Synthesis, antimicrobial, and alkylating properties of 3-phosphonic derivatives of chromone

Article abstract of DOI:10.1002/1521-4184(200112)334:12<381::AID-ARDP381>3.0.CO;2-0

Dimethyl 2,6-dimethyl-4-oxo-4H-chromen-3-yl-phosphonate (1a) and dimethyl 6-methyl-2-phenyl-4-oxo-4H-chromen-3-yl-phosphonate (1b) were synthesized and reacted with primary aliphatic amines to yield title compounds 4-6. Their antibacterial properties against Gram-positive and Gram-negative bacteria strains were tested by the MIC method. Four of seventeen tested compounds (1d, 3, 4a, and 4b) exhibit detectabre activity against S. aureus. Some representative exampres of newly synthesized compounds were tested for their alkylating properties in vitro in the Preussmann test. Compounds 1a, 1c, 1d, 3, 5d, and 6a possess highly alkylating activity toward standard derivative 4-(4′-nitrobenzyl)pyridine (NBP).

Full text of DOI:10.1002/1521-4184(200112)334:12<381::AID-ARDP381>3.0.CO;2-0

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
Elzbieta Budzisza), Ewa Nawrotb),
3-Phosphonic derivatives of chromone 381
Synthesis, antimicrobial, and alkylating
Magdalena Maleckac)
properties of 3-phosphonic derivatives of
chromone
a)
Institute of Chemistry, Faculty of
Pharmacy, Medical University of Lodz,
90-151 Lodz, Muszynskiego 1, Poland
Dimethyl 2,6-dimethyl-4-oxo-4H-chromen-3-yl-phosphonate (1a) and dimethyl 6-
methyl-2-phenyl-4-oxo-4H-chromen-3-yl-phosphonate (1b) were synthesized and re-
acted with primary aliphatic amines to yield title compounds 4–6. Their antibacterial
properties against Gram-positive and Gram-negative bacteria strains were tested by the
MIC method. Four of seventeen tested compounds (1d, 3, 4a, and 4b) exhibit detectable
activity against S. aureus. Some representative examples of newly synthesized com-
pounds were tested for their alkylating properties in vitro in the Preussmann test.
Compounds 1a, 1c, 1d, 3, 5d, and 6a possess highly alkylating activity toward standard
derivative 4-(4′-nitrobenzyl)pyridine (NBP).
b)
Institute of Chemistry, Department
of Pharmaceutical Microbiology, Fac-
ulty of Pharmacy, Medical University
of Lodz, 90-151 Lodz, Poland
c) Department of Crystallography, Uni-
versity of Lodz, 90-236 Lodz, Poland
Key Words: Chromone; Phosphonic acids; Antibacterial and alkylating activity
Received: May 17, 2001 [FP582]
Introduction
Results
The phosphorus-containing antibiotics, especially phos-
phonic acids, steadily increasing in number, represent an
interesting group of antimicrobial agents. Some of these
compounds are produced by total chemical synthesis, but
many represent products of microbial origin. Up to the
seventies there were no major developments concerning
antimicrobial aminophosphonic acids[1–4]. However for the
last three decades numerous aminophosphonic and ami-
nophosphinic acid derivatives have been identified as
biologically active compounds [5]. Fosmidomycin
[HCON(OH)-(CH2)3-PO3H2], one of four struc-
Chemistry
Synthesis of dimethyl 2,6-dimethyl-4-oxo-4H-chromen-3-
yl-phosphonate 1a and dimethyl 6-methyl-2-phenyl-4-oxo-
4H-chromen-3-yl-phosphonate 1b was achieved by acyl-
ation of 2′-hydroxyacetophenone in basic conditions, sub-
sequent bromination of keto-methyl group and heating of
[13, 14]
bromo-derivative 2
with trimethyl phosphite under
conditionsofArbuzovreaction [15] (Scheme 1). When acetyl
chloride was used as an acylating agent, and a crude
turally related phosphonic acid derivatives, iso-
lated from Streptomyces, is the most interest-
ing agent among natural phosphonate anti-
O
C
O
C
1. 4 MeONa/MeOH
2. RCOCl
OH
O
R
O
R
Br2/CCl4
P(OMe)3
115°C
biotics for use in therapy [6]. On the other hand,
the derivatives of chromones or flavones are
another group of biologically interesting com-
pounds. They exhibit diverse biological proper-
ties including anticonvulsant [7], antimicro-
biological [8], and antitumor activities [9]. Little
is known about the biological activity of new
chromone derivatives containing phospho-
rus [10]. Synthesis of such benzo-γ-pyrone de-
rivatives containing phosphonic acid residue
was previously presented by us [11] and others
[12]. This paper is a continuation of our research
on the derivatives of chromone modified with
phosphonic acid residue (1a and 1b), their
chemical synthesis and reactions with primary
amines, as well the studies on antimicrobial
and alkylating properties of the obtained com-
pounds.
H3C
C
O
CH3
H3C
C
O
CH3
H3C
C
O
CH2Br
2
O
C
O
R
O
O
R
i
CH2P(O)(OCH3)2
H3C
C
O
H3C
P(O)(OCH3)2
1
3
1a R = CH3
1b R = Ph
i:
i:
spontanous cyclisation during chromatographic purification for R = CH3,
190°C /5h for R = Ph
Scheme 1
reaction product was purified on a silica gel column, the title
compound 1a was isolated as the only reaction product with
overall yield of 29%. In the case of acylation of 2′-hydroxy-
acetophenone with benzyl chloride we were able to isolate
by-product 3, which underwent further cyclisation into de-
sired product 1b after additional heating at 190 °C for 5 h.
