Synthesis and biological screening of 2-substituted 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles and of their intermediates

Article abstract of DOI:10.1002/1522-2675(200202)85:2<559::AID-HLCA559>3.0.CO;2-A

To evaluate the effect of substituents on biological activities of electron-rich N-containing heterocycles, the variably 2-substituted 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles 26-33 were synthesized and evaluated for antibacterial, antifungal, and enzyme-inhibition activities. The target compounds were obtained from alkyl 4- or 3-hydroxy benzoates 1 and 2, respectively, and from methyl indoleacetate 3. The phenolic OH group of benzoates 1 and 2 were substituted with p-toluenesulfonyl (→ 4 and 5), benzoyl (→ 6 and 7), and benzyl groups (→ 8 and 9) and then converted to 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles. To establish structure-activity relationships (SAR), a pharmacological screening of the intervening intermediates was also conducted, which revealed that the intermediate hydrazide 11 possesses significant antimicrobial and MAO-A inhibiting properties and intermediates 12, 24, 28, and 29 appreciable antifungal activities. Compound 7 inhibits α-chymotrypsin.

Full text of DOI:10.1002/1522-2675(200202)85:2<559::AID-HLCA559>3.0.CO;2-A

Helvetica Chimica Acta Vol. 85 (2002)
559
Synthesis and Biological Screening of 2-Substituted 5,6-Dihydro-5-oxo-
4H-1,3,4-oxadiazine-4-propanenitriles and of Their Intermediates
by Khalid Mohammed Khan*, Shagufta Rahat, Muhammad Iqbal Choudhary, Atta-ur-Rahman, Usman Ghani,
Shahnaz Perveen, Shagufta Khatoon, Ahsana Dar, and Abdul Malik
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi-
75270, Pakistan
(Phone: (92-21)-9243224, 9243211; fax: (92-21)-9243190-91; e-mail:hassaan2@super.net.pk)
To evaluate the effect of substituents on biological activities of electron-rich N-containing heterocycles, the
variably 2-substituted 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles 26 33 were synthesized and
evaluated for antibacterial, antifungal, and enzyme-inhibition activities. The target compounds were obtained
from alkyl 4- or 3-hydroxy benzoates 1 and 2, respectively, and from methyl indoleacetate 3. The phenolic OH
group of benzoates 1 and 2 were substituted with p-toluenesulfonyl ( ! 4 and 5), benzoyl ( ! 6 and 7), and
benzyl groups ( ! 8 and 9) and then converted to 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles. To
establish structure-activity relationships (SAR), a pharmacological screening of the intervening intermediates
was also conducted, which revealed that the intermediate hydrazide 11 possesses significant antimicrobial and
MAO-A inhibiting properties and intermediates 12, 24, 28, and 29 appreciable antifungal activities. Compound 7
inhibits a-chymotrypsin.
Introduction. A diversity of biological effects is associated with 1,3,4-oxadiazine
and its derivatives, they exhibit cardiovascular [1 3], antibacterial [4], plant-growth
regulating [5], miticidal and nematocidal [6], acricidal [7], and insecticidal activities [8]
and monoamine-oxidase (MAO) inhibition [9]. The promising therapeutic potential of
this class of compounds prompted us to synthesize and biologically screen a series of
structural variants of 4H-1,3,4-oxidiazine-5(6H)-ones. To establish structure activity
relationships (SAR), the activities of the intervening intermediates were also
determined. Thus, the readily available alkyl benzoates 1, 2, and 4 9 and the
indoleacetate 3 were transformed to the target 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-
4-propanenitriles 26 33 in excellent yields. The structures of 26 33 were established
by spectroscopic techniques, and their biological activities were determined.
Results and Discussion.
The synthesis of the 5,6-dihydro-5-oxo-4H-1,3,4-
oxadiazine-4-propanenitriles 26 33 is outlined in the Scheme. The phenolic OH group
of the benzoates 1 and 2 was substituted with p-toluenesulfonyl ( ! 4 and 5), benzoyl
( ! 6 and 7) and benzyl groups ( ! 8 and 9). The benzoates 1, 2, and 4 9, and the
indoleacetate 3 were then treated with hydrazine hydrate in EtOH to afford the
hydrazides 10 17. Alkylation of a hydrazide occurs at the N(1) or N(2) atom
depending upon the conditions; thus, in neutral medium, the terminal N(2) atom [10] is
alkylated, whereas, in the presence of a strong base like Na or NaOMe, the position of
alkylation is strongly dependent on the nature of the solvent [11]: aprotic solvents like

560
Helvetica Chimica Acta Vol. 85(2002)
Scheme
i) N2H4, H2O, EtOH, reflux, 48 h. ii) H2CˆCHCN, EtOH, reflux, 48 h. iii) ClCH2COCl, CHCl3, reflux, 1 h.
iv) K2CO3, EtOH, reflux, 18 24 h.

Helvetica Chimica Acta Vol. 85(2002)
561
Et2O and benzene favor N(1) substitution, while, in protic solvents like EtOH, N(2)
substitution predominates. Michael reaction between hydrazides 10 17 and acryloni-
trile led exclusively to N(2)-substituted derivates 18 25. Under no circumstances was
the formation of double Michael products detectable. The cyclization of the 2-(2-
cyanoethyl)hydrazides 18 25 was brought about with chloroaectyl chloride in the
presence of K2CO3 to furnish the target 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-
propanenitriles 26 33 in excellent yields.
