Safety and Efficacy of New 3,6-diaryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine Analogs as Potential Phosphodiesterase-4 Inhibitors in NIH-3T3 Mouse Fibroblastic Cells

Article abstract of DOI:10.1111/j.1747-0285.2011.01167.x

A novel series of potential phosphodiesterase-4 (PDE-4) inhibitors, 6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines, were developed. Different concentrations of the synthesized compounds were tested on cultured NIH-3T3 cells to determine their safety and efficacy in NIH-3T3 mouse fibroblastic cells in comparison with rolipram, a selective PDE-4 inhibitor. The viability of cells was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazoliumbromide (MTT) assay. The PDE inhibition rate was measured indirectly by determination of concentrations of extracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using enzyme-linked immunoassay technique. The results showed that all tested compounds caused a marked increase in the concentration of cAMP, whereas the concentration of cGMP stayed approximately unchanged. The cytotoxic IC₅₀ of all synthesized compounds was approximately twofold greater than their required concentration for inhibition of PDE-4 (in terms of elevation of cAMP), and thus, these structures could be used to develop potent and safe inhibitors of PDE-4 enzyme.

Full text of DOI:10.1111/j.1747-0285.2011.01167.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01167.x
Chem Biol Drug Des 2011; 78: 438–444
Research Article
Safety and Efficacy of New 3,6-diaryl-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine Analogs as
Potential Phosphodiesterase-4 Inhibitors in NIH-
3T3 Mouse Fibroblastic Cells
Maryam Baeeri1,2, Alireza Foroumadi1,3
Maryam Motamedi1, Azadeh Yahya-
,
Received 2 June 2010, revised 3 June 2011 and accepted for publica-
tion 27 June 2011
Meymandi3, Loghman Firoozpour3, Seyed
N. Ostad1, Abbas Shafiee1, Saeid
Souzangarzadeh2 and Mohammad
Abdollahi1,*
Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes in
charge for the hydrolysis of cyclic adenosine monophosphate
(cAMP) and cyclic guanosine monophosphate (cGMP) playing a key
role in many physiological and pathological processes in mammalian
organs. PDE inhibitors have revealed numerous worthy pharmaco-
logical properties in various organs like cardiovascular, central ner-
vous system, respiratory tract, gastrointestinal tract (GI), and as
antioxidants (1–4). PDE-4 inhibitors are especially useful for the
treatment of inflammation in illnesses like asthma and chronic
obstructive pulmonary disorders (COPD, emphysema, and bronchitis),
as well as for the treatment of depression, psychosis, and memory
problems (5). As a result, diverse synthetic PDE-4 inhibitors are
under development (5–7) and several natural PDE inhibitors have
been derived from medicinal plants (4).
1Faculty of Pharmacy, and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran 1417614411,
Iran
2Department of Chemistry, Shahr-e-Rey Branch, Islamic Azad
University, Tehran 18735-334, Iran
3Drug Design and Development Research Center, Tehran University
of Medical Sciences, Tehran, Iran
*Corresponding author: Mohammad Abdollahi,
mohammad@tums.ac.ir
A novel series of potential phosphodiesterase-4
(PDE-4) inhibitors, 6-(3-(cyclopentyloxy)-4-meth-
oxyphenyl)-3-aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazines,
were
developed.
Different
As the development of PDE-4 inhibitors, such as rolipram 1
(Figure 1), and structurally related compounds are restricted by
some side effects, the development of unique, powerful, and
selective PDE-4 inhibitors with lower side effects is an urgent
need. Recently, several alkoxy-substituted 3,6-diphenyl-7H-
[1,2,4]triazolo[1,3,4]thiadiazines, including 2 (Figure 1), as potent
PDE-4 inhibitors, have been reported (5–7). To extend the struc-
ture activity relationship (SAR) of this series of compounds, we
report the synthesis, efficacy, and safety of some new 6-(3-(cyclo-
pentyloxy)-4-methoxyphenyl)-3-aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazines 3 (Figure 1) while aryl= (2-methoxyphenyl in 6a), (3-
methoxyphenyl in 6b), (p-Tolyl in 6c), and (Phenyl in 6d) tested
in NIH-3T3 cells. Preliminary SAR very much suggests that these
compounds are among the catechol class of PDE-4 inhibitors;
hence, rolipram as a potent and selective PDE-4 inhibitor from
this class was used for comparison. Previous study showed that
company of a non-large 3,4-functionality placed at the 5 position
of the 3,6-dihydro-2H-1,3,4-thiadiazine ring, but not the phenyl ring
attached at the 3 position of the 1,2,4-triazole, is essential (7).
