The novel phosphoramidate derivatives of NSAID 3-hydroxypropylamides: Synthesis, cytostatic and antiviral activity evaluations

Article abstract of DOI:10.1016/j.ejmech.2008.03.037

The target phosphoramidates 5a-e were prepared in one step from 3-hydroxypropyl derivatives 3a-e of nonsteroidal anti-inflammatory drugs (fenoprofen, ketoprofen, ibuprofen, indomethacin, diclofenac). The products 3a-e and 5a-e were evaluated for their cyt

Full text of DOI:10.1016/j.ejmech.2008.03.037

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 143e151
http://www.elsevier.com/locate/ejmech
Original article
The novel phosphoramidate derivatives of NSAID
3-hydroxypropylamides: Synthesis, cytostatic and
antiviral activity evaluations
a
a
b
b
c
c
c
K. Wittine , K. Benci , Z. Rajic , B. Zorc , M. Kralj , M. Marjanovic , K. Pavelic ,
´
´
´
E. De Clercq d, G. Andrei d, R. Snoeck d, J. Balzarini d, M. Mintas a,
*
a
´
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20, HR-10000 Zagreb, Croatia
b
´
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovaꢀcica 1, HR-10000 Zagreb, Croatia
c
´
ꢀ
Division of Molecular Medicine, Rudjer Bosˇkovic Institute, Bijenicka cesta 54, HR-10000 Zagreb, Croatia
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 22 January 2008; received in revised form 5 March 2008; accepted 18 March 2008
Available online 8 April 2008
Abstract
The target phosphoramidates 5aee were prepared in one step from 3-hydroxypropyl derivatives 3aee of nonsteroidal anti-inflammatory
drugs (fenoprofen, ketoprofen, ibuprofen, indomethacin, diclofenac). The products 3aee and 5aee were evaluated for their cytostatic and an-
tiviral activity against malignant tumour cell lines and normal human fibroblasts (WI 38). All phosphoramidate derivatives 5aee possess sig-
nificantly greater inhibitory activities than the corresponding 3-hydroxypropyl derivatives 3aee, whereby compound 5a showed the most potent
inhibitory activities against cervical, pancreatic and colon carcinoma cell lines (IC50 ¼ 5 ꢀ 7 mM).
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Nonsteroidal anti-inflammatory drugs (NSAID); 3-Hydroxypropylamides; Phosphoramidates; Cytostatic activity
1. Introduction
prodrugs of antineoplastic agents showed a substantial en-
hancement of potency versus colon and prostate cancer cell
lines [5].
The phosphoramidate approach has been conceived as
a means to improve cellular penetration of antiviral nucleo-
tides and to bypass the first step of kinase-mediated activation
of nucleosides [1]. Alkyl and aryloxy phosphoramidate and
specially substituted aryl phosphoramidate derivatives of the
anti-HIV drugs stavudine and zidovudine demonstrated an el-
evation of in vitro potency relative to that of the parent nucle-
osides [2]. Exhaustive modifications to the amino acid moiety
in phosphoramidate derivatives established L-alanine as the
moiety for optimal antiviral activity. In addition, WHI-05
and WHI-07, bromo-methoxy-substituted aryl phosphate de-
rivatives of zidovudine proved to be potent dual-action contra-
ceptive agents [3,4], while the aryloxy phosphoramidate
In this paper the phosphoramidate strategy has been applied
to nonsteroidal anti-inflammatory drugs (NSAID). Numerous
experimental, epidemiologic and clinical studies provide evi-
dence that NSAIDs are promising anticancer drugs [6].
Thus, ibuprofen, indomethacin and some other NSAIDs are ef-
fective chemopreventive agents against carcinogen-induced
and genetically manipulated animal models of colon carcino-
genesis [6e9]. NSAIDs may also be associated with reduced
risk of cancers of bladder, breast, oesophagus, lung, ovary,
prostate, stomach, liver, pancreas, tongue and glioblastoma
multiforme [10].
Furthermore, it has been demonstrated that modification of
the NSAID by amidation provides COX-2 selective inhibitors
[11], while fenoprofen and ketoprofen amidation significantly
enhance antiproliferative activity of the parent compounds
[12]. On the other hand, diclofenac with bisphosphonic moiety
* Corresponding author. Tel.: þ385 1 4597 214; fax: þ385 1 4597 250.
E-mail addresses: mladen.mintas@fkit.hr, mmintas@fkit.hr (M. Mintas).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.037

144
K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
is convenient prodrug for bone-specific delivery [13,14]. This
led us to synthesize a series of new phosphoramidate deriva-
tives (5aee) of NSAID 3-hydroxypropylamides (fenoprofen,
ketoprofen, ibuprofen, indomethacin and diclofenac) (3aee).
Here we report their synthesis and evaluation of their cyto-
static and antiviral activity potency.
