Synthesis of bioactive polyheterocyclic ring systems as 5α-reductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.07.053

Simple synthetic strategies for the hitherto unreported [1,2,4]triazolo[4,3-a]pyrido[4,3-d]pyrimidines 8 and [1,2,4]triazolo[4′, 3′:1,2]pyrimido[4,5-b][1,6]naphthyridine-5-one 15 are described based on reaction of thione 3 and 12 with hydrazonoyl chloride 1a-h, respectively. The structures of products 8 and 15 were confirmed by spectroscopic and X-ray crystallographic analyses. Also, the mechanism of such reactions was discussed. In addition, reaction of compound 12 with bromoacetic acid and hydrazine hydrate was investigated. Compounds were screened against 5α-reductase and showed activities with good LD₅₀ and LD₉₀ for all compounds.

Full text of DOI:10.1016/j.ejmech.2010.07.053

European Journal of Medicinal Chemistry 45 (2010) 4838e4844
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of bioactive polyheterocyclic ring systems as 5
a-reductase inhibitors
,
c
d
Naglaa A. Abdel Hafez a, Thoraya A. Farghaly b , Mohamed A. Al-Omar , Mohamed M. Abdalla
*
a Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo, Egypt
b Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
c Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
d MAPCO Pharmaceuticals Ltd., Balteem, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Simple synthetic strategies for the hitherto unreported [1,2,4]triazolo[4,3-a]pyrido[4,3-d]pyrimidines 8
and [1,2,4]triazolo[40,30:1,2]pyrimido[4,5-b][1,6]naphthyridine-5-one 15 are described based on reaction
of thione 3 and 12 with hydrazonoyl chloride 1aeh, respectively. The structures of products 8 and 15
were confirmed by spectroscopic and X-ray crystallographic analyses. Also, the mechanism of such
reactions was discussed. In addition, reaction of compound 12 with bromoacetic acid and hydrazine
Received 14 July 2010
Received in revised form
24 July 2010
Accepted 27 July 2010
Available online 6 August 2010
hydrate was investigated. Compounds were screened against 5
good LD50 and LD90 for all compounds.
a-reductase and showed activities with
Keywords:
Hydrazonoyl halides
Ó 2010 Elsevier Masson SAS. All rights reserved.
5a-Reductase inhibitors
Polyheterocycles
Piperidone
1. Introduction
analyses and spectroscopic methods (IR, 1H NMR and MS) (see
Experimental). Compound 2 was then reacted with thiourea in
ethanol in presence of potassium hydroxide to yield 6-ethyl-8-(4-
methylphenylmethylene)-4-(4-methylphenyl)-3,4,5,6,7,8-hexahy-
dropyrido [4,3-d]pyrimidine-2(1H)-thione 3 (cf. Scheme 1). Also,
reaction of 3 with methyl iodide in dimethylformamide in presence
of anhydrous potassium carbonate afforded 6-ethyl-8-(4-methyl-
phenylmethylene)-2-(methylthio)-4-(4-methylphenyl)-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidine 4 (cf. Scheme 1). The structure
of products 3 and 4 were evidenced based on mass, IR and 1H NMR
spectral data (see Experimental).
Heterocycles bearing pyrimidine and pyridopyrimidine moie-
ties are reported to show a broad spectrum of pharmacological
properties such as antimicrobial [1e5], central nervous system
(CNS), analgesic, anti-inflammatory [6e9], and anti-HIV [10]. In
addition, [1,2,4]triazolo[4,3-a]pyrimidines are pharmacological
scaffold that represent a wide range of biological activities such as
antitumor [11], and anti-inflammatory [12]. Based on these find-
ings, we report herein the synthesis of [1,2,4]triazolo[4,3-a]pyrido
[4,3-d]pyrimidines and [1,2,4]triazolo[40,30:1,2]pyrimido [4,5-b]
[1,6]naphthyridine-5-ones that have not been reported hitherto.
Also, we study the biological activity of the newly synthesized
Reaction of 3 with the appropriate hydrazonoyl chlorides 5 in
dioxane in the presence of triethylamine under reflux was found to
give in each case one isolable product (TLC) that was identified to be
compounds against 5a-reductase activity.
[1,2,4]triazolo[4,3-a]pyrido[4,3-d]pyrimidines
8 rather than its
isomeric structure [1,2,4]triazolo[4,3-a]pyrido[3,4-e]pyrimidines 9
(Scheme 2). The direct formation of products 8 from the reaction of
compound 3 with hydrazonoyl chloride 5 indicates that the inter-
mediate thiohydrazonate esters 6 underwent Smiles rearrange-
ment [13] to give the corresponding thiohydrazides 7, which in situ
underwent cyclization with concurrent elimination of hydrogen
sulfide gas to give 8 as end products (cf. Scheme 2). All attempts to
isolate the intermediates 6 and 7 were failed since they were
consumed as soon as they were formed under the employed reac-
tion conditions. Alternatively, the formation of 8 from 2-methylthio
derivative 4 and hydrazonoyl chloride 5 can be accomplished via
2. Chemistry
1-Ethyl-4-piperidinone
1
was reacted with 4-methyl-
benzaldehyde in ethanol and potassium hydroxide to yield 1-ethyl-
3,5-bis(4-methylphenylmethylene)-piperidin-4-one 2 (Scheme 1).
The structure of the isolated product 2 was verified by elemental
* Corresponding author. Tel.: þ20 27494447.
