General Chemistry
Reagents, starting materials, and solvents were purchased from commercial suppliers (Sigma, St.
Louis, MO, USA) and used as received. All organic solutions were dried over sodium sulfate. The
melting points of the synthesized molecules were determined by open end capillary tube method
in melting point apparatus (Thiele tube) and are uncorrected. 1H NMR spectra were recorded with
Avance-II (Bruker), FT NMR Spectrometer. Chemical shifts are reported in ppm, using the solvent
as internal standard. Infrared spectra (IR) were recorded as KBr discs with a Perkin–Elmer
Spectrum RX-I, FTIR Spectrometer. Mass spectra (m/z) were recorded on a Waters Micromass QTof Micro, Mass Spectrometer with ESI as ionization method. Merck F-254 commercial plates
were used for analytical TLC to follow the course of reaction. Silica Gel 60 (Merck 70– 230 mesh)
was used for column chromatography.
Synthesis of 2-[(4-Iodo-2-methyl-phenylamino)-methylene]-malonic acid diethyl ester (1). A
mixture of 4-Iodo-2-methyl-phenylamine (0.01mol) and diethyl ethoxy methylene malonate
(0.01mol) was heated at 120-130°C for two hrs. The remaining ethanol was evaporated off. The
crude solid was filtered, dried and recrystallized from n-hexane and used in following step.
Yield: 88%, M.R.: 54-56oC, Rf = 0.63 (CHCl3 : MeOH, 99 : 1), IR (KBr, cm-1) υ: 3337 (-NH-),
1733 (-CO-, ester), 1570 (-C=C-, aromatic), 535 (C-I); 1H NMR (CDCl3, 300 MHz) δ in ppm:
7.51-7.79 (4H, m), 4.19 (2H, s), 4.0 (1H, s), 2.35 (3H, s), 1.3 (3H, s); MS(m/z, %): M+ (374)
Synthesis of 6-Iodo-8-methyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl ester (2).
Diphenyl ether was heated under stirring at 240°C. 0.1 mol of 2-[(4-Iodo-2-methyl-phenylamino)methylene]-malonic acid diethyl ester was added slowly to the boiling diphenyl ether for about 15
minutes after adding the mixture was refluxed in oil bath for two hrs. The mixture was cooled,
filtered and washed twice with 200 ml pet ether. The crude solid obtained was dried and purified
by recrystallization twice from DMF.
Yield: 78%, M.R.: 68-71oC, Rf = 0.45 (CHCl3 : MeOH, 99 : 1), IR (KBr, cm-1) υ: 3348 (-NH-),
1710 (-CO-, ketone), 1578 (-C=C-, aromatic), 526 (C-I); 1H NMR (CDCl3, 300 MHz) δ in ppm:
7.47-7.79 (3H, m), 4.22 (2H, s), 4.1 (1H, s), 2.4 (3H, s), 1.34 (3H, s); MS(m/z, %): M+ (357)
Synthesis of 4-Chloro-6-iodo-8-methyl-1,4-dihydro-quinoline-3-carboxylic acid amide (3). 6Iodo-8-methyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl ester (0.1 mol) was dissolved
in ethanol (5 ml) and 2M sodium hydroxide (5 ml) and heated under reflux for three hrs. After
cooling the solvent was removed under vacuum and the residue dissolved in water and washed
with ethyl acetate. The aqueous layer was acidified with 2M hydrochloric acid. The precipitates
were filtered washed with water and dried under vacuum. The solid obtained, DMF 1 ml, and
thionyl chloride (0.01 mol) was refluxed using benzene as a solvent on water bath at 80°C for 5–
6 h in anhydrous condition with the help of calcium chloride guard tube, until the HCl gas evolution
was ceased and then solvent and thionyl chloride were removed by distillation. The solid material
obtained was cooled and ammonia was added drop wise to it and the suspension was stirred at
room temperature for 10 hrs. The resulting precipitate was filtered off and washed with water and
dried under vacuum.
