Shen F et al / Acta Pharmacol Sin 2002 Aug; 23 (8): 762-768

Inhibitory effect of 3,4-diaryl-3-pyrrolin-2-one derivatives on cyclooxygenase 1 and 2 in murine peritoneal macrophages

SHEN Fang, BAI Ai-Ping, GUO Zong-Ru, CHENG Gui-Fang¹

Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Received 2001-09-25 Accepted 2002-05-31

KEY WORDS cyclooxygenase 1; cyclooxygenase 2; peritoneal macrophages; rofecoxib; indomethacin

ABSTRACT

AIM: To develop a whole-cell assay based on murine peritoneal macrophages and evaluate the inhibitory effect of candidate compounds on cyclooxygenase-1 (COX-1) and COX-2. METHODS: Macrophages were stimulated with calcimycin or lipopolysaccharide (LPS) for various periods. Their abilities to convert endogenous arachidonic acid to 6-keto-PGF_1¦Á or PGE₂ were examined by radioimmunoassay (RIA). RNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and COX-1/2 was detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers. RESULTS: Rofecoxib selectively inhibited LPS-induced, COX-2-derived PGE₂ synthesis with an IC₅₀ value of (4.7±0.5) nmol/L compared with maximum inhibitory ratio of 17.3 % for the inhibition of calcimycin induced, COX-1-derived 6-keto-PGF_1¦Á synthesis. Indomethacin exhibited dual inhibitory effects on COX-1 and COX-2 with IC₅₀ of (4.7±1.1) nmol/L and (7.1±1.2) nmol/L, respectively. Two series of 17 compounds were tested. Most of compounds in series II showed comparable inhibitory activities to rofecoxib on COX-2. The relative position of the sulfonylphenyl group to the lactam carbonyl group has important effects on COX-2 inhibitory activity. CONCLUSION: The established whole cell assay is appropriate for drug-design oriented in vitro assay. 3,4-Diaryl-3-pyrrolin-2-one derivatives were proved to be prospective new type of COX-2 selective inhibitors.

INTRODUCTION

Prostanoids are important mediators of a wide variety of physiological processes. Cyclooxygenase (COX) plays a important role in prostaglandin synthesis^[1]. Two different cyclooxygenase isozymes have been identified in mammals: COX-1 (EC 1.14.99.1) and COX-2^[2]. COX-1 is expressed constitutively in stomach, kidney collecting tubules, platelets, vascular endothelial cells, and macrophages, exerting certain physiologic housekeeping functions including normal renal function, gastric mucosal integrity, and hemostasis. On the other hand, COX-2 is virtually undetectable in most tissues under physiological conditions, but it may be dramatically induced by a variety of stimulus. COX-2 is postulated to be involved in the generation of prostanoids in certain stages of cell proliferation and differentiation^[1]. Recently, many research show that COX-2 is also over-expressed in colon cancer, which lead to further studies on COX-2 functions in cancer and development^[3].

Nonsteroidal antiinflammatory drugs (NSAID) are known to mitigate pain and inflammation by blocking COX isozymes. Classical NSAID such as aspirin and indomethacin nonspecifically inhibit COX-1/2 at standard antiinflammatory doses. The benefits of anti-
inflammation occur through inhibition of COX-2, but the gastrointestinal toxicity occurs as a result of concurrent inhibition of COX-1^[4]. Selective COX-2 inhibitors such as celecoxib (Celebrex, Searle & Pfizer)^[5] and rofecoxib (Vioxx, Merck)^[6] are now widely accepted as promising agents to treat inflammations without side-effects associated with classical NSAID^[7].

Macrophages are known to release prostanoids in two kinetically distinct patterns: the immediate and delayed phase^[8]. In the immediate phase, 6-keto-PGF_1¦Á and TXB₂ were the major arachidonic acid metabolites in response to Ca²⁺ stimulation. In the delayed phase, the PGE₂ production in response to lipopolysaccharide (LPS) over long-term culture is dependent on induced COX-2. In this study, we tried to develop a whole-cell assay based on murine peritoneal macrophages and evaluate the inhibitory effect of candidate compounds on COX-1/2.

MATERIALS AND METHODS

LPS (E coli 055:B5) and calcimycin (A23187) were from Sigma. Brewer thioglycollate medium was from Difco. RPMI-1640, M-MLV reverse transcriptase, and Trizol reagent were from GIBCO-BRL. Random hexamers were from Promega. dNTPs mixture and TaKaRa Tag were from TaKaRa. Newborn calf serum was from HyClone. 6-keto-PGF_1¦Á and PGE₂ RIA kit were from PLA General Hospital.

