O
O
A
NH2
H+
N
N
COOEt
H
COOEt
O
O
O
O
OEt
N
COOEt
O
OH
Scheme 4 Proposed reaction mechanism for the amide-ester transformation
during the synthesis of
1.
Scheme 3 Examination of the reactivity of amides 1a and 5a with sodium ethoxide.
Based on these results, we suggested a different reaction
mechanism for the amide-ester transformation, as illustrated in
Scheme 4, which involves the generation of a pyrrolidinetrione
intermediate (A) during the course of the reaction followed by
opening of the cyclic imide via the attack of ethoxide, and
subsequent cyclocondensation to form the amide-ester
transformation product.
Assuming that the amide-ester transformation reaction occurs
via this mechanism, bulking of the terminal amide is considered
to prevent the imide formation and consequently suppress the
amide-ester transformation reaction. Thus, we used a new
synthetic route, in which the N-tritylacrylamide, 7 25
employed as a Michael acceptor (Scheme 5). The reaction of
with benzylamines 4a gave the corresponding 8a , which
were subsequently reacted with diethyl oxalate in the presence
of sodium ethoxide to provide 9a in moderate to good yields.
,
was
7
–
e
–e
–e
It should be emphasized that no amide-ester transformation
product was observed. These results obviously supported the
suggested mechanism in Scheme 4. Compounds 9a
detritylated under acidic conditions to obtain the desired
products, 1a , in satisfactory yields.
–e were
–e
Scheme 5 Improved synthetic route to zinc(II) complexes 10a–e.
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Then, 1a
–
e
were reacted with zinc sulfate in the presence of inhibitory activity23, whereas the ester group at C3 was not
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DOI: 10.1039/C7NJ00970D
lithium hydroxide to afford the corresponding zinc(II) expected to affect the activity. However, their AR inhibitory
complexes, 10a–e (Scheme 5). In all cases, the reactions activity was greatly decreased compared to that of EBPC. Based
provided the desired products in good yields. The structures of on these findings, we suggested that not only the 4-hydroxyl
the complexes were assigned on the basis of their spectroscopic group but also the ester moiety at the 3-position was essential
1
data and combustion analysis. In H-NMR spectra, each ligand for the high AR inhibitory activity of the 2,5-dihydro-5-oxo-1H-
showed two broad peaks around 7 ppm, assignable to the non- pyrrole derivatives.
equivalent amide NH protons, while these signals disappeared
on complexation with zinc(II) ion, as exemplified by the spectra Table 1. The in vitro AR inhibitory activities of 1a–e
for 1b (Fig. 3). Some zinc(II)-amide complexes with the amide
Compounds
R1 R2 Inhibition rate
carbonyl oxygens coordinating with zinc(II) ion showed
discernible signals for the amide NH protons in their H-NMR
spectra.26, 27 Based on these facts, the coordination mode of the
ligands in 10a–e was determined as that the zinc(II) ion was
configured with the amide nitrogen and the hydroxide oxygen
at C3 position as depicted in Scheme 5.
1a
1b CH3
1c
H
H
H
H
H
16% at 30 µM
17% at 30 µM
23% at 32 µM
26% at 31 µM
R2
1
O
N
F
R1
NH2
O
1d Cl
R2
OH
1e
H
-
CF3 28% at 31 µM
-
-
epalrestata
EBPC
50% at 0.066 µM
50% at 0.20 µM
-
aEpalrestat was used as a reference compound.
In order to better interpret these findings, molecular docking
simulations of EBPC and 1a in the active site of the enzyme were
performed. In these experiments, the protein structure coded
as 2FZD in the Protein Data Bank (PDB) was employed because
this protein structure has been frequently used in the docking
studies of the AR inhibitors.30-33 The results of molecular
docking are depicted in Fig. 4. EBPC was predicted to interact
with His110 and Trp111, which form the active site of AR
together with Tyr48, via formation of hydrogen bonds with the
hydroxide anion at C4 and with the 5-oxo moiety, respectively,
besides the interaction of the carbonyl oxygen of the ester
group with Trp20, present in the vicinity of the active site (Fig.
4a). The active site of AR is frequently called an “anion-binding
pocket” because His110 is positively charged at physiological pH
and therefore attracts the negatively charged substituents.34
Hence, the predicted docking pose well explained the high AR
inhibitory activity of EBPC. On the contrary, compound 1a
formed hydrogen bonds with His110 and NADP+ through the C3-
carboxamide moiety, whereas the anionic substituent at C4 was
left out of the anion-binding active site (Fig. 4b). This suggested
that the binding of 1a to the active site was weaker than that of
EBPC, which rationally explained the observed decrease in the
AR inhibitory effects.
Fig. 3 1H NMR spectra (400 MHz at 25 °C in DMSO-d6) of selected 1b and its zinc(II)
complex 10b
.
In vitro aldose reductase inhibitory activities of 1a–e
The AR inhibitory activities of 1a were evaluated in vitro via
measurement of their inhibitory effects on the reduction of
–e
D,L-
glyceraldehyde, which was catalyzed by recombinant human AR
in the presence of NADPH as a cofactor.28 The assays were
based on spectrophotometric monitoring of NADPH
consumption, which was proven a reliable method.29 The results
are presented in Table 1.