The main purpose of this study was to study the safety and effi-
cacy of 6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-aryl-7H-[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazines in inhibition of cAMP- or cGMP PDE in
NIH-3T3 cells.
concentrations of the synthesized compounds
were tested on cultured NIH-3T3 cells to deter-
mine their safety and efficacy in NIH-3T3 mouse
fibroblastic cells in comparison with rolipram, a
selective PDE-4 inhibitor. The viability of cells was
determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazoliumbromide (MTT) assay. The PDE
inhibition rate was measured indirectly by
determination of concentrations of extracellular
cyclic adenosine monophosphate (cAMP) and cyc-
lic guanosine monophosphate (cGMP) using
enzyme-linked
immunoassay
technique.
The
results showed that all tested compounds caused
a marked increase in the concentration of cAMP,
whereas the concentration of cGMP stayed
approximately unchanged. The cytotoxic IC50 of
all synthesized compounds was approximately
twofold greater than their required concentration
for inhibition of PDE-4 (in terms of elevation of
cAMP), and thus, these structures could be used
to develop potent and safe inhibitors of PDE-4
enzyme.
Key words: cAMP, cGMP, drug discovery, phosphodiesterase
inhibitor
438

Safety and Efficacy of New Analogs of PDE-4 Inhibitors
R2
OR1
MeO
O
OMe
MeO
N
N
N
N
O
S
NH
2
R1 = OMe,Cyclopentyl
2 = H,OMe
1
R
N
N
S
N
N
R
O
3
Figure 1: Rolipram 1, alkoxy-s-
ubstituted 3,6-diphenyl-7H-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazines 2,
some new 6-(3-(cyclopentyloxy)-4-
methoxyphenyl)-3-aryl-7H-[1,2,4]tria-
zolo[3,4-b][1,3,4]thiadiazines 3.
OMe
3a: R = 2-OMe
3b: R = 3-OMe
3c: R = 4-Me
3d: R = H
6-(3-(cyclopentyloxy)-4-methoxyphenyl)-
3-(2-methoxyphenyl)-7H-[1,2,4]triazolo
[3,4 b][1,3,4]thiadiazine
Methods
General chemistry
(6a)
Melting points were determined on a Kofler hot stage apparatus.
The IR spectra were recorded on a Shimadzu 470 spectrophotome-
ter (potassium bromide disks). H-NMR spectra were recorded on a
White solid; Yield 82%; mp: 153.9-155.7 ꢀC; IR (KBr, per cm):
3425.2, 2970.9, 1669; 1HNMR (CDCl3, 400 MHz) d: 1.52-2.05 (m,
8H), 3.93 (s, 3H), 3.94 (s, 3H), 4.90 (s, 2H), 4.85 (m, 1H), 6.91 (d,
1H), 7.06 (m, 2H), 7.49 (t, 1H), 7.56 (s, 1H), 7.68 (d, 1H), 7.87 (d,
1H); 13CNMR (125 MHz, CDCl3) d: 24.08, 32.73, 41.21, 55.94,
56.14, 80.60, 110.60, 111.89, 112.69, 113.48, 120.70, 123.23,
127.96, 130.29, 133.06, 147.91, 155.1, 157.75, 163.62, 164.63,
173.33; MS m ⁄ z [%]: 436 [M+] (44), 404 (11), 368 (100), 353 (47),
336 (31).
1
Varian unity 400 spectrometer, and chemical shifts were expressed
as d (ppm) with tetramethylsilane as internal standard. MS spectra
were obtained with a Finnigan MAT TSQ-70 spectrometer (Finnigan
Mat, Bremen, Germany). The purity of synthesized compounds was
confirmed by thin layer chromatography using various solvents of
different polarities.
Preparation of substituted 6-(3-
6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-
(3-methoxyphenyl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine
(cyclopentyloxy)-4-methoxyphenyl)-3-(aryl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6a–d)
(6b)
Ethanol (50 mL) was added to a mixture of substituted 4-amino-5-
(aryl)-2,3-dihydro-1,2,4-triazine-3-thion (5a–d) (11 mmol) and 2-
bromo-1-(3-(cyclopentyloxy)-4-methoxyphenyl)ethanone (11.5 mmol).