All compounds synthesized were assessed for enzyme-inhibitory activity against
monoamine oxidase (MAO), serine proteases (a-chymotrypsin, trypsin, and elastase),
cysteine protease papain, and tyrosinase enzymes. Significant inhibition was observed
for monoamine oxidase and a-chymotrypsin; the other enzymes were found to be
either resistant or only partially sensitive to the compounds tested.
MAO Activities in the absence of test compounds (control enzyme) were found to
be 18 Æ 1.7 nmol of H2O2 hÀ1 mgÀ1 of protein. This activity was inhibitied by hydrazide
11 in a concentration-dependent manner and similarly to the monoamine oxidase A
(MAO-A) inhibitors clorgyline and moclobemide [12]; clorgyline, moclobemide, and
11 caused complete enzyme inhibition at 0.1, 10000, and 100 mm, respectively (Fig. 1).
The inhibitor concentrations required for 50% inhibition of enzyme (IC50) decreased in
the order moclobemide > hydrazide 11 > clorgyline (Table 1). Although 11 was ca.
60 times less potent than clorgyline (a nonselective inhibitor of MAO-A); it was ca. 80
times more potent than moclobemide, a clinically effective, selective inhibitor of MAO-
A. This suggests that hydrazide 11 may have potential for future research aimed at the
synthesis of a better antidepressant agent. On the other hand, compound 33 failed to
inhibit enzyme activity up to the highest tested concentration (100 mm). Since
hydrazide 10 containing a p-OH group and hydrazide 14 bearing a m-TsO group
failed to inhibit MAO, these groups do probably not interfere with the enzyme. The
dramatic inhibition of MAO by hydrazide 11 containing a p-TsO group suggests that
this group is likely to be responsible for the observed interaction with the enzyme.
However, the conversion of the hydrazide to the oxadiazine ring (11 ! 27) resulted in
complete loss of activity, implying that the N(2) atom of the hydrazide plays a crucial
role. Thus, simultaneous occurrence of a hydrazide moiety and a p-TsO group at a
phenyl ring are prerequisites for MAO inhibition.
The most potent among the inhibitors of a-chymotrypsin was benzoate 7 containing
a m-BzO group (IC50 ˆ 29.34 Æ 1.15 mm), while other compounds of the series showed
moderate to weak activity (Table 2, Fig. 2). The active site of a-chymotrypsin contains
a primary specificity pocket that interacts with the bulky side chains of the amino acids
tyrosine, phenylalanine, and tryptophan, suggesting that the inhibitory activity of 7
might be due to its aryl rings. To determine which aryl ring of 7 is critical for inhibition,
its inhibitory activity was compared with that of benzoate 5 containing a m-TsO group.
A significant decrease in the activity was observed in the latter case (see Table 2 and
Fig. 2).
A further decrease of a-chymotrypsin inhibition was observed when the hydrazide
moiety was converted to a 2-(2-cyanoethyl)hydrazide function. Moreover, none of the
target oxadiazine derivatives were significantly active, probably due to affinity or steric
factors. Thus, the presence of a m-BzO group at the benzoate plays an important role in
the inhibition of a-chymotrypsin.

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Helvetica Chimica Acta Vol. 85(2002)
Fig. 1. Effect of various
concentrations of the in-
hibitors a) clorgyline,
b) hydrazide 11, and
c) moclobemide on
MAO-A activity. A sam-
ple of synaptosomes
(28 mg/ml protein) was
pre-incubated with the
inhibitor for 20 min at
378 before the addition
of 5-hydroxytryptamine
(500 mm). Inhibition of
enzyme activity is ex-
pressed as % of remain-
ing activity of the con-
trol sample pre-incubat-
ed without the inhibitor.
Each bar is the mean of
2
6 determinations,
each in duplicate or
triplicate.

Helvetica Chimica Acta Vol. 85(2002)
563
Table 1. IC50 Values of Monoamine-Oxidase Inhibitors (clorgyline and moclobemide) andof Compound 11
Clorgyline
0.05275
Moclobemide
11
IC50 [mm]
3.2
Table 2. Inhibition of a-Chymotrypsin by 2-Substituted5,6-Dihydro-5-oxo-4 H-1,3,4-oxadiazine-4-propaneni-
triles andof Their Intermediates
IC50 [mm]
Percentage inhibition at 1 mm
Percentage inhibition at 100 mm
7
22
21
25
33
4
5
6
8
27
32
29.34 Æ 1.15
1160 Æ 37.12
77.11
45.92
48.68
35.16
64.40
58.47
27.63
28.81
26.31
-
Antibacterial activity of various synthesized compounds was tested against six
Gram-positive and four Gram-negative bacterial strains (Table 3). The results were
compared with the standard drugs amoxillin and ampicillin. Hydrazide 11 having a p-
TsO substituent at the phenyl ring was found to be the most active against a broad
range of both Gram-positive and Gram-negative bacteria. Interestingly, hydrazide 10
having a p-OH group showed significant activity against Pseudomonas aeroginosa and
Salmonella typhi and a modest activity against Shigella boydii and Streptococcus
pyogenes. The corresponding 2-(2-cyanoethyl)hydrazide 18 showed varying activities
against Escherichia coli, Klebsiella pneumoniae, P. aeroginosa, S. typhi, S. aureus, and S.