2.1.4. Indomethacin 3-hydroxypropylamide (3d)
The synthesis of compound 3d was previously published
but no spectral data were available [17]. Mp 130e131 ꢁC;
IR (KBr, n/cmꢀ1) 3428, 3314, 3093, 2928, 2884, 1673,
1
1620, 1561, 1478, 1317, 1071, 835, 755; H NMR (DMSO-
d6) d 8.02 (t, 1H, NH), 7.71e7.63 (q, 4H, 13, 14, 16, 17),
7.12e7.11 (s, 1H, 9), 6.96e6.93 (d, 1H, 7), 6.72e6.69 (dd,
1H, 6), 4.43 (t, 1H, OH), 3.77 (s, 3H, 20), 3.49 (s, 2H, 2),
3.42e3.37 (q, 2H, 300), 3.15e3.08 (q, 2H, 100), 2.23 (s, 3H,
10), 1.60e1.51 (m, 2H, 200).
2. Materials and methods
2.1. Synthesis
2.1.1. Materials and general methods
2.1.5. Diclofenac 3-hydroxypropylamide (3e)
Melting points were determined on a Stuart SMP 3 melting
point apparatus (Barloworld Scientific, UK) and were uncor-
rected. IR spectra were recorded on a FTIR Perkin Elmer Par-
agon 500 spectrometer. 1H and 13C NMR spectra were
recorded on a Varian Gemini 300 spectrometer, operating at
300, 75.5 and 243 MHz for the 1H, 13C and 31P nuclei, respec-
tively. Samples were measured in DMSO-d6 solutions at 20 ꢁC
in 5 mm NMR tubes. Chemical shifts (d) in ppm were referred
to TMS. Precoated Merck silica gel 60 F254 plates were used
for thin-layer chromatography. Solvent systems were cyclo-
hexane/ethyl acetate (1:1), ethyl acetate, dichloromethane/
methanol (50:1). Spots were visualized by shortwave UV light
and iodine vapour. Column chromatography was performed on
silica gel (0.063e0.200 mm), with dichloromethane/methanol
(50:1) as eluent.
Ketoprofen and indomethacin were purchased from Belupo
(Croatia), fenoprofen from Eli Lilly Company (USA), diclofe-
nac from Pliva (Croatia), benzotriazole and 3-hydroxypropyl-
amine from Merck (Germany), whereas N-methylimidazole,
L-alanine methyl ester hydrochloride and p-chlorophenyl
phosphochloridate were purchased from Sigma Aldrich
(Germany). All solvents were of analytical grade purity and dry.
A solution of diclofenac benzotriazolide 2e (0.795 g,
2.00 mmol), 3-hydroxypropylmine (0.165 g, 2.20 mmol), tri-
ethylamine (0.202 g, 2.00 mmol) and sodium dithionite
(0.010 g) in ethyl acetate (10 ml) was stirred for 1 h at room
temperature. The solvent was evaporated under reduced pres-
sure. The residue was dissolved in acetone/water (1:1) mixture
and acidified with HCl to pH 1. Acetone was evaporated under
reduced pressure. The precipitated product was filtered off,
several times washed with water and triturated with petrol
ether. Yield: 0.676 g (96%); mp 130e133 ꢁC; IR (KBr, n/
cmꢀ1) 3495, 34323, 3297, 3253, 3082, 2947, 2882, 1618,
1
1565, 1508, 1453,1286, 1070, 775, 747; H NMR (DMSO-
d6) d 8.45 (s, 1H, 9), 8.35 (t, 1H, NH), 7.52e7.7.49 (d, 2H,
12, 14), 7.20e7.12 (m, 2H, 4, 6), 7.04 (t, 1H, 5), 6.85 (t,
1H, 13), 6.30e6.28 (d, 1H, 7), 4.45 (t, 1H, OH), 3.57 (s,
1H, 2), 3.45e3.39 (q, 2H, 300), 3.17e3.10 (q, 2H, 100), 1.62e
1.53 (m, 2H, 200). Anal. (C17H18Cl2N2O2) C, H, N.
2.1.6. p-Chlorophenyl (methoxy-L-alaninyl)phospho-
chloridate (4)
Compound 4 was prepared in accord with a procedure
given in the literature [5].
2.1.2. Fenoprofen 3-hydroxypropylamide (3a) and
ketoprofen 3-hydroxypropylamide (3b)
2.1.7. Phosphoramidate derivatives (5aee):
general procedure
Compounds 3a and 3b were prepared following the pub-
lished procedures [15,16].
N-methylimidazole (NMI) (5.00 mmol) was added drop-
wise with vigorous stirring to a cold solution of NSAID 3-hy-
droxypropylamide
3
(1.00 mmol) and p-chlorophenyl
2.1.3. Ibuprofen 3-hydroxypropylamide (3c)
(methoxyalaninyl)phosphochloridate 4 (5.00 ꢀ 6.00 mmol) in
anhydrous THF (3 ꢀ 4 ml) at ꢀ70 ꢁC. After 15 min the tem-
perature was left to rise to ambient temperature with stirring
over a period of 12 h. The solvent was removed under reduced
pressure. The obtained oil was dissolved in dichloromethane
and washed with 0.5 M HCl and water. The organic layer
was dried over MgSO4, filtered, evaporated to dryness and pu-
rified by column chromatography.
