Synthesis and potent antimicrobial activity of some novel 2-phenyl or methyl-4H-1-benzopyran-4-ones carrying amidinobenzimidazoles

Article abstract of DOI:10.1016/j.bmc.2004.12.006

Series of flavones and methyl-4H-1-benzopyran-4-ones carrying mono or diamidinobenzimidazoles at different positions were synthesized and evaluated for antibacterial and antifungal activities against E. coli, S. aureus, MRSA (methicillin-resistant S. aure

Full text of DOI:10.1016/j.bmc.2004.12.006

Bioorganic & Medicinal Chemistry 13 (2005) 1707–1714
Synthesis and potent antimicrobial activity of some novel
2-phenyl or methyl-4H-1-benzopyran-4-ones
carrying amidinobenzimidazoles
a
c
Hakan Goker,a,b, David W. Boykin and Sulhiye Yıldız
*
¨
aDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
cDepartment of Microbiology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
Received 2 April 2004; accepted 3 December 2004
Available online 29 December 2004
Abstract—Series of flavones and methyl-4H-1-benzopyran-4-ones carrying mono or diamidinobenzimidazoles at different positions
were synthesized and evaluated for antibacterial and antifungal activities against E. coli, S. aureus, MRSA (methicillin-resistant S.
aureus), MRSE (methicillin-resistant S. epidermidis), S. faecalis and C. albicans, C. krusei. The results showed that while all diami-
dines are inactive, the compounds having monoamidinobenzimidazoles at the C-6 position of the 2-phenyl-4H-1-benzopyran-4-one
have potent antibacterial activities, particularly, against Gram-positive bacteria. Compounds 23 and 22 exhibited the best inhibitory
activity with MIC values of 1.56 lg/mL against S. aureus, MRSA, MRSE and 3.12 lg/mL against C. albicans, respectively.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
256 lg/mL. In addition several flavonolignans and sim-
ple alkylated flavones were prepared and were shown
to be potent inhibitors of the NorA MDR(multi drug
resistance) efflux pump in S. aureus.4 In other study, a
series of flavones was synthesized for their DNA-gyrase
inhibitory and antibacterial activities.5 This led to the
identification of compounds with potent Escherichia coli
DNA-gyrase inhibitory activity, and significant antimi-
crobial activity The most active compound, ellagic acid
has an IC50 = 3.3 lg/mL, which is comparable to some
of the currently marketed 4-quinolone antibacterials.
Later, a series of novel aza-analogues of flavones were
reported as topoisomerase inhibitors from the same lab-
oratory.6 Recently, possible modes of antimicrobial ac-
tion mechanism of flavonoids have been discussed in a
review.7 The antimicrobial properties of dicationic aryl-
amidines against a number of pathogens including pro-
tozoa, bacteria and fungi have been reported.8 Among
the dicationic molecules pentamidine, has seen signifi-
cant clinical use. Pentamidine has been used clinically
against African trypanosomiasis, antimony-resistant
leishmaniasis and P. carinii pneumonia.8 This class of
dicationic molecules has been shown to bind to the min-
or-groove of DNA at AT-rich sites. This binding has
been postulated to be important in the mode of
antimicrobial action of these compounds possibly
leading to inhibition of DNA dependent enzymes
In our efforts to discover novel antimicrobial agents, we
have screened series of mono and diamidinobenzimidaz-
oles linked to various flavone nuclei. Flavonoids are a
groupof benzo- c-pyrone derivatives which exhibit a
wide spectrum of biological activity. Several recent pa-
pers report the presence of antibacterial activity for this
class of compounds.1 Thus, the retrochalcone licochal-
cone is active against Staphylococcus aureus with a
MIC of 6.25 lg/mL. Also, 5,7-dihydroxy-3,8-dimeth-
oxyflavone has an MIC of 50 lg/mL against S. epidermi-
dis. Another of the flavonone2 derivatives inhibits the
growth of S. aureus with MIC of 1.56–6.25 lg/mL,
and is effective against MRSA (methicillin-resistant
strains of S. aureus) as well. A series of 38 plant-derived
flavonoids representing seven different structural groups
were tested against antibiotic-resistant bacteria MRSA,
VRE (vancomycin-resistant enterococci), multidrug-
resistant Burkholderia cepacia and Klebsiella pneumo-
niae.3 Among them myricetin displayed the best inhibi-
tory activity with MIC values ranging from 32 to
Keywords: Methicillin-resistant S. aureus; Methicillin-resistant S. epi-
dermidis; 4H-1-Benzopyran-4-one; Amidinobenzimidazoles.
*
Corresponding author. Tel.: +90 312 222 0471; fax: +90 312 213
1081; e-mail: goker@pharmacy.ankara.edu.tr
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.006

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H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
(i.e., topoisomerases, nucleases) or possibly by direct
inhibition of transcription.8,9 In our previous work
many dicationic amidines have been found to be highly
effective against P. carinii, Cryptosporidium parvum, C.
albicans, Cryptococcus neoformans,8,9 Trypanosoma b.
rhodesiense,10 Trypanosoma cruzi11 and Leishmania
donovani.11 On the other hand, mono amidinobenzimid-
azoles have been identified as inhibitors of the bacterial
KinA/SpoOF two-component system TCS (which in-
clude a histidine protein kinase HPK and a response
regulator RR) which are responsible for adapting bacte-
ria to the environmental changes and survival within the
host.12 Many of these inhibitors display good in vitro
antibacterial activity in particular, against Gram-posi-
tive bacteria (i.e., S. aureus, MRSA, VRE).
using a modified Pinner method, and the imidate ester
was used directly without characterization to make the
amidines. The dicationic amidine derivatives 6 and 7
were obtained by reacting the imidate esters with appro-
priate amines (Scheme 1). 2-(4-Methylphenyl)-7-meth-
oxy-4H-1-benzopyran-4-one 10, was synthesized from
8 and 9, by a similar procedure and is outlined in
Scheme 2. Compound 10 was converted into 40-bro-
momethylflavone 11, by reaction with N-bromosuccin-
imide in the presence of benzoyl peroxide. Treatment
of this compound with hexamethylenetetramine in gla-
cial acetic acid gave 2-(4-formylphenyl)-7-methoxy-4H-
1-benzopyran-4-one 12. The known 4H-1-benzopyran-
4-one aldehydes 13,15 17,16 1915 and 2615 were reacted
with N-alkyl substituted-3,4-diaminobenzamidines in
the presence of 1,4-benzoquinone to give the targeted
benzimidazoles (Scheme 3).
