New isochromans. 1. Synthesis and antimicrobial activity of 4-substituted (±)-1H-spiro[benzo[c]pyran-3(4H), 1′-cyclohexane]-1-ones

Article abstract of DOI:10.1002/jhet.5570440325

(Chemical Equation Presented) The reaction between homophthalic anhydride and cyclohexanone was examined both in the presence of DMAP or BF ₃?Et₂O complex as a catalyst. The latter yielded (±)-1-oxo-1H-spiro[benzo[c]pyran-3(4H), 1′-cyclohexane]-4- carboxylic acid (3) in a higher yield (82 %). A series of new (±)-4-(N,N-disubstituted-1-carbamoyl)-1H-spiro[benzo[c]pyran-3(4H), 1′-cyclohexane]-1-ones (5a-h) were synthesized from the parent acid 3 by a two-step reaction. Differentiating microbial screening was performed for most of the synthesized compounds against twelve microorganisms belonging to different taxonomic groups. The spiro acid 3 was active against all bacterial strains with MIC ≤ 20 μg/ml against B. subtillis and P. vulgaris. E. coli was the most sensitive strain to the antibacterial effect of the tested compounds.

Full text of DOI:10.1002/jhet.5570440325

May-Jun 2007
673
New Isochromans. 1. Synthesis and Antimicrobial Activity of
4-Substituted (±)-1H-Spiro[benzo[c]pyran-3(4H),
1'-cyclohexane]-1-ones
Milen G. Bogdanova, Blagovesta T. Gochevab, Darina B. Dimitrovab
and Mariana D. Palamarevaa*
aUniversity of Sofia, Faculty of Chemistry, 1 James Bouchier Blvd., Sofia 1164, Bulgaria
bUniversity of Sofia, Faculty of Biology, 8 Dragan Tsankov Blvd., Sofia 1164, Bulgaria
*E-mail: mpalamareva@chem.uni-sofia.bg
Received September 26, 2006
X
O
O
O
O
O
O
O
1
3,4,5a-h
2
X = OH, OCH3, cyclic amino groups
The reaction between homophthalic anhydride and cyclohexanone was examined both in the presence of
DMAP or BF3•Et2O complex as a catalyst. The latter yielded (±)-1-oxo-1H-spiro[benzo[c]pyran-3(4H), 1'-
cyclohexane]-4-carboxylic acid (3) in a higher yield (82 %). A series of new (±)-4-(N,N-disubstituted-1-
carbamoyl)-1H-spiro[benzo[c]pyran-3(4H),1'-cyclohexane]-1-ones (5a-h) were synthesized from the
parent acid 3 by a two-step reaction. Differentiating microbial screening was performed for most of the
synthesized compounds against twelve microorganisms belonging to different taxonomic groups. The spiro
acid 3 was active against all bacterial strains with MIC ꢀ 20 μg/ml against B. subtillis and P. vulgaris. E.
coli was the most sensitive strain to the antibacterial effect of the tested compounds.
J. Heterocyclic Chem., 44, 673 (2007).
has been performed in the presence of different basic or
acidic catalysts [12-16]. In contrast to aldehydes, the
cyclization of 1 with ketones has been less documented
and the only data available are for acyclic and aromatic
ketones in the presence of BF3•Et2O [13]. Until now,
cyclic ketones are not used in this reaction.
INTRODUCTION
The naturally occurring 1H-benzo[c]pyran-1-ones (3,4-
dihydroisocoumarins) as well as their synthetic analogues
[1,2] present a class of compounds possessing a broad
spectrum of pharmacological activities such as antiulcer,
[3] antiallergic, [4] antitumor, [5] antibacterial, [6]
antifungal [7] etc. Thus, this class of compounds can be
considered as a challenging target for the development of
synthetic strategies and the evaluation of the biological
activity of the derivatives.
Recently, we have demonstrated that the reaction be-
tween 1 and a series of aromatic aldehydes proceeds at
mild conditions (rt, 2-3 h) in the presence of 4-
dimethylaminopyridine (DMAP) as a catalyst, yielding
benzopyrano cycloadducts [12]. DMAP has been proved
to be a better catalyst than pyridine because of reduction
of the by-products. In this paper we continue our attempts
to specify further the scope and limitations of the reaction
between homophthalic anhydride and compounds
containing activated double bonds [17-21]. Our attention
was focused on the reaction between homophthalic
anhydride and cyclohexanone in the presence of different
catalysts. Furthermore, in the context to define the
influence of different substituents on the microbial
activity, we synthesized some new compounds with a
spiro coupling at position 3 and different substituents at
position 4 of the dihydroisocoumarin core and carried out
a comparative evaluation of their antimicrobial activity
against twelve microorganisms belonging to different
taxonomic groups.