Compounds 1a–e showed 16–28% inhibition at a concentration
of 30 µM, thus their half maximal inhibitory concentration (IC50)
values were not determined owing to their low efficacy. These
compounds were expected to exhibit the same inhibitory
activity as that of EBPC because they possess the hydroxyl group
at the C4-position, which was considered crucial for a high AR
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results of the assay are summarized in Table 2. All the
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DOI: 10.1039/C7NJ00970D
complexes, 10a–d, exhibited a slightly greater activity than zinc
sulfate, which was used as a positive control.
Fig.
5 The inhibitory effects of zinc(II) complexes, 10a–d, on epinephrine-
stimulated FFA release from rat adipocytes. The letter “b” stands for “blank” and
“c” for “control.”
Moreover, the activities of the studied complexes were the
same as those of the previously investigated zinc(II) complexes
based on EBPC (0.26–0.82 mM).22 Regarding the coordination
mode of the ligands toward zinc(II) ion, no significant
differences were observed between the IC50 values of the
studied complexes and those of the previously investigated
zinc(II) complexes with the corresponding esters. The present
complexes exhibited higher activity than ZnSO4, thereby they
are potential candidates for hypoglycemic agents.
Table 2. The in vitro insulin-mimetic activities of 10a–d
Zn complex
10a
IC50 (mM)
0.37
10b
0.36
10c
0.39
10d
ZnSO4
0.41
0.44†
† Data was taken from reference 17
Fig. 4 Depiction of the predicted docking poses for (a) EBPC and (b) 1a in the active site
of 2FZD. The inhibitors are depicted in green, and NADP+ in yellow.
Conclusions
To synthesize the 2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole-3-
carboxamide derivatives (1a ), the problem of amide-ester
Evaluation of the insulin-mimetic activity of the zinc(II)
complexes
–
e
transformation during the cyclocondensation step was
encountered. After careful examination, we figured out the
cause and mechanism of the amide-ester transformation
reaction. We were able to effectively obtain the desired amides
using an appropriate protecting group. This could help the
future synthesis of the series of the 2,5-dihydro-4-hydroxy-5-
oxo-1H-pyrrole derivatives. All the newly synthesized
complexes with Zn(N2O2) coordination mode demonstrated a
sufficient insulin-mimetic activity that allows their probable
classification as hypoglycemic agents because their IC50 values
were slightly higher than that of ZnSO4 and comparable to those
of the corresponding Zn(O4)-type complexes based on the EBPC
derivatives. These results provided useful information about
the structure-activity relationship of zinc(II) complexes based on
To evaluate the insulin-mimetic activity of the synthesized
zinc(II) complexes (10a–e) in vitro, we investigated the
inhibitory effects of the complexes on free fatty acid (FFA)
release from rat adipose cells stimulated with epinephrine. This
method was proven simple and reliable.14, 22, 35 As shown in Fig.
4, all complexes, except 10e, inhibited the FFA release in a dose-
dependent manner, which showed that complexes 10a–d
exhibited insulin-mimetic activity. Compound 10e was hardly
soluble in the assay medium, therefore, its inhibitory activity
could not be determined. The quantitative evaluation of the
inhibitory activity of 10a–d was carried out via calculation of the
IC50 value, which was defined as the concentration of the
complex that resulted in 50% inhibition of the FFA release. The
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2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole derivatives, which 36.7, 45.4, 54.0, 70.3, 126.9, 127.3, 128.0, 128.4, V1ie2w8A.7rti,cl1e2O8nl.in9e,
could aid the development of new chemotherapeutics for 139.2, 145.2, 171.7. IR (KBr) νmax/cm–1 3D4O55I: 1(0ν.N10-3H9)/,C37N2J70509(7ν0ND-
treating diabetes.
H), 3024–2853 (νC-H), 1664 (νC=O), 1551 (δN-H). MS (FAB+,
DTT/TG = 1/1) m/z 421 [M+H]+. HRMS (positive ESI) Calcd for
C29H29N2O+ [M+H]+: 421.2274. Found: 421.2296.
Acknowledgements
3-((4-Methylbenzyl)amino)-N-tritylpropanamide (8b
)
This research was supported by JSPS KAKENHI Grant Number
JP25410179. The partial financial support to RS by the MEXT-
Supported Program for the Strategic Research Foundation at
Private Universities at Toho University, 2012-2016 (S1201034),
is gratefully appreciated. YY and HY were financially supported,
in part, by a grant from the MEXT-Supported Program for the
Strategic Research Foundation at Private Universities at Kyoto
Pharmaceutical University, 2012-2016 (S1201008). The authors
would like to thank Editage (www.editage.jp) for English
language editing.