Based on the above mentioned studies, we have synthe-
sized a series of compounds carrying either mono- or di-
N-alkyl substituted amidinobenzimidazoles linked to the
4H-1-benzopyran-4-one nucleus in order to investigate
their antimicrobial activity.
2,6-Dimethylchromone 30,17 was prepared from 2-ace-
toacetyl-4-methylphenol 29,13 as described in the litera-
ture (Scheme 4). Mono bromination of this compound
with one equivalent of N-bromosuccinimide gave the
31. The reaction of this compound with hexamethylene-
tetramine afforded 32 as previously described for 12.
The aldehyde was reacted with 3,4-diamino-N-isopropyl
or butylbenzamidine in the presence of 1,4-benzoqui-
none to give the benzimidazoles 33 and 34, respectively.
2. Chemistry
For the synthesis of 4H-1-benzopyran-4-one (3), the
Baker–Venkataraman13 rearrangement, a general proce-
dure for the preparation of flavones, was chosen
(Scheme 1). 50-Bromo-20-hydroxy-acetophenone was
first converted into a 4-bromobenzoyl ester (1) and this
intermediate was then treated with KOH/pyridine,
which formed 1,3-diketone (2). Treatment of the dike-
tone with concd H2SO4 lead to formation of the dibro-
minated flavone (3).14 The reaction of the dibromide
with copper(I) cyanide gave the corresponding bis nitrile
(4). The bis nitrile was converted into the imidate ester 5,
3. Microbiology
The benzimidazoles 6–34 were tested in vitro for anti-
bacterial activity against Gram-positive S. aureus, methi-
cillin-resistant S. aureus (MRSA, clinical isolate),
methicillin-resistant S. epidermidis (MRSE, clinical iso-
late), S. faecalis, Gram-negative E. coli bacteria and
Br
Br
OH
O
O
Br
OH
CH3
a
c
b
+
Cl
O
O
Br
Br
O
O
CH3
O
Br
2
NH
1
CN
e
Br
OC2H5
O
O
O
d
C2H5O
NC
N
Br
NH
O
O
O
3
5
4
5
NH
g
f
N
N
O
O
O
O
N
N
NH
N
7
6
Scheme 1. Reagents: (a) pyridine; (b) powder KOH/pyridine; (c) concd sulfuric acid; (d) CuCN; (e) HCl gas; (f) ethylenediamine; (g) isopropylamine.

H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
1709
CH3
MeO
CH3
O
MeO
OH
CH3
OH
O
CH3
a
c
+
b
MeO
O
O
CH3
O
O
Cl
O
8
9
H
Br
O
CH3
MeO
O
MeO
O
O
MeO
O
O
d
e
O
12
11
10
Scheme 2. Reagents: (a) pyridine; (b) powder KOH/pyridine; (c) concd sulfuric acid; (d) N-bromosuccinimide; (e) hexamethylenetetramine.
O
NH
N
H
NHR
R'
O
O
N
H
a
R'
O
O
14 R : R' : H
15 R : i-Pr R' : H
12 R' : OMe
13 R' : H
16 R : i-Pr R' : OMe
N
O
H
O
O
S
N
H
NH
O
O
a
S
N
H
18
17
20 R : H
21 R : i-Pr
O
22 R : n-butyl
23 R : cyclohexyl
24 R : benzyl
N
NH
O
a
RHN
NH
O
O
O
N
H
NH
O
O
a
25
N
19
NH
NH
N
O
NHR
N
H
H
O
O
a
O
O
N
H
NH
O
27 R : H
28 R : i-Pr
RHN
26
NH
Scheme 3. Reagents: (a) 1,4-benzoquinone/corresponding 3,4-diaminobenzamidine derivatives.
for antifungal activity against Candida albicans by the
macro-broth dilution18,19 assay to determine the MICs
(listed in Table 1). The synthesized compounds and ref-
erence drugs were dissolved in DMSO–water (50%), at
a concentration of 400 g/mL. The concentration was
adjusted to 100 lg/mL by 4-fold dilution with media
culture and bacteria solution. Data were not taken
for the initial solution because of the high DMSO
concentration (12.5%). As shown in Table 1, none of
the compounds have inhibitory effect against E. coli.
However, compounds carrying amidinobenzimidazoles
at position C-6 of 2-phenyl-4H-1-benzopyran-4-ones

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H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
O
O
CH3
OH
b
a
OH
CH3
c
CH3
H3C
H3C
H3C
O
O
30
O
29
O
CH3
N
O
O
CH3
N
O
O
CH3
e
d
HN
O
Br
33 R : i-Pr
34 R : n-butyl
NHR
H
O
32
31
Scheme 4. Reagents: (a) sodium hydride/EtOAc; (b) CH3COOH/HCl; (c) N-bromosuccinimide; (d) hexamethylenetetramine; (e) 1,4-benzoquinone/
3,4-diamino-N-isopropylbenzamidine or 3,4-diamino-N-butyllbenzamidine.