Several approaches are used for synthesis of
compounds containing 3,4-dihydroisocoumarin core. The
most important of them are: a) epoxide-opening reaction
of ortho-metallated benzamide derivatives, [8] b) Diels-
Alder/retro-Diels-Alder sequence of acetylenic esters, [9]
c) ring-cleavage of phthalides, [10] d) reaction of homo-
phthalic acids with acidic chlorides and subsequent trans-
formations [11] and e) reaction of homophthalic anhydri-
des with carbonyl compounds [12]. The cyclization
proceeds in one step but usually the starting compounds
are not easily prepared. Moreover, hard conditions are
required for successful performance of the majority of the
reactions. The only exception is the reaction between
homophthalic anhydride 1 and a commercial carbonyl
compound. The reaction proceeds under mild conditions
and requires a suitable catalyst. Since now, the reaction

674
M. Bogdanov, B. Gocheva, D. Dimitrova and M. Palamareva
Vol 44
RESULTS AND DISCUSSION
The structures of the nucleophiles (Nu) and the yields of
the target compounds 5a-h are presented in Table 1.
Moreover, we converted acid 3 into the corresponding
methyl ester 4 by treatment with an etherial solution of
diazomethane, since the traditional esterification lead to
compounds with acyclic structure [12]. All synthesized
compounds were isolated in solid state after recrystal-
lization and were characterized by spectral methods (1H
nmr- and ir-spectra) and elemental analysis. The
interpretation of the 1H nmr spectra of the newly
synthesized compounds is in agreement with the literature
data for this class of compounds [27,29,30] and showed
the following characteristic signals: the signals for H-4
proton appear as a singlet within 4.52-4.61 ppm; the
signals for aliphatic protons from the spiro coupled chain
appear in the region 1.10-2.00 ppm; the signals for proton
H-8 appear at lower field (7.90-8.04) compared to the
other aromatic protons. This phenomenon is an indication
for proximity of H-8 to the lactone carbonyl group.
Trying to perform for the first time the reaction
between homophthalic anhydride and cyclic ketones, we
first used cyclization conditions similar to those applied
for the reaction with aldehydes [12]. Thus, the reaction
with cyclohexanone
2 was carried out at room
temperature in the presence of the Lewis base DMAP in
chloroform for 3 hours (Scheme 1). The consumption of 1
was been monitored by tlc and formation of a complex
reaction mixture was detected. It is worth noting that the
target acid 3 is already known by another protocol: it is
obtained in a lower yield (42 %) and considerably greater
reaction time (12 h) by cyclization of a halfester of cyclo-
hexylidene homophthalic acid by refluxing it in a mixture
of hydrobromic acid, acetic acid, and water [22]. In our
case, the expected spiro acid 3 was isolated in a moderate
yield (55 %) after column chromatography. Alternatively,
the reaction was carried out in the presence of BF3•Et2O
as a Lewis acid catalyst. After 8 h, the only product of the
reaction (tlc) was the spiro acid 3 isolated in 82 % yield.
Consequently, the BF3•Et2O complex has been proven to
be a better catalyst than DMAP in the reaction of 1 with
cyclic ketones. This phenomenon can be explained by the
ability of the boron atom to form coordinative bonds [23].
According to the results, the reaction can be mechanic-
stically considered to proceed via initial formation of a
six-membered cyclic transition state as expected by the
Zimmerman-Traxler model [24,25]. Thus, BF3 activates
both homophthalic anhydride and cyclohexanone through
coordination with their Lewis base centers (the oxygen
atoms). Alternatively, the Lewis base DMAP activates
only the homophthalic anhydride by reaction with its
acidic proton in position 4.