The reaction mixture was refluxed for 4–5 h. The solvent was
removed under reduced pressure, and the residue was treated with
HCL (2N, 100 mL). The precipitate was filtered, and the filtrate was
treated with NaOH (2N, 100 mL). The product was filtered and
crystallized from ethanol–water to give the substituted 6-(3-(cyclo-
pentyloxy)-4-methoxyphenyl)-3-(aryl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazine (6a–d) in 81% yield.
White solid; Yield 87%; mp: 147.3-178.8 ꢀC; IR (KBr, per cm):
3440.0, 2951.9, 1607; 1HNMR (CDCl3, 400 MHz) d: 1.8-2.05 (m,
8H), 3.86 (s, 3H), 3.93 (s, 3H), 3.97 (s, 2H), 4.83 (m, 1H), 6.93 (d,
1H), 7.03 (d, 1H),7.36 (s, 1H), 7.38 (t, 1H), 7.64 (s, 1H), 7.76 (d,
2H); 13CNMR (125 MHz, CDCl3): 22.79, 24.2, 32.81, 55.43, 56.14,
80.65, 110.96, 112.29, 113.46, 116.11, 120.63, 120.95, 125.69,
127.46, 129.43, 148.1, 148.32, 152.18, 153.14, 153.67, 159.61;
MS m ⁄ z [%]: 436 [M+] (83), 404 (22), 368 (100), 353 (77), 336
(46).
Chem Biol Drug Des 2011; 78: 438–444
439

Baeeri et al.
6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-p-
tolyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6c)
humidified atmosphere with 5% CO2 in air. NIH-3T3 cells were
grown in RPMI medium with L-glutamine, supplemented with
10% fetal bovine serum and 1% antibiotic, 100 units of penicillin
G ⁄ mL, and 100 lg of streptomycin ⁄ mL. Cells were seeded at 24
multi-well plate, and four wells were harvested using trypsin
each day until stationary phase was reached. The population
doubling time of these cells (26 h) was determined by cell
counting.
Light yellow solid, Yield 85%; mp: 196.1–201 ꢀC; IR (KBr, cm)1):
3540.0, 2851.9, 1607;1HNMR (CDCl3, 400 MHz) d: 1.68–2.13 (m,
8H), 2.42 (s, 3H), 3.93 (s, 3H), 3.96 (s, 2H), 4.83 (m, 1H), 6.93 (d,
1H, J = 8.2 Hz), 7.28 (d, 1H, J = 8 Hz) 7.36 (d, 1H, J = 8.2 Hz), 7.62
(s, 1H, H2), 8.05 (d, 1H, J = 8 Hz); 13CNMR (125 MHz, CDCl3): 22.81,
24.13 29.69, 32.79, 56.13, 80.65, 111.00, 112.33, 120.92, 123.52,
125.80, 128.18, 129.16, 140.38, 148.29, 151.95, 152.95, 153.58; MS
m ⁄ z [%]: 422 [M+] (3), 404 (20), 368 (100), 353 (77), 336 (49), 325
(10), 307 (5).
Treatments
Concerning estimated ED50 of rolipram (0.3 lM) (8), five concentra-
tions of 0.05, 0.1, 0.5, 1, and 5 lM were used as control positive
groups. As well by considering the average ED50 of substituted 3,6-
6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-
phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
(6d)
diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
(20 lM)
(7),
concentrations of 1, 10, 20, 40, and 80 lM of each synthesized
compound were prepared in dimethylsulfoxide (DMSO). The cell
monolayers in exponential growth were harvested by means of
0.25% trypsin, and at that point, single-cell suspensions were
acquired by frequent pipetting. The number of cells was calculated
by a hemocytometer, and single-cell suspensions having cell mass
of 1 · 103 cells per 100 lL medium ⁄ well were added to 96-well
plates. Then, cells were protected overnight at 37 ꢀC before treat-
ment. All of the set concentrations of rolipram and created com-
pounds were added to three wells of each plate, together with a
group of control which did not receive any drugs. Afterward, the
cells were kept warm at 37 ꢀC. At 1, 6, and 24 h after treatment,
and the culture medium of every one well of a 96-well plate was
moved to a microtube for measurement of cAMP and cGMP
concentrations, and then, (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phen-
yltetrazoliumbromide (MTT) assay was performed.