Cell culture Adherent macrophages were harvested from the peritoneal cells of male mice (C57BL-6J, Grade II, from Experimental Animal Center, Academy of Military Medical Science) 3 d after the injection (ip) of brewer thioglycollate medium (50 mL/kg body weight) as described previously^[9]. Shortly, peritoneal cells obtained from 3-4 mice were mixed and seeded in 48-well cell culture cluster (Costar) at a cell density of 1×10⁹/L in RPMI-1640 supplemented with 5 % (v/v) newborn calf serum, penicillin 100 kU/L, and streptomycin 100 g/L. After settlement for 2-3 h, non-adherent cells were washed by D-Hanks' balanced salt solution. Then macrophages were cultured in RPMI-1640 without serum. Almost all of adherent cells were macrophages as assessed by Giemsa staining. Cell viability was examined by Trypan blue dye exclusion. All incubation procedures were performed with 5 % CO₂ in humidified air at 37 ¡æ.

COX-1 assay Macrophages were incubated with test compound at different concentrations or solvent (Me₂SO) for 1 h and were stimulated with calcimycin 1 mmol/L for 1 h. The amount of 6-keto-PGF_1¦Á (a stable metabolite of PGI₂) in supernatant was measured by radioimmunoassay (RIA) according to manufacturer's guide. The inhibitory ratio (IR) was calculated as

C refer to 6-keto-PGF_1¦Á concentration in supernatants of calcimycin, test compound, and control groups, respectively.

COX-2 assay Macrophages were incubated with test compound at different concentrations or solvent (Me₂SO) for 1 h and were stimulated with LPS 1 mg/L for 9 h. The amount of PGE₂ in supernatants was measured by RIA. The inhibitory ratio was calculated using the same formula as in COX-1 assay section. C refer to PGE₂ concentration in supernatants of LPS, test compound, and control groups, respectively.

RT-PCR The GAPDH primer used as 5'-GAGGGGCCATCCACAGTCTTC-3' and 5'-CATCACCATCTTCCAGGAGCG-3'. The COX-1 primer used as 5'-AGTGCGGTCCAACCTTATCC-3' and 5'-CCGCAGGTGATACTGTCGTT-3'. The COX-2 primer used as 5'-GGGAAGCCTTCTCCAACC-3' and 5'-GAACCCAGGTCCTCGCTT-3'. Total RNA was extracted from macrophages using Trizol reagent. First strain cDNA was synthesized from 2 ¦Ìg of total RNA with M-MLV reverse transcriptase and random hexamer. Equal amount of RT product (1 ¦ÌL) were amplified by PCR with TaKaRa Tag for 28 cycles consist of 94 ¡æ for 30 s, 55 ¡æ for 30 s, and 72 ¡æ for 45 s. The amplified cDNA were solved on 2 % (w/v) agarose gel electrophoresis and visualized by ethidium bromide.

Inhibitors Rofecoxib, indomethacin, and all other compounds used in this study were synthesized by our group. Structures of compounds are shown (Fig 1, Tab 1). All tested samples were prepared in stock solution 0.01 mol/L with Me₂SO and stored at -20 ¡æ. Before use, stock solutions were diluted to appropriate concentrations in RPMI-1640.

Fig 1. Structure of rofecoxib, indomethacin, and 3,4-diaryl-3-pyrrolin-2-ones.

Tab 1. Substitution of 3,4-diaryl-3-pyrrolin-2-ones.

	R1	R2	R3
I-1	CH3	CH3	CH3
I-2	CH3	p-F	CH2CH2CH3
I-3	CH3	m-Cl	CH3
I-4	CH3	m-Cl	CH2CH2CH3
I-5	NH2	m-Br	CH2CH2CH3
I-6	NH2	m-Cl	CH2CH2CH3
I-7	NH2	m-F	CH2CH2CH3
I-8	NH2	m-Cl	Cyclopropyl
II-1	CH3	H	CH3
II-2	CH3	H	CH2CH2CH3
II-3	NH2	p-CH3	Cyclopropyl
II-4	CH3	p-Cl	CH3
II-5	CH3	p-F	CH2CH2CH3
II-6	CH3	p-Cl	CH2CH2CH3
II-7	CH3	p-CH3	Cyclopropyl
II-8	CH3	p-Cl	Cyclohexyl
II-9	CH3	p-Br	CH3

Statistical analysis Data were expressed as mean±SD of more than three independent experiments. Dose-inhibitory effect curves were fit through "uphill dose response curves, variable slope" using Prism, GraphPad version 3.00.