A solution of ibuprofen benzotriazolide 2c (0.615 g,
2.00 mmol), 3-hydroxypropylamine (0.165 g, 2.20 mol) and
triethylamine (0.202 g, 2.00 mmol) in ethyl acetate (10 ml)
was stirred for 1 h at room temperature. The reaction mixture
was extracted several times with sodium hydroxide solution
(pH 8). The organic layer was washed with water, dried over
anhydrous sodium sulphate and evaporated under reduced
pressure. The obtained crude product was triturated three
times with petrol ether. Yield: 0.450 g (85%); oil; IR (film,
n/cmꢀ1) 3299, 3088, 2954, 2932, 2869, 1652, 1548, 1512,
2.1.8. Fenoprofen 3-hydroxypropylamide (p-chlorophenyl
(methoxy-L-alaninyl)phosphate) (5a)
1
1065, 849; H NMR (DMSO-d6) d 7.88 (t, 1H, NH), 7.22e
Compound 5a was prepared according to general procedure
using fenoprofen 3-hydroxypropylamide (3a) (0.170 g,
0.57 mmol), phosphochloridate 4 (1 g, 3.20 mmol) and N-
methylimidazole (271 ml, 3.42 mmol). Yield: 0.130 g (40%);
7.19 (d, 2H, 5, 9), 7.08e7.06 (d, 2H, 6, 8), 4.39 (t, 1H,
OH), 3.57e3.50 (q, 1H, 2), 3.38e3.32 (q, 2H, 300), 3.13e
3.01 (m, 2H, 100), 2.41e2.39 (d, 2H, 10), 1.87e1.74 (m, 1H,
11), 1.56e1.47 (m, 2H, 200), 1.31e1.29 (d, 3H, 3), 0.87e
0.85 (d, 6H, 12, 13). Anal. (C16H25NO2) C, H, N.
1
oil; 31P NMR (DMSO-d6) d 4.65, 4.40; H NMR (CDCl3)
d 7.37e6.86 (m, 13H, 5, 7e9, 20-60, 500, 600, 800, 900),

K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
145
4.05e3.92 (m, 4H, 2, 300, AlaeCH), 3.71 (s, 3H, AlaeOCH3),
3.32e3.16 (m, 2H, 100), 1.83e1.69 (m, 2H, 200), 1.49 (d, 3H, 3),
1.38 (d, 3H, AlaeCH3). Anal. (C28H32ClN2O7P) C, H, N.
(d, 1H, 7), 4.04 (m, 2H, 40), 3.81 (m, 1H, AlaeCH), 3.58
(m, 5H, 2, AlaeCH, AlaeOCH3), 3.16 (m, 2H, 20), 1.78 (m,
2H, 30), 1.21 (d, 3H, AlaeCH3). Anal. (C27H29Cl3N3O6P) C,
H, N.
2.1.9. Ketoprofen 3-hydroxypropylamide (p-chlorophenyl
(methoxy-L-alaninyl)phosphate) (5b)
2.2. Biological tests
Compound 5b was prepared according to general procedure
using ketoprofen 3-hydroxypropylamide (3b) (0.144 g,
0.46 mmol), phosphochloridate 4 (0.720 g, 2.31 mmol) and
N-methylimidazole (220 ml, 2.78 mmol). Yield: 0.150 g
(55%); oil; 31P NMR (DMSO-d6) d 4.64, 4.39; 1H NMR
(DMSO-d6) d 8.11 (C]OeNH) 7.73e7.15 (m, 13H, 5, 7e
9, 20-60, 500, 600, 800, 900), 4.02e3.91 (m, 3H, 2, 300), 3.69 (d,
2H, AlaeNH), 3.57 (s, 3H, AlaeOCH3), 3.14e3.04 (m, 2H,
100), 1.77e1.65 (m, 2H, 200), 1.35 (d, 3H, 3), 1.20 (d, 3H,
AlaeCH3). Anal. (C29H32ClN2O7P) C, H, N.
2.2.1. Cytostatic activity assays
Thecytostatic experiments werecarried outon nine humancell
lines, eight of which are derived from eight cancer types and one
normal, fibroblast cell line. The following cell lines were used:
murine leukaemia (L1210), human T-lymphocytes (Molt4/C8,
CEM), cervical carcinoma (HeLa), pancreatic carcinoma (Mia-
PaCa-2), colon carcinoma (SW 620), breast carcinoma (MCF-
7), lung carcinoma (H 460) and diploid fibroblasts (WI 38).
