Design, synthesis and structure-activity relationship studies of novel and diverse cyclooxygenase-2 inhibitors as anti-inflammatory drugs

http://informahealthcare.com/enz

ISSN: 1475-6366 (print), 1475-6374 (electronic)

J Enzyme Inhib Med Chem, Early Online: 1–22

2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.864650

!

ORIGINAL ARTICLE

Design, synthesis and structure–activity relationship studies of novel

and diverse cyclooxygenase-2 inhibitors as anti-inflammatory drugs

Shigeo Hayashi, Naomi Ueno, Akio Murase, and Junji Takada

Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., Taketoyo, Aichi, Japan

Abstract

Keywords

Because of the pivotal role of cyclooxygenase (COX) in the inflammatory processes, non-

steroidal anti-inflammatory drugs (NSAIDs) that suppress COX activities have been used

clinically for the treatment of inflammatory diseases/syndromes; however, traditional NSAIDs

exhibit serious side-effects such as gastrointestinal damage and hyper sensitivity owing to their

COX-1 inhibition. Also, COX-2 inhibition-derived suppressive or preventive effects against

initiation/proliferation/invasion/motility/recurrence/metastasis of various cancers/tumours such

as colon, gastric, skin, lung, liver, pancreas, breast, prostate, cervical and ovarian cancers are

significant. In this study, design, synthesis and structure–activity relationship (SAR) of various

novel {2-[(2-, 3- and/or 4-substituted)-benzoyl, (bicyclic heterocycloalkanophenyl)carbonyl or

cycloalkanecarbonyl]-(5- or 6-substituted)-1H-indol-3-yl}acetic acid analogues were investigated

to seek and identify various chemotypes of potent and selective COX-2 inhibitors for

the treatment of inflammatory diseases, resulting in the discovery of orally potent agents in

the peripheral-inflammation model rats. The SARs and physicochemical properties for the

analogues are described as significant findings. For graphical abstract: see Supplementary

Material. (www.informahealthcare.com/enz)

Carrageenan-induced oedema, COX-2/COX-1

selectivity, inflammation, NSAID, orally

active COX-2 inhibitor,

History

Received 25 June 2013

Revised 29 October 2013

Accepted 30 October 2013

Published online 11 February 2014

Introduction

inflammatory-diseases and/or -symptoms^4,5,25but it is not involved

in the COX-1-mediated GI tract events^15–17. As well, the inhibitory

actions of traditional NSAIDs that include aspirin (acetylsalicylic

acid) against COX-1 can be crucial problems in clinical pharma-

cotherapy, such as respiratory- and cutaneous-hypersensitivities

Cyclooxygenase (COX, or prostaglandin G/H synthase or endo-

peroxidase; EC 1.14.99.1) is

a key rate-limiting enzyme

for prostaglandin (PG) cascades that produce prostanoids^1–3, and

inhibition of COX activity is able to attenuate the levels of

prostanoids that are involved in the inflammatory symptoms such

as swelling, flare, heat and pain. Therefore, COX inhibition has

been considered as a significant target of drug candidates for the

treatment of inflammatory diseases/syndromes that include

rheumatoid arthritis (RA) and osteoarthritis (OA)⁴. Non-steroidal

anti-inflammatory drugs (NSAIDs) that suppress COX activities

have been used clinically^4–14; however, traditional NSAIDs that are

non-selective inhibitors against COX isoforms, COX-1 (EC

1.14.99.1) and COX-2 (EC 1.14.99.1), exhibit serious side-effects

such as gastrointestinal damage^15–17. COX-1 is predominantly

expressed ubiquitously and constitutively and serves a housekeep-

ing role in processes such as GI mucosa protection. COX-2 is

absent or exhibits a low level of expression in most tissues, and is

highly upregulated in response to inflammatory or tissue-injury

stimuli/signals, growth factors, endotoxin, viruses, proinflamma-

tory cytokines, activated T lymphocytes, carcinogens/tumour-

promoters and hyperglycemia-induced mitochondrial reactive-

oxygen species in the various types of organs, tissues and

cells^{2,6,7,10,18–24}(Ref. 25 by Hayashi et al.). Significantly, COX-2

is associated with prostaglandin E₂(PGE₂) and prostacyclin (PGI₂)

production that evokes or sustains systemic or peripheral

and cross-reactivities^26–28

.

Furthermore, it has been elucidated that COX-2 has key and

pivotal roles in (i) induction and proliferation of various types of

cancers or tumours in various regions such as colon (colorec-

tal)^29,30, gastric^31,32, esophageal^33,34, skin (non-melanoma/melan-

oma)^35–37, lung^38–40, liver^41–44, pancreatic^45–47, bile duct⁴⁸,

gallbladder^48,49, urinary bladder⁵⁰, breast^51,52, prostate^53,54, cer-

vical (uterine cervix)^55,56, ovarian^57,58, nasopharyngeal⁵⁹and

oral⁶⁰cancers, and in (ii) metastasis of cancers such as lymph

node^61,62, haematogenous^63,64, gastric⁶⁵, lung⁶⁶, liver^67–69, mela-

noma^70–72and bone^73,74metastases that are correlated with

COX-2 upregulation in cancer/tumour cells by inflammatory- and

mitogenic-stimuli and growth factors^{22,38,52,61,65,75}and by other

reasons (as mentioned below). In patients with various cancers,

adverse prognosis, disease relapse and poor survival rates

have been associated with overexpression of COX-2^{30,37–40,49,}

^{56,60,63,69,70,76,77}, but not with expression of COX-1^30,56,69,76. Also,

it has been widely studied and demonstrated that COX-2

inhibition has great clinical potentials of chemotherapeutic and

chemopreventive treatments, with its anti-inflammatory and

mortality-reduction effects, for cancer/tumour patients, owing to

its suppressive and preventive effects against COX-2-dependent

(i) stimulation or increases in pro-mitogenic factors, carcinogen-

esis, tumour angiogenesis and lymph angiogenesis and (ii) pro-

motion, growth, resistance to apotosis, proliferation, invasion,

spreading, migration, drug-resistance, recurrence and metastasis

Address for correspondence: Shigeo Hayashi, Pfizer Global Research and

Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo,

Aichi 470-2393, Japan. E-mail: Shigeo_Hayashi@nifty.com

2

S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

of cancer/tumour cells/tissues^{29–31,36–38,40,44–46,48,51–54,57,58,60–69,}intrinsic COX-2 inhibitions. In this study, design, synthesis

^{71–75,78–82}. Actually, the cancer/tumour progression mechanisms and SAR of diverse novel {2-[(2-, 3- or 4-substituted, or 2,4- or

including initiation, proliferation, invasion, spreading, migration, 3,4-disubstituted)-benzoyl, (bicyclic heterocycloalkanophenyl)

drug-resistance and metastasis of cancer/tumour cells are caused carbonyl or cycloalkanecarbonyl]-(5- or 6-substituted)-1H-indol-

by (i) COX-2-derived mediators including prostanoids such as 3-yl}acetic acid analogues were investigated to seek and identify

PGE₂, prostaglandin F_2a(PGF_2a) and thromboxane A₂(TXA₂) various chemotypes of orally potent and selective COX-2 inhibitors

and growth factors^{37,38,40,43,44,53,54,60–62,65,69,75,79,81,83–87}

,

by as potential agents for the treatment of inflammatory diseases.

(ii) DNA oxidative damage resulting from COX-2 activ- Based on the results of these studies of in vitro and in vivo SARs

ity^54,75,88,89and/or by (iii) other COX-2-derived signal path- and physicochemical properties for the analogues together with

ways^{42,45,52,57,78}, which are dependent on COX-2 upregulation the previously reported studies by us, molecular mechanisms

and activation that result from (i) chronic inflammatory stimuli or of actions for the in vivo anti-inflammatory efficacies of the

carcinogens/tumour-promoters or from (ii) inflammatory- and present analogues as COX-inhibitors are also discussed herein.

mitogenic-stimuli in and growth-factors in cancer/tumour cells/

tissues, as mentioned, or from (iii) other promoters/inducers, for Experimental

example, increased/accumulated ultraviolet (UV) light (UVA,

Chemistry

UVB and UVC) exposures^{35,36,71,90,91}, radiation exposures⁹²,

various chronic infections such as Helicobacter pylori (H. pylori) General

infection^32,83, hepatitis B virus (HBV) or hepatitis C virus (HCV)

In general, reagents, solvents and other chemicals were used as

infection^41,42and human papillomavirus (HPV) infection⁵⁵,

purchased without further purification. All reactions with air- or

cigarette (tobacco) smoke²³and chronic morphine-treatment⁸²,

moisture-sensitive reactants and solvents were carried out under

or from (iv) other cancer/tumour cells-derived mechan-

nitrogen atmosphere unless noted otherwise. Flash column

isms^{40,45,53,71,72}. COX-2 inhibitors can boost immune systems of

chromatography (medium pressure liquid chromatography) puri-

patients and/or augment other immunotherapies; hence, immuno-

fications were carried out using Merck silica gel 60 (230–400

therapy effects of COX-2 inhibitors have been clinically important

mesh ASTM; Merck KGaA, Darmstadt, Land Hessen, Germany).

to treat cancer/tumour and to prevent metastasis as well⁹³.

Preparative thin-layer chromatography (PTLC) purifications were

On the other hand, it has been reported that COX-2

carried out on Merck silica gel 60 F₂₅₄pre-coated glass plates at a

inhibitors or traditional NSAIDs showed no or agent structure/

thickness of 0.5 or 1.0 mm (Merck KGaA). The structures of all

character-dependent potential cardiovascular-risk in long-term

isolated compounds were ensured by NMR, IR, MS or elementary

clinical studies with attention to permanent blockade against

1

analysis. H nuclear magnetic resonance (¹H NMR) data were

COX-2^94–96, and that COX-2 selective inhibition improved

determined at 270 MHz on a JNM-LA 270 (JEOL Ltd., Akishima,

endothelial function in coronary artery disease derived from

Tokyo, Japan) spectrometer. Chemical shifts are expressed in ꢀ

vascular-inflammation and oxidative-stress in clinical study⁹⁷,

(ppm). ¹H NMR chemical shifts were determined relative to

and attenuated cardiovascular-failure and liver-damage induced

tetramethylsilane (TMS) as internal standard. NMR data are

by endotoxin in rat model study⁹⁸, which show importance of

reported as follows: chemical shift, number of atoms, multi-

safety-index assessment for respective COX-2 inhibitors in long-

plicities (s, singlet; d, doublet; t, triplet; m, multiplet; dd, double

term use and other clinical potential of COX-2 inhibitors for the

doublet; ddd, double double doublet; br, broadened), and coupling

treatment of cardiovascular- and liver-diseases as already

constants. Infrared spectra were measured by an IR-470

mentioned in the previous report by Hayashi et al.²⁵

(Shimadzu Co., Nakagyo-ku, Kyoto, Japan) infrared spectrometer.