Table 1. Antibacterial and antifungal activity of compounds 6–34 (MIC, minimum inhibitory concentration lg/mL)
Compound E. coli ATCC 25922 S. aureus ATCC 29213 MRSA* MRSE** S. faecalis ATCC 29212 C. albicans
C. krusei
ATCC 10231 ATCC 10231
6
>50
50
>50
>50
50
>50
50
NT
NT
NT
25
NT
NT
NT
50
>50
>50
25
>50
>50
>50
7
14
>50
>50
>50
>50
50
50
12.5
15
16
12.5
50
6.25
12.5
12.5
50
25
25
50
50
>50
>50
50
18
20
25
12.5
12.5
12.5
12.5
12.5
12.5
25
12.5
25
25
21
22
>50
25
6.25
3.12
25
6.25
1.56
6.25
3.12
3.12
1.56
1.56
3.12
12.5
12.5
3.12
12.5
6.25
6.25
>50
6.25
23
24
>50
25
1.56
3.12
25
25
6.25
25
27
>50
>50
>50
>50
>50
25
25
25
12.5
50
50
50
50
NT
NT
NT
50
>50
28
33
NT
50
>50
25
50
50
50
>50
50
>50
50
34
50
50
25
Ampicillin
Fluconazol
Gentamisin
Sultamisilin
0.39
50
0.78
3.12
50
0.78
0.78
25
3.12
1.56
MRSA*: methicillin-resistant S. aureus (clinical isolate).
MRSE**: methicillin-resistant S. epidermidis (clinical isolate).
NT: not tested.
20–24, showed significant activity, in particular against
S. aureus, methicillin-resistant S. aureus (MRSA) and
methicillin-resistant S. epidermidis (MRSE). Among
them compound 23 is the most active with a MIC value
of 1.56 lg/mL against S. aureus and both of the drug-
resistant bacteria. While ampicillin and sultamisilin is
practically inactive against MRSA (50 and 25 lg/mL),
all the compounds belonging to the series 20–24 are
effective towards MRSA. Less activity was noted against
S. faecalis, the most potent compound was 24 with
6.25 lg/mL MIC value. According to the obtained re-
sults, bulky alkylated amidines such as cyclohexyl or
n-butyl results yield increased activity. Interestingly,
compounds in the isomeric series, where the benzimid-
azole system is linked to the 2-phenyl ring rather than
the benzopyran-4-one (14–16, 18), were not active. In
contrast to the broad spectrum antimicrobial activity
noted for other diamidines, compounds 6, 7, 27 and 28
show no significant activity against bacteria and fungi.
Meanwhile, compound 22 gave the best inhibitory activ-
ity with MIC values of 3.12 and 6.25 lg/mL against C.
albicans and C. krusei, respectively. Generally, all of
the compounds showed somewhat less antifungal than
antibacterial activity. Compounds 33 and 34, which
have a 2-methyl instead of 2-phenyl on the benzopy-
ran-4-one moiety, show much less activity than the cor-
responding compounds 21 and 22. This result
demonstrates the importance of the 2-phenyl group in
this series. Furthermore, in one of our recently pub-
lished article,20 we have reported that this class of the
cationic 4H-1-benzopyran-4-one-substituted benzimi-
dazoles exhibited no cytotoxic effects.
4. Conclusion
This work demonstrates that monoamidines in the benz-
imidazole flavone series show a good activity profile ver-
sus Gram-positive bacteria. Compounds 22 and 23,
having N-bulky alkyl substituted amidinobenzimidaz-
oles at the position C-6, of 2-phenyl-4H-1-benzopyran-
4-one exhibited the greatest activity against S. aureus,

H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
1711
methicillin-resistant S. aureus (MRSA) and methicillin-
resistant S. epidermidis (MRSE) with MIC values of
1.56 lg/mL. In vivo and mode of action mechanism
studies for compounds 22 and 23 are necessary to fully
understand and to expand upon their potent antibacte-
rial activities.
was heated 3 h under nitrogen. The reaction mixture
was poured into ice water. The green coloured precipi-
tate was filtered off and washed with water. The solid
was stirred in water containing ethylenediamine
(15 mL) for 1 h, filtered and washed with water, then
the solid was stirred in a solution of KCN (7.5 g) in
water (100 mL) for overnight. The dinitrile was filtered,
washed with water (2.98 g), dried, dissolved in (600 mL)
the mixture of CHCl3–EtOAc (30:1) and chromato-
graphed over neutral Al2O3 to yield a white fluffy solid
5. Experimental
1
Uncorrected melting points were measured on a Mel
Temp 3.0 capillary melting point apparatus. 1H
NMR and 13C NMR spectra were recorded employing
Varian GX400 spectrometer, chemical shifts ( d) are in
ppm relative to TMS, and coupling constants (J) are re-
ported in hertz. FAB, MS(CI) and ESI mass spectra
were recorded at Georgia Institute of Technology,
Atlanta, GA and Ankara University Faculty of Phar-
macy (Waters Micromass ZQ), respectively. Microanal-
yses were performed by Atlantic Microlab Inc.
(Norcross, GA) and were within 0.4% of calculated
values. All chemicals and solvents were purchased from
Aldrich Chemical Co., or Fischer Scientific.
2.27 g (70.7%): mp339–340 °C; H NMR (DMSO-d6)
d 7.28 (s, 1H), 8.0 (d, 1H, J = 8.73), 8.05 (d, 2H,
J = 8.25), 8.24 (dd, 1H, Jo = 8.7, Jm = 2.1), 8.3 (d, 2H,
J = 8.3), 8.43 (d, 1H, J = 2.1); Anal. (C17H8N2O2Æ0.25
H2O) C, H, N.
