Synthesis of certain pyrimidine derivatives as antimicrobial agents and anti-inflammatory agents

Article abstract of DOI:10.3390/molecules15031882

A variety of novel bicyclic and tricyclic pyrimidine derivatives was obtained via reaction of 6-amino-2-thioxo-1H-pyrimidine-4-one (1) with a different reagents. The antimicrobial and anti-inflammatory activities of some of the synthesized compounds were

Full text of DOI:10.3390/molecules15031882

Molecules 2010, 15, 1882-1890; doi:10.3390/molecules15031882
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Certain Pyrimidine Derivatives as Antimicrobial
Agents and Anti-Inflammatory Agents
Mosaad S. Mohamed, Samir M. Awad and Amira Ibrahim Sayed *
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University,
Helwan, Egypt
* Author to whom correspondence should be addressed; E-Mail: amiraelsaedy@yahoo.com.
Received: 11 January 2010; in revised form: 11 March 2010 / Accepted: 12 March 2010 /
Published: 15 March 2010
Abstract: A variety of novel bicyclic and tricyclic pyrimidine derivatives was obtained via
reaction of 6-amino-2-thioxo-1H-pyrimidine-4-one (1) with a different reagents. The
antimicrobial and anti-inflammatory activities of some of the synthesized compounds were
tested.
Keywords: pyrimidine; 6-amino-2-thioxo-1H-pyrimidine-4-one; antimicrobial; anti-
inflammatory
1. Introduction
Pyrimidines exhibit a range of pharmacological activity such as antibacterial [1–3], antifungal [4,5],
anticancer [6,7], anti-inflammatory [8,9] and cardioprotective effects [10]. These observations led us to
attempt the synthesis of some new pyrimidine derivatives using 6-amino-2-thioxo-2,3-dihydro-1H-
pyrimidine-4-one (1) as starting material.
2. Results and Discussion
The requisite starting material 1 was prepared by the condensation of thiourea with
ethylcyanoacetate in sodium ethoxide according to the known procedure [11]. This compound was
previously used in the synthesis of several pyrimidines, for example 6-{[1-phenyl or (4-

Molecules 2010, 15
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methoxyphenyl)-methylidene]-amino}-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one [12] and 7-amino-5-
(4-chlorophenyl)- or (4-methoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-
carbonitrile [13].
Stirring of 1 with primary amines and excess formalin in methanol in presence of acetic acid
afforded 6-aryl-2-thioxo-2,3,5,6,7,8-hexahydro-1H-pyrimido[4,5-d]pyrimidine-4-ones 2a-c (Scheme
1) and not the 8-hydroxymethyl derivatives of 2a-c, as reported earlier [11], a conclusion supported by
1
the absence of signals corresponding to an 8-CH2OH group in the H-NMR spectra of the
products 2a-c.
Reaction of 1 with aromatic aldehydes in methanol containing few drops of hydrochloric acid
(pH = 5-6) led to the formation of 9-substituted-1,3,6,8,9,10-hexahydro-2,7-dithioxopyrido[2,3-d:6,5
d`] dipyrimidine-4,6-diones 3a-e. In this reaction, the polar and acidic condition of solvent make the
reaction occur through the formation of a 6-imino group that leads to higher nucleophilicity of C-5
whereby attack occurs at the aldehyde carbonyl group (Scheme 1).
Scheme 1. Synthetic pathway of compounds (1–5).
O
S
5
4
3
Ar
-C6H5
a , Ar =
b, Ar = o -CH3-C6H4-
-CH -C H -
2 H2C=O/ArNH2
MeOH/AcOH
HN
N 6
H2N
NH2
2
7
c, Ar =
m
3
6
4
+
S
1 N
N 8
H
H
O
2a-c
O
NC
O
Ar
3
O
OC2H5
p -Br-C H -
a,Ar =
b,Ar =
6
4
5
4
1
4
5
6
3
HN
ArCHO
3,4,5 -(OCH3)3-C6H2-
9
HN
NH
EtONa
HCl/ MeOH
2
c,Ar = 3-indolyl
d,Ar =
2
S
N
H
NH2
6
10
7
S
N 8
H
N
H
3a-e
N
S
p -N(CH3)2-C6H4-
H1
1
ArCHO
DMF
e,Ar=
p -NO2-C6H4-
dps of AcOH
O
3
5
NC
NC
H
C
X
4
1
HN
Ar
Ar
2
6
S
N
H
NH
4a`,b`
O
3
4
O
Ar
5
HN
4
5
3
CN
1
HN
6
-C H
a, Ar =
6
5
6
2
S
N
H
N
1
b,Ar = 3-indolyl
7
2
S
N
H
N
NH2
8
Ar
4a,b
5a,b
a,Ar = p -OCH3-C6H4-
p -N(CH ) -C H -
b,Ar =
3 2
6
4
On the other hand, when we used a non-polar and slightly acidic solvent (DMF and few drops of
acetic acid , pH = 6.5–7), the imino group wasn't formed so the reaction proceeds via nucleophilic