White solid; Yield 88%; mp: 192.7–195.6 ꢀC; IR (KBr, per cm):
1
3725.2, 2967.9, 2862, 1660; HNMR (CDCl3, 400 MHz) d: 1.8-2.1 (m,
8H), 3.86 (s, 3H), 3.94 (s, 3H), 3.98 (s, 2H), 4.82 (m, 1H), 6.94 (d,
1H), 7.37 (s, 1H), 7.49 (m, 2H), 7.63 (s, 1H), 7.89 (s, 1H), 8.18 (d,
2H); 13CNMR (125 MHz, CDCl3): 24.5, 34.9, 36.3, 55.94, 56.2, 79.3,
114.50, 111.89, 121.8, 126.9, 127.48, 128.8, 129.3, 130.7, 146.7,
148.0, 152.23, 164.6; MS m ⁄ z [%]: 406 [M+] (49), 388 (2), 368 (3.1),
338 (100), 323 (64), 295 (10).
Cell line
The NIH-3T3 mouse fibroblastic cell line was purchased from the
Institute of Pasteur at Tehran and was cultured as monolayers
on polystyrene culture dishes and maintained at 37 ꢀC in a
Table 1: Concentrations
of
cAMP concentration (pmol ⁄ cell)
Time (h)
cAMP after 1, 6, and 24 h of expo-
sure of NIH-3T3 cells to different
concentration of 6a–6d in com-
parison with those of rolipram
Groups
1
6
24
Concentration
Mean € SEM
Mean € SEM
Mean € SEM
Drug name
(lM)
(percent of increase)
(percent of increase)
(percent of increase)
Rolipram
Rolipram
Rolipram
Rolipram
Rolipram
6a
0.05
0.1
0.5
1
76.3 € 2.3* (58)
78 € 2* (59)
75.6 € 15.9* (72)
80.1 € 4.5* (73)
136 € 2.5* (84)
166.6 € 13.3* (87)
247.3 € 6.9* (91)
49.1 € 3.7* (56.8)
60.6 € 1.2* (65)
106.3 € 5* (80)
110 € 4.9* (80)
136.6 € 7.3* (84)
157.3 € 8.8* (86)
110 € 4* (80)
162.3 € 12.5* (87)
206.6 € 7.4* (89)
106.6 € 4.2* (80)
156.6 € 6.6* (86)
189.6 € 9.3* (88)
21.2 € 5
117.3 € 0.9* (76)
132.3 € 13.9* (78.8)
201.6 € 6.17* (86)
222.6 € 8.17* (87)
309.3 € 18.3* (90)
40.6 € 1.45 (31)
41 € 2.08 (31)
51 € 1.7 (45)
43.6 € 1.45 (35)
95 € 4.6* (70)
173.3 € 58* (83)
35.3 € 2.02 (20)
38.6 € 1.6 (27)
68.3 € 2.02* (59)
48.3 € 1.76 (42)
79.6 € 5.3* (64)
130 € 5.7* (78)
28 € 3.5
145.3 € 1.3* (78)
164.6 € 2.9* (80)
220 € 6.4* (85)
85 € 18.9* (62)
99.6 € 2.7* (68)
115.6 € 2.3* (72)
104.2 € 2.1* (69)
123.3 € 1.15* (74)
132.6 € 2.9* (76)
124.1 € 17* (74)
157.5 € 4.5* (80)
159.4 € 4.7* (80)
139.5 € 5.57* (77)
165.6 € 7.8* (81)
239.8 € 2.4* (86)
31.6 € 7.3
5
10
20
40
10
20
40
10
20
40
10
20
40
–
6a
6a
6b
6b
6b
6c
6c
6c
6d
6d
6d
Control
*Significantly different from control group at p < 0.05.
440
Chem Biol Drug Des 2011; 78: 438–444

Safety and Efficacy of New Analogs of PDE-4 Inhibitors
Safety test
cells. The base of test was on the competition between the enzyme
conjugate and the cAMP or cGMP in the sample for a narrow num-
ber of binding sites.
