Design, synthesis, and biological evaluation of 1,4-diaryl-1,4- dihydropyrazines as novel 11-βHSD1 inhibitors

Article abstract of DOI:10.1248/bpb.b14-00070

The inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has demonstrated potential for the treatment of various components of metabolic syndrome. In this study, a series of 1,4-diaryl-1,4-dihydropyrazines were designed as inhibitors of 11β-HSD1 based on the structure-activity relationship of known 11β-HSD1 inhibitors through docking simulations. The docking simulation results supported the initial pharmacophore hypothesis: the docking results of the known inhibitors with 11β-HSD1 suggested a similar interaction of 1,4-diaryl-1,4- dihydropyrazines with the catalytic site of 11β- HSD1. Twelve of these compounds were synthesized through the cyclization of N, N-dialkylanilines with anilines, and their structures were determined by 1H-NMR, 13C- NMR, high resolution (HR)-MS, and single-crystal X-ray diffraction. The inhibitory activities of these compounds against human 11β-HSD1 were investigated in vitro through a scintillation proximity assay using microsomes containing 11β- HSD1.

Full text of DOI:10.1248/bpb.b14-00070

840
Regular Article
Biol. Pharm. Bull. 37(5) 840–846 (2014)
Vol. 37, No. 5
Design, Synthesis, and Biological Evaluation of 1,4-Diaryl-1,4-
dihydropyrazines as Novel 11β-HSD1 Inhibitors
Xiaowei Duan, Hongxing Xin, and Hong Yan*
College of Life Science and Bio-engineering, Beijing University of Technology; Pingleyuan Street No. 100, Chaoyang
District, Beijing 100124, P.R. China.
Received January 22, 2014; accepted February 20, 2014
The inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has demonstrated potential for
the treatment of various components of metabolic syndrome. In this study, a series of 1,4-diaryl-1,4-dihydro-
pyrazines were designed as inhibitors of 11β-HSD1 based on the structure–activity relationship of known
11β-HSD1 inhibitors through docking simulations. The docking simulation results supported the initial phar-
macophore hypothesis: the docking results of the known inhibitors with 11β-HSD1 suggested a similar in-
teraction of 1,4-diaryl-1,4-dihydropyrazines with the catalytic site of 11β-HSD1. Twelve of these compounds
were synthesized through the cyclization of N,N-dialkylanilines with anilines, and their structures were de-
1
termined by H-NMR, 13C-NMR, high resolution (HR)-MS, and single-crystal X-ray diffraction. The inhibi-
tory activities of these compounds against human 11β-HSD1 were investigated in vitro through a scintillation
proximity assay using microsomes containing 11β-HSD1.
Key words 1,4-diaryl-1,4-dihydropyrazine; synthesis; structure–activity relationship; 11β-hydroxysteroid
dehydrogenase type 1 (11β-HSD1) inhibitor
11β-Hydroxysteroid dehydrogenase type
1 (11β-HSD1)
is a reduced nicotinamide adenine dinucleotide phosphate
(NADPH)-dependent reductase responsible for catalyzing the
conversion of inactive cortisone into active glucocorticoid
cortisol.1) Excessive levels of glucocorticoids can cause meta-
bolic syndrome, including insulin resistance, visceral obesity,
and type 2 diabetes.2–5) Thus, 11β-HSD1 is hypothesized to
be critical for the development of metabolic syndrome.6) In
the past few years, researchers have designed and synthesized
a large number of 11β-HSD1 inhibitors, such as arylsulfonyl
piperazine 1, benzenesulfonamide 2, and diaryl sulfone 37–10)
(Fig. 1).
Based on the molecular modeling of the interactions
between the known inhibitors and 11β-HSD1, the develop-
ment of small-molecule inhibitors of 11β-HSD1 was initiated
with 1,4-dihydropyrazines. The interaction of the designed
1,4-diaryl-1,4-dihydropyrazines (4) with the catalytic site
of 11β-HSD1 was similar to that of the known inhibitors
(Fig. 2). The molecular structure of 4 can fold at an angle
of 107.18–113.15° into a V shape and has the potential to fit
into the enzyme’s V-shaped hydrophobic pocket. To test this
hypothesis, a series of 4 was synthesized through the cycli-
zation of N,N-dialkyl aniline with aniline in the presence of
catalytic hydrochloride (Chart 1). The inhibition of 11β-HSD1
by the enzymatic activities of 4 was determined through a
scintillation proximity assay using microsomes containing
11β-HSD1.11,12)
Fig. 1. Representative 11β-HSD1 Inhibitors
Fig. 2. Structure of 1,4-Diaryl-1,4-dihydropyrazine 4
and double-bonded oxygen in the middle of the structure, ad-
opted a V-shaped crystal structure in the docking models. A
detailed examination of the inhibitor binding site of 11β-HSD1
RESULTS AND DISCUSSION
Structure Designs Based on the X-ray crystal struc- with 1, 2, and 3 showed that the hydrogen bond donated by
ture of 11β-HSD1-inhibitor complexes (PDB code: 3H6K),13) sulfonyl oxygen with Tyr183 and the enzyme’s V-shaped
AutoDock 4.0 (Scripps Research Institute, La Jolla, CA, hydrophobic pockets, which contain residues 170 to 220, are
U.S.A.) was used to simulate the known inhibitors (1–3) and 4 critical for the inhibitory effect in the catalytic cycle. For
with 11β-HSD1. As illustrated in Fig. 3, the known inhibitors, example, the docking models of 1, 2, and 3 with 11β-HSD1
which contain two hydrophobic groups at the two terminals reveal that the amido hydrogen of Tyr183 donates a hydrogen
bond to the sulfonyl oxygen and the molecules are folded at an
angle of 100.71°, 100.11° and 106.11° into a V shaped to match
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: hongyan@bjut.edu.cn
© 2014 The Pharmaceutical Society of Japan

May 2014
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Chart 1. Synthesis of 1,4-Diaryl-1,4-dihydropyrazines 4
Fig. 3. The Binding Mode of 1, 2, 3, and 4-22 to Human 11β-HSD1 (PDB Code: 3H6K)
The amino acid residues forming hydrogen bonds with molecules are shown in green, the hydrogen bond in red dashed.