4-Methylbenzylamine (600 µl, 4.75 mmol) was added dropwise
to N-trithylacrylamide (1.01 g, 3.21 mmol) in chloroform (12 ml)
at r.t., and then the mixture was stirred at 60 °C for 2 days. After
evaporation of the solvent, the obtained yellow solid was
purified by column chromatography on silica gel (46–50 µm,
chloroform:ethyl acetate = 4:1, 1;1) to afford the product as
1
light yellow solid (1.26 g, 90%). Mp 159.5–160.0 °C. H NMR
(400 MHz, CDCl3) δ/ppm 2.34 (s, 3H), 2.39–2.44 (m, 2H), 2.91–
2.96 (m, 2H), 3.72 (s, 2H), 6.95 (d, J = 7.7 Hz, 2H), 7.04 (d, J = 7.7
Hz, 2H), 7.18–7.29 (m, 15H), 9.42 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ/ppm 21.2, 36.9, 45.3, 53.7, 70.3, 126.9, 127.9, 128.3,
128.9, 129.3, 136.2, 136.9, 145.3, 171.8. IR (KBr) νmax/cm–1 3455
(νN-H), 3240 (νN-H), 3060–2826 (νC-H), 1644 (νC=O), 1531 (δN-
H). MS (FAB+, DTT/TG = 1/1) m/z 435 [M+H]+. HRMS (positive
ESI) Calcd for C30H31N2O+ [M+H]+: 435.2431. Found: 435.2412.
Experimental
General
1H-NMR spectra were recorded using an ECP-400 spectrometer
(JEOL Ltd., Japan). Chemical shifts () are expressed in ppm
employing tetramethylsilane (TMS) or a non-deuterated solvent
as internal standards in the deuterated solvent used. Coupling
constants (J) were given in Hz. 13C-NMR spectra were recorded
using the ECP-400 spectrometer (JEOL Ltd., Japan). Chemical
shift multiplicities were expressed as s = singlet, d = doublet, t =
triplet, q = quintet, m = multiplet. Infrared (IR) spectra were
obtained using FT/IR-4100, FT/IR-660 plus, or FT/IR-460 plus
spectrophotometers (JASCO., Ltd, Japan). Fast-atom
bombardment (FAB) mass spectra were recorded using a JMS-
600-H mass spectrometer (JEOL Ltd., Japan). Xenon was used as
a bombardment gas, and all analyses were carried out in a
positive ion mode with the ionization energy and accelerating
voltage set at 70 eV and 3 kV, respectively. A mixture of
dithiothreitol (DTT) and α-thioglycerol (TG) (1:1) was used as a
liquid matrix.
3-((4-Fluorobenzyl)amino)-N-tritylpropanamide (8c
)
4-Fluorobenzylamine (600 µl, 4.84 mmol) was added dropwise
to N-trithylacrylamide (1.04 g, 3.33 mmol) in chloroform (12 ml)
at r.t., and then the mixture was stirred at 60 °C for 3 days. After
evaporation of the solvent, the obtained yellow solid was
purified by column chromatography on silica gel (46–50 µm,
chloroform:ethyl acetate = 4:1, 1;1) to afford the product as
colorless solid (1.22 g, 84%). Mp 173.8–174.0 °C. 1H NMR (400
MHz, CDCl3) δ/ppm 2.40–2.46 (m, 2H), 2.89–2.96 (m, 2H), 3.72
(s, 2H), 6.89 (t, J = 8.7, 2H), 6.96–7.03 (m, 2H), 7.17–7.29 (m,
15H), 9.14 (s, 1H). 13C NMR (100 MHz, CDCl3) δ/ppm 36.9, 45.3,
53.3, 70.4, 115.4 (d, 2JCF = 21 HZ), 127.0, 128.0, 128.9, 129.9 (d,
3JCF = 8 HZ), 135.0, 145.2, 162.1 (d, 1JCF = 246 HZ), 171.6. IR (KBr)
νmax/cm–1 3474 (
N-H), 3267 (νN-H), 3145–2901 (νC-H), 1660
(νC=O), 1545 (δN-H), 1218 (νC-F). MS (FAB+, DTT/TG = 1/1) m/z
439 [M+H]+. HRMS (positive ESI) Calcd for C29H28FN2O+ [M+H]+:
439.2180. Found: 439.2161.
All conventional chemicals used in the present study are
commercially available and were used as received. Column
chromatography was carried out on silica gel (particle size, 46–
50 μm; Fuji Silysia Chemical Ltd., Japan). Recombinant human
aldose reductase (AR), produced by ATGen Co., Ltd., was
purchased from Cosmo Bio Co., Ltd.
3-((4-Chlorobenzyl)amino)-N-tritylpropanamide (8d
)
4-Cholorobenzylamine (300 µl, 2.45 mmol) was added dropwise
to N-trithylacrylamide (501 mg, 1.60 mmol) in chloroform (3 ml)
at r.t., and then the mixture was stirred at 60 °C for 2 days. After
evaporation of the solvent, the obtained yellow solid was
purified by column chromatography on silica gel (46–50 µm,
chloroform:ethyl acetate = 1;1) to afford the product as
colorless solid (645 mg, 89%). Mp 177.0–177.5 °C. 1H NMR (400
MHz, CDCl3) δ/ppm 2.40–2.45 (m, 2H), 2.89–2.95 (m, 2H), 3.72
(s, 2H), 6.97 (d, J = 8.2 Hz, 2H), 7.14–7.31 (m, 17H), 9.06 (s, 1H).
13C NMR (100 MHz, CDCl3) δ/ppm 37.0, 45.3, 53.3, 70.4, 127.0,
128.0, 128.7, 128.9, 129.7, 133.0, 137.8, 145.1, 171.4. IR (KBr)
νmax/cm–1 3446 (νN-H), 3244 (νN-H), 3030–2828 (νC-H), 1643
(νC=O), 1530 (δN-H). MS (FAB+, DTT/TG = 1/1) m/z 455 [M+H]+.