Yield: 75%, M.R.: 120-125oC, Rf = 0.43 (Hexane : Ethyl Acetate, 90 : 10), IR (KBr, cm-1) υ: 3428
(-NH2, Antisymmetric), 3357 (-NH2, Symmetric), 1678 (Amide I), 1614 (Amide II), 1530 (-C=C, aromatic), 1265 (C-N), 650 (C-Cl), 516 (C-I); 1H NMR (CDCl3, 300 MHz) δ in ppm: 7.00-7.20
(3H, m), 6.0 (2H, s), 5.44 (1H, s), 4.0 (1H, s), 2.36 (3H, s); MS(m/z, %): M+ (348)
Synthesis of 4-(4-Dimethylamino-phenylamino)-6-iodo-8-methyl-1,4-dihydro-quinoline-3carboxylic acid amide (4). Product obtained in previous step was dissolved in acetonitrile and
heated at 80oC for 2 hrs. The resulting precipitate was filtered off and washed with ether. Physical
and chromatographic characterisation: Yield: 84 %, M.R.: 180-182oC, Rf
=
0.54 (Hexane : Ethyl
Acetate, 90 : 10)
IR (KBr, cm-1) υ: 3512 (-NH2, antisymmetric), 3201 (-NH2, symmetric), 1670 (Amide I), 1640
(Amide II), 1563 (-C=C-, aromatic), 1269 (C-N), 548 (C-I); 1H NMR (CDCl3, 300 MHz) δ in
ppm: 6.25-7.02 (7H, m), 6.0 (2H, s), 4.7 (1H, s), 4.4 (2H, s), 2.89 (6H, s), 2.38 (3H, s); MS(m/z,
%): M+ (448)
Synthesis of Q1-Q6. CuI (0.05 mmol) and K3PO4 (2.0 mmol) were added to a screw-capped test
tube with a Teflon lined septum. DMPU (1.0 mL), N,N-diethylsalicylamide (2.0 mmol), 4-(4Dimethylamino-phenylamino)-6-iodo-8-methyl-1,4-dihydro-quinoline-3-carboxylic acid amide
(4) (1.0 mmol), and respective thiol (RSH) (1.2 mmol) were added by syringe at room temperature.
The reaction mixture was heated at 80 °C to furnish a pale yellow suspension. The reaction mixture
was allowed to cool to room temperature after heating for the time specified. Water was added,
and the mixture was extracted with diethyl ether (4 x 10 mL). The combined organic phases were
washed with brine and dried over Na2SO4. The solvent was removed under vacuum to yield the
crude product as a deep yellow residue that was purified by column chromatography on silica gel.
Potassium peroxomonosulfate (Oxone) (0.1 mol) and DMF (25 mL) are mixed and cooled to 5°C.
A solution of the crude product obtained in previous step in DMF (25 ml) is placed in the addition
funnel and added in a slow stream to the stirring slurry. After addition of the sulfide, the reaction
mixture is stirred at room temperature for 4 hr, and the DMF is removed on a rotary evaporator.
The colorless solid is dried under vacuum (0.1 mm) at room temperature followed by
recrystallization from hexane.
4-(4-Dimethylamino-phenylamino)-8-methyl-6-(toluene-4-sulfonyl)-1,4-dihydro-quinoline3-carboxylic acid amide (Q1). Yield: 77 %, M.R.: 132-136oC, Rf
=
0.48 (Hexane : Ethyl Acetate,
90 : 10), IR (KBr, cm-1) υ: 3485 (-NH2, antisymmetric), 3373 (-NH2, symmetric), 1686 (Amide I),
1645 (Amide II), 1585 (-C=C-, aromatic), 1351(O=S=O, antisymmetric), 1281(C-N),
1150(O=S=O, symmetric), 752 (N-H, wag); 1H NMR (CDCl3, 300 MHz) δ in ppm: 6.48-7.78
(11H, m), 6.0 (2H, s), 4.7 (1H, s), 4.5 (2H, s), 2.85 (6H, s), 2.34 (6H, s); MS(m/z, %): M+ (476)
4-(4-Dimethylamino-phenylamino)-6-(3,4-dimethyl-benzenesulfonyl)-8-methyl-1,4-dihydroquinoline-3-carboxylic acid amide (Q2). Yield: 75 %, M.R.: 129-133oC, Rf
=
0.45 (Hexane :
