Design and synthesis of new 8-anilide theophylline derivatives as bronchodilators and antibacterial agents

Article abstract of DOI:10.1007/s12272-012-0805-4

Theophylline derivatives have long been recognized as potent bronchodilators for the relief of acute asthma. Recently, it was found that bacterial infection has a role in asthma pathogenesis. The present work involves the design and synthesis of 8-substituted theophylline derivatives as bronchodilators and antibacterial agents. The chemical structures of these compounds were elucidated by IR, 1H-NMR, mass spectrometry, and elemental analyses. The bronchodilator activity was evaluated using acetylcholine-induced bronchospasm in guinea pigs, and most of the compounds showed significant anti-bronchoconstrictive activity in comparison with standard aminophylline. In addition, the antibacterial activity of all the target compounds was investigated in vitro against Gram-positive and Gram-negative bacteria using ampicillin as a reference drug. Results showed that some of the tested compounds possessed significant antibacterial activity. A pharmacophore model was computed to obtain useful insight into the essential structural features of bronchodilator activity. A structure activity relationship was also discussed.

Full text of DOI:10.1007/s12272-012-0805-4

Arch Pharm Res Vol 35, No 8, 1355-1368, 2012
DOI 10.1007/s12272-012-0805-4
Design and Synthesis of New 8-Anilide Theophylline Derivatives as
Bronchodilators and Antibacterial Agents
Alaa M. Hayallah1, Ahmad A. Talhouni2, and Abdel Alim M. Abdel Alim1
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt and
2Department of Applied Pharmaceutical Sciences, Faculty of Pharmacy, Al-Israa University, Amman, Jordan
(Received September 25, 2011/Revised November 21, 2011/Accepted November 24, 2011)
Theophylline derivatives have long been recognized as potent bronchodilators for the relief of
acute asthma. Recently, it was found that bacterial infection has a role in asthma pathogene-
sis. The present work involves the design and synthesis of 8-substituted theophylline deriva-
tives as bronchodilators and antibacterial agents. The chemical structures of these compounds
1
were elucidated by IR, H-NMR, mass spectrometry, and elemental analyses. The bronchodi-
lator activity was evaluated using acetylcholine-induced bronchospasm in guinea pigs, and
most of the compounds showed significant anti-bronchoconstrictive activity in comparison
with standard aminophylline. In addition, the antibacterial activity of all the target com-
pounds was investigated in vitro against Gram-positive and Gram-negative bacteria using
ampicillin as a reference drug. Results showed that some of the tested compounds possessed
significant antibacterial activity. A pharmacophore model was computed to obtain useful
insight into the essential structural features of bronchodilator activity. A structure activity
relationship was also discussed.
Key words: Theophylline, 1,3,8-Trisubstituted-purine-2,6-diones, Antibacterial activity, Bron-
chodilators
ease (COPD) (Sethi et al., 2002). The response of asth-
matics to antibiotics also suggests the importance of
INTRODUCTION
Asthma is a heterogeneous syndrome of intermittent acute and chronic bacterial infections in the patho-
wheezing and airway inflammation that affects 300 genesis of disease (Blasi and Johnston, 2007). There is
million individuals worldwide. Although its causes are epidemiologic evidence that the prevalence of asthma
unknown, many studies suggest a role of microbiota in and allergic diseases has significantly increased over
its etiology (Cookson, 2004). Viral infections are im- the last three decades, particularly in children and
portant inducers of seasonal exacerbations of asthma young adults, and thus making allergic diseases a very
(Johnston et al., 1995), but there is circumstantial common chronic health problem (Burr et al., 1989;
evidence that bacterial infections may also play a role. Yunginger et al., 1992; Woolcock and Peat, 1997;
Asymptomatic neonates whose throats are colonized von Mutius et al., 1999; Jedrychowski et al., 2004;
with Streptococcus pneumoniae
,
Haemophilus influenzae
,
Pawlinska-Chmara et al., 2008).
or Moraxella catarrhalis are at increased risk for
Searching for new bronchodilators with antimicro-
recurrent wheezing and asthma early in life (Bisgaard bial effect remains an important and challenging target
et al , 2007). These same bacteria have consistently due to the emergence of resistant strains of bacteria to
been associated with exacerbations of both asthma the major classes of antibacterial agents.
.
(Kraft, 2000) and chronic obstructive pulmonary dis-
It has previously been reported that several 8-aralkyl-
thiotheophylline (Fig. 1 I) and triazole-based theophyl-
line derivatives showed either more or equipotent
activity to aminophylline and ampicillin as reference
drugs (Elgaher et al., 2009; Hayallah et al., 2011).
Based on these data, and in view of a pharmacophore
Correspondence to: Alaa A. K. M. Hayallah, Department of
Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
Tel: 2-183410110, Fax: 2-882332776
E-mail: alaa_hayalah@yahoo.com
1355

1356
A. M. Hayallah et al.
Fig. 1. Reported and targeted derivatives.
model, we designed and synthesized several 8-sub- meter (IR-470) as potassium bromide discs at the
stituted theophylline derivatives (Fig. 1 II, and III) as Faculty of Pharmacy, Assiut University. NMR spectra
novel bronchodilators with potent antibacterial activity. were recorded on a Varian EM-360 60 MHz spectro-
meter at the Faculty of Pharmacy, Assiut University.
DMSO-d6 was used as a solvent, unless otherwise
specified, the chemical shifts were given in (ppm),
and coupling constants ( ) were in Hertz (Hz). Chemical
MATERIALS AND METHODS
δ
J
Chemistry
Reagents used for synthesis were purchased from shifts are expressed either relative to tetramethylsi-
Sigma-Aldrich and MERCK. All solvents were obtained lane (TMS) as an internal standard or to the chemical
from commercial suppliers and used without further shifts of the remaining protons of DMSO-d6: 1H:
δ 2.49
purification. Melting points (mp) were determined on ppm. Protons of NH and OH groups were confirmed
an electrothermal Stuart Scientific SMP1 melting point by D2O. The EI-MS were determined using or JOEL
apparatus and were uncorrected. Thin-layer chroma- JMS600 mass spectrometer at the Unit of Microanaly-
tography (TLC, Rf values) was carried out using TLC sis, Assiut University. The microanalyses for C, H, N,
aluminum sheets kieselgel 60 F254 (MERCK) and and S were performed on Perkin-Elmer 240 elemental
dichloromethane-methanol (9.5:0.5) as a mobile phase. analyzer at the Unit of Microanalysis, Faculty of Sci-
Visualization was effected with ultraviolet lamp Spec- ence, Assiut University.
troline ENF-240C/F at short wavelength (λ = 254 nm).
Not all chemical yields were optimized and they gen-
erally represented the findings of a single experiment.
IR spectra were recorded on a Shimadzu spectrophoto-
General method for synthesis of compounds
22-38
In a stirred solution of compound
4 (0.5 g, 2.35 mmol)
Table I. Physical and microanalytical data of compounds 22-38
Microanalyses
Calcd. %
mp (ºC)
Mol. formula
(Mol. wt)
No.
22
R
Yield %
82
Crystal. solvent
Found %
C
H
N
S
53.47
4.77
19.49
--
53.23
5.02
19.37
--
238-240
Ethanol
C16H17N5O3S
(359.41)
C
H
N
S
43.85
3.68
43.35
4.11
265-267
Ethanol
C16H16BrN5O3S
(438.31)
23*
24*
83
85
15.98
7.32
15.88
7.05
C
H
N
S
46.66
4.41
17.00
7.78
46.43
4.92
16.78
7.93
C16H16ClN5O3S.
H2O
277-279
aq. ethanol 30%
(411.87)