Scheme 1
O
X
O
O
Catalyst
O
O
CHCl3, rt
O
O
SOCl2/C6H6
3
: X = OH
1
2
CH2N2
4
: X = OCH3
O
O
Nu
Cl
NuH
CH2Cl2
O
O
O
O
Scheme 1 also gives the route of acid 3 to the target
compounds 5a-h. The presence of various pharma-
cophoric groups at position 4 of the isocoumarin moiety
gives the possibility to enhance the chemical diversity of
this class of compounds and is an additional point that can
modify the expected biological activity. Amides of type 5
including 5b are prepared through a different synthetic
strategies. DCC has been used as an activation agent for
the conversion of 3,4-dihydroisocoumarin-4-carboxylic
acids to amides by Yu et al. [13]. Alternatively, com-
pounds of type 5 have been obtained by reaction of 3-N-
substituted 1H-benzo[c]pyran-1-ones and correspondding
carbonyl compounds in boiling acetic acid [26,27]. Using
the latter method Boyd et al. [27] synthesize compound
5b for 9 days in 18 % yield. In our case, the transforma-
tion of 3 to the target compounds 5a-h was performed
similarly to the protocol of Haimova et al. [28] applied to
the tetrahydroisoquinoline series. Thus, the acid 3 was
converted into acylchloride in boiling benzene in the
presence of thionyl chloride. Treatment of the later with
cyclic secondary amines (NuH) gave the target products.
5a-h
Table 1
Structures of the Nu group and the yields of compounds 5a-h.
Microbial assay: Seven of the newly synthesized
compounds 3,4,5a-d,f were initially screened for their in

May-Jun 2007
Synthesis and Antimicrobial Activity of SpiroIsochroman-1-ones
675
Table 2
In Vitro Antimicrobial activity. Diameter of inhibition zone [mm] as a criterion for the antimicrobial activitya of the tested compounds.
Compounds
Microorganisms
Bacterial strains
Fungal strains
Gram positive
Gram negative
Phytopathogens
B.
S.
P.
C.
A.
A.
F.
P.
S. lutea
E. coli
subtilis aureus
vulgaris
albicans orizae niger oxysporum notatum
E. amylo-
vora
P. syrin-
gae
3
4
13b
12
–
–
–
11
–
12
–
10
11
–
10
13b
–
–
–
12
12
14b
–
–
9
10
15b
11
–
–
–
11
–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10
–
–
–
–
–
10
–
11
–
–
10
11
–
–
–
–
–
–
5a
5b
5c
5d
5f
11b
–
–
–
–
–
–
12
12
–
–
–
–
11b
–
Penicillin G
32
55
57
20
21
13
ndc
ndc
ndc
ndc
ndc
ndc
a Concentration 200μg/disc if not stated otherwise.b MICꢀ 20 μg/mlc Not Determine.
vitro antibacterial and antifungal activity against various
test-microorganisms belonging to different taxonomic
groups. The antibacterial activity was tested against the
bacterial strains: Bacillus subtillis (1049 NBIMCC),
Staphylococcus aureus (744 NBIMCC), Sarcina lutea
(Gram-positive); Esherichia coli (1752 NBIMCC),
Proteus vulgaris (1393 NBIMCC) and the phytopatho-
genic bacteria Erwinia amylovora (2331 NBIMCC) and
Pseudomonas syringae patovar siringae (2420 NBIMCC)
(Gram-negative). Antifungal activity was tested against
Candida albicans (72 NBIMCC), Aspergillus orizae (118
NBIMCC), Aspergillus niger (1107 NBIMCC), Fusarium
oxysporum (124 NBIMCC) and Penicillium notatum.
The microbial assay was carried out by the agar
diffusion method. The compounds were tested both at
concentration 20 and 200 μg/disc and the activity was
evaluated by the diameter of the inhibition zone in mm.
Penicillin G was used as a standard drug for comparison
of the antibacterial activity. A solvent control was kept.
The results obtained are summarized in Table 2.
In general, the tested compounds showed higher anti-
bacterial than antifungal activity. E. coli was the most
sensitive strain to the antibacterial effect of the tested
compounds (5 of 7 compounds active) followed by P.
vulgaris and S. aureus (4 compounds active), S. lutea and
B. subtillis (2 compounds active.) whereas E. amylovora
and P. siringae were the least sensitive organisms (1
compound active). The spiro acid 3 was active against all
bacterial strains with MIC ꢀ 20 μg/ml against B. subtillis
and P. vulgaris. Also, the antibacterial screening data
indicated that only two compounds of 4,5a-d,f have a
higher activity (MIC ꢀ 20 μg/ml) than the parent acid 3,
namely, methyl ester 4 against Gram-positive S. lutea and
amide 5a against Gram-negative E. coli. Thus in the
majority of the cases, the presence of an amide or ester
group instead of the carboxylic function at position 4 in
the dihydroisocoumarin core did not evoke antibacterial
activity against the strains tested. It is worth noting that
none of the tested compounds showed superior activity to
the standard drug Penicillin G.