pyogenes, and the corresponding 4H-1,3,4-oxadiazine derivative 26 displayed weak
activity against P. mirabilis, S. typhi, S. boydii, S. aureus, and S. pyogenes but was
significantly active against K. pneumoniae, P. aeroginosa, and B. cerous. Compounds 12,
20, and 28 all having a p-BzO group, predominantly exhibited weak to moderate
activity against some of the bacterial strains. Hydrazide 13 demonstrated moderate
activity against K. pneumoniae, P. mirabilis, P. aeroginosa, S. typhi, S. boydii, S. aureus,
and S. pyogenes, while its cyanoethyl derivative 21 displayed the same degree of activity
against E. coli, K. pneumoniae, P. aeroginosa, S. typhi, S. boydii, and S. aureus, and no
activity against P. mirabilis, P. cerous, C. diptheriae, and S. pyogenes as compared to its
precursor. The corresponding 4H-oxadiazine derivative 29 failed to show activity
against E. coli, P. mirabilis, S. boydii, B. cerous, S. aureus, and S. pyogenes, moderately
active against K. pneumoniae, P. aeroginosa, and C. diptheriae, and significant activity
against S. typhi. Compounds 19 and 27, the 2-(2-cyanoethyl)hydrazide and oxadiazine
derivatives of hydrazide 11, respectively, were less active compared with 11. The
important bactericidal activity of 11 could be related to the strong electron-with-
drawing nature of the p-TsO substituent at the phenyl ring. In general, poor activity of
the oxadiazine derivatives is possibly due to the steric factors being responsible for
retarding passive diffusion of the molecule into the bacterial cell. It has been suggested

564
Helvetica Chimica Acta Vol. 85(2002)
Fig. 2. Effect of various compounds as inhibitors of a-chymotrypsin: a) 1 mm of 21, 25, and 33 and b) 100 mm of
4, 5, 6, 8, 27, and 32
that less substitution at an aromatic ring promotes passive diffusion. The electronic
nature of the substituent at the p-position and steric factors promotes passive diffusion.
The electronic nature of the substituent at the p-position and steric factors associated
with the six-membered oxadiazine ring thereby may be responsible for the poor
bactericidal activity.
Hydrazide 11 containing a p-TsO group was the most potent bactericidal agent as
compared to its parent Me ester 4 and all the tested compounds. However, alkylation at
the N(2)-atom of the hydrazide moiety attenuated the antibacterial activity, revealing
that the N(2)-atom of the hydrazide moiety is indispensable for the bacterial inhibition.
Cyclization caused a further decline in the antibacterial activity, indicating that the H-
atom at N(2) in open-chain compounds is probably responsible for it.

Helvetica Chimica Acta Vol. 85(2002)
565
Table 3. Antibacterial Activity of 2-Aryl-Substituted5,6-Dihydro-5-oxo-4 H-1,3,4-oxadiazine-4-propanenitriles
a
andof Their Intermediates
)
4
6
10 11 12
13 18
19
20
21
26
27
28
29
Gram-negative:
Escherichia coli
7.0 7.0 0.511
000.59.0 00
7.0 6.0 8.0 6.0 00
6.0 7.0 .05 0.5 7.0 0.5
7.0 8.0
00 0.500
Klebsiella pneumoniae
Proteus mirabilis
Pseudomonas aeroginosa
Salmonella typhi
Shigella boydii
00
00
00
00
00
0.512
00 10
6
6.0 6.0
0.500
00
00
00
00
6.0 00
8.0 00
6.0 9.0 12
6.0 7.0 6.0 7.0 7.0 8.0 0.57.0 7.0
7.0 6.0 00
6.0 0.56.0 00
8.0 00
8.0 11
6.0 10
00
6.0 0.56.0 7.0 8.0
7.0 0.57.0 7.0 0.5
00
00
Gram-positive:
Bacillus cerous
Corynebacterium diptheriae 6.0 8.0 0.511
Staphylococcus aureus
Streptococus pyogenes
6.0 00
00
10
00
00
0.5 0.500
0.7 00 00
00
00
8.0 00
8.0 0.500
6.0 00
0.5
00
6.0
00
8.0 0.5 00
7.0 7.0 00
00 5.5 7.0 11
9.0
7.0 6.0 6.0 6.0 6.0 6.0 00
7.0 7.0 6.0 6.0 00 00 00
6.0 00
a) < 5: weak; 5 7: moderate; > 7: significant.
The 2-aryl-substituted 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles
and their synthetic intermediates were also tested for their in vitro antifungal activity
against the eleven fungal strains Aspergillus flavus, Trichophyton schoenleinii,
Trichophyton rubrum, Pseudallescheria boydii, Candida albicans, Aspergillus niger,
Microsporum canis, Tricophyton simii, Trichophyton mentagrophytes, Fusarium oxy-
sporum, and Fusarium solani (see Table 4). The results were compared with the
standard drugs micoanazole, ketocanozole, benlate, and nabam. Hydrazide 11 having a
p-TsO substituent at the phenyl ring showed strong growth-inhibitory activity against
C. albicans and P. boydii in an agar-dilution assay, indicating that it can be considered a
lead for design of drug against candidiasis. Hydrazide 12 containing a p-BzO group
showed significant activity against P. boydii and F. oxysporum, whereas 24 containing a
m-BnO group and a side chain at the N(2) atom was active against P. boydii. The p-
BzO-substituted 4H-1,3,4-oxadiazine derivative 28 appeared to be active against T.
simii, whereas the p-BnO-substituted 4H-1,3,4-oxadiazine derivative 29, showed
activity against M. canis. In general, the oxadiazine derivatives were very weakly active.