Ethyl Acetate, 90 : 10), IR (KBr, cm-1) υ: 3480 (-NH2, antisymmetric), 3371 (-NH2, symmetric),
1686 (Amide I), 1602 (Amide II), 1380(O=S=O, antisymmetric), 1289(C-N), 1200(O=S=O,
symmetric), 780 (N-H, wag); 1H NMR (CDCl3, 300 MHz) δ in ppm: 6.75-7.68 (13H, m), 5.6 (2H,
s), 4.6 (1H, s), 4.4 (2H, s), 2.82 (6H, s), 2.36 (6H, s); MS(m/z, %): M+ (490)
4-(4-Dimethylamino-phenylamino)-8-methyl-6-(toluene-2-sulfonyl)-1,4-dihydro-quinoline3-carboxylic acid amide (Q3). Yield: 77 %, M.R.: 132-136oC, Rf
=
0.48 (Hexane : Ethyl Acetate,
90 : 10), IR (KBr, cm-1) υ: 3469 (-NH2, antisymmetric), 3381 (-NH2, symmetric), 1698 (Amide I),
1630 (Amide II), 1547 (-C=C-, aromatic), 1357(O=S=O, antisymmetric), 1276(C-N),
1101(O=S=O, symmetric), 784 (N-H, wag); 1H NMR (CDCl3, 300 MHz) δ in ppm: 6.32-7.69
(11H, m), 5.8 (2H, s), 4.6 (1H, s), 4.1 (2H, s), 2.80 (6H, s), 2.35 (6H, s),; MS(m/z, %): M+ (476)
4-(4-Dimethylamino-phenylamino)-6-(2,5-dimethyl-benzenesulfonyl)-8-methyl-1,4-dihydroquinoline-3-carboxylic acid amide (Q4). Yield: 79 %, M.R.: 106-111oC, Rf
=
0.40 (Hexane :
Ethyl Acetate, 90 : 10), IR (KBr, cm-1) υ: 3312 (-NH2, antisymmetric), 3245 (-NH2, symmetric),
1697 (Amide I), 1645 (Amide II), 1543 (C=C, aromatic) 1356(O=S=O, antisymmetric), 1260(CN), 1150(O=S=O, symmetric), 756 (N-H, wag); 1H NMR (CDCl3, 300 MHz) δ in ppm: 7.25-7.78
(13H, m), 6.2 (2H, s), 4.8 (1H, s), 4.5 (2H, s), 2.79 (6H, s), 2.32 (6H, s); MS(m/z, %): M+ (490)
4-(4-Dimethylamino-phenylamino)-6-(2,4-dimethyl-benzenesulfonyl)-8-methyl-1,4-dihydroquinoline-3-carboxylic acid amide (Q5). Yield: 83 %, M.R.: 128-34oC, Rf = 0.47 (Hexane : Ethyl
Acetate, 90 : 10), IR (KBr, cm-1) υ: 3437 (-NH2, antisymmetric), 3368 (-NH2, symmetric), 1676
(Amide I), 1618 (Amide II), 1515 (C=C, aromatic) 1354(O=S=O, antisymmetric), 1245(C-N),
1148(O=S=O, symmetric), 787 (N-H, wag); 1H NMR (CDCl3, 300 MHz) δ in ppm: 7.10-7.92
(13H, m), 6.3 (2H, s), 4.7 (1H, s), 4.4 (2H, s), 2.79 (6H, s), 2.30 (6H, s); MS(m/z, %): M+ (490)
4-(4-Dimethylamino-phenylamino)-6-(2,3,4-trimethyl-benzenesulfonyl)-8-methyl-1,4dihydro-quinoline-3-carboxylic acid amide (Q6). Yield: 84 %, M.R.: 138-143oC, Rf
=
0.51
(Hexane : Ethyl Acetate, 90 : 10), IR (KBr, cm-1) υ: 3438 (-NH2, antisymmetric), 3348 (-NH2,
symmetric), 1676 (Amide I), 1610 (Amide II), 1512 (C=C, aromatic) 1354(O=S=O,
antisymmetric), 1296(C-N), 1169(O=S=O, symmetric), 743 (N-H, wag); 1H NMR (CDCl3, 300
MHz) δ in ppm: 6.35-7.47 (13H, m), 6.2 (2H, s), 4.8 (1H, s), 4.5 (2H, s), 2.75 (6H, s), 2.32 (8H,
s); MS(m/z, %): M+ (504)
Pharmacological Evaluation
Animals
The studies were carried out at Pinnacle Biomedical Research Institute, Bhopal. Male and female
Dunkin Hartley guinea pigs weighing between 400-700 g and Male Albino Mice of weight
between 25–30 g were obtained from the animal facility of the institute a few days before use. The
animals were kept in an environmentally controlled animal room with temperature control and a
12 hour light darkness cycle. Ethical clearance was obtained for the use of the guinea pigs and
Albino mice from the Institutional Ethical committee (protocol approval reference number:
PBRI/IAEC/12/PN-183).
