A new series of 2-substituted 3-phosphonic derivatives of chromone. Part II. Synthesis, in vitro alkylating and in vivo antitumour activity

Article abstract of DOI:10.1039/b205800f

Products of the reaction of (±)-O-acetylmandeloyl chloride with, respectively, sodium 2-hydroxy- or 2-hydroxy-5-methyl-acetophenone were brominated and coupled with trimethyl phosphite to give the Perkov products 4a and 4b, the Wittig-type products 6a and 6b and the title 3-phosphonic derivatives of chromone, 7a {2-[1-(±)-acetoxybenzyl]-3-(dimethoxyphosphoryl)-4-oxo-4H-chromene} and 7b {2-[1-(±)-acetoxybenzyl]-3-(dimethoxyphosphoryl)-4-oxo-6-methyl-4H- chromene}. Esters 7a and 7b were subjected to acidic hydrolysis to give the corresponding phosphonic acids 8a and 8b, and the unexpected phosphonolactones 9a and 9b. They were also treated with benzylamine forming the corresponding salts of the cyclic phosphonolactones (10a and 10b). Derivatives 4a,b, 6a,b-10a,b were tested for in vitro alkylating activity while compounds 7a, 7b and 9a were tested for in vivo antitumor activity. As determined by in vitro Preussmann tests, compounds 4, 6 and 7 possess strong alkylating activity. Compounds 10 have moderate potential for alkylation, whereas the remaining compounds 8 and 9 are only weakly active. The derivatives 7a, 7b and 9a demonstrated low in vivo antitumour activity against lymphocytic leukaemia L1210, whereas compound 7b exhibited significant antitumour activity against leukaemia P388 in mice.

Full text of DOI:10.1039/b205800f

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A new series of 2-substituted 3-phosphonic derivatives of chromone.
Part II. Synthesis, in vitro alkylating and in vivo antitumour activity
Elzbieta Budzisz,*a Julita Graczyk-Wojciechowska,b Remigiusz Ziebab and Barbara Nawrotc
a
Chair of Medical Chemistry, Faculty of Pharmacy, Medical University of Lodz,
Muszynskiego 1, 90-151, Lodz, Poland. E-mail: elora@ich.pharm.am.lodz.pl;
Fax: +48 42 678 93 83; Tel: +48 42 677 92 17
Institute of Pharmacology, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1,
b
90-151, Lodz, Poland. E-mail: julita@mail.bp.am.lodz.pl, remusz@wp.pl;
Fax: +48 42 678 93 83; Tel: +48 42 677 91 79, 677 92 98
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of
c
Bioorganic Chemistry, Sienkiewicza 112, 90-363, Lodz, Poland. E-mail: bnawrot@bio.cbmm.lodz.pl;
Fax: +48 42 681 54 83; Tel: +48 42 681 69 70
Received (in Montpellier, France) 14th June 2002, Accepted 5th September 2002
First published as an Advance Article on the web 31st October 2002
Products of the reaction of (ꢀ)-O-acetylmandeloyl chloride with, respectively, sodium 2-hydroxy- or
2-hydroxy-5-methyl-acetophenone were brominated and coupled with trimethyl phosphite to give the Perkov
products 4a and 4b, the Wittig-type products 6a and 6b and the title 3-phosphonic derivatives of chromone,
7a {2-[1-(ꢀ)-acetoxybenzyl]-3-(dimethoxyphosphoryl)-4-oxo-4H-chromene} and 7b {2-[1-(ꢀ)-acetoxybenzyl]-
3-(dimethoxyphosphoryl)-4-oxo-6-methyl-4H-chromene}. Esters 7a and 7b were subjected to acidic hydrolysis
to give the corresponding phosphonic acids 8a and 8b, and the unexpected phosphonolactones 9a and 9b.
They were also treated with benzylamine forming the corresponding salts of the cyclic phosphonolactones
(10a and 10b). Derivatives 4a,b, 6a,b–10a,b were tested for in vitro alkylating activity while compounds 7a, 7b
and 9a were tested for in vivo antitumor activity. As determined by in vitro Preussmann tests, compounds 4, 6
and 7 possess strong alkylating activity. Compounds 10 have moderate potential for alkylation, whereas the
remaining compounds 8 and 9 are only weakly active. The derivatives 7a, 7b and 9a demonstrated low in vivo
antitumour activity against lymphocytic leukaemia L1210, whereas compound 7b exhibited significant
antitumour activity against leukaemia P388 in mice.
Chromones and their structural analogues are of great interest
on account of their anticonvulsant,1 antimicrobial,2,3 and anti-
tumour activities.4 On the other hand phosphonic acids, and
their salts and esters, attract significant attention because of
their role as both agrochemical (herbicides, pesticides, growth
regulators in plants) and medical (antibiotics, antivirals,
enzyme inhibitors) products with a broad spectrum of applica-
tions.5–9 The alkylating properties of phosphonic esters have
been exhaustively described in many studies10,11 and have been
a subject of many patents.12–14 Incorporation of a C–P bond
into the chromone system offers a new class of compounds
of potential importance. Since alkylating agents have a long
history in the treatment of cancer, recent interest has focused
on these aspects of their activity.