F. oxysporum was the most sensitive fungal test strain
which was inhibited by compounds 5a,d with MIC ꢀ 20
μg/ml. Compounds 3,5a,d,f exhibited very weak
inhibition effect against P. notatum and methyl ester 4
against A. niger. None of the tested compounds exhibited
any inhibitory effect against C. albicans and A. orizae.
Consequently, the antifungal activity of the tested
compounds is limited.
EXPERIMENTAL
Melting points were determined on a Kofler microscope
Boetius PHMK 0.5 and are uncorrected. The ir spectra were
acquired in chloroform, if not stated otherwise, on a Specord 75
1
and are reported in reciprocal centimeters. The H nmr spectra
were obtained on a DRX Bruker Avance NMR spectrometer at
250 MHz in corresponding solvent given in parentheses. The
chemical shift is given in ppm (ꢀ) relative to tetramethylsilane as
internal standard. Elemental analyses were obtained in the
relevant laboratories at the Faculty of Chemistry, University of
Sofia. The tlc was done on precoated 0.2 mm Merck silica gel
60F254 plates. Merck silica gel 60 (0.040-0.063 mm) was used for
column chromatography. The strains S. lutea and P. notatum
were available in the Department of General and Industrial
Microbiology, Biological Faculty, Sofia University. The
remaining test microorganism strains were taken from National
Bank for Industrial Microorganisms and Cell Cultures
(NBIMCC), Bulgaria.
(±)-1-Oxo-1H-spiro[benzo(c)pyran-3(4H),1'-cyclohexane]-
4-carboxylic acid (3). DMAP catalyzed reaction: To a mixture
of homophthalic anhydride (1.24 g, 7.65 mmol) and 1.1 equiv.
of cyclohexanone (0.84 g, 8.46 mmol) in dry chloroform (10 ml)
DMAP 1 equiv. was added. The reaction mixture was stirred for

676
M. Bogdanov, B. Gocheva, D. Dimitrova and M. Palamareva
Vol 44
3 h at room temperature. At the end of the reaction (tlc) the
obtained carboxylic acids were extracted with 10 % sodium
hydrogen carbonate. The aqueous layer was acidified (pH = 3)
with 10% hydrochloric acid and extracted with ethyl acetate.
The organic layer was dried (sodium sulfate), filtered and the
solvent was then evaporated under reduced pressure. The
carboxylic acid 3 was obtained after column chromatography
and crystallization from benzene. Yield: 1.10 g (55 %), mp 165-
168° [Lit. mp 183-186° (from ethyl acetate-heptane)] [22]; ir
(nujol): 1590-1620 cm-1 (ArH), 1710 cm-1 (C=O), 1740 cm-1
(C=O); 1H nmr (DMSO-d6): ꢀ 1.15-1.90 (m, 10H, CH2-
aliphatic), 4.61 (s, 1H, H-4), 7.10 (d, 1H, J = 7.5 Hz, Ph-H),
7.40-7.50 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 7.60 (dt, 1H, J =
1.3 and 7.5 Hz, Ph-H), 7.92 (dd, 1H, J = 1.5 and 7.5 Hz, Ph-H).
Anal. Calcd. for C15H16O4: C, 69.22; H, 6.20. Found: C, 69.55;
H, 6.14.
BF3•Et2O catalyzed reaction: To a mixture of homophthalic
anhydride (1.24 g, 7.65 mmol) and cyclohexanone (0.79 ml,
7.65 mmol) in 10 ml dry chloroform BF3•Et2O (9.6 ml, 10
equiv.) was added. The reaction mixture was stirred for 8 h at
room temperature. At the end of the reaction (tlc) the reaction
mixture was diluted with ethyl acetate and was washed with 10
% hydrochloric acid and then with water (pH = 7). The organic
layer was dried (sodium sulfate), filtered and the solvent was
then evaporated under reduced pressure. The carboxylic acid 3
was obtained as white crystals after crystallization from
benzene. Yield: 1.64g (82 %), mp 165-168°.