After transmitting the medium of each well into a microtube,
100 lL of fresh medium and 20 lL of 5 mg ⁄ mL MTT dissolved in
phosphate-buffered saline were added. One set of wells were allo-
cated as the control group which contained an adjusted planting
cell number in log phase in the culture medium holding 0.1%
DMSO. Cells were incubated for 3.5 h at 37 ꢀC. Subsequently,
media was removed and 150 lL DMSO was added to each well of
the 96-well plates to solubilize formazan crystals. The absorbance
was measured at the test wavelength of 570 nm and the reference
wavelength of 690 nm by a microplate reader. The percentage of
viable cells was calculated using the formula: percent of con-
trol = [1 ) (A570 test ⁄ A570 cont)] · 100 where A570 test = absor-
bance of test sample, A570 cont = absorbance of control sample.
Dose–response curves were plotted from percentage of control val-
ues against concentrations of inhibitors as lM (x-axis, log scale).
The values of 50% inhibition of cell proliferation (IC50) were
calculated by locating the x-axis values corresponding to one-half
the absorbance values of control containing an adjusted seeding
cell number and 0.1% DMSO without addition of any inhibitors
(Table 1).
Statistical analysis
Results were expressed as mean € SEM and analyzed by Student's
t-test. Statistical significance level was defined at p < 0.05. Stats-
Direct statistical software version 2.7.8 was used.
Results
Chemical synthesis
Reaction sequence employed for the synthesis of substituted 6-
(3-(cyclopentyloxy)-4-methoxyphenyl)-3-(aryl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6a–d) is shown in Scheme 1. The starting
material substituted benzoic acid (1a–d) was converted into
substituted ethyl benzoates (2a–d). Substituted arylhydrazides
(3a–d) were achieved by refluxing (2a–d) with hydrazine
hydrate in methanol. Compound (3a–d) on reaction with carbon
disulfide in methanolic potassium hydroxide yielded potassium 3-
aryldithiocarbazates (4a–d) in worthy yield and was right used
for the next stage without additional purification. The triazoles 5
were synthesized by refluxing (4a–d) with hydrazine hydrate.
Compression of (5a–d) with phenacyl bromide in ethanol under
reflux state provided replaced 6-(3-(cyclopentyloxy)-4-methoxyphe-
nyl)-3-(aryl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a–d) in good
yield.
Measurement of cAMP and cGMP
concentrations
Medium of each well was transferred to a microtube at certain
intervals after treating with inhibitors. For precipitation of interfer-
ing proteins, 10 lL of trichloroacetic acid was added to each sam-
ple. Then, microtubes were put in the ice for 15 min and
centrifuged subsequently at 11 490 g for 10 min. The acid content
was neutralized with KOH 2 lM. Microtubes were kept at )20 ꢀC.
The ELISA kit from Bender MedSystems (Vienna, Austria) was used
to assay non-acetylated samples. The optical densities were deter-
mined at 405 nm. The concentrations of cAMP or cGMP were cal-
culated according to the standard curve generated in each assay.
The results of each group were normalized by dividing the calcu-
lated cyclic nucleotides concentrations by the number of viable
Cell proliferation
NIH-3T3 cells were multiplied quickly with a cell doubling time of
26 h. To prompt adequate cell death, the dose of rolipram was
increased up to 500 lM. Rolipram was less cytotoxic than other
synthesized compounds with IC50 values of 160, 353, and 500 lM
after 1, 6, and 24 h of exposure, respectively.
O
Br
CHO
i-iv
N
N
O
HO
S
OMe
OMe
N
N
X
1
ix
5
O
OMe
NH
COOH
N
S
6a-d
v-viii
N
6a: X = 2-OMe
6b: X = 3-OMe
6c: X = 4-Me
6d: X = H
X
NH2
X
5a-d
1a-d
Scheme 1. Synthesis of substituted 6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-(aryl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (6a-d). Condi-
tions: (i) cyclopentyl bromide (1.5 equiv), K2CO3 (1.2 equiv), DMF, 60 ꢀC, 12 h, 84%; (ii) MeMgBr (1.3 equiv), Et2O, 0–25 ꢀC, 1 h, 76%; (iii)
MnO2, Et2O, 0–25 ꢀC, 4 h, 73%; (iv) CuBr2, EtOAc, reflux, 3.5 h, 74%; (v) cat. H2SO4, MeOH, rt, 12 h; (vi) hydrazine, EtOH, reflux 12 h.; (vii)
KOH, EtOH; then CS2 rt, 12 h; (viii) hydrazine monohydrate, H2O, reflux, 3 h, then Conc. HCl; (ix) EtOH, reflux ꢀC, 4 h.