———
Correspondence: Dr Elzbieta Budzisz, Institute of Chemistry,
Faculty of Pharmacy, Medical University of Lodz, 90-151 Lodz,
Muszynskiego 1, Poland.
E-mail: elora@ich.pharm.am.lodz.pl
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/1212/0381 $17.50 +.50/0

382 3-Phosphonic derivatives of chromone
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
In addition the structure of the derivatives 4b and 4d was
confirmed by X-ray analysis (Fig. 1a and Fig. 1b, respec-
tively). Although the oxaphosphinane rings of both deriva-
tives are not ideally planar (the atom deviating most from
weighted least-squares plane passing through O1, P2, C3,
C4, C10, C9 is O1–0.242 (2) Å in 4b and C3–0.058 (3) Å
in 4d), the C3, C4, C10, C9 fragment is perfectly planar,
which indicates structural similarity to nalidixic acid. More-
over, the intramolecular hydrogen bonds N32-H32...O4
also leads to planarity of the oxaphosphinane ring. Geome-
try of hydrogen bond: N32-H32: 0.82 (3) Å 3b, 0.93 (4) Å
4d; H32...O4: 1.89 (3)Å 4b, 1.76 (4)Å 3d; N32...O4:
2.59 (1) Å 4b, 2.56 (1) Å 4d, and N32-H32...O4: 144 (2)°
4b, 142 (4)° 4d.
2
O
O
O
O
R
P
OCH3
3
+ R NH2
1
R
1
2
R
P(OMe)2
C
R
O
3
O
NH
R
4a-d, 5a-d, 6a-d
1a-d
&RPSG
5ꢀ
5ꢁ
&+ꢀ
3K
5ꢂ
²
ꢀD
ꢀE
ꢀF
ꢀG
ꢃD
ꢃE
ꢃF
ꢃG
ꢄD
ꢄE
ꢄF
ꢄG
ꢅD
ꢅE
ꢅF
ꢅG
&+ꢀ
&+ꢀ
+
²
&+ꢀ
3K
²
²
+
&+ꢀ
&+ꢀ
+
&+ꢀ
3K
&+ꢁ3K
&+ꢁ3K
&+ꢁ3K
&+ꢁ3K
&+ꢀ
&+ꢀ
&+ꢀ
&+ꢀ
&+ꢀ
3K
+
&+ꢀ
&+ꢀ
+
&+ꢀ
3K
&+ꢀ
3K
+
&+ꢀ
&+ꢀ
+
&+ꢀ
3K
&+ꢀ
3K
ꢂ&+ꢁꢃꢁ2+
ꢂ&+ꢁꢃꢁ2+
ꢂ&+ꢁꢃꢁ2+
ꢂ&+ꢁꢃꢁ2+
Pharmacology
+
Scheme 2
In these studies we determined antibacterial activity of all
the new phosphonic acid chromone derivatives, including
compounds described in our previous paper [11]. Thus,
compounds 1a–d, 3, 4a–d, 5a–d and 6a–d were investi-
gated with regard to their antibacterial activity against
standard Gram-positive bacteria, Staphylococcus aureus
and Bacillus subtilis, and against two Gram-negative bac-
teria, Escherichia coli and Pseudomonas aeruginosa, the
Compound 1b was isolated with 47% yield. The structure
of 1a and 1b was unequivocally confirmed by spectral
analysis detailed in Experimental. Compounds 1a and 1b,
as well as previously obtained derivatives 1c and 1d [11]
,
were reacted with primary aliphatic amines i.e. benzy-
lamine, methylamine and ethanolamine, as previously de-
scribed [16]. Several derivatives of 2-methoxy-3-[1-alkyl-
amino)ethylidene or benzylidene]-2,3-dihydro-2,4-dioxo-
2λ5-benzo[e][1,2]oxaphosphinane (4–6) were obtained
(Scheme 2). 1H, 31P and 13C NMR, mass spectrometry, IR,
and elemental analysis elucidated the structure of com-
pounds 4, 5, and 6. All spectral data were in accordance
with those expected for structures 4, 5, and 6. The IR
spectra of all the investigated derivatives exhibited γ-py-
strains of different drug resistance recommended by Na-
[17]
tional Committee for Laboratory Standards (NCCLS)
.
Minimum inhibitory concentrations (MIC) were measured
using a standard method of dilution on solid support (Muel-
ler-Hinton agar, Difco). We used nalidixic acid, an active
therapeutic against Gram-negative bacteria, as a control
reference. The results were collected after incubation of
bacteria strain with solutions of the investigated com-
pounds on the agar plate for 18 h at 35 °C. Subsequent
dilutions in average range from 1000 to 31 µg/mL were
applied for preparation of the samples. Used concentra-
tions of nalidixic acid were much lower and ranged from
125 to 3.1 µg/mL.
rone C=O stretching bonds at 1580–1618 cm–1. In the 31
P
NMR spectra characteristic signals of phosphonic phos-
phorus atom appeared at 19.13–24.73 ppm. Reaction
yields and characteristics of derivatives 4–6 are collected
in Table 1.
Table 1. Data of the synthesized compounds.
O
CH3
2
O
O
O
P
R
1
R
3
R
NH
————————————————————————————————————————————————————————————–
Comp. R1
R2
R3
Mp
[°C]
Yield Solvent
[%] for cryst.