This work is a continuation of the earlier studies of our groups
focused on the synthesis and biological properties of 3-phospho-
nic derivatives of chromone.15–17 Here we present the synthesis,
isolation and characterisation of a new series of 2-substituted
3-phosphonic derivatives of chromone, 7a,b–9a,b, and studies
on their in vitro alkylating and in vivo antitumour activity.
previously reported by us,18 gave excellent yields of the esters
2a and 2b, respectively (Series a: R ¼ H; series b: R ¼ Me,
Scheme 1). Selective bromination of the methyl group of the
acetophenone moiety of 2, performed in carbon tetrachloride
according to Hercouet et al.,19 led to the bromo derivatives
3. On treatment with trimethyl phosphite at 110–115 ꢁC for
30 min these compounds were transformed into a mixture of
at least three major products (a: Rf 0.86, 0.63, 0.39 and b: Rf
0.84, 0.62, 0.33), as detected by TLC analysis (chloroform–
acetone, 9 : 1 v/v mixture). Traces of products of Rf 0.55 (a)
and 0.57 (b) were also visible. After removal of excess trimethyl
phosphite, the reaction mixture was chromatographed over
silica gel eluting with a chloroform–acetone, 5 : 1 v/v mixture.
The two fast-eluting components of the mixture were isolated
in ca. 10 and 40% yield. Their structures were assigned as 6a/b
(Rf 0.86 and 0.84) and 4a/b (Rf 0.63 and 0.62) (Scheme 1) on
the basis of their spectroscopic properties (see Experimental).
However, the products of Rf ¼ 0.39 (a) and 0.33 (b) were
not eluted from the column. Instead, we isolated derivatives
of much higher mobility [Rf 0.55 (a) and 0.57 (b)] in ca. 40%
yield. The 1H, 13C and 31P NMR analysis, as well as IR spectra
and MS data, indicate that these compounds are depicted by
structures 7a and 7b, respectively (Scheme 1). It seems prob-
able that compounds 7a/b are formed by transformation of
the polar components of the reaction mixtures on the column
(see Discussion).
Results
Chemistry
Reaction of the sodium salt of 2-hydroxyacetophenone (1a) or
2-hydroxy-5-methyl-acetophenone (1b) with racemic (ꢀ)-O-
acetylmandeloyl chloride, carried out according to the method
The racemic mixtures 7a (R ¼ H) and 7b (R ¼ Me) were
subjected to acidic hydrolysis with 30% HBr in acetic acid
DOI: 10.1039/b205800f
New J. Chem., 2002, 26, 1799–1804
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This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2002

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Scheme 2
quantified spectrophotometrically at 560 nm. Thus, the com-
pounds 4a,b, 6a,b–10a,b were screened for their alkylating
activity toward NBP at a concentration of 1.67 ꢂ 10ꢃ4
–
1 ꢂ 10ꢃ3 M in 2-methoxyethanol. For comparison we quote
the data of alkylating activity of trimethyl phosphite, trimethyl
phosphate, and the widely accepted alkylating drug cyclophos-
phamide.20,21 The results obtained are presented in Table 1.
Compounds 4, 6 and 7 possess strong alkylating properties
(+++) while compounds 10 are comparable to cyclophos-
phamide. The remaining derivatives 8 and 9 are very poor
alkylating agents.
Antitumour activity. The two phosphonic derivatives of
chromone, 7a and 7b, demonstrating the strongest alkylating
activity in vitro, and the derivative 9a, representing a new class
of phosphonolactone derivatives of chromone, were selected
for biological studies in an animal model (hybrid male
CD2F1 mice, weighing 22–30 g, 8–12 weeks old). The antineo-
plastic activity of the test compounds was examined against
two murine leukaemias: L1210 and P388. L1210 or P388
Table 1 NBP test results
Scheme 1
Alkylation
Absorbance Aa activityb
Compound
Conc/10ꢃ3
M
(Scheme 2). When the molar ratio of hydrobromic acid to
the substrate 7a or 7b was 2 : 1 we obtained the corresponding
acids 8a and 8b. When the molar ratio was 3 : 1 we obtained a
cyclic derivative 9a or its methyl analogue 9b. Treatment of
compounds 7 with 2 equiv. of benzylamine gave the benzyl-
ammonium salts 10, which, when treated with concentrated
(ca. 20%) aqueous hydrochloric acid produced the acid
derivatives 9.
4a
4b
0.5
0.5
0.7243
0.7633
0.5438
0.5693
0.9840
1.1268
0.0627
0.0626
0.0680
0.0745
0.1025
0.1037
0.1200
0.5300
0.9000
+++
+++
+++
+++
+++
+++
+
6a
6b
0.167
0.167
7a
7b
1
1
1
1
1
1
1
1
1
1
1
8a
8b
+
9a
9b
+
+
++
Pharmacology
10a
10b
Alkylating activity. For determination of the alkylating
properties of the new compounds 4, 6–10 we chose an in vitro
Preussmann20 test employing 4-(4-nitrobenzyl)pyridine (NBP)
as the target molecule. Alkylation of the nitrogen atom of
the pyridine ring of NBP results in formation of a quaternary
salt, which under the alkaline conditions is transformed into a
neutral coloured compound. The extent of alkylation can be
++
++
Cyclophosphamide
Trimethyl phosphite
Trimethyl phosphate
+++
+++
a
b
Means from 3 determinations. According to Preussmann20: (ꢃ)
A < 0.05, (+) A ¼ 0.05–0.10, (++) A ¼ 0.10–0.50, (+++) A > 0.50.