5.5. 6-(2-Imidazolinyl)-2-4-(2-imidazolinyl)phenyl-4H-1-
benzopyran-4-one hydrochloride (6)
The bis nitrile 4 (0.78 g, 2.87 mmol) was suspended in a
mixture of absolute EtOH and CH2Cl2 (30 mL, 50%),
cooled in ice bath and dry HCl gas was passed through
it. When the suspension was saturated with HCl(g), the
flask was stoppered and the contents were stirred at
room temperature until the TLC monitored the disap-
pearance of the bis nitrile (7 days). Ether was added,
then the suspension was collected by filtration, washed
with anhydrous Et2O and dried in vacuo to give bis imi-
date ester HCl, 5. A mixture of ethylenediamine (0.23 g,
3.8 mmol) and 5 (0.64 g, 1.68 mmol) in 15 mL of EtOH
was heated at reflux for 12 h. A white solid separated
which was filtered and washed with EtOH. The solid
was treated with water and the mixture was basified with
dilute NaOH solution, the solid filtered, washed with
water and dried, mp>350 °C. Free base was suspended
in absolute EtOH saturated with HCl gas and the mix-
ture was heated for 10 min. After cooling ether was
added and the solid was collected by filtration, washed
with ether and dried in vacuo, yield 0.2 g (26.7%) of
white powder: mp >370 °C; 1H NMR (DMSO-d6) d
4.19 (8H), 7.25 (s, 1H), 8.09 (d, 1H), 8.11 (d, 2H,
J = 8.8), 8.33 (dd, 1H, Jo = 8.7, Jm = 2.1), 8.37 (d, 2H,
J = 8.6), 8.66 (d, 1H, J = 2.2); 13C NMR(DMSO-d6):
178.3, 165.5, 165.1, 163.01, 159.6, 136.7, 134.6, 130.09,
128.3, 127.5, 125.9, 124.2, 121.4, 120.5, 109.9, 45.6;
MS (FAB) m/z 358.8 (M+); Anal. (C21H18N4O2Æ2HClÆ-
HOH) C, H, N.
5.1. 4-Bromobenzoic acid 20-acetyl-40-bromophenyl ester
(1)
p-Bromobenzoyl chloride (5 g, 22.7 mmol) was added to
a mixture of 5-bromo-2-hydroxy-acetophenone (4.89 g,
22.7 mmol) in pyridine (10 mL) and heated for 0.5 h,
at 80 °C. The mixture was cooled and poured into the
water, washed with dilute Na2CO3 solution and water,
recrystallization from EtOH to give 1 (8.2 g, 88.7%) as
a white needle: mp139–140 °C; Anal. (C15H10Br2O3)
C, H.
5.2. 1-(5-Bromo-2-hydroxyphenyl)-3-(4-bromophenyl)-
1,3-propanedione (2)
Compound 1 (8 g, 20 mmol) in 60 mL pyridine and
powdered KOH (2 g) were stirred for 1 h at 60 °C, the
reaction mixture was cooled, water was added and the
solution pH adjusted to 5 with HCl acid. The yellow
precipitate was filtered and washed with water. Recrys-
tallization from acetone–MeOH to give 2 (5.94 g,
74.3%): mp155–157 °C; Anal. (C15H10Br2O3) C, H.
5.3. 6-Bromo-2-(4-bromophenyl)-4H-1-benzopyran-4-one
(3)
5.6. 6-Isopropylamidino-2-(4-isopropylamidinophenyl)-
4H-1-benzopyran-4-one hydrochloride (7)
A mixture of 2 (5.5 g, 13.8 mmol) and concd H2SO4
(15 mL) was stirred at room temperature for 20 min.
Water was added and precipitate was collected, recrys-
tallization from EtOH to give 3 (4.69 g, 89.5%) as white
needles: mp253 °C; 1H NMR (DMSO-d6) d 7.01 (s, 1H),
7.79 (3H), 7.99 (dd, 1H, Jo = 8.8, Jm = 2.3), 8.05 (d, 2H),
8.11 (d, 1H, J = 2.3); Anal. (C15H8Br2O2) C, H.
A mixture of freshly distilled isopropylamine (0.355 g,
6 mmol) and 5 (0.64 g, 1.68 mmol) in 15 mL of EtOH
was heated at reflux for 12 h. A white solid separated
and was filtered and washed with EtOH. The solid was
treated with water and the mixture was basified with di-
lute NaOH solution, the solid filtered, washed with
water and dried, mp358 °C. Free base was suspended
in absolute EtOH saturated with HCl and the mixture
was heated for 5 min. After cooling dry ether was added
and the solid collected by filtration, washed with ether
and dried in vacuo, yield 0.643 g (46.03%) of white pow-
5.4. 6-Cyano-2-(4-cyanophenyl)-4H-1-benzopyran-4-one
(4)
A mixture of 3 (4.5 g, 11.8 mmol) and copper(I) cyanide
(2.2 g, 24.71 mmol) in 1-methyl-2-pyrolidinone (25 mL)
1
der: mp381–382 °C; H NMR (DMSO-d6) d 1.31 and

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H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
1.33 (12H), 4.16 (m, 2H, J = 6.6), 7.37 (s, 1H), 7.99 (d,
2H, J = 8.42), 8.08 (d, 1H, J = 8.5), 8.22 (dd, 1H,
Jo = 8.2, Jm = 2.7), 8.37 (d, 2H, J = 8.24), 8.44 (d, 1H,
J = 1.95), 9.37 (d, 1H), 9.72 (s, 1H), 9.89 (d, 1H); 13C
NMR (DMSO-d6) d 179.3, 164.1, 163.1, 162.7, 159.7,
136.4, 135.5, 133.6, 130.6, 128.8, 128.07, 127.5, 124.59,
121.69, 110.19, 47.2, 22.6; MS (FAB): m/z 390.9 (M+);
Anal. (C23H26N4O2Æ2HClÆ0.5C2H5OH) C, H, N.
was collected. Recrystallization of the solid from
EtOAc–n-hexane (50%) gave 12 as white powder, yield
(0.5 g, 58.4%): mp183–184 °C; MS (EI) m/z 280
(M+,100), 252 (52.4), 237 (56.9), 209 (6.3), 150 (23.6);
Anal. (C17H12O4) C, H.