Formula*
Molecular
weight
————————————————————————————————————————————————————————————–
4a
4b
4d
5a
5b
6a
6b
6d
CH3
CH3
H
CH3
Ph
CH2Ph
CH2Ph
CH2Ph
CH3
94.0–95.0
62.0
57.1
90.0
46.3
75.0
69.6
64.5
57.9
methanol
C19H20NO4P
C24H22NO4P
C23H20NO4P
C13H16NO4P
C18H18NO4P
C14H18N05P
C19H20N05P
C18H18N05P
357.33
419.39
405.37
281.24
343.30
311.26
373.33
359.30
169.0–171.0
192.0–194.0
157.0–160.0
123.0–125.0
144.0–146.0
147.5–150.0
176.5–178.0
methanol
Ph
methanol
CH3
CH3
CH3
CH3
H
CH3
Ph
methanol
CH3
methanol
CH3
Ph
(CH2)20H
(CH2)20H
(CH2)20H
methanol:ethyl ether
benzene
Ph
acetone:ethyl ether
————————————————————————————————————————————————————————————–
*All new compounds gave satisfactory elemental analyses (±0.4%).

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
3-Phosphonic derivatives of chromone 383
a)
b)
C 8
O 1
C 7
C 6
C 23
C 9
C 8
C 7
C 2 3
O 1
O 22
O 2 2
C 9
O 21
C 6
P2
C 6 1
C 5
O 2 1
C 10
C 5
P 2
C 3
C 1 0
C 315
C 316
C 314
C 4
C 3 16
C 3 15
C 313
C 4
C 3
C 31
C 311
O 4
C 3 14
C 312
C 3 1
N 32
C 3 11
C 3 12
N 3 2
C 3 3
C 336
C 3 13
C 33
C 3 36
C 335
C 3 35
C 331
C 334
C 333
C 3 31
C 332
C 3 34
C 3 32
C 3 33
Figure 1. The molecular structure and atomic numbering scheme of compounds 4b (a) and 4d (b). Displacement ellipsoids are drawn at
40% probability level.
Table 2.
————————————————————————————————————————————————————————————–
Minimum inhibitory concentrations (MIC), µg/mL
———————————————————————————————————————————————————
Gram-positive
Gram-negative
—————————————————————————————
———————————————————
Cmpd Conc.
S. aureus
ATCC
6538P
S. aureus
ATCC*
29213
S. aureus
ATCC *
25923
E. faecalis
ATCC*
29212
B. subtilis
ATCC
6633
E. coli
ATCC*
35218
E. coli
ATCC
25922
Ps. aeruginosa
ATCC
27853
µg/mL
————————————————————————————————————————————————————————————–
1d
3
4a
4b
700–31
1000–31
500–31
700–31
500
250
62
125
62
600
250
62
125
62
600
250
250
250
62
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
250
125
250
≤3.1
> 500
–
nali- 125–3.1
dixic
62
≤3.1
≤3.1
55
acid
————————————————————————————————————————————————————————————–
(–) Lack of inhibiting activity.
* Strains of high drug-resistance.
The obtained results of minimum inhibitory concentrations
are listed in Table 2. Only very few of the investigated
derivatives (1d, 3, 4a and 4b) showed any inhibitory effect
in used concentrations. The remaining investigated com-
pounds showed no inhibitory effect against any of the
microorganisms tested. The most sensitive species to-
wards the tested compounds are two strains of Staphylo-
coccus aureus (ATCC 6538P and 29213). The values of
MIC for 4a are close to those ones determined for nalidixic
acid. However, none of the investigated compounds show-
ed any inhibitory effect toward Gram-negative bacteria.
cules, and of proteins. Such covalent cross-link of DNA
prevents unwinding of nucleic acids functionally important
in various biological processes, including replication and
transcription [19]. One of the most known and useful meth-
ods for determination of alkylating properties of tested
compounds is the in vitro test of Preussmann [20]. In this
test 4-(4′-nitrobenzyl)pyridine (NBP) is used as alkylating
target molecule. Nitrogen atom of pyridine ring of NBP is
alkylated giving respective quarternary salt, which in alka-
line conditions transforms into neutral coloured compound.
The level of alkylation can be quantified spectrophotomet-
rically in the range of 560 nm. Thus, compounds 1–6 of the
a, c, and d series (Table 3) were screened for their alkylat-
ing activity toward NBP. The screening was carried out at
a concentration of 0.005 mM/mL in 2-methoxyethanol. The
obtained results are presented in Table 3.
Alkylating agents belong to the first class of cytostatics used
for therapy [18]. In in vivo conditions these agents alkylate
nucleophilic centres of nucleobases and of amino acids,
resulting in cross-linking of a double stranded DNA mole-

384 3-Phosphonic derivatives of chromone
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
Table 3. NBP test results
————————————————————————————–
due its structural proximity to the investigated phosphonic
acid derivatives (Figure 2). Nowadays therapeutic applica-
tion of nalidixic acid is remarkably limited due to the fast
development of drug-resistant bacterial strains. This com-
pound, however, is still used as a common reference for
comparison of antibacterial activity of newly investigated
compounds [21, 22, 23]. Antibacterial activity of nalidixic acid
is coupled with its high affinity to double stranded DNA. The
presence of C-4 carbonyl group and C-3 carboxyl group,
both positioned in one plane, in nalidixic acid structure
seems to be responsible for its biological activity in respect
of the nucleic acid biomolecule [24]. In our studies com-
pound 1d is a close structural analogue of nalidixic acid in
which phopsphonic acid residue is introduced into the
chinolone system instead of carboxylic acid group. How-
ever, the two other compounds, 4a and 4b, posses the
cis-enone structural motif with enamine at its C-terminus.