1800
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Table 2 Antitumour activity of derivatives 7a, 7b and 9a in in vivo tests against L1210 and P388 leukaemias in mice
L1210
P388
Compd.
Dosea /mg kgꢃ1
MSTb /days
ILSc (%)
ALD50d /mg kgꢃ1
MST/days
ILS (%)
ALD50/mg kgꢃ1
7a
750
500
14
14
14
11
14
14
2
0
10
10
11
10
10
14
2
ꢃ9
ꢃ9
0
0
1000
2000
1000
2000
250
0
7b
9a
1500
1000
500
ꢃ21
ꢃ9
ꢃ9
+27
Toxic
Toxic
0
0
0
Toxic
Toxic
800*
400*
200
4
1200
2
1200
14
14
0
–
11
11
Control (untreated)
–
–
a
Dose administered the 1st, 3rd and the 5th day after inoculation, except for *, where the drug was administered once on the first day after inocu-
b
lation. Median survival time. Increase of lifespan. Approximate lethal dose.
c
d
leukaemia cells (3 ꢂ 105) were intraperitoneally implanted. The
test compounds were administered three times: on the first,
third and fifth day after inoculation of the leukaemia cells.
The results are collected in Table 2. Compounds 7a and 9a
are not effective in the treatment of the L1210 and P388 leu-
kaemias in mice. Those mice treated with various doses of 7a
and 9a lived as long as or shorter than the mice of the control
group. In contrast, compound 7b exhibits significant antitu-
mour activity against P388 leukaemia after administration of
500 mg kgꢃ1 three times (the 1st, 3rd and 5th day after inocu-
lation). Increase of the lifespan (ILP) of the P388 infected mice
is estimated at around 27%. Compound 7b demonstrates anti-
tumour activity only in the case of the cell line P388 at a dose
of 500 mg kgꢃ1. The results of treating infected mice with 9a
indicate that it is significantly more toxic than 7a and 7b. These
experiments also demonstrate that the approximate lethal dose
the alpha protons more acidic. The presence of intermediate
50 was observed by 31P magnetic resonance spectroscopy of
a crude reaction mixture in series b. A resonance signal at
23.72 ppm (integration of 46%) does not appear in any of
the isolated products. Instead, a new signal at 15.94 ppm
appears in the spectrum of product 7, isolated in 35.5% yield
after column chromatography.
In general alkylating agents are the first class of cytostatics
used for therapy.27 Under in vivo conditions these agents alky-
late nucleophilic centres of nucleobases and amino acids,
resulting either in cleavage or cross-linking of double-stranded
DNA molecules or proteins. Such cleavage causes damage of
DNA while covalent cross-links prevent unwinding of nucleic
acids, functionally important in replication and transcription
processes.28 The alkylating properties of the test derivatives
can be determined by an in vitro Preussmann test.20 This test
permits the estimation of the direct ability of the compound
to alkylate the model target molecule 4-(4-nitrobenzyl)pyridine
and it provides a useful indication of alkylation potential for
nucleophilic centres of aminoacids and nucleobases. However,
due to its simplicity, the Preussmann test does not allow the
determination of the mode of DNA alkylation. Despite this
drawback, this test has been used to determine the alkylating
potential of several trialkyl phosphate, phosphite and phos-
phonate derivatives as well as cyclophosphamide toward
NBP.20 We used this test to determine the alkylating properties
of the derivatives 4, 6–10 (Table 1). Compounds 4, 6, and 7
possess strong alkylating activity. Compounds 10 have moder-
ate potential for alkylation, whereas the remaining compounds
8 and 9 are only weakly active. The test compounds 4, 6 and 7
exhibit higher alkylating activity in vitro than trimethyl phos-
phite and cyclophosphamide, and comparable alkylating prop-
erties to trimethyl phosphate.20 It should be noted that
cyclophosphamide, which is a widely known alkylating drug,
has significantly stronger alkylating activity in vivo due to its
metabolic transformation into an active alkylating species.21
It is interesting to correlate the alkylating properties of the
test compounds with their in vivo antitumour activity. The
phosphonic derivatives of chromone, 7a and 7b, demonstrating
the strongest alkylating activity in vitro, and compound 9a,
representing a new class of phosphonolactone derivatives of
chromone, were selected for antitumour studies in leukaemia
L1210 and P388 infected mice. Only compound 7b at a dose
of 500 mg kgꢃ1 demonstrates antitumour activity in the P388
cell line (Table 2). The results of treating infected mice with
9a indicate that it is significantly more toxic than 7a and 7b.
The interesting biological activity of 7b encouraged us to
investigate further this class of compounds. Thus, we asked
the question whether the pure enantiomers of compound 7b
would differ in their biological properties. Starting from
(ALD50) of derivative 7b is 2000 mg kgꢃ1
.
Discussion
Alkyl phosphites react with bromomethylketones by two pos-
sible mechanisms.22,23 One mechanism involves nucleophilic
attack either at halogen, to give an intermediate salt that rear-
ranges, or at the methylene carbon (the Arbusov reaction24).
