NO-donors (VII [1]): Synthesis and cyclooxygenase inhibitory properties of N- and S-nitrooxypivaloyl-cysteine derivatives of naproxen - A novel type of NO-NSAID

Article abstract of DOI:10.1002/1521-4184(200211)335:8<363::AID-ARDP363>3.0.CO;2-S

Nitric oxide (NO) has been reported to subserve many of the same mucosal protection mechanisms as prostaglandins and is sufficient for acute gastroprotection and ulcer healing. In fact, NO-donating NSAID hybrid compounds such as the nitrooxybutyl ester of naproxen show reduced ulcerogenic activity while maintaining anti-inflammatory activity. We introduce two prototypes of novel triple-hybrid compounds consisting of cysteine which is known to enhance the activity of organic nitrates and to reduce nitrate tolerance, an NSAID (naproxen), and an organic nitrate (nitrooxypivaloic acid). L-Cysteine ethyl ester first was N-acylated in a CH₂Cl₂/H₂O two-phase system using the acid chlorides of naproxen or nitrooxypivaloic acid, respectively, and sodium acetate, or alternatively using the DCC-activated nitrooxy acid in absolute CH₂Cl₂. The N-acylated intermediates were subsequently S-acylated using the acid chlorides or alternatively the carbonyldiimidazole (CDI)-activated acids again. The two naproxen-cysteine-nitrate hybrid prodrugs were screened in vitro for their cyclooxygenase inhibitory properties relative to naproxen. In this screening the N-nitrooxyacylcysteine derivative was found to be inactive in the concentration range of 0.1-10 μmol/L against both COX-1 and COX-2, while the S-nitrooxyacylcysteine derivative had only weak activity against COX-1.

Full text of DOI:10.1002/1521-4184(200211)335:8<363::AID-ARDP363>3.0.CO;2-S

Arch. Pharm. Pharm. Med. Chem. 2002, 8, 363–366
Nitrooxypivaloyl-cysteine Derivatives of Naproxen 363
Rahmana E. Kartasasmitaa,b
Stefan Lauferc,
,
NO-Donors (VII [1]): Synthesis and Cyclooxygenase
Inhibitory Properties of N- and
Jochen Lehmanna
S-Nitrooxypivaloyl-cysteine Derivatives of Naproxen
– A Novel Type of NO-NSAID
a
Institute of Pharmacy,
University of Bonn, Bonn,
Germany
Nitric oxide (NO) has been reported to subserve many of the same mucosal protec-
tion mechanisms as prostaglandins and is sufficient for acute gastroprotection and
ulcer healing. In fact, NO-donating NSAIDhybrid compounds such as the nitrooxy-
butyl ester of naproxen show reduced ulcerogenic activity while maintaining anti-in-
flammatory activity. We introduce two prototypes of novel triple-hybrid compounds
consisting of cysteine which is known to enhance the activity of organic nitrates and
to reduce nitrate tolerance, an NSAID(naproxen), and an organic nitrate (nitro-
oxypivaloic acid). L-Cysteine ethyl ester first was N-acylated in a CH2Cl2/H2O two-
phase system using the acid chlorides of naproxen or nitrooxypivaloic acid, respec-
tively, and sodium acetate, or alternatively using the DCC-activated nitrooxy acid in
absolute CH2Cl2. The N-acylated intermediates were subsequently S-acylated us-
ing the acid chlorides or alternatively the carbonyldiimidazole (CDI)-activated acids
again.The two naproxen-cysteine-nitrate hybrid prodrugs were screened in vitro for
their cyclooxygenase inhibitory properties relative to naproxen.In this screening the
N-nitrooxyacylcysteine derivative was found to be inactive in the concentration
range of 0.1–10 µmol/L against both COX-1 and COX-2, while the S-nitrooxyacyl-
cysteine derivative had only weak activity against COX-1.