X-Ray crystallographic data of compound 4b support an
almost planar structure of the oxaphosphinane ring, indi-
cating a close structural similarity between the nalidixic acid
and derivatives 4a and 4b.
No
Cmpds
Absorbance [A]a
Alkylation
activityb
————————————————————————————–
1
2
3
4
1a
1c
1d
3
0.8916
1.3118
0.6819
1.2191
0.1251
0.2263
0.3476
0.2588
0.2083
0.9821
0.7487
0.1213
0.0893
0.1200
+++
+++
+++
+++
++
++
++
++
++
+++
+++
++
+
++
5
6
7
8
8
9
10
11
12
13
4a
4c
4d
5a
5c
5d
6a
6c
6d
Cyclo-
phosphamideb
————————————————————————————–
a) means from 3 determinations, b) according to ref.[20] : (–) A <
0.05, (+) A = 0.05–0.1, (++) A = 0.1– 0.5, (+++) A > 0.5
Alkylating properties of compounds 1–6 of the a, c, and d
series (Table 3) were determined by the in vitro Preuss-
mann test [20]. The level of alkylation was quantified spec-
trofotometrically in the range of 560 nm. Most of the tested
compounds possess very high (+++) and high (++) alkyla-
tion activity toward NBP. For comparison the activity of
cyclophosphamide, the well know DNA alkylating agent,
used in the clinical treatment of some kinds of cancer, is
Discussion
Several new derivatives of chromone modified with phos-
phonic acid residue were chemically synthesized by the
three-step procedure outlined in Scheme 1. We obtained
3-phosphonic derivatives of chromone substituted with
various alkyl or aryl substituents at position 2 and/or 6 of
the benzo-γ-pyrone ring. These derivatives were further
functionalised by their reaction with aliphatic amines
(Scheme 2). Nucleophilic addition of nitrogen atom on C-2
of a pyrone ring resulted in formation of an open interme-
diate, which after subsequent oxy-anion nucleophilic attack
on phosphorus atom resulted in oxaphosphinane ring for-
mation. The detailed mechanism of this reaction was dis-
cussed in our previous paper [16]. All the newly synthesized
compounds (1a, 1b, 3, 4a, 4b, 4d, 5a, 5b, 6a, 6b, and 6d)
shown [20, 25]
.
In summary, we present here the chemical synthesis, and
spectral characteristics of new chromone analogues pos-
sessing an aminophosphonic acid residue. Biological activ-
ity of the obtained compounds was tested in two aspects.
As determined, the tested derivatives are very efficient
alkylating agants; however, they do not exhibit any signifi-
cant antibacterial activity. Further screenings for cytotoxic-
ity of compounds 1–6 in in vivo systems are in progress.
as well as previously described ones (1c, 1d, 4c, 5c, 5d,
[11, 16]
and 6c)
were tested for their antibacterial activity
toward standard Gram-positive bacteria Staphylococcus
aureus and Bacillus subtilis, and against two Gram-nega-
tive bacteria, Escherichia coli and Pseudomonas aerugi-
nosa, the strains of different drug resistance. Most of the
investigated derivatives showed no inhibitory effect in the
concentrations used. Only compounds 1d, 3, 4a, and 4b
show selective antibacterial activity against Gram-positive
bacteria of different drug-resistance strains. The values of
MIC (Table 2) are in the range of 600 to 62 µg/mL and
among the lowest values close to those determined for
nalidixic acid. We used nalidixic acid as a control reference
Acknowledgements
Financial support from Medical University of Lódz (grant No
502-13-516(178) to E.B.) is gratefully acknowledged. I am
grateful to Dr Habil. Barbara Nawrot for the help in preparation
of this manuscript. We thank Mrs. Agnieszka Zdolska, Mrs.
Joanna Róg, and Mrs. Agnieszka Rybarczyk for skillful experi-
mental assistance.
Experimental
The melting points were determined using an Electrothermal
1A9100 apparatus and they are uncorrected. The IR spectra
were recorded on a Pye-Unicam 200G Spectrophotometer in
KBr. The 1H NMR spectra were registered at 300 MHz on a
Varian Mercury spectrometer. 31P NMR spectra were recorded
on a Varian 75 MHz spectrometer. Positive chemical shift
values are assigned to compounds absorbing downfield of
phosphoric acid. The MS data were obtained on a LKB 2091
mass spectrometer (70 eV ionisation energy). Satisfactory ele-
mental analyses (±0.4% of the calculated values) were ob-
tained for the new compounds. The Microanalytical Laboratory
O
O
O
O
P
N
N
O
O
R2
CH3
P(O)(OCH3)2
COOH
O
N
H
4
nalidixic acid
1d
a R =CH
2
3
b R =Ph
2
Figure 2.

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
3-Phosphonic derivatives of chromone 385
of the Institute of Chemistry performed elemental analyses
using a Perkin Elmer PE 2400 CHNS analyser.
134 (35.43), 106 (14.90), 78 (15.30). Anal. (C, H, N) C18H17O5P
(344.29).