8-Anilide Theophylline Derivatives as Bronchodilators
1357
Table I. Continued
Microanalyses
Calcd. %
mp (ºC)
Mol. formula
(Mol. wt)
No.
R
Yield %
84
Crystal. solvent
Found %
C
H
N
S
48.79
4.09
17.78
8.14
48.54
4.47
17.68
7.95
266-268
aq. methanol
20%
C16H16ClN5O3S
(393.85)
25
C
H
N
S
48.79
4.09
48.91
4.24
258-260
aq. methanol
20%
C16H16ClN5O3S
(393.85)
26
27
80
87
78
79
84
77
73
79
80
90
82
80
87
17.78
8.14
17.55
8.22
C
H
N
S
54.68
5.13
18.75
8.59
54.24
5.35
18.54
8.40
246-248 dec.
aq. methanol
20%
C17H19N5O3S
(373.44)
C
H
N
S
54.68
5.13
18.75
8.59
54.30
5.45
18.45
8.10
234-236 dec.
aq. methanol
20%
C17H19N5O3S
(373.44)
28*
29
C
H
N
S
52.43
4.92
17.98
8.23
52.65
5.25
17.80
7.95
258-260 dec.
aq. ethanol 30%
C17H19N5O4S
(389.44)
C
H
N
S
45.49
4.30
19.89
7.59
45.20
4.43
19.95
7.20
C16H16N6O5S.
H2O
263-265
aq. ethanol 20%
30*
31*
32
(422.43)
C
H
N
S
50.61
4.25
17.36
7.95
50.15
4.68
17.40
8.14
275-277 dec.
aq. ethanol 20%
C17H17N5O5S
(403.42)
C
H
N
S
50.61
4.25
17.36
7.95
50.30
4.70
17.50
7.65
255-257
aq. ethanol 20%
C17H17N5O5S
(403.42)
C
H
N
S
52.66
4.92
17.06
7.79
52.20
4.95
16.80
7.65
C18H19N5O4S.
1/2 H2O
270-272
ethanol
33*
34
(410.55)
C
H
N
S
54.68
5.13
18.75
8.59
55.12
4.88
18.53
8.34
222-224 dec.
ethanol
C17H19N5O3S
(373.44)
C
H
N
S
58.67
4.68
17.10
7.83
58.32
4.79
17.30
8.35
249-251
ethanol
C20H19N5O3S
(409.47)
35
C
H
N
S
55.80
5.46
18.07
8.28
55.42
5.73
18.15
7.90
230-232
ethanol
C18H21N5O3S
(387.46)
36
C
H
N
S
55.80
5.46
18.07
8.28
55.37
5.85
17.86 8.40
237-239 dec.
ethanol
C18H21N5O3S
(387.46)
37
C
H
N
S
50.12
6.57
18.26
8.36
49.80
6.90
18.10
8.55
C16H23N5O3S.
H2O
257-269 dec.
ethanol
38
(383.48)
*
These compounds were further confirmed by mass spectrometry.

1358
A. M. Hayallah et al.
in aqueous NaOH 1% (20 mL), an appropriate amount N3-CH3), 4.6 (q,
J
= 6.9 Hz, 1H, CH), 7.6-8.4 (m, 4H,
of N-(substituted)aryl/aralkyl or cycloalkyl-2-bromo- phenyl C-H), 10.6 (s, 1H, amide N-H), 14.7 (br s, 1H,
propio-namides 21 (2.35 mmol) dissolved in the least N7-H).
5
-
amount of ethanol was added. The reaction mixture
was stirred at the ambient temperature of 12 h, and N-(3-Chlorophenyl)-2-(1,3-dimethyl-2,6-dioxo-2,3,
then cooled in a refrigerator for 3 h. The product was 6,7-tetrahydro-1H-purin-8-ylsulfanyl)propiona-
filtered, washed with water, diethyl ether, dried, and mide 26
crystallized from the appropriate solvent to afford the IR cm−1: 3445 (N-H); 3220 (N-H amide); 3040 (Ar-H);
desired products 22-38. Physical and micro-analytical 2870 (C-H aliphatic); 1695, 1645 (C=O); 1525 (N-H);
data are given in Table I.
782, 741 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.8 (d,
= 7 Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3), 3.7 (s, 3H, N3-
CH3), 4.7 (q, = 7 Hz, 1H, CH), 7.7-8.3 (m, 4H, phenyl
C-H), 10.9 (s, 1H, amide N-H), 14.6 (br s, 1H, N7-H).
J
2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
H
-
J
purin-8-ylsulfanyl)-N-phenyl propionamide 22
IR cm−1: 3470 (N-H); 3265 (N-H amide); 3045 (Ar-H);
2870 (C-H aliphatic); 1693, 1643 (C=O); 1529 (N-H); 2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
745, 710 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.5 (d,
purin-8-ylsulfanyl)-N- -tolyl propionamide 27
= 7 Hz, 3H, CH3), 3.3 (s, 3H, N1-CH3), 3.5 (s, 3H, N3- IR cm−1: 3450 (N-H); 3245 (N-H amide); 3040 (Ar-H);
CH3), 4.5 (q, = 6.9 Hz, 1H, CH), 7.9-7.7 (m, 5H, phenyl 2870 (C-H aliphatic); 1695, 1650 (C=O); 1530 (N-H);
H-
J
p
J
C-H), 10.6 (s, 1H, amide N-H).
805 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.8 (d,
Hz, 3H, CH3), 2.5 (s, 3H, 4'-CH3), 3.5 (s, 3H, N1-CH3), 3.7
(s, 3H, N3-CH3), 4.9 (q, = 6.8 Hz, 1H, CH), 7.6 (d,
8.5 Hz, 2H, phenyl C-H), 8.1 (d, = 8.5 Hz, 2H, phenyl
C-H), 10.9 (s, 1H, amide N-H), 14.7 (br s, 1H, N7-H).
J = 6.8
N-(4-Bromophenyl)-2-(1,3-dimethyl-2,6-dioxo-2,3,
J
J =
6,7-tetrahydro-1
mide 23
H
-purin-8-ylsulfanyl)propiona-
J
IR cm−1: 3450 (N-H); 3230 (N-H amide); 3080 (Ar-H);
2940 (C-H aliphatic); 1711, 1643 (C=O); 1529 (N-H); 2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
835 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.6 (d,
= 7 purin-8-ylsulfanyl)-N-m-tolyl propionamide 28
Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3), 3.7 (s, 3H, N3- IR cm−1: 3460 (N-H); 3255 (N-H amide); 3045 (Ar-H);
CH3), 4.6 (q, = 6.9 Hz, 1H, CH), 7.2 (d, = 8.8 Hz, 2890 (C-H aliphatic); 1695, 1655 (C=O); 1530 (N-H);
2H, phenyl C-H), 7.7 (d,
= 8.8 Hz, 2H, phenyl C-H), 782, 742 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.6 (d,
10.8 (s, 1H, amide N-H). EI-MS (m/z, % base): 438.49 = 6.8 Hz, 3H, CH3), 2.25 (s, 3H, 3'-CH3), 3.3 (s, 3H, N1-
(M+, 1.2), 440.56 (M++2, 0.9), 265.61 (100), 237.76 CH3), 3.5 (s, 3H, N3-CH3), 4.45 (q,
= 6.8 Hz, 1H,
H-
J
J
J
J
J
J
(24.7), 199.91 (50.8), 197.81 (53.2).
CH), 6.8-7.4 (m, 4H, Ar-H), 10.7 (s, 1H, amide-H), 14.7
(br s, 1H, N7-H). EI-MS (m/z, % base): 373.25 (M+,
1.5), 239.02 (42.5), 150.04 (68.9), 134.07(100).
N-(4-Chlorophenyl)-2-(1,3-dimethyl-2,6-dioxo-2,3,
6,7-tetrahydro-1
mide 24
H-purin-8-ylsulfanyl)propiona-
2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
IR cm−1: 3450 (N-H); 3280 (N-H amide); 3110 (Ar-H); purin-8-ylsulfanyl)-N-(4-methoxyphenyl)propio-
2965 (C-H aliphatic); 1715, 1650 (C=O); 1530 (N-H); namide 29
833 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.8 (d,
J
= 6.9 IR cm−1: 3465 (N-H); 3265 (N-H amide); 3035 (Ar-H);
Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3), 3.7 (s, 3H, N3- 2930 (C-H aliphatic); 1693, 1640 (C=O); 1535 (N-H);
CH3), 4.6 (q, = 6.9 Hz, 1H, CH), 7.7 (d,
= 8.7 Hz, 1248, 1040 (C-O); 815 (Ar). 1H-NMR (60 MHz, DMSO-
2H, phenyl C-H), 8.3 (d, = 8.7 Hz, 2H, phenyl C-H), d6): 1.7 (d, = 6.9 Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3),
11 (s, 1H, amide N-H). EI-MS (m/z, % base): 393.59 3.7 (s, 3H, N3-CH3), 3.8 (s, 3H, 4'-OCH3), 4.9 (q,
(M+, 1.7), 395.06 (M++2, 0.6), 265.75 (100), 237.81 6.9 Hz, 1H, CH), 6.9 (d,
= 8.5 Hz, 2H, phenyl C-H),
= 8.5 Hz, 2H, phenyl C-H), 10.2 (s, 1H, amide
J
J
J
J
J
=
J
(24.9), 126.85 (56.2).
7.4 (d, J
N-H), 14.6 (br s, 1H, N7-H).
N-(2-Chlorophenyl)-2-(1,3-dimethyl-2,6-dioxo-2,3,
6,7-tetrahydro-1
mide 25
H
-purin-8-ylsulfanyl)propiona-
2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
purin-8-ylsulfanyl)-N-(4-nitrophenyl)propiona-
IR cm−1: 3455 (N-H); 3205 (N-H amide); 3030 (Ar-H); mide 30
2860 (C-H aliphatic); 1705, 1655, 1640 (C=O); 1530 IR cm−1: 3395 (N-H); 3275 (N-H amide); 3110 (Ar-H);
(N-H); 748 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.7 (d, 2875 (C-H aliphatic); 1710, 1688 (C=O); 1538 (N-H);
J
= 6.9 Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3), 3.7 (s, 3H, 1488, 1326 (NO2); 854 (Ar). 1H-NMR (60 MHz, DMSO-