b
Department of Pharmacy,
Bandung Institute of
Technology, Bandung,
Indonesia
Institute of Pharmacy,
University of Tübingen,
Tübingen, Germany
c
Keywords: Naproxene; Organic nitrates; Cysteine; Cyclooxygenase inhibition;
Inflammation
Received: December 6, 2001 [FP653]
was reported to prevent specific tolerance to glyceryl ni-
trate [9]. Cysteine activates organic nitrates probably by
Introduction
The application of non-steroidal anti-inflammatory drugs
(NSAID’s) is limited by their side effects on the gastroin-
testinal tract (GIT) [2–4]. One approach to improve the
safety of NSAIDs is inhibition of COX-2 instead of
COX-1. Another one is nitric oxide (NO) donation, as the
latter subserves many of the same protective mecha-
nisms in GIT defense as prostaglandins do [4]. NO in-
duces vasodilatation and modulation of vascular factors,
and is sufficient for acute gastroprotection and for ulcer
healing [5,6].NO donors enhance collagen deposition at
a wound site [7] and even accelerate gastric ulcer heal-
ing [8]. NO-donating NSAIDhybrid compounds such as
the nitrooxybutyl ester of naproxen were found to have
reduced ulcerogenic activity compared to the parent
drug while maintaining full anti-inflammatory activity [6].
However, organic nitrates generally depress blood pres-
sure and rapidly induce nitrate tolerance. Co-treatment
with cysteine or N-acetyl-L-cysteine in a group of rats
reducing them to NO and by serving as a NO-store and
NO-carrier. Nitrate-cysteine hybrid compounds repre-
sent NO-donors nearly free of tolerance and only moder-
ately decrease blood pressure [10].All this led us to syn-
thesize the first nitrate-cysteine-NSAIDtriple-hybrids
representing potential GIT-safe NSAIDs. The new pro-
drugs 8 and 9, consisting of naproxen, cysteine ethyl es-
ter, and nitrooxypivaloic acid may show good anti-inflam-
matory activity, less GIT-damage, less tolerance, and less
decrease in blood pressure in-vivo. This paper presents
the synthesis and a preliminary in-vitro screening.
Results and discussion
Chemistry
The synthesis of the target compounds 8 and 9 involves
chemoselective N- and S-acylations. N-Acylation of L-
cysteine ethyl ester or its hydrochloride salt 1 has been
reported [11, 12].Also the N-acylation of L-cysteine ethyl
ester with nitrooxypivaloic acid using dicyclohexylcarbo-
diimide (DCC) as activating reagent has been patented
Correspondence: Jochen Lehmann, Institute of Pharmacy,
University of Bonn, An der Immenburg 4, D-53121 Bonn, Ger-
many. Phone: +49 (0) 228 735 250, Fax: +49 (0) 228 737 929,
e-mail: J.Lehmann@uni-bonn.de.
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/08/0363 $ 17.50+.50/0

364 Kartasasmita et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 363–366
Scheme 1
[12]. Applying this procedure to the acylation of L-
cysteine ethyl ester with naproxen (7) did not yield an N-
acylation product, but rather an inseparable mixture,
containing a high amount of unreacted naproxen.On the
other hand, 1 has been N-acylated in a CH2Cl2/water
two-phase system using of 3- and 4-nitrooxymethylben-
zoyl chloride and sodium acetate [13]. According to this
procedure, 1 was acylated with both acid chlorides of ni-
trooxypivaloic acid (2) and naproxen (4), respectively,
the latter being prepared as reported [14]. The reaction
occurred selectively at the amine group and gave satis-
factorily pure 5 and 6.Due to their sensitivity towards oxi-
dation these N-acylated intermediates were subse-
quently S-acylated using the acid chlorides again or al-
ternatively the carbonyldiimidazole (CDI)-activated acid
7, analogously to a reported procedure [15]. Again, the
acylation with acid chlorides gave better results with re-
Biology
The intermediate 6 and the target compounds 8 and 9
were tested for their cyclooxygenase inhibitory proper-
ties relative to naproxen.They were tested for COX-1 in-
hibition in a human platelet cell assay [16] and for COX-2
inhibition in mononuclear cells from whole blood [17].
Naproxen inhibits COX-1 with an IC50 of 0.1 µM and
COX-2 with an IC50 of 9.0 µM. 6 showed no inhibition of
COX-2 and only weakly inhibited COX-1 (IC50 = 6.0 µM).