Dimethyl 2-methyl-4-oxo-4H-chromen-3-yl-phosphonate (1c),
dimethyl 2-phenyl-4-oxo-4H-chromen-3-yl-phosphonate (1d),
2-methoxy-3-[1-(alkylamino)ethylidene]-2,3-dihydro-2,4-dioxo-
2λ5-benzo[e][1,2]oxaphosphinane 3c, 4c, 5c, and 2-methoxy-
3-[1-(methylamino)benzylidene]-2,3-dihydro-2,4-dioxo-2λ5-
benzo[e][1,2]oxaphosphinane 4d were prepared as described
[11, 16]. All solvents were purified using standard methods.
General procedure for synthesis of 2-methoxy-3-[1-(alkyl-
amino)ethylidene or benzylidene]-2,3-dihydro-2,4-dioxo-
2λ5-benzo[e][1,2]oxaphosphinane (4–6)
To the solution of respective derivative of chromone (1a, 1b, 1c
or 1d) (10 mmol), in methanol (5 mL) a solution of benzylamine,
methylamine or ethanolamine (10 mmol) in methanol (0.5 mL)
were added. The mixture was left overnight at room tempera-
ture, and then cooled to –10 °C. The precipitated crude solid
was filtered off, dried, and crystallised. Colourless products 4–6
were obtained in 57–95% yields. The properties of the com-
pounds 4–6 are listed in Table 1.
Dimethyl 2,6-dimethyl-4-oxo-4H-chromen-3-yl-phosphon-
ate (1a)
To 10 mmol of 2′-bromo-2-acyloxy-5-methylacetophenone
melted in a flask, 12 mmol of trimethyl phosphite were added
dropwise at 110–115 °C. After 30 min heating the excess of
phosphite was removed by distillation and the resulting yellow
oil was applied on a silica gel column. The column was eluted
with a mixture of solvents chloroform: acetone = 5:1. The crude
product was crystallised by addition of a little methanol to yield
0.96 g of pure 1a (29.0%), mp 73.5–75.0 °C. Rf = 0.45 in ethyl
acetate:acetone 5:1.
2-Methoxy-6-methyl-3-[1-(benzylamine)ethylidene]-2,3-
dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane (4a)
IR (KBr): ν (cm–1) = 3432 (NH), 1579 (C=O), 1265 (P=O), 1014
(COC); 1H NMR (CDCl3): δ (ppm) = 2,35 (s, 3H, CH3), 2.58 (s,
3H, CH3), 3.73 (d, 6H, OCH3, 3JPH = 11.7 Hz), 4.65 (d, 2H, CH2,
J = 5.95), 6.92–7.83 (m, 8H, aromat.), 13.71 (broad, 1H, NH);
3
13C NMR (CDCl3): δ (ppm) = 18.57 (d, C=C-CH3, JPC = 3.15
IR (KBr): ν (cm–1) = 1650 (C=O), 1237 (P=O), 1026 (C-O-C),
1H NMR (CDCl3): δ (ppm) = 2.44 (s, 3H, CH3), 2.86 (s, 3H, CH3),
3.18 (d, 6H, OCH3, 3JPH=11.7 Hz), 7.27–7.93 (m, 3H, aromat.);
13C NMR (CDCl3): δ (ppm) = 21.04 (s, CH3), 21.37 (s, CH3),
Hz), 20.97 (CH3), 48.00 (CH2), 52.80 (d, OCH3, 2JPC = 7.16 Hz),
1
91.32( d, C=C-CH3, JPC = 196.10 Hz), 172.78 (d, C=C-CH3,
2JPC = 18.61 Hz), 182.85 (d, C=O, 2JPC = 14.87 Hz); 31P NMR
(CDCl3): δ (ppm) = 21.85; MS (70 eV): m/z (%) 357 (100%) [M+],
340 (16.97), 266 (30.70), 131 (29.82), 106 (29.65), 91 (67.68).
2
1
53.07 (d, OCH3, JPC = 5.73 Hz), 109.46 (d, C=C-P, JPC
=191.2 Hz), 176.25 (C=O, 2JPC = 25.19 Hz); 31P NMR (CDCl3):
δ (ppm) = 18.191; MS (70 eV): m/z (%) = 282 (100) [M+], 267
(23), 236 (11), 188 (27.56), 174 (52), 158 (15.85), 134 (24.0),
78 (6.03). Anal. (C, H, N) C13H15O5P (282.22).
2-Methoxy-6-methyl-3-[1-(benzylamine)benzylidene]-
2,3-dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane
(4b)
IR (KBr): ν (cm–1) = 3417 (NH), 1618 (C=O), 1240 (P=O), 1053
Dimethyl 5-methyl-2-oxo-phenylethyl-phosphonate (3)
(COC); 1H NMR (CDCl3): δ (ppm) = 2.36 (s, 3H, CH3), 3.32 (d,
To the 10 mmol of 2′-bromo-5-methyl-2-benzyloxyacethophen-
one melted in a flask, 1.5 g (12 mmol) of trimethyl phosphite
were added dropwise at 110–120 °C. After about 30 minutes of
heating the excess of phosphite was distilled off. The obtained
yellow oil was purified by column chromatography with chloro-
form: acetone 5:1, to yield 1.99 g of compound 3 (55%, mp
126.5–128.7 °C).