Accordingly, discovery of potent and selective diverse-

Low-resolution mass spectral data (EI) were obtained on an

chemotypes of COX-2 inhibitors has become increasingly

Automass 120 (JEOL Ltd.) mass spectrometer or an Integrity

important to meet the above various clinical needs with no or

mass spectrometer (Waters Corporation, Milford, MA). Low-

minimal/controllable adverse effects, and to reveal the molecular

resolution mass spectral data (ESI) were obtained on a Quattro II

mechanisms of enzyme inhibition by COX-2 inhibitor in vitro and

(Micromass, Milford, MA) mass spectrometer–Agilent 1100

in vivo. As approaches for elucidation of ligand–COX-2 inter-

HPLC system (Agilent Technologies, Inc., Santa Clara, CA).

action mechanisms and for (further) drug-design of potent and

Melting points were obtained using an Exstar 6000 (Seiko

selective COX-2 inhibitors, the studies of X-ray protein-crystal-

Instruments Inc., Chiba, Japan) and were uncorrected. All general

lography and molecular-structure/-docking calculation for COX

chemicals were the highest available grade.

isozymes with or without ligands have been reported^99–103. In the

The detailed original-procedures and obtained data for the

studies, the respective ligand structure-dependent various inter-

respective compounds are described as follows and in the

actions with COX-2, that is, respective binding modes in the

Appendix of the Supplementary Material.

complexes, respective time-dependent enzyme inhibitions and

respective conformational changes of the enzyme by the ligands

[6-Chloro-2-(2-methylbenzoyl)-1H-indol-3-yl]acetic acid (8a):

have been displayed or proposed. To clarify further detailed

general procedure of Method A

molecular mechanisms, various design and SAR studies for potent

and selective COX-2 inhibitors are highly needed.

Step 1. 6-Chloro-1-(phenylsulfonyl)-1H-indole (2). A mixture of

Besides, it has been described already, design, synthesis and 6-chloroindole (Lancaster Synthesis, Heysham, Lancashire, UK,

in vitro and in vivo structure–activity relationship (SAR) studies 52.52 g, 346.4 mmol), n-Bu₄NHSO₄(Wako Pure Chemical

for novel [2-{[(4-substituted or 4,5-disubstituted)-pyridin-2- Industries, Ltd., Chuo-ku, Osaka, Japan, 11.76 g, 34.64 mmol),

yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-1H-indol- 50% aqueous KOH (Wako Pure Chemical Industries, Ltd., 200 mL)

3-yl]acetic acid analogues were investigated by Hayashi and benzene (Wako Pure Chemical Industries, Ltd., 500 mL) was

et al.^25,104, which resulted in the discoveries of novel acid-types stirred for 10 min at room temperature under N₂, then cooled to

of COX-2 inhibitors as a new-class of orally potent anti-pyretic 0 ^ꢀC, benzenesulfonyl chloride (Tokyo Chemical Industry Co.,

and/or anti-inflammatory drugs in vivo; also, it has been shown Ltd., Chuo-ku, Tokyo, Japan, 66.3 mL, 519.5 mmol) was added

in the studies that the mechanisms of orally potent anti-pyretic dropwise at 0 ^ꢀC under N₂. The reaction mixture was allowed to

effect and anti-oedematous effect in the in vivo models for the room temperature, and stirred under N₂for 3 h. Some of the solvent

analogues were due to their respective highly potent and selective, were removed, and the resulting suspension was filtered with

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors

3

anhydrous Et₂O (Wako Pure Chemical Industries, Ltd.). The Pure Chemical Industries, Ltd., 16.0 mL) was degassed then

organic layer of the filtrate was separated, and the aqueous layer purged with N₂at room temperature, and then Mn(OAc)₃ꢃ2H₂O

was extracted with Et₂O (Wako Pure Chemical Industries, Ltd., (Sigma-Aldrich, St. Louis, MO, 1.40 g, 5.22 mmol) was

200 mL ꢁ 3). The organic layers were combined, dried over added^106,107. The reaction mixture was stirred at room tempera-

anhydrous MgSO₄(Wako Pure Chemical Industries, Ltd.), filtered ture under N₂for 5 min, warmed to 80 ^ꢀC, and stirred under N₂.

and concentrated in vacuo. The resulting brown crystalline solid After several minutes, the dark-brown mixture turned to black.

was washed with EtOH (Wako Pure Chemical Industries, Ltd.) After being stirred for 1 h, more Mn(OAc)₃ꢃ 2H₂O (1.40 g,

to afford 78.93 g of the title product 2 in 78% yield as a slight 5.22 mmol) was added to the resulting brown mixture that

pinkish-white crystalline solid. ¹H NMR (270 MHz, CDCl₃) ꢀ contained several amount of starting material detected by TLC

8.03–8.02 (1 H, m), 7.89–7.86 (2 H, m), 7.57–7.41 (5 H, m), 7.20 analysis. The reaction mixture was stirred at 80 ^ꢀC for 1 h, cooled

(1 H, dd, J ¼ 8.40 Hz, J ¼ 1.81 Hz), 6.63 (1 H, d, J ¼ 3.62 Hz).

Step 2. [6-Chloro-1-(phenylsulfonyl)-1H-indol-2-yl](2- suspended in AcOEt (50 mL)–brine (50 mL) with vigorously

methylphenyl)methanone (4a). To stirred solution of stirring, and then filtered. After the organic layer of the filtrate

to room temperature, and concentrated in vacuo. The residue was

a

6-chloro-1-(phenylsulfonyl)-1H-indole 2 (2.000 g, 6.855 mmol) was separated, the aqueous layer was extracted with AcOEt

in anhydrous THF (Dojindo Laboratories, Mashikimachi, (50 mL ꢁ 2). The organic layers were combined, dried over

Kumamoto, Japan, 32.0 mL) was added dropwise tert-BuLi anhydrous MgSO₄, filtered and concentrated in vacuo. The

(Kanto Chemical Co., Inc., Chuo-ku, Tokyo, Japan, 1.64 M in residue was purified by flash column chromatography (silica gel,

pentane, 4.60 mL, 7.54 mmol) via gas-tight syringe at ꢂ78 ^ꢀC hexane/AcOEt ¼ 3:1) to afford 578.2 mg of the title product 6a

1

under N₂. The solution was then stirred at the same temperature in 70% yield as a brown amorphous solid. H NMR (270 MHz,

under N₂for 45 min under N₂. To the resulting yellow anion CDCl₃) ꢀ 8.53 (1 H, br s), 7.78 (1 H, d, J ¼ 8.88 Hz), 7.49–7.13

solution was added dropwise a solution of 2-toluoyl chloride (6 H, m), 4.31–4.18 (4 H, m), 2.52 (3 H, s), 1.96 (3 H, s), 1.22

(Wako Pure Chemical Industries, Ltd., 1.20 mL, 9.20 mmol) in (6 H, t, J ¼ 7.26 Hz).

anhydrous THF (5.0 mL) at ꢂ78 ^ꢀC under N₂¹⁰⁵. The reaction Step 5. Diethyl [6-chloro-2-(2-methylbenzoyl)-1H-indol-

solution was stirred at the same temperature under N₂for 2 h, 3-yl]malonate (7a). To a stirred solution of diethyl 2-acetoxy-2-

gradually warmed up to 0 ^ꢀC, and then stirred for 45 min. After [6-chloro-2-(2-methylbenzoyl)-1H-indol-3-yl]malonate

6a

saturated aqueous NH₄Cl (Wako Pure Chemical Industries, Ltd., (569.8 mg, 1.173 mmol) in dry CH₂Cl₂(Wako Pure Chemical

50 mL) was added to the reaction mixture at 0 ^ꢀC, the resulting Industries, Ltd., 8.5 mL) was added TFA (Wako Pure Chemical

mixture was extracted with AcOEt (Wako Pure Chemical Industries, Ltd., 108 mL, 1.41 mmol) followed by Et₃SiH (Tokyo

Industries, Ltd., 50 mL ꢁ 3). The combined extracts were dried Chemical Industry Co., Ltd., 749 mL, 4.69 mmol) at room

over anhydrous MgSO₄, filtered and concentrated in vacuo to temperature under N₂¹⁰⁷. The reaction solution was warmed up

afford 3.820 g of the title product 4a as a pale gray solid (crude). to reflux conditions using an oil bath, stirred under N₂for 17 h,

TLC: R_f¼ 0.34, AcOEt/hexane ¼ (1:10) ꢁ 2 (hexane: Wako Pure then more TFA (451 mL, 5.85 mmol) was added, and stirred for

1

Chemical Industries, Ltd.). H NMR (270 MHz, CDCl₃) ꢀ 8.19– 7 h. The reaction solution was cooled to 0 ^ꢀC, ice-cooled saturated

6.79 (13 H, m), 2.54 (3 H, s).

Step 3. (6-Chloro-1H-indol-2-yl)(2-methylphenyl)methanone was stirred at 0 ^ꢀC for 15 min. The organic layer was separated,

(5a). mixture of [6-chloro-1-(phenylsulfonyl)-1H-indol-2- and the aqueous layer was extracted with CH₂Cl₂(Wako Pure

aqueous NaHCO₃(20 mL) was added, and the resulting mixture

A

yl](2-methylphenyl)methanone 4a (crude, 1.170 g) and anhydrous Chemical Industries, Ltd., 20 mL ꢁ 2). The combined extracts

K₂CO₃(Wako Pure Chemical Industries, Ltd., 1.996 g, were dried over anhydrous MgSO₄, filtered and concentrated

14.44 mmol) in THF (Wako Pure Chemical Industries, Ltd., in vacuo. The residue was purified by flash column chromatog-

16.0 mL)–MeOH (Wako Pure Chemical Industries, Ltd., 8.0 mL)– raphy (silica gel, hexane/AcOEt ¼ 4:1) to afford 491.8 mg of the

pure H₂O (Wako Pure Chemical Industries, Ltd., 4.0 mL) was title product 7a in 98% yield as a brown oil. ¹H NMR (270 MHz,

warmed up to reflux conditions using an oil bath, stirred under N₂CDCl₃) ꢀ 9.02 (1 H, br s), 7.74 (1 H, d, J ¼ 8.88 Hz), 7.73–7.25

for 14 h, then cooled to room temperature. The mixture was (5 H, m), 7.12 (1 H, dd, J ¼ 8.75 Hz, J ¼ 1.81 Hz), 4.90 (1 H, s),

extracted with AcOEt (50 mL ꢁ 3). The combined extracts were 4.20–4.08 (4 H, m), 2.35 (3 H, s), 1.19 (6 H, t, J ¼ 7.10 Hz).

washed with saturated aqueous NaHCO₃and brine (saturated Step 6. [6-Chloro-2-(2-methylbenzoyl)-1H-indol-3-yl]acetic

aqueous NaCl) (NaHCO₃, NaCl; Wako Pure Chemical Industries, acid (8a). A mixture of diethyl [6-chloro-2-(2-methylbenzoyl)-

Ltd.), dried over anhydrous MgSO₄, filtered and concentrated 1H-indol-3-yl]malonate 7a (491.8 mg, 1.15 mmol) and 2 N NaOH

in vacuo. The residue was recrystallised from AcOEt to afford (Wako Pure Chemical Industries, Ltd., 2.00 mL, 4.00 mmol) in

181.4 mg of the title product a white crystalline solid. The mother EtOH (13.0 mL) was warmed up to reflux conditions using an oil

liquor was also repurified by flash column chromatography (silica bath, and stirred under N₂for 4 h. The reaction solution was cooled

gel, hexane/AcOEt ¼ 10:1), and the fraction that contained to room temperature, concentrated in vacuo, then the residue was

desired product was recrystallised from AcOEt–hexane to afford partitioned between 2 N HCl (Wako Pure Chemical Industries,

335.6 mg of the title product a white crystalline solid. In total, Ltd., 50 mL)–Et₂O (50 mL) with acidification. The organic layer

517.0 mg of the title product 5a was obtained in 91% yield was separated, and the aqueous layer was extracted with AcOEt

[theoretical yield of two steps from compound 2 calculated with (40 mL ꢁ 3). The combined extracts were dried over anhydrous

the ratio for (obtained weights in step 2)/(used weights in step 3) MgSO₄, filtered and concentrated in vacuo. The residue was

of compound 4a]. ¹H NMR (270 MHz, CDCl₃) ꢀ 9.37 (1 H, br s), recrystallised from AcOEt–hexane to afford 178.0 mg of the