Chemicals and equipment
All the drugs, chemicals and solvents used were procured from Sigma, St. Louis, MO, USA.
Solution of Lipopolysaccharide was prepared in pyrogen free saline. Drug solutions were prepared
freshly at the beginning of each experiment.
Experimental protocol
Two top scoring molecules were subjected to pharmacological screening as per the details
presented hereunder. The assays were selected on the basis of considerations of main effects of
PDE4 inhibition. Three assays namely guinea pig tracheal chain relaxation, inhibition of
lipopolysaccharide induced endotoxemia (TNF-α), neutrophilia and eosinophilia were used for the
purpose.
2.5.3.1 Inhibition of histamine-induced contractions of the isolated guinea pig trachea
Guinea pigs were weighed and sacrificed by overdose of thiopental sodium (50 mg/kg body
weight). The trachea was removed from the animal and placed in a petri-dish containing cold
Kreb’s (KRB) solution, the excess tissue and fat was trimmed off using a razor blade and the
muscle opened by cutting longitudinally through the cartilage rings, diametrically opposite the
trachealis muscle. The flat tissue was pinned onto a corkboard (still immersed in the cold KRB
solution) and cut into zig-zag strips by making transverse slits at equal intervals in the tissue.
Finally, the strip was divided into 4 equal pieces, and a cotton thread inserted at each end and was
mounted under an applied load of 1.5 grams in the isolated organ bath system having a reservoir
(34 ml) containing KRB solution and continuously aerated using 5% CO2/95% O2. The KRB
solution consisted of (mM): NaCl 118.5, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.9, NaHCO3
25.0, and glucose 10.1.
Dose response curves of histamine in plain KRB solution, KRB solution containing 10-4-10-8 M of
molecule Q1, KRB solution containing 10-4-10-8 M of molecule Q2, and KRB solution containing
10-4-10-8 M of Roflumilast were obtained. Percent of maximum contractile responses were plotted
to record dose response curves of histamine in the absence and presence of synthesized molecules
(Q1 & Q2).
Responses to Q1 and Q2 (mean ± SEM) are expressed as % inhibition of histamine-induced
contraction.
LPS
induced
mice
endotoxemia,
neutrophilia
and
eosinophilia
Model
The dose of Q1 and Q2 were determined as per the OECD guidelines for acute oral toxicity studies.
At the beginning of the experiment, male albino mice were divided into six groups of six animals
each and treated as follows daily for seven days:

Group I received vehicle (4% DMSO) and then challenged with phosphate-buffered saline
(PBS) after 4.5 hrs. This group served as negative control.

Group II received equivalent volume of vehicle and then challenged with
lipopolysaccharide (LPS) (0.5 mg/kg) in phosphate-buffered saline after 4.5 hrs.

Group III received Q1 (10 mg/kg, i.p.) and challenged with LPS (0.5 mg/kg) in phosphatebuffered saline after 4.5 hrs.

Group IV received with Q2 (10 mg/kg, i.p.) and challenged with LPS (0.5 mg/kg) in
phosphate-buffered saline after 4.5 hrs.

Group V received with Roflumilast (10 mg/kg, i.p.) and challenged with LPS (0.5 mg/kg)
in phosphate-buffered saline after 4.5 hrs.
The concentration s of LPS, sample and standard drug solutions were adjusted so that not more
than 0.3-0.4 ml of solution was injected to the animal.
On the seventh day of treatment, blood samples from all the groups were collected by puncturing
the retro-orbital plexus under mild ether anesthesia. Blood was collected in vials, pre-treated with
disodium EDTA, and analyzed for total leukocyte count (TLC) and differential leukocyte count
(DLC), by fixing blood extractars and staining with Field stain I and Leishman's stain. After the
initial counts, blood samples were incubated with nylon fiber (80 mg/ml of blood sample) for 15
min at 37°C. The incubated blood samples were again analyzed for TLC and DLC.
The plasma was separated from the blood samples withdrawn from mice and mouse TNF-α
enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences Pharmigen) was used to detect
TNF- α in the cell supernatants. TNF- α concentration was determined by extrapolation from the
TNF- α standard curve, according to the manufacturer's protocol.
Statistical analysis
Results were expressed as mean±SEM. Statistical significance of differences between means of
groups was determined by ANOVA followed by Students t-test, and probabilities of, <0.05 was
considered statistically significant.