Cytostatic activity against L1210, Molt4/C8 and CEM cell
lines were measured essentially as originally described [18].
After 48 (L1210) or 72 (CEM, Molt4/C8) hours, the tumour
cell number was counted by a Coulter counter.
2.1.10. Ibuprofen 3-hydroxypropylamide (p-chlorophenyl
(methoxy-L-alaninyl)phosphate) (5c)
Compound 5c was prepared according to general procedure
using ibuprofen 3-hydroxypropylamide (3c) (0.150 g,
0.57 mmol), phosphochloridate 4 (0.900 g, 2.85 mmol) and
N-methylimidazole (271 ml, 3.42 mmol). Yield: 0.131 g
The cytostatic activity against HeLa, MiaPaCa-2, SW 620,
MCF-7, H 460 and WI 38 cell lines was assessed as described
previously, according to the slightly modified procedure of the
National Cancer Institute, Developmental Therapeutics Pro-
gram [12,19]. Briefly, the cells were cultured as monolayers
and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS),
2 mM L-glutamine, 100 U mlꢀ1 penicillin and 100 mg mlꢀ1
streptomycin in a humidified atmosphere with 5% CO2 at
37 ꢁC. The cells were inoculated onto a series of standard
96-well microtiter plates on day 0, at 1 ꢂ 104 to
3 ꢂ 104 cells/ml, depending on the doubling times of specific
cell line. Test agents were then added in five 10-fold dilutions
(10ꢀ8 to 10ꢀ4 M) and incubated for a further 72 h. Working di-
lutions were freshly prepared on the day of testing. The sol-
vent was also tested for eventual inhibitory activity by
adjusting its concentration to be the same as in the working
concentrations. The cell growth rate was evaluated by per-
forming the MTT assay after 72 h of incubation, which detects
mitochondrial dehydrogenase activity in viable cells.
Each test point was performed in quadruplicate in three in-
dividual experiments, except for WI 38 cells, whereby only
one experiment was performed, and L1210, CEM and
Molt4/C8 cells for which two independent experiments were
performed. The results were expressed as IC50, a compound
concentration necessary for 50% of inhibition. The IC50 values
for each compound are calculated from doseeresponse curves
using linear regression analysis by fitting the test concentra-
tions that give percentage-of-growth values above and below
the reference value (i.e., 50%). If, however, for a given cell
line all of the tested concentrations produce PGs exceeding
the respective reference level of effect (e.g., PG value of
50), then the highest tested concentration is assigned as the de-
fault value, which is preceded by a sign >.
1
(43%); oil; 31P NMR d 3.87, 3.63; H NMR d 7.95 (t, 1H,
10), 7.43e7.37 (d, 2H, 5, 9), 7.20e7.18 (m, 4H, 80, 90, 100,
110), 7.07e7.04 (d, 2H, 6, 8), 4.02e3.93 (q, 1H, 20), 3.86e
3.80 (m, 2H, AlaeCH, AlaeNH), 3.59 (t, 2H, 40), 3.58 (s,
3H, AlaeOCH3), 3.14e3.03 (m, 2H, 20), 2.38 (d, 2H, 10),
1.80e1.67 (m, 3H, 11, 30), 1.29 (d, 3H, 3), 1.22 (d, 3H,
AlaeCH3), 0.85e0.83 (d, 6H, 12, 13). Anal.
(C26H36ClN2O6P) C, H, N.
2.1.11. Indomethacin 3-hydroxypropylamide (p-chloro-
phenyl (methoxy-L-alaninyl)phosphate) (5d)
Compound 5d was prepared according to general procedure
using indomethacin 3-hydroxypropylamide (3d) (0.159 g,
0.38 mmol), phosphorchloridate 4 (0.600 g, 1.92 mmol) and
N-methylimidazole (183 ml, 2.30 mmol). Yield: 0.112 g
(41%); oil; 31P NMR (DMSO-d6) d 4.70, 4.46; 1H NMR
(DMSO-d6): d 8.07 (t, 1H, 10), 7.70e7.62 (q, 4H, 13, 14, 16,
17), 7.42e7.39 (d, 2H, 90, 130), 7.21e7.18 (d, 2H, 100, 120),
7.10 (s, 1H, 9), 6.95e6.93 (d, 1H, 7), 6.72e6.69 (dd., 1H,
6), 4.04e3.96 (m, 2H, AlaeCH, AlaeNH), 3.75 (s, 3H, 200),
3.57 (s, 3H, AlaeOCH3), 3.50 (s, 2H, 2), 3.31 (s, 2H, 40),
3.16e3.11 (q, 2H, 20), 2.22 (s, 3H, 100), 1.79e1.71 (m, 2H,
30), 1.20 (d, 3H, AlaeCH3). Anal. (C32H34Cl2N3O8P) C, H, N.