7.59–7.56 (2 H, m), 7.48 (1 H, m), 7.44–7.41 (1 H, m), 7.34–7.27 title product 8a in 47% yield as a yellow crystalline solid. Mp: 150–

(2 H, m), 7.12 (1 H, dd, J ¼ 8.56 Hz, J ¼ 1.81 Hz), 6.87 (1 H, m), 152 ^ꢀC. ¹H NMR (270 MHz, DMSO-d₆) ꢀ 11.68 (1 H, s), 7.68 (1 H,

2.44 (3 H, s).

d, J ¼ 8.72 Hz), 7.51–7.30 (5 H, m), 7.09 (1 H, dd, J ¼ 8.72 Hz,

Step 4. Diethyl 2-acetoxy-2-[6-chloro-2-(2-methylbenzoyl)- J ¼ 1.97 Hz), 3.55 (2 H, s), 2.23 (3 H, s). IR (KBr): 3321, 1717,

1H-indol-3-yl]malonate (6a). A mixture of (6-chloro-1H-indol- 1624, 1602, 1568, 1531, 1431, 1319, 1249, 1230 cm^ꢂ1. MS

2-yl)(2-methylphenyl)methanone 5a (455.3 mg, 1.69 mmol), (EI direct) m/z: M^þ327. Anal. (C₁₈H₁₄ClNO₃) C, H, N.

diethyl malonate (Wako Pure Chemical Industries, Ltd.,

1.29 mL, 8.45 mmol) and AcONa (Wako Pure Chemical For the subsections below, see ‘‘Experimental S1’’

Industries, Ltd., 693.2 mg, 8.45 mmol) in glacial AcOH (Wako section in the Appendix of the Supplementary Material for the

4

S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

detailed procedures and obtained data for the respective up to reflux conditions using an oil bath, and stirred under N₂for

compounds:

15 h. The reaction solution was cooled to 0 ^ꢀC, ice-cooled

saturated aqueous NaHCO₃(20 mL) was added, and the resulting

mixture was stirred at 0 ^ꢀC for 15 min. The organic layer was

separated, and the aqueous layer was extracted with CH₂Cl₂

(20 mL ꢁ 3). The combined extracts were dried over anhydrous

MgSO₄, filtered and concentrated in vacuo. The residue was

purified by flash column chromatography (silica gel, hexane/

AcOEt ¼ 5:1) to afford 316.8 mg of the title product 13 in 18%

yield [four steps from compound 2 calculated with the ratios

for (obtained weights in the preparation step)/(used weights in

the next step) of the respective intermediates] as a brown solid.

[6-Chloro-2-(3-methylbenzoyl)-1H-indol-3-yl]acetic acid (8b)

[6-Chloro-2-(4-methylbenzoyl)-1H-indol-3-yl]acetic acid (8c)

{6-Chloro-2-[4-(trifluoromethyl)benzoyl]-1H-indol-3-yl}acetic

acid (8d)

[6-Chloro-2-(2-chlorobenzoyl)-1H-indol-3-yl]acetic acid (8e)

[6-Chloro-2-(2,4-dichlorobenzoyl)-1H-indol-3-yl]acetic acid (8f)

[6-Chloro-2-(cyclohexanecarbonyl)-1H-indol-3-yl]acetic acid (14)

Step 1. [6-Chloro-1H-indol-2-yl](cyclohexyl)methanone (11). ¹H NMR (270 MHz, CDCl₃) ꢀ 8.91 (1 H, br s), 7.72 (1 H, d,

To a stirred solution of compound 2 (1.3133 g, 4.501 mmol) in J ¼ 8.88 Hz), 7.35 (1 H, d, J ¼ 1.81 Hz), 7.11 (1 H, dd,

anhydrous THF (8.0 mL) was added dropwise tert-BuLi (1.64 M J ¼ 8.88 Hz, J ¼ 1.81 Hz), 5.70 (1 H, s), 4.30–4.16 (4 H, m),

in pentane, 3.30 mL, 5.41 mmol) via gas-tight syringe at ꢂ78 ^ꢀC 3.03–2.93 (1 H, m), 1.96–1.22 (16 H, m, including 6 H at

under N₂. The resulting solution was then stirred at the same 1.23 ppm, t, 7.07 Hz).

temperature under N₂for 20 min. The resulting yellow solution Step 3. [6-Chloro-2-(cyclohexanecarbonyl)-1H-indol-3-yl]ace-

was slowly cannulated directing into a stirred solution of tic acid (14). A mixture of diethyl [6-chloro-2-(cyclohexanecar-

cyclohexanecarbonyl chloride (Wako Pure Chemical Industries, bonyl)-1H-indol-3-yl]malonate 13 (302.7 mg, 0.7209 mmol) and

Ltd., 722 mL, 5.40 mmol) in anhydrous THF (3.0 mL) at ꢂ78 ^ꢀC 2 N NaOH (2.00 mL, 4.00 mmol) in EtOH (13.0 mL) was warmed

under N₂over 10 min. After being stirred at the same temperature up to reflux conditions using an oil bath, stirred under N₂for 14 h,

under N₂for 2 h, the reaction mixture was poured into ice-cooled cooled to room temperature and concentrated in vacuo. The

aqueous saturated NH₄Cl (20 mL). The resulting mixture was residue was mixed with H₂O (30 mL) and Et₂O (30 mL), then the

extracted with AcOEt (20 mL ꢁ 3), and the combined extracts resulting mixture was vigorously stirred for 15 min. The organic

were dried over anhydrous MgSO₄, filtered and concentrated layer was separated to remove impurity. The aqueous layer was

in vacuo to afford 2.2561 g of [6-chloro-1-(phenylsulfonyl)-1H- further washed with Et₂O (30 mL), acidified by adding 2 N HCl

indol-2-yl](cyclohexyl)methanone 10 as a brown solid (crude). (5 mL), then extracted with AcOEt (30 mL ꢁ 3). The combined

TLC: R_f¼ 0.38, AcOEt/hexane ¼ 1:4.

extracts were dried over anhydrous MgSO₄, filtered and

To a suspension of [6-chloro-1-(phenylsulfonyl)-1H-indol- concentrated in vacuo. The residue was recrystallised from

2-yl](cyclohexyl)methanone 10 (crude, 2.2036 g) in EtOH AcOEt–hexane to afford 73.0 mg of the title product 14 in 32%

1

(19.0 mL) was added 2 N NaOH (14.8 mL, 29.6 mmol) at room yields as a solid. Mp: 206–209 ^ꢀC. H NMR (270 MHz, DMSO-

temperature under N₂. The mixture was dissolved in THF d₆) ꢀ 11.77 (1 H, s), 7.70 (1 H, d, J ¼ 8.56 Hz), 7.45 (1 H, d,

(10.0 mL) at 40 ^ꢀC. The resulting solution was warmed up to J ¼ 1.81 Hz), 7.07 (1 H, dd, J ¼ 8.56 Hz, J ¼ 1.81 Hz), 4.00 (2 H,

reflux conditions, and stirred under N₂for 30 min. The reaction s), 3.20 (1 H, m), 1.89–1.13 (10 H, m). IR (KBr): 3314, 2924,

solution was cooled to room temperature, and concentrated 2856, 1734, 1650, 1537, 1396, 1248 cm^ꢂ1. Anal. (C₁₇H₁₈ClNO₃)

in vacuo. The residue was partitioned between H₂O (30 mL) and C, H, N.

AcOEt (30 mL). The organic layer was separated, and the aqueous

layer was extracted with AcOEt (30 mL ꢁ 3). The combined

[6-Chloro-2-(3-fluorobenzoyl)-1H-indol-3-yl]acetic acid (22a):

extracts were dried over anhydrous MgSO₄, filtered and concen-

general procedure of Method B

trated in vacuo to afford 1.3981 g of the title product 11 as a brown

1

solid (crude). H NMR (270 MHz, CDCl₃) ꢀ 10.08 (1 H, br s), Step 1. 6-Chloro-1-(phenylsulfonyl)-1H-indole-2-carboxylic

7.59–7.03 (4 H, m), 3.21–3.11 (1 H, m), 2.28–1.20 (10 H, m).

acid (15). To a stirred solution of dry iso-Pr₂NH (Wako Pure

Step 2. Diethyl [6-chloro-2-(cyclohexanecarbonyl)-1H-indol-3- Chemical Industries, Ltd., 23.5 mL, 168 mmol) in anhydrous THF

yl]malonate (13). A mixture of [6-chloro-1H-indol-2-yl](cyclo- (150 mL) was added dropwise n-BuLi (Kanto Chemical Co., Inc.,

hexyl)methanone 11 (crude, 1.370 g), diethyl malonate (3.60 mL, 1.54 M in hexane, 109 mL, 168 mmol) below ꢂ60 ^ꢀC under N₂.

23.7 mmol) and AcONa (1.846 g, 22.5 mmol) in glacial AcOH The resulting solution was stirred at 0 ^ꢀC under N₂for 1.5 h, then

(44.0 mL) was degassed then purged with N₂at room temperature cooled to ꢂ78 ^ꢀC. To a stirred solution of compound 2 (refer

and then Mn(OAc)₃ꢃ 2H₂O (3.73 g, 13.9 mmol) was added. The Step 1 of ‘‘[6-Chloro-2-(2-methylbenzoyl)-1H-indol-3-yl]acetic

reaction mixture was stirred at room temperature under N₂for acid (8a): general procedure of Method A’’ section; 43.77 g,

5 min, warmed to 80 ^ꢀC, stirred under N₂for 2 h, and then more 150.0 mmol) in anhydrous THF (440 mL) was added the above

Mn(OAc)₃ꢃ 2H₂O (1.80 g, 6.71 mmol) was added. The reaction cooled solution of lithium diisopropylamide (LDA) through

mixture was stirred at 80 ^ꢀC for 4 h, cooled to room temperature, cannula at ꢂ78 ^ꢀC under N₂for 15 min. After being stirred at

then poured into brine (30 mL). The resulting mixture was the same temperature under N₂for 1 h, CO₂gas was bubbled into

extracted with AcOEt (30 mL ꢁ 3). The organic layers were the reaction solution at ꢂ78 ^ꢀC for 1.5 h. Saturated aqueous

combined, dried over anhydrous MgSO₄, filtered and concen- NH₄Cl (300 mL) that was cooled in an ice-cooled water bath was

trated in vacuo. The residue was roughly purified by flash column added to the stirred reaction solution at ꢂ78 ^ꢀC, and the resulting

chromatography (silica gel, hexane/AcOEt ¼ 4:1) to afford mixture was allowed to warm to room temperature with stirring,

946.0 mg of diethyl 2-acetoxy-2-[6-chloro-2-(cyclohexanecarbo- and then H₂O (600 mL) was added. The resulting mixture was

nyl)-1H-indol-3-yl]malonate 12 as a brown solid containing extracted with Et₂O (800 mL). The ethereal layer was separated,

impurity.

and the aqueous layer was acidified with 2 N HCl (30 mL), then

To a stirred solution of diethyl 2-acetoxy-2-[6-chloro-2- extracted with Et₂O (400 mL). The ethereal layers were

(cyclohexanecarbonyl)-1H-indol-3-yl]malonate 12 (containing combined, extracted with 2 N NaOH (250 mL ꢁ 3). The aqueous

impurity, 920.0 mg) in dry CH₂Cl₂(33.0 mL) was added TFA layers were combined, acidified with 2 N HCl (900 mL), then

(1.16 mL, 15.1 mmol) followed by Et₃SiH (1.15 mL, 7.20 mmol) extracted with Et₂O (900 mL ꢁ 1, then 500 mL ꢁ 1). The ethereal

at room temperature under N₂. The reaction solution was warmed layers were combined, then AcOEt (300 mL) was added to