RESULTS

Conversion of endogenous arachidonic acid to 6-keto-PGF_1¦Á immediately after calcimycin stimulation The ability of macrophages to metabolize endogenous arachidonic acid to 6-keto-PGF_1¦Á was dependent on calcimycin dosage. When cells were incubated with calcimycin 0, 0.125, 0.25, 0.5, 1, 2, or 4 ¦Ìmol/L for 1 h, the concentrations of 6-keto-PGF_1¦Á in supernatant reached (125±21), (119±33), (107±26), (447±116), (1423±244), (1748±152), or (2497±255) ¦Ìg/L, respectively (n=5) (Fig 2).

Fig 2. Efficacy of calcimycin on 6-keto-PGF_1¦Á production in macrophages utilizing endogenous arachidonic acid. n=5 independent experiments. Mean±SD. ^cP<0.01 vs control group (Me₂SO).

Conversion of endogenous arachidonic acid to PGE₂ after long-term culture with LPS stimulation The inducible production of PGE₂ in macrophages utilizing endogenous arachidonic acid was dependent on both LPS dosage and time. When cells were incubated with LPS 0, 0.0625, 0.125, 0.25, 0.5, 1, or 2 mg/L for 9 h, the concentrations of PGE₂ in supernatant reached (2.6±0.3), (4.6±0.8), (5.1±0.7), (7.1±0.7), (9.6±1.2), (11.1±0.8), or (12.7±1.6) ¦Ìg/L, respectively (n=5) (Fig 3). When cells were incubated with LPS 1 mg/L for 3, 6, 9, or 12 h, the concentration of PGE₂ in supernatant reached (2.4±0.9), (22±3), (73±4), or (75±4) ¦Ìg/L, respectively (n=5). There was a latent period for 9 h. The PGE₂ production increased about 28-fold after 9 h culture with LPS 1 mg/L stimulation (Fig 3).

Fig 3. Efficacy and time course of LPS on PGE₂ production in macrophages utilizing endogenous arachidonic acid.Macrophages (5×10⁵ cells per well) were incubated with LPS in different concentration for 9 h (A) or LPS 1 mg/L for different time (B). n=5 independent experiments. Mean±SD. ^cP<0.01 vs control group (Me₂SO).

The elevation of COX-2 mRNA level after LPS stimulation Macrophages were cultured in a medium containing LPS 1 mg/L. Total RNA was extracted from cells at different time. mRNA level of GAPDH and COX-1/2 was detected by RT-PCR using specific primers. After incubation for 0, 3, 6, 9, and 12 h, an accumulation of COX-2 mRNA was observed, while the mRNA level of COX-1 were almost not changed (Fig 4).

Fig 4. The mRNA level of GAPDH, COX-1, and COX-2 in macrophage with LPS stimulation.

The inhibitory effect of rofecoxib and indomethacin on COX-1/2 The inhibitory ratio of rofecoxib on COX-1 at a concentration of 1 mmol/L was 17.3 % (n=5), while that of indomethacin at the same concentration was 104.2 % (n=5). The IC₅₀ of indomethacin on COX-1 was (4.7±1.1) nmol/L. The IC₅₀ of rofecoxib and indomethacin on COX-2 were(4.7±0.5) nmol/L and (7.1±1.2) nmol/L, respectively (Fig 5).

Fig 5. Does-response curve of rofecoxib and indomethacin on COX-1 and COX-2. The inhibitory ratios of indomethacin (¡ö), rofecoxib (¡õ) on COX-1 and rofecoxib (¡ð), indomethacin(¡ñ) on COX-2 at different concentrations were measured. n=4-6 repetitive wells in cell culture.

The inhibitory effect of substituted 3,4-diaryl-3-pyrrolin-2-ones on COX-1/2 Rofecoxib possesses two aryl rings in cis-position connecting with a third ring and one aromatic moiety is substituted by a sulfonyl group. To explore the effect of the position of sulfonylphenyl group relative to the lactam carbonyl group on activity and the influence of different substituents on nitrogen atom, 17 compounds in two series were designed and synthesized. The IC₅₀ of candidate compounds on COX-1 and COX-2 were obtained. The selectivity of compounds was expressed as the
IC_50,COX-1/IC_50,COX-2 (Tab 2). In our system, rofecoxib exhibits over 213-fold selectivity on COX-2 over COX-1, while indomethacin shows more selectivity on COX-1. Most of compounds in series II showed comparable inhibitory effect to rofecoxib on COX-2 and some compounds have high selectivity. In series I, the IC_50,COX-2 of compounds are 10-100 fold lower than that of compounds in series II. The induction of 6-keto-PGF_1a production by 0.1 % Me₂SO was also observed in our model (data not shown). Because of solubility, the IC₅₀ of some compounds on COX-1 were not measured.