Chem Biol Drug Des 2011; 78: 438–444
441

Baeeri et al.
Table 2: Concentrations
of
cGMP concentration (pmol ⁄ cell)
Time (h)
cGMP after 1, 6, and 24 h of expo-
sure of NIH-3T3 cells to different
concentration of 6a–6d in com-
parison with those of rolipram
Groups
1
6
24
Concentration
Mean € SEM
Mean € SEM
Mean € SEM
Drug name
(lM)
(percent of increase)
(percent of increase)
(percent of increase)
Rolipram
Rolipram
Rolipram
Rolipram
Rolipram
6a
0.05
0.1
0.5
1
2.3 € 1.2 (–)
2.5 € 1.1 (–)
2.4 € 0.9 (–)
2.5 € 1.1 (–)
2.6 € 1.1 (4)
2.5 € 0.9 (–)
3 € 1.3 (20)
3.3 € 1.1 (32)
2.3 € 0.9 (–)
3.4 € 1.5 (36)
3.6 € 0.9 (44)
3 € 1.3 (20)
3 € 1.1 (20)
3.4 € 1.2 (36)
2.5 € 1.1 (–)
2.6 € 1 (4)
2.6 € 1.3 (3%)
2.5 € 1 (–)
2.4 € 0.9 (–)
2.5 € 1.2 (–)
2.6 € 1.1 (4)
3.3 € 1.5 (20)
3 € 1.55 (20)
2.3 € 1.1 (–)
3 € 1 (20)
3.2 € 1.6 (28)
2.5 € 1.2 (–)
3.6 € 1.5 (44)
3.5 € 1.2 (40)
3 € 1.35 (20)
3.4 € 1.2 (36)
3.5 € 1.3 (44)
2.6 € 1.3 (4)
3 € 1.4 (20)
3.1 € 1.6 (24)
2.5 € 1.2
2.4 € 0.8 (–)
2.5 € 1.2 (–)
2.8 € 1.2 (12)
2.3 € 1.1 (–)
3 € 1.4 (20)
3 € 1.5 (20)
3 € 1.4 (20)
3.5 € 1.6 (40)
3.6 € 1.4 (44)
3.5 € 1.45 (40)
3.6 € 1.5 (44)
3.6 € 1.1 (44)
2.5 € 1.2 (–)
2.8 € 1.3 (12)
3 € 1.5 (20)
2.5 € 1.3
5
10
20
40
10
20
40
10
20
40
10
20
40
–
6a
6a
6b
6b
6b
6c
6c
6c
6d
6d
6d
Control
2.7 € 0.9 (8)
2.5 € 1.2
Level of cAMP and cGMP
of response on NIH-3T3 cell line after 6 h of contact. Thus, the IC50
reduced from early >80 lM at 1 h to 73 and 40 lM after 6 and
24 h of exposure, respectively. The IC50 value of 6b at 24 h was
found 2-fold higher than that of 1 h. Sensitivity of cell line to 6c
was augmented with IC50 values of 74, 42, and 41 lM, after 1, 6,
and 24 h postexposure, respectively. Compound 6d was the most
cytotoxic among the tested compounds owing to the strong inhibi-
tion of cell proliferation with IC50 values of 66, 59, and 40 lM,
respectively, after 1, 6 and 24 h of exposure. After 1-h exposure,
the NIH-3T3 cell line displayed the maximum toxicity in response to
80 lM dose of 6d and 6c, whereas the cells showed little
response to 6a and 6b in the equal duration. After 6 h of experi-
ence, the cell reaction to 6b cytotoxicity amplified especially in
upper doses close to 80 lM. By increasing the contact duration, the
variance between the cytotoxicity of 6d, 6c, and 6b and that of
6a appeared in lower doses. As a result, a noteworthy difference
was found between toxic effects of 6b, 6c, 6d, and 6a in doses
greater than 30 lM after 24 h (Table 4).
All synthesized compounds produced a major increase in cAMP
level (Table 1), although the concentration of cGMP remained
roughly unchanged (Table 2). The potency of rolipram and each of
four synthesized compounds were calculated as cAMP and cGMP
ED50 (Table 3). In this regard, 6d showed specificity for PDE-4 with
the lowest cAMP ED50 of 13 lM and the highest cGMP ED50 of
49.8 lM (Table 3).