HRMS (positive ESI) Calcd for C29H28ClN2O+ [M+H]+: 455.1885.
Found: 455.1871.
Synthesis
3-(Benzylamino)-N-tritylpropanamide (8a
)
Benzylamine (160 µl, 1.46 µmol) was added dropwise to N-
trithylacrylamide (309 mg, 985 µmol) in chloroform (4 ml) at r.t.,
and then the mixture was stirred at 60 °C for 2 days. After
evaporation of the solvent, the obtained yellow solid was
purified by column chromatography on silica gel (46–50 µm,
hexane:ethyl acetate = 9:1, chloroform:ethyl acetate = 1;1) to
afford the product as light yellow solid (299 mg, 72%). Mp
168.0–168.9 °C. 1H NMR (400 MHz, CDCl3) δ/ppm 2.40–2.4 (m,
2H), 2.91–2.97 (m, 2H), 3.74 (s, 2H), 7.04–7.09 (m, 2H), 7.19–
7.28 (m, 18H), 9.32 (s, 1H). 13C NMR (100 MHz, CDCl3) δ/ppm
6 | New J. Chem., 2017, 00, 1-3
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ARTICLE
(νC=O), 1541 (δN-H), 1254 (νC-O). HRMS (negativeVEieSwI)ACrtiacllecOdnflioner
–
–
DOI: 10.1039/C7NJ00970D
3-((3,5-Bis(trifluoromethyl)benzyl)amino)-N-tritylpropanamide
8e
3,5-Bis(trifluoromethyl)benzylamine (583 mg, 2.40 mmol) was 1-(4-Fluorobenzyl)-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-
added dropwise to N-trithylacrylamide (501 mg, 1.60 mmol) in pyrrole-3-carboxamide (9c
chloroform (3 ml) at r.t., and then the mixture was stirred at 60 To a mixture of 3-((4-fluorobenzyl)amino)-N-tritylpropanamide
°C for 9 days. After evaporation of the solvent, the obtained 8c: 1.17 g, 2.67 mmol) and diethyl oxalate (430 µl, 3.17 mmol)
C32H27N2O3 [M–H] : 487.2027. Found: 487.2043.
(
)
)
(
yellow solid was purified by column chromatography on silica was added ethanolic sodium ethoxide, prepared by dissolving
gel (46–50 µm, chloroform:ethyl acetate = 1;1) to afford the sodium (123 mg, 5.35 mmol) in dry ethanol (30 ml), and the
product as colorless solid (747 mg, 84%). Mp 143.0–144.0 °C. 1H mixture was stirring at 80 °C for 2 days. After cooling to room
NMR (400 MHz, CDCl3) δ/ppm 2.47–2.52 (m, 2H), 2.92–2.97 (m, temperature, the resulting precipitate was collected and then
2H), 3.86 (s, 2H), 3.98 (s, 1H), 7.18–7.29 (s, 15H), 7.69 (s, 2H), dissolved in water. The aqueous solution was acidified with 10%
7.77 (s, 1H), 7.89 (s, 1H). 13C NMR (100 MHz, CDCl3) δ/ppm 37.1, HCl to pH = 3. The resulting precipitate was collected and dried
3
1
45.5, 53.2, 70.6, 121.3 (q, JCF = 4 HZ), 123.4 (q, JCF = 274 HZ), in vacuo to afford the aimed compound as a beige solid. (1.24 g,
127.1, 128.0, 128.2 (q, 3JCF = 4 HZ), 128.7 131.8 (q, 2JCF = 33 HZ), 94%). Mp 167.5–168.5 °C. 1H NMR (400 MHz, CDCl3) δ/ppm 3.92
142.4, 144.9, 171.1. IR (KBr) νmax/cm–1 3438 (νN-H), 3240 (νN- (s, 2H), 4.60 (s, 2H), 7.00 (t, J = 8.6 Hz, 2H), 7.18–7.29 (m, 17H),
H), 2849-3029 (νC-H), 1644 (νC=O), 1532 (δN-H), 1281, 1134 8.25 (s, 1H), 10.42 (br. s, 1H). 13C NMR (100 MHz, CDCl3) δ/ppm
2
(νC-F). MS (FAB+, DTT/TG = 1/1) m/z 557 [M+H]+. HRMS (positive 46.7, 47.3, 70.6, 115.3, 115.8 (d, JCF = 22 HZ), 127.1, 128.1,
ESI) Calcd for C31H27F6N2O+ [M+H]+: 557.2022. Found: 557.2046. 128.8, 130.3 (d, 3JCF = 8 HZ), 131.6 (d, 4JCF = 4 HZ), 144.8, 147.9,
1
161.4, 162.7 (d, JCF = 248 HZ), 166.6. IR (KBr) νmax/cm–1 3518
1-Benzyl-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-pyrrole-3-
carboxamide (9a
(νN-H), 3364 (νO-H), 3060–2921 (νC-H), 1694, 1668 (νC=O),
1549 (δN-H), 1255 (νC-O). HRMS (negative ESI) Calcd for
)
To a mixture of 3-(benzylamino)-N-tritylpropanamide (8a: 201 C31H24FN2O3– [M–H]–: 491.1776. Found: 491.1755.