Table 4. Antifungal Activity of 2-Aryl-Substituted5,6-Dihydro-5-oxo-4 H-1,3,4-oxadiazine-4-propanenitriles and
of Their Intermediates
4
6
8
10
11 12 13 18 19 20 21
24 26 27 28 29
Aspergillus flavus
00 00 00 00
16 00 33 50
00 00 00 56
00 00 00 33
00 57 00 00 00 00 00
58 00 00 00 00
00 50 50 00 34
00 67 33 00 00
Trichophton schoenleinii
Trichophyton rubrum
Pseudallescheria boydii
Candida albicans
Aspergillus niger
Microsporum canis
Trichopyton simii
00 00
3.0 42 00 00 61
00 00 00 00 7.0 00 00
82 9500 00 00 00 00 100 00 4500 00
00 00 00
7.0 100
7.0 00 00 00 00 00
3.0 9.0 00 14 00 00 7.0
6.0 52 00 33 00 00 54
00 00 00 15 00 00 38
00 29 00 00 00 00 00
1500 00 00 00
12 00 36 00
00 00 50 00
25 00 5.0 50
6.0 50 00 00 15
00 71 14 00 88
00 30 60 100 30
00 00 00 00 00
Trichophytonmentagrophytes 00 00 00 00
Fusarium oxysporum
Fusarium solani
5
0
00
5
0
00
29 83
5
0
5
0
00 00
58 00 25 12 30
5
0
19 25
14
7 30
30 00 48 00
00 31 60 56 00 00 54

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Helvetica Chimica Acta Vol. 85(2002)
Conclusions. The structure-activity relationship (SAR) study conducted with 2-
aryl-substituted 5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles for selected
biological activities indicated that these compounds were slightly active, in contrast to
our hypothesis. Interestingly, some intermediates of their syntheses exhibited biological
activities. Particularly, hydrazide 11 bearing a p-TsO group demonstrated significant
monoamine-oxidase inhibition and bactericidal properties, suggesting that the elec-
tron-withdrawing nature of p-TsO probably enhances the interaction with the active
sites of the enzyme and its entry into the bacterial cell. Compounds 11, 12, 24, 28, and 29
demonstrated excellent antifungal activities against a few fungal strains. The m-BzO-
substituted benzoate strongly inhibited a-chymotrypsin, the m-BzO group in this type
of ester probably playing an important role in the inhibition process. Possibilities also
exist for further exploring the structure-activity relationships of these compounds.
Experimental Part
General. Anh. CHCl3 and EtOH were prepared by standard methods. All other solvents and reagents were
reagent grade and used directly without purification, except for chloroacetyl chloride and benzoyl chloride,
which were distilled before use. M.p.: open capillaries, B¸chi-535 apparatus; uncorrected. Column
chromatography: silica gel (70 230 mesh, E. Merck). IR: Shimadzu IR-460 for KBr pellets and Jasco A-302
spectrophotometer for CHCl3 solns.; in cmÀ1. 1H-NMR: Bruker apparatus; at 300 and 400 MHz; d in ppm rel. to
SiMe4 (ˆ0 ppm) as internal standard. EI-MS: Finnigan-MAT 311-A apparatus; m/z (rel. %).
Preparation of Synaptosomes. Adult Wistar rats (both sexes, locally bred) were sacrificed by rapid
decapitation, followed by quick removal of brains. Homogenate was prepared in 20 volumes of 0.32m ice-cold
sucrose by means of polytron (8000 minÀ1 Â 8 for 3 s). The homogenate was centrifuged at 1000 g for 10 min at
48, and the supernatant was further centrifuged at 17000 g for 30 min. The pellet obtained was resuspended in 10
volumes of sucrose and homogenized by means of polytron as described above. Synaptosomes were used either
for the determination of MAO-A activity immediately or aliquoted and stored at À508 till further use [13].
Monoamine Oxidase Assay: MAO-A Activity was determined by the fluorometric method [14][15] with 5-
hydroxytryptamine (500 mm) as a substrate. Synaptosomes (20 ml) were pre-incubated either with buffer or test
compound (11 or 33 in 10% DMSO in d/w) for 30 min at 378, followed by the addition of substrate (50 ml). Assay
tubes were incubated for 20 min with a final reaction volume of 200 ml. An adrenaline/peroxidase system was
used to form fluorescent adrenolutine; the intensity of the fluorescence was determined at an emission
wavelength of 550 nm with an excitation wavelength of 405 nm. In all of the experiments, H2O2 (2 nmol) was
used as a standard that gave a fluorescence of 251 Æ 3.5. The control enzyme activity was expressed in nmol of
H2O2 formed hÀ1 mgÀ1 protein. The % inhibition of enzyme activity in the presence of test compound was
determined by comparing it with the control value. Clorgyline (an irreversible MAO-A inhibitor) at 0.1 mm
caused complete enzyme inhibition, confirming the presence of MAO-A in the reaction mixture. Protein
concentrations were determined by the method of Lowry et al. [16] with bovine serum albumin as a standard.