1.5 and 7.5 Hz, Ph-H), 8.02 (dd, 1H, J = 1.5 and 7.5 Hz, Ph-H).
Anal. Calcd. for C20H25NO3: C, 73.37; H, 7.40. Found: C, 73.05;
H, 7.75.
(±)-4-(Morpholine-4-ylcarbonyl)-1H-spiro[benzo(c)pyran-
3(4H),1'-cyclohexane]-1-one (5b). This compound was
obtained as white crystals from methanol (yield: 0.21 g, 37 %),
mp 271-273° (Lit. mp 269-271°) [27]; NuH: morpholine; ir:
1
1590-1620 cm-1 (ArH), 1640 cm-1 (C=O), 1720 cm-1 (C=O); H
nmr (DMSO-d6): ꢀ 1.15-1.85 (m, 10H, CH2-aliphatic), 3.40-
3.60 (m, 4H, H-11, H-12), 3.62-3.70 (m, 2H, H-9), 3.80-3.98 (m,
2H, H-10), 4.52 (s, 1H, H-4), 7.32 (d, 1H, J = 7.5 Hz, Ph-H),
7.45 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 7.60 (dt, 1H, J = 1.5 and
7.5 Hz, Ph-H), 7.90 (dd, 1H, J = 1.3 and 7.5 Hz, Ph-H). Anal.
Calcd. for C19H23NO4: C, 69.28; H, 7.04. Found: C, 69.36; H,
7.00.
(±)-4-(4-Methylpiperazine-1-ylcarbonyl)-1H-spiro[benzo-
(c)pyran-3(4H),1'-cyclohexane]-1-one (5c). This compound
was obtained as white crystals from methanol (yield: 0.21 g, 51
%), mp 256-259°; NuH: 4-methylpiperazine; ir: 1590-1620 cm-1
(ArH), 1640 cm-1 (C=O), 1720 cm-1 (C=O); 1H nmr (DMSO-d6):
ꢀ 1.15-1.85 (m, 10H, CH2-aliphatic), 2.21 (s, 3H, NCH3), 2.25-
2.55 (m, 4H, H-11, H-12), 3.60-3.70 (m, 2H, H-9), 3.70-4.00 (m,
2H, H-10), 4.54 (s, 1H, H-4), 7.29 (d, 1H, J = 7.5 Hz, Ph-H),
7.44 (t, 1H, J = 7.5 Hz, Ph-H), 7.60 (t, 1H, J = 7.5 Hz, Ph-H),
7.90 (d, 1H, J = 7.5 Hz, Ph-H). Anal. Calcd. for C20H26N2O3: C,
70.15; H, 7.65. Found: C, 70.48; H, 7.88.
(±)-4-(4-(2-Hydroxyethyl)-piperazine-1-ylcarbonyl)-1H-
spiro[benzo(c)pyran-3(4H),1'-cyclohexane]-1-one (5d). This
compound was obtained as yellow crystals from methanol (yield:
0.20 g, 50 %), mp 236-239°; NuH: 2-piperazinoethanol; ir: 1590-
Methyl ester of (±)-1-Oxo-1H-spiro[benzo[c]pyran-3(4H),
1'-cyclohexane]-4-carboxylic acid (4). A stirring solution of 3
(0.50 g, 1.92 mmol) in 1 ml chloroform was treated with ether
solution of diazomethane (1.13 mmol/ml). The mixture was
stirred at room temperature for an hour. The excess of
diazomethane and chloroform was removed under reduced
pressure giving an oil. The later afforded 4 as colorless crystals
(from ethyl acetate). Yield: 0.41 g (78 %), mp 98-100° (Lit. mp
102-103°) [30]; ir (nujol): 1590-1620 cm-1 (ArH), 1710 cm-1
1
1620 cm-1 (ArH), 1640 cm-1 (C=O), 1720 cm-1 (C=O); H nmr
(methanol-d4): ꢀ 1.20-2.00 (m, 10H, CH2-aliphatic), 2.50-2.76 (m,
8H, H-11, H-12, H-13, H-14), 3.19-3.23 (m, 1H, -OH), 3.56-3.65
(m, 2H, H-9), 3.85-4.10 (m, 2H, H-10), 4.53 (s, 1H, H-4), 7.32 (d,
1H, J = 7.5 Hz, Ph-H), 7.47 (t, 1H, J = 7.5 Hz, Ph-H), 7.61 (t, 1H, J
= 7.5 Hz, Ph-H), 8.03 (d, 1H, J = 7.5 Hz, Ph-H). Anal. Calcd. for
C20H25NO3: C, 73.37; H, 7.40. Found: C, 73.05; H, 7.75.