8-Anilide Theophylline Derivatives as Bronchodilators
1359
d6): 1.9 (d,
(s, 3H, N3-CH3), 4.8 (q,
8.6 Hz, 2H, phenyl C-H), 8.3 (d,
C-H), 10.9 (s, 1H, amide N-H), 14.7 (br s, 1H, N7-H). 2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
EI-MS (m/z, % base): 404.31 (M+, 6.0), 265.90 (100), purin-8-ylsulfanyl)-N-naphthalen-1-yl propiona-
J
= 7 Hz, 3H, CH3), 3.5 (s, 3H, N1-CH3), 3.7 phenyl C-H), 9.1 (t,
= 7 Hz, 1H, CH), 7.9 (d, s, 1H, N7-H).
= 8.6 Hz, 2H, phenyl
J
= 6 Hz, 1H, amide N-H), 14.4 (br
J
J =
J
H-
236.95 (17.1), 137.96 (38.6).
mide 35
IR cm−1: 3465 (N-H); 3210 (N-H amide); 3045 (Ar-H);
2875 (C-H aliphatic); 1698, 1640 (C=O); 1532 (N-H);
4-[2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
purin-8-ylsulfanyl)-propionylamino]-benzoic acid 782 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.7 (d,
J
= 6.9
31
Hz, 3H, CH3), 3.4 (s, 3H, N1-CH3), 3.5 (s, 3H, N3-CH3),
IR cm−1: 3395 (N-H); 3330-2530 (broad O-H); 1710, 4.9 (q,
J = 6.9 Hz, 1H, CH), 7.3-7.9 (m, 7H, naphthyl
1664, 1640 (C=O); 1520 (N-H); 849 (Ar). 1H-NMR (60 C-H), 10.3 (s, 1H, amide N-H), 14.5 (br s, 1H, N7-H).
MHz, DMSO-d6): 1.6 (d,
N1-CH3), 3.5 (s, 3H, N3-CH3), 4.6 (q,
CH), 7.5 (d, = 8.7 Hz, 2H, phenyl C-H), 7.9 (d,
8.7 Hz, 2H, phenyl C-H), 10.9 (s, 1H, amide N-H), 12.7 36
(br s, 1H, COOH), 14.7 (br s, 1H, N7-H). EI-MS (m/z
IR cm−1: 3470 (N-H); 3260 (N-H amide); 3050 (Ar-H);
J
= 7 Hz, 3H, CH3), 3.3 (s, 3H,
= 7 Hz, 1H, 2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
purin-8-ylsulfanyl)-N-phenethyl prpoionamide
J
H-
J
J =
,
% base): 402.95 (M+, 5.3), 356.04 (13.8), 223.84 (28.6), 2945 (C-H aliphatic); 1695, 1645 (C=O); 1535 (N-H);
177.92 (100), 126.90 (5.5).
739, 692 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.2-1.7
(m, 5H, CH3 & CH2CH2Ph), 3.4 (s, 3H, N1-CH3), 3.7
2-[2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
purin-8-ylsulfanyl)-propionylamino]-benzoic acid 1H, CH), 7.5 (br. s, 5H, phenyl C-H), 8.6 (t,
H
-
(m, 5H, N3-CH3 & HNCH2 CH2), 4.8 (q,
J
= 6.9 Hz,
= 6 Hz,
J
32
1H, amide-H), 14.7 (br s, 1H, N7-H).
IR cm−1: 3450 (N-H); 3325-2520 (broad O-H); 1698,
1666, 1640 (C=O); 1531 (N-H); 750 (Ar). 1H-NMR (60 2-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1
H-
MHz, DMSO-d6): 1.6 (d,
3H, N1-CH3), 3.4 (s, 3H, N3-CH3), 4.5 (q,
1H, CH), 7.2-7.5 (m, 2H, phenyl C-H), 7.9 (d,
Hz, 1H, phenyl C-H), 8.4 (d, = 8.6 Hz, 1H, phenyl C- 2870 (C-H aliphatic); 1701, 1633 (C=O); 1535 (N-H);
H), 10.4 (br s, 1H, COOH), 10.7 (s, 1H, amide N-H), 742, 690 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.5 (d,
J
= 6.9 Hz, 3H, CH3), 3.3 (s, purin-8-ylsulfanyl)-N-(1-phenyl ethyl) prpoiona-
= 6.9 Hz, mide 37
= 8.6 IR cm−1: 3460 (N-H); 3265 (N-H amide); 3055 (Ar-H);
J
J
J
J
14.3 (br s, 1H, N7-H).
= 7 Hz, 3H), 2.2 (d,
J = 7 Hz, 3H), 3.4 (s, 3H, N1-CH3),
3.6 (s, 3H, N3-CH3), 4.6-5.0 (m, 2H, CH & CH), 7.3 (br
N-(4-Acetylphenyl)-2-(1,3-dimethyl-2,6-dioxo-2,3,
6,7-tetrahydro-1H-purin-8-ylsulfanyl)propiona-
mide (33)
s, 5H, phenyl C-H), 8.5 (d,
14.4 (br s, 1H, N7-H).
J = 7 Hz, 1H, amide-H),
IR cm−1: 3455 (N-H); 3245 (N-H amide); 3100 (Ar-H); N-Cyclohexyl-2-(1,3-dimethyl-2,6-dioxo-2,3,6,7-te-
2970 (C-H aliphatic); 1715, 1680, 1665 (C=O); 1525 trahydro-1
H-purin-8-yl-sulfanyl) prpoionamide
(N-H); 825 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.6 (d, 38
J
= 7 Hz, 3H, CH3), 2.5 (s, 3H, 4'-CH3CO), 3.3 (s, 3H, IR cm−1: 3440 (N-H); 3275 (N-H amide); 2900 (C-H
N1-CH3), 3.5 (s, 3H, N3-CH3), 4.5 (q,
J
= 7 Hz, 1H, aliphatic); 1710, 1640 (C=O); 1530 (N-H). 1H-NMR (60
J = 8.5 Hz, 2H, phenyl C-H), 7.8 (d, J = MHz): 0.74-2.17 (m, 13H, CH3 and cyclohexyl-(CH2)5),
CH), 7.6 (d,
8.5 Hz, 2H, phenyl C-H), 10.7 (s, 1H, amide N-H), 14.6 3.49 (s, 3H, N1-CH3), 3.74 (s, 4H, N3-CH3 & HNCH),
(br s, 1H, N7-H). EI-MS (m/z, % base): 401.24 (M+, 8.64 (s, 1H, amide N-H), 14.44 (br s, 1H, N7-H).
4.5), 355.82 (11.1), 223.84 (28.6), 265.63 (100), 237.92
(36.6), 134.93 (43.0).
General method for synthesis of compounds
43-46
N-Benzyl-2-(1,3-dimethyl-2,6-dioxo-2,3,6,7-tetra-
hydro-1H-purin-8-ylsulfanyl) propionamide 34
In a stirred solution of compound
in aqueous NaOH 1% (20-25 mL), an appropriate
IR cm−1: 3478 (N-H); 3265 (N-H amide); 3040 (Ar-H); amount of N-(substituted)aryl-3-chloropropionamides
2865 (C-H aliphatic); 1704, 1638 (C=O); 1535 (N-H); 39 42 (2.35 mmol) dissolved in the least amount of
738, 691 (Ar). 1H-NMR (60 MHz, DMSO-d6): 1.6 (d,
ethanol was added. The reaction mixture was stirred
4 (0.5 g, 2.35 mmol)
-
J
= 7 Hz, 3H, CH3), 3.3 (s, 3H, N1-CH3), 3.4 (s, 3H, N3- at an ambient temperature for 36-48 h and the reac-
CH3), 4.4-4.6 (m, 4H, CH & NH-CH2), 7.2 (br s, 5H, tion was monitored using TLC chromatography. The