Chymotrypsin-Inhibition Assay. Protease inhibitory activity of selected synthesized compounds was
determined according to [17]. Increasing concentrations of compounds were incubated with a-chymotrypsin
(9 U/ml) in 50 mm Tris ¥ HCl buffer (pH 7.6) at 258 for 30 min. Release of 4-nitroaniline was continuously
monitored at 405nm in a SprctraMax-340 microplate reader (Molecular Devices, USA) immediately after the
addition of 50 ml of substrate N-succinyl-phenylalanine-4-nitroanilide (2 mg/ml). Results were compared with
chymostatin, a standard inhibitor of chymotrypsin [18]. The % inhibition was calculated as the difference of
absorbance values with and without compounds (see Table 2).
Antibacterial Activity. The synthesized compounds were tested against six Gram-negative (E. coli, K.
pneumoniae, P. mirabilis, P. aeroginosa, S. typhi, and S. boydii) and four Gram-positive (B. cerous, C. diptheriae,
S. aureus, and S. pyogenes) bacterial strains by the agar-well-diffusion method [19]. The wells were dug in the
media with the help of a sterile metallic borer with centers at least 24 mm apart. Two- to eight-hours-old
bacterial inocula containing approximately 104 106 colony-forming units (CFU)/ml were spread on the surface
of nutrient agar with the help of a sterile cotton swab. The recommended concentration of the test sample (2 mg/
ml in DMSO) was introduced in respective wells. Other wells supplemented with DMSO and reference
antibacterial drugs served as negative and positive controls, respectively. The plates were incubated immediately

Helvetica Chimica Acta Vol. 85(2002)
567
at 378 for 20 h. Activity was determined by measuring the diameter of zones showing complete inhibition (mm).
Growth inhibition was calculated with reference to positive control.
Antifungal Activity. The synthesized compounds were tested for antifungal activity against six human
pathogens (A. flavus, T. schoenleinii, T. rubrum, P. boydii, C. albicans, and A. niger), three animal pathogens (M.
canis, T. simii, and T. mentagrophytes), and two plant pathogens (F. oxysporum and F. solani) by means of the
tube diffusion test [20]. Micoanazole (75 mg/ml), ketocanozole (75 mg/ml), benlate (100 mg/ml), and nabam
(50 mg/ml) were used as standard drugs. Stock solns. of pure compounds (12 mg/ml) were prepared in sterile
DMSO. Sabouraud dextrose agar was prepared by mixing sabouraud (32.5g), glucose agar (4%) and agar-agar
(4 g) in 500 ml of distilled H2O followed by steam dissolution; the media (4 ml) was dispensed into screw-caped
tubes and autoclaved at 1218 for 15min. Test compound (66.6 ml) was added from the stock soln. to non-
solidified sabouraud agar media (508). Tubes were allowed to solidify at r.t. and inoculated with 4-mm diameter
of inocula derived from seven-days-old respective fungal culture. For nonmycelial growth, an agar surface streak
was employed. The tubes incubated at 27 298 for 7 10 days and the growth in the compound-containing media
was determined by measuring the linear growth (mm) and growth inhibition with reference to the respective
control.
Benzoates 4 and 5: General Procedure. TsCl (0.04 mol) was added in small portions to a cold soln. of 1 or 2
(0.03 mol) in pyridine (0.06 mol) at <108. After stirring for 3 h at <108, the mixture was kept overnight at 58,
neutralized with 2m HCl, and filtered. The solid was washed with H2O and recrystallized from MeOH: 4 or 5,
resp.
Methyl 4-{[(4-Methylphenyl)sulfonyl]oxy}benzoate (4): Yield 89%. M.p. 858. IR (KBr): 3000, 1725, 1440.
1H-NMR (300 MHz, (D6)DMSO): 2.42 (s, 3 H); 3.82 (s, 3 H); 7.12 7.95( m, 8 arom. H). Anal. calc. for
C15H14O5S (306.24): C 58.83, H 4.61; found: C 58.73, H 4.59.
Ethyl 3-{[(4-Methylphenyl)sulfonyl]oxy}benzoate (5): Yield 79%. M.p. 378. IR (KBr): 3000, 1725, 1375.
1H-NMR (300 MHz, (D6)DMSO): 1.34 (t, 3 H); 2.43 (s, 3 H); 4.32 (q, 2 H); 7.32 7.93 (m, 8 arom. H). Anal.
calc. for C16H16O5S (320.27): C 60.00, H 5.03, found: C 59.91, H 5.00.
Benzoates 6 and 7: General Procedure. To a mixture of 1 or 2 (0.03 mol) in pyridine (0.06 mol), benzoyl
chloride (0.05mol) was added dropwise and the mixture stirred overnight. The mixture was then poured in
crushed ice and acidified with dil. HCl soln. The resulting crystalline solid was filtered, washed with MeOH
(20 ml) and H2O (20 ml), and air-dried: 6 or 7, resp.
Methyl 4-(Benzoyloxy)benzoate (6): Yield 73%. M.p. 1268. IR (KBr): 3000, 1725, 1680. 1H-NMR
(300 MHz, (D6)DMSO): 3.81 (s, 3 H); 7.41 8.13 (m, 9 arom. H). Anal. calc. for C15H12O4 (256.25): C 70.30,
H 4.72; found: C 70.24, H 4.68.