N-3-nitrooxypivaoyl-S-(+)-2-(6-methoxy-2-naphthyl)-
propanoyl-L-cysteine ethyl ester (8) was inactive in the
concentration range of 0.1–10 µM against both COX-1
and COX-2, while N-(+)-2-(6-methoxy-2-naphthyl)pro-
panoyl-S-3-nitrooxypivaloyl-L-cysteine ethyl ester (9)
exhibited no inhibition of COX-2 in the same concentra-
tion range and a low inhibition activity against COX-1
with an IC50 of 5.6 µM. These results are in accordance
with the prodrug character of 6, 8, and 9.Antiinflammato-
ry activity certainly affords the metabolic liberation of
naproxen. In-vivo experiments (rats) are ongoing; the
results will be reported later.
1
gard to yield and purity of the crude products. The H-
NMR spectra show D2O-exchangeable signals for the
thiol groups at 2.41 ppm (5) and 2.46 ppm (6). All reac-
tions are summarized in Scheme 1.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 363–366
Nitrooxypivaloyl-cysteine Derivatives of Naproxen 365
was washed with 25 mL 1N hydrochloric acid solution, with
25 mL saturated sodium bicarbonate solution and 25 mL water
(twice), dried over anhydrous MgSO4 and evaporated under re-
duced pressure. The crude oily product was dissolved and
stirred in ethanol (20 mL). To the solution was added slowly
Ϸ 20 mL of water from a dropping funnel. The precipitated
white solid was separated by suction and dried in a desiccator
under reduced pressure. Yield 1.87 g (74 %).
Acknowledgement
The authors are grateful to Deutscher Akademischer
Austausch Dienst (DAAD) for sponsoring a doctoral pro-
gram in Germany for the first-named author.
Procedure 2: A solution of naproxen (7, 1.73 g, 7.5 mmol) in
30 mL absolute dimethylformamide (DMF) was cooled to –10
to –15 °C, treated portionwise with carbonyldiimidazole (CDI)
(1.28 g, 7.5 mmol ) under argon atmosphere, and stirred for
2 hours.To the mixture was slowly added a solution of 5 (2.94 g,
10 mmol) in 20 mL absolute DMF.The mixture was then stirred
for additional 2–3 hours at –10 °C. After the addition of 50 mL
ethyl acetate, the mixture was washed three times with 30 mL
of a saturated solution of sodium chloride, dried over Na2SO4
and evaporated under reduced pressure. The crude product
was purified by flash chromatography (ethyl acetate :n-hexane
1 : 1). Yield 0.29 g (7.6 %), white solid.
Experimental part
Chemistry
Thin layer chromatography was performed by using precoated
silica gel plates (Merck 60 F254) which were detected by short
UV light. Melting points were measured on a Gallenkamp melt-
ing point apparatus and are uncorrected. NMR spectra were
performed on Brucker WH 90 (90 MHz) and Brucker WM 500
(500 MHz), using DMSO-d6 as solvent and TMS as an internal
standard. Chemical shifts (δ) are expressed in ppm downfield
from TMS. Elemental analyses were carried out on a Heraeus
elemental analyzer, results were within 0.4 % of the theoreti-
cal values.Column chromatography was performed on 70–230
mesh silica gel from Merck.
All analytical and spectral data of the products obtained by both
procedures are identical:
Mp 74–77 °C, 1H-NMR (DMSO-D6, 500 MHz, δ ppm): 1.07,
1.09 (2 x s, 6 H, (CH3)2C<), 1.12 (t, J = 7.1 Hz, 3 H, –O–CH2–
CH3), 1.49 (d, J = 7.3 Hz, 3 H, >CH–CH3), 3.10 and 3.32 (2 x dd,
J = 13.7/9.6 Hz, J = 13.7/5.0 Hz, 2 H, >CH–CH2–S–), 3.86 (s,
3 H, –O–CH3), 4.03 (m, 2 H, –O–CH2–CH3), 4.11 (q, J = 7.3 Hz,
1 H, >CH–CH3), 4.27–4.33 (m, 1 H, >CH–CH2–S–), 4.58 (2 × d,
2 H, J = 10.1 Hz, –CH2–ONO2), 7.15 (dd, J = 9.1/2.5 Hz, 1 H, ar-
omat. H), 7.28 (d, J = 2.5 Hz, 1 H, aromat. H), 7.36 (dd, J = 8.5/
1.9 Hz, 1 H, aromat.H), 7.73 (d, J = 1.3 Hz, 1 H, aromat.H), 7.76
(d, J = 8.5 Hz, 1 H, aromat. H), 7.79 (d, J = 9.1 Hz, 1 H, aromat.