mg, 477 µmol) and diethyl oxalate (78.0 µl, 576 µmol) was
added ethanolic sodium ethoxide, prepared by dissolving 1-(4-Chlorobenzyl)-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-
sodium (21.9 mg, 953 µmol) in dry ethanol (4 ml), and the pyrrole-3-carboxamide (9d
mixture was stirring at 80 °C for 4h. After cooling to room To a mixture of 3-((4-chlorobenzyl)amino)-N-tritylpropanamide
temperature, the resulting precipitate was collected and then 8d: 306 mg, 671 µmol) and diethyl oxalate (110 µl, 738 µmol)
)
(
dissolved in water. The aqueous solution was acidified with 10% was added ethanolic sodium ethoxide, prepared by dissolving
HCl to pH = 3. The resulting precipitate was collected and dried sodium (30.9 mg, 1.34 mmol) in dry ethanol (6 ml), and the
in vacuo to afford the aimed compound as a beige solid. (223 mixture was stirring at 80 °C for 3h. After cooling to room
mg, 98%). Mp 186.5–1187.0 °C. 1H NMR (400 MHz, CDCl3) temperature, the resulting precipitate was collected and then
δ/ppm 3.92 (s, 2H), 4.62 (s, 2H), 7.18–7.33 (m, 20H), 8.26 (s, 1H). dissolved in water. The aqueous solution was acidified with 10%
13C NMR (100 MHz, CDCl3) δ/ppm 47.3, 47.5, 70.6, 114.9, 127.1, HCl to pH = 3. The resulting precipitate was collected and dried
128.1, 128.3, 128.5, 128.8, 129.1, 135.7, 144.9, 148.3, 161.6, in vacuo to afford the aimed compound as a beige solid. (214
166.6. IR (KBr) νmax/cm–1 3384 (νN-H), 3032 (νO-H), 2638 (νC-H), mg, 63%). Mp 184.2–184.5 °C. 1H NMR (400 MHz, CDCl3) δ/ppm
1668 (νC=O), 1521 (δN-H), 1240 (νC-O). HRMS (negative ESI) 3.92 (s, 2H), 4.60 (s, 2H), 7.16 (d, J = 8.1 Hz, 2H), 7.23–7.29 (m,
Calcd for C31H25N2O3– [M–H]–: 473.1871. Found: 473.1901.
17H), 8.16 (s, 1H), 10.11 (br. s, 1H). 13C NMR (100 MHz, CDCl3)
δ/ppm 46.8, 47.3, 70.7, 115.0, 127.2, 128.1, 128.8, 129.3, 129.8,
134.2, 134.3, 144.8, 148.0, 161.5, 166.4. IR (KBr) νmax/cm–1 3527
(νN-H), 3392 (νO-H), 3025–2941 (νC-H), 1664 (νC=O), 1532 (δN-
4-Hydroxy-1-(4-methylbenzyl)-5-oxo-N-trityl-2,5-dihydro-1H-
pyrrole-3-carboxamide (9b
)
–
To mixture of
a
3-((4-methylbenzyl)amino)-N- H), 1252 (νC-O). HRMS (negative ESI) Calcd for C31H24ClN2O3
tritylpropanamide (8b: 1.14 g, 2.61 mmol) and diethyl oxalate [M–H]–: 507.1481. Found: 507.1462.
(425 µl, 3.14 mmol) was added ethanolic sodium ethoxide,
prepared by dissolving sodium (120 mg, 5.22 mmol) in dry 1-(3,5-Bis(trifluoromethyl)benzyl)-4-hydroxy-5-oxo-N-trityl-2,5-
ethanol (30 ml), and the mixture was stirring at 80 °C for 2 days. dihydro-1H-pyrrole-3-carboxamide (9e
)
After cooling to room temperature, the resulting precipitate To a mixture of 3-((3,5-bis(trifluoromethyl)benzyl)amino)-N-
was collected and then dissolved in water. The aqueous solution tritylpropanamide (8e: 354 mg, 6.36 µmol) and diethyl oxalate
was acidified with 10% HCl to pH = 3. The resulting precipitate (100 µl, 738 µmol) was added ethanolic sodium ethoxide,
was collected and dried in vacuo to afford the aimed compound prepared by dissolving sodium (29.2 mg, 1.27 mmol) in dry
as a beige solid. (1.26 g, 99%). Mp 175.0–176.0 °C. 1H NMR (400 ethanol (6 ml), and the mixture was stirring at 80 °C for 16 h.