2.1.12. Diclofenac 3-hydroxypropylamide (p-chlorophenyl
(methoxy-L-alaninyl)phosphate) (5e)
Compound 5e was prepared according to general procedure
using diclofenac 3-hydroxypropylamide (3e) (0.150 g,
0.43 mmol), phosphochloridate 4 (0.530 g, 1.70 mmol) and
N-methylimidazole (202 ml, 2.55 mmol). Yield: 0.159 g
(60%); oil; 31P NMR (DMSO-d6) d 4.64, 4.39; 1H NMR
(DMSO-d6) d 8.42 (s, 1H, NH-9), 8.38 (t, 1H, 10), 7.52e
7.49 (d, 2H, 12, 14), 7.45e7.42 (d, 2H, 9, 90), 7.23e7.13
(m, 4H, 4, 6, 10, 100), 7.04 (t, 1H, 5) 6.83 (t, 1H, 13), 6.29
2.2.2. Antiviral activity assays
Antiviral activity against HSV-1, HSV-2, vaccinia virus, ve-
sicular stomatitis virus (VSV) in human embryonic lung

146
K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
(HEL) cell cultures, Coxsackie virus B4, Sindbis virus, Punta
Toro virus, parainfluenza-3 virus and reovirus-1 in Vero cell
cultures, and respiratory syncytial virus in HeLa cell cultures
was determined. After a 2-h incubation period with 100
CCID50 of the respective viruses, residual virus was removed
and the infected cells were further incubated with the medium
containing different concentrations of the tested compounds.
After incubation for 3 days at 37 ꢁC, virus-induced cytopatho-
genicity was monitored microscopically. Antiviral activity was
expressed as the concentration required to reduce virus-in-
duced cytopathogenicity by 50% (EC50).
as EC50, the compound concentration required to reduce viral
plaque formation after 5 days by 50% as compared with un-
treated controls.
For the HIV-1(IIIB) and HIV-2(ROD) assays, virus-induced
cytopathicity was recorded in CEM cell cultures as described
[18].
3. Results and discussion
3.1. Chemistry
For the CMV assays, confluent HEL fibroblasts were grown
in 96-well microtiter plates and infected with the human cyto-
megalovirus strains Davis and AD-169 at 100 PFU per well.
After a 2-h incubation period, residual virus was removed
and the infected cells were further incubated with medium
containing different concentrations of the test compounds (in
duplicate). After incubation for 7 days at 37 ꢁC, virus-induced
cytopathogenicity was monitored microscopically after etha-
nol fixation and staining with Giemsa. Antiviral activity was
expressed as the EC50 or compound concentration required
to reduce virus-induced cytopathogenicity by 50%.
For the VZV assays, the laboratory wild-type VZV strain
Oka and the thymidine kinase-deficient VZV strain 07/1
were used. Confluent HEL cells grown in 96-well microtiter
plates were inoculated with VZV at an input of 20 PFU per
well. After a 2-h incubation period, residual virus was re-
moved and varying concentrations of the test compounds
were added (in duplicate). Antiviral activity was expressed
NSAID amides 3aee were synthesized by method devel-
oped by us from 3-hydroxypropylamine and NSAID benzo-
triazolides 2aee [15,16]. Compounds 2aee were prepared
by reaction of the corresponding NSAID drugs 1aee (fenopro-
fen, ketoprofen, ibuprofen, indomethacin, diclofenac) and
N-1-benzotriazole carboxylic acid chloride (BtcCl). Phospho-
chloridate 4 was synthesized from p-chlorophenyl phosphoro-
dichloridate (CPD) and L-alanine amino acid ester
hydrochloride [5]. The target phosphoramidates 5aee were
prepared in one step from the corresponding NSAID 3-hy-
droxypropylamides 3aee and compound 4 (Scheme 1). The
reaction was performed in THF in the presence of N-methyl-
imidazole at low temperature, rising to room temperature.
Evaporation, extraction and column chromatography on silica
gave the desired compounds 5aee in 40e60% yields. The
products were isolated as roughly 50:50 mixtures of diastereo-
isomers at the phosphate centre. These isomers do not easily
O
O
O
NH2
OH , TEA
TEA
-CO2
+
BtcCl
-BtH
R
Bt
R
N
H
OH
R
OH
1a-e
2a-e
3a-e
O
Cl
O
Cl
N
O
NMI
Cl
N
N
P
Btc
HN
O
O
O
4
O
O
P
R
N
H
O
NH
5a-e
O
O
a
b
c
d
e
O
Cl
H
N
O
O
R
N
Cl
O
Cl
Scheme 1. Synthesis of NSAID 3-hydroxypropylamides 3aee and their phosphoramidate derivatives 5aee.

K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
147
Table 1
13C NMR data and atom enumeration for compounds 3cee and 5aee
O
O
1
8''
5''
1''
3''
1''
3''
7'' Cl
6''
R1
2
R1
2
9''
O
N
H
OH
N
H
O
1
2''
2''
P
R2
R2
HN
O 4''
H3C
3c-e
5a-e
O
Compd.