1360
A. M. Hayallah et al.
Table II. Physical and microanalytical data of compounds 43-46
Microanalyses
mp (ºC)
Crystal. solvent
Mol. formula
(Mol. wt)
No.
R
Yield %
Calcd. %
Found %
C
H
N
S
53.47
4.77
19.49
−
53.23
5.02
19.37
−
238-240
Ethanol
C16H17N5O3S
(359.41)
43
4-Br
78
C
H
N
S
43.85
3.68
15.98
7.32
43.35
4.11
15.88
7.05
265-267
Ethanol
C16H16BrN5O3S
(438.31)
44
45
46
4-Cl
77
80
81
C
H
N
S
46.66
4.41
17.00
7.78
46.43
4.92
16.78
7.93
C16H16ClN5O3S.
H2O
277-279
aq. ethanol 30%
4-COOH
2-COOH
(411.87)
C
H
N
S
48.79
4.09
17.78
8.14
48.54
4.47
17.68
7.95
266-268
aq. methanol 20%
C16H16ClN5O3S
(393.85)
reaction mixture was cooled in a refrigerator for 6-8 h. 3H, N1-CH3), 3.5 (s, 3H, N3-CH3), 3.8 (t, 2H,
The product was filtered, washed with water, diethyl Hz, CH2), 7.6 (d, = 8.5 Hz, 2H, phenyl C-H), 7.9 (d,
ether, dried, and crystallized from the appropriate = 8.5 Hz, 2H, phenyl C-H), 10.8 (s, 1H, amide N-H),
solvent to afford the desired products 43 46. Physical 12.6 (br s, 1H, COOH), 14.5 (br s, 1H, N7-H).
J = 7.1
J
J
-
and micro-analytical data are given in Table II.
2-[3-(2,3,6,7-Tetrahydro-1,3-dimethyl-2,6-dioxo-1
H-
3-(2,3,6,7-Tetrahydro-1,3-dimethyl-2,6-dioxo-1H-
purin-8-ylthio)-N-(4-bromo-phenyl)propionamide
purin-8-ylthio)propion-amido]benzoic acid 46
IR cm−1: 3450 (N-H); 3325-2620 (broad O-H); 1698,
1666, 1640 (C=O); 1531 (N-H); 750 (Ar). 1H-NMR (60
43
IR cm−1: 3455 (N-H); 3285 (N-H amide); 3110 (Ar-H); MHz, DMSO-d6): 2.8 (t, 2H,
J = 7.3 Hz, CH2), 3.3 (s,
2970 (C-H aliphatic); 1715, 1650 (C=O); 1535 (N-H); 3H, N1-CH3), 3.4 (s, 3H, N3-CH3), 3.7 (t, 2H,
840 (Ar). 1H-NMR (60 MHz, DMSO-d6): 2.8 (t, 2H,
Hz, CH2), 7.2-7.4 (m, 2H, phenyl C-H), 7.9 (d,
7.2 Hz, CH2), 3.5 (s, 3H, N1-CH3), 3.7 (s, 3H, N3-CH3), Hz, 1H, phenyl C-H), 8.5 (d,
3.8 (t, 2H, = 7.2 Hz, CH2), 7.4 (d, = 8.6 Hz, 2H, H), 10.5 (br s, 1H, COOH), 10.7 (s, 1H, amide N-H),
J
= 7.3
J
=
J
= 8.6
J
= 8.6 Hz, 1H, phenyl C-
J
J
phenyl C-H), 8.1 (d,
1H, amide N-H).
J
= 8.6 Hz, 2H, phenyl C-H), 11 (s, 14.4 (br s, 1H, N7-H).
Bronchodilator activity
The bronchodilator activity was carried out following
3-(2,3,6,7-Tetrahydro-1,3-dimethyl-2,6-dioxo-1
H-
purin-8-ylthio)-N-(4-chloro-phenyl)propionamide Kesler and Canning’s method (Kesler and Canning,
44
1999) with minor modifications (Grosa et al., 1989).
IR cm−1: 3450 (N-H); 3265 (N-H amide); 3070 (Ar-H); Male Hartley guinea pigs, 300-400 g (House of Labora-
2890 (C-H aliphatic); 1710, 1655, 1645 (C=O); 1530 tory Animals, Faculty of Medicine, Assiut University)
(N-H); 810 (Ar). 1H-NMR (60 MHz, DMSO-d6): 2.7 (t, were anesthetized with urethane (1 g/kg i.p.) and posi-
2H,
J
= 7.2 Hz, CH2), 3.5 (s, 3H, N1-CH3), 3.6 (s, 3H, tioned ventral side up on a wooden pad. The trachea
= 7.2 Hz, CH2), 7.1 (d, = 8.5 was connected to a pump for artificial respiration, stain-
N3-CH3), 3.7 (t, 2H,
J
J
Hz, 2H, phenyl C-H), 7.9 (d,
H), 10.8 (s, 1H, amide N-H).
J
= 8.5 Hz, 2H, phenyl C- less steel hooks were passed between two cartilage
rings on either side of the trachea, one hook was su-
tured to a fixed bar, and the other hook was sutured to
4-[3-(2,3,6,7-Tetrahydro-1,3-dimethyl-2,6-dioxo-1
H
-
an isometric force transducer (Universal oscillograph).
When the animals were stabilized, a bronchospasm
purin-8-ylthio)propion-amido]benzoic acid 45
IR cm−1: 3395 (N-H); 3330-2580 (broad O-H); 1710, was stimulated with acetylcholine (0.2 mg/kg i.p.).
1664, 1640 (C=O); 1520 (N-H); 849 (Ar). 1H-NMR (60 After two similar responses to spasm inducing injec-
MHz, DMSO-d6): 2.7 (t, 2H,
J = 7.1 Hz, CH2), 3.3 (s, tions, target compounds (dissolved in distilled water