H), 8.03 (d, 1 H, –C(=O)–NH–), 13C-NMR (DMSO-D6, δ ppm):
13.98 (–O–CH2–CH3), 18.20 (>CH–CH3), 22.05 and 22.11
(CH3)2C<), 29.55 (>CH–CH2–S–), 41.32 ((CH3)2C<), 51.70
(>CH–CH2–S–), 53.18 (>CH–CH3), 55.32 (–O–CH3), 60.98
(–O–CH2–CH3), 78.28 (–CH2–ONO2), 105.89 Ct, 118.6 Ct,
126.60 Ct, 126.61 Ct, 127.14 Ct, 128.51 Cq, 129.35 Ct, 133.70
Cq, 134.72 Cq, 157.49 Cq, 170.10 (–C(=O)–O–), 173.96
(–C(=O)–NH–), 200.53 (–C(=O)–S–), anal. (C24H30N2O8S) C,
H, N.
3-Nitrooxy-2,2-dimethylpropanoic acid (3) and (+)-2-(6-meth-
oxy-2-naphthyl)propanoic acid chloride (4) were prepared as
described previously [12, 14].
3-Nitrooxy-2,2-dimethylpropanoic acid chloride (2)
To a mixture of thionyl chloride (4.1 mL, 57.2 mmol, d = 1.64)
and chloroform (10 mL), cooled with an ice bath, were added
3-nitrooxy-2,2-dimethylpropanoic acid (3, 6.53 g, 40 mmol) and
two drops of absolute dimethylformamide. The mixture was
heated slowly under stirring up to 50 °C and was kept at this
temperature until evolution of gas had finished. After cooling,
dry dichloromethane (100 mL) and active carbon (40 mg) were
added, the mixture was stirred for a few minutes, filtered, and
the filtrate was evaporated under reduced pressure.The prod-
uct was used for further reaction without purification. 7.26 g
(100 %), yellowish oil.
N-3-Nitrooxy-2,2-dimethylpropanoyl-L-cysteine ethyl ester (5)
To a solution of L-cysteine ethyl ester hydrochloride (1, 5.12 g,
27.5 mmol) and sodium acetate (4.10 g, 50.0 mmol) in water
(50 mL) were added 25 mL dichloromethane.This mixture was
then stirred strongly under argon, a solution of 2 (4.54 g, 25.0
mmol) in dichloromethane (25 mL) was added dropwise, and
stirring was maintained for 1.5 hours. The organic phase was
separated, washed with 50 mL water, 50 mL 5 % sodium bicar-
bonate solution, and 50 mL water again (twice), dried over an-
hydrous magnesium sulphate, evaporated under reduced
pressure, and the remaining yellowish oil was dried in vacuo
and subsequently used for further reaction.Yield 5.45 g (74 %).
1H-NMR (DMSO-D6, 500 MHz, δ ppm): 1.17 (t, J = 7.1 Hz, 3 H,
–O–CH2–CH3), 1.20 (2 × s, 6 H, (CH3)2C<), 2.41 (t, J = 8.4 Hz,
1 H, –SH), 2.75–2.92 (m, 2 H, >CH–CH2–SH), 4.08 (q, J =
7.1 Hz, 2 H, –O–CH2–CH3), 4.30–4.35 (m, 1 H, >CH–CH2–
SH), 4.58 (s, 2 H, –CH2–ONO2), 8.03 (d, J = 7.7 Hz, 1 H,
–C(=O)–NH–).