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors

5

dissolve solid parts of the product in the organic extracts. The degassed then purged with N₂at room temperature, and then

resulting solution was washed with brine (400 mL ꢁ 2), dried over Mn(OAc)₃ꢃ 2H₂O (2.82 g, 10.5 mmol) was added. The reaction

anhydrous MgSO₄, filtered, then concentrated in vacuo to afford mixture was stirred at room temperature under N₂for 5 min,

42.44 g of the title product 15 in 84% yield as a white crystalline warmed to 80 ^ꢀC, stirred under N₂for 3 h, and then more

solid. TLC: R_f¼ 0.27, CH₂Cl₂/MeOH ¼ 5:1. ¹H NMR (270 MHz, Mn(OAc)₃ꢃ 2H₂O (1.41 g, 5.26 mmol) was added. The reaction

DMSO-d₆) ꢀ 8.01–7.38 (10 H, m).

mixture was stirred at 80 ^ꢀC for 2.5 h, cooled to room temperature,

Step 2. 6-Chloro-1H-indole-2-carboxylic acid (16). A mixture then poured into brine (50 mL). The resulting mixture was

of 6-chloro-1-(phenylsulfonyl)-1H-indole-2-carboxylic acid 15 extracted with AcOEt (50 mL ꢁ 3). The combined extracts were

(35.83 g, 106.7 mmol), 2 N NaOH (500 mL, 1.00 mol) and EtOH washed with brine (50 mL), dried over anhydrous MgSO₄, filtered

(500 mL) was warmed up to reflux conditions using an oil bath, and concentrated in vacuo. The residue was purified by flash

and stirred under N₂for 11 h. The resulting solution was column chromatography (silica gel, hexane/AcOEt ¼ 3:1) to

concentrated in vacuo to a half-volume. The resulting white afford 1.273 g of the title product 20a in 76% yield as a yellow

1

suspension was partitioned between H₂O (750 mL) and Et₂O crystalline solid. H NMR (270 MHz, CDCl₃) ꢀ 9.15 (1 H, br s),

(750 mL) with shaking vigorously. The aqueous layer was 8.83 (1 H, d, J ¼ 8.72 Hz), 7.66–7.27 (5 H, m), 7.17 (1 H, dd,

separated, acidified with 2 N HCl (750 mL) and then extracted J ¼ 8.72 Hz, J ¼ 2.00 Hz,), 4.25–4.13 (4 H, m), 1.75 (3 H, s), 1.19

with Et₂O (1 L). The organic layer was washed with brine (6 H, t, J ¼ 7.07 Hz).

(300 mL), dried over anhydrous MgSO₄, filtered and concentrated Step 6. Diethyl [6-chloro-2-(3-fluorobenzoyl)-1H-indol-3-

in vacuo to afford 19.74 g of the title product 16 in 95% yield as a yl]malonate (21a). To a stirred solution of diethyl 2-acetoxy-

1

white crystalline solid. TLC: R_f¼ 0.17, CH₂Cl₂/MeOH ¼ 5:1. H 2-[6-chloro-2-(3-fluorobenzoyl)-1H-indol-3-yl]malonate

20a

NMR (270 MHz, DMSO-d₆) ꢀ 11.94 (1 H, s), 7.69 (1 H, d, (1.233 g, 2.517 mmol) in dry CH₂Cl₂(26.0 mL) was added TFA

J ¼ 8.56 Hz), 7.50 (1 H, m), 7.16 (1 H, m), 7.12–7.09 (1 H, m). (1.360 mL, 17.65 mmol) followed by Et₃SiH (1.610 mL,

Step 3. 6-Chloro-N-methoxy-N-methyl-1H-indole-2- 10.08 mmol) at room temperature under N₂. The reaction solution

carboxamide (17). To a stirred suspension of 6-chloro-1H- was warmed up to reflux conditions using an oil bath, and stirred

indole-2-carboxylic acid 16 (7.00 g, 35.8 mmol) in SOCl₂(Wako under N₂for 8 h. The reaction solution was cooled to 0 ^ꢀC, and

Pure Chemical Industries, Ltd., 30 mL) was added dropwise dry ice-cooled saturated aqueous NaHCO₃(40 mL) was added. The

DMF (Wako Pure Chemical Industries, Ltd., 1.0 mL) at room resulting mixture was stirred at 0 ^ꢀC for 15 min. The organic layer

temperature under N₂, with heating by handy-drier several times. was separated, and the aqueous layer was extracted with CH₂Cl₂

The resulting solution was stirred at room temperature under N₂(40 mL ꢁ 3). The combined extracts were dried over anhydrous

for 30 min, then concentrated in vacuo. The residue was dissolved MgSO₄, filtered and concentrated in vacuo. The residue was

in dry CH₂Cl₂(100 mL) at room temperature under N₂, N,O- purified by flash column chromatography (silica gel, hexane/

dimethylhydroxylamine hydrochloride (Sigma-Aldrich, 6.98 g, AcOEt ¼ 5:1) to afford 784.5 mg of the title product 21a in 72%

71.6 mmol) was added with stirring, cooled to 0 ^ꢀC_,then dry yield as a yellow viscosity oil. ¹H NMR (270 MHz, CDCl₃) ꢀ 8.91

pyridine (Wako Pure Chemical Industries, Ltd., 15.0 mL, (1 H, br s), 7.77–7.12 (7 H, m), 5.21 (1 H, s), 4.25–4.11 (4 H, m),

185 mmol) was added¹⁰⁸. After being stirred at 0 ^ꢀC under N₂1.22 (6 H, t, J ¼ 7.07 Hz).

for 2 h, the mixture was quenched with H₂O (100 mL), and then Step 7. [6-Chloro-2-(3-fluorobenzoyl)-1H-indol-3-yl]acetic

extracted with CH₂Cl₂(150 mL ꢁ 2). The combined organic acid (22a). A mixture of diethyl [6-chloro-2-(3-fluorobenzoyl)-

extracts were washed with 2 N HCl (100 mL), saturated aqueous 1H-indol-3-yl]malonate 21a (753.4 mg, 1.74 mmol) and 2 N NaOH

NaHCO₃(100 mL) and brine (100 mL), then dried over anhydrous (3.00 mL, 6.00 mmol) in EtOH (20.0 mL) was warmed up to reflux

MgSO₄, filtered and concentrated in vacuo to afford 8.20 g of the conditions using an oil bath, and stirred under N₂for 16 h.

title product 17 in 96% yield as a yellow solid. ¹H NMR (CDCl₃) The reaction solution was concentrated in vacuo, then the residue

ꢀ 9.48 (1 H, br s), 7.60 (1 H, d, J ¼ 8.6 Hz), 7.46–7.41 (1 H, m), was partitioned between 2 N HCl (40 mL) and AcOEt (40 mL)

7.22–7.18 (1 H, m), 7.11 (1 H, dd, J ¼ 8.6 Hz, J ¼ 1.8 Hz), 3.85 with acidification. The organic layer was separated, and the

(3 H, s), 3.44 (3 H, s).

aqueous layer was extracted with AcOEt (40 mL ꢁ 3). The

Step 4. (6-Chloro-1H-indol-2-yl)(3-fluorophenyl)methanone combined extracts were dried over anhydrous MgSO₄, filtered

(19a). To a stirred solution of 6-chloro-N-methoxy-N-methyl- and concentrated in vacuo. The residue was recrystallised

1H-indole-2-carboxamide 17 (1.193 g, 5.000 mmol) and 1-bromo- from AcOEt–hexane to afford 446.7 mg of the title product 22a

1

3-fluorobenzene (Wako Pure Chemical Industries, Ltd., 1.68 mL, in 77% yield as a yellow crystalline solid. Mp: 278–281 ^ꢀC. H

15.0 mmol) in anhydrous THF (40.0 mL) was added n-BuLi NMR (270 MHz, DMSO-d₆) ꢀ 11.79 (1 H, br s), 7.76–7.48 (6 H,

(1.54 M in hexane, 9.74 mL, 15.0 mmol) at ꢂ78 ^ꢀC under m), 7.13 (1 H, dd, J ¼ 8.56 Hz, J ¼ 1.97 Hz), 3.80 (2 H, s). IR

N₂^109,110. The reaction solution was stirred at the same tempera- (KBr): 3385, 1697, 1638, 1583, 1541, 1508, 1420, 1400, 1315,

ture under N₂for 2 h. The reaction solution was poured into ice- 1261, 1236 cm^ꢂ1

.

MS (EI direct) m/z: M^þ331. Anal.

cooled saturated aqueous NH₄Cl (40 mL), then extracted with (C₁₇H₁₁ClFNO₃) C, H, N.

AcOEt (40 mL ꢁ 3). The combined extracts were washed with 2 N

HCl (40 mL) followed by saturated aqueous NaHCO₃(40 mL),

then dried over anhydrous MgSO₄, filtered and concentrated

in vacuo. The residue was purified by flash column chromatog-

raphy (silica gel, hexane/AcOEt ¼ 12:1) to afford 943.0 mg of the

title product 19a in 69% yield as a yellow solid. ¹H NMR

(270 MHz, CDCl₃) ꢀ 9.28 (1 H, br s), 7.79–7.75 (1 H, m), 7.68–

7.63 (2 H, m), 7.56–7.48 (2 H, m), 7.36–7.30 (1 H, m), 7.17–7.14

(2 H, m).

For the subsections below, see ‘‘Experimental S1’’

section in the Appendix of the Supplementary Material for the

detailed procedures and obtained data for the respective

compounds:

[6-Chloro-2-(4-fluorobenzoyl)-1H-indol-3-yl]acetic acid (22b)

{6-Chloro-2-[3-(trifluoromethyl)benzoyl]-1H-indol-3-yl}

acetic acid (22c).

Step 5. Diethyl 2-acetoxy-2-[6-chloro-2-(3-fluorobenzoyl)-

1H-indol-3-yl]malonate (20a). A mixture of (6-chloro-1H-

[2-(4-Chlorobenzoyl)-5-fluoro-1H-indol-3-yl]acetic acid (29)

indol-2-yl)(3-fluorophenyl)methanone

19a

(930.9 mg,

3.401 mmol), diethyl malonate (2.58 mL, 17.0 mmol) and Step 1. 5-Fluoro-N-methoxy-N-methyl-1H-indole-2-carboxa-

AcONa (1.395 g, 17.0 mmol) in glacial AcOH (34.0 mL) was mide (24). According to the procedure described in step 3 of

6

S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

general procedure of Method B (refer ‘‘[6-Chloro-2-(3-fluoro- 0.520 mmol); that was synthesised by us at Pfizer Global

benzoyl)-1H-indol-3-yl]acetic acid (22a): general procedure of Research & Development, Nagoya Laboratories, Pfizer Japan

Method B’’ section), the title product 24 was prepared from Inc. (Aichi, Japan) according to the reported procedure by

,

5-fluoro-1H-indole-2-carboxylic acid (Sigma-Aldrich) instead Hayashi et al.^{104 1}H NMR (270 MHz, CDCl₃) ꢀ 7.64 (1 H, d,

of 6-chloro-1H-indole-2-carboxylic acid 16 in the procedure. J ¼ 8.40 Hz), 7.46–7.19 (6 H, m), 6.94–6.87 (1 H, m), 6.77 (1 H,

¹H NMR (270 MHz, CDCl₃) ꢀ 10.15 (1 H, br s), 7.39 (1 H, dd, br s), 6.13 (1 H, d, J ¼ 15.8 Hz), 3.79 (3 H, s), 2.38 (3 H, s)},

J ¼ 8.88 Hz, J ¼ 4.62 Hz), 7.32 (1 H, dd, J ¼ 9.23 Hz, 4-chlorophenacyl bromide (Tokyo Chemical Industry Co., Ltd.,

J ¼ 2.48 Hz), 7.20 (1 H, dd, J ¼ 2.13 Hz, J ¼ 0.81 Hz), 7.05 (1 H, 182.1 mg, 0.780 mmol) and anhydrous K₂CO₃(718.7 g,

ddd, J ¼ 9.23 Hz, J ¼ 9.05 Hz, J ¼ 2.48 Hz), 3.84 (3 H, m), 3.47 5.20 mmol) in dry acetone (Wako Pure Chemical Industries,

(3 H, s).