E-mail address: thoraya-f@hotmail.com (T.A. Farghaly).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.053

N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
4839
O
O
CHO
KOH / EtOH
CH
3
2
H C
3
+
N
N
Et
1
2
Et
CH
3
NH -CS-NH
2
KOH
2
CH
3
CH
3
H
Et
N
N
H
Et
MeI
K CO / DMF
2
3
N
N
N
H
S
N
SMe
4
3
H C
3
H C
3
Scheme 1. Synthesis of compounds 3 and 4.
cyclization of the intermediate amidrazone 10 with concurrent
elimination of methanethiol to give 8 as end products (cf. Scheme
2). The structure of products 8 cannot be distinguished from the
discarded products 9 by Ms, IR, 1H NMR or elemental analyses. So,
we turned to X-ray crystallographic analysis as an exclusive
evidence for elucidating the structure of products 8. The ORTEP
drawing of 8b isolated from the reaction of 3 with 5b, taken as
a typical example of the series prepared, is shown in Fig. 1 with
selected bond lengths and bond angles depicted in Table 1 [14].
Fig. 1. The ORTEP drawing of 8b.
Reaction of arylidene 2 with 6-amino-2-thioxo-pyrimidine-4
(3H)-one 11 in acetic acid under reflux afforded 7-ethyl-9-(4-
methylphenylmethylene)-5-(4-methylphenyl)-2-thioxo-1,2,6,7,8,9-
hexahydro-pyrimido[4,5-b][1,6]naphthyridine-4(3H)-one 12. The
structure of compound 12 was established on the basis of Ms, IR and
1H NMR spectral data (see Experimental).
Reaction of the thione 12 with hydrazonoyl chloride 5a,c,h in
dioxane in presence of triethylamine afforded in each case a single
product. Both spectroscopic data and elemental analyses were
consistent with either compound 15 or 16 (Scheme 3). An imme-
diate distinction between these two structures was reached by
comparison of the 13C NMR spectral data with those of similar
annelated pyrimidinones. Literature reports [15e18] have shown
that the chemical shift for the carbonyl carbon in 4-pyrimidinone
Ar
Ar
H
Et
H
N
N
Et
N
N
N
H
S
N
SMe
Ar
3
4
Ar
R-C(Cl)=N-NH-Ar' / TEA
R-C(Cl)=N-NH-Ar'/ TEA
5
5
Ar
R
Ar
H
Et
N
Et
N
N
N
N
N
H
Ar'
R
N
S
N
SMe
N
Table 1
Ar
10
HN
6
Ar
Selected bond lengths and bond angles in the ORTEP of compound 8b in the crystal.
The crystallographic numbering does not reflect systematic numbering.
Ar'
-MeSH
Bond length, Å
Bond length, Å
Bond length, Å
C11eC29, 1.742
O2eC28, 1.211
N3eN5, 2.171
N3eN8, 1.370
N3eC26, 1.393
N3eC30, 1.416
N4eC16, 1.424
N4eC26, 1.279
N5eC14, 1.474
N6eC15, 1.458
N6eC18, 1.455
C7eC10, 1.381
C7eC14, 1.519
N8eC13, 1.302
C11eC24, 1462
C12eC15, 1.505
C12eC16, 1.473
C20eC36, 1.501
C22eC27, 1.390
N6eH15A, 1.988
N6eH20A, 2.007
Ar
Ar
R
H
Et
Et
HS
N
N
N
N
N
N
R
N
N
N
N
Ar'
-H S
2
Ar'
Ar
Ar
8
7
-H S
Angle (
u
)
Angle (
u)
Angle (u)
2
Ar = 4-CH C H
4
3
6
C24eC12eH15A, 119.1
C24eC12eH15B, 99.9
C7eC14eH14, 107.8
N6eC15eH15A, 108.8
C12eC15eH15A, 108.3
C31eC17eH31, 24.6
C33eC17eH31, 143
C40eC17eH31, 95.7
C40eC17eH33, 97.4
C40eC17eH40A, 25.9
H31eC17eH40A, 121.6
H31eC17eH40B, 82.1
H33eC17eH40A, 71.5
H40eC17eH40B, 44.66
C36eC20eH20A, 108.8
C36eC20eH20B, 107.3
C36eC20eH36C, 26.45
C27eC21eH21, 118.3
C27eC21eH37, 145.3
C37eC21eH21, 120.5
C9eC22eH9, 23.9
C13eN5eH14, 111.3
C14eN5eH14, 26.56
C26eN5eH14, 135.1
C15eN6eH15A, 27.20
C15eN6eH15B, 27.24
C18eN6eH15A, 95.1
C7eC10eH10, 118.7
C24eC11eH24, 23.67
C15eC12eH15A, 26.78
Ar
Ar' = XC H
6
4
Et
R/X : a, CH CO / H; b, CH CO / 4-Cl; c, EtOCO / H;
3
3
N
N
d, EtOCO / 4-CH ;e, EtOCO / 4-Cl; f, PhNHCO / H;
3
Ar'
g, PhNHCO / 4-CH ; h, PhNHCO / 4-Cl
N
N
3
N
Ar
R
9
Scheme 2. Synthesis of compound 8.

4840
N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
O
Ar
O
O
Et
H
S
Ar
H
S
Ar
N
N
N
AcOH
+
N
N
N
H
H N
2
N
H
Et
12
Ar
2
11
R-C(Cl)=N-NH-Ar' / TEA
5
Ar
O
Ar
O
H
HS
Et
Et
H
N
R
N
N
N
R
N
N
N
N
N
N
S
H
N
N
Ar'
14
Ar
Ar
Ar'
13
Ar
O
Ar
O
R
Et
N
Et
N
N
N
N
N
Ar'
N
N
N
N
N
Ar'
N
15
Ar
R
Ar
16
Ar = 4-CH C H
4
3
6
Ar' = XC H
6
4
R/X : a, CH CO / H; c, EtOCO / H; h, PhNHCO / 4-Cl
3
Scheme 3. Synthesis of compound 15.
derivatives is markedly affected by the nature of the adjacent
nitrogen (N3) (pyrrole type in structure 15 and pyridine type as in
structure 16). For example, the 13C NMR spectra of 15a taken as
typical example of the series prepared, revealed the signal of the
a constant standard diet. Twenty-three groups, each of 12 male
SpragueeDawley rats in the postnatal third days, were treated
subcutaneously with the 5a-reductase inhibitor (tested compound
or reference standard). The tested compounds were dissolved in 5%
Tween 80 in water. The solvent was used for both standard and
negative control group, beginning on the postnatal third day until
the age of seven weeks.