5.12. General method for the synthesis of 14–16, 18, 20–
25
5.7. 4-Methylbenzoic acid 20-acetyl-50-methoxyphenyl
ester (8)
A solution of related mono aldehyde derivatives
(1.5 mmol), the corresponding N-substitued-amidino-
1,2-phenylenediamine HClÆ0.5HOH (1.5 mmol), and
1,4-benzoquinone (1.5 mmol) in EtOH (20 mL) was
heated at reflux for 12 h. The reaction mixture was
cooled to room temperature, and the blue coloured solid
was collected by filtration, washed with cold EtOH and
the solid stirred in dilute sodium hydroxide (1 M,
40 mL) for 5 h. The precipitate was washed with water,
until the colour completely disappeared and the solid
was dried. Free base was dissolved in hot EtOH
(200 mL), boiled with charcoal and filtered. The filtrate
volume was reduced to 50 mL and acidified with HCl
gas and the white powder solid was collected.
A mixture of 2-hydroxy-4-methoxyacetophenone (5 g,
30 mmol) and p-toluoyl chloride (4.65 g, 30 mmol) in
pyridine (10 mL) were heated for 0.5 h at 80 °C. The
mixture was cooled and poured into ice water, acidified
with HCl acid, precipitate was filtered washed with
water, recrystallization from EtOH gave 8 (7.8 g,
91.5%), as white crystals: mp66–67 °C; Anal.
(C17H16O4) C, H.
5.8. 1-(2-Hydroxy-4-methoxyphenyl)-3-(4-methylphenyl)-
1,3-propanedione (9)
Compound 8 (7.1 g, 25 mmol) in 40 mL pyridine and
powdered KOH (3 g) were stirred for 1 h at 60 °C, the
reaction mixture was cooled, water was added and the
solution pH adjusted to 5 with HCl. The yellow precipi-
tate was filtered and washed with water. Recrystalliza-
tion from EtOH gave 9 (4.5 g, 63.4%), as yellow
crystals: mp123–125 °C; Anal. (C17H16O4) C, H.
5.12.1. 2-[4-(5(6)-Amidinobenzimidazoyl)phenyl]-4H-1-
benzopyran-4-one hydrochloride (14). Yield 0.27 g,
38.2%, white: mp>350 °C; 1H NMR (DMSO-d6) d
7.17 (s, 1H), 7.5 (m, 1H), 7.83 (m, 3H), 7.91 (d, 1H,
J = 8.1), 8.05 (dd, 1H), 8.3 (d, 1H, J = 1.4), 8.35 (d,
2H, J = 8.5), 8.6 (d, 2H, J = 8.7), 9.29 (s, 2H), 9.52 (s,
2H); 13C NMR d 177.1, 165.6, 161, 155.6, 151.4, 138.6,
135.5, 134.4, 134.1, 128.5, 128.3, 127.1, 125.6, 124.7,
124, 123.5, 123.3, 118.6, 115.8, 114.7, 107.9; MS
(FAB) m/z 380.8 (M+); Anal. (C23H16N4O2Æ2HClÆHOH)
C, H, N.
5.9. 7-Methoxy-2-(4-methylphenyl)-4H-1-benzopyran-4-
one (10)
Compound 9 (4 g, 14 mmol) in sulfuric acid (98%,
20 mL) were stirred for 1 h at room temperature. Water
was added, the precipitate was collected and recrystalli-
zation from EtOH gave 10 (3.37 g, 90%) as white pow-
der: mp133–134 °C; Anal. (C17H14O3) C, H.
5.12.2. 2-[4-(5(6)-Isopropylamidinobenzimidazoyl)-
phenyl]-4H-1-benzopyran-4-one hydrochloride (15). Yield
0.267 g, 35.6%, white; mp>350 °C; H NMR (DMSO-
1
d6) d 1.31 (d, 6H, J = 6.4), 4.12 (m, 1H, J = 6.3), 7.19
(s, 1H), 7.5 (m, 1H), 7.67 (dd, 1H), 7.85 (m, 3H), 8.05
(dd, 1H), 8.12 (d, 1H), 8.35 (d, 2H, J = 8.6), 8.58 (d,
2H, 8.7), 9.12 (s, 1H), 9.5 (s, 1H), 9.65 (d, 1H); 13C
NMR (DMSO-d6) d 177.1, 162.1, 161.3, 155.7, 151.7,
139.3, 136.3, 134.4, 133.8, 129.5, 128.2, 127.1, 125.5,
124.8, 124.5, 123.9, 123.4, 118.6, 116.1, 114.7, 107.9,
45.2, 21.3; MS (FAB) m/z 422.8 (M+); Anal.
(C26H22N4O2Æ2HClÆ0.25HOH) C, H, N.