Ltd., 5.2 mL) was stirred at room temperature under N₂for 22.5 h,

Step 2. (4-Chlorophenyl)(5-fluoro-1H-indol-2-yl)methanone then 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; Wako Pure

(26). According to the procedure described in step 4 of general Chemical Industries, Ltd., 233 mL, 1.56 mmol) was added. The

procedure of Method B (refer ‘‘[6-Chloro-2-(3-fluorobenzoyl)- reaction mixture was stirred at the same temperature under N₂for

1H-indol-3-yl]acetic acid (22a): general procedure of Method B’’ 17 h, then concentrated in vacuo. The residue was partitioned

section), 474 mg of the title product 26 was prepared in 64% yield between H₂O (40 mL) and AcOEt (40 mL). The organic layer was

as a slight yellow solid from 5-fluoro-N-methoxy-N-methyl-1H- separated and the aqueous layer was extracted with AcOEt

indole-2-carboxamide 24 (600 mg, 2.70 mmol) and 1-bromo-4- (40 mL ꢁ 4). The combined extracts were dried over anhydrous

chlorobenzene (Tokyo Chemical Industry Co., Ltd., 1.55 g, MgSO₄, filtered and concentrated in vacuo. The residue was

8.10 mmol). ¹H NMR (270 MHz, CDCl₃) ꢀ 9.27 (1 H, br s), purified by flash column chromatography (silica gel, hexane/

7.94 (2 H, d, J ¼ 8.4 Hz), 7.52 (2 H, d, J ¼ 8.4 Hz), 7.45–7.40 (1 H, AcOEt ¼ 6:1) to afford 152.4 mg of the title product 43a in 85%

1

m), 7.37–7.33 (1 H, m), 7.21–7.12 (1 H, m), 7.10–7.09 (1 H, m). yield as a yellow solid. H NMR (270 MHz, CDCl₃) ꢀ 9.10 (1 H,

Step 3. Diethyl 2-acetoxy-2-[2-(4-chlorobenzoyl)-5-fluoro- br s), 7.76–7.44 (5 H, m), 7.02–6.90 (2 H, m), 3.81 (2 H, s), 3.66

1H-indol-3-yl]malonate (27). According to the procedure (3 H, s).

described in step 5 of general procedure of Method B (refer Step 2. [2-(4-Chlorobenzoyl)-6-fluoro-1H-indol-3-yl]acetic

‘‘[6-Chloro-2-(3-fluorobenzoyl)-1H-indol-3-yl]acetic acid (22a): acid (44a). To a stirred mixture of methyl [2-(4-chlorobenzoyl)-

general procedure of Method B’’ section), 500 mg of the title 6-fluoro-1H-indol-3-yl]acetate 43a (152.4 mg, 0.441 mmol) in

product 27 was prepared in 59% yield as a yellow solid from MeOH (10.0 mL)–THF (20.0 mL) was added 2 N NaOH

(4-chlorophenyl)(5-fluoro-1H-indol-2-yl)methanone 26 (474 mg, (1.10 mL, 2.20 mmol) at room temperature under N₂. The reaction

1.73 mmol). ¹H NMR (270 MHz, CDCl₃) ꢀ 9.01 (1 H, br s), mixture was warmed up to reflux conditions using an oil bath,

7.80–7.77 (2 H, m), 7.58–7.54 (1 H, m), 7.45–7.41 (2 H, m), stirred under N₂for 2.5 h, cooled to room temperature, then

7.36–7.27 (1 H, m), 7.12–7.01 (1 H, m), 4.29–4.15 (4 H, m), 1.74 concentrated in vacuo. The residue was partitioned between H₂O

(3 H, s), 1.28–1.17 (6 H, m).

(40 mL) and Et₂O (40 mL), and the organic layer was separated

Step 4. Diethyl [2-(4-chlorobenzoyl)-5-fluoro-1H-indol-3- to remove impurity. The aqueous layer was acidified by adding

yl]malonate (28). According to the procedure described in step 2 N HCl (3.0 mL), then extracted with AcOEt (40 mL ꢁ 3). The

6 of general procedure of Method B (refer ‘‘[6-Chloro-2-(3- combined extracts were dried over anhydrous MgSO₄, filtered and

fluorobenzoyl)-1H-indol-3-yl]acetic acid (22a): general proced- concentrated in vacuo. The residue was recrystallised from

ure of Method B’’ section), 361 mg of the title product 28 was AcOEt–hexane to afford 114.0 mg of the title product 44a in

prepared in 84% yield as a slight yellow solid from diethyl 2- 78% yield as a brownish-yellow solid. Mp: 214 ^ꢀC. ¹H NMR

acetoxy-2-[2-(4-chlorobenzoyl)-5-fluoro-1H-indol-3-yl]malonate

(270 MHz, DMSO-d₆) ꢀ 11.73 (1 H, brs), 7.78–7.72 (3 H, m),

27 (486 mg, 0.992 mmol). ¹H NMR (270 MHz, CDCl₃) ꢀ 8.98 (1 H, 7.66–7.63 (2 H, m), 7.17 (1 H, dd, J ¼ 9.72 Hz, J ¼ 2.30 Hz), 7.19

br s), 7.75 (2 H, ddd, J ¼ 8.72 Hz, J ¼ 2.13 Hz, J ¼ 1.97 Hz), 7.49 (1 H, ddd, J ¼ 9.72 Hz, J ¼ 8.96 Hz, J ¼ 2.48 Hz), 3.84 (2 H, s). IR

(2 H, ddd, J ¼ 8.75 Hz, J ¼ 2.16 Hz, J ¼ 1.97 Hz), 7.40 (1 H, dd, (KBr): 3335, 1699, 1618, 1605, 1587, 1531, 1425, 1327, 1267,

J ¼ 9.72 Hz, J ¼ 2.48 Hz), 7.16 (1 H, dd, J ¼ 8.88 Hz, J ¼ 4.13 Hz), 1231, 1134, 1094, 1001 cm^ꢂ1. MS (EI direct) m/z: M^þ331. Anal.

7.00 (1 H, ddd, J ¼ 8.99 Hz, J ¼ 8.88 Hz, J ¼ 2.48 Hz), 5.29 (1 H, (C₁₇H₁₁ClFNO₃) C, H, N.

s), 4.25–4.07 (4 H, m), 1.24 (6 H, t, J ¼ 7.10 Hz).

Step 5. [2-(4-Chlorobenzoyl)-5-fluoro-1H-indol-3-yl]acetic

acid (29). According to the procedure described in step 7 of

general procedure of Method B (refer ‘‘[6-Chloro-2-(3-fluoro-

benzoyl)-1H-indol-3-yl]acetic acid (22a): general procedure of

Method B’’ section), 240 mg of the title product 29 was prepared

in 87% yield as a yellow crystalline solid from diethyl [2-(4-

chlorobenzoyl)-5-fluoro-1H-indol-3-yl]malonate 28 (360 mg,

For the subsections below, see ‘‘Experimental S1’’ section in

the Appendix of the Supplementary Material for the detailed

procedures and obtained data for the respective compounds:

[5-(tert-Butyl)-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid (44b)

[2-(4-Chlorobenzoyl)-6-methoxy-1H-indol-3-yl]acetic acid (44c)

[2-(4-Chlorobenzoyl)-5-methoxy-1H-indol-3-yl]acetic acid (44d)

[2-(4-Chlorobenzoyl)-5-(trifluoromethoxy)-1H-indol-3-yl]acetic

acid (44e)

1

0.834 mmol). Mp: 233–238 ^ꢀC. H NMR (270 MHz, DMSO-d₆)

ꢀ 12.21 (1 H, br s), 11.73 (1 H, br s), 7.79–7.74 (2 H, m), 7.67–

7.63 (2 H, m), 7.52–7.44 (2 H, m), 7.22–7.19 (1 H, ddd,

J ¼ 9.23 Hz, J ¼ 9.05 Hz, J ¼ 2.48 Hz), 3.84 (2 H, s). IR (KBr):

3317, 1707, 1624, 1609, 1587, 1526, 1458, 1408, 1344, 1263,

1242 cm^ꢂ1. MS (EI direct) m/z: M^þ331. Anal. (C₁₇H₁₁ClFNO₃)

C, H, N.

[6-Chloro-2-(4-chloro-3-fluorobenzoyl)-1H-indol-3-yl]acetic acid

(44f)

Step 1. 1-(4-Chloro-3-fluorophenyl)ethanone (34). A solution

of n-BuLi (Kanto Chemical Co., Inc., 1.55 M in hexane, 6.77 mL,

10.5 mmol) was added to a stirring solution of 1-bromo-4-chloro-

3-fluorobenzene (Fluorochem Ltd., Hadfield, Derbyshire, UK,

2.09 g, 9.98 mmol) in anhydrous Et₂O (12.0 mL) at ꢂ78 ^ꢀC under

[2-(4-Chlorobenzoyl)-6-fluoro-1H-indol-3-yl]acetic acid (44a):

general procedure of Method C

Step 1. Methyl [2-(4-chlorobenzoyl)-6-fluoro-1H-indol-3- N₂over 5 min, warmed to ꢂ20 ^ꢀC, then stirred for 45 min. To the

yl]acetate (43a). A mixture of methyl (2 E)-3-(4-fluoro-2-{[(4- above solution, a solution of dry N,N-dimethylacetamide (DMA;

methylphenyl)sulfonyl]amino}phenyl)acrylate 30a {(181.7 mg, Wako Pure Chemical Industries, Ltd., 1.04 mL, 11.2 mmol) in

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors

7

anhydrous Et₂O (1.5 mL) was added at the same temperature J ¼ 8.40 Hz, J ¼ 1.81 Hz), 7.46 (1 H, d, J ¼ 1.81 Hz), 7.12 (1 H,

under N₂over 5 min. The reaction solution was stirred at the dd, J ¼ 8.56 Hz, J ¼ l.81 Hz), 3.83 (2 H, s). MS (EI direct) m/z:

temperature for 1 h, allowed to room temperature, and stirred for M^þ365. Anal. (C₁₇H₁₀Cl₂FNO₃) C, H, N.

3 h. The reaction solution was poured into ice-cooled saturated

aqueous NH₄C1 (20 mL), then extracted with Et₂O (30 mL ꢁ 3).

(6-Chloro-2-{4-[(methylsulfonyl)amino]benzoyl}-1H-indol-3-yl)

The organic layers were combined, washed with 2 N HCl (20 mL),

acetic acid (44g)

saturated aqueous NaHCO₃(20 mL) and brine (20 mL), then dried

over anhydrous MgSO₄, filtered and concentrated in vacuo to Step 1. N-(4-Acetylphenyl)methanesulfonamide (37). To a

afford 1.6203 g of the title product 34 as a yellow oil in 94% yield stirred solution of 4⁰-aminoacetophenone (Wako Pure Chemical

(that was calculated with the gross yields containing a small Industries, Ltd., 1.3517 g, 10.0 mmol) in dry CH₂Cl₂(60.0 mL)

amount of impurity). This compound was used for the next was added dry pyridine (34.0 mL) at room temperature under N₂.

step without further purification. ¹H NMR (270 MHz, CDCl₃) The solution was cooled to 0 ^ꢀC, then methanesulfonyl chloride

ꢀ 7.74–7.66 (2 H, m), 7.53–7.47 (1 H, m), 2.59 (3 H, s).