Twenty-one groups were used to test the activities, of which one
was used as the positive control for anastrozole and another served
as the negative control group. After, scarifying blood was
carbonyl carbon of the pyrimidinone ring residue at
d 162 ppm.
Such chemical shift value is similar to that of annelated pyr-
imidinones of pyrrole type rather than that of the pyridine type. On
the basis of this similarity, the isolated products were assigned
structure 15 and structure 16 was therefore excluded.
Finally, compound 12 reacted with 2-bromoacetic acid in acetic
acid and acetic anhydride inpresence of fused sodium acetateto give
thiazolo[30,20:1,2]pyrimido[4,5-b][1,6]naphthyridine-3,5-dione 17.
Treatment of the later product 17 with 3,4-dimethoxy-benzalde-
hyde afforded 2-(3,4-dimethoxyphenylmethylene)-thiazolo[30,20:
1,2]pyrimido[4,5-b][1,6]naphthyridine-3,5-dione 18. Compound 18
was also prepared by one step reaction via 2-bromoethanoic acid
and 3,4-dimethoxy-benzaldehyde under the same reaction condi-
tions (Scheme 4).
Ar
O
O
Et
BrCH COOH
2
N
N
S
AcOH
N
N
17
Ar'CHO
Ar
Ar
O
Ar
O
O
Et
Reaction of compound 12 with hydrazine hydrate led to
formation of 2-hydrazino derivatives 19. The structure of all prod-
ucts 17e19 was established on the basis of mass, IR, 1H NMR and
elemental analyses.
Ar'
Et
H
S
BrCH COOH
2
N
N
N
N
S
N
N
H
Ar'CHO
N
N
18
Ar
Ar
12
Ar
O
3. Pharmacology
Et
H
Ar = 4-CH C H
4
N
N
3
6
NH NH
2
2
3.1. Biological assay
N
N
NHNH
2
Ar' =
19
OMe
Ar
Treatment of animals e Animals were obtained from the animal
house colony of the National Research Center, Cairo, Egypt. All
animals were allowed free access to water and were kept on
OMe
Scheme 4. Reaction of compound 12 with bromoethanoic acid and hydrazine hydrate.

N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
4841
withdrawn for testosterone and dihydrotestosterone (DHT) deter-
mination [19]. Moreover, intraprostatic concentrations of testos-
terone and DHT were determined [20].
The biological experiments were performed according to the
official standards.
All the tested compounds showed
5
a
-reductase inhibitor
activities with good LD50 and LD90 for all compounds were high
enough to provide good therapeutic windows and a soft profile
margin. The ascending order of activity is 3, 8d, 8g, 8a, 8b, 15h, 12,
17, 15c, 8e and 8f.
Radioimmuno assay for testosterone and dihydrotestosterone e
Serum testosterone and dihydrotestosterone were measured by
radioimmunoassay in serum extracts using specific antisera
without prior chromatography. Serum samples of 0.5 mL were
extracted with 2 mL of freshly purified peroxide-free diethyl ether
by shaking for 60 s on a Vortex mixer. The aqueous phase was
frozen at ꢀ70 ꢁC, the ether phase containing steroids was trans-
ferred to a conical test tube and evaporated in BSA/phosphate
buffer (pH ¼ 7.4) containing (1,2,6,7-3H)-testosterone or (1,2,6,7-
3H)-dihydrotestosterone and then specific antisera were added and
incubated over a period of 24 h at 4 ꢁC under non-equilibrium
conditions. Bound hormone and free hormone were separated by
adsorption on dextran-coated charcoal. The activity of each sample
was determined in a Beckman-counter (USA) using a commercially
available scintillation cocktail (Mini-RIA, Zinsser, Spain). As for
other steroid hormones, commercially available KIA-kits, e.g.,
Biermann GmbH, Germany, can be used. The hormone level in the
sample was calculated from a standard curve by means of
a computer program (KIA-Calc, LKB, Canada), using appropriate
control sera. Steroid levels of rats treated with different doses of 5-
reductase inhibitors were compared with vehicle-treated controls
(Table 1). The relative potency was calculated by dividing the ED50
(dose that causes 50% of pharmacological response in the test) of
anastrozole by that of a tested compound.
4. Conclusion
In conclusion, simple synthetic strategies for the synthesis of tri
and tetra heterocyclic ring systems namely [1,2,4]triazolo[4,3-a]
pyrido[4,3-d]pyrimidines 8 and [1,2,4]triazolo[40,30:1,2]pyrimido
[4,5-b][1,6]naphthyridine-5-one 15 were described. The structures
of compounds 8 and 15 were confirmed by spectroscopic and X-ray
crystallographic analyses. Also, the mechanism of such reactions
was discussed. In addition, all synthesized compounds were tested
for their 5
showed 5
a
-reductase activity in vivo. All the tested compounds
a-reductase activities with good LD50 and LD90 for all
compounds were high enough to provide good therapeutic
windows and a soft profile margin.
5. Experimental protocol
5.1. Chemistry
All melting points were determined on an electrothermal Gal-
lenkamp apparatus. Solvents were distilled and dried by standard
literature procedures prior to use. The IR spectra were measured on
a Pye-Unicam SP300 instrument in potassium bromide discs. The
1H NMR and 13C NMR spectra were recorded on a Varian Mercury
(500 MHz for 1H NMR and 125 MHz for 13C NMR) and the chemical
shifts were related to that of the solvent DMSO-d6. The mass spectra
were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A
HP spectrometers, the ionizing voltage was 70 eV. Elemental
analyses were carried out by the Microanalytical Center of Cairo
University, Giza, Egypt. Hydrazonoyl halides 5aeh were prepared
following literature methods [22,23].