Molecules 2010, 15
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attack of the amino group on the aldehyde carbonyl group leading to the 6-{[1-aryl-methylidene]-
amino}-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one condensation products 4a,b and not 4a′,b′, as
reported earlier [12]. The formation of the isomeric imino compounds 4a′,b′ is not plausible because
the amino group is much more nucleophilic than the CH at position 5 of 6-amino-2-thioxo-1H-
1
pyrimidine-4-one (1); in addition the H-NMR showed one proton signal at δ = 5.26 ppm for the
pyrimidine H-5 and one proton signal at δ = 7.84 ppm for the azamethine proton (N=CH), confirming
fortmation of the structures 4a,b.
Refluxing compound 1 with arylidene malononitriles in absolute ethanol containing a few drops of
triethylamine afforded 7-amino-5-aryl-4-oxo-2-thioxo-1,2,3,4- tetrahydro -pyrido[2,3-d] pyrimidine-6-
carbonitriles 5a,b (Scheme 1). The structures of all new synthesized compounds were confirmed by
IR, 1H-NMR, MS and elemental analysis results.
2.1. Antimicrobial activity
The in vitro antimicrobial activity of the newly synthesized compounds against Escherichia coli
(ATCC 25922) as an example of Gram negative bacteria, Bacillus subtilis (ATCC-6633) as a Gram
positive bacteria and Candida albicans (ATCC-10231) as a fungus was investigated using the disk-
diffusion method [14] at the Department of Microbiology and Immunology, Faculty of Pharmacy,
Helwan University, Helwan, Egypt. Ampicillin was used as an antibacterial standard and nystatin as a
standard antifungal agent. The results are given in Table 1.
Table 1. Antimicrobial activity of the synthesized compounds expressed as size of the
inhibition zone (mm/mg sample).
Species tested
Compd.
E. coli
B. subtilis
C. albicans
(ATCC-25922) (ATCC-6633)
(ATCC-10231)
Control (DMSO)
-
-
-
13
-
-
-
2a
2b
-
-
2c
-
-
-
3a
8
-
-
-
3b
8
-
3c
9
-
10
-
10
-
3d
3e
4a
-
-
-
13
14
-
20
22
-
-
4b
-
5a
-
5b
-
-
-
Ampicillin
Nystatin
11
-
18
-
-
12
- : No activity, DMSO: dimethylsulfoxide (used as solvent)