N-(+)-2-(6-methoxy-2-naphthyl)propanoyl-L-cysteine ethyl
ester (6)
To a solution of L-cysteine ethyl ester hydrochloride (1, 2.04 g,
11.0 mmol) and sodium acetate (1.64 g, 20.0 mmol) in water
(20 mL) was added dichloromethane (15 mL).This mixture was
stirred vigorously under argon atmosphere and a solution of 4
(2.49 g, 10,0 mmol) in dichloromethane (15 mL) was added
dropwise. Stirring was maintained for 1.5 hours. The organic
phase was separated, washed with 20 mL water, 20 mL 5 % so-
dium bicarbonate solution (twice) and 20 mL water (twice),
dried over MgSO4 and evaporated under reduced pressure to
1
dryness. Yield 2.66 g (74 %), white solid, mp 104–105 °C, H-
NMR (DMSO-D6, 90 MHz, δ ppm): 1.08 (t, 3 H, –O–CH2–CH3),
1.42 (d, 3 H, >CH–CH3), 2.46 (s, 1 H, –SH), 3.31–3.58 (m, 2 H,
>CH–CH2–SH), 3.85 (s, 3 H, –O–CH3), 3.85–4.15 (m, 3 H,
–O–CH2–CH3 and >CH–CH3), 4.27–4.54 (m, 1 H, >CH–CH2–
S–), 7.04–7.81 (m, 6 H, aromatic), 8.39 (d, 1 H, –C(=O)–NH–),
anal. (C19H23NO4S) C, H, N.
N-3-Nitrooxy-2,2-dimethylpropanoyl-S-(+)-2-(6-methoxy-2-
naphthyl)propanoyl-L-cysteine ethyl ester (8)
N-(+)-2-(6-methoxy-2-naphthyl)propanoyl-S-3-nitrooxy-2,2-
dimethylpropanoyl-L-cysteine ethyl ester (9)
Procedure 1: To a solution of 5 (1.62 g, 5.5 mmol) in dichlo-
romethane (30 mL) was added under ice cooling and argon at-
mosphere a solution of 4 (1.24 g, 5.0 mmol) and triethylamine
(0.51 g, 5.0 mmol) in dry dichloromethane (30 mL).The mixture
was stirred overnight at room temperature. Then, the mixture
To a solution of 6 (1.99 g, 5.5 mmol) in dichloromethane (30 mL)
was added under ice cooling and argon atmosphere a solution
of 3-nitrooxy-2,2-dimethylpropanoic acid chloride (2, 0.91 g,

366 Kartasasmita et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 363–366
5.0 mmol) and triethylamine (0.51 g, 5.0 mmol) in dry dichlo-
romethane (30 mL).The mixture was stirred overnight at room
temperature, washed with 25 mL 1N hydrochloric acid solution,
25 mL saturated sodium bicarbonate solution and 25 mL water
(twice), dried over MgSO4 and evaporated under reduced pres-
sure.The crude oily product was dissolved and stirred in etha-
nol (20 mL).To the solution was added the same volume of wa-
ter from a dropping funnel. The precipitated white solid was
separated and dried in a desiccator under reduced pressure.