Ethyl 3-(Benzoyloxy)benzoate (7): Yield 80%. M.p. 528. IR (KBr): 3000, 1720, 1680. 1H-NMR (300 MHz,
(D6)DMSO): 1.33 (t, 3 H), 4.35( q, 2 H); 7.61 8.02 (m, 9 arom. H). Anal. calc. for C16H14O4 (270.30): C 71.10,
H 5.22; found: C 71.08, H 5.18.
Benzoates 8 and 9. General Procedure. To a mixture of 1 or 2 (0.02 mol) and K2CO3 (0.04 mol) in acetone
(30 ml), benzyl bromide (0.02 mol) was added dropwise at r.t. The mixture was refluxed at 608 for 3 h and then
filtered. The filtrate was evaporated and the residue crystallized from hot hexane: 8 or 9, resp.
Methyl 4-(Benzyloxy)benzoate (8): Yield 62%. M.p. 948. IR (KBr): 3300, 1725, 1600. 1H-NMR (300 MHz,
(D6)DMSO): 3.75( s, 3 H); 5.14 (s, 2 H); 7.12 7.91 (m, 9 arom. H). Anal. calc. for C15H14O3 (242.27): C 74.37,
H 5.82; found: C 74.32, H 5.79.
1
Ethyl 3-(Benzyloxy)benzoate (9): Yield 82%. M.p. 698. IR (KBr): 3300, 1725, 1600. H-NMR (300 MHz,
(D6)DMSO): 1.32 (t, 3 H); 3.82 (s, 3 H); 5.25 (s, 2 H); 4.32 (q, 2 H); 7.21 7.65( m, 9 arom. H). Anal. calc. for
C16H16O3 (256.30): C 74.98, H 5.82; found: C 74.93, H 5.75.
Hydrazides 10 17: General Procedure. To 99% hydrazine hydrate (0.01 mol) in EtOH (3.7 ml), 1 or one of
3
9 (0.06 mol) was added in small portions and then heated under reflux for 28 h. After evaporation, the oily
residue crystallized overnight in the cold.
4-Hydroxybenzoic Acid Hydrazide (10): Yield 80%. M.p. 2688. IR (KBr): 3350, 3000, 1620. 1H-NMR
(300 MHz, (D6)DMSO): 6.75 7.62 ( dd, 4 H); 9.52 (s, 1 H). Anal. calc. for C7H8N2O2 (152.15): C 55.26, H 5.3,
N 18.41; found: C 55.22, H 5.32, N 18.37.
4-{[(4-Methylphenyl)sulfonyl]oxy}benzoic Acid Hydrazide (11): Yield 63%. M.p. 1058. IR (KBr): 3350,
1690, 1640, 1380. 1H-NMR (300 MHz, (D6)DMSO): 2.38 (s, 3 H); 6.75 7.90 ( m, 8 arom. H); 9.49 (s, 1 H).
Anal. calc. for C14H14N2O4S (306.24): C 54.90, H 4.60, N 9.14; found: C 54.87, H 4.51, N 9.11.
4-(Benzoyloxy)benzoic Acid Hydrazide (12): Yield 69%. M.p. 2558. IR (KBr): 3350, 1700, 1690, 1640.
1H-NMR (300 MHz, (D6)DMSO): 6.80 7.68 (m, 9 arom. H); 9.46 (s, 1 H). Anal. calc. for C14H12N2O3 (256.26):
C 65.61, H 4.72, N 10.93; found: C 65.60, H 4.68, N 10.90.

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4-(Benzyloxy)benzooic Acid Hydrazide (13): Yield 73%. M.p. 1408. IR (KBr): 3290, 1680, 1600. 1H-NMR
(300 MHz, (D6)DMSO): 5.15 (s, 2 H); 7.03 7.85( m, 9 arom. H); 9.38 (s, 1 H). Anal. calc. for C14H14N2O2
(242.27): C 69.40, H 5.82, N 11.56; found: C 69.37, H 5.84, N 11.47.
3-{[(4-Methylphenyl)sulfonyl]oxy}benzoic Acid Hydrazide (14): Yield 72%. M.p. 1558. IR (KBr): 3300,
1660, 1610, 1375. 1H-NMR (300 MHz, (D6)DMSO): 2.42 (s, 3 H); 7.16 7.90 (m, 8 arom. H); 9.91 (s, 1 H). Anal.
calc. for C14H14N2O4S (306.24): C 54.90, H 4.60, N 9.14; found: C 54.87, H 4.52, N 9.17.
3-(Benzoyloxy)benzoic Acid Hydrazide (15): Yield 60%. M.p. 1548. IR (KBr): 3300, 1710, 1680, 1620.
1H-NMR (300 MHz, (D6)DMSO): 7.62 8.15( m, 9 arom. H); 9.65( s, 1 H). Anal. calc. for C14H12N2O3 (256.26):
C 65.62, H 4.72, N 10.93; found: C 65.63, H 4.67, N 10.90.
1
3-(Benzyloxy)benzoic Acid Hydrazide (16): Yield 85%. M.p. 1278. IR (KBr): 3300, 1640, 1610. H-NMR
(300 MHz, (D6)DMSO): 5.21 (s, 2 H); 7.25 7.63 ( m, 9 arom. H); 9.82 (s, 1 H). Anal. calc. for C14H14N2O2
(242.27): C 69.40, H 5.82, N 11.56; found: C 69.29, H 5.86, N 11.49.