Both routes lead to the same product, a phosphonate. Alkyl
phosphites can also react with the carbon of the carbonyl
group of bromomethylketones according to the Perkov
mechanism, resulting in formation of enol-phosphate deriva-
tives.25 In our experiments the reaction of trimethyl phosphite
with bromoderivatives 3 (a: R ¼ H, b: R ¼ Me, Scheme 1) led
to products 4, 6 and 7. Product 4, obtained in ca. 40% yield, is
a typical Perkov product while 6 and 7 (ca. 10% and 40% yield,
respectively), are the products of intramolecular cyclisation
and elimination. The structures of 6 and 7 suggest that the first
step of the reaction involves nucleophilic attack of phosphite
at either halogen or at the methylene carbon of 3, in a typical
Arbusov process, leading to the unstable phosphonate 5. This
compound cyclises to a mixture of diastereoisomers 50 that
subsequently either lose dimethyl phosphate to give the
Wittig-type26 product 6 or undergo 1,2-trans-elimination of
water on the column to give the 3-phosphonic chromone deri-
vative 7. A stable Arbusov product of type 5 was isolated when
derivative 3 had a benzoyl instead of an O-acetylmandeloyl
moiety.15,16 However, the presence of the O-acetyl substituent
in 5 significantly increases the partial positive charge on the
carbonyl carbon atom of the mandelic residue, facilitating
intramolecular cyclisation of this derivative. Cyclisation of 5
to 50 is additionally facilitated by the presence of a carbonyl
group at the beta position to the phosphonic residue, making
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enantiomerically pure mandelic acid precursors, we obtained
both enantiomers of 7a and both enantiomers of 7b. X-Ray
analysis showed that the levorotatory product has the R con-
figuration of the chiral centre. The results of preliminary eva-
luation of biological activity indicate significant differences
between the enantiomers of both derivatives 7a and 7b. These
results will be published elsewhere.
(%): 327 (3, M+), 239 (17), 225 (10), 177 (78), 151 (29), 149
(100), 135 (43). Anal. found: C, 69.56; H 5.53. Calcd. for
C19H18O5 (326.33): C, 69.92; H 5.56%.
Synthesis of compounds 3a and 3b. Bromine (0.01 mol) was
added dropwise to a carbon tetrachloride solution (50 ml) of
compound 2a or 2b (0.01 mol). The mixture was refluxed for
15 min. The solvent was evaporated under reduced pressure
and the semisolid residue was suspended in 50 ml of diethyl
ether. The resultant colourless crystalline product 3a or 3b
was filtered off and washed with diethyl ether.
Conclusions
The 3-dimethoxyphosphonyl derivatives 7a and 7b have been
prepared. Hydrolysis afforded the corresponding acetates 8a
and 8b or phosphonolactones 9a and 9b. These compounds
were tested for their in vitro alkylating and selected ones for
their in vivo antitumour activity. Most of the tested com-
pounds possess strong or moderate alkylating activity, while
compound 7b demonstrates significant in vivo antitumour
activity against leukaemia P388 cells in an animal model.
(ꢀ)-Acetoxyphenylacetic acid 2-(2-bromoacetyl)phenyl ester
.
1773.4 (C=O); 1741.2 (C=O); 1702.3 (C=O) cmꢃ1 1H NMR
(CDCl3): d 2.23 (s, 3H, CH3); 4.09 (s, 2H, CH2); 6.17 (s, 1H,
CH); 7.08–7.82 (m, 6H, arom.). EI MS m/z (%): 391 (9,
M+), 333 (6), 304 (9), 211 (22), 177 (96), 135 (100), 107 (13).
Anal. found: C, 55.46; H, 3.91. Calcd. for C18H15O5Br
(391.20): C, 55.26; H, 3.86%.
(3a). Yield 79%. M.p. ¼ 90.1–93.1 ꢁC. IR (KBr): n ¼
(ꢀ)-Acetoxyphenylacetic acid 2-(2-bromoacetyl)-4-methylphe-
nyl ester (3b). Yield 73.6%. M.p. ¼ 89.1–91.0 ꢁC. IR (KBr):
n 1779.9 (C=O); 1737.4 (C=O); 1700.0 (C=O); 1040.0 (C–O–
Experimental
C) cmꢃ1 1H NMR (CDCl3): d 2.24 (s, 3H, CH3); 2.36 (s,
.
Chemistry: materials and methods
3H, CH3); 4.05 (s, 2H, CH2); 6.14 (s, 1H, CH); 6.96–8.01 (m,
8H, aromat.). EI MS m/z (%): 406 (58, M+), 335 (100), 228
(35), 177 (63), 149 (13), 107 (78), 77 (16). Anal. found: C,
56.04; H, 3.99. Calcd. for C19H17O5Br (405.23): C, 56.31; H,
4.23%.
The (ꢀ)-O-acetylmandelic acid used in this study was pur-
chased from Aldrich. Purity of all compounds was analysed
on silica gel PF254 plates (Merck). Column chromatography
was carried out using silica gel 30–60 mm (Baker), and a mix-
ture of chloroform–acetone (5 : 1, v/v) as eluent. The melting
points were determined using an Electrothermal 1A9100 appa-
ratus and they are uncorrected. The IR spectra were recorded
on a Pye-Unicam 200G Spectrophotometer in solid KBr.