8-Anilide Theophylline Derivatives as Bronchodilators
1361
with a minimal amount of 1 N NaOH) (Baziard- method (Scott, 1989) for compounds having moderate
Mouysset et al., 1995) or aminophylline as a reference to strong antibacterial activity. The squares of inhibi-
standard were administered (2.5-10 mg/kg i.p.), and tion zone diameters were plotted against log concen-
acetylcholine was administered again three to five trations of the test compounds. Extrapolation of the
minutes later. The effects of the test compounds were resulting straight line intersecting with the log con-
expressed as means of percentage inhibition of five centration scale in the curve corresponds to log MIC,
experiments ± S.E.M. of the induced bronchospasm for and MIC was obtained as antilog (Hewitt, 1977).
three doses (2.5, 5, and 10 mg/kg body weight). The
ID50 value in each case was calculated by linear re-
gression (Raeburn et al., 1994). At the end of each ex-
periment, animals were killed by cervical dislocation.
Acute toxicity (LD50)
Groups of five male adult albino mice, 18-22 g
(House of Laboratory Animals, Faculty of Medicine,
Assiut University), were injected i.p. with 4 graded
doses of the test compounds suspended in 0.5% car-
boxymethylcellulose. LD50 was calculated based on the
Antibacterial activity
Organisms and culture conditions
Four bacterial species representing both Gram number of animals showing decreased muscle tone,
positive and Gram negative strains were used to test and labored respiration signs using the Litchfield and
the antibacterial activity of the target compounds: Wilcoxon’s method (Litchfield and Wilcoxon, 1949;
methicillin resistant Staphylococcus aureus (MRSA), Grosa et al., 1989; Akhila et al., 2007).
Bacillus cereus
moniae (clinical isolates were obtained from Infection
Control Unit, Assiut University Hospital, Faculty of fication
, Escherichia coli, and Klebsiella pneu-
Receptor building and pharmacophore identi-
Medicine, Assiut University).
All the computational works were carried out at the
Department of Medicinal Chemistry, Faculty of Phar-
macy, Assiut University, Assiut, Egypt. Receptor build-
µL of ing and pharmacophore identification were performed
Agar cup diffusion method
The sterile Petri dishes were seeded with 100
the microorganism: a specified amount of the molten on the Molecular Operating Environment (MOE) ver-
Mueller-Hinton (MH) agar medium (45-50ºC) was sion 2007.09, Chemical Computing Group Inc., 1010
poured into the seeded Petri dishes to give a depth of Sherbrooke St. West, Suite 910, Montreal, Quebec,
3-4 mm and allowed to solidify. Cylindrical plugs were H3A 2R7, Canada. The program operated under the
removed from the agar using a sterile cork borer. One “Microsoft Windows XP” operating system installed on
hundred µL of the test compounds or ampicillin sodium an Intel Pentium IV PC with a 2.8 GHz processor and
(20 mg/mL in DMSO), or the solvent, were added to 512 Mb of RAM.
the wells in triplicate. The seeded plates were incubat-
ed at 37°C for 24 h, the average diameters of the
inhibition zones were then measured in millimeters.
RESULTS AND DISCUSSION
Chemistry
Minimum inhibitory concentration
The key intermediate 1,3-dimethyl-8-thioxo-3,7,8,9-
The MIC was determined using the two-fold dilution tetrahydro-1H-purine-2,6-dione (4) was prepared from
Fig. 2. Synthetic pathway for compound 4.

1362
A. M. Hayallah et al.
compound
3
, as illustrated in Fig. 2, in reaction with around 3455 cm−1, presence of strong absorption bands
CS2 in anhydrous DMF, based on previous reported at 1712-1619 cm−1 corresponding to C=O stretching
methods (Hayallah, 2007, 2011; Elgaher et al., 2009). bands, and also the absence of C=S stretching band.
The structure was proven using IR and 1H-NMR.
1H-NMR spectra of these compounds showed the
The intermediates (un)substituted 2-bromo-propio- appearance of a doublet around
namides 21 and -substituted-3-chloropropionamides ponding to -S-CH-CH3 protons; the quartet around
39 42 were prepared according to reported methods 4.5-4.9 was equivalent to one proton of -S-CH-CH3
(Malatesta et al., 1962; Sadanandam et al., 1972; proton. In addition, the appearance of singlet at
δ 1.5-1.9 ppm corres-
5-
N
δ
-
δ
Snatzke and El-Abadelah, 1973; Snatzke et al., 1973; 10.66 ppm was equivalent to one proton corresponding
Fontanella et al., 1981; El-Abadelah, 1982; Malatesta to the monosubstituted amide group; broad singlet
et al., 1982; El-said et al., 1993), and their structures around
were verified using 1H-NMR.
multiplet at
Alkylation of with -(substituted)aryl/aralkyl/ aromatic protons of most of the derivatives that also
cyclo-alkyl 2-bromo-propionamides
21 in the pre- appeared. The aforementioned 1H-NMR data are proof
sence of aqueous ethanolic NaOH 1% furnished 2- in the formation of the new derivatives.
[(1,3-Dimethylxanthin-8-yl)thio]- -substituted-2-pro- [(1,3-Dimethylxanthin-8-yl)thio]- -substituted-3-pro-
pionamides 22 38 (Fig. 3). Structures of the new pionamides 43 46, (Fig. 4) were prepared by alkyla-
compounds 22 with -(substituted)
38 were verified by IR, 1H-NMR, mass tion of theophylline-8-thione
spectroscopy, and elemental analyses, as illustrated aryl-3-bromo-propionamides 39 42 in the presence of
δ
14.49 ppm corresponding to N7-H, and a
δ
6.92-8.94 ppm corresponding to the
4
N
5-
N
N
-
-
-
4
N
-
above and displayed in Table I. The IR spectra of the aqueous ethanolic NaOH 1% (Dietz and Burgison,
newly synthesized compounds showed the appearance 1966) with slight modifications. Their structures were
1
1
of a new N-H stretching band around 3260 cm−
verified by IR, H-NMR, and elemental analysis as
1
characteristic of monosubstituted amides, in addition illustrated above. The H-NMR spectra of compounds
to the original N7-H stretching band of xanthine 43-46 are characterized by the appearance of a pair of
Fig. 3. Synthetic pathway for the preparation of compounds 22-38.

8-Anilide Theophylline Derivatives as Bronchodilators
1363
Fig. 4. Synthetic pathway for the preparation of compounds 43-46.
Table III. Inhibitory effects of the test compounds on
acetylcholine induced bronchospasm in anaesthetized
guinea pigs
triplet around
δ 2.7-2.8 and δ 3.7-3.8 corresponding to
OC-CH2-CH2-S respectively. In addition, the introduced
aromatic moiety and the presence of N7-H signal is a
strong support for an S-alkylation reaction rather
than N-one. It is known that mercaptopurines undergo
S-alkylation at a lower temperature, while N7-H is
also attacked when the temperature is elevated
(Lister, 1971). The preparation of new derivatives 43
46 takes a longer reaction time when compared to
derivatives 22 38 due to the lower reactivity of
substituted-3-chloropropionamides 39 42 in compari-
Dose % Decrease of acetylcholine
Com-
pound
ID50
mg/kg i.p.
mg/kg
induced bronchospasm in
guinea pigs
i.p.
2.5
5
10
22.5 ± 1.1
48.6 ± 1.4
78.8 ± 1.1
Amino-
phylline
5.8
>20
6.7
-
-
N-
2.5
5
10
5.8 ± 0.5
15.7 ± 1.19
18.0 ± 1.37
-
22
23
24
25
26
27
28
29
son to the isomers 5-21.
2.5
5
10
18.5 ± 0.7
40.7 ± 1.6
67.7 ± 1.2
Pharmacology
Bronchodilator activity
Following Kesler and Canning’s method, all the
synthesized compounds were investigated for in vivo
anti-bronchospatic activity on acetylcholine-induced
bronchospasm in anaesthetized guinea pigs in com-
parison to aminophylline as a reference drug. The anti-
bronchoconstrictive effect was expressed as a per-
centage inhibition (mean ± S.E.M.) of bronchospasm
for three doses (2.5, 5, and 10 mg/kg body weight). The
ID50 value (the dose of the drug causing 50% inhibi-
tion of bronchospasm) in each case was calculated by
linear regression. Results are shown in Table III.
Eight compounds (23
exhibited an anti-bronchoconstrictive activity nearly
similar to aminophylline. Compounds 24 32 37, and
38 showed either a more significant or equivalent
effect to aminophylline.
2.5
5
10
22.2 ± 1.3
47.0 ± 1.7
76.0 ± 1.9
6
2.5
5
10
20.5 ± 1.27
38.6 ± 1.49
61.3 ± 1.67
7.7
2.5
5
10
−
>20
8.5
7.8 ± 0.56
11.9 ± 0.94
2.5
5
10
19.7 ± 1.3
35.7 ± 1.57
56.0 ± 1.85
, 24, 31, 32, 37, 38, 45, and 46)
,
,
2.5
5
10
−
>20
7.3
7.9 ± 0.51
12.3 ± 1.07
Regarding the results of
(theophyllin-8-ylthio)-2-propionamides series 22
the meta-substituted derivatives 26 and 28, and the
para-acetyl 33 revealed a very weak activity (ID50 >20
mg/kg). These results are similar to 2-[(1,3-Dimethyl-
N-(substituted)phenyl-2-
-
38
,
2.5
5
10
20.6 ± 1.2
39.2 ± 1.5
64.7 ± 1.9