the cavity of the enzyme’s active site. In these cases, sulfonyls stituent groups to the phenyl substituent and docked with
are presumed to donate hydrogen bonds to the catalytic site. 11β-HSD1. The docking results are shown in Table 1.
To better fit into the enzymatic active site, these compounds
adjust their configurations to a V shape with an angle that capacity with 11β-HSD1 compared with the known inhibitors;
ranges from 100° to 115°.
in fact, the free energy values ΔG and inhibition constant
As shown in Table 1, compounds 4 exhibit a better binding
The docking of 1,4-diphenyl-dihydropyrazine 4-22 with values Ki of most compounds 4 are between −8.48 and
11β-HSD1 using the same method used for the docking of 1, −5.49kcal/mol and between 0.605 and 131.72µM, respectively.
2 and 3 revealed that the carbonyl oxygen of the ester of 4-22 The binding capacities of the V-shaped 4 were similar to
accepts a hydrogen bond from the amides of Tyr183, as was those obtained from the re-docking of 1, 2, and 3 (ΔG=−9.25,
observed with the known inhibitors. The hydrophobic aryl at −6.73, and −6.10kcal/mol, Ki=0.166, 11.63, and 33.88µM, re-
the N1 and N4 positions of the pyrazine ring can be modu- spectively). The hydrophilic and the steric effect of the substit-
lated to fit the V-shaped hydrophobic pocket of the enzyme. uents (R1 and R2) were speculated to affect the binding capac-
The stacked plots between 1,4-diphenyl-1,4-dihydropyrazines ity. The docking results obtained with hydrophilic substituents
and the known inhibitors are very similar.
are better than those obtained with hydrophobic substituents,
More than 60 of the 1,4-diaryl-1,4-dihydropyrazines (4) such as 4-64 (R1=4-COOH, R2=4-OH, ΔG=−7.81kcal/mol,
were grown in silico through the addition of different sub- Ki=1.88µM) and 4-31 (R1=4-CF3, R2=4-CF3, ΔG=−5.29kcal/

842
Vol. 37, No. 5
Table 1. Molecular Docking Results of 4 and the Known Inhibitors with
11β-HSD1
mol, Ki=131.73 µM). If the R is a small group, such as H, 4-Cl,
and 4-CH3, the values of Ki are between 0.65 and 19.04µM and
similar to those of the known inhibitors, as was observed with
4-20 (R1=4-CH3, R2=4-Cl, ΔG=−7.57 kcal/mol, Ki=2.83µM).
If R is a large group, such as 4-CO2CH2CH3, the values of Ki
are between 12.56 and 68.60µM and slightly larger than those
of the known inhibitors, as was observed with 4-58 (R1=4-
COOC2H5, R2=4-OCH3, ΔG=−5.80kcal/mol, Ki=56.48µM),
which might explain why the ethoxycarbonyl group is too
large to fit the V-shaped cavity of 11β-HSD1.
Synthesis of 1,4-Diaryl-1,4-dihydropyrazine Based on
previously reported reaction conditions,14) the 1,4-diaryl-1,4-
dihydropyrazines 4 were synthesized through the cyclization
reactions of N,N-dialkyl-anilines 5 with anilines, as depicted
in Chart 1. The intermediates 5 were prepared by the N-
alkylation of aniline with ethyl 2-diazo acetoacetate in the
presence of catalytic rhodium acetate dimer. Under a nitro-
gen atmosphere, intermediates 5 were obtained at acceptable
yields (49–69%). It is worth mentioning that an amount of by-
product is generated by the oxidation of some intermediates
that are not isolated from air.15) The synthesis of 4-aryl-1,4-
dihydropyrazine reported by Chorvat and Rorig14) and im-
proved by Sit et al.16) and He et al.15) was used for the cycliza-
tion of N,N-bisalkylated anilines 5 with ammonium acetate.