5.10. 2-(4-Bromomethylphenyl)-7-methoxy-4H-1-benzo-
pyran-4-one (11)
The mixture of 10 (2 g, 7.52 mmol), N-bromosuccin-
imide (1.34 g, 7.52 mmol) and catalytic amount of ben-
zoyl peroxide in CCl4 (30 mL) was heated at reflux for
6 h. The mixture was filtered while it was hot and
CCl4 was evaporated. Recrystallization of the solid res-
idue from EtOAc–n-hexane (50%) two times, gave pure
compound as white needles, yield (0.86 g, 33.2%): mp
187–188 °C; MS (ESI) m/z 345 and 347 (M+1, 31), 289
(100), MS (APCI) m/z 345 and 347 (M+1, 48), 141
(100); Anal. (C17H13BrO3) C, H.
5.12.3. 2-[4-(5(6)-Isopropylamidinobenzimidazoyl)-
phenyl]-7-methoxy-4H-1-benzopyran-4-one hydrochloride
1
(16). Yield 0.28 g, 36.7%, white: mp>330–332 °C; H
NMR (DMSO-d6) d 1.296 (d, 6H, J = 6.2), 3.92 (s,
3H), 4.12 (m, 1H, J = 6.3), 7.13 (s, 1H), 7.19 (s, 1H),
7.35 (m, 1H), 7.56 (d, 1H), 7.8 (d, 1H), 7.92 (dd, 1H),
8.14 (s, 1H), 8.32 (d, 2H, J = 8.6), 8.45 (d, 2H, 8.7),
8.99 (s, 1H), 9.4 (s, 1H), 9.5 (d, 1H); MS (FAB) m/z
453 (M+1); Anal. (C27H24N4O3ÆHClÆ0.25EtOHÆ0.5-
HOH) C, H, N.
5.11. 2-(4-Formylphenyl)-7-methoxy-4H-1-benzopyran-4-
one (12)
A mixture of 11 (0.85 g, 2.46 mmol) and hexamethylene-
tetramine (4 g, 28.6 mmol) in 30 mL of acetic acid (50%)
was heated at reflux for 4 h. HCl (10 mL, 50%) was
added and refluxing was continued for 0.5 h. The reac-
tion mixture was diluted with water and precipitate
5.12.4. 2-[5-(5(6)-Isopropylamidinobenzimidazoyl)thio-
phenyl]-4H-1-benzopyran-4-one hydrochloride (18). Yield
1
0.31 g, 40.3%, yellow: mp>350 °C: H NMR(DMSO-

H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
1713
d6) d 1.27 (d, 6H, J = 6.53), 7.01 (s, 1H), 7.47 (td, 1H),
7.53 (dd, 1H), 7.70 (d, 1H), 7.75 (d, 1H), 7.81 (td,
1H), 7.97 (s, 1H), 8.01 (dd, 1H), 8.25 (d, 1H), 8.28 (d,
1H), 8.9 (s, 1H), 9.45 (s, 1H), 9.5 (d, 1H); MS (FAB)
m/z 429 (M+1). Anal. (C24H20N4O2SÆ2HClÆ0.75HOH)
C, H, N.
J = 8.7), 8.04 (d, 1H, J = 8.8), 8.14 (m, 3H), 8.79 (dd,
1H, Jo = 8.5, Jm = 2), 8.95 (d, 1H, J = 2.1), 10.1 (s,
2H); MS (ESI) m/z 421 (M+1, 100); Anal.
(C26H20N4O2Æ2HClÆ1.2HOH) C, H, N.
5.13. 6-[2-(5(6)-Amidinobenzimidazoyl)]-2-[4-(5(6)-amid-
inobenzimidazoyl)phenyl]-4H-1-benzopyran-4-one (27)
5.12.5. 6-[2-(5(6)-Amidinobenzimidazoyl)]-2-phenyl-4H-
1-benzopyran-4-one hydrochloride (20). Yield 0.28 g,
40.6%, white: mp>350 °C; 1H NMR (DMSO-d6) d
7.08 (s, 1H), 7.62 (3H), 7.76 (dd, 1H, Jo = 8.5,
Jm = 1.9), 7.83 (d, 1H, J = 8.6), 7.97 (d, 1H, J = 8.73),
8.12 (2H), 8.24 (d, 1H, Jm = 1.8), 8.75 (dd, 1H,
Jo = 8.7, Jm = 2.1), 8.94 (1H), 9.2 (br s, 1H), 9.4 (br s,
This compound was prepared by the general method of
14 with 26 (1 mmol), 3,4-diamino-benzamidine HClÆ0.5-
HOH (2 mmol), and 1,4-benzoquinone (2 mmol) in
EtOH (20 mL) by refluxing for 24 h, yield 0.13 g,
1
20.2%, light yellow coloured: mp>350 °C; H NMR
(DMSO-d6) d 7.03 (s, 1H), 7.41 (m, 3H), 7.54 (m, 3H),
7.82 (d, 1H, J = 8.6), 8.1 (m, 6H), 8.45 (d, 2H,
J = 8.2), 8.68 (d, 1H, J = 8.2), 8.91 (s, 1H); MS (FAB)
1H);
MS
(FAB)
m/z
380.8
(M+).
Anal.
(C23H16N4O2Æ2HClÆ0.3HOH) C, H, N.
m/z 538.8
1.5C2H5OH) C, H, N.
(M+);
Anal.
(C31H22N8O2Æ2HOHÆ
5.12.6. 6-[2-(5(6)-Isopropylamidinobenzimidazoyl)]-2-phen-
yl-4H-1-benzopyran-4-one hydrochloride (21). Yield
1
0.16 g, 21.6%, white: mp>350 °C; H NMR (DMSO-
5.14. 6-[2-(5(6)-Isopropylamidinobenzimidazoyl)]-2-[4-(5(6)-
isopropylamidinobenzimidazoyl)phenyl]-4H-1-benzo-
pyran-4-one hydrochloride (28)
d6) d 1.342 (d, 6H, J = 6.4), 4.2 (m, 1H), 7.09 (s, 1H),
7.61 (3H), 7.68 (dd, 1H, Jo = 8.5, Jm = 1.9), 7.85 (d,
1H, J = 8.6), 8.05 (d, 1H, J = 8.73), 8.13 (3H), 8.83
(dd, 1H, Jo = 8.7, Jm = 2.1), 8.97 (d, 1H, J = 2), 9.2 (br
s, 1H), 9.52 (br s, 1H), 9.66 (br s, 1H); MS (FAB) m/z
422.8 (M+); Anal. (C26H22N4O2Æ2HCl) C, H, N.