3.2. Determination of acute toxicity
LD50 and LD90 were determined by using male albino rats and
injecting them with different increasing doses of agents. Doses that
killed 50% and 90% of the tested animals, respectively, were
calculated according to Austen et al. [21] (Table 2).
5.2. Crystallographic analysis
3.3. Pharmacological screening
The crystals were mounted on a glass fiber. All measurements
were performed on an ENRAF NONIUS FR 590. The data were
Circulating testosterone and dihydrotestosterone hormone
levels or tissue concentrations were measured after administration
collected at a temperature of 25 ꢁC using the
u scanning technique
of 5
a
-reductase inhibitor radioimmuno assays. All synthesized
-reductase inhibitor activity in
to a maximum of a 20 of 22.986ꢁ. The structure was solved by direct
method using SIR 92 and refined by full-matrix least squares. Non-
hydrogen atoms were refined anisotropically. Hydrogen atoms
were located geometrically and were refined isotropically.
compounds were tested for their 5
a
vivo; the ED50, LD50 and LD90 data were determined and are given in
Table 2.
5.2.1. Crystal data
Table 2
Evaluation of ED50, LD50, LD90 and 5
anastrozole.
a
-reductase inhibitor activities relative to
For compound 8b: C33H32ClN5O, M ¼ 550.106, monoclinic,
a ¼ 12.5470 (6), b ¼ 9.6393 (4), c ¼ 24.1177 (14) Å, v ¼ 2904.3 (2),
a
b
c
a
¼
g
¼ 90.00ꢁ,
b
¼ 95.326 (2), space group: P21/c, Z ¼ 4,
Compd.
No.
ED50
g kgꢀ1
LD50
g kgꢀ1
LD90
Potency relative to
Anastrozole
(
m
)
(
m
)
(m
g kgꢀ1
434.76
)
Dx ¼ 1.258 Mg mꢀ3 reflection 1,0911 measured, qmax ¼ 27.43ꢁ. Fig. 1
illustrates the structure as determined. Full data can be obtained on
request from the CCDC [14].
3
8d
1.88
1.00
222.23
224.33
0.579787
1.09
5 14.33
417.34
419.23
735.34
848.56
937.34
1148.50
1226.89
1438.60
1950.12
3.69
8g
8a
8b
15h
12
17
15c
8e
0.925
0.911
0.856
0.811
0.795
0.766
0.611
0.441
0.432
1.09
23 6.28
257.23
3 12.34
413.34
447.88
516.56
823.45
712.55
6534.45
1.178378
1.196487
1.273364
1.34402
1.371069
1.422977
1.783961
2.471655
2.523148
1.00
5.3. Preparation of 1-ethyl-3,5-bis(4-methylbenzylidene)piperidin-
4-one (2)
To a mixture of compound 1 (1.27 g, 0.01 mol) and 4-methyl-
benzaldehyde (2.40 g, 0.02 mol) in ethanol (100 mL) was added
potassium hydroxide (1 g) in few drops of water. The mixture was
stirred at room temperature for 30 min, the solid formed was
collected, washed with ethanol and crystallized from ethanol to
give compound 2 as yellow crystals, yield (95%), mp 210 ꢁC 1H NMR
8f
Anastrozole
2.415
a
ED50: Dose caused 50% of pharmacological response in the test.
LD50: Dose killed 50% of the tested animals.
LD90: Dose killed 90% of the tested animals.
b
c
(CDCl3) at
d
¼ 0.80 (t, J ¼ 7 Hz, 3H, CH3), 2.31 (q, J ¼ 7 Hz, 2H, CH2),

4842
N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
2.53 (s, 6H, 2CH3), 3.12e3.40 (4d, 4H, 2CH2), 7.61 (s, 2H,
benzyliceH), 7.63e7.80 (m, 8H, ArH), IR (KBr, cmꢀ1) 1676. MS m/z
(%) 332 (Mþþ1, 14), 331 (Mþ, 100), 104 (61), 91 (95), 77 (93). Anal.
Calcd. For C23H25NO (331.46): C, 83.34; H, 7.60; N, 4.23. Found C,
83.21; H, 7.36; N, 4.10%.
products identical in all respects (mp, mixed mp and IR) with that
were formed from Method A.
5.6.1. 3-Acetyl-1-phenyl-5-(4-methylphenyl)-9-(4-
methylphenylmethylene)-7-ethyl-5H-6,7,8,9-tetrahydro[1,2,4]
triazolo[4,3-a]pyrido[4,3-d]pyrimidine (8a)
Yellow crystal, yield (85%) mp 200e202 ꢁC (Ethanol). IR (KBr,
cmꢀ1) 1651 (CO), MS m/z (%) 516 (Mþþ1, 2), 515 (Mþ, 8), 445 (20),
251(100), 208 (80), 83 (12). Anal. Calcd. For C33H33N5O(515.66): C,
76.87; H, 6.45; N, 13.58, Found C, 76.59; H, 6.30; N, 13.42%.
5.4. Preparation of 6-ethyl-8-(4-methylphenylmethylene)-4-(4-
methylphenyl)-3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2
(1H)-thione (3)
To a boiling mixture of compound 2 (3.3 g, 0.01 mol) in ethanol
(100 mL) containing potassium hydroxide (1 g) in water (0.5 mL)
was added thiourea (0.76 g, 0.01 mol) and the reaction mixture was
refluxed for 3 h, allowed to cool and the solid formed was filtered
off, dissolved in water and precipitated by HCl. The solid formed
was filtered off and crystallized from ethanol to afford compound 3
as white crystals. Yield (85%) mp 260 ꢁC 1H NMR (DMSO-d6) at
5.6.2. 3-Acetyl-7-ethyl-1-(4-chlorophenyl)-5-(4-methylphenyl)-9-
(4-methylphenylmethylene)-5H-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]pyrido[4,3-d] pyrimidine (8b)
Golden yellow crystal, yield (82%) mp 194e196 ꢁC (Ethanol). 1H
NMR (DMSO-d6) at
d
¼ 0.95 (t, J ¼ 7 Hz, 3H, CH3), 2.29 (s, 6H, 2CH3),
2.36 (s, 3H, CH3), 2.41 (q, J ¼ 7 Hz, 2H, CH2), 2.75e3.76 (4d, 4H,
2CH2), 6.22 (s, 1H, pyrimidineeH), 7.25e8.34 (m, 13H,
ArH þ benzyliceH). IR (KBr, cmꢀ1) 1695 (CO). MS m/z (%) 552
(Mþ þ 2, 8), 551 (Mþ þ 1, 15), 550 (Mþ, 56), 548 (100), 129 (25), 105
(34), 91 (37). Anal. Calcd. For C33H32ClN5O (550.11): C, 72.05; H,
5.86; N, 12.73. Found C, 71.96; H, 5.63; N, 12.54%.