NMR spectra were registered on a Varian Mercury spectro-
meter (1H 300 MHz; 13C 75.5 MHz; 31P 121.5 MHz). In the
31P spectra positive chemical shifts are assigned to resonances
downfield of phosphoric acid. The MS data were obtained on a
Finnigan Matt mass spectrometer (100 eV ionisation energy)
with isobutane as reagent. UV/VIS spectra were obtained at
800–400 nm on a Lambda 19 Perkin Elmer instrument. Satis-
factory elemental analyses (ꢀ0.4% of the calculated values)
were obtained for the new compounds. Elemental analyses
were performed by the Microanalytical Laboratory of the
Institute of Chemistry using a Perkin Elmer PE 2400 CHNS
analyser.
Synthesis of compounds 4, 6 and 7. To 10 mmol of the corre-
sponding acetate 3a or 3b melted in a flask, 12 mmol of tri-
methyl phosphite were added dropwise at 110–115 ꢁC. After
30 min of heating, the excess of phosphite was removed by dis-
tillation, and the resulting yellow oil was applied on a silica gel
column. The column was eluted with a mixture of chloroform–
acetone (5 : 1, v/v). Compounds 4a,b were obtained as oils
while the crude products 6 and 7 were purified by crystal-
lisation.
(ꢀ)-Acetoxyphenylacetic acid 2-[1-(dimethoxyphosphoryloxy)-
vinyl]phenyl ester (4a). Yield 38%; Rf ¼ 0.63, oil. IR (KBr):
1
n ¼ 1744 (C=O), 1258 (P=O), 1104 (P–O–C) cmꢃ1. H NMR
(CDCl3): d 2.20 (s, 3H, CH3); 3.71 (d, 3H, O–CH3 , 3JPH ¼ 11.31
11.31 Hz); 3.74 (s, 3H, O–CH3 , 3JPH ¼ 11.31 Hz); 4.77 (t, 1H,
4
4
CH2 , JPH ¼ 2.4 Hz); 5.02 (t, 1H, CH2 , JPH ¼ 2.4 Hz); 6.17
(s, 1H, CH); 7.02–7.58 (m, 9H, aromat.). 13C NMR (CDCl3): d
20.65, 54.74 (d, 2JPC ¼ 6.01 Hz); 103.0; 102.94; 122.36, 127.41;
167.1 (C=O); 170.3 (C=O). 31P NMR (CDCl3): d ꢃ3.9. EI MS
(70 eV) m/z (%): 244 (100) 245 (8), 135 (19), 127 (96), 118 (35),
109 (21), 107 (37), 105 (8), 90 (17); Anal. found: C, 56.89; H,
5.03, P, 7.70. Calcd. for C20H21O8P (420.34): C, 57.14; H, 5.04,
P, 7.37%.
Syntheses
Synthesis of compounds 2a and 2b. Racemic O-acetylmande-
loyl chloride (22.6 mmol) was added dropwise with intensive
stirring into a cooled (ice-bath) solution of sodium 2-hydroxy-
or 2-hydroxy-5-methyl-acetophenone 1a or 1b (22.6 mmol) dis-
solved in 150 ml of diethyl ether. The stirring was continued
for an additional hour. Then water (20 ml) was added to the
reaction mixture and it was extracted with chloroform
(4 ꢂ 50 ml) and with aqueous sodium bicarbonate. The col-
lected organic layers were dried with MgSO4 and concentrated
under reduced pressure. Products 2a and 2b were obtained as
pale yellow oils.
(ꢀ)-Acetoxyphenylacetic acid 2-[1-(dimethoxyphosphoryl-
oxy)-vinyl]-4-methylphenyl ester (4b). Yield 36%, Rf ¼ 0.62;
oil. IR (KBr): n ¼ 1774.2, 1746.9 (C=O), 1232.9 (P=O),
1048.9 (C–O–C), 1006 (P–O–C) cmꢃ1 1H NMR (CDCl3): d
.
2.20 (s, 3H, CH3); 2.32 (s, 3H, CH3); 3.72 (d, 3H, O–CH3 ,
3
3JPH ¼ 11.31 Hz); 3.74 (d, 3H, O–CH3 , JPH ¼ 11.31 Hz);
4
4.72 (t, 1H, CH2 , JPH ¼ 2.4 Hz); 4.99 (t, 1H, CH2 ,
(ꢀ)-Acetoxyphenylacetic acid 2-acetylphenyl ester (2a). Yield
4JPH ¼ 2.4 Hz); 6.15 (s, 1H, CH); 6.89–7.58 (m, 8H, aromat.);
2
84.2%. Oil. IR (KBr): n ¼ 1781.7, 1741.1, 1709.1 (C=O)
13C NMR (CDCl3): d 20.67, 20.88; 54.74 (d, JPC ¼ 6.01 Hz);
cmꢃ1
.
1H NMR (CDCl3): d 2.17 (s, 3H, CH3); 2.35 (s, 3H,
103.0; 102.94; 122.36, 127.41; 167.1 (C=O); 170.3 (C=O). 31P
NMR (CDCl3): d ꢃ3.93. EI MS m/z (%): 258 (100), 243 (3),
149 (12), 132 (62), 127 (22), 107 (16); Anal. found: C, 57.75;
H, 5.33, P, 7.33. Calcd. for C21H23O8P (434.36): C, 58.06; H,
5.34, P, 7.13%.