Molecules 2010, 15
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2.2. Anti-inflammatory activity [15]
The anti-inflammatory activity of compounds 2c, 3c, 4b, 5b was investigated in comparison with
ibuprofen as standard anti-inflammatory agent (Table 2, Figure 1).
Table 2. % Inhibition of acute inflammation.
% inhibition of acute inflammation
Compd.
1 h
2 h
3 h
4 h
2c
3c
4b
5b
RF
8.695652 18.92308 51.85185*
55.55556***
25.21739 27.69231 78.67647** 81.26984***
1.73913 26.92308 68.38235** 77.14286***
19.13043 20.76923 68.38235** 62.85714***
6.086957 45.38462 60.66176** 69.52381***
Rf: Ibuprofen; Using ANOVA method, at 3H and 4 H all compounds are statistically significant
from control * P < 0.05, ** P < 0.01, *** P < 0.001.
Figure 1. Anti-inflammatory test result.
% Inhibition for tested compounds
100
80
60
1 Hr
2 Hr
40
3 Hr
4 Hr
20
0
2c
3c
4b
5b
RF
Tested compounds
The carrageenan-induced rat paw oedema assay was carried out using a modified Winter’s method
as a preliminary screening test [16]. The rats (in groups of five animals weighing 120–170 g, young
adult male Sprague–Dawley) were housed in a controlled environment and provided with standard
rodent chow and water for 24 h before a dose of the test compounds (50 and 70 mg/kg sc) was
administered. One hour later, the volume of the right hind paw was measured, and 0.05 mL of a 1%
suspension of carrageenan in sterile pyrogen-free 0.9% NaCl solution was injected subcutaneously into
planter aponeurosis of the hind paw. One hour after the injection of carrageenan, the paw volume was
again measured by a water pletysmometer. The mean increase of paw volume at each time interval was
compared with that of control group (five rats treated with carrageenan, but without test compounds) at
the same time intervals. The percentage inhibition values were calculated according to the formula: %
Anti-inflammatory activity = (1- Rt/Rc)*100 (Rt = result of tested group; Rc = result of control group).

Molecules 2010, 15
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All experiments involving animals were carried out using protocols approved by the Animal
Committee, University of Barcelona (Spain). Animal care was in compliance with Generalitat of
Catalunya regulations on protection of animals used for experimental and other scientific purposes.
3. Experimental
3.1. General
All melting points are uncorrected. Elemental analyses were carried out in the Microanalytical
Center, Cairo University, Giza, Egypt. IR spectra (KBr) were recorded on Pye Unicam SP 1200
1
Spectrophotometer. H-NMR spectra were recorded in DMSO-d6 on a 90 MHz Varian NMR
spectrometer using TMS as an internal standard and chemical shifts are expressed as δ ppm units. The
mass spectra were determined with HP model MS-5988 at electron energy 70 eV. The homogeneity of
all compounds synthesized was checked by TLC on 2.0 cm × 6.0 cm aluminum sheets coated to a
thickness of 0.25 µm with silica gel 60 containing a fluorescent indicator. Characterization data of the
various compounds prepared are given in Tables 3 and 4.
Table 3. Physical properties and elemental analyses of the new compounds.
Analysis % Calc. (Found)
Yield %
m.p.oC
Formula
(m w)
Compd.
2a
C
H
N
21.52
85
C12H12N4OS
(260.32)
55.37
4.65
260
82
(55.15)
56.92
(4.98)
5.14
(21.32)
20.42
2b
2c
3a
3b
3c
3d
3e
4a
4b
5a
5b
C13H14N4OS
(274.35)
240
86
(56.68)
56.92
(5.37)
5.14
(20.65)
20.42
C13H14N4OS
(274.35)
245
78
(57.14)
41.29
(5.02)
2.31
(20.64)
16.05
C15H10BrN5O2S2
(436.31)
380
80
(41.05)
48.31
(2.56)
3.83
(16.27)
15.65
C18H17N5O5S2
(447.49)
310
75
(48.65)
51.50
(3.65)
3.05
(15.32)
21.20
C17H12N6O2S2
(396.45)
360
78
(51.27)
50.99
(3.37)
4.03
(21.49)
20.98
C17H16N6O2S2
(400.48)
370
77
(50.67)
44.77
(3.88)
2.50
(20.69)
20.88
C15H10N6O4S2
(402.41)
330
88
(44.53)
57.13
(2.25)
3.92
(20.65)
18.17
C11H9N3OS
(231.28)
278
81
(57.01)
57.76
(3.76)
3.73
(18.39)
20.73
C13H10N4OS
(270.31)
272
72
(57.95)
55.38
(3.96)
3.41
(20.50)
21.53
C15H11N5O2S
(325.35)
380
74
(55.29)
56.79
(3.34)
4.17
(21.37)
24.84
C16H14N6OS
(338.39)
270
(56.42)
(3.98)
(25.01)