1364
A. M. Hayallah et al.
Table III. Continued
Table IV. Antibacterial activity of the test compounds
In vitro activity-inhibition zone in mm
(MIC in µg/mL)
Dose % Decrease of acetylcholine
Com-
pound
ID50
mg/kg i.p.
mg/kg
i.p.
induced bronchospasm in
guinea pigs
Com-
pound
Methicillin
resistant
Staphylococcus cereus
Bacillus Escherichia Klebsiella
2.5
5
10
11.5 ± 1.1
31.7 ± 1.6
41.5 ± 1.8
coli
pneumoniae
30
31
32
33
34
35
36
37
38
11.9
6.6
aureus (MRSA)
a
DMSO
Ampicillin
Nalidi-
xic acid
−
−
−
−
2.5
5
21.8 ± 1.1
44.2 ± 1.51
69.8 ± 1.46
22 (70)
22 (60)
27 (50)
20 (20)
27 (30)
20 (67)
27 (25)
30 (20)
10
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
43
44
45
46
17 (−)
−
17
22
2.5
5
10
26.1 ± 1.19
47.1 ± 1.54
75.5 ± 1.83
32 (22)
10
28 (31)
21 (73)
20 (75)
29 (33)
20
17 (120)
17 (110)
18 (126)
17
18 (125)
21 (63)
27 (45)
21
5.9
2.5
5
7.4 ± 0.65
15.8 ± 1.11
24.5 ± 1.38
−
>20
17.1
8.7
2
11
14
−
10
35 (20)
23 (66)
11
16 (140)
−
18 (130)
−
−
−
2.5
5
10
7.8 ± 0.47
15.7 ± 1.27
29.4 ± 1.4
−
−
−
17 (125)
16 (153) 15 (158)
15 (158)
19 (75)
32 (25)
−
−
−
−
22 (72)
32 (23)
29 (43)
17 (143)
−
17 (168)
31 (25)
−
−
2.5
5
19.0 ± 1.4
35.7 ± 1.6
54.8 ± 1.47
32 (27)
28 (30)
18 (115)
−
10
−
2.5
5
10
12.8 ± 1.04
23.5 ± 1.24
37.5 ± 1.46
−
−
−
−
−
13.7
5.8
−
−
−
31 (22)
12
19 (120)
16 (122)
21 (75)
22 (75)
17 (133) 14 (148)
20 (78) 19 (162)
18 (110)
16 (90)
19 (115)
23 (63)
16 (159)
18 (78)
2.5
5
26.7 ± 1.48
47.4 ± 1.54
76.2 ± 1.8
10
a
2.5
5
10
22.3 ± 1.33
45.7 ± 1.50
75.5 ± 1.91
(
−
) means no antibacterial activity at the studied concen-
tration.
6.1
and 7.8 mg/kg respectively) (Elgaher et al., 2009). For
naphthyl derivative 35 and 1-phenethyl derivative 37
N-substituted-acetamide derivatives the ID50 values were markedly enhanced from >20 to
,
xanthin-8-yl)thio]-
(Elgaher et al., 2009), which ensures that the meta- 8.7 and from 7.3 to 5.8 mg/kg, respectively, in com-
substituted derivatives are the weakest derivatives. parison with acetamide derivatives (Elgaher et al.,
The biological activities of these derivatives are also in 2009). On the other hand, 2-phenethyl derivative 36
agreement with their calculated RMSD values (0.2950, demonstrated a lower activity (ID50 value: 13.7 mg/kg)
0.2815, and 0.2553, respectively) (Table V). Among the when compared to the same analogue of acetamide
various para-substituted phenyl groups, the
p-bromo derivative (ID50 value: 9.1 mg/kg, Elgaher et al., 2009).
23 -chloro 24, and p-COOH 31, derivatives have The selected ortho-substituted derivatives (25 and 32)
,
p
significant activities (ID50 values: 6.7, 6.0, and 6.6 mg/ showed significant activity (ID50 values: 7.7 and 5.9
kg, respectively). Several derivatives of p-substituted mg/kg, respectively). Accordingly, the best position for
derivatives of
N-(substituted)phenyl-2-(theophyllin-8- substitution at the phenyl group is the para and ortho,
ylthio)-2-propionamides series, such as p-CH3 27, p- which leads to active derivatives. Activity may be due
OCH3 29, and p-COOH 31, showed enhanced ID50 to the non-planar orientation of the phenyl group with
values (8.5, 7.3, and 6.6 mg/kg, respectively) when the 2-(theophyllin-8-ylthio)acetamide or propionamide
compared to 2-[(1,3-Dimethylxanthin-8-yl)thio]-N-sub- moiety due to the presence of a bulky group at the
stituted-acetamide derivatives (ID50 values: >20, 9.6 ortho position. This assumption is in agreement with

8-Anilide Theophylline Derivatives as Bronchodilators
1365
Table V. RMSD values of some representative compounds
preferable to the straight chain one.
Compound
RMSD
Microbiology
22
23
24
26
28
31
32
33
43
44
45
46
0.2328
0.2138
0.2073
0.2950
0.2815
0.2053
0.2096
0.2553
0.3320
0.2464
0.4147
0.2207
Antibacterial activity of all the synthesized target
compounds was investigated in vitro against the Gram-
positive bacteria methicillin resistant Staphylococcus
aureus (MRSA) and Bacillus cereus, and the Gram-
negative bacteria Escherichia coli and Klebsiella pneu-
moniae using the agar diffusion assay (Clark et al.,
1981). MIC of the active compounds was also calculat-
ed in comparison to nalidixic acid and ampicillin as a
reference drug (Table IV).
Analysis of the antibacterial results showed that
compounds 23
parable or better activities (MIC 22-30
MRSA than ampicillin and nalidixic acid. They were
Baziard-Mouysset et al. (1995) for the bronchodilator also active against B. cereus (MIC 23-75 g/mL), sev-
activity of various 8-substituted theophylline derivatives. eral of which, including 25 33, and 34, were even
[(1,3-Dimethylxanthin-8-yl)thio]- -substituted-3-pro- more potent than ampicillin and nalidixic acid (MIC
pionamides 43 46 were synthesized according to the 23-43 g/mL). Compound 33 was the most active
calculated RMSD values (Table V) for these compounds compound against all bacterial strains (MIC 23-27 g/
and their isomers [(1,3-Dimethylxanthin-8-yl)thio]- mL).
substituted-2-propionamide series, which showed signi- These compounds showed a significant activity
ficant activity. These derivatives 43 46 showed a de- (MIC 45-158 g/mL) against Gram-negative bacteria
crease of the bronchodilator activity of three derivatives strains E. coli and K. pneumoniae except for com-
43 44, and 45 (ID50 values: 8.8, 6.7, and 8.7 mg/kg, pound 34, which did not show any activity against
respectively), in comparison with 23 24, and 31 of the Gram-negative bacteria. These results indicate that
[(1,3-Dimethylxanthin-8-yl)thio]- -substituted-2-pro- these derivatives showed better activity against
,
25
,
28
,
33
,
34, and 43 exhibited com-
µg/mL) against
µ
,
N
-
µ
µ
N-
-
µ
,
,
N
pionamide series which showed ID50 values 6.7, 6.0, Gram-positive bacteria when compared to the Gram-
and 6.6 mg/kg, respectively (Table III). The ortho- negative one.
substituted carboxy derivative 46 is the only one that
Compounds 44-46 showed a significant to moderate
retained its high bronchodilator activity (ID50 values: activity (MIC 63-159
µg/mL) against both Gram-
6.2 mg/kg) if compared to its isomer 32 (ID50 values: positive and Gram-negative bacterial strains, while
6.0 mg/kg). This may be because one of the best posi- compound 35 showed a moderate activity (MIC 115-
tions for substitution at the phenyl group is the ortho 143
position, which leads to active derivatives (Elgaher et and B. cereus) and compound 24 was active against all
al., 2009). The activity may be due to a non-planar orien- bacteria strains except B. cereus
tation of the phenyl group with the 2-(theophyllin-8- In a trial to find a possible mechanism of action for
µg/mL) against only Gram-positive bacteria (MRSA
.
ylthio)acetamide and 2-(theophyllin-8-ylthio)-propi- the title compounds, the following reported studies
onamide moiety due to the presence of a bulky group on the interaction of DNA or RNA with xanthine
at the ortho position. This assumption is in agreement derivatives were considered. It was found that caf-
with that of (Baziard-Mouysset et al., 1995), for the feine inhibits the incorporation of adenine and thymi-
bronchodilator activity of various 8-substituted theo- dine in the synthesis of DNA (Labbe and Nolan, 1987);
phylline derivatives. In addition, the hydrogen bond of it also inhibits the synthesis of DNA (Sandlie et al.,
carboxy group at this position may play a vital role for 1980) and RNA (Pérez et al., 1994). Among the three
the biological activity of these derivatives as new xanthine derivatives, theophylline showed a greater
potential bronchodilator agents. Finally, an increase binding efficacy with RNA than theobromine and
in the carbon chain between the 8-sulfur and the amide caffeine (Johnson et al., 2003). G-C and A-U bases of
function group may lead to more potent bronchodilator RNA were the targets for binding. At the same time,
agents of this series. The pharmacophore study, the complexation occurred with the G-C and A-T bases
calculating both RMSD and biological results, also and PO2 group of DNA through hydrogen bonding
indicated that the increase of carbon chain length (Nafisi et al., 2008). Considering these data, we sug-
from acetamide to iso-propionamide derivatives is gest that the mechanism of action of the target xanthine