CH3); 6.71 (s, 1H, CH); 7.21–8.21 (m, 9H, aromat.). EI MS
m/z (%): 312 (13, M+), 253 (20), 225 (52), 177 (43), 135 (45),
121 (53), 107 (61). Anal. found: C, 69.34; H, 5.30. Calcd. for
C18H16O5 (312.31): C, 69.22; H, 5.16%.
(ꢀ)-Acetoxyphenylacetic acid 2-acetyl-4-methyl-phenyl ester
.
1643.0 (C=O), 1050 (C–O–C) cmꢃ1 1H NMR (CDCl3):
d 2.24 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.48 (s, 3H, CH3),
6.18 (s, 1H, CH), 7.02–7.81 (m, 8H, aromat.). EI MS m/z
(ꢀ)-(4-Oxo-4H-chromen-2-yl)phenylmethyl acetate (6a).
Yield 9%, M.p. ¼ 131.4–132.0 ꢁC (diethyl ether); Rf ¼ 0.86.
IR (KBr): n ¼ 1764.1, 1658.8 (C=O), 1620 (C=C), 1034 (C–
(2b). Yield 92.6%. Oil. IR (KBr): n ¼ 1746.1, 1687.7 (C=O),
O–C) cmꢃ1 1H NMR (CDCl3): d 2.22 (s, 3H, CH3); 6.48
.
(s, 1H, CH); 6.62 (s, 1H, CH); 7.42–7.58 (m, 9H, aromat.).
1802
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13C NMR (CDCl3): d 20.54 (CH3); 74.43 (CH); 76.59 (CH);
123.98; 128.08; 131.16; 146.22; 170.39 (C=O); 189.82 (C=O).
EI MS m/z (%): 295 (100, M+ + 1); 245 (16). Anal. found:
C, 73.57; H, 4.89. Calcd. for C18H14O4 (294.29): C, 73.46; H,
4.79%.
found: C, 59.02; H, 4.32; P, 7.93. Calcd. for C19H17O7P
(388.30): C, 58.77; H, 4.41; P, 7.98%.
Synthesis of compounds 9a and 9b. Method A. A 30% hydro-
bromic acid solution in glacial acetic (2.1 mmol) was added to
0.7 mmol of the corresponding acetate 7a or 7b. The reaction
mixture was kept at room temperature for 24 h. The precipi-
tated product was collected and washed with acetone.
(ꢀ)-(6-Methyl-4-oxo-4H-chromen-2-yl)phenylmethyl acetate
(6b). Yield 10.5%, M.p. ¼ 73.5–75.0 ꢁC (diethyl ether);
Rf ¼ 0.84. IR (KBr): n ¼ 1751.2, 1652.3 (C=O), 1616 (C=C),
1045 (C–O–C) cmꢃ1
.
1H NMR (CDCl3): d 2.21 (s, 3H,
(ꢀ)-1-Hydroxy-1-oxo-3-phenyl-1,3-dihydro-1l5-2,1-oxaphos-
pholo[4,5-b]-4H-1-benzopyran-4-one (9a). Yield 67.5%,
M.p. 261.5–262 ꢁC (acetone). IR (KBr): n ¼ 3349 (P–OH);
CH3); 2.23 (s, 3H, CH3); 6.45 (s, 1H, CH); 6.62 (s, 1H, CH);
7.27–7.95 (m, 8H, aromat.). 13C NMR (CDCl3): d 20.98
(CH3); 21.1 (CH3); 74.89 (CH); 76.80 (CH); 122.95; 128.08;
130.16; 167.22; 170.60 (C=O); 191.02 (C=O). CI MS m/z
(%): 309 (100, M+ + 1); 251 (27). Anal. found: C, 73.88; H,
5.40. Calcd. for C19H16O4 (308.32): C 74.01, H, 5.23%.
(ꢀ)-2-(1-Acetoxybenzyl)-3-(dimethoxyphosphoryl)-4-oxo-4H-
chromene (7a). Yield 40.5%, M.p. 128.6–129.1 ꢁC (ethyl ether–
methanol, 25 : 2), Rf ¼ 0.56. IR (KBr): n ¼ 1746.1 (C=O);
1648.2 (C=O); 1238.6 (P=O); 1024 (P–O–C); 1045 (C–O–C)
1
1759.4 (C=O); 1249.2 (P=O) cmꢃ1. H NMR (DMSO-d6): d
3
6.49 (d, 1H, CH, JP–H ¼ 8.3 Hz); 7.28–8.13 (m, 8H, aro-
mat.); 12.01 (sbroad , 1H, OH). 13C NMR (DMSO-d6): d
1
76.78 (CH–Ph); 107.72 (d, C–P, JPC ¼ 173.49 Hz), 118.55;
2
128.89; 173.12 (d, C=O, JPC ¼ 12.31 Hz). 31P NMR
(DMSO-d6): d 25.1. FAB-MS: 315 [M + H]+. Anal. found:
C, 60.87; H, 3.19; P, 9.71. Calcd. for C16H11O5P (314.14):
C, 61.18; H, 3.50; P, 9.86%.
cmꢃ1
.