To determine the synthesis mechanism of 1,4-diaryl-1,4-dihy-
dropyrazines 4, 4-1 was used as a reactant in a model reaction
(Chart 1). The cyclization of N,N-bisalkylated anilines 5 with
anilines yielded 4-1 at markedly lower yields. The use of a
proton or Lewis acid as the catalyst produced 4-1 at higher
yields (Table 2). The catalyst amount was 0.2–0.4mol% based
on the synthesis of 1,4-dihydropyridines.17)
ΔGa)
(kcal/mol)
Compound
R1
R2
Kib) (µM)
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
H
H
H
H
H
H
H
H
H
4-Cl
4-OH
4-CH3
4-CF3
4-OCH3
4-CH2OH
4-COOH
4-COOC2H5
H
4-Cl
4-CH3
4-CF3
4-OCH3
4-CH2OH
4-COOH
4-COOC2H5
4-OH
−6.95
−7.16
−6.94
−7.07
−6.19
−6.56
−6.44
−7.54
−5.73
−6.44
−6.89
−6.81
−6.23
−6.81
−6.64
−7.77
−6.25
−7.15
−7.00
−7.57
−7.26
−7.08
−6.34
−6.51
−6.75
−6.16
−5.82
−5.84
−5.63
−5.68
−5.29
−5.49
−5.19
−6.79
−5.64
−7.78
−7.32
−7.03
−6.04
−7.11
−6.56
−7.45
−5.94
−8.01
−8.03
−7.81
−8.48
−5.65
−6.55
−8.08
−7.81
−6.24
−6.38
−6.00
−5.68
−6.19
−6.09
−5.80
−5.92
−6.11
−6.74
−9.25
−6.73
−6.10
8.02
5.68
8.14
6.56
28.98
15.64
19.04
2.95
63.24
19.04
8.90
10.18
27.10
10.17
13.59
2.02
26.26
5.79
7.38
2.83
4.79
4-9
H
4-10
4-11
4-12
4-13
4-14
4-15
4-16
4-17
4-18
4-19
4-20
4-21
4-22
4-23
4-24
4-25
4-26
4-27
4-28
4-29
4-30
4-31
4-32
4-33
4-34
4-35
4-36
4-37
4-38
4-39
4-40
4-41
4-42
4-43
4-44
4-45
4-46
4-47
4-48
4-49
4-50
4-51
4-52
4-53
4-54
4-55
4-56
4-57
4-58
4-59
4-60
4-61
1
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OH
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CF3
4-CF3
4-CF3
H
4-Cl
4-OH
4-CH3
6.43
4-CF3
22.71
16.96
11.22
30.62
53.73
52.62
74.23
68.86
131.73
94.02
157.74
10.61
73.78
2.00
4.27
7.01
37.58
6.19
15.60
3.46
4-OCH3
4-CH2OH
4-COOC2H5
H
4-Cl
4-OH
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CH3
4-CF3
The data in Table 2 show that the use hydrochloric acid
(HCl) as the catalyst synthesizes 4-1 in the least time (3.5h)
and at the highest yield (58%) compared with the other acid
catalysts tested. The catalytic mechanism through which 4-1
formed is thus likely similar to that responsible for the syn-
thesis of 1-aryl-1,4-dihydropyridines.16,17) In the presence of
hydrochloric acid as the catalyst, the hydroxyl of 5 was easily
protonated and thus easily cyclized with aniline. Based on
the synthesis mechanism of 4-1, a series of compounds 4 was
synthesized by the cyclization of bis-alkylated anilines in the
presence of catalytic hydrochloride (Table 3).
4-OCH3
4-CH2OH
4-COOH
4-COOC2H5
H
4-Cl
4-OH
4-CF3
4-OCH3
4-CH2OH
4-COOH
4-COOC2H5
H
4-Cl
4-OH
4-CH3
4-CF3
4-CH2OH
4-CH2OH
4-CH2OH
4-CH2OH
4-CH2OH
4-CH2OH
4-CH2OH
4-CH2OH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOC2H5
44.10
1.35
1.30
1
The structures of 4 were confirmed by H-NMR, 13C-NMR,
high resolution (HR)-MS, and single-crystal X-ray diffrac-
tion. Compound 4-1 will be used as a representative example
to discuss the structural details of these compounds. The
1H-NMR spectrum of 4-1 exhibit the signals from the two
ethyl methanoate groups as a triplet at δ 1.26ppm and a quar-
tet at δ 4.26ppm and the signals from the two methyl groups
at δ 2.17ppm. The signals corresponding to the five protons
1.88
0.605
72.02
15.70
1.19
4-OCH3
4-CH2OH
4-COOH
4-COOC2H5
H
1.88
26.83
21.14
39.87
68.60
29.02
34.57
56.48
45.48
12.56
11.58
0.166 (0.003c))
11.63 (0.063c))
33.88 (0.012c))
4-COOC2H5 4-Cl
4-COOC2H5 4-OH
4-COOC2H5 4-CH3
4-COOC2H5 4-CF3
4-COOC2H5 4-OCH3
4-COOC2H5 4-CH2OH
4-COOC2H5 4-COOC2H5
Table 2. Yields of 4-1 Catalyzed by Different Acidsa)
Catalysts
Reaction time (h)
Yield (%)b)
Acetic acid
5.0
4.5
3.5
4.0
40
45
58
49
Sulfuric acid
Hydrochloric acid
Zinc chloride
3-NO2
3-NO2
—
—
—
—
—
—
2
3
a) Anilines (1.2mmol), N,N-dialkylanilines 5b (1mmol), catalyst (0.01mmol),
tert-butyl alcohol (15mL) at reflux. b) Isolated yields, purified by chromatography on
silica gel (petroleum ether–ethyl acetate, 20:1).
a) Binding free energy. b) Inhibition constant. c) Experimental value (µM).