d
¼ 0.83 (t, J ¼ 7 Hz, 3H, CH3), 2.29 (q, J ¼ 7 Hz, 2H, CH2), 2.49 (s, 6H,
2CH3), 2.61e3.47 (4d, 4H, 2CH2), 4.82 (s, 1H, pyrimidineeH), 7.08 (s,
1H, benzyliceH), 7.17e7.18 (m, 8H, ArH), 9.04 (s, 1H, NH,
exchangeable with D2O), 9.40 (s, 1H, 1NH, exchangeable with D2O).
13C NMR (DMSO-d6) 12.07, 20.69, 20.79, 50.35, 51.68, 51.84, 56.83,
111.83, 122.51, 125.94, 126.17, 126.66, 128.92, 129.03, 129.22, 133.50,
136.30, 137.15, 139.65, 174.01. MS m/z (%) 389 (Mþ, 100), 287 (20),
104 (45), 91 (90), 77 (72). Anal. Calcd. For C24H27N3S (389.57): C,
74.00; H, 6.99; N, 10.79. Found C, 73.92; H, 6.84; N, 10.65%.
5.6.3. 3-Ethoxycarbonyl-7-ethyl-1-phenyl-5-(4-methylphenyl)-9-
(4-methylphenylmethylene)-5H-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]pyrido[4,3-d]pyrimidine (8c)
Orange crystal, yield (85%) mp 164e166 ꢁC (Ethanol). 1H NMR
(DMSO-d6) at
d
¼ 1.07 (t, J ¼ 7 Hz, 3H, CH3), 1.20 (t, 3H, CH3), 2.24 (s,
5.5. Preparation of 6-ethyl-8-(4-methylphenylmethylene)-2-
(methylthio)-4-(4-methyphenyl)-3,4,5,6,7,8-hexahydropyrido[4,3-
d]pyrimidine (4)
3H, CH3), 2.28 (s, 3H, CH3), 2.56 (q, J ¼ 7 Hz, 2H, CH2), 3.19e3.38 (4d,
4H, 2CH2), 4.21 (q, 2H, CH2), 5.85 (s, 1H, pyrimidineeH), 7.02e8.01
(m, 14H, ArH þ benzyliceH). IR (KBr, cmꢀ1) 1731 (CO). MS m/z (%)
546 (Mþ þ 1, 2), 545 (Mþ, 6), 445 (21), 184 (17), 118 (27), 91 (100).
Anal. Calcd. For C34H35N5O2 (545.69): C, 74.84; H, 6.47; N, 12.83.
Found C, 74.60; H, 6.35; N, 12.56%.
To a stirred solution of compound 3 (1.9 g, 5 mmol) in DMF
(20 mL) was added anhydrous potassium carbonate (0.70 g,
5 mmol) and methyl iodide (0.71 g, 5 mmol). The reaction mixture
was stirred overnight at room temperature then poured onto ice-
water. The solid formed was filtered off, washed with water, dried
and crystallized from ethanol to give compound 4 as yellow solid.
5.6.4. 3-Ethoxycarbonyl-7-ethyl-1-(4-methylphenyl)-5-(4-
methylphenyl)-9-(4-methylphenylmethylene)-5H-6,7,8,9-
tetrahydro[1,2,4]triazolo[4,3-a]pyrido[4,3-d] pyrimidine (8d)
Yellow crystal, yield (75%), mp 220e222 ꢁC (Ethanol). 1H NMR
Yield (68%) mp 244 ꢁC 1H NMR (DMSO-d6) at
d
¼ 1.10 (t, J ¼ 7 Hz,
3H, CH3), 2.25(s, 3H, SCH3), 2.35 (q, J ¼ 7 Hz, 2H, CH2), 2.41 (s, 6H,
2CH3), 2.64e3.46 (m, 4H, 2CH2), 4.96 (s, 1H, pyrimidineeH),
7.25e7.50 (m, 9H, ArH þ benzyliceH), 8.62 (s, 1H, NH, exchangeable
with D2O). IR (KBr, cmꢀ1) 3424 (NH) MS m/z (%) 403 (Mþ, 25), 401
(91), 400 (100), 386 (56), 296 (66), 142 (75), 118 (44), 77(40). Anal.
Calcd. For C25H29N3S (403.59): C, 74.40; H, 7.24; N, 10.41. Found C,
74.21; H, 7.06; N, 10.20%.
(DMSO-d6) at
d
¼ 0.83 (t, J ¼ 7 Hz, 3H, CH3), 1.18 (t, 3H, CH3), 2.22 (s,
3H, CH3), 2.28 (s, 3H, CH3), 2.32 (s, 3H, CH3), 3.13 (q, J ¼ 7 Hz, 2H,
CH2), 3.29e3.53 (4d, 4H, 2CH2), 4.22 (q, 2H, CH2), 6.17 (s, 1H,
pyrimidineeH), 7.13e8.11 (m, 13H, ArH þ benzyliceH). IR (KBr,
cmꢀ1) 1725 (CO). Anal. Calcd. For C35H37N5O2 (559.72): C, 75.11; H,
6.66; N, 12.51. Found C, 75.34; H, 6.29; N, 12.61%.