3
6H, CH3, JPH = 11.7 Hz), 4.29 (d, 2H, CH2, J = 6.15 Hz),
6.96–7.85 (m, 13H, aromat.), 13.64 (s, 1H, NH); 13C NMR
(CDCl3): δ (ppm) = 20.98 (s, CH3), 49.04 (CH2), 52.69 (d, OCH3,
2JPC = 6.87 Hz), 93.84 (d, C=C-CH3, 1JPC = 197.82 Hz), 172.86
2
(d,C=C-CH3, JPC = 18.04 Hz), 183.5(d, C=O, 2JPC = 5.17 Hz);
31P NMR (CDCl3): δ (ppm) = 19.24; MS (70 eV): m/z (%) = 419
(100%) [M+], 402 (10.32), 313 (12.34), 193 (53.18), 106 (17.23),
91 (34.15).
IR (KBr): ν (cm–1) = 1730, 1677 (C=O), 1274 (P=O), 1060
(C-O-C); 1H NMR (CDCl3): δ (ppm) = 2.43 (s, 3H, CH33), 3.59
2
(d, 2H, CH2, JPH = 22.2 Hz), 3.7 (d, 6H, O-CH3, JPH
=
2-Methoxy-3-[1-(benzylamine)benzylidene]-2,3-dihydro-
11.31 Hz), 7.13–8.21 (m, 8H, aromat.); 13C NMR (CDCl3): δ
2,4-dioxo-2λ5-benzo[e][1,2]-oxaphosphinane (4d)
1
(ppm) = 21.14(CH3), 40.47 (d, CH2, JPC =131.19 Hz), 53.26
IR (KBr): ν (cm–1) = 3414.0 (NH), 1590 (C=O), 1261 (P=O); 1H
2
(d, OCH3, JPC = 6.59 Hz), 165.10 (s, C=O), 191.44 (d, C=O,
3
2JPC = 6.87 Hz), 31P NMR (CDCl3): δ (ppm) = 23.54, MS (70eV):
m/z(%) = 362 (20.3) [M+], 105 (100), 77 (28.67). Anal. ( C, H,
N) C18H19O6P (362.30).
NMR (CDCl3): δ (ppm) = 3.32 (d, 3H, OCH3, JPH = 11.9 Hz),
4.30 (d, 2H, CH2, J = 6.15 Hz), 7.06–8.08 (m, 14H, aromat.),
13.65 (sbroad, 1H, NH); 13C NMR (CDCl3): δ (ppm) = 49.02
2
2
(CH2), 52.75 (d, JPC = 7.16 Hz), 93.78 (d, JPH =196.64 Hz),
118.94 (d, J = 8.90), 172.93 (d, J = 18.03 Hz), 183.26 (d, J =
15.07 Hz); 31P NMR (CDCl3): δ (ppm) = 19.13 Hz; MS (70 eV):
m/z (%) = 405 (100) [M+], 388 (9.60), 299 (10.68), 193 (52.57),
106 (18.96), 91 (38.74).
Dimethyl 6-methyl-2-phenyl-4-oxo-4H-chromen-3-yl-phos-
phonate (1b)
Compound 3 (6 mmol) was heated in an oil bath at 190–200 °C
for 5 h. The obtained dense brownish oil was purified on a silica
gel column with chloroform: acetone 9:1. The product was
crystallised by addition of a small quantity of methanol. 0.98g
of 1b (47.4% yield) was obtained (mp 143.5–145 °C. Rf = 0.46
ethyl acetate:acetone 9 : 1).
2-Methoxy-6-methyl-3-[1-(methylamine)ethylidene]-2,3-di
hydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane (5a)
IR (KBr): ν (cm–1) = 3433 (NH), 1590 (C=O), 1257 (P=O), 1010
(COC); 1H NMR (CDCl3): δ (ppm) = 2.36 (s, 3H, CH3), 2.54 (d,
3H, CH3, 4JPH = 0.99 Hz), 3.14 (d, 3H, NCH3, J = 5.16 Hz), 3.73
IR (KBr): ν (cm–1) = 1164 (C=O), 1618 (C=C), 1242 (P=O), 1027
3
(C-O-C); 1H NMR (CDCl3): δ (ppm) = 2.47 (s, 3H, CH3), 3.64
(d, 3H, O-CH3, JPH = 11.7 Hz), 6.99–7.82 (m, 3H, aromat.),
3
13.29 (sbroad, 1H, NH); 13C NMR (CDCl3): δ (ppm) = 18.21 (d,
(d, 2H, OCH3, JPH = 11.5 Hz), 7.26–8.01 (m, 8H, aromat.);
13C NMR (CDCl3): δ (ppm) = 20.97 (s, CH3), 53.27 (d, OCH3,
C=C-CH3, 3JPC = 2.86 Hz), 21.00 (s, CH3), 30.23 (d, NCH3, 4JPC
1
2
2JPC = 5.76 Hz), 100.76 (d, C=C-P, JPC = 196.2 Hz), 175.25
= 1.15 Hz), 52.79 (d, O-CH3, JPC = 7.16 Hz), 91.00 (d, -C=C,
2
1JPC = 196.39 Hz), 182.50 (d, C=O, JPC = 14.89 Hz), 173.50
2
(C=O, JPC = 24.19 Hz); 31P NMR (CDCl3): δ (ppm) = 16.61;
2
MS (70 eV): m/z (%) = 344 (32.72) [M+], 329 (37.73), 249 (100),
(d, C=C-CH3, JPC = 18.61 Hz); 31P NMR (CDCl3): δ (ppm) =

386 3-Phosphonic derivatives of chromone
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
21.97 Hz. MS (70 eV): m/z (%) = 281 (100%) [M+], 264 (40.55),
237 (11.49), 234 (7.87), 134 (12.0), 56 (23.49).