May 2014
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Table 3. Yields of 1,4-Diaryl-1,4-dihydropyrazines 4a)
Table 4. Crystal Data and Structure Refinement for 4-12
Entry
R1
R2
Time (h)
Yieldb) (%)
Empirical formula
Formula weight
C25H27ClN2O4
454.94
4-1
H
H
2
56
55
55
63
62
60
59
33
31
29
68
62
Temperature
113(2)K
4-2
H
4-Cl
2
Wavelength
0.71073Å
Monoclinic, P2(1)/n
a=10.160(2)
b=17.522(4)
c=13.821(3)
α=90.00°
4-4
H
4-CH3
H
2.5
2
Crystal system, space group
Unit cell dimensions
4-10
4-11
4-12
4-14
4-19
4-20
4-24
4-60
4-61
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
1.5
2.5
2.5
3.0
2
4-CH3
4-OCH3
H
4-CH3
β=104.93(3)°
γ=90.00°
4-CH3
4-Cl
4-CH3
4-OCH3
4-COOC2H5
3-NO2
3.5
1.5
1.5
Volume
1438.3(3)Å3
4
4-COOC2H5
3-NO2
Z
Calculated density
Absorption coefficient
F(000)
1.271gm−3
0.194mm−1
960
a) Anilines (1.2mmol), N,N-dialkylanilines
(0.01mmol), tert-butyl alcohol (15mL) at reflux. b) Isolated yields, purified by chro-
matography on silica gel (petroleum ether–ethyl acetate, 20:1).
5 (1mmol), hydrochloric acid
θ Range for data collection
Index ranges
1.917–27.906°
−12≤h≤12, −14≤k≤20, −16≤l≤16
Observed reflections
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
R indices (all data)
Extinction coefficient
17128
4184
4184/124/328
1.1
R1=0.0510, wR2=0.1416
0.275
Fig. 4. ORTEP of Compound 4-12 (One of Two Independent Molecules
with 50% Probability)
from the benzene group at N1 of 4-1 are in the range of δ
7.17–7.37, and the signals corresponding to the five protons
from the benzene group at N4 of 4-1 are in the range of δ
678–6.90. In the 13C-NMR spectra of 4-1, there are only 14
signals due to the symmetry of the benzene ring and the pyr-
azine ring. The two carbonyl carbons resides at δ 166.8ppm,
the two signals from the pyrazine ring appear at δ 112.1 and
113.3ppm, and the eight signals from the two benzene rings
are found in the range of δ 128.5–150.5. The remaining signals
are ascribed to the two ethoxyl groups and two methyl groups
(δ 14.4, 16.4, 60.4ppm). The HR-MS (electrospray ionization
(ESI)) of 4-1 shows a molecular ion peak at 407.1983 m/z,
which is consistent with the calculated 407.1971 m/z for 4-1
[M+H]+. The single-crystal X-ray diffraction of 4-12 further
Fig. 5. The Binding Mode of 4-61 to Human 11β-HSD1 (PDB Code:
3H6K)
The amino acid residues forming hydrogen bonds with molecules are shown in
green, the hydrogen bond in red dashed.
proves that 4-1 is 3,5-dimethyl-1,4-diphenyl-1,4-dihydropyr- 10 µM of each molecule. The results showed that 4 exhibited
azine-2,6-diethylmethanoate (Fig. 4). Structure parameters no inhibitory activity at a concentration of 1µM and inhibited
for 4-12 are given in Table 4 and crystallographic data were approximately 10% of the 11β-HSD1 enzymatic activity at
deposited with the Cambridge Crystallographic Data Centre a concentration of 10µM. Specifically, 4-11 and 4-61 inhib-
under deposition number CCDC 775906.
ited more than 20% of the activity of human 11β-HSD1. This
Biological Activity of 1,4-Diaryl-1,4-dihydropyrazines result was different from that obtained through molecular
The inhibitory properties of the synthesized molecules were docking simulations. Although the V-shaped structure of 4
evaluated through a scintillation proximity assay with human is similar to that of 1, 2, and 3, the substituents on the aryl
11β-HSD1 (from HEK293 cells transfected with a full-length group also affect the inhibitory effect of the compound on
pcDNA3-derived expression plasmid). The percentage inhibi- the activity of 11β-HSD1. The known inhibitors 1, 2, and 3
tion of 11β-HSD1 was measured at concentration of 1µM and have donating hydrogen-bond groups (–NO2, –F, and –CN,

844
Vol. 37, No. 5
respectively) at two terminals, so do 4-11 (4-Cl, 4-Cl) and GAUSSVIEW 3.09 software (Gaussian, Inc., Wallingford, CT,
4-61 (3-NO2, 3-NO2). The docking mode of 4-61 explains the U.S.A.). The geometry optimizations were conducted using
reason of its better inhibitory activities than the other synthe- the semi-empirical Austin Model 1 (AM1) method, and the
sized compounds for its additional hydrogen bonds formed by output files were then minimized using the density functional
NO2 with Leu217 excepts the ester group with Tyr183 (Fig. 5). (DFT) method by applying the B3 LYP (Becke, Lee, Yang,
The biological activities obtained show that the structures of and Parr) correlation function during the second optimization.