(Wako Pure Chemical Industries, Ltd., 0.851 mL, 11.0 mmol) was

Step 2. 2-Bromo-1-(4-chloro-3-fluorophenyl)ethanone (35). added dropwise under N₂. The reaction mixture was allowed to

A mixture of 1-(4-chloro-3-fluorophenyl)ethanone 34 (1.0355 g, room temperature, and stirred for 23 h. The volatiles were then

6.00 mmol, estimated as 100% purity) and tetrabutylammonium removed in vacuo, azeotroping with H₂O. The residue was

tribromide (Tokyo Chemical Industry Co., Ltd., 3.182 g, partitioned between 1 N HCl (Wako Pure Chemical Industries,

6.60 mmol) in dry CH₂Cl₂(18.0 mL)–dry MeOH (9.0 mL) was Ltd., 100 mL) and AcOEt (100 mL). The organic layer was

stirred at room temperature under N₂for 19 h^111,112. The reaction separated, and the aqueous layer was extracted with AcOEt

solution was poured into H₂O (20 mL), then extracted with Et₂O (100 mL ꢁ 2). The combined extracts were washed with brine,

(30 mL ꢁ 2). The combined extracts were washed with H₂O dried over anhydrous MgSO₄, filtered and concentrated in vacuo

1

(30 mL ꢁ 2), dried over anhydrous MgSO₄, filtered and concen- to afford 2.14 g of the title product 37 in 100% yield. H NMR

trated in vacuo. The residue was washed with iso-Pr₂O (Wako (270 MHz, CDCl₃) ꢀ 7.97 (2 H, d, J ¼ 8.75 Hz), 7.26 (2 H, d,

Pure Chemical Industries, Ltd.) and AcOEt to afford 224.0 mg of J ¼ 8.75 Hz), 6.75 (1 H, br s), 3.10 (3 H, s), 2.59 (3 H, s).

the title product 35 in 15% yield as a colourless crystalline solid. Step 2. N-[4-(2-Bromoacetyl)phenyl]methanesulfonamide (38).

¹H NMR (270 MHz, CDCl₃) ꢀ 7.80–7.71 (2 H, m), 7.55 (1 H, dd,

J ¼ 8.24 Hz, J ¼ 6.94 Hz), 4.38 (2 H, s).

A

(1.0663 g, 5.00 mmol) and tetrabutylammonium tribromide

Step 3. Methyl [6-chloro-2-(4-chloro-3-fluorobenzoyl)- (2.652 g, 5.50 mmol) in dry CH₂Cl₂(15.0 mL)–dry MeOH

1H-indol-3-yl]acetate (43f). A mixture of methyl 3-{4-chloro- (7.5 mL) was stirred at room temperature under N₂for 19 h.

mixture of N-(4-acetylphenyl)methanesulfonamide 37

2-[(phenylsulfonyl)amino]phenyl}acrylate

30f

{279.8 mg, The reaction solution was poured into H₂O (15 mL), then

0.795 mmol; that was synthesised by us at Pfizer Global extracted with Et₂O (40 mL ꢁ 2). The combined extracts were

Research & Development, Nagoya Laboratories, Pfizer Japan washed with H₂O (30 mL ꢁ 2), dried over anhydrous MgSO₄,

Inc. according to the reported procedure by Hayashi et al.²⁵, filtered and concentrated in vacuo to afford 873.8 mg of the title

1

¹H NMR (270 MHz, CDCl₃) ꢀ 7.75–7.72 (2 H, m), 7.58–7.36 product 38 in 60% yield as a white solid. H NMR (270 MHz,

(6 H, m), 7.20 (1 H, dd, J ¼ 8.56 Hz, J ¼ 2.13 Hz), 7.14 (1 H, br s), CDCl₃) ꢀ 8.01 (2 H, d, J ¼ 8.72 Hz), 7.28 (2 H, d, J ¼ 8.72 Hz),

6.15 (1 H, d, J ¼ 15.8 Hz), 3.78 (3 H, s)}, 2-bromo-1-(4-chloro-3- 6.60 (1 H, br s), 4.40 (2 H, s), 3.13 (3 H, s).

fluorophenyl)ethanone 35 (220.0 mg, 0.875 mmol) and anhydrous Step 3. Methyl (6-chloro-2-{4-[(methylsulfonyl)amino]ben-

K₂CO₃(329.8 mg, 2.39 mmol) in dry acetone (6.0 mL) was stirred zoyl}-1H-indol-3-yl)acetate (43g). A mixture of methyl 3-{4-

at room temperature under N₂for 16 h, then DBU (357 mL, chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate 30f (351.8 mg,

2.39 mmol) was added. The reaction mixture was stirred at the 1.00 mmol), N-[4-(2-bromoacetyl)phenyl]methanesulfonamide 38

same temperature under N₂for 11 h, then cooled to 0 ^ꢀC. The (350.6 mg, 1.20 mmol) and anhydrous K₂CO₃(414.6 mg,

reaction mixture was poured into ice-cooled 2 N HCl (20 mL), 3.00 mmol) in dry acetone (7.0 mL) was stirred at room tempera-

then the resulting mixture was extracted with AcOEt (20 mL ꢁ 3). ture under N₂for 4 h, then DBU (449 mL, 3.00 mmol) was added.

The organic layers were combined, washed with 2 N HCl The reaction mixture was stirred at the same temperature under

(20 mL ꢁ 2), saturated aqueous NaHCO₃(20 mL) and brine N₂for 24 h. The reaction mixture was diluted with AcOEt

(20 mL), then dried over anhydrous MgSO₄, filtered and (30 mL), washed with 2 N HCl (20 mL ꢁ 2), saturated aqueous

concentrated in vacuo. The residue was purified by flash NaHCO₃(20 mL ꢁ 2) and brine (20 mL ꢁ 2), then dried over

column chromatography (silica gel, hexane/AcOEt ¼ 6:1) fol- anhydrous MgSO₄, filtered and concentrated in vacuo. The

lowed by recrystallisation from 2-propanol (Wako Pure Chemical residue was purified by flash column chromatography (silica gel,

Industries, Ltd.)–hexane to afford 150.5 mg of the title product hexane/AcOEt ¼ 2:1 to 1:1) to afford 182.0 mg of the title product

1

43f in 50% yield as a yellow solid. H NMR (270 MHz, CDCl₃) 43g in 43% yield as a yellow solid. ¹H NMR (270 MHz, CDCl₃) ꢀ

ꢀ 8.87 (1 H, br s), 7.66–7.35 (5 H, m), 7.25–7.12 (1 H, m), 3.80 8.86 (1 H, br s), 7.82 (2 H, d, J ¼ 8.72 Hz), 7.58 (1 H, d,

(2 H, s), 3.68 (3 H, s).

Step 4. [6-Chloro-2-(4-chloro-3-fluorobenzoyl)-1H-indol- J ¼ 8.72 Hz), 7.17 (1 H, dd, J ¼ 8.72 Hz, J ¼ 1.81 Hz), 6.80 (1 H,

J ¼ 8.72 Hz), 7.42 (1 H, d, J ¼ 1.49 Hz), 7.30 (2 H, d,

3-yl]acetic acid (44f). A mixture of methyl [6-chloro-2-(4- br s), 3.83 (2 H, s), 3.67 (3 H, s), 3.14 (3 H, s).

chloro-3-fluorobenzoyl)-1H-indol-3-yl]acetate 43f (150.5 mg, Step

0.396 mmol) and 2 N NaOH (1.00 mL, 2.00 mmol) in EtOH 1H-indol-3-yl)acetic acid (44g).

4.

(6-Chloro-2-{4-[(methylsulfonyl)amino]benzoyl}-

mixture of methyl

A

(7.0 mL) was warmed up to reflux conditions using an oil bath, (6-chloro-2-{4-[(methylsulfonyl)amino]benzoyl}-1H-indol-3-yl)

and stirred under N₂for 1.5 h. The reaction mixture was cooled to acetate 43g (174.7 mg, 415 mmol) and 2 N NaOH (1.00 mL,

room temperature, 2 N HCl (1.0 mL) was added, and the resulting 2.00 mmol) in EtOH (7.0 mL) was warmed up to reflux conditions

mixture was concentrated in vacuo. The residue was dissolved in using an oil bath, stirred under N₂for 20 h, cooled to room

AcOEt, dried over anhydrous MgSO₄, filtered and concentrated temperature and concentrated in vacuo. The residue was parti-

in vacuo. The residue was recrystallised from AcOEt–hexane to tioned between H₂O (30 mL) and Et₂O (30 mL), then the organic

afford 126.5 mg of the title product 44f in 87% yield as a yellow layer was separated to remove impurity. The aqueous layer

1

solid. Mp: 179–182 ^ꢀC. H NMR (270 MHz, DMSO-d₆) ꢀ 12.28 was further washed with Et₂O (30 mL ꢁ 3), acidified by adding

(1 H, br s), 11.79 (1 H, br s), 7.83–7.71 (3 H, m), 7.58 (1 H, dd, 2 N HCl (30 mL), then extracted with AcOEt (30 mL ꢁ 4).

8

S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

The combined extracts were dried over anhydrous MgSO₄, The residue was recrystallised from AcOEt–hexane to afford

filtered and concentrated in vacuo. The residue was recrystallised 61.0 mg of the title product 44h in 65% yield as a pale-yellow

1

from AcOEt–hexane to afford 124.0 mg of the title product 44g solid. Mp: 233 ^ꢀC. H NMR (270 MHz, DMSO-d₆) ꢀ 11.71 (1 H,

in 73% yield as a yellow solid. Mp: 207–210 ^ꢀC. ¹H NMR br s), 7.68 (1 H, d, J ¼ 8.75 Hz), 7.46 (1 H, d, J ¼ 1.65 Hz), 7.29

(270 MHz, DMSO-d₆) ꢀ 11.72 (1 H, br s), 7.75 (2 H, d, (1 H, dd, J ¼ 8.40 Hz, J ¼ 2.00 Hz), 7.24 (1 H, d, J ¼ 1.97 Hz),

J ¼ 8.88 Hz), 7.70 (1 H, d, J ¼ 8.56 Hz), 7.45 (1 H, J ¼ 1.65 Hz), 7.11 (1 H, dd, J ¼ 8.72 Hz, J ¼ 1.97 Hz), 7.02 (1 H, d,

7.33 (1 H, J ¼ 8.72 Hz), 7.11 (1 H, dd, J ¼ 8.72 Hz, J ¼ 1.65 Hz), J ¼ 8.40 Hz), 4.36–4.29 (4 H, m), 3.80 (2 H, s). IR (KBr): 3321,

3.80 (2 H, s), 3.12 (3 H, s). IR (KBr): 3333, 3248, 1715, 1603, 1707, 1612, 1568, 1429, 1317, 1288, 1263, 1225, 1119, 1065,

1570, 1529, 1508, 1394, 1323, 1259, 1231, 1159, 1061 cm^ꢂ1. MS 1005, 922, 895 cm^ꢂ1. MS (EI direct) m/z: M^þ371. Anal.

(EI direct) m/z: M^þ406. Anal. (C₁₈H₁₅ClN₂O₅S) C, H, N.

(C₁₉H₁₄ClNO₅) C, H, N.

{2-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carbonyl)-5-

(trifluoromethyl)-1H-indol-3-yl}acetic acid (44i)

{6-Chloro-2-(2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-

1H-indol-3-yl}acetic acid (44h)

Step

carbonyl)-5-(trifluoromethyl)-1H-indol-3-yl}acetate

mixture of methyl (2 E)-3-{2-[(phenylsulfonyl)amino]-5-

1.