Yield 1.96 g (77 %), mp 50–51 °C, 1H-NMR (DMSO-D6,
500 MHz, δ ppm): 1.06 (t, J = 7.1 Hz, 3 H, –O–CH2–CH3), 1.18,
1.23 (2 × s, 6 H, (CH3)2C<), 1.40 (d, J = 7.1 Hz, 3 H, >CH–CH3),
3.12 and 3.35 (2 × dd, J = 13.6/8.5 Hz, J = 13.6/5.4 Hz, 2 H,
>CH–CH2–S–), 3.79 (q, J = 7.1 Hz, 1 H, >CH–CH3), 3.85 (s,
3 H, –O–CH3), 4.00 (q, J = 7.1 Hz, 2 H, –O–CH2–CH3),
4.39–4.45 (m, 1 H, >CH–CH2–S–), 4.58 (2 × d, J = 2 × 10.0 Hz,
2 H, –CH2–ONO2), 7.13 (dd, J = 9.0/2.7 Hz, 1 H, aromat. H),
7.26 (d, J = 2.5 Hz, 1 H, aromat.H), 7.41 (dd, J = 8.5/1.6 Hz, 1 H,
aromat. H), 7.69 (s, 1 H, aromat. H), 7.73 (d, J = 8.5 Hz, 1 H, ar-
omat. H), 7.75 (d, J = 9.1 Hz, 1 H, aromat. H), 8.48 (d, 1 H,
–C(=O)–NH–), 13C-NMR (DMSO-D6, δ ppm): 13.94 (–O–CH2–
CH3), 18.61 (>CH–CH3), 22.07 and 22.25 ((CH3)2C<), 29.68
(>CH–CH2–S–), 48.79 ((CH3)2C<), 51.28 (>CH–CH2–S–),
44.76 (>CH–CH3), 55.28 (–O–CH3), 60.99 (–O–CH2–CH3),
77.47 (–CH2–ONO2), 105.85 Ct, 118.65 Ct, 125.52 Ct, 126.62
Ct, 126.65 Ct, 128.50 Cq, 129.19 Ct, 133.30 Cq, 136.94 Cq,
157.16 Cq, 170.06 (–C(=O)–O–), 173.67 (–C(=O)–NH–), 202.3
(–C(=O)–S, anal. (C24H30N2O8S) C, H, N.
References
[1] A.Horstmann, L.Menzel, R.Gäbler, A.Jentsch, W. Urban,
J. Lehmann, NITRIC OXIDE 2002, 6, 135–141.
[2] C.Allison, A.G.Howatson, C.J.Torrance, F.D.Lee, R.Rus-
sel, N. Engl. J. Med. 1992, 327, 749–754.
[3] G. Singh, G. Triadafilopoulos, J. Rheumatol. 1999, 26,
18–24.
[4] C. J. Hawkey, Gastroenterology 2000, 119, 521–535.
[5] M. T. Donnely, C. J. Hawkey, Aliment. Pharmacol. Ther.
1997, 11, 227–236.
[6] N. M. Davies, A. G. Roseth, C. B. Appleyard, W. McKnight,
P. Del Soldato, A. Calignano, G. Cirino, J. L. Wallace,
Aliment. Pharmacol. Ther. 1997, 11, 69–79.
[7] M. N. Muscara, W. McKnight, S. Asfaha, J. L.Wallace, B. J.
Pharmacol. 2000, 129, 681–686.
[8] J. L. Wallace, M. J. S. Miller, Gastroenterology 2000, 119,
512–520.
[9] C. M. Newman, J. B.Warren, G.W.Taylor, A. R. Boobis, D.
S. Davies, B. J. Pharmacol. 1990, 99, 825–829.
[10] J. Lehmann, Exp. Opin. Ther. Patents 2000, 10, 559–574
[11] W. Hanefeld, Arch. Pharm. 1985, 318, 375–377.
[12] Schwarz Pharma, AG, E.P. 362 575 A1, 1990; ref. CA:
1990, 113, 212672x,
[13] B. Böttcher, J. Lehmann, unpublished result.
[14] F. Turconi, E.P. 036 684 A1, 1981; ref. CA 1982, 96,
142854n,
Biology
[15] S.Endres, A.Hacker, E.Noack, G.Kodja, J.Lehmann, Eur.
J. Med. Chem. 1999, 34, 895–901.
COX-1 inhibition was determined in platelets isolated from
fresh human blood according to a reported method [16]. Plate-
lets were incubated in the presence and absence of inhibitors
for 10 minutes in IKP buffer. Prostaglandin E2 as a key interme-
diate was determined using an ELISA assay method.COX-2 in-
hibition was determined in mononuclear cells isolated from hu-
man fresh blood according to Laufer et al. [17]. COX-2 was in-
duced over 5 hours by LPS.PGE2 determination again was as-
sayed with an ELISA method.
[16] S. Laufer, H. Striegel, K. Neher, P. Zechmeister, C. Donat,
K. Stolingwa, S. Baur, S. Tries, G. Dannhardt, T. Kammer-
meier, W. Kiefer, Arch. Pharm. Pharm. Med. Chem. 1997,
330, 307–312.
[17] S.Laufer, P. Zechmeister, T.Klein, Inflamm.Res. 1999, 48,
13313-8.