Tab 2. The inhibitory effect of substituted 3,4-diaryl-3-pyrrolin-2-ones on COX-1 and COX-2.

Compounds	IC50 /nmol¡¤L-1		IC50,COX-1/ IC50,COX-2
	COX-1	COX-2
I-1	>1000	1950¡À685	>0.51
I-2	>1000	185¡À47	>5.4
I-3	>1000	305¡À89	>3.28
I-4	>1000	859¡À252	>1.16
I-5	>1000	933¡À306	>1.07
I-6	493¡À168	259¡À88	1.90
I-7	>1000	403¡À99	>2.48
I-8	>1000	1730¡À546	>0.58
II-1	933¡À256	16.7¡À2.3	55.9
II-2	149¡À52	24.7¡À7.9	6.03
II-3	>1000	18¡À4.2	>55.5
II-4	155¡À53	30.8¡À8.2	5.03
II-5	513¡À198	40.7¡À9.2	12.59
II-6	10.3¡À4.1	32.8¡À9.8	0.31
II-7	243¡À92	84.3¡À22	2.88
II-8	182¡À76	129¡À49	1.41
II-9	56.8¡À23	15¡À4	3.86
Rofecoxib	>1000	4.7¡À0.5	>213
Indomethacin	4.7¡À1.2	7.1¡À1.2	0.67

DISCUSSION

Recently, many models have been established to evaluate the inhibitory effect of candidate compounds on both COX isozymes, for example, the human whole blood assay^[10], Chinese hamster ovary cells expressing human COX-2 and COX-1^[11], purified recombinant human COX-1/2 enzyme^[12], and microsomal COX
assays^[13]. In our study, macrophages spontaneously secreted low level of 6-keto-PGF_1a and PGE₂. Stimulation with calcimycin or LPS promoted a dramatic enhancement of prostanoids production. Calcimycin is a calcium ionophore. Calcimycin stimulus will cause an influx of calcium in macrophages, activation of calcium-dependent PLA₂, release of arachidonic acid from membrane, and then conversion of arachidonic acid to PGH₂ by constitutively expressed COX-1. 6-keto-PGF_1a was one of the major arachidonic acid metabolites in this phase^[14]. The 6-keto-PGF_1a production in response to immediate calcimycin stimulation is COX-1 dependent^[8]. In the delayed phase, the inducible PGE₂ production in response to LPS over long-term culture is correlated with expression of COX-2. Due to the functional coupling of PLA₂, COX-2 and PGE₂ synthase, COX-2 tend to utilize low concentration of arachidonic acid despite of coexistence of COX-1 in the same
cell^[8]. The induced PGE₂ production has not been diminished even if COX-1 was blocked by pre-incubating cells with aspirin, which irreversiblely inhibits COX-1 through acetylation of a serine residue within COX active center^[15].

Compared to other system, our model exhibit following advantages: Firstly, macrophages were used as the source of both COX assays. This made it possible to determine the IC₅₀ of compounds on COX-1/2 on same cells. Secondly, to retain the original signal transduction pathway and functional coupling of PLA₂, COX and terminal synthase, the intact macrophages instead of purified enzymes or microsomal were used. Thirdly, the endogenous instead of exogenous arachidonic acid were used. This helps to retain original physiological concentration of arachidonic acid.

Rofecoxib and indomethacin showed different inhibitory pattern on COX-1/2 in our system, which were in line with values in other published literatures. The differences in activity between the two series of compounds shows that the position of sulfonylphenyl group relative to a, b-unsaturated lactam carbonyl group has important effects on COX-2 inhibitory activity. In
series II, SO₂NH₂ type compounds tended to have
stronger activity for inhibiting COX-2 activity than SO₂CH₃ type compounds. In series I, the inhibitory effect of SO₂NH₂ and SO₂CH₃ on COX-2 inhibitory activity was uncertain. In both series, a para-orientation electrophilic group on R₂ was favorable for inhibiting COX-2 activity. The compounds with large volume substituents on the nitrogen atom in lactam carbonyl group inclined to lose COX-2 selectivity.

The present paper shows that the whole-cell assay based on murine peritoneal macrophages was successfully established. It is appropriate for drug-design oriented in vitro assay. It provides opportunities to determine the IC₅₀ of candidate compounds on COX-1/2 in same cells. 3,4-Diaryl-3-pyrrolin-2-one derivatives were proved to be prospective new type of COX-2 selective inhibitors.

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