Molecules 2010, 15
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Table 4. IR, 1H-NMR, and MS of the new compounds.
IR (cm -1) 1H-NMR (δ, ppm) and/or MS
Compd.
2a
3143 (NH), 1663 (C=O),
1240 (C=S).
3.5 [s, 2H, CH2(5)], 3.9 [d, 2H, CH2(7)], 7.1
(s, 2H, 2NH), 7.3-7.6 (m, 5H, Ar-H), 10.7 [s, 1H, NH(3)].
MS: (m/z) = 260 (M+, 6.7 %).
2b
3187 (NH), 1671(C=O), 1262 2.1 (s, 3H, CH3), 3.5 [s, 2H, CH2(5)], 3.8 [s, 2H, CH2(7)], 7.0
(C=S).
(s, 2H, 2NH), 7.3-7.5 (m, 4H, Ar-H), 10.9 [s, 1H, NH(3)].
MS: (m/z) = 274 (M+, 2.7 %).
MS: (m/z) = 274 (M+, 5 %).
2c
3a
3187 (NH), 1671 (C=O),
1262 (C=S).
3329 (NH), 1650 (C=O),
1234 (C=S).
5.3 [s, 1H, CH(9)], 6.8 [s, 1H, NH(10)], 7.1 (dd, 4H, Ar-H),
11.88-12.11 (s, 4H, 4NH).
MS: (m/z) = 436 (M+, 10.2 %).
3b
3c
3d
3e
4a
4b
5a
5b
3390 (NH), 1660 (C=O),
1235 (C=S).
3.8 (s, 9H, 3OCH3), 4.7 [s, 1H, CH(9)], 6.6 [s, 1H, NH(10)],
7.1 (s, 2H, Ar-H), 11.9-12.2 (4H, s, 4NH).
MS: (m/z) = 447 (M+, 100 %).
3225 (NH), 1665 (C=O),
1242(C=S).
4.8 [s, 1H, CH(9)], 6.7 [s, 1H, NH(10)], 7.2-7.5 (m, 5H, Ar-
H), 10.9, 11.4, 11.6 (s, 5H, 5NH).
MS: (m/z) = 396 (M+, 6.5 %).
3412 (NH), 1678 (C=O),
1260(C=S).
3.1 (s, 6H, 2CH3), 5.1 [s, 1H, CH(9)], 6.5 [s, 1H, NH(10)],
7.1-7.3 (dd, 4H, Ar-H), 11.8-12.0(s, 4H, 4NH).
MS: (m/z) = 400 (M+, 7.8 %), 385 (M-CH3, 100%).
MS: (m/z) = 402 (M+, 7.5 %).
3337 (NH), 1675 (C=O),
1338-1541 (NO2), 1235
(C=S).
3386 (NH), 1654 (C=O),
1182 (C=S)
5.2 (s, 1H, H-5 pyrimidine), 6.9–7.1 (m, 5H, Ar-H), 7.8 (s,
1H, N=CH), 11.4-11.8 (s, 2H, 2NH).
MS (m/z) = 231 (M+, 22.4%).
3360 (NH), 1650 (C=O),
1160 (C=S).
5.2 (s, 1H, H-5 pyrimidine), 7.6-8.2 (m, 5H, Ar-H), 7.7 (s,
1H, N=CH), 11.2, 11.4, 11.8 (s, 3H, 3NH).
MS (m/z) = 270 (M+, 16%).
3324-3424 (NH2), 2225
(CN),1680 (C=O), 1169
(C=S).
3.81 (s, 3H, OCH3), 6.9-7.3(dd, 4H, Ar-H), 7.6 (s, 2H, NH2),
12.07-12.7 (s, 2H, 2NH).
3333-3422 (NH2), 2214 (CN), 3.1 (s, 6H, 2CH3), 7.1-7.3 (dd, 4H, Ar-H), 7.5 (s, 2H, NH2),
1662 (C=O), 1197 (C=S).
12.2-12.6 (s, 2H, 2NH).
MS (m/z) =338 (M+, 31%).
3.2. 6-Aryl-2-thioxo-2,3,5,6,7,8-hexahydro-1H-pyrimido[4,5-d]pyrimidine-4-ones 2a-c
A mixture of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1, 0.002 mol) which was
prepared as described in the literature [11] in methanol (20 mL) and acetic acid (2 mL) was heated to
o
40 C and then primary aromatic amines (0.002 mol) in methanol (5 mL) and formalin (40%,
0.004 mol) were added dropwise with stirring until a clear solution was obtained. The product was
filtered, washed with ethanol and dried to give compounds 2a-c.