Methyl {2-(2,3-dihydrobenzo[b][1,4]dioxine-6-

Step 1. 2-Bromo-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)etha-

none (40). To a stirred solution of 1-(2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)ethanone (Sigma-Aldrich, 2.138 g, 12.00 mmol) in dry

CHCl₃(Wako Pure Chemical Industries, Ltd., 10.0 mL) was

added 25% HBr/AcOH (Wako Pure Chemical Industries, Ltd.,

25.0 mL) at room temperature under N₂. The reaction mixture was

cooled to 0 ^ꢀC, then a solution of bromine (Wako Pure Chemical

Industries, Ltd., 0.68 mL, 13.2 mmol) in glacial AcOH (5.4 mL)

was added dropwise at 0 ^ꢀC under N₂over 15 min. The reaction

mixture was allowed to room temperature, stirred under N₂for

1.5 h and concentrated in vacuo. The residue was diluted with Et₂O

(80 mL), then basified by adding saturated aqueous 2 N NaOH. The

ethereal layer was separated and the aqueous layer was extracted

with Et₂O (50 mL ꢁ 2). The ethereal layers were combined, dried

over anhydrous MgSO₄, filtered and concentrated in vacuo to

afford 3.42 g of the title product 40 as a pale-green solid (crude). ¹H

NMR (270 MHz, CDCl₃) ꢀ 7.64–7.51 (2 H, m), 6.96–6.92 (1 H, m),

4.37 (2 H, s), 4.36–4.28 (4 H, m).

(43i).

A

(trifluoromethyl)phenyl}acrylate 30g {536.6 mg, 1.39 mmol; that

was synthesised by us at Pfizer Global Research & Development,

Nagoya Laboratories, Pfizer Japan Inc. according to the reported

procedure by Hayashi et al.²⁵, ¹H NMR (CDCl₃, 270 MHz) ꢀ

7.79–7.75 (2 H, m), 7.66–7.54 (5 H, m), 7.50–7.44 (2 H, m), 7.18

(1 H, br s), 6.26 (1 H, d, J ¼ 15.8 Hz), 3.81 (3 H, s)}, bromo-1-

(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanone

40

(1.548 g,

6.00 mmol) and anhydrous K₂CO₃(2.948 g, 21.3 mmol) in dry

acetone (20.0 mL) was stirred at room temperature under N₂for

11 h, then DBU (718 mL, 4.80 mmol) was added. The reaction

mixture was stirred at the same temperature under N₂for 14 h,

then concentrated in vacuo. The residue was partitioned between

H₂O (30 mL) and AcOEt (40 mL). The organic layer was

separated, and the aqueous layer was extracted with AcOEt

(30 mL ꢁ 3). The organic layers were combined, dried over

anhydrous MgSO₄, filtered and concentrated in vacuo. The

residue was purified by flash column chromatography (silica gel,

hexane/AcOEt ¼ 2:1) to afford 119.3 mg of the title product 43i in

20% yield as a yellowish-white solid. ¹H NMR (270 MHz, CDCl₃)

ꢀ 9.19 (1 H, br s), 7.95 (1 H, m), 7.58–7.47 (2 H, m), 7.38 (1 H, s),

7.37 (1 H, dd, J ¼ 7.75 Hz, J ¼ 2.16 Hz), 6.98–6.95 (1 H, m), 4.37–

4.29 (4 H, m), 3.91 (2 H, s), 3.70 (3 H, s).

Step 2. {2-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carbonyl)-5-

(trifluoromethyl)-1H-indol-3-yl}acetic acid (44i). To a stirred

solution of methyl {2-(2,3-dihydrobenzo[b][1,4]dioxine-6-car-

bonyl)-5-(trifluoromethyl)-1H-indol-3-yl}acetate 43i (119.3 mg,

0.284 mmol) in MeOH (7.0 mL)–THF (7.0 mL) was added 2 N

NaOH (0.700 mL, 1.40 mmol) at room temperature under N₂. The

reaction mixture was warmed up to reflux conditions using an oil

bath, stirred under N₂for 5 h, cooled to room temperature and

concentrated in vacuo. The residue was partitioned between H₂O

(30 mL) and Et₂O (30 mL), and the organic layer was separated to

remove impurity. The aqueous layer was further washed with

Et₂O (30.0 mL), acidified by adding 2 N HCl and then extracted

with AcOEt (40 mL ꢁ 3). The combined extracts were dried

over anhydrous MgSO₄, filtered and concentrated in vacuo. The

residue was recrystallised from AcOEt–hexane to afford 90.4 mg

of the title product 44i in 79% yield as a pale-yellow solid. Mp:

4250 ^ꢀC. ¹H NMR (270 MHz, DMSO-d₆) ꢀ 12.03 (1 H, br s), 8.11

(1 H, s), 7.64–7.52 (2 H, m), 7.33–7.26 (2 H, m), 7.03 (1 H, d,

J ¼ 8.40 Hz), 4.37–4.29 (4 H, m), 3.88 (2 H, s). IR (KBr): 3304,

1701, 1624, 1578, 1508, 1333, 1288, 1236, 1165, 1109, 1049,

1011, 893, 820 cm^ꢂ1. MS (EI direct) m/z: M^þ405. Anal.

(C₂₀H₁₄F₃NO₅) C, H, N.

Step 2. Methyl {6-chloro-2-(2,3-dihydrobenzo[b][1,4]dioxine-

6-carbonyl)-1H-indol-3-yl}acetate (43h).

methyl 3-{4-chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate

A

mixture of

30f (211.1 mg, 0.600 mmol), bromo-1-(2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)ethanone 40 (crude, 348.5 mg) and anhydrous K₂CO₃

(1.106 g, 8.00 mmol) in dry acetone (7.0 mL) was stirred at room

temperature under N₂for 11 h, then DBU (269 mL, 1.80 mmol)

was added. The reaction mixture was stirred at the same

temperature under N₂for 14 h, and concentrated in vacuo. The

residue was partitioned between H₂O (30 mL) and AcOEt

(40 mL). The organic layer was separated, and the aqueous layer

was extracted with AcOEt (30 mL ꢁ 3). The organic layers were

combined, dried over anhydrous MgSO₄, filtered and concen-

trated in vacuo. The residue was purified by flash column

chromatography (silica gel, hexane/AcOEt ¼ 2:1) to afford

97.3 mg of the title product 43h in 42% yield as an orange-

yellowish white solid. ¹H NMR (270 MHz, CDCl₃) ꢀ 8.85 (1 H, br

s), 7.56 (1 H, d, J ¼ 8.56 Hz), 7.41–7.34 (3 H, m), 7.15 (1 H, dd,

J ¼ 8.56 Hz, J ¼ 1.81 Hz), 6.98–6.94 (1 H, m), 4.38–4.29 (4 H, m),

3.88 (2 H, s), 3.68 (3 H, s).

Step 3. {6-Chloro-2-(2,3-dihydrobenzo[b][1,4]dioxine-6-car-

bonyl)-1H-indol-3-yl}acetic acid (44h). To a stirred solution of

methyl

{6-chloro-2-(2,3-dihydrobenzo[b][1,4]dioxine-6-car-

bonyl)-1H-indol-3-yl}acetate 43h (97.3 mg, 0.252 mmol) in

MeOH (5.0 mL)–THF (5.0 mL) was added 2 N NaOH

(0.600 mL, 1.20 mmol) at room temperature under N₂. The

reaction mixture was warmed up to reflux conditions using an

oil bath, stirred under N₂for 6 h, cooled to room temperature and

concentrated in vacuo. The residue was partitioned between H₂O

(20 mL)–Et₂O (20 mL), and the organic layer was separated to

remove impurity. The aqueous layer was further washed with

Et₂O (20 mL), acidified by adding 2 N HCl, and then extracted

[6-Chloro-2-(2,3-dihydrobenzofuran-5-carbonyl)-1H-indol-3-

yl]acetic acid (44j)

with AcOEt (30 mL ꢁ 3). The combined extracts were dried Step 1. 2-Bromo-1-(2,3-dihydrobenzofuran-5-yl)ethanone (42).

over anhydrous MgSO₄, filtered and concentrated in vacuo. To a stirred solution of 1-(2,3-dihydrobenzofuran-5-yl)ethanone

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors

9

(Lancaster Synthesis, 486.6 mg, 3.00 mmol) in dry CHCl₃(15.0 mL)–dry MeOH (7.5 mL) was stirred at room temperature

(6.0 mL) was added 25% HBr/AcOH (6.3 mL) at room tempera- under N₂for 30 h, then concentrated in vacuo. The residue was

ture under N₂. The reaction mixture was cooled to 0 ^ꢀC, then a partitioned between H₂O (30 mL) and Et₂O (30 mL). The ethereal

solution of bromine (155 mL, 3.01 mmol) in glacial AcOH layer was separated, and the aqueous layer was extracted with

(1.4 mL) was added dropwise at 0 ^ꢀC under N₂over 10 min. Et₂O (30 mL). The ethereal layers were combined, washed with

The reaction mixture was allowed to room temperature, stirred H₂O (30 mL ꢁ 2), dried over anhydrous MgSO₄, filtered and

under N₂for 1.5 h and concentrated in vacuo. The residue was concentrated in vacuo to afford 1.608 g of 2-bromo-1-cyclobuty-

diluted with Et₂O (30 mL), then basified by adding saturated lethanone 46a as an oil (crude). TLC: R_f¼ 0.65, AcOEt/

aqueous 2 N NaOH. The ethereal layer was separated and the hexane ¼ 1:2.

aqueous layer was extracted with Et₂O (30 mL ꢁ 4). The ethereal

A mixture of methyl 3-{4-chloro-2-[(phenylsulfonyl)amino]-

layers were combined, dried over anhydrous MgSO₄, filtered phenyl}acrylate 30f (492.5 mg, 1.40 mmol), 2-bromo-1-cyclobu-

and concentrated in vacuo to afford 1.108 g of the title product 42 tylethanone 46a (crude, 424.9 mg) and anhydrous K₂CO₃

1

as a green oil (crude). H NMR (270 MHz, CDCl₃) ꢀ 7.98–6.81 (580.5 mg, 4.20 mmol) in dry acetone (12.0 mL) was stirred at

(3 H, m), 4.73–4.67 (2 H, m), 4.39 (2 H, s), 3.32–3.25 (2 H, m).

Step 2. Methyl [6-chloro-2-(2,3-dihydrobenzofuran-5- 4.20 mmol) was added. The reaction mixture was stirred under

room temperature under N₂for 6 h, then DBU (628 mL,

carbonyl)-1H-indol-3-yl]acetate (43j). A mixture of methyl N₂at the same temperature under N₂for 24 h, then cooled to 0 ^ꢀC.