R1
R2
13C NMR (DMSO-d6, d ppm)a
3c
3CH3
173.52 (1), 139.79 (4), 139.25 (7), 128.85 (5, 9), 127.03 (6, 8), 58.46 (300), 44.86 (2),
44.37 (10), 35.87 (100), 32.50 (200), 29.75 (11), 22.32 (12, 13), 18.74 (3)
12
13
H3C
CH3
6
5
11
O
4
4
10
7
8
9
9
2'
H3C
8
10
3
1'
CH3
7
N
11
12
17
5
3d
3e
H
H
169.82 (1), 168.31 (11), 156.01 (8), 138.01 (15), 135.56 (4), 134.74 (5), 131.61 (13,
17), 131.35 (12), 130.74 (10), 129.49 (14, 16), 115.02 (9), 114.90 (3), 111.75 (7),
102.25 (6), 58.85 (300), 55.87 (20), 36.41 (100), 32.89 (200), 31.66 (2), 13.84 (10)
6
13
14
15
O
Cl
16
9
H
N
Cl
15
3
10
11
14
13
4
5
172.03 (1), 143.43 (8), 137.65 (10), 130.82 (4), 129.83 (11, 15), 129.65 (12, 14),
127.63 (6), 126.03 (3), 125.44 (13), 121.11 (7), 116.40 (5), 58.77 (300), 40.04 (100),
36.45 (200), 32.67 (2)
8
7
Cl
12
3'
6
2'
1'
4'
5'
O
6
5a
3CH3
174.15 (C]O 1), 174.02 (C]O Ala), 157.56 (10), 156.96 (6), 149.27 (4), 143.80 (400),
143.63 (700), 130.02 (60), 129.78 (500, 900), 129.67 (8), 123.39 (600, 800), 122.34 (40),
121.54 (9), 118.88 (30, 50), 118.13 (5), 117.25 (7), 64.40 (300), 52.54 (CH3OeAla),
50.14 (CHeAla), 46.94 (2), 35.43 (100), 29.60 (200), 20.88 (CH3eAla), 18.34 (3)
6'
5
7
4
8
9
3'
2'
1'
4'
5'
O
3CH3
196.19 (C]O), 174.15 (C]O 1), 174.02 (C]O Ala), 150.13 (10), 150.03 (6), 143.19
(4), 137.51 (400), 137.32 (700), 132.14 (60), 132.05 (500, 900), 130.27 (8), 129.91 (600, 800),
129.14 (40), 128.56 (9), 122.53 (30, 50), 122.49 (5), 122.42 (7), 64.57 (300), 52.27
(CH3OeAla), 50.14 (CHeAla), 45.36 (2), 35.66 (100), 30.29 (200), 20.08 (CH3eAla),
19.01 (3)
6'
5b
6
5
7
4
8
9
12
CH3
13
H3C
6
5
5c
3CH3
174.08 (C]O Ala), 173.92 (C]O 1), 150.06 (400), 140.01 (4), 139.62 (7), 129.90 (500,
900), 129.20 (5, 9), 128.90 (700), 127.34 (6, 8), 122.43 (600, 800), 64.62 (300), 52.29
(CH3OeAla), 50.15 (CHeAla), 45.23 (2), 44.70 (10), 35.61 (100), 30.38 (200), 30.07
(11), 22.63 (12, 13), 20.12 (CH3eAla), 19.06 (3)
11
4
10
7
8
9
9
2'
O
3
10
8
H3C
1'
CH3
13
4
7
N
5
5d
H
174.10 (C]O Ala), 169.91 (1), 168.30 (11), 156.02 (8), 150.03 (80), 138.01 (15),
135.60 (4), 134.71 (5), 131.69 (13, 17), 131.62 (12), 131.34 (10), 129.93 (14, 16),
129.53 (90, 130), 128.91 (110), 122.55 (100, 120), 115.04 (9), 114.74 (3), 111.74 (7),
102.21 (6), 64.61 (300), 55.87 (20), 52.30 (CH3OeAla), 50.13 (CHeAla), 35.72 (100),
31.63 (200), 30.43 (2), 20.05 (CH3eAla), 13.82 (10)
6
11
12
17
14
O
15
Cl
16
9
Cl
12
H
N
15
3
10
5e
H
174.10 (C]O Ala), 172.13 (1), 150.07 (400), 143.42 (8), 137.62 (10), 130.82 (4),
129.91 (12, 14), 129.84 (11, 15), 129.65 (500, 900), 128.92 (700), 127.68 (6), 125.90 (3),
125.49 (13), 122.48 (600, 800), 121.14 (7), 116.42 (5), 64.53 (300), 52.31 (CH3OeAla),
50.15 (CHeAla), 40.07 (100), 35.82 (200), 30.16 (2), 20.08 (CH3eAla)
14
13
4
5
8
11
7
Cl
6
a
The spectrum of 5a was recorded in CDCl3 whereas the spectra of 5bee in DMSO-d6.