MHz, CDCl3) δ/ppm 2.33 (s, 3H), 3.88 (s, 2H), 4.58 (s, 2H), 7.12 After cooling to room temperature, the resulting precipitate
(s, 4H), 7.21–7.30 (m, 15H), 7.94 (br. s, 1H). 13C NMR (100 MHz, was collected and then dissolved in water. The aqueous solution
CDCl3) δ/ppm 21.3, 47.1, 47.2, 70.6, 114.9, 127.1, 128.0, 128.5, was acidified with 10% HCl to pH = 3. The resulting precipitate
128.8, 129.8, 132.7, 138.1, 144.9, 148.2, 161.6, 166.3. IR (KBr) was collected and dried in vacuo to afford the aimed compound
1
νmax/cm–1 3526 (νN-H), 3367 (νO-H), 3056–2917 (νC-H), 1669 as a beige solid. (287 mg, 74%). Mp 178.0–178.8 °C. H NMR
(400 MHz, CDCl3) δ/ppm 3.95 (s, 2H), 4.74 (s, 2H), 7.20–7.26 (m,
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ARTICLE
15H), 7.68 (s, 2H), 7.85 (s, 1H), 8.24 (s, 1H), 10.20 (br. s, 1H). 13
NMR (100 MHz, CDCl3) δ/ppm 46.6, 47.5, 70.7, 115.8, 122.5 (q, 1-(4-Chlorobenzyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
3JCF = 4 HZ), 123.1 (q, 1JCF = 274 HZ), 127.2, 128.1, 128.5 (q, 3JCF carboxamide (1d
NJC
C
View Article Online
DOI: 10.1039/C7NJ00970D
)
= 3 HZ), 128.7, 132.6 (q, 2JCF = 34 HZ), 138.4, 144.7, 147.6, 161.3, 1-(4-Chlorobenzyl)-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-
166.8. IR (KBr) νmax/cm–1 3391 (νN-H), 2928-3060 (νO-H, νC-H), pyrrole-3-carboxamide (9d: 119 mg, 234 µmol) in 40% TFA in
1671 (νC=O), 1539 (δN-H), 1280 (νC-O), 1247, 1134 (νC-F). CH2Cl2 (2 ml) was stirried at r.t. for 30 min. The resulting solution
HRMS (negative ESI) Calcd for C33H23F6N2O3– [M–H]–: 609.1618. was epaporated and washing with CHCl3 to afford the product
1
Found: 609.1614.
as a colorless solid (41.6 mg, 67%). Mp 212 °C (decomp.). H
NMR (400 MHz, DMSO-d6) δ/ppm 3.82 (s, 2H), 4.58 (s, 2H), 6.97
(br, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.48 (br,
1H). 13C NMR (100 MHz, DMSO-d6) δ/ppm 45.2, 46.1, 112.1,
128.7, 129.6, 132.1, 136.1, 150.7, 164.6, 165.1. IR (KBr) νmax/cm–
1-Benzyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
carboxamide (1a
)
1-Benzyl-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-pyrrole-3-
carboxamide (9a: 102 mg, 215 µmol) in 40% TFA in CH2Cl2 (2 ml)
1
3464 (νasN-H), 3334 (νsN-H), 1694, 1672 (νC=O), 1456 (νC-N),
was stirried at r.t. for 30 min. The resulting solution was 1325, 1251 (νC-O). MS (FAB+, DTT/TG = 1/1) m/z 267 [M+H]+.
epaporated and washing with CHCl3 to afford the product as a Anal. Calcd. for C12H11ClN2O3: C, 54.05; H, 4.16; N, 10.50. Fond:
1
colorless solid (41.9 mg, 84%). Mp 214 °C (decomp.). H NMR C, 53.85; H, 4.07; N, 10.59.
(400 MHz, DMSO-d6) δ/ppm 3.80 (s, 2H), 4.59 (s, 2H), 6.95 (br,
1H), 7.22–7.38 (m, 5H), 7.45 (br, 1H). 13C NMR (100 MHz, DMSO- 1-(3,5-Bis(trifluoromethyl)benzyl)-4-hydroxy-5-oxo-2,5-dihydro-
d6) δ/ppm 45.8, 46.0, 111.9, 127.5, 127.7, 128.7, 137.0, 150.8, 1H-pyrrole-3-carboxamide (1e
)
164.6, 165.0. IR (KBr) νmax/cm–1 3434 (νasN-H), 3158 (νsN-H), 1-(3,5-Bis(trifluoromethyl)benzyl)-4-hydroxy-5-oxo-N-trityl-2,5-
1688, 1670 (νC=O), 1457 (νC-N), 1332, 1259 (νC-O). MS (FAB+, dihydro-1H-pyrrole-3-carboxamide (9e: 119 mg, 234 µmol) in
DTT/TG = 1/1) m/z 233 [M+H]+. Anal. Calcd. for C12H12N2O3: C, 40% TFA in CH2Cl2 (2 ml) was stirried at r.t. for 30 min. The
62.06; H, 5.21; N, 12.06. Fond: C, 61.50; H, 5.09; N, 11.65.