5.6.5. 3-Ethoxycarbomyl-7-ethyl-1-(4-chlorophenyl)-9-(4-
methylphenylmethylene)-5H-6,7,8,9-tetrahydro[1,2,4]triazolo[4,3-
a]pyrido[4,3-d]pyrimidine (8e)
5.6. Preparation of [1,2,4]triazolo[4,3-a]pyrido[4,3-d]pyrimidines
(8aeh)
Yellow solid, yield (78%) mp 228 ꢁC (Ethanol). 1H NMR (DMSO-
Method A: To a mixture of equimolar amount of 3 and the
appropriate hydrozonyl chloride 5aeh (2.5 mmol of each) in
dioxane (20 mL) was added triethylamine (0.35 mL, 2.5 mmol). The
reaction mixture was refluxed till all of the starting materials have
disapppeared and hydrogen sulfide gas ceased to evolve (10 h
monitored by TLC). The solvent was evaporated and the residue was
treated with methanol. The solid that formed was filtered off and
crystallized from the appropriate solvent to give compounds 8aeh.
Method B: To a mixture of equimolar amount of 4 and the
appropriate hydrozonyl chlorides 5b,d,f (2.5 mmol of each) in
dioxane (20 mL) was added triethylamine (0.35 mL, 2.5 mmol). The
reaction mixture was refluxed till all methanthiol gas ceased to
evolve (20 h, monitored by TLC). The solvent was evaporated and
the residue was treated with methanol. The solid that formed was
filtered off and crystallized from the appropriate solvent to give
d6) at
d
¼ 1.12 (t, J ¼ 7 Hz, 3H, CH3), 1.28 (t, J ¼ 7 Hz, 3H, CH3), 2.24 (s,
3H, CH3), 2.28 (s, 3H, CH3), 2.55e3.72 (m, 6H, 3CH2), 4.01 (q,
J ¼ 7 Hz, 2H, CH2), 5.94 (s, 1H, pyrimidineeH), 7.02e8.00 (m, 13H,
ArH þ benzyliceH). IR (KBr, cmꢀ1) 1694 (CO). MS m/z (%) 581
(Mþ þ 1, 10), 580 (Mþ, 30), 576 (25), 564 (35), 430 (66),111 (100), 90
(70). Anal. Calcd. For C34H34ClN5O2 (580.14): C, 70.39; H, 5.91; N,
12.07. Found C, 70.18; H, 5.82; N, 12.00%.
5.6.6. 7-Ethyl-3-phenylcarbamoyl-1-phenyl-5-(4-methylphenyl)-
9-(4-methylphenylmethylene)-5H-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]pyrido[4,3-d] pyrimidine (8f)
Orange solid, yield (80%), mp 182e184 ꢁC (Ethanol/Dioxane), IR
(KBr, cmꢀ1) 3381(NH), 1645 (CO). MS m/z (%) 593 (Mþ þ 1, 25), 592
(Mþ, 100), 105 (48), 91 (50), 77 (85). Anal. Calcd. For C38H36N6O

N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
4843
(592.75): C, 77.00; H, 6.12; N, 14.18. Found C, 76.92; H, 6.05; N,
14.03%.
118.24, 124.50, 126.51, 126.45, 128.92, 129.04, 129.91, 130.27, 131.09,
132.11, 135.14, 136.0, 138.99, 139.25, 140.09, 141.52, 143.53, 146.23,
148.42, 154.33, 155.12, 158.70, 159.06, 162.0, 198.16. IR (KBr, cmꢀ1
)
5.6.7. 7-Ethyl-3-phenylcarbamoyl-1-(4-methylphenyl)-5-(4-
methylphenyl)-9-(4-methylphenylmethylene)-5H-6,7,8,9-
tetrahydro[1,2,4]triazolo[4,3-a]pyrido[4,3-d] pyrimidine (8g)
Yellow solid, yield (72%) mp 158 ꢁC (Ethanol). 1H NMR (DMSO-
1710, 1682 (2CO), MS m/z (%) 581(Mþ þ 1, 10), 580 (Mþ, 34), 422
(20), 91 (100), 105 (38), 77 (82). Anal. Calcd. For C36H32N6O2
(580.70): C, 74.46; H, 5.55; N, 14.47. Found: C, 74.25; H, 5.20; N,
14.65%.
d6) at
d
¼ 0.95 (t, J ¼ 7 Hz, 3H, CH3), 2.23, 2.26 (2s, 6H, 2CH3), 2.30 (s,
3H, CH3), 2.33e3.42 (br, 6H, 3CH2), 6.31 (s, 1H, pyrimidineeH),
5.8.2. 3-Ethoxycarbonyl-1-phenyl-6-(4-methylphenyl)-10-(4-
methylphenyl-methylene)-8-ethyl-7,9-dihydro[1,2,4]triazolo
[40,30:1,2]pyrimido[4,5-b][1,6] naphthyridine-5-one (15c)
Yellow solid, yield (73%) mp 224e226 ꢁC (Ethanol). 1H NMR
7.14e8.17 (m, 18H, ArH
þ
benzyliceH), 10.61 (s, 1H, NH,
exchangeable with D2O). IR (KBr, cmꢀ1) 3398 (NH), 1653 (CO). MS
m/z (%) 607 (Mþ þ 1, 14), 606 (Mþ, 35), 105 (28), 90 (75), 77 (100).
Anal. Calcd. For C39H38N6O (606.78): C, 77.20; H, 6.31; N, 13.85.
Found C, 76.99; H, 6.22; N, 13.66%.