X-ray crystallographic details for 4b compound [29], Fig. 1a
C24H22NO4P, Mr = 419.39, triclinic, space group P-1, a =
10.645(1) b = 11.520(1) c = 9.643(1)Å α = 110.91(1) β =
97.39(1) γ = 98.58(1)°, V = 1070.9(1) Å3 , Z = 2, Dx = 1.301 Mg
2-Methoxy-6-methyl-3-[1-(methylamine)benzylidene]-
2,3-dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane
(5b)
m
–3, F(000) = 440, T = 293 K µ (CuKα) = 1.390 mm–1, colour-
less crystal of dimensions 0.2 × 0.3 × 0.4 mm, cell dimension
from 18 reflections in the range θ = 39.85–39.97°. The intensi-
ties were collected on a Rigaku AFC5S diffractometer using
graphite-monochromated CuKα radiation, λ = 1.54178Å, ω
IR (KBr): ν (cm–1) = 3433 (NH), 1589 (C=O), 1257 (P=O), 1010
(COC); 1H NMR (CDCl3): δ (ppm) = 2.47 (s, 3H, CH3), 2.82 (d,
3
[23]
3H, NCH3, J = 5.16 Hz), 3.64 (d, 3H, OCH3, JPH = 11.5 Hz),
scans. The intensities were corrected for the absorption
6.95–8.00 (m, 8H, aromat.), 13.22 (sbroad, 1H, NH); 13C NMR
effect with Tmin = 0.5664 and Tmax = 0.7892 and the Lorentz
and polarization efect. Intensities of 3 standard reflections
checked every 150 reflections: no decay, θ range 4.2–72.6°,
4
(CDCl3): δ (ppm) = 21.00 (s, CH3), 30.23 (d, N-CH3, JPC
=
1.15 Hz), 53.39 (d, O-CH3, 2JPC = 7.15 Hz), 96.70 (d, C=C, 1JPC
2
= 195.37 Hz), 184.50 (d, C=O, JPC = 15.81 Hz), 172.32 (d,
4277 measured reflections of which 4052 were unique (Rint
=
C=C-CH3, JPC = 19.53 Hz); 31P NMR (CDCl3): δ (ppm) =
2
0.022) The structure was solved by direct methods using
SHELXS86[27], which revealed the positions of non-H atoms.
All H-atoms were located in a difference Fourier map. The
structure was refined on F2 by full-matrix least-squares meth-
ods using SHELX97[28] non-H atoms refined anisotropically,
H-atoms fixed in calculated positions excluding H32. The re-
finement was carried out on 304 parameters using 2840 ob-
served reflections with I>2σ (I) gave R1 = 0.048 wR2 = 0.1465
(w =1/[σ2(Fo2)+(0.0876P)2], where P = (Fo2+2Fc2)/3, S = 1.018,
max and min residual electron density 0.280, –0.289 e Å–3
19.60 Hz; MS (70 eV): m/z (%) = 343 (29.35) [M+], 326 (100),
282 (54.23), 234 (13.78), 77 (18.96).
2-Methoxy-6-methyl-3-[1-(ethanolamine)ethylidene]-
2,3-dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane
(6a)
IR (KBr): ν (cm–1) = 3371 (NH), 1594 (C=O), 1257 (P=O), 1013
(CO-C); 1H NMR (CDCl3): δ (ppm) = 2.34 (s, 3H, CH3), 2.56 (d,
3H, CH3, 4JPH = 0.99 Hz), 2.84(sbroad, 1H, OH), 3.67 (t, 2H, CH2,
X-ray crystallographic details for 4d compound [29], Fig. 1b
3
JHH = 3.17), 3.71 (d, 3H, OCH3, JPH = 11.9 Hz), 3.91(t, 2H,
C23H20NO4P, Mr = 405.39, triclinic, space group P-1, a =
9.994(5) b = 11.682(3) c = 9.085(1)Å, α = 95.24(1) β =
101.90(2) γ = 70.20(3) °, V = 976.3(6) Å3, Z = 2, Dx = 1.379 Mg
CH2, JHH = 5.35), 7.00–7.82 (m, 3H, aromat.), 13.54 (sbroad, 1H,
3
NH); 13C NMR (CDCl3): δ (ppm) = 18.61 (d, C=C-CH3, JPC
=
3.15 Hz), 20.98 (s, CH3), 46.26 (d, CH2), 4JPC = 1.15 Hz), 52.90
m
–3, F(000) = 424, T = 293 K, µ (CuKα) = 1.506 mm–1, colour-
(d, OCH3, 2JPC = 7.16 Hz), 91.00 (d, -C=C, 1JPC = 196.39 Hz),
182.50 (d, C=O, 2JPC = 14.89 Hz), 173.50 (d, C=CCH3, 2JPC
=
less crystal of dimensions 0.5 × 0.2 × 0.1mm, AFC5S Rigaku
four-circle diffractometer, graphite-monochromated CuKα ra-
diation, λ = 1.54178Å, ω scans, cell constants from 12 reflec-
tions in the range θ = 38.36–39.81°, intensities of 3 standard
reflections checked every 150 reflections, no decay, θ range
4.79–72.63°, 3924 measured reflections of which 3691 were
18.61 Hz); 31P NMR (CDCl3): δ (ppm) = 22.04 Hz; MS (70 eV):
m/z (%) = 311 (25.76) [M+], 280 (42.91), 268 (100), 251 (99.37),
227 (8.99), 145 (9.16).