resulting solution was epaporated and washing with CHCl3 to
afford the product as a colorless solid (34.1 mg, 47%). Mp 212
°C (decomp.). 1H NMR (400 MHz, DMSO-d6) δ/ppm 3.92 (s, 2H),
4.78 (s, 2H), 6.96 (br, 1H), 7.47 (br, 1H) 7.96 (s, 2H), 8.05 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ/ppm 45.3, 46.6, 112.7, 121.4
4-Hydroxy-1-(4-methylbenzyl)-5-oxo-2,5-dihydro-1H-pyrrole-3-
carboxamide (1b
)
4-Hydroxy-1-(4-methylbenzyl)-5-oxo-N-trityl-2,5-dihydro-1H-
pyrrole-3-carboxamide (9b: 102 mg, 208 µmol) in 40% TFA in (q, JCF = 4 HZ), 123.3 (q, JCF = 273 HZ), 128.8 (q, JCF = 3 HZ),
3
1
3
2
CH2Cl2 (2 ml) was stirried at r.t. for 30 min. The resulting solution 130.5 (q, JCF = 33 HZ), 141.0, 150.7, 164.8, 165.8. IR (KBr)
was epaporated and washing with CHCl3 to afford the product νmax/cm–1 3461 (νasN-H), 3307 (νsN-H), 1684 (νC=O), 1382 (νC-
as a colorless solid (35.7 mg, 70%). Mp 202 °C (decomp.). H N), 1282 (νC-F), 1173, 1129 (νC-O). MS (FAB+, DTT/TG = 1/1) m/z
1
NMR (400 MHz, DMSO-d6) δ/ppm 2.28 (s, 1H), 3.77 (s, 2H), 4.54 369 [M+H]+. Anal. Calcd. for C14H10F6N2O3: C, 45.66; H, 2.74; N,
(s, 2H), 6.96 (br, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 7.61. Fond: C, 44.87; H, 3.21; N, 7.65.
2H), 7.44 (br, 1H). 13C NMR (100 MHz, DMSO-d6) δ/ppm 20.7,
45.6, 45.8, 111.8, 127.8, 129.3, 133.9, 136.7, 150.9, 164.7, Bis(1-benzyl-3-carbamoyl-2,5-dihydro-5-oxo-1H-pyrrol-4-
164.9. IR (KBr) νmax/cm–1 3455 (νasN-H), 3196 (νsN-H), 2921 (νC- olato)zinc(II) (10a
)
H), 1673, 1638 (νC=O), 1501 (νC-N), 1332, 1252 (νC-O). MS 1-Benzyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
(FAB+, DTT/TG = 1/1) m/z 247 [M+H]+. Anal. Calcd. for carboxamide (1a: 107 mg, 462 µmol) and lithium hydroxide
C13H14N2O3: C, 63.40; H, 5.73; N, 11.38. Fond: C, 63.15; H, 5.69; monohydrate (33.5 mg, 798 µmol) were dissolved in water/THF
N, 11.19.
mixture solution (1:1, 4 ml), and then 1M ZnSO4 aq. (220 µl, 220
µmol) was added to the solution. The mixture was stirred for 2
hours. The resulting precipitate was collected by suction
filtration, and dried in vacuo to afford the aimed compound as
1-(4-Fluorobenzyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
carboxamide (1c
1-(4-Fluorobenzyl)-4-hydroxy-5-oxo-N-trityl-2,5-dihydro-1H-
)
1
a colorless solid (91.4 mg, 79%). H NMR (400 MHz, DMSO-d6)
pyrrole-3-carboxamide (9c: 74.2 mg, 151 µmol) in 40% TFA in δ/ppm 3.75 (s, 4H), 4.62 (s, 4H), 7.19–7.23 (m, 4H), 7.26–7.39
CH2Cl2 (1.5 ml) was stirried at r.t. for 30 min. The resulting (m, 6H). IR (KBr) νmax/cm–1 3397 (νasN-H), 3208 (νsN-H), 1655,
solution was epaporated and washing with CHCl3 to afford the 1624 (νC=O), 1339, 1261 (νC-O). Anal. Calcd. for
product as a colorless solid (22.8 mg, 60%). Mp 202 °C C24H22N4O6Zn•2.0H2O: C, 51.12; H, 4.65; N, 9.94. Fond: C, 51.04;
1
(decomp.). H NMR (400 MHz, DMSO-d6) δ/ppm 3.81 (s, 2H), H, 4.62; N, 9.64.
4.58 (s, 2H), 6.95 (br, 1H), 7.18 (td, J = 8.8, 3.0 Hz, 2H), 7.26–7.31
(m, 2H), 7.45 (br, 1H). 13C NMR (100 MHz, DMSO-d6) δ/ppm Bis{3-Carbamoyl-2,5-dihydro-1-(4-methylbenzyl)-5-oxo-1H-
2
3
45.1, 45.9, 112.0, 115.5 (d, JCF = 21 HZ). 129.9 (d, JCF = 8 HZ), pyrrol-4-olato}zinc(II) (10b
)
4
1
133.22 (d, JCF = 3 HZ), 150.7, 161.5 (d, JCF = 243 HZ), 164.6, 4-Hydroxy-1-(4-methylbenzyl)-5-oxo-2,5-dihydro-1H-pyrrole-3-
165.0. IR (KBr) νmax/cm–1 3476 (νasN-H), 3315 (νsN-H), 1682, carboxamide (1b: 83.7 mg, 340 µmol) and lithium hydroxide
1634 (νC=O), 1501 (νC-N), 1337, 1252 (νC-O). MS (FAB+, DTT/TG monohydrate (23.6 mg, 562 µmol) were dissolved in water/THF
= 1/1) m/z 251 [M+H]+. Anal. Calcd. for C12H11FN2O3: C, 57.60; mixture solution (1:1, 3 ml), and then 1M ZnSO4 aq. (170 µl, 170
H, 4.43; N, 11.20. Fond: C, 57.52; H, 4.39; N, 11.20.