(DMSO-d6) at
d
¼ 0.84 (t, J ¼ 7 Hz, 3H, CH3), 1.25 (t, J ¼ 7 Hz, 3H,
CH3), 2.35, 2.41 (2s, 6H, 2CH3), 2.49 (q, J ¼ 7 Hz, 2H, CH2), 3.25e3.72
(br, 4H, 2CH2), 4.38 (q, J ¼ 7 Hz, 2H, CH2), 7.09e8.20 (m, 14H,
ArH þ benzyliceH). IR (KBr, cmꢀ1) 1749, 1712 (2CO), MS m/z (%) 610
(Mþ, 20), 608 (4), 607 (4), 482 (16), 435 (24), 119 (65), 105 (60), 91
(98), 77 (100). Anal. Calcd. For C37H34N6O3 (610.72): C, 72.77; H,
5.61; N, 13.76. Found C, 72.54; H, 5.48; N, 13.61%.
5.6.8. 7-Ethyl-3-phenylcarbamoyl-1-(4-chlorophenyl)-5-(4-
methylphenyl)-9-(4-methylphenylmethylene)-5H-6,7,8,9-
tetrahydro[1,2,4]triazolo[4,3-a]pyrido[4,3-d] pyrimidine (8h)
Yellow solid, yield (71%) mp ¼ 236 ꢁC (Ethanol/Dioxane). 1H
NMR (DMSO-d6) at
d
¼ 0.85 (t, J ¼ 7 Hz, 3H, CH3), 2.21(s, 3H, CH3),
2.31 (s, 3H, CH3), 2.50 (q, 2H, CH2), 3.31e3.54 (4d, 4H, 2CH2), 6.21 (s,
1H, pyrimidineeH), 7.12e8.42 (m, 18H, ArH þ benzyliceH), 10.66 (s,
1H, NH, exchangeable with D2O). 13C NMR (DMSO-d6) 12.17, 20.70,
20.74, 50.90, 52.40, 52.38, 59.28, 108.43, 120.35, 120.50, 122.95,
124.65, 126.57, 128.42, 128.81, 128.88, 129.09, 129.41, 131.74, 133.36,
134.21, 135.95, 136.71, 137.29, 137.96, 138.53, 139.26, 141.40, 143.60,
146.50, 153.88. IR (KBr, cmꢀ1) 3371 (NH), 1703 (CO). MS m/z (%) 628
(Mþ þ 1, 21), 627 (Mþ, 52), 626 (25), 564 (51), 625 (99),152 (32),119
(90), 105 (46), 91 (100). Anal. Calcd. For C38H35ClN6O (627.20): C,
72.77; H, 5.62; N, 13.40. Found C, 72.52; H, 5.43; N, 13.21%.
5.8.3. 3-Phenylcarbamoyl-1-(4-chlorophenyl)-6-(4-methylphenyl)-
10-(4-methylphenyl-methylene)-8-ethyl-7,9-dihydro[1,2,4]triazolo
[40,30:1,2]pyrimido[4,5-b][1,6] naphthyridine-5-one (15h)
Orange solid, yield (72%) mp ¼ 262 ꢁC (Dioxane). 1H NMR
(DMSO-d6) at
d
¼ 0.85 (t, J ¼ 7 Hz, 3H, CH3), 2.23, 2.32 (2s, 6H,
2CH3), 2.36 (q, J ¼ 7 Hz, 2H, CH2), 2.80, 3.1, 3.52 (2d, 4H, 2CH2),
7.05e8.02 (m, 19H, ArH þ benzyliceH), 11.94 (s, 1H, NH). IR (KBr,
cmꢀ1) 3426 (NH), 1662 (CO); MS m/z (%) 694 (Mþ þ 2, 2), 693
(Mþ þ 1, 2), 692 (Mþ, 4), 119 (100), 91 (74), 77 (17). Anal. Calcd. For
C41H34ClN7O2 (692.23): C, 71.14; H, 4.95; N, 14.16. Found: C, 71.05;
H, 4.71; N, 14.38%.
5.7. Preparation of 7-ethyl-9-(4-methylphenylmethylene)-5-(4-
methylphenyl)-2-thioxo-2,3,6,7,8,9-hexahydro-pyrimido[4,5-b][1,6]
naphthyridine-(4H)-one (12)
5.9. Preparation of thiazolo[30,20:1,2]pyrimido[4,5-b][1,6]
naphthyridine-3,5-dione (17)
A mixture of compound 2 (3.3 g, 10 mmol) and 6-amino-2-thi-
oxo-2,3-dihydropyrimidin-4(1H)-one (1.43 g, 10 mmol) in glacial
acetic acid (40 mL) was refluxed for 20 h. The reaction mixture was
cooled and poured onto ice-cold water and the solid product was
collected by filtration and crystallized from dioxane to give
compound 12 as yellow solid. Yield (92%), mp 235 ꢁC (Ethanol/
A mixture of compound 12, bromoacetic acid (1.4 g, 0.01 mol),
and fused sodium acetate (6 g) in glacial acetic acid (30 mL) and
acetic anhydride (10 mL) was refluxed for 3 h, left to cool, then
poured gradually with stirring onto cold water, the solid formed
was filtered off, washed with water and crystallized from ethanol to
give compound 17 as red solid, yield (60%), mp 226e228 ꢁC
Dioxane).1H NMR (DMSO-d6) at
d
¼ 0.80 (t, J ¼ 7 Hz, 3H, CH3), 2.22 (s,
3H, CH3), 2.35 (s, 3H, CH3), 2.50 (q, 2H, CH2), 3.13e3.54 (4d, 4H,
2CH2), 6.90e7.32 (m, 9H, ArH), 10.21(s, 1H, NH, exchangeable with
D2O), 12.0 (s, 1H, NH, exchangeable with D2O). IR (KBr, cmꢀ1) 3420,
3356 (2NH),1660(CO). MSm/z (%)454 (Mþ, 23), 453 (15),142(15), 98
(46), 91 (100), 89 (39), 77(39). Anal. Calcd. For C27H26N4OS (454.60):
C, 71.34; H, 5.76; N, 12.32. Found: C, 71.16; H, 5.60; N, 12.19%.