1H-Indole-3-acetic Acid Hydrazide (17): Yield 68%. M.p. 1378. IR (KBr): 3300, 1640, 1620. 1H-NMR
(300 MHz, (D6)DMSO): 4.27 (s, 2 H); 6.90 7.58 (m, 5arom. H); 9.81 ( s, 1 H). Anal. calc. for C10H11N3O
(189.21): C 63.48, H 5.86, N 22.21; found: C 63.41, H 5.80, N 22.17.
2-(2-Cyanoethyl)hydrazides 18 25: General Procedure. To one of the hydrazides 10 17 (0.5mol) in EtOH
(172 ml), acrylonitrile (1.5mol) was added and the mixture heated under reflux for 48 h. After evaporation, the
residue was chromatographed (silica gel, hexane/AcOEt 7:3): crystalline 18 25.
4-Hydroxybenzoic Acid 2-(2-Cyanoethyl)hydrazide (18): Yield 75%. M.p. 1358. IR (KBr): 3500, 3350, 2350,
1660, 1610. 1H-NMR (300 MHz, (D6)DMSO): 2.62 (t, 2 H); 3.05( t, 2 H); 6.90 7.74 (dd, 4 H); 10.0 (s, 1 H).
Anal. calc. for C10H11N3O2 (205.21): C 58.53, H 5.40, N 20.48; found: C 58.46, H 5.28, N 20.56.
4-{[(4-Methylphenyl)sulfonyl]oxy}benzoic Acid 2-(2-Cyanoethyl)hydrazide (19): Yield 61%. M.p. 1038. IR
(KBr): 3300, 2240, 1690, 1620. 1H-NMR (300 MHz, (D6)DMSO): 2.40 (s, 3 H); 2.62 (t, 2 H); 3.05( t, 2 H); 7.15
7.82 (m, 8 arom. H); 10.1 (d, 1 H). Anal. calc. for C17H17N3O4S (359.31): C 56.83, H 4.77, N 11.70; found:
C 56.84, H 4.69, N 11.71.
4-(Benzoyloxy)benzoic Acid2-(2-Cyanoethyl)hydrazide (20): Yield 50%. M.p. 1288. IR (KBr): 3300, 2230,
1725, 1670, 1625. 1H-NMR (300 MHz, (D6)DMSO): 2.63 (t, 2 H); 3.05( t, 2 H); 7.15 7.82 ( m, 9 arom. H); 10.1
(d, 1 H). Anal. calc. for C17H15N3O3 (309.32): C 66.01, H 4.89, N 13.59; found: C 66.03, H 4.81, N 13.61.
4-(Benzyloxy)benzoic Acid 2-(2-Cyanoethyl)hydrazide (21): Yield 66%. M.p. 1398. IR (KBr): 3310, 2240,
1680, 1630. 1H-NMR (300 MHz, (D6)DMSO): 2.62 (t, 2 H); 3.0 (t, 2 H); 5.15 (s, 2 H); 7.05 7.85( m, 9 arom. H);
10.01 (d, 1 H). Anal. calc. for C17H17N3O2 (295.34): C 69.14, H 5.80, N 14.23; found: C 69.10, H 5.86, N 14.17.
3-{[(4-Methylphenyl)sulfonyl]oxy}benzoic Acid2-(2-Cyanoethyl)hydrazide (22): Yield 64%. M.p. 398. IR
1
(KBr): 3300, 2300, 1680, 1600, 1375. H-NMR (300 MHz, (D6)DMSO): 2.42 (s, 3 H); 2.6 (t, 2 H); 3.0 (t, 2 H);
7.2 7.91 (m, 8 arom. H); 10.1 (s, 1 H). Anal. calc. for C17H17N3O4S (359.31): C 56.83, H 4.77, N 11.70; found:
C 56.80, H 4.81, N 11.67.
3-(Benzoyloxy)benzoic Acid 2-(2-Cyanoethyl)hydrazide (23): Yield 55%. M.p. 1928. IR (KBr): 3350, 2230,
1670, 1600. 1H-NMR (300 MHz, (D6)DMSO): 2.62 (t, 2 H); 2.91 (t, 2 H); 6.90 8.05( m, 9 arom. H); 10.15
(s, 1 H). Anal. calc. for C17H15N3O3 (309.32): C 66.01, H 4.89, N 13.59; found: C 65.96, H 4.87, N 13.58.
3-(Benzyloxy)benzoic Acid 2-(2-Cyanoethyl)hydrazide (24): Yield 72%. M.p. 688. IR (KBr): 3300, 2240,
1680, 1620. 1H-NMR (300 MHz, (D6)DMSO): 2.62 (t, 2 H); 3.25( t, 2 H); 5.15 (s, 2 H); 7.15 7.55 ( m, 9 ar-
om. H); 10.1 (d, 1 H). Anal. calc. for C17H17N3O2 (295.34): C 69.14, H 5.80, N 14.23; found: C 69.12, H 5.75,
N 14.19.