µmol) was added to the solution. The mixture was stirred for 2
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ARTICLE
hours. The resulting precipitate was collected by suction Molecular docking
View Article Online
DOI: 10.1039/C7NJ00970D
filtration, and dried in vacuo to afford the aimed compound as The optimized structures and atomic charges of the selected
a colorless solid (99.4 mg, quant.). 1H NMR (400 MHz, DMSO-d6) ligands were estimated using the semi-empirical PM7
δ/ppm 2.28 (s, 6H), 3.73 (s, 4H), 4.56 (s, 4H), 7.10 (d, J = 7.8 Hz, calculation method,36 prior to conducting docking simulations.
4H), 7.16 (d, J = 7.8 Hz, 4H). IR (KBr) νmax/cm–1 3399 (νasN-H), All docking experiments were carried out using GOLD Suite
3220 (νsN-H), 1655, 1625 (νC=O), 1336, 1260 (νC-O). Anal. Calcd. software package (ver. 5.2) obtained from the Cambridge
for C26H26N4O6Zn•1.2H2O: C, 54.07; H, 4.96; N, 9.70. Fond: C, Crystallographic Data Centre (CCDC).37 ChemPLP scoring
54.16; H, 4.89; N, 9.64.
function38 was used for pose prediction, and the obtained poses
were rescored using GOLDscore.39 The best pose for each ligand
was determined by considering the results obtained using both
scoring functions. All docking results were visualized by Hermes
software package (ver. 1.6) provided by CCDC.40 All calculations
Bis{3-Carbamoyl-1-(4-fluorobenzyl)-2,5-dihydro-5-oxo-1H-
pyrrol-4-olato}zinc(II) (10c
1-(4-Fluorobenzyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
)
carboxamide (1c: 86.7 mg, 346 µmol) and lithium hydroxide were conducted using a PC with an Intel CoreTM i3-3220
monohydrate (17.8 mg, 424 µmol) were dissolved in water/THF processor (3.30 GHz) and 8GB RAM.
mixture solution (1:1, 3 ml), and then 1M ZnSO4 aq. (170 µl, 170
µmol) was added to the solution. The mixture was stirred for 2 In vitro insulin-mimetic activity16, 17
hours. The resulting precipitate was collected by suction Male Wistar rats were sacrificed under anesthesia with ether.
filtration, and dried in vacuo to afford the aimed compound as The adipose tissues were removed, chopped with scissors, and
1
a colorless solid (95.6 mg, 97%). H NMR (400 MHz, DMSO-d6) digested with collagenase for 60 min at 37 °C in Krebs-Ringer
δ/ppm 3.75 (s, 4H), 4.60 (s, 4H), 7.15–7.21 (m, 4H), 7.23–7.29 bicarbonate buffer (120 mM NaCl, 1.27 mM CaCl2, 1.2 mM
(m, 4H). IR (KBr) νmax/cm–1 3399 (νasN-H), 3216 (νsN-H), 1651, MgSO4, 4.75 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3, pH 7.4)
1624 (νC=O), 1337, 1261 (νC-O). Anal. Calcd. for containing 2% bovine serum albumin. The adipocytes were then
C24H20F2N4O6Zn•1.8H2O: C, 48.35; H, 3.99; N, 9.40. Fond: C, separated from the undigested tissues and washed with buffer
48.29; H, 4.16; N, 9.23.
three times. The isolated adipocyte solutions were pre-
incubated at 37 °C for 30 min with various concentrations of the
zinc(II) complexes or ZnSO4 in saline containing 5 mM glucose.
Then, a solution of epinephrine was added to the reaction
mixtures (final concentration: 10 mM) and the resulting
Bis{3-carbamoyl-1-(4-chlorobenzyl)-2,5-dihydro-5-oxo-1H-
pyrrol-4-olato}zinc(II) (10d
1-(4-Chlorobenzyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-
)
carboxamide (1d: 73.7 mg, 276 µmol) and lithium hydroxide solutions were incubated at 37 °C for 3 h. The reaction was
monohydrate (27.6 mg, 657 µmol) were dissolved in water/THF stopped by soaking in ice water and the mixtures were
mixture solution (1:1, 3 ml), and then 1M ZnSO4 aq. (140 µl, 140 centrifuged at 3000 rpm (600 g) at 4 °C for 10 min. The level of
µmol) was added to the solution. The mixture was stirred for 2 FFA in the extracellular fluid was determined using an FFA kit
hours. The resulting precipitate was collected by suction (NEFA C-test Wako, Wako Pure Chemicals, Osaka, Japan).
filtration, and dried in vacuo to afford the aimed compound as All animal experiments were approved by the Experimental
1
a colorless solid (58.0 mg, 70%). H NMR (400 MHz, DMSO-d6) Animal Research of Kyoto Pharmaceutical University (KPU) and
δ/ppm 3.75 (s, 4H), 4.61 (s, 4H), 7.23 (d, J = 8.2 Hz, 4H), 7.41 (d, were performed according to the Guideline for Animal
J = 8.2 Hz, 4H) . IR (KBr) νmax/cm–1 3398 (νasN-H), 3216 (νsN-H), Experimentation of KPU.
1651, 1625 (νC=O), 1338, 1262 (νC-O). Anal. Calcd. for
C24H20Cl2N4O6Zn•1.5H2O: C, 46.21; H, 3.72; N, 8.98. Fond: C,
46.27; H, 3.81; N, 8.61.
Notes and references
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