4 needs to be further modified by changing the hydrophobic The Gasteiger partial charges were assigned using AutoDock
properties of R1 and R2 into hydrophilic groups to enhance the Tools. The crystal structure of human 11β-HSD1 complexed
interaction of the resulting compound with 11β-HSD1.
with a sulfonyl-piperazine inhibitor (PDB code: 3H6K) was
retrieved from the Brookhaven Protein Data Bank. After the
removal of the inhibitor from the complex, the polar hydrogen
atoms and the Gasteiger charges were added to the macromol-
CONCLUSION
Novel 1,4-diaryl-1,4-dihydropyrazines (4) were rationally ecule.
designed as 11β-HSD1 inhibitors based on docking simula-
Modeling and Analysis of the Docked Data For
tions. The docking results indicated that the novel compounds the docking, the size of the receptor grid box was set to
exhibit a similar V shape and similar ΔG and Ki values to 50×50×50, and its grid spacing was 0.375Å. Given the
those of the known inhibitors 1, 2, and 3. The compounds 4 known location of the sulfonyl-piperazine binding site, the
were synthesized through the cyclization of N,N-dialkyl-ani- cubic grid box was centered in the catalytically active region,
lines with anilines in the presence of catalytic hydrochloride which encompassed the binding site, and the grid center was
at yields in the range of 29 to 68%. The inhibitory activities designated at dimensions x, y, and z of 5.317, 38.734, and
of these compounds against 11β-HSD1 (microsome fractions −6.249, respectively. The docked conformations were gener-
from HEK-293 cells) were evaluated. The results showed ated using the LGA with an initial population size of 150
that most of the compounds inhibited less than 10% of the structures. The rest of the parameters were set to their default
11β-HSD1 activity at a concentration of 10µM, whereas 4-11 values. The docking assessment depends on the size of the
(4-Cl, 4-Cl) and 4-61 (3-NO2, 3-NO2) inhibited more than data, the estimated free energy of binding (ΔG bind), and the
20% of the activity.
estimated inhibition constant (Ki). The model analyses were
performed using the ACCELRYS DS VISUALIZER 3.0 soft-
ware (Accelrys, Inc., San Diego, CA, U.S.A.).
MATERIALS AND METHODS
General Procedure for the Preparation of N,N-Dialkyl-
All of the chemicals were used as purchased without further anilines (5) The compound 5 were synthesized by Sing-
purification. All of the solvents were reagent grade and, when Yuen Sit method.16) The aromatic amines (5mmol) and the
necessary, were purified and dried using standard methods. rhodium(II) acetate dimer (5.5mg) were heated at reflux
The melting points were determined using an X-5 apparatus temperature in anhydrous benzene (10mL) with fast stirring
1
(open capillaries, uncorrected values). The H- and 13C-NMR under an atmosphere of nitrogen. A solution of ethyl diazo-
spectra were obtained using a Bruker ARX-400MHz spec- acetoacetate in anhydrous benzene was then added dropwise.
trophotometer, and the chemical shift values are expressed The reaction was monitored by TLC and chromatographed on
in δ values (ppm) relative to tetramethylsilane (TMS) as the silica gel (petroleum ether–ethyl acetate, 20:1) to provide the
internal standard. The coupling constants are given in Hz, N,N-dialkyl aromatic amines 5.
and the signals are quoted as follows: s (singlet), d (doublet), t
Diethyl
2,2′-(Phenylazanediyl)bis(3-hydroxybut-2-enoate)
(triplet), and m (multiplet). All of the compounds were routine- (5a): Orange crystals (64%); mp 147.2–148.6°C; 1H-NMR
1
ly checked by TLC, H- and 13C-NMR. TLC was performed (400MHz, CDCl3) δ: 1.25 (t, J=7.2Hz, 6H, –CO2CH2CH3),
using coated silica gel plates (GF254, Haiyang Chemical 1.68 (s, 6H, CH3), 4.27 (q, J=7.2Hz, 4H, –CO2CH2CH3),
Plant, Qingdao, China). The developed chromatograms were 6.94–7.59 (m, 4H, Ar-H), 12.87 (s, 2H, –OH), and 12.90 (s,
visualized under ultraviolet light at 254nm or by iodine vapor. 1H, –OH).
Molecular Modeling Study The automated docking
Diethyl
2,2′-(Phenylazanediyl)bis(3-hydroxybut-2-enoate)
studies were performed with AutoDock 4.0. This automated (5b): Colorless crystals (49%); mp 82.0–82.8°C; 1H-NMR
ligand-docking program uses the Lamarckian genetic algo- (400MHz, CDCl3) δ: 1.25 (t, J=7.2Hz, 6H, –CO2CH2CH3),
rithm (LGA) to explore the full range of ligand conforma- 1.85 (s, 6H, CH3), 4.24 (q, J=7.2Hz, 4H, –CO2CH2CH3),
tional flexibility with partial flexibility of the receptor. The 6.63–7.20 (m, 5H, Ar-H), 12.82 (s, 2H, –OH).
Lamarckian genetic algorithm is a hybrid of a genetic algo-
Diethyl 2,2′-((4-Chlorophenyl)azanediyl)bis(3-hydroxybut-2-
rithm and a local search algorithm. This algorithm first builds enoate) (5c): Colorless crystals (67%); mp 128.1–129.0°C;
a population of individuals (genes), in which each gene is a 1H-NMR(400MHz, CDCl3) δ: 1.23 (t, J=7.2Hz, 6H,
different random conformation of the docked compound. The –CO2CH2CH3), 1.83 (s, 6H, CH3), 4.22 (q, J=7.2Hz, 4H,
local search algorithm then performs energy minimizations on –CO2CH2CH3), 6.55–7.12 (m, 4H, Ar-H), 12.79 (s, 2H, –OH).
a user-specified proportion of the population of individuals. If
Diethyl 2,2′-(p-Tolylazanediyl)bis(3-hydroxybut-2-enoate)
the energy of the new individual is lower than that of the old (5d): Colorless crystals (45%); mp 46.5–47.7°C; 1H-NMR
individual, the new one is automatically accepted as the next (400MHz, CDCl3) δ: 1.25 (t, J=7.2Hz, 6H, –CO2CH2CH3),
step in the docking simulation.