Molecules 2010, 15
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3.3. 9-Substituted-1,3,6,8,9,10-hexahydro-2,7-dithioxopyrido[2,3-d:6,5d′]dipyrimidine-4,6-diones
3a-e
To 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1, 0.002 mol) in methanol (10 mL) and
concentrated hydrochloric acid (0.4 mL), the appropriate aromatic aldehyde (0.001 mol) was added
and stirred at room temperature for 4.0 h, the product was collected by filtration and recrystallized
from aqueous acetic acid.
3.4. 6-{[1-Arylmethylidene]amino}-2-thioxo-2,3-dihydro-1H-pyrimidine-4-ones 4a,b
To a solution of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1, 0.01 mol) in DMF (30
mL), an equivalent amount of an aromatic aldehyde (0.01 mol) and few drops of acetic acid were
added, and the reaction mixture was heated under reflux for 4 h. The reaction mixture was allowed to
cool and poured on to ice/water, the product was filtered and crystallized from ethanol/dioxane
mixture (1:1).
3.5. 7-Amino-5-aryl-4-oxo-2-thioxo-1,2,3,4- tetrahydropyrido[2,3-d] pyrimidine-6-carbonitriles 5a,b
A mixture of equimolar amounts of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1) and the
appropriate arylidene malononitrile (0.002 mol) in absolute ethanol (10 mL) containing triethylamine
(5 drops) was heated at reflux for 3–6 h. The reaction mixture was concentrated and cooled. The solid
obtained was filtered off, washed with ethanol and crystallized from DMF/ethanol (2:1).
4. Conclusions
The results show that compounds 2a, 3a,b,c, 4a,b are active as antibacterials. Compound 3c is
active as an antifungal agent, while the other compounds are inactive toward the tested organisms.
From the obtained results, we note that cyclisation at the 5,6-positions of 6-amino-2-thioxo-2,3-
dihydro-1H-pyrimidine-4-one (1) may abolish or decrease the antimicrobial activity, as suggested by
the activities of the bicyclic pyrimido[4,5-d]pyrimidine-4-one (2a-c) and pyrido[2,3-d] pyrimidine-6-
carbonitrile (5a,b) and the tricyclic pyrido[2,3-d:6,5 d`] dipyrimidine (3a-e) systems.
Concerning the anti-inflammatory activity, analysis of the results obtained using one-way analysis
of variance (ANOVA) shows that there is a significant difference of all compounds from control at 3 h
and 4 h At 3 h, compounds 3c, 4b and 5b show higher activity than ibuprofen, while compound 2c
shows less activity. At 4 h, compounds 3c, 4b show higher activity than ibuprofen while compound 5b
joined 2c in showing less activity. This lesser activity of compounds 2c, 5b may be due to cyclisation
of the 5,6-positions of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1) and the higher activity
of compounds 3c, 4b may be due to the presence of an indolyl group as substituent.
References
1. Wyrzykiewicz, E.; Bartkowiak, G.; Kedzia, B. Synthesis and anti-microbial properties of S-
substituted derivatives of 2-thiouracil. Farmaco. Poland 1993, 48, 979–988.