This compound was prepared by the general method of
14 with 26 (1 mmol), 3,4-diamino-N-isopropylbenzami-
dine HClÆ0.5HOH (2 mmol), and 1,4-benzoquinone
(2 mmol) in EtOH (20 mL) by refluxing for 24 h, yield
0.21 g, 25.9%, grey coloured (base colour is yellow):
mp>350 °C; 1H NMR(DMSO-d6) d 1.35 (d, 12H,
J = 5.49), 4.13 (m, 2H), 7.22 (s, 1H), 7.57 (t, 2H,
J = 8.1), 7.78 (t, 2H, J = 8.75), 7.99 (d, 1H, J = 8.8),
8.09 (d, 2H, J = 8.4), 8.29 (d, 2H, J = 8.6), 8.51 (d,
2H, J = 8.4), 8.72 (dd, 1H, Jo = 8.35, Jm = 1.9), 8.88
(d, 1H, J = 2), 9.1 (s, 1H), 9.47 (s, 1H), 9.6 (s, 1H);
MS (FAB) m/z 623.6 (M+1); Anal. (C37H34N8O2Æ4HClÆ-
HOHÆ0.5C2H5OH) C, H, N.
5.12.7. 6-[2-(5(6)-Butylamidinobenzimidazoyl)]-2-phenyl-
4H-1-benzopyran-4-one hydrochloride (22). Yield 0.36 g,
1
44.7%, white: mp345–346 °C; H NMR (DMSO-d6) d
0.97 (t, 3H, J = 7.1), 1.41 (m, 2H, J = 7.2), 1.65 (m,
2H, J = 7.1), 3.45 (2H), 7.14 (s, 1H), 7.61 (4H), 7.85
(d, 1H, Jo = 8.5), 8.04 (d, 1H, J = 8.7), 8.1 (3H), 8.75
(dd, 1H, Jo = 8.7, Jm = 2), 8.77 (d, 1H, J = 2), 9.0 (br
s, 1H), 9.42 (br s, 1H), 9.76 (br s, 1H); MS (ESI) m/z
437 (M+1, 100); Anal. (C27H24N4O2Æ2HClÆ0.5C2H5OHÆ
0.25HOH) C, H, N.
5.15. 6-Bromomethyl-2-methyl-4H-1-benzopyran-4-one
(31)
5.12.8. 6-[2-(5(6)-Cyclohexylamidinobenzimidazoyl)]-2-
phenyl-4H-1-benzopyran-4-one hydrochloride (23). Yield
0.31 g, 34.4%, white: mp>340 °C; H NMR (DMSO-
d6) d 1.36–2.0 (m, 10H), 3.78 (d, 1H), 7.14 (s, 1H),
7.62 (4H), 7.84 (d, 1H, Jo = 8.5), 8.04 (d, 1H, J = 8.6),
8.08 (s, 1H), 8.14 (m, 2H), 8.75 (d, 1H, Jo = 8.7,
Jm = 2.1), 8.95 (d, 1H, J = 2), 9.11 (br s, 1H), 9.43 (br
s, 1H), 9.55 (1H); MS (ESI) m/z 463 (M+1, 100); Anal.
(C29H26N4O2Æ2HClÆ0.25C2H5OHÆ2.3HOH) C, H, N.
1
A mixture of 30 (0.8 g, 4.6 mmol) and N-bromosuccin-
imide (0.82 g, 4.6 mmol) in CCl4 (20 mL) was heated
at reflux overnight. The mixture was filtered while it
was hot and CCl4 was evaporated. The solid residue
was crystallized from EtOAc–n-hexane, mp115–
121 °C, a second time crystallization from EtOAc gave
pure cubic crystal compound, yield (0.55 g, 47.4%): mp
1
139–140 °C lit21 132 °C; H NMR (DMSO-d6) d 2.4 (s,
5.12.9. 6-[2-(5(6)-Benzylamidinobenzimidazoyl)]-2-phen-
yl-4H-1-benzopyran-4-one hydrochloride (24). Yield
0.345 g, 40.6%, white colour: mp340–341 °C; 1H
NMR(DMSO-d6) d 4.77 (d, 2H), 7.13 (s, 1H), 7.35–7.62
(m, 8H), 7.72 (dd, 1H), 7.87 (d, 1H, Jo = 8.5), 8.0 (d,
1H, J = 8.6), 8.15 (m, 2H), 8.19 (s, 1H), 8.76 (dd, 1H,
Jo = 8.6, Jm = 1.9), 8.95 (d, 1H, J = 2), 9.31 (s, 1H), 9.68
(s, 1H), 10.4 (t, 1H, J = 4.9); MS (ESI) m/z 471 (M+1,
100); Anal. (C30H22N4O2Æ2HClÆ0.5C2H5OH) C, H, N.
3H), 4.86 (s, 2H), 6.26 (s, 1H), 7.59 (d, 1H, J = 8.6),
7.83 (dd, 1H, Jo = 8.7, Jm = 2.2), 8.08 (d, 1H, J = 2.1);
MS (ESI) m/z 253 and 255 (M+1, 8), 173 (100); Anal.
(C11H9BrO2) C, H, N.