Preparation of the Receptor and Ligand Molecules –CO2CH2CH3), 6.61–7.12 (m, 4H, Ar-H), 12.80 (s, 2H, –OH).
The three-dimensional structures of the ligands were con-
Diethyl 2,2′-((4-(Ethoxycarbonyl)phenyl)azanediyl)bis(3-
structed using standard bond lengths and bond angles in the hydroxybut-2-enoate) (5e): Colorless crystals (69%); mp
1.85 (s, 6H, CH3), 3.80 (s, 3H, Ar-CH3), 4.21 (q, J=7.2Hz, 4H,

May 2014
845
105.3–107.9°C; 1H-NMR (400MHz, CDCl3) δ: 1.23 (t, δ: 1.27 (t, J=7.2Hz, 6H, –CO2CH2CH3), 2.16 (s, 6H, –CH3),
J=7.2Hz, 6H, –CO2CH2CH3), 1.22 (t, J=7.2Hz, 3H, 2.31 (s, 3H, Ar-CH3), 4.26 (q, J=7.2Hz, 4H, –CO2CH2CH3),
–ArCO2CH2CH3), 1.76 (s, 6H, CH3), 4.20 (q, J=7.2Hz, 2H, 6.67–7.37 (m, 8H, Ar-H); 13C-NMR (100MHz, CDCl3) δ:
–ArCO2CH2CH3), 4.14 (q, J=7.2Hz, 4H, –CO2CH2CH3), 14.4, 16.4, 55.5, 111.3, 114.3, 114.5, 124.0, 128.3, 129.3, 130.6,
7.79–7.97 (m, 4H, Ar-H), 12.78 (s, 2H, –OH).
General Procedure for the Preparation of 1,4-Diaryl-1,4- C25H27ClN2O4: 454.1659 (Calcd), 454.1668 (Found).
dihydropyrazines (4) The aromatic amines 5 (1.2mmol) and 3,5-Dimethyl-1-(4-chlorophenyl)-4-(4-methoxyphenyl)-1,4-
134.2, 149.7, 153.4, 159.7, 167.2; HR-MS (ESI) (m/z): [M+] for
N,N-dialkyl aromatic amine (1mmol) were heated at reflux dihydropyrazine-2,6-diethylmethanoate (4-14): Yellowish
temperature in tert-butyl alcohol (TBA; 15mL) with fast stir- green solid (59%); mp 128.6–129.3°C; 1H-NMR (400MHz,
ring. Then, 1mL of hydrochloric acid (0.01mol/L) was added. CDCl3) δ: 1.28 (t, J=7.2Hz, 6H, –CO2CH2CH3), 2.20 (s,
The reaction was monitored by TLC and chromatographed on 6H, –CH3), 3.83 (s, 3H, Ar-OCH3), 4.26 (q, J=7.2Hz, 4H,
silica gel (petroleum ether–ethyl acetate, 20:1) to produce the –CO2CH2CH3), 6.68–7.28 (m, 8H, Ar-H); 13C-NMR (100MHz,
desired compounds.
CDCl3) δ: 14.4, 16.4, 55.5, 60.3, 111.3, 114.3, 114.5, 124.0,
3,5-Dimethyl-1,4-diphenyl-1,4-dihydropyrazine-2,6-diethyl- 128.3, 129.3, 130.6, 131.2, 149.7, 151.4, 159.7, 166.2; HR-MS
methanoate (4-1): Light yellow solid (56%); mp 226.5–227.7°C; (ESI) (m/z): [M]+ for C25H27ClN2O5: 470.1608 (Calcd),
1H-NMR (400MHz, CDCl3) δ: 1.26 (t, J=7.2Hz, 6H, 470.1613 (Found).
–CO2CH2CH3), 2.17 (s, 6H, –CH3), 4.26 (q, J=7.2Hz, 4H,
3,5-Dimethyl-1-(4-methylphenyl)-4-phenyl-1,4-dihydropyr-
–CO2CH2CH3), 6.78–6.90 (m, 5H, Ar-H), 7.17–7.37 (m, 5H, azine-2,6-diethylmeth-anoate (4-19): Yellow solid (33%);
Ar-H); 13C-NMR (100MHz, CDCl3) δ: 14.4, 16.4, 60.4, 112.1, mp 220.3–221.5°C; 1H-NMR (400MHz, CDCl3) δ: 1.26 (t,
113.3, 128.5, 128.9, 129.0, 129.3, 130.4, 138.4, 148.9, 150.5, J=7.2Hz, 6H, –CO2CH2CH3), 2.17 (s, 6H, –CH3), 2.27 (s, 3H,
166.8; HR-MS (ESI) m/z: [M+H]+ for C24H27N2O4: 407.1971 Ar-CH3), 4.25 (q, J=7.2Hz, 4H, –CO2CH2CH3), 6.67–7.37 (m,
(Calcd), 407.1968 (Found).
9H, Ar-H); 13C-NMR (100MHz, CDCl3) δ: 14.4, 16.5, 20.5,
3,5-Dimethyl-1-phenyl-4-(4-chlorophenyl)-1,4-dihydropyr- 60.3, 112.1, 113.3, 128.5, 128.9, 129.0, 129.3, 130.4, 138.4,
azine-2,6-diethylmethanoate (4-2): Yellowish green solid 148.9, 150.5, 166.8; HR-MS (ESI) (m/z): [M]+ for C25H28N2O4:
1
(55%); mp 200.1–201.3°C; H-NMR (400MHz, CDCl3) δ: 1.26 420.2049 (Calcd), 420.2058 (Found).