Molecules 2010, 15
1889
2. Sharma, P.; Rane, N.; Gurram, V. K. Synthesis and QSAR studies of pyrimido[4,5-d]pyrimidine-
2,5-dione derivatives as potential antimicrobial agents. Bioorg. Med. Chem. Lett. 2004, 14,
4185–4190.
3. Elkholy, Y.M.; Morsy, M.A. Facile Synthesis of 5, 6, 7, 8-Tetrahydropyrimido[4,5-b]-quinoline
Derivatives. Molecules 2006, 11, 890–903.
4. Holla, B.S.; Mahalinga, M.; Karthikeyan, M.S.; Akberali, P.M.; Shetty, N.S. Synthesis of some
novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents. Bioorg. Med.
Chem. 2006, 14, 2040–2047.
5. Ingarsal, N.; Saravanan, G.; Amutha, P.; Nagarajan, S. Synthesis, in vitro antibacterial and
antifungal evaluations of 2-amino-4-(1-naphthyl)-6-arylpyrimidines. Eur. J. Med .Chem. 2007,
42, 517–520.
6. Zhao, X.-L.; Zhao, Y.-F.; Guo, S.-C.; Song, H.-S.; Wang, D. Gong, P. Synthesis and anti-tumour
activities of novel [1,2,4]triazolo[1,5-a]pyrimidines. Molecules 2007, 12, 1136–1146.
7. Cordeu, L.; Cubedo, E.; Bandrés, E.; Rebollo, A.; Sáenz, X.; Chozas, H.; Domínguez, M.V.;
Echeverría, M.; Mendivil, B.; Sanmartin, C.; Palop, J.A.; Font, M.; García-Foncillas, J. Biological
profile of new apoptotic agents based on 2,4,-pyrido[2,3-d]pyrimidine derivatives. Bioorg. Med.
Chem. 2007, 15, 1659–1669.
8. Sondhi, S. M.; Singh, N.; Johar, M.; Kumar, A. Synthesis, anti-inflammatory and analgesic
activities evaluation of some mono, bi, tricyclic pyrimidine derivatives. Bioorg. Med. Chem.
2005, 13, 6158–6166.
9. Amin, K.M.; Hanna, M.M.; Abo-Youssef, H.E.; George, R.F. Synthesis, analgesic and anti-
inflammatory activities of some bi-, tri- and tetracyclic condensed pyrimidines. Eur. J. Med.
Chem. 2009, XXX, 1–13.
10. Khalifa, N.M.; Ismail, N.S.; Abdulla, M.M. Synthesis and reactions of fused cyanopyrimidine
derivatives and related glycosides as antianginal and cardio protective agents. Egypt. Pharm. J.
2005, 4, 277–288.
11. Youssif, S. 6-aminouracil as precursors for the synthesis of fused di- and tricyclic pyrimidines. J.
Chem. Res. 2004, 341–343.
12. Mohamed, N.R.; El-Saidi, M.M.T.; Ali, Y.M.; Elnagdi, M.H. Utility of 6-amino-2-thiouracil as
precursor for the synthesis of bioactive pyrimidine derivatives. Bioorg. Med. Chem. 2007, 15,
6227–6235.
13. Youssif, S; El-Bahaie, S; Nabih, E. A facile one-pot synthesis of pyrido[2,3-d]-pyrimidines and
pyrido[2,3-d: 6,5-d']dipyrimidines. J. Chem. Res.1999, 112–113.
14. Bauer, A.W.; Kirby, W.M.M.; Sherris, J.C.; Turck, M. Antibiotic susceptibility testing by a
standardized single disk method. Amer. J. Clin. Pathol. 1966, 45, 493–496.
15. Harrak, Y.; Rosell, G.; Daidone, G.; Plescia, S.; Schillaci, D.; Pujol, M.D. Synthesis and biological
activity of new anti-inflammatory compounds containing the 1,4-benzodioxine and/or pyrrole
system. Bioorg. Med. Chem. 2007, 15, 4876–4890.

Molecules 2010, 15
1890
16. Winter C.A.; Risley E.A.; Nuss G.W. Carrageenin-induced oedema in hind paw of rat as the assay
for antiinflammatory drugs. Proc. Soc. Exp. Biol. NY 1962, 3, 544–547.
Sample Availability: Contact the authors.
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