1H-Indole-3-acetic Acid 2-(2-Cyanoethyl)hydrazide (25): Yield 75%. M.p. 1238. IR (KBr): 3300, 2250, 1660,
1620. 1H-NMR (300 MHz, (D6)DMSO): 2.62 (t, 2 H); 3.05( t, 2 H); 4.21 (s, 2 H); 6.90 7.50 (d, 1 H); 10.01
(s, 1 H). Anal. calc. for C13H14N4O (242.28): C 64.45, H 5.82, N 23.13; found: C 64.41, H 5.75, N 23.09.
5,6-Dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitriles 26 33: General Procedure. To a soln. of one of
the (cyanoethyl)hydrazides 18 25 (0.01 mol) in anh. CHCl3 (135ml), chloroacetyl chloride (0.02 mol) was
added dropwise, and the mixture was refluxed for 3 h. After cooling and evaporation, the residue was dissolved
in anh. EtOH (120 ml), K2CO3 (0.04 mol) added, and the mixture refluxed overnight under stirring. Filtration
and evaporation gave, in each case, an oily residue, which was chromatographed (silica gel, hexane/AcOEt 7:3):
26 33.
5,6-Dihydro-2-(4-hydroxyphenyl)-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (26): Yield 67%. M.p. 2098.
IR (KBr): 3500, 2360, 1660. 1H-NMR (300 MHz, (D6)DMSO): 2.90 (t, 2 H); 3.91 (t, 2 H); 4.80 (s, 2 H); 6.82
7.65( dd, 4 arom. H). Anal. calc. for C12H11N3O3 (245.23): C 58.77, H 4.52, N 17.13; found: C 58.71, H 4.49,
N 17.17.

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5,6-Dihydro-2-{4-{[(4-methylphenyl)sulfonyl]oxy}phenyl}-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile
(27): Yield 66%. M.p. 1098. IR (KBr): 2320, 1660. 1H-NMR (300 MHz, (D6)DMSO): 2.42 (s, 3 H); 2.92 (t, 2 H);
3.90 (t, 2 H); 4.85( s, 2 H); 7.15 7.82 ( m, 8 arom. H). Anal. calc. for C19H17N3O5S (399.33): C 57.17, H 4.29,
N 10.52; found: C 57.10, H 4.19, N 10.57.
2-[4-(Benzoyloxy)phenyl]-5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (28): Yield 68%. M.p.
1
1538. IR (KBr): 2240, 1680. H-NMR (300 MHz, (D6)DMSO): 2.90 (t, 2 H); 3.91 (t, 2 H); 4.99 (s, 2 H); 6.81
7.42 (m, 9 arom. H). Anal. calc. for C19H15N3O4 (349.34): C 65.32, H 4.33, N 12.03; found: C 65.27, H 4.26,
N 12.00.
2-[4-(Benzyloxy)phenyl]-5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (29): Yield 76%. M.p.
1408. IR (KBr): 2240, 1680. 1H-NMR (300 MHz, (D6)DMSO): 2.90 (t, 2 H); 3.91 (t, 2 H); 4.84 (s, 2 H); 5.22
(s, 2 H); 7.20 7.35( m, 9 arom. H). Anal. calc. for C19H17N3O3 (335.36): C 68.05, H 5.11, N 12.53; found:
C 68.00, H 5.15, N 12.48.
5,6-Dihydro-2-{3-{[(4-methylphenyl)sulfonyl]oxy}phenyl}-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile
(30): Yield 60%. M.p. 2778. IR (KBr): 2260, 1690. 1H-NMR (300 MHz, (D6)DMSO): 2.39 (s, 3 H); 2.82 (t, 2 H);
4.12 (t, 2 H); 4.78 (s, 2 H); 7.05 7.45 ( m, 8 arom. H). Anal. calc. for C19H17N3O5S (399.33): C 57.18, H 4.29,
N 10.52; found: C 57.14, H 4.30, N 10.48.
2-[3-(Benzoyloxy)phenyl]-5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (31). Yield 65%. M.p.
1
2288. IR (KBr): 2260, 1698, 1665. H-NMR (300 MHz, (D6)DMSO): 2.82 (t, 2 H); 3.95( t, 2 H); 4.85( s, 2 H);
6.90 7.91 (m, 9 arom. H). Anal. calc. for C19H15N3O4 (349.34): C 65.32, H 4.33, N 12.03; found: C 65.27, H 4.35,
N 12.00.
2-[3-(Benzyloxy)phenyl]-5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (32): Yield 63%. M.p.
958. IR (KBr): 2100, 1660. 1H-NMR (300 MHz, (D6)DMSO): 2.85( t, 2 H); 3.91 (t, 2 H); 4.85( s, 2 H); 5.16
(s, 2 H); 7.10 7.45( m, 9 arom. H). Anal. calc. for C19H17N3O3 (335.36): C 68.05, H 5.11, N 12.53; found:
C 68.08, H 5.07, N 12.47.
2-(1H-Indol-3-ylmethyl)-5,6-dihydro-5-oxo-4H-1,3,4-oxadiazine-4-propanenitrile (33): Yield 85%. M.p.
1
898. IR (KBr): 2350, 1685. H-NMR (300 MHz, (D6)DMSO): 2.82 (t, 2 H); 3.82 (t, 2 H); 4.40 4.70 (m, 4 H);
6.90 7.65( m, 5arom. H). Anal. calc. for C 15H14N4O2 (282.30): C 63.82, H 5.00, N 19.85; found: C 63.76, H 4.93,
N 19.88.
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ReceivedJuly 26, 2001