Cytotoxicity
The cytotoxicity of 6a on NIH-3T3 cells was assessed with IC50
value of >80 lM, after 24 h. Compound 6b produced an abrupt rise
Table 3: ED50 values of compounds for inhibition of cAMP- and
cGMP PDE after 1, 6, and 24 h of exposure
Time (h)
Groups
1
6
24
cAMP (lM)
Rolipram
Molecular modeling
1.459
22.6
23
23
38
1.2
29
24.34
36.2
27.4
0.8
38.1
22
21.6
13
The initial model of phosphodiesterase-4d (PDE-4D) for docking
studies was built based on the X-ray crystal structure of human
PDE-4D with the regulatory domain in complex with 1-(3-nitrophe-
nyl)-3-(pyridin-4-ylmethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
which was obtained from the Protein Data Bank (PDB entry 3G4G)
(9, 10). The original ligand was removed while water molecules
present in the PDB file were maintained in their position. 3D struc-
tures of 4d was generated and optimized by MarvinSketch, Marvin
Beans package 5.0.7. AUTODOCK 4 package was used to perform
docking simulations, which adopts the hybrid Lamarckian Genetic
Algorithm as searching algorithm and allows full flexibility of the
6a
6b
6c
6d
GMP (lM)
Rolipram
Inactive
44
Inactive
45.7
Inactive
48.6
6a
6b
6c
6d
46.2
45.3
49.7
48.4
49.9
49.2
48.5
46.4
49.8
442
Chem Biol Drug Des 2011; 78: 438–444

Safety and Efficacy of New Analogs of PDE-4 Inhibitors
Table 4: Cytotoxicity of tested compounds after 1 and 24 h of
exposure of NIH-3T3 cells to different concentration of 6a–6d in
comparison with that of rolipram
thiadiazine ring are serious functionalities for effective PDE-4 inhibi-
tion. However, the phenyl ring attached at the 3 position of the
1,2,4-triazole seems to be less involved in the pharmacophore of
this structure. As stated, the 3,4-alkoxy moiety plays a crucial role
in many PDE-4 inhibitors, as clarified in the structure of rolipram
(7).
Cytotoxicity (percent of control) (mean € SEM)
Concentrations (lM)
After 1 h
After 6 h
After 24 h
Rolipram (0.05)
Rolipram (0.1)
Rolipram (0.5)
Rolipram (1)
Rolipram (5)
Rolipram (25)
Rolipram (125)
Rolipram (250)
Rolipram (500)
6a (1)
6a(10)
6a (20)
6a (40)
6a (80)
6b (1)
6b (10)
6b (20)
6b (40)
6b (80)
6c (1)
6c (10)
6c (20)
6c (40)
6c (80)
6d (1)
6d (10)
82 € 2
82 € 3
82 € 1
82 € 4
79 € 2
76 € 4
69 € 3
61 € 5
50 € 2
81 € 5
81 € 4
78 € 4.2
73 € 5
68.6 € 1.7
86 € 6
84 € 3
79 € 4
75 € 3
74 € 3
77 € 5
75 € 3
74 € 3
73 € 5
46 € 6
81 € 7
80 € 6
61 € 5
68 € 4
42 € 3
80 € 5
77 € 2
76 € 2
70 € 2
65 € 3
62 € 5
58 € 3
53 € 5
46 € 7
79 € 5
78 € 3
74 € 2
68 € 2
65 € 1.3
80 € 5
72 € 5
64 € 3
57 € 3
50 € 2*
78 € 4
63 € 5
57 € 2
51 € 1
46 € 4*
74 € 2
69 € 1
64 € 3
61 € 2
41 € 5*
77 € 4
76 € 4
73 € 6
69 € 3
67 € 3
61 € 4
52 € 3
46 € 4
40 € 4
78 € 3
77 € 3
73 € 5
68 € 3
65 € 5
79 € 5
72 € 4
63 € 3
50 € 3*
45 € 6*
73 € 5
63 € 7
59 € 6
51 € 4*
43 € 7*
74 € 9
65 € 5
61 € 4
50 € 5*
41 € 9*
We synthesized a novel series of inhibitors, with a 3-cyclopentyl-
oxy-4-methoxyphenyl group at the 5 position of the 3,6-dihydro-2H-
1,3,4-thiadiazine ring. Our findings revealed that such moieties on
the 5 position of the 3,6-dihydro-2H-1,3,4-thiadiazine ring do not
improve the inhibitory effects of these compounds. As a matter of
fact' the calculated ED50s of the synthesized compounds (22 lM for
6a, 6b, 6c, and 13 lM for 6d) are much higher than those of the
previously synthesized 3-(2,5-dimethoxyphenyl)-6-(3,4-dimethoxyphe-
nyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine with ED50 of 0.01 lM
(7).