148
K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
Table 2
Inhibitory effects of NSAID 3-hydroxypropylamides 3aee (cf. Scheme 1) and their phosphoramidate derivatives 5aee on the growth of malignant tumour cell
lines in comparison with their effects on the growth of normal human diploid fibroblast (WI 38)
a
Tumour cell growth [IC50 (mM)]
Compd.
L1210
Molt4/C8 CEM
HeLa
MIA PaCa-2 SW 620 MCF-7 H 460
WI 38
N.T.b
3a
230 ꢃ 6 99 ꢃ 18
71 ꢃ 1
>100
>100
>100
>100
>100
O
O
N
H
OH
CH3
O
3b
273 ꢃ 14 97 ꢃ 18
71 ꢃ 9
>100
>100
>100
>100
>100
N.T.
O
N
H
OH
CH3
O
C
H3C
N
OH
H
3c
213 ꢃ 16 93 ꢃ 7
55 ꢃ 3
>100
ꢄ100
>100
>100
>100
N.T.
H3C
H3C
O
N
OH
H
O
H3C
CH3
3d
179 ꢃ 93 113 ꢃ 68 78 ꢃ 26 26 ꢃ 16 44 ꢃ 20
47 ꢃ 2
22 ꢃ 14 >100
N.T.
N
O
Cl
O
Cl
N
OH
H
H
3e
200 ꢃ 35 215 ꢃ 12 212 ꢃ 23 46 ꢃ 10 54 ꢃ 25
36 ꢃ 3
39 ꢃ 29 68 ꢃ 34 N.T.
N
Cl
O
O
5a
24 ꢃ 17 39 ꢃ 4
38 ꢃ 5
6 ꢃ 5
5 ꢃ 3
7 ꢃ 6
12 ꢃ 2 13 ꢃ 5
20
Cl
O
N
O
HN
H
P
O
CH3
OCH3
H3C
O

K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
149
Table 2 (continued)
a
Tumour cell growth [IC50 (mM)]
Compd.
L1210
Molt4/C8 CEM
HeLa
MIA PaCa-2 SW 620 MCF-7 H 460
WI 38
5b
O
O
44 ꢃ 7
40 ꢃ 2
40 ꢃ 2
19 ꢃ 2
14 ꢃ 2
17 ꢃ 2
21 ꢃ 7
15 ꢃ 2
14 ꢃ 3 18 ꢃ 1
30
Cl
O
N
H
O
P
HN
O
OCH3
CH3
H3C
O
O
Cl
H3C
C
O
N
O
H
P
HN
O
OCH3
5c
64 ꢃ 59 48 ꢃ 7
90 ꢃ 67 15 ꢃ 0.4 13 ꢃ 4
14 ꢃ 2 19 ꢃ 0.3 21
H3C
O
H3C
H3C
O
Cl
O
N
H
O
P
O
HN
O
H3C
CH3
OCH3
5d
24 ꢃ 7
28 ꢃ 16
11 ꢃ 4
17 ꢃ 1
8 ꢃ 7
16 ꢃ 1 18 ꢃ 0.5 16
H3C
N
O
O
Cl
O
Cl
O
Cl
N
O
HN
H
N
H
P
5e
13 ꢃ 8
9.7 ꢃ 0.8 22 ꢃ 17 16 ꢃ 1
16 ꢃ 2
16 ꢃ 0.6 9 ꢃ 7
17 ꢃ 1
18
O
OCH3
H3C
Cl
O
a
IC50 e the concentration that causes 50% growth inhibition.
NT e not tested.
b
separate by column chromatography, but were readily distin-
guished by 31P NMR.
effects on the growth of human normal fibroblasts (WI 38)
(Table 2 and Fig. 1).
The results of the in vitro cytostatic activity evaluations
showed that virtually all phosphoramidate derivatives 5aee
1
Structures of new compounds were deduced from their H,
13C and 31P NMR as well as IR spectra and confirmed by el-
emental analysis. The spectral data are given in experimental
part and in Table 1.