2-Methoxy-6-methyl-3-[1-(ethanolamine)benzylidene]-
2,3-dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane
(6b)
unique (Rint= 0.013 ). The intensities were corrected for absorp-
[22]
tion
efect with Tmin = 0.5382 and Tmax = 0.8705. Structure
solution by direct methods using SHELXS86[23], and refined
296 parameters on F2 by full-matrix least-square methods using
SHELX97[20], non-H atoms refined anisotropically, H-atoms
fixed in calculated positions and refined using a riding model.
Final R1 = 0.0501 and wR2 = 0.1293 (w = 1/[σ2(Fo2) +
(0.0731 P)2], where P = (Fo2 + 2Fc2)/3, S = 0.786, max and min
residual electron density 0.47, –0.408 e Å–3
IR (KBr): ν (cm–1) = 3311 (NH, OH), 1617 (C=O), 1230 (P=O),
1029 (COC); 1H NMR (CDCl3): δ (ppm) = 1,26 (sbroad, 1H, OH),
2,37 (s, 3H, CH3), 3.27 (d, 2H, CH3), 3.33 (d, 6H, OCH3,
3JPH=11.7 Hz), 3.74 (t, 2H, CH2, J = 5.35), 6.96–8.11 (m, 8H,
aromat.), 13.46 (sbroad, 1H, NH); 13C NMR (CDCl3): δ (ppm) =
21.03 (CH3), 47.34 (CH2), 52.75 (d, OCH3, 2JPC = 7.16), 49.04
(d, C-O-C, 2J=4.87 Hz), 61.16 (CH2), 93.44 (d, C=C-Ph, 1JPC
=
2
Pharmacology
198.11 Hz), 173.14 (d, C=C-Ph, JPC = 18.32 Hz), 183.22 (d,
C=O, 2JPC = 15.46 Hz); 31P NMR (CDCl3): δ (ppm) = 24.73; MS
(70 eV): m/z (%): 373 (20.69) [M+], 342 (55.35), 330 (100), 313
(17.93), 227 (10.4), 179 (11.17), 104 (7.49).
Antibacterial activity
The new synthesised compounds were tested. Minimal inhibi-
tory concentrations (MIC) were measured using standard
method of dilution on solid support (Mueller-Hinton agar, Difco).
Bacterial suspension (104 CFU/mL) was spotted on the support
with Steer’s replicator. Simultaneously, MIC for nalidixic acid
was measured. MHA plates with bacterial suspension alone
were used as a control. The plates were analysed after 18 h
incubation at 35 °C. DMSO solutions of all analysed com-
pounds were prepared with the concentration of 1000 to
31 µg/mL.
2-Methoxy-6-methyl-3-[1-(ethanolamine)benzylidene]-
2,3-dihydro-2,4-dioxo-2λ5-benzo[e][1,2]oxaphosphinane
(6d)
IR (KBr): ν (cm–1) = 3337.0 (NH, OH), 1583 (C=O), 1220 (P=O),
1056 (C-O-C); 1H NMR (CDCl3): δ (ppm) = 2.43 (sbroad, 1H, OH),
3.25 (t, 2H, CH2, J = 5.36), 3.32 (d, 3H, OCH3, 3JPH = 11.9 Hz),
3.71 (t, 2H, CH2, J = 5.36), 7.05–8.24 (m, 9H, aromat.), 13.45 (s,
1H, NH); 13C NMR (CDCl3): δ (ppm) = 47.39 (s, CH2-CH2-OH),
52.82 (d, OCH3, 2JPC = 6.87 Hz), 61.06 (s, CH2-CH2OH), 93.34 (d,
C=CPh, JPC = 198.1 Hz), 151.04 (d, 2JPC = 4.88 Hz), 173.18 (d,
Alkylating properties (NBP test)
The tested compounds were dissolved in 2-methoxyethanol
(1 mL, 0.005 mmol) and NBP (1 mL, 5% 2130-methoxyethanol
solution) was added. The samples were heated at 100 ± 0.5 °C
for 1 h and cooled quickly to temp. 20 °C. To the samples
2-methoxyethanol (2.5 mL) and piperidine (0.5 mL) were added
3
2
C=C-Ph, JPC = 18.32 Hz), 182.90 (d, C=O, JPC = 15.17 Hz);
31P NMR (CDCl3): δ (ppm): 19.42; MS (70 eV): m/z (%) = 359
(17.26) [M+], 315 (78.40), 283 (13.51), 235 (100), 120 (28.40), 92
(25.78), 77 (38.48).

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 381–387
3-Phosphonic derivatives of chromone 387
[13] A. Hercouet, M. Le Corre, Y. Le Floc’h, Synthesis 1982, 7,
to give a total volume of 5 mL. The final concentration was 1.0 ×
10–3 mol/L. After 90 s the absorbance was measured at λ =
560 nm in glass cells (1 cm), in the presence of 2-methoxy-
ethanol (Table 3)
597–598.
[14] J. A. Donnelly, P. Kerr, P. O’Boyle, Tetrahedron 1973, 29,
3979–3983.
[15] D. E. C. Corbridge in Phosphorus – An Outline of its Chem-
istry, Biochemistry and Technology. Elsevier, The Nether-
lands, 1990, Chapter 4.
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