1H NMR (CDCl3): d 2.20 (s, 3H, CH3); 3.85 (d, 3H,
(ꢀ)-1-Hydroxy-7-methyl-1-oxo-3-phenyl-1,3-dihydro-1l5-2,1-
oxaphospholo-[4,5-b]-4H-1-benzopyran-4-one (9b). Yield
80.5%, M.p. 207.8–209.5 ꢁC (acetone). IR (KBr): n ¼ 3419.8
3
3
–OCH3 , JPH ¼ 11.7 Hz); 3.92 (d, 3H, –OCH3 , JPH ¼ 11.7
Hz), 6.75 (s, 1H, CH), 7.26–7.78 (m, 9H, aromat.). 31P NMR
(CDCl3): d 16.78. 13C NMR (CDCl3): d 20.69 (CH3); 53.34
1
(P–OH); 1762.2, 1645 (C=O); 1226.1 (P=O) cmꢃ1. H NMR
2
3
(d, O–CH3 , JPC ¼ 5.70 Hz); 72.28 (CH); 117.49; 125.42;
(DMSO-d6): d 2.21 (s, 3H, CH3); 6.41 (d, 1H, CH, JP–H
8.3 Hz); 6.98–7.91 (m, 8H, aromat.); 12.01 (sbroad , 1H, OH).
¼
2
127.70; 170.10 (C=O); 172.42 (d, C=O, JPC ¼ 25.35 Hz). EI
MS (70 eV) m/z (%): 402 (0.3, M+); 359 (9); 342 (27); 329
(25); 328 (100); 313; 295; 105. Anal. Found: C, 59.88; H, 4.70;
P, 7.81. Calcd. for C20H19O7P (402.32): 59.70; H, 4.76; P,
7.70%.
13C NMR (DMSO-d6): d 20.19 (CH3), 77.18 (CH–Ph);
1
107.52 (d, C–P, JPC ¼ 173.50 Hz); 118.35; 128.29; 172.12
(d, C=O, JPC ¼ 12.31 Hz). 31P NMR (DMSO-d6):
d
2
24.31. FAB-MS: 388 [M]+. Anal. C, 62.38; H, 4.08; P,
9.48. Calcd. for C19H17O7P (388.30): C, 62.20; H, 3.99; P,
9.44%.
(ꢀ)-2-(1-Acetoxybenzyl)-3-(dimethoxyphosphoryl)-6-methyl-
4-oxo-4H-chromene (7b). Yield 35.5%, M.p. 137.0–137.9 ꢁC
(acetone), Rf ¼ 0.57. IR (KBr): n ¼ 1743 (C=O); 1649
1
(C=O); 1236 (P=O); 1024 (P–O–C); 1045 (C–O–C) cmꢃ1. H
Synthesis of compound 9a. Method B. To a 50% aqueous
methanolic solution of benzylammonium salt 10a (0.1 mmol),
a solution of hydrochloric acid was added to pH 6. The preci-
pitated solid was filtered off, dried and crystallised from acet-
one. Compound 9a was obtained in 56% yield.
NMR: (CDCl3) d 2.19 (s, 3H, CH3); 2.44 (s, 3H, CH3); 3.74
3
(d, 3H, –OCH3 , JPH ¼ 11.7 Hz); 3.92 (d, 3H, –OCH3 ,
3JPH ¼ 11.9 Hz). 31P NMR (CDCl3): d 16.78. 13C NMR
(CDCl3): d 20.69 (CH3); 21.00 (CH3); 53.34 (d, O–CH3 ,
2JPC ¼ 5.70 Hz); 72.28 (CH); 117.49; 125.42; 127.70; 170.10
2
(C=O); 172.42 (d, C=O, JPC ¼ 25.35 Hz). EI MS (70 eV)
Synthesis of compounds 10a and 10b. To a solution of 7a or
7b (1.00 mmol) in methanol (2 ml), 2.0 mmoles of benzylamine
were added. The mixture (after standing overnight at room
temperature in the case of 7b) was then cooled to ꢃ10 ꢁC to
initiate crystallisation. The colourless product was obtained
in 75–78% yield. The crude product was purified by further
crystallisation from acetone.
m/z (%): 416 (100, M+), 384 (40), 342 (70), 105 (3). Anal.
found: C, 60.77; H, 5.28; P, 7.54. Calcd. for C21H21O7P
(416.35): C, 60.58; H, 5.08; P, 7.44%.
Synthesis of the racemic acids 8a and 8b. A 30% solution of
hydrobromic acid in acetic acid was added at room tempera-
ture to 0.7 mmol of the corresponding ester 7a or 7b. The
reaction mixture was stirred at room temperature for 24 h.
The precipitated solid was filtered and washed with water
and acetone.
1-Hydroxy-1-oxo-3-(ꢀ)-3-phenyl-1,3-dihydro-1k5-2,1-oxaphos-
pholo-[4,5-b]-4H-1-benzopyran-4-on benzylammonium salt
(10a). Yield 67%, M.p. 214–216 ꢁC (methanol). IR (KBr):
n ¼ 3442.03–3035.67 (–NH3+–CH2Ph); 1648.74 (C=O),
(ꢀ)-[4-Oxo-3-phosphono-4H-chromen-2-yl]phenylmethyl acet-
1204.2 (P=O) cmꢃ1
.