1
(C=O), 1740 cm-1 (C=O); H nmr (DMSO-d6): ꢀ 1.15-1.90 (m,
(±)-4-(4-Phenylpiperazine-1-ylcarbonyl)-1H-spiro[benzo-
(c)pyran-3(4H),1'-cyclohexane]-1-one (5e). This compound
was obtained as colorless prisms from ethyl acetate (yield:
0.35g, 70 %), mp 234-236°; NuH: phenylpiperazine; ir: 1590-
10H, CH2-aliphatic), 3.65 (s, 3H, OCH3), 4.60 (s, 1H, H-4), 7.10
(d, 1H, J = 7.5 Hz, Ph-H), 7.40-7.50 (dt, 1H, J = 1.5 and 7.5 Hz,
Ph-H), 7.60 (dt, 1H, J = 1.3 and 7.5 Hz, Ph-H), 7.92 (dd, 1H, J =
1.5 and 7.5 Hz, Ph-H). Anal. Calcd. for C16H18O4: C, 70.06; H,
6.61. Found: C, 69.89; H, 6.75.
1
1620 cm-1 (ArH), 1645 cm-1 (C=O), 1730 cm-1 (C=O); H nmr
(DMSO-d6): ꢀ 1.15-1.90 (m, 10H, CH2-aliphatic), 3.04-3.16 (m,
2H, H-11), 3.24-3.31 (m, 2H, H-12), 3.58-3.63 (m, 2H, H-10),
3.90-4.10 (m, 2H, H-9), 4.61 (s, 1H, H-4), 6.83 (t, 1H, J = 7.3
Hz, Ph-H), 6.99 (m, 2H, Ph-H), 7.21-7.28 (m, 2H, Ph-H), 7.34
(d, 1H, J = 7.5 Hz, Ph-H), 7.45 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-
H), 7.60 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 7.91 (dd, 1H, J = 1.3
and 7.8 Hz, Ph-H). Anal. Calcd. for C25H28N2O3: C, 74.23; H,
6.98. Found: C, 74.11; H, 6.77.
(±)-4-(4-(2-Methoxyphenyl)-piperazine-1-ylcarbonyl)-1H-
spiro[benzo(c)pyran-3(4H),1'-cyclohexane]-1-one (5f). This
compound was obtained as colorless prisms from methanol
(yield: 0.23 g, 58 %), mp 180-183°; NuH: 2-methoxyphenyl-pi-
perazine; ir: 1590-1620 cm-1 (ArH), 1645 cm-1 (C=O), 1710 cm-1
(C=O); 1H nmr (methanol-d4): ꢀ 1.25-1.90 (m, 10H, CH2-
aliphatic), 2.85-3.20 (m, 4H, H-11, H-12), 3.60-3.80 (m, 2H, H-
10), 3.88 (s, 3H, OCH3), 3.95-4.18 (m, 2H, H-9), 4.58 (s, 1H, H-
4), 6.91-7.05 (m, 4H, Ph-H), 7.35 (d, 1H, J = 7.8 Hz, Ph-H),
7.48 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 7.62 (dt, 1H, J = 1.5 and
7.5 Hz, Ph-H), 8.04 (d, 1H, J = 7.8 Hz, Ph-H). Anal. Calcd. for
C26H30N2O4: C, 71.87; H, 6.96. Found: C, 72.15; H, 7.18.
General procedure for the synthesis of compounds 5a-h.
To a suspension of acid 3 in dry benzene, thionyl chloride (4
equiv.) was added. The reaction mixture was stirred at 80° for 3
h. Then, the solvents were evaporated under reduced pressure
and the residue was dissolved in dichloromethane. The cooled
solution (ice bath: 0°) was treated with the corresponding amine
(3 equiv.) and was stirred for an hour. At the end of the reaction
(tlc), the reaction mixture was diluted with ethyl acetate and was
washed with water (pH = 7). The organic layer was dried
(sodium sulfate), filtered and the solvent was then evaporated
under reduced pressure. The products 5a-h was isolated after
recrystallization.