5.16. 2-Methyl-4H-1-benzopyran-4-one-6-carboxyalde-
hyde (32)
A mixture of 31 (0.55 g, 2.17 mmol) and hexamethylene-
tetramine (2.2 g, 15.7 mmol) in 10 mL of acetic acid
(50%) was heated at reflux for 2 h. HCl acid (5 mL,
50%) was added and refluxing was continued for 0.5 h.
The reaction mixture was diluted with water and satu-
rated with NaCl, then extracted with EtOAc. The organ-
ic layer was evaporated and the residue was crystallised
5.12.10. 2-Phenyl-6-[5(6)-(1,4,5,6-tetrahydro-pyrimidin-
2yl)-1H-benzimidazol-2-yl]-4H-1-benzopyran-4-one hydro-
1
chloride (25). Yield 0.48 g, 62%, white: mp>340 °C; H
NMR (DMSO-d6) d 2.01, 2.48 and 3.52 (m, 6H), 7.13 (s,
1H), 7.63 (m, 3H), 7.68 (d, 1H, J = 8.4), 7.84 (d, 1H,

1714
H. Go¨ker et al. / Bioorg. Med. Chem. 13 (2005) 1707–1714
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from EtOH–water, yield (0.28 g, 68.6%): mp171–
1
172 °C; H NMR (CDCl3) d 2.43 (s, 3H), 6.36 (s, 1H),
7.78 (d, 1H, J = 8.6), 8.21 (dd, 1H, Jo = 8.5, Jm = 2),
8.57 (d, 1H, J = 1.9), 10.1 (s, 1H); MS (ESI) m/z 189
(M+1); Anal. (C11H8O3) C, H.
7. Havsteen, B. H. Pharmacol. Ther. 2002, 96, 67.
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460.
9. Stephens, C. E.; Tanious, F.; Kim, S.; Wilson, D. W.;
Schell, W. A.; Perfect, J. R.; Franzblau, S. G.; Boykin, D.
W. J. Med. Chem. 2001, 44, 1741.
10. Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F.;
Wilson, W. D.; Boykin, D. W. J. Med. Chem. 2003, 46,
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11. Stephens, C. E.; Brun, R.; Salem, M. M.; Werbovetz, K.
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5.17. 6-[2-(5(6)-Isopropylamidinobenzimidazoyl)]-2-meth-
yl-4H-1-benzopyran-4-one hydrochloride (33)
This compound was prepared by the general method of
14 with 32 as a starting material, yield 0.29 g, 42.9%,
1
light pink: mp >320 °C; H NMR (DMSO-d6) d 1.36
(d, 6H, J = 6.4), 2.47 (s, 3H), 4.12 (m, 1H, J = 6.2),
6.38 (s, 1H), 7.71 (dd, 1H, Jo = 6.8, Jm = 1.7), 7.87 (d,
1H), 7.90 (d, 1H), 8.14 (d, 1H, Jm = 1.1), 8.64 (dd, 1H,
Jo = 8.7, Jm = 2.2), 8.92 (d, 1H, J = 2.19), 9.17 (s, 1H),
9.55 (s, 1H), 9.7 (d, 1H); MS (CI) m/z 361 (M+1); Anal.
(C21H20N4O2Æ2HClÆHOH) C, H, N.
5.18. 6-[2-(5(6)-Butylamidinobenzimidazoyl)]-2-methyl-
4H-1-benzopyran-4-one hydrochloride (34)
12. Weidner-Wells, M. A.; Ohemeng, K. A.; Nguyen, V. N.;
Fraga-Spano, S.; Macielag, M. J.; Werblood, H. M.;
Foleno, B. D.; Webb, G. C.; Barrett, J. F.; Hlasta, D. J.
Bioorg. Med. Chem. Lett. 2001, 11, 1545.
This compound was prepared by the general method of
14 with 32 as a starting material, yield 0.21 g, 30%, light
13. Baker, W. J. Chem. Soc. 1933, 1381.
1
grey: mp305–309 °C; H NMR (DMSO-d6) d 0.94 (t,
14. Marder, M.; Viola, H.; Bacigaluppo, J. A.; Colombo, M.
I.; Wasowski, C.; Wolfman, C.; Medina, J. H.; RuVeda,
E. A.; Paladini, A. C. Biochem. Biophys. Res. Commun.
1998, 249, 481.
15. Wurm, G.; Nordmann, M. Arch. Pharm. (Weinheim)
1988, 321, 355.
3H, J = 7.3), 1.43 (m, 2H, J = 7.2), 1.66 (m, 2H,
J = 7.2), 2.48 (s, 3H), 3.45 (q, 2H, J = 7), 6.34 (s, 1H),
7.70 (d, 1H), 7.82 (d, 1H, J = 8.4), 7.85 (d, 1H), 8.1 (s,
1H), 8.67 (d, 1H, J = 8.7), 8.89 (s, 1H), 9.01 (br s, 1H),
9.46 (br s, 1H), 9.76 (br s, 1H); MS (ESI) m/z 374
(M+1); Anal. (C22H22N4O2Æ2HClÆ1.2HOH) C, H, N.
16. Goker, H.; Suzen, S.; Kußs, C.; Boykin, D. W. Hetereocycl.
¨
Commun. 2000, 6, 385.
17. Angyal, S. J.; Morris, P. J.; Tetaz, J. R.; Wilson, J. G. J.
Chem. Soc. 1950, 2141.
Acknowledgements
18. Shadomy, S.; Pfaller, M. A. Laboratory Studies with
Antifungal Agents: Susceptibility Tests and Quantitation
in Body Fluids. In Manual of Clinical Microbiology, 5th
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Chapter 117, p 1173.
This work was supported by NIH Grant NIAID AI-
46365.
References and notes
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