1H NMR (DMSO-d6): d 4.04 (s, 2H,
3
ate (8a). Yield 68%, M.p. 208.5–209 ꢁC (acetone). IR (KBr):
NH–CH2), 5.96 (d, 1H, JHP ¼ 8.22 Hz), 7.28–7.67 (m, 9H,
arom.), 8.71 (s, 3H, –NH3+). 31P NMR (DMSO-d6): d 20.23.
MS (70 eV): m/z (%) 421 (5, M+), 258 (100), 149 (19), 132
(62), 127 (21), 43 (15). Anal. found: C, 65.78; H, 4.98; N,
3.43; P, 7.56. Calcd. for C23H20NO5P (421.37): C, 65.56; H,
4.78; N, 3.32; P, 7.35%.
n 3100–2560 (OH), 1752.8, 1620 (C=O); 1244 (P=O) cmꢃ1
.
1H NMR (DMSO-d6): d 2.22 (s, 3H, CH3); 6.22 (sbroad , 2H,
OH); 7.42–7.58 (m, 9H, aromat.); 8.39 (s, 1H, CH). 13C
NMR (DMSO-d6): d 20.51 (CH3); 71.06 (CH); 114.07 (d,
1
C–P, JPC ¼ 177.71 Hz); 121.45; 122.01; 128.37; 152.78;
2
167.54 (d, C=O, JPC ¼ 20.94 Hz); 169.24; 176.09. 31P NMR
1-Hydroxy-7-methyl-1-oxo-3-(ꢀ)-3-phenyl-1,3-dihydro-1l5-
2,1-oxaphospholo-[4,5-b]-4H-1-benzopyran-4-on benzylammo-
nium salt (10b). Yield 75%, M.p. 176–177 ꢁC (acetone). IR
(KBr): n ¼ 3393.3–3035.67 (–NH3+–CH2Ph); 1597.2 (C=O),
(DMSO-d6): d 7.9. FAB-MS: 375 [M]+. Anal. found: C,
58.10; H, 3.83; P, 8.15. Calcd. for C18H15O4P (374.27): C,
57.76; H, 4.04; P, 8.27%.
(ꢀ)-[6-Methyl-4-oxo-3-phosphono-4H-chromen-2-yl]phenyl-
methyl acetate (8b). Yield 62.96%, M.p. 201.5–202.5 ꢁC
(acetone). IR (KBr): n 1761.2, 1630.3 (C=O); 1225.4 (P=O)
1578 (C=C) 1204.8 (P=O), 1047.0 (P–O–C) cmꢃ1 1H NMR
.
(DMSO-d6): d 2.20 (s, 3H, CH3), 3.80 (s, 2H, NH–CH2),
3
5.91 (d, 1H, JHP ¼ 11.12 Hz), 7.03–7.79 (m, 13H, aromat.),
1003 (P–O); 3070.1–2813.0 (OH) cmꢃ1
.
1H NMR (DMSO-
10.05 (s, 3H, –NH3+). 31P NMR (DMSO-d6): d 29.88. 13C
NMR (DMSO-d6): d 20.19 (CH3), 40.07 (CH2–N), 74.77
(CH), 119.5, 127.15, 128.32, 130.14, 134.93, 154.13, 179.1
(C=O). MS (70 eV): m/z (%) 435 (3, M+), 328 (9), 310 (20),
248 (15), 107 (51), 106 (88), 91 (100), 77 (40). Anal. found:
C, 66.48; H, 4.98; N, 3.43; P, 7.36. Calcd. for C24H22NO5P
(435.40): C, 66.20; H, 5.09; N, 3.22; P, 7.13%.
d6): d 2.18 (s, 3H, CH3); 2.50 (s, 3H, CH3); 7.33–7.78 (m,
8H, aromat.); 8.32 (s, 1H, CH). 13C NMR (DMSO-d6): d
20.37 (CH3), 20.68 (CH3), 71.12 (CH), 112.03 (d, C–P,
1JPC ¼ 177.71 Hz), 117.82, 124.11, 126.76, 128.43, 135.82,
2
135.91, 136.47, 152.73, 169.14 (d, C=O, JPC ¼ 20.86 Hz).
31P NMR (DMSO-d6): d 6.49. FAB-MS: 388 [M]+. Anal.
New J. Chem., 2002, 26, 1799–1804
1803

View Article Online
Pharmacology
gratefully acknowledged. We thank Mrs. Agnieszka Zdolska
for skillful experimental assistance.
Alkylating properties (NBP test). The test compounds
(0.835–5.00 mmol) were dissolved in 2-methoxyethanol (1 ml)
and a solution of NBP in 2-methoxyethanol (5% solution,
1 ml) was added. The samples were heated at 100 ꢀ 0.5 ꢁC
for 1 h and then quickly cooled to 20 ꢁC. 2-Methoxyethanol
(2.5 ml) and piperidine (0.5 ml) were added to the samples to
give a total volume of 5 ml. The final concentration of the test
compound was 1.67 ꢂ 10ꢃ4–1 ꢂ 10ꢃ3 M. After 90 s the absor-
bance was measured at l ¼ 560 nm in a glass cell (1 cm), in
the presence of 2-methoxyethanol (Table 1).
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Financial support from the Medical University of Lodz
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New J. Chem., 2002, 26, 1799–1804