(±)-4-(Piperidine-1-ylcarbonyl)-1H-spiro[benzo(c)pyran-
3(4H),1'-cyclohexane]-1-one (5a). This compound was
obtained as colorless prisms from methanol (yield: 0.23 g, 58
%), mp 253-255°; NuH: piperidine; ir: 1590-1620 cm-1 (ArH),
1
1640 cm-1 (C=O), 1720 cm-1 (C=O); H nmr (methanol-d4): ꢀ
1.20-2.00 (m, 16H, CH2-aliphatic, H-11, H-12, H-13), 3.35-4.00
(m, 4H, H-9, H-10), 4.64 (s, 1H, H-4), 7.29 (d, 1H, J = 7.5 Hz,
Ph-H), 7.47 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 7.58 (dt, 1H, J =

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677
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(±)-4-(4-(3-Chlorophenyl)-piperazine-1-ylcarbonyl)-1H-
spiro[benzo(c)pyran-3(4H),1'-cyclohexane]-1-one (5g). This
compound was obtained as colorless prisms from methanol
(yield: 0.26 g, 67 %), mp 190-192°; NuH: 3-chlorophenyl-
piperazine; ir: 1590-1620 cm-1 (ArH), 1640 cm-1 (C=O), 1730
1
cm-1 (C=O); H nmr (methanol-d4): ꢀ 1.25-2.05 (m, 16H, CH2-
aliphatic), 3.00-3.15 (m, 2H, H-11), 3.20-3.30 (m, 2H, H-12),
3.60-3.80 (m, 2H, H-10), 3.90-4.20 (m, 2H, H-9), 4.57 (s, 1H,
H-4), 6.82-6.98 (m, 3H, Ph-H), 7.20 (t, 1H, J = 7.5 Hz, Ph-H),
7.34 (d, 1H, J = 7.5 Hz, Ph-H), 7.49 (t, 1H, J = 7.5 Hz, Ph-H),
7.62 (dt, 1H, J = 1.5 and 7.5 Hz, Ph-H), 8.04 (d, 1H, J = 7.5 Hz,
Ph-H). Anal. Calcd. for C25H27ClN2O3: C, 68.41; H, 6.20. Found:
C, 68.41; H, 6.19.
(±)-4-(4-(3-Trifluoromethylphenyl)-piperazine-1-ylcarbo-
nyl)-1H-spiro[benzo(c)pyran-3(4H),1'-cyclohexane]-1-one
(5h). This compound was obtained as colorless prisms from
methanol (yield: 0.22 g, 58 %), mp 170-173°; NuH: 3-trifluoro-
methylphenyl-piperazine; ir: 1590-1620 cm-1 (ArH), 1645 cm-1
1
(C=O), 1720 cm-1 (C=O); H nmr (DMSO-d6): ꢀ 1.15-1.90 (m,
10H, CH2-aliphatic), 3.10-3.32 (m, 2H, H-11), 3.35-3.45 (m,
2H, H-12), 3.52-3.69 (m, 2H, H-10), 4.03-4.10 (m, 2H, H-9),
4.61 (s, 1H, H-4), 7.10 (d, 1H, Ph-H), 7.20-7.38 (m, 3H, Ph-H),
7.40-7.50 (m, 2H, Ph-H), 7.60 (dt, 1H, J = 1.3 and 7.5 Hz, Ph-
H), 7.92 (dd, 1H, J = 1.5 and 7.5 Hz, Ph-H). Anal. Calcd. for
C26H27F3N2O3: C, 66.09; H, 5.76. Found: C, 66.09; H, 5.75.
Agar diffusion method: Methanol solution was prepared for each
compound. The pure culture suspensions of the studied micro-
organisms in concentration 108 CFU/ml are inoculated into 12 ml
molten and tempered to 45°C nutrient agar (Difco) for the bacteria,
beer agar for the yeast and moulds and potato dextrose agar
(Oxoid) for the phytopathogenic bacteria. The inoculated media is
poured into sterile petri dishes. After it set, 6 holes have been cut in
each plate using a heat-sterilized cork borer. Corresponding
quantities of the methanol solutions of each chemical compound
have been spilled into the holes. These solutions should diffuse into
the agar medium so the petri dishes stay 2 hours in refrigerator at
4°. Afterwards they are put into thermostat for 1 to 5 days at
appropriate temperature for growing the test microorganisms.
Antimicrobial activity is determined as diameter of the inhibitory
zone, obtained after incubating in millimeters.
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