1366
A. M. Hayallah et al.
derivatives as antibacterial agents is through the
inhibition of nucleic acid synthesis.
Acute toxicity (LD50)
Acute toxicity (LD50) study was performed in mice
via intraperitoneal (i.p.) injection for the most active
derivatives (compounds 24, 32, 37, and 38) and com-
pared to aminophylline as a reference drug. The ob-
tained experimental data showed that not all of the
test compounds recorded significant toxicity with LD50
= 300 mg/kg in comparison with the standard drug
aminophylline LD50 = 180 mg/kg (Peikov et al., 1994)
(Table VI). The maximal toxicity was observed after
12 h, when the animals showed decreased muscle tone
and strenuous respiration signs.
Fig. 5. Mapping of compound 32 onto hypothetical model.
contains RMSD, i.e., the root mean square distance
between the query features and their corresponding
ligand target points. The smaller the RMSD, the better
Receptor building and pharmacophore identi-
fication
It is known that the actual molecular mechanism of fitting the query compound has.
action of xanthine derivatives as bronchodilators is
The mapping of compound 32 onto the pharmaco-
controversial (Howell, 1990; Barnes and Pauwels, phore model is shown in Fig. 5. It can be seen that the
1994). The inhibition of phosphodiesterase III and IV chemical functionalities of the hypothesis are all
isoenzymes relaxes smooth muscles in pulmonary matched by the chemical groups of the molecule: N1
arteries and air ways (Hall, 1993), while antagonists atom, imidazole ring, and C6 carbonyl group fitted the
of adenosine A2B receptors were proposed to have region of ML/HydS/HydP/AccP/AccS/ DonP/DonP, F1;
potential use as antiasthmatic agents (Feoktistov et C2 carbonyl group fitted the region of AccP/ML, F2;
al., 1998). However, it is necessary to determine the N9 fitted the region of ML/HydP/AccP, F3; N3 methyl
important attributes of the active site to design better group fitted the region of HydS/HydP, F4; N1-methyl
drugs. One way to suggest the properties of the active group fitted the HydS, F5; sulfur atom and methylene
sites is to assume that they are complementary to group fitted the region of HydS/HydP/ML/AccP, F6.
active lead molecules. Before the receptor model can
be built, the lead molecules must be aligned so that
the active functional groups of the molecules overlap
in space. All the computational works were performed
ACKNOWLEDGEMENTS
The authors are greatly indebted to Prof. Dr. Ahmed
on the Molecular Operating Environment (MOE) ver- Osman, Department of Pharmacology, Faculty of
sion 2007.09, Chemical Computing Group Inc. Thirteen Medicine, Assiut University, for his kind supervision
reported active ligands were selected as the training of the bronchodilator activity studies. Great thanks
set. Two of them, theophylline and bamifylline, were are also expressed to Helal F. Hetta, Department of
in therapeutic use. The receptor model and the phar- Microbiology and Immunology, Faculty of Medicine,
macophore query were built as reported before (Elgaher Assiut University, Assiut, for his sincere help in the
et al., 2009).
screening of the antibacterial activity, and to Walid
A pharmacophore search was done for our target Elgaher, Department of Pharm. Org. Chemistry, As-
compounds: the output of the pharmacophore search siut University for his sincere help in the pharma-
cophore study.
Table VI. Acute toxicity in mice following ip injection of
compounds 24, 32, 37, and 38
REFERENCES
Compound
LD50 mg/kg
Akhila, J. S., Shyamjith, D., and Alwar, M. C., Acute toxicity
studies and determination of median lethal dose. Curr.
Sci., 93, 917-920 (2007).
Barnes, P. J. and Pauwels, R. A., Theophylline in the mana-
gement of asthma: time for reappraisal? Eur. Respir. J., 7,
579-591 (1994).
24
32
37
38
300
300
>300
300
Aminophylline
aLD50 value as reported (Peikov et al., 1994).
180a