possess significantly greater inhibitory activities (IC50
¼
5e90 mM) than the corresponding parent NSAID 3-hydroxy-
propylamides (3aee), whose IC50 concentrations mostly
exceed 100 mM (Table 2). The increased antiproliferative
activity was up to >20-fold depending on the nature of the
drug and the tumour cell line evaluated. Among all investi-
gated compounds, compound 5a showed the most pronounced
inhibitory activity against the cervical carcinoma (HeLa), pan-
creatic carcinoma (MIA PaCa-2) and colon carcinoma (SW
620) cell lines (IC50 ¼ 5e7 mM). Additionally, compounds
5aec showed the most pronounced antiproliferative effect
against the solid tumour cell lines HeLa, MIA PaCa-2, SW
620, MCF-7, and H 460. Instead, the increase of cytostatic
3.2. Biological evaluations
3.2.1. Cytostatic activity
Phosphoroamidate derivatives 5aee and NSAID 3-hydroxy-
propylamides 3aee were evaluated in vitro on their cytostatic
effects against malignant tumour cell lines: murine leukaemia
(L1210) cells, human T-lymphocytes (Molt4/C8 and CEM),
cervical carcinoma (HeLa), pancreatic carcinoma (MIA
PaCa-2), colon carcinoma (SW 620), breast carcinoma
(MCF-7), H 460 (lung carcinoma) and compared with their

150
K. Wittine et al. / European Journal of Medicinal Chemistry 44 (2009) 143e151
3a
5a
A
B
150
100
50
150
100
50
WI 38
HeLa
HeLa
SW 620
H 460
0
SW 620
H 460
0
MCF-7
MiaPaCa-2
-50
-100
-50
-100
MCF-7
MiaPaCa-2
-9
-8 -5
log10 concentration (M)
-7
-6
-4
-3
-9
-8 -4
log 10 concentration (M)
-7
-6
-5
-3
3d
5d
C
D
150
100
50
150
100
50
WI38
HeLa
HeLa
SW 620
H 460
0
SW 620
H 460
0
MCF-7
MiaPaCa-2
-50
-100
-50
-100
MCF-7
MiaPaCa-2
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)
log10 concentration (M)
Fig. 1. Doseeresponse profiles for fenoprofen 3-hydroxypropylamide 3a (A), indomethacin 3-hydroxypropylamide 3d (C) and the corresponding phosphoramidate
derivatives 5a (B) and 5d (D) tested on various human cell lines in vitro. The cells were treated with the compounds at different concentrations, and percentage-of-
growth (PG) was calculated.
activity of compounds 5aec compared to 3aee was much less
pronounced against the human T-lymphocyte Molt4/C8 and
CEM cells (at most 2-fold, and in case of 5c even a w2-
fold lower cytostatic activity). It is currently unclear, however,
why the lymphocytic suspension cells do behave somewhat
different from the solid (monolayer) tumour cells. Different
levels of uptake and/or hydrolyzing enzymes in the solid ver-
sus suspension tumour cells may be likely. The preferential
effect of 5aec for solid tumour cells versus suspension tumour
cells is not observed for compounds 5d and 5e. Still, it should
be additionally evaluated whether the more pronounced cyto-
static activity of the phosphoramidate derivatives is due to an
increased uptake (increased intracellular delivery) of the com-
pounds and/or to the release of the phosphorylated parent
compound. It would therefore be interesting to synthesize
the phosphorylated derivatives of the compounds for testing
against the tumour cell lines.
and 5b that showed some slight inhibitory activity against
HCMV and VZV at 30e77 mM.
4. Conclusions
This study is a continuation of our research and synthetic
optimisation of novel NSAID derivatives as potential prodrugs
for anticancer therapy or chemopreventive applications with
less toxic side effects. In this paper we have proved that phos-
phoramidate derivatives of fenoprofen, ketoprofen, ibuprofen,
indomethacine and diclofenac (5aee) possess significantly
higher antiproliferative activities than the corresponding
NSAID 3-hydroxypropylamides, probably due to a better
cell uptake. The most evident increase in the cytostatic activity
was demonstrated for fenoprofen phosphoroamidate deriva-
tive. Evaluation of NSAID phosphoramidate derivatives as os-
teotropic drug delivery systems for bone-located inflammatory
and malignant diseases is planned.
3.2.2. Antiviral activity
Acknowledgement
Compounds 3aee and 5aee were evaluated for their inhib-
itory activity against human immunodeficiency virus type
1(IIIB) and type 2(ROD) in CEM cell cultures, herpes simplex
virus type 1 and 2, vaccinia virus, varicella-zoster virus, cyto-
megalovirus (CMV) and vesicular stomatitis virus (VZV) in
HEL cell cultures; parainfluenza-3 virus, reovirus-1, sindbis
virus, Coxsackie virus B4 and Punta Toro virus in Vero cell
cultures and respiratory syncytial virus (RSV), VSV and Cox-
sackie virus B4 in HeLa cell cultures. No specific antiviral ef-
fects (i.e. the minimal antiviral effective concentration less
than 5-fold lower than the minimal cytotoxic concentration)
were noted for any of the compounds against any of the vi-
ruses that were evaluated (data not shown), except for 3d
Support for this study was provided by Ministry of Science,
Education and Sports of the Republic of Croatia (Projects 006-
0000000-3216, 125-0982464-2922, 098-0982464-2514 and
098-0982464-2393) and the K.U.Leuven (GOA 05/15). We thank
Mrs. Lizette van Berckelaer, Leen Ingels, Leentje Persoons,
Vicky Broeckx, Frieda De Meyer, Anita Camps, Lies Vanden-
heurck and Steven Carmans for dedicated technical help.
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