(Ethanol). 1H NMR (DMSO-d6) at
d
¼ 1.03 (t, J ¼ 7 Hz, 3H, CH3), 2.24
(s, 3H, CH3), 2.31 (s, 3H, CH3), 2.56 (q, J ¼ 7 Hz, 2H, CH2), 3.19e3.38
(4d, 4H, 2CH2), 4.25 (q, 2H, CH2), 7.02e7.24 (m, 9H,
ArH þ benzyliceH). MS m/z (%) 494 (Mþ,6), 284 (12), 251 (21), 185
(13), 149 (30), 119 (51), 91 (40), 87 (89), 77 (11). Anal. Calcd. For
C29H26N4O2S (494.62): C, 70.42; H, 5.30; N, 11.33. Found: C, 70.62;
H, 5.05; N, 11.11%.
5.8. Preparation of compound 15a, c, h
To a mixture of equimolar amount of 12 and the appropriate
hydrozonyl chloride 5a,c,h (2.5 mmol of each) in ethanol (50 mL)
was added TEA (0.35 mL, 2.5 mmol). The reaction mixture was
refluxed till all hydrogen sulfide gas ceased to evolve (10 h moni-
tored by TLC). The solvent was evaporated and the residue was
treated with methanol. The solid that formed was filtered off and
crystallized from the appropriate solvent to give compounds
15aeh.
5.10. Preparation of 2-(3,4-dimethoxyphenylmethylene)-thiazolo
[30,20:1,2]pyrimido[4,5-b][1,6]naphthyridine-3,5-dione (18)
Method A: A mixture of compound 12 (4.5 g, 0.01 mol), bro-
moacetic acid (1.39 g, 0.01 mol) and fused sodium acetate (6 g) in
glacial acetic acid (30 mL), acetic anhydride (10 mL) and aromatic
aldehyde (1.66 g, 0.01 mol) was refluxed for 3 h, the reaction
mixture was cooled and poured onto cold water, the solid formed
was collected and crystallized from ethanol to give compound 18 as
yellow solid, yield (65%), mp 238 ꢁC (AcOH). IR (KBr, cmꢀ1) 1672,
1658 (CO), MS m/z (%) 642(Mþ, 15), 476 (15), 152 (34), 119 (46), 117
(32), 110 (34), 105 (32), 98 (44), 91 (44), 78 (59), 77 (34), 57 (100).
Anal. Calcd. For C38H34N4O4S (642.78): C, 71.01; H, 5.33; N, 8.72.
Found: C, 71.35; H, 5.09; N, 8.47%.
5.8.1. 3-Acetyl-1-phenyl-6-(4-methylphenyl)-10-(4-
methylphenylmethylene)-8-ethyl-7,9-dihydro[1,2,4]triazolo
[40,30:1,2]pyrimido[4,5-b][1,6] naphthyridine-5-one (15a)
Yellow solid, yield (82%) mp 240e242 ꢁC (Ethanol). 13C NMR
(DMSO-d6) 12.24, 18.12, 21.0, 21.18, 49.58, 50.57, 59.04, 110.24,

4844
N.A. Abdel Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4838e4844
[6] S.M. Sondhi, V.K. Sharma, R.P. Verma, N. Singhal, R. Shukla, R. Raghubir,
M.P. Dubey, Synthesis (1999) 878e884.
5.11. Preparation of 7-ethyl-9-(4-methylphenylmethylene)-5-(4-
methylphenyl)-2-hydrazino-2,3,6,7,8,9-hexahydro-pyrimido[4,5-b]
[1,6]naphthyridine-(4H)-one (19)
[7] D.L. Boyle, E.A. Kowaluk, M.F. Jarvis, C.H. Lee, S.S. Bhagwat, M. Williams,
G.S. Firestein, J. Pharmacol. Exp. Ther. 296 (2001) 495e500.
[8] C.H. Lee, M. Jiang, M. Cowart, G. Gfesser, R. Perner, K.H. Kim, Y.G. Gu,
M. Williams, M.F. Jarvis, E.A. Kowaluk, A.O. Stewart, S.S.S. Bhagwat, J. Med.
Chem. 44 (2001) 2133e2138.
[9] H.N. Hafez, N.S. Abbas, A.B.A. El-Gazzar, Acta Pharm. 58 (2008) 359e378.
[10] R.K. Rawal, R. Tripathi, S.B. Katti, C. Pannecouquec, E. De Clercq, Bioorg. Med.
Chem. 15 (2007) 3134e3142.
[11] H.N. Hafez, A.B.A. El-Gazzar, Bioorg. Med. Chem. Lett. 19 (2009) 4143e4147.
[12] R. Gupta, A.K. Gupta, S. Paul, P.L. Kachroo, Indian J. Chem. 37B (1998)
1211e1215.
[13] A.J. Elliott, P.D. Callaghan, M.S. Gibson, S.T. Nemeth, Can. J. Chem. 53 (1975)
1484e1488.
A mixture of compound 12 (4.5 g, 0.01 mol) and hydrazine hydrate
(85%, 5 mL) in ethanol (50 mL) was refluxed for 10 h. The reaction
mixture was cooled and poured onto cold water and the solid formed
was filtered off and crystallized from ethanol to give compound 19 as
white solid, yield (60%), mp 275 ꢁC (Ethanol). IR (KBr, cmꢀ1) 3416,
3328, 3240 (NH, NH2), 1685 (CO), MS m/z (%) 453 (Mþ þ 1, 47), 452
(Mþ, 85), 364 (67), 318 (47), 222 (33),198 (73),154 (100),140 (67), 89
(80), 81 (40), 78 (13). Anal. Calcd. For C27H28N6O (452.56): C, 71.66; H,
6.24; N, 18.57. Found: C, 71.54; H, 6.35; N, 18.29%.
[14] All crystallographic data have been deposited at the Cambridge Crystallo-
graphic Data Center as supplementary publication no. CCDC 749447. Copies of
the data can be obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK.
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