Our findings showed that all synthesized compounds cause a sub-
stantial intensification in the concentration of extracellular cAMP,
whereas the concentration of cGMP stays approximately unaffected.
These findings enlightened the possible selectivity of these com-
pounds against cAMP-specific PDE enzymes (Table 3). Among these
analogs, 6d showed the highest potential for elevation of cAMP.
Also, the time course effect of 6d bears a resemblance to that of
rolipram. It is obvious that the p-Tolyl moiety on the 5 position
of the 3,6-dihydro-2H-1,3,4-thiadiazine ring potentiates the activity
A
6d (20)
6d (40)
6d (80)
*Significant difference exists between the cytotoxicity of 6a (known as the
least cytotoxic compound) and the cytotoxicity of 6b, 6c, and 6d.
ligand. Solvation parameters and Kollman charges for all atoms in
PDE4D were assigned using AUTODOCK Tools 1.5.4. The grid for
energy evaluation was centered at residue Phe196 in PDE4D with
grid points in the x-, y-, z-axes set to 50 · 50 · 50 and separated
by 0.375 ꢀ. The initial population size and maximum number of
energy evaluations were set to 100 and 1.0 · 107, respectively.
The docked result within an RMSD of 2 ꢀ was clustered, and the
final was selected considering both the embedded empirical binding
free energy evaluation and the clustering analysis.
B
Discussion
On the basis of the recent studies, mercapto- and thione-substi-
tuted 1,2,4-triazole ring systems have been reported for having anti-
bacterial, antifungal, and anti-inflammatory properties (11,12).
Figure 2: Docking study of compound 4d (atom type base col-
ored) and the three-dimensional structure of PDE-4D (PDB ID: 3G4G)
(A) ribbon view of active site in complex with 4d, (B) Hydrogen
bond is shown with dotted green line between triazol and side
chain of Gln535.
Substituted
3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
are potent inhibitors of PDE-4 (5–7). This fundamental structure
signifies an innovative chemotype capable of PDE-4 inhibition. Excit-
ingly, the exchanges on the 5 position of the 3,6-dihydro-2H-1,3,4-
Chem Biol Drug Des 2011; 78: 438–444
443

Baeeri et al.
of compound 6d, whereas methoxy substitutions have less effects
in terms of SAR. However, the methoxy group will intensify the
steadiness of the compound when attached at the 3 position of
phenyl ring. Compound 6c showed two peak elevations in cAMP
levels at 1 and 24 h postexposure (Table 3). Interestingly, the cyto-
toxicity of 6d was directly proportional to its PDE inhibitory effect.
It is worthy to mention that the cytotoxic IC50 of all synthesized
compounds was approximately twofold greater than their required
concentration for inhibition of PDE-4 (in terms of elevation of cAMP)
telling that these structures could be used to develop potent and
safe inhibitors of PDE-4 enzyme.
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To ensure that the elevation of cAMP is mediated through the inhi-
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studied by molecular docking simulation using the AUTODOCK pro-
gram in MGL Tools package software based on the X-ray crystal
structure of PDE-4D (11, 12). As shown in Figure 2, compound 4d
has a nice fit along the active site of PDE-4D. The figures show
that 4d makes several principal interactions along the active site
of PDE-4D. Compound 4d is located in a strong hydrophobic pocket
of PDE-4D, in which the two substituted-phenyl groups are involved
in hydrophobic interactions with Phe538, Phe506, Met523, and
Met439. Moreover, one hydrogen bond was formed by nitrogen of
triazol ring with residue Gln535, with bond length of 2.47 ꢀ.
The precise function of 6-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-
aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
remains
to
be
completed by further investigation into their selectivity for different
intact cAMP PDE isoforms.
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43:808–815.
Acknowledgment
This study was partly supported by a grant from Pharmaceutical
Sciences Research Center, Tehran University of Medical Sciences
and INSF.
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