8-Anilide Theophylline Derivatives as Bronchodilators
1367
Baziard-Mouysset, G., Rached, A., Younes, S., Tournaire, C.,
Stigliani, J. L., Payard, M., Yavo, J. C., and Advenier, C.,
Synthesis and in vitro bronchospasmolytic activity of 8-
aryl, heteroaryl or arylalkyl theophyllines. Eur. J. Med.
Chem., 30, 253-260 (1995).
Bisgaard, H., Hermansen, M. N., Buchvald, F., Loland, L.,
Halkjaer, L. B., Bønnelykke, K., Brasholt, M., Heltberg,
A., Vissing, N. H., Thorsen, S. V., Stage, M., and Pipper,
C. B., Childhood asthma after bacterial colonization of the
airway in neonates. N. Engl. J. Med., 357, 1487-1495
(2007).
Blasi, F. and Johnston, S. L., The role of antibiotics in asthma.
Int. J. Antimicrob. Agents, 29, 485-493 (2007).
Burr, M. L., Butland, B. K., King, S., and Vaughan-Williams,
E., Changes in asthma prevalence: two surveys 15 years
apart. Arch. Dis. Child., 64, 1452-1456 (1989).
Clark, A. M., El-Feraly, F. S., and Li, W. S., Antimicrobial
activity of phenolic constituents of Magnolia grandiflora
L. J. Pharm. Sci., 70, 951-952 (1981).
Cookson, W., The immunogenetics of asthma and eczema: a
new focus on the epithelium. Nat. Rev. Immunol., 4, 978-
988 (2004).
Dietz, A. J., Jr., and Burgison, R. M., The synthesis of some
8-alkylthio-2-thiotheophyllines and 8-alkylthio-6-thiotheo-
phyllines. J. Med. Chem., 9, 160 (1966).
Hayallah, A. M., Elgaher, W. A., Salem, O. I., and Alim, A. A.,
Design and synthesis of some new theophylline derivatives
with bronchodilator and antibacterial activities. Arch.
Pharm. Res., 34, 3-21 (2011).
Hewitt, W., Microbiological assay an introduction to quanti-
tative principles and evaluation. Academic Press, New
York, pp. 17-69, (1977).
Howell, R. E., Multiple mechanisms of xanthine actions on
airway reactivity. J. Pharmacol. Exp. Ther., 255, 1008-
1014 (1990).
Jedrychowski, W., Wojtyniak, B., Szafraniec, K., and
Gorynski, P., Trends in hospitalization rates of childchood
asthma in Poland. Central Europ. J. Occup. Environ.
Med., 10, 275-282 (2004).
Johnson, I. M., Kumar, S. G., and Malathi, R., RNA binding
efficacy of theophylline, theobromine and caffeine. J.
Biomol. Struct. Dyn., 20, 687-692 (2003).
Johnston, S. L., Pattemore, P. K., Sanderson, G., Smith, S.,
Lampe, F., Josephs, L., Symington, P., O'Toole, S., Myint,
S. H., Tyrrell, D. A. J., and Holgate, S. T., Community
study of role of viral infections in exacerbations of asthma
in 9-11 year old children. BMJ, 310, 1225-1229 (1995).
Kesler, B. S. and Canning, B. J., Regulation of baseline
cholinergic tone in guinea-pig airway smooth muscle. J.
Physiol., 518, 843-855 (1999).
Eder, W., Ege, M. J., and von Mutius, E., The asthma epi-
demic. N. Engl. J. Med., 355, 2226-2235 (2006).
El-Abadelah, M. M., Optical rotatory power of some halo-
propionanilides. Egyptian Journal of Chemistry, 16, 401-
409 (1973).
Kraft, M., The role of bacterial infections in asthma. Clin.
Chest. Med., 21, 301-313 (2000).
Labbe, R. G. and Nolan, L. L., Inhibition of macromolecular
synthesis by caffeine in Clostridium perfringens. Can. J.
Microbiol., 33, 589-592 (1987).
Elgaher, W. A., Hayallah, A. M., Salem, O. I. A., and Abdel
Alim, A. M., Synthesis, anti-bronchoconstrictive, and anti-
bacterial activities of some new 8-substituted-1,3-dimeth-
Lister, J. H., The Chemistry of Heterocyclic Compounds:
Brown, D. J. (Ed). Fused Pyrimidines, Wiley - Interscience,
New York, pp. 278-281, (1971).
ylxanthine derivatives. Bull. Pharm. Sci. Assiut University
32, 153-187 (2009).
,
Litchfield, J. T., Jr., and Wilcoxon, F., A simplified method of
evaluating dose-effect experiments. J. Pharmacol. Exp.
Ther., 96, 99-113 (1949).
Malatesta, P., Migliaccio, G., and Aliberti, U., Synthesis of
some new anilides with local anesthetic activity. Farmaco
Sci., 17, 601-610 (1962).
El-Said, M. K., Aly, S. M. E., Romeih, F. A., Barsoum, F. F.,
and Hassan, A. B., Synthesis and pharmacological screen-
ing of some new phenothiazine derivatives. Bulletin of the
Faculty of Pharmacy (Cairo University), 31, 181-186 (1993).
Feoktistov, I., Polosa, R., Holgate, S. T., and Biaggioni, I.,
Adenosine A2B receptors: a novel therapeutic target in
asthma? Trends Pharmacol. Sci., 19, 148-153 (1998).
Fontanella, L., Corsico, N., Diena, A., Galliani, G., and Glässer,
A., Synthesis of new psychotropic 2-imidazolidinones.
Farmaco Sci., 36, 3-12 (1981).
Grosa, G., Caputo, O., Ceruti, M., Biglino, G., Franzone, J.
S., and Cirillo, R., Synthesis and antibronchospastic activ-
ity of theophylline thioacetal derivatives. Eur. J. Med.
Chem., 24, 635-638 (1989).
Hall, I. P., Isoenzyme selective phosphodiesterase inhibi-
tors: potential clinical uses. Br. J. Clin. Pharmacol., 35, 1-
7 (1993).
Malatesta, P., Bianchi, B., Aliberti, U., and Malatesta, C.,
Synthesis of basic anilides with raised local anesthetic
activity. III. Boll. Chim. Farm., 121, 443-455 (1982).
Nafisi, S., Manouchehri, F., Tajmir-Riahi, H.-A., and Varavi-
pour, M., Structural features of DNA interaction with
caffeine and theophylline, J. Mol. Struct., 875, 392-399
(2008).
Pawlinska-Chmara, R., Wronka, I., and Muc, M., Prevalence
and correlates of allergic diseases among children. J.
Physiol. Pharmacol., 59 Suppl 6, 549-556 (2008).
Peikov, P. T., Zlatkov, A. B., Markov, M. T., Danchev, N. D.,
Ivanov, D. I., and Panova, J. T., Synthesis, toxicological and
pharmacological assessment of 7-substituted derivatives of
1,3-dimethylxanthine. Eur. J. Med. Chem., 29, 295-299
(1994).
Hayallah, A. M., Design and synthesis of new 1,8-disubsti-
tuted purine-2,6-diones and 3,6-disubstituted thiazolo[2,3-
f
]purine-2,4-diones as potential antinociceptive and anti-
Pérez, C., Pelayo, F., Vilaboa, N. E., and Aller, P., Caffeine
attenuates the action of amsacrine and etoposide in U-937
inflammatory agents. Pharmacia, 54, 3-13 (2007).

1368
A. M. Hayallah et al.
cells by mechanisms which involve inhibition of RNA
synthesis. Int. J. Cancer, 57, 889-893 (1994).
ive pulmonary disease. N. Engl. J. Med., 347, 465-471
(2002).
Raeburn, D., Underwood, S. L., Lewis, S. A., Woodman, V.
R., Battram, C. H., Tomkinson, A., Sharma, S., Jordan, R.,
Souness, J. E., Webber, S. E., and Karlsson, J.-A., Anti-
inflammatory and bronchodilator properties of RP 73401,
a novel and selective phosphodiesterase type IV inhibitor.
Br. J. Pharmacol., 113, 1423-1431 (1994).
Snatzke, G. and El-Abadelah, M. M., Synthesis of optically
active halopropionanilides. Chem. Ber., 106, 2072-2075
(1973).
Snatzke, G., El-Abadelah, M. M., and Nazer, M. Z., Circular
dichroism. LVII. Chiroptical properties of some
α-halo-
propionanilides. Tetrahedron, 29, 487-495 (1973).
Sadanandam, Y. S., Sattur, P. B., and Sidhu, G. S., Synthe-
sis and pharmacology o N-chloracyl-benzylamines. Arch.
Pharm. (Weinheim), 305, 891-901 (1972).
Sandlie, I., Solberg, K., and Kleppe, K., The effect of caf-
feine on cell growth and metabolism of thymidine in
von Mutius, E., Illi, S., Hirsch, T., Leupold, W., Keil, U., and
Weiland, S. K., Frequency of infections and risk of asthma,
atopy and airway hyperresponsiveness in children. Eur.
Respir. J., 14, 4-11 (1999).
Woolcock, A. and Peat, J. K., Evidence for the increase in
asthma worldwide. In: The Rising Trends in Asthm,
Chadwick, D. J. and Cardew, G. (Eds). Chichester, Ciba
Foundation, J. Wiley and Sons, pp. 122-134, (1997).
Yunginger, J. W., Reed, C. E., O'Connell, E. J., Melton, L. J.,
3rd, O'Fallon, W. M., and Silverstein, M. D., A communi-
ty-based study of the epidemiology of asthma. Incidence
rates, 1964-1983. Am. Rev. Respir. Dis., 146, 888-894
(1992).
Escherichia coli. Mutat. Res., 73, 29-41 (1980).
Scott, A. C., Laboratory control of antimicrobial therapy, In
Collee, J. G., Duguid, J. P., Fraser, A. G., and Marmion, B.
P. (Eds.). Mackie & McCartney Practical Medical Micro-
biology. Churchill Livingstone, Edinburgh, pp. 161-181,
(1989).
Sethi, S., Evans, N., Grant, B. J., and Murphy, T. F., New
strains of bacteria and exacerbations of chronic obstruct-