(t, 6H, J=7.2Hz, –CO2CH2CH3), 2.18 (s, 6H, –CH3), 4.26
3,5-Dimethyl-1-(4-methylphenyl)-4-(4-chlorophenyl)-1,4-di-
(q, J=7.2Hz, 4H, –CO2CH2CH3), 6.74–7.35 (m, 9H, Ar-H); hydropyrazine-2,6-diethylmethanoate (4-20): Yellowish green
1
13C-NMR (100MHz, CDCl3) δ: 14.4, 16.4, 60.4, 112.4, 113.2, solid (31%); mp 153.2.1–154.6°C; H-NMR (400MHz, CDCl3)
119.5, 128.5, 129.6, 131.7, 135.1, 136.9, 150.2, 150.9, 166.4; δ: 1.28(t, J=7.2Hz, 6H, –CO2CH2CH3), 2.18 (s, 6H, –CH3),
HR-MS (ESI) (m/z): [M+H]+ for C24H26ClN2O4: 441.1581 2.29 (s, 3H, Ar-CH3), 4.26 (q, J=7.2Hz, 4H, –CO2CH2CH3),
(Calcd), 441.1587 (Found).
6.67–7.36 (m, 8H, Ar-H); 13C-NMR (100MHz, CDCl3) δ: 14.3,
3,5-Dimethyl-1-phenyl-4-(4-methylphenyl)-1,4-dihydropyr- 16.4, 20.5, 60.3, 112.8, 113.3, 128.6, 129.1, 129.5, 129.6, 131.7,
azine-2,6-diethylmethanoate (4-4): Light yellow solid (55%); 135.0, 137.0, 148.8, 150.0, 166.6; HR-MS (ESI) (m/z): [M]+ for
mp 201.5–202.7°C; 1H-NMR (400MHz, CDCl3) δ: 1.28 (t, C25H27ClN2O4: 454.1659 (Calcd), 454.1672 (Found).
J=7.2Hz, 6H, –CO2CH2CH3), 2.20 (s, 6H, –CH3), 2.37 (s,
3,5-Dimethyl-1-(4-methylphenyl)-4-(4-methoxyphenyl)-1,4-
3H, Ar-CH3), 4.27 (q, J=7.2Hz, 4H, –CO2CH2CH3), 6.77–7.28 dihydropyrazine-2,6-diethylmethanoate (4-24): Light yellow
1
(m, 9H, Ar-H); 13C-NMR (100MHz, CDCl3) δ: 14.4, 16.4, solid (29%); mp 171.1–172.4°C; H-NMR (400MHz, CDCl3) δ:
21.1, 60.2, 111.6, 113.2, 119.2, 128.4, 129.9, 130.0, 135.7, 139.0, 1.26 (t, J=7.2Hz, 6H, –CO2CH2CH3), 2.17 (s, 6H, –CH3), 2.27
150.8, 151.1, 166.6; HR-MS (ESI) (m/z): [M]+ for C25H28N2O4: (s, 3H, Ar-CH3), 3.80 (s, 3H, Ar-OCH3), 4.24 (q, J=7.2Hz, 4H,
420.2049 (Calcd), 420.2054 (Found).
–CO2CH2CH3), 6.66–7.26 (m, 8H, Ar-H); 13C-NMR (100MHz,
3,5-Dimethyl-1-(4-chlorophenyl)-4-phenyl-1,4-dihydropyr- CDCl3) δ: 14.4, 16.4, 20.5, 55.5, 60.2, 112.0, 113.3, 114.3,
azine-2,6-diethylmethanoate (4-10): Light yellowish green 128.4, 129.0, 130.9, 131.3, 149.0, 150.9, 159.6, 166.8; HR-MS.
1
solid (63%); mp 217.5–218.3°C; H-NMR (400MHz, CDCl3) δ: (ESI) (m/z): [M]+ for C26H30N2O5: 450.2155 (Calcd), 450.2173
1.28 (t, J=7.2Hz, 6H, –CO2CH2CH3), 2.20 (s, 6H, –CH3), 4.27 (Found).
(q, J=7.2Hz, 4H, –CO2CH2CH3), 6.69–7.41 (m, 9H, Ar-H);
3,5-Dimethyl-1,4-(4-ethoxycarbonylphenyl)-1,4-dihydropyr-
13C-NMR (100MHz, CDCl3) δ: 14.4, 16.5, 60.1, 111.4, 114.3, azine-2,6-diethylmethanoate (4-60): Light yellow solid (68%);
124.0, 128.3, 129.0, 129.4, 130.3, 149.2, 149.4, 150.5, 166.1; mp 180.1–182.3°C; 1H-NMR (400MHz, CDCl3) δ: 1.25 (t,
HR-MS (ESI) (m/z): [M+] for C24H25ClN2O4: 440.1503 (Calcd), J=7.2Hz, 6H, Ar-CO2CH2CH3), 1.38 (t, 6H, –CO2CH2CH3),
440.1517 (Found).
2.21 (s, 6H, CH3), 4.25 (q, J=7.2Hz, 4H, –CO2CH2CH3),
3,5-Dimethyl-1,4-dis(4-chlorophenyl)-1,4-dihydropyr- 4.36 (q, J=7.2Hz, 4H, Ar-CO2CH2CH3), 6.70–8.07 (m, 8H,
azine-2,6-diethylmethanoate (4-11): Light yellowish green Ar-H); 13C-NMR (100MHz, CDCl3) δ: 14.8, 16.5, 16.7, 59.8,
1
solid (62%); mp 214.3–216.4°C; H-NMR (400MHz, CDCl3) δ: 60.5, 61.5, 110.5, 116.1, 116.5, 121.3, 122.4, 124.7, 125.6, 131.3,
1.28 (t, J=7.2Hz, 6H, –CO2CH2CH3), 2.20 (s, 6H, –CH3), 4.26 134.9, 149.8, 153.3, 159.7, 161.8; HR-MS-ESI (m/z): [M]+ for
(q, J=7.2Hz, 4H, –CO2CH2CH3), 6.67–7.39 (m, 8H, Ar-H); C30H34N2O8: 550.2315 (Calcd), 550.2328 (Found).
13C-NMR (100MHz, CDCl3) δ: 14.3, 16.4, 60.4, 112.1, 114.3,
3,5-Dimethyl-1,4-dis(3-nitrophenyl)-1,4-dihydropyr-
124.2, 128.4, 129.7, 131.6, 135.2, 136.7, 149.4, 150.5, 166.0; azine-2,6-diethylmethanoate (4-61): Light yellow solid (62%);
HR-MS (ESI) (m/z): [M+] for C24H24Cl2N2O4: 474.1113 (Calcd), mp 66.5–67.3°C; 1H-NMR (400MHz, CDCl3) δ: 1.25–1.28
474.1138 (Found).
(t, J=7.2Hz, 6H, –CO2CH2CH3), 2.24 (s, 6H, –CH3), 4.29
3,5-Dimethyl-1-(4-chlorophenyl)-4-(4-methylphenyl)-1,4-di- (q, J=7.2Hz, 4H, –CO2CH2CH3), 7.01–7.73 (m, 8H, Ar-H);
hydropyrazine-2,6-diethylmethanoate (4-12): Yellowish green 13C-NMR (100MHz, CDCl3) δ: 14.8, 16.5, 60.4, 103.6,
1
solid (60%); mp 222.2–223.4°C; H-NMR (400MHz, CDCl3) 104.2, 106.5, 107.8, 112.1, 124.6, 128.3, 129.5, 145.6, 146.9,

846
Vol. 37, No. 5
149.4, 150.8, 151.3, 152.5, 166.3; HR-MS-ESI (m/z): [M]+ for
C24H24N4O8: 496.1594 (Calcd), 496.1586 (Found).
4) Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ,
Seckl JR, Flier JS. A transgenic model of visceral obesity and the
metabolic syndrome. Science, 294, 2166–2170 (2001).
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Fatty acid regulation of hepatic gene transcription. J. Nutr., 135,
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nase type 1 as a promising therapeutic target. Drug Discov. Today,
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X, Jaen JC, Labelle M, Liu J, Ma J, Miao S, Sudom A, Tang L, Tu
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11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Bioorg. Med.
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Biological Assay The inhibition of the enzymatic activ-
ity of human 11β-HSD1 was determined through the scintil-
lation proximity assay (SPA) using microsomes containing
11β-HSD1.11,18) Briefly, the full-length cDNAs of human
11β-HSD1 were isolated from cDNA libraries provided by
the NIH Mammalian Gene Collection and cloned into the
pcDNA3 expression vector (Invitrogen, Carlsbad, CA, U.S.A.)
by polymerase chain reaction (PCR). HEK293 cells were
transfected with the pcDNA3-derived expression plasmids and
selected by cultivation in the presence of 700µg/mL G418.
The microsomal fractions overexpressing 11β-HSD1 were
prepared from HEK293 cells that were stably transfected
with 11β-HSD1 and used as the enzyme source for SPA. The
assay was performed in a 96-well microtiter plate. Different
concentrations of the compounds were added, followed by the
addition of 80µL of 50mmol/L N-(2-hydroxyethyl)piperazine-
N′-2-ethanesulfonic acid (HEPES) buffer, pH 7.4, containing
25 nmol/L [1,2-(n)3H]cortisone (Amersham, Buckinghamshire,
U.K.) and 1.25mmol/L NADPH. The reactions were initiated
by the addition of the 11β-HSD1 enzyme preparation as mi-
crosomal fractions from HEK293 cells to a final concentration
of 80µg/mL 11β-HSD1. After incubation for 60min at 37°C,
the reaction was stopped by the addition of 35µL of 10mg/mL
protein A-coated SPA beads (GE, Piscataway, NJ, U.S.A.)
suspended in Superblock® Blocking Buffer (Pierce, Rockford,
IL, U.S.A.) with 3µg/mL of murine monoclonal cortisol anti-
body (East Coast Biologics, North Berwick, Maine, U.S.A.)
and 314µmol/L glycyrrhetinic acid (Sigma-Aldrich, St. Louis,
MO, U.S.A.). The plates were incubated under plastic film on
an orbital shaker for 120min at room temperature. The [3H]-
cortisol generated in the enzymatic reaction was captured
by the beads and measured in a liquid scintillation counter
equipped to read microplates. The percentage inhibition was
calculated relative to an uninhibited control. The data were
obtained from at least three independent experiments.
Acknowledgments This work was financially supported
by the Key Projects of the National Science and Technology
Pillar Program (No. 2012ZX10001007-008-002) and the Doc-
toral Fund of Innovation of Beijing University of Technology.
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