3-{4-chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate

30f The reaction mixture was partitioned between AcOEt (30 mL) and

(361.6 mg, 1.03 mmol), 2-bromo-1-(2,3-dihydrobenzofuran-5- 2 N HCl (30 mL). The organic layer was separated and the

yl)ethanone 42 (crude, 1.108 g) and anhydrous K₂CO₃(4.15 g, aqueous layer was extracted with AcOEt (30 mL). The organic

30.0 mmol) in dry acetone (20.0 mL) was stirred at room layers were combined, washed with 2 N HCl (30 mL ꢁ 2),

temperature under N₂for 20 h, then DBU (1.00 mL, 6.69 mmol) saturated aqueous NaHCO₃(30 mL) and brine (30 mL), then

was added. The reaction mixture was stirred at the same dried over anhydrous MgSO₄, filtered and concentrated in vacuo

temperature under N₂for 22 h, then concentrated in vacuo. The to afford 440.7 mg of the title product 47a as a yellow solid

residue was partitioned between H₂O (50 mL) and AcOEt (crude). ¹H NMR (270 MHz, CDCl₃) ꢀ 9.35 (1 H, br s), 7.53 (1 H,

(50 mL). The organic layer was separated, and the aqueous layer d, J ¼ 8.56 Hz), 7.19 (1 H, d, J ¼ 1.81 Hz), 7.07 (1 H, dd,

was extracted with AcOEt (50 mL ꢁ 3). The organic layers were J ¼ 8.56 Hz, J ¼ 1.81 Hz), 4.10 (2 H, s), 3.77 (3 H, s), 3.77–3.71

combined, dried over anhydrous MgSO₄, filtered and concen- (1 H, m), 2.44–1.86 (6 H, m).

trated in vacuo. The residue was purified by flash column Step 2. 2-(6-Chloro-2-cyclobutanecarbonyl-1H-indol-3-yl)

chromatography (silica gel, hexane/AcOEt ¼ 7:3) to afford acetic acid (48a). A mixture of methyl (6-chloro-2-cyclobutane-

154.1 mg of the title product 43j in 40% yield as a pale-yellow carbonyl-1H-indol-3-yl)acetate 47a (crude, 440.7 mg) and 2 N

1

solid. H NMR (270 MHz, CDCl₃) ꢀ 9.03 (1 H, br s), 7.73–7.64 NaOH (3.50 mL, 7.00 mmol) in EtOH (24.5 mL) was warmed up

(2 H, m), 7.55 (1 H, d, J ¼ 8.72 Hz), 7.39 (1 H, d, J ¼ 1.65 Hz), to reflux conditions using an oil bath, and stirred under N₂for

7.13 (1 H, dd, J ¼ 8.72 Hz, J ¼ 1.81 Hz), 6.84 (1 H, d, 20 h. The reaction mixture was cooled to room temperature, and

J ¼ 8.40 Hz), 4.69 (2 H, t, J ¼ 8.72 Hz), 3.85 (2 H, s), 3.67 (3 H, concentrated in vacuo. The residue was partitioned between H₂O

s), 3.26 (2 H, t, J ¼ 8.72 Hz).

(40 mL) and Et₂O (40 mL), then the organic layer was separated

Step 3. [6-Chloro-2-(2,3-dihydrobenzofuran-5-carbonyl)- to remove impurity. The aqueous layer was further washed

1H-indol-3-yl]acetic acid (44j). To a stirred solution of with Et₂O (40 mL ꢁ 3), acidified by adding 2 N HCl (30 mL) and

methyl

[6-chloro-2-(2,3-dihydrobenzofuran-5-carbonyl)-1H- then extracted with AcOEt (40 mL ꢁ 3). The combined extracts

indol-3-yl]acetate 43j (154.1 mg, 0.417 mmol) in MeOH were dried over anhydrous MgSO₄, filtered and concentrated

(10.0 mL)–THF (10.0 mL) was added 2 N NaOH (1.00 mL, in vacuo. The residue was recrystallised from AcOEt–hexane

2.00 mmol) at room temperature under N₂. The reaction mixture to afford 221.4 mg of the title product 48a in 54% yield

was warmed up to reflux conditions using an oil bath, stirred (two steps from compound 30f) as a yellow solid. Mp: 225–

under N₂for 5 h, cooled to room temperature and concentrated 228 ^ꢀC. ¹H NMR (270 MHz, DMSO-d₆) ꢀ 12.26 (1 H, br s), 11.63

in vacuo. The residue was partitioned between H₂O (40 mL) and (1 H, br s), 7.71 (1 H, d, J ¼ 8.72 Hz), 7.46 (1 H, d, J ¼ 1.97 Hz),

Et₂O (40 mL), and the organic layer was separated to remove 7.09 (1 H, dd, J ¼ 8.72 Hz, J ¼ 1.97 Hz), 4.04 (2 H, s), 4.07–3.95

impurity. The aqueous layer was further washed with Et₂O (1 H, m), 2.30–1.78 (6 H, m). IR (KBr): 3303, 2954, 1705,

(40 mL ꢁ 3), acidified by adding 2 N HCl and then extracted with 1632, 1564, 1529, 1437, 1412, 1335, 1242, 1213, 1188,

AcOEt (50 mL ꢁ 3). The combined extracts were dried over 1157, 1056, 1024 cm^ꢂ1. MS (EI direct) m/z: M^þ291. Anal.

anhydrous MgSO₄, filtered and concentrated in vacuo. The (C₁₅H₁₄ClNO₃) C, H, N.

residue was recrystallised from AcOEt–hexane to afford 35.6 mg

of the title product 44j in 24% yield as a yellow solid. Mp: 217 ^ꢀC.

(6-Chloro-2-cyclopropanecarbonyl-1H-indol-3-yl)acetic

¹H NMR (270 MHz, DMSO-d₆) ꢀ 11.73 (1 H, br s), 7.70–7.59

acid (48b)

(3 H, m), 7.47–7.46 (1 H, m), 7.12 (1 H, dd, J ¼ 8.59 Hz,

Step 1. Methyl (6-chloro-2-cyclopropanecarbonyl-1H-indol-

J ¼ 1.81 Hz), 6.92 (1 H, d, J ¼ 8.24 Hz), 4.67 (2 H, t,

3-yl)acetate (47b). A mixture of cyclopropyl methyl ketone

J ¼ 8.72 Hz), 3.78 (2 H, s), 3.26 (2 H, t, J ¼ 8.72 Hz). IR (KBr):

(Tokyo Chemical Industry Co., Ltd., 1.20 mL, 12.1 mmol) and

tetrabutylammonium tribromide (6.365 g, 13.2 mmol) in dry

CH₂Cl₂(18.0 mL)–dry MeOH (9.0 mL) was stirred at room

temperature under N₂for 30 h, then concentrated in vacuo. The

residue was partitioned between H₂O (30 mL) and Et₂O (30 mL),

then the ethereal layer was separated. The aqueous layer was

extracted with Et₂O (30 mL). The ethereal layers were combined,

washed with H₂O (30 mL ꢁ 2), dried over anhydrous MgSO₄,

filtered and concentrated in vacuo to afford 2.879 g of 2-bromo-1-

cyclopropylethanone 46b as an oil (crude). TLC: R_f¼ 0.57,

AcOEt/hexane ¼ 1:2.

3368, 1699, 1618, 1605, 1578, 1570, 1541, 1329, 1265, 1250,

1097, 1059, 939 cm^ꢂ1. MS (EI direct) m/z: M^þ355. Anal.

(C₁₉H₁₄ClNO₄) C, H, N.

2-(6-Chloro-2-cyclobutanecarbonyl-1H-indol-3-yl)acetic

acid (48a)

Step 1. Methyl (6-chloro-2-cyclobutanecarbonyl-1H-indol-3-

yl)acetate (47a). A mixture of cyclobutyl methyl ketone (Tokyo

Chemical Industry Co., Ltd., 1.09 mL, 10.0 mmol) and tetra-

butylammonium tribromide (5.30 g, 11.0 mmol) in dry CH₂Cl₂

10 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

A mixture of methyl 3-{4-chloro-2-[(phenylsulfonyl)amino]- In vitro studies

phenyl}acrylate 30f (492.5 mg, 1.40 mmol), 2-bromo-1-cyclopro-

Human cell-based COX-1 assay. Platelets were prepared from

human peripheral blood obtained from healthy adult volunteers

under informed consent. Fresh blood was collected into

vacutainers (Becton Dickinson, Franklin Lakes, NJ) containing

1/10 volume of anticoagulant solution (3.8% sodium citrate), and

centrifuged at 200 ꢁ g for 10 min. The supernatant (platelet-rich

plasma) was mixed with 50% volume of 0.14 M NaCl containing

12 mM Tris–HCl, 1.2 mM EDTA, pH 7.4. This mixture was

centrifuged at 750 ꢁ g for 10 min, and the pellet was suspended in

platelet buffer (Ca^2þ-free Hanks buffer containing 20 mM

HEPES, pH 7.4 and 0.2% BSA). After centrifugal washing with

the platelet buffer, the resulting pellet, referred to as HWPs, was

resuspended in the platelet buffer at a cell concentration of

2.85 ꢁ 10⁸cells/mL, then stored at room temperature until use.

Immediately prior to assay, 10 mL of 12.6 mM CaCl₂was added

to 70 mL HWPs suspension (2.0 ꢁ 10⁷cells/mL in a 96-well

U bottom plate). Platelets were pre-incubated in the absence or

presence of 10 mL of test compound dissolved in DMSO at

final concentrations varying 0.01–10 mM (final concentration,

less than 0.1%) for 20 min before stimulation with 10 mL of

A23187 (final concentration, 10 mM). After further 15 min

incubation at 37 ^ꢀC with A23187, the reaction was stopped by

the addition of EDTA (final concentration, 7.7 mM), and the

reaction medium was quantitated for TXB₂by a radioimmuno-

assay (RIA) kit (Amersham, Little Chalfont, Buckinghamshire,

pylethanone 46b (crude, 391.2 mg) and anhydrous K₂CO₃

(580.5 mg, 4.20 mmol) in dry acetone (12.0 mL) was stirred at

room temperature under N₂for 6 h, then DBU (628 mL,

4.20 mmol) was added. The reaction mixture was stirred under

N₂at the same temperature under N₂for 24 h. The reaction

mixture was partitioned between AcOEt (30 mL) and 2 N HCl

(30 mL). The organic layer was separated, and the aqueous layer

was extracted with AcOEt (30 mL). The organic layers were

combined, washed with 2 N HCl (30 mL ꢁ 2), saturated aqueous

NaHCO₃(30 mL) and brine (30 mL), then dried over anhydrous

MgSO₄, filtered and concentrated in vacuo to afford 384.4 mg

of the title product 47b in 94% yield (from compound 30f)

1

as an oil. H NMR (270 MHz, CDCl₃) ꢀ 9.55 (1 H, br s), 7.58

(1 H, d, J ¼ 8.56 Hz), 7.26 (1 H, d, J ¼ 1.65 Hz), 7.10 (1 H,

dd, J ¼ 8.56 Hz, J ¼ 1.65 Hz), 4.17 (2 H, s), 3.73 (3 H, s),

2.58–2.49 (1 H, m), 1.30–l.25 (2 H, m), 1.09–1.02 (2 H, m).

{Recrystallisation study: Compound 47b was recrystallised from

hexane to afford a crystalline solid. ¹H NMR spectrum of the

crystalline solid was the same as that of the above compound 47b.

MS (ESI positive) m/z: [MþH]^þ291.9. MS (ESI negative) m/z:

[M–H]^ꢂ289.9. MS (EI direct) m/z: M^þ291.10. Mp: 158.0 ^ꢀC.

Anal. (C₁₅H₁₄ClNO₃) C, H, N}.

Step 2. (6-Chloro-2-cyclopropanecarbonyl-1H-indol-3-yl)

acetic acid (48b). A mixture of methyl (6-chloro-2-cyclopropa-

necarbonyl-1H-indol-3-yl)acetate 47b (384.4 mg, 1.318 mmol)

and 2 N NaOH (3.20 mL, 6.40 mmol) in EtOH (22.4 mL) was

warmed up to reflux conditions using an oil bath, stirred under N₂

for 20 h, cooled to room temperature and concentrated in vacuo.

The residue was partitioned between H₂O (40 mL) and Et₂O

(40 mL). The organic layer was separated to remove impurity. The

aqueous layer was further washed with Et₂O (40 mL ꢁ 3),

acidified by adding 2 N HCl (30 mL) and then extracted with

AcOEt (40 mL ꢁ 3). The combined extracts were dried over

anhydrous MgSO₄, filtered and concentrated in vacuo. The

residue was recrystallised from AcOEt–hexane to afford 147.3 mg

of the title product 48b in 40% yield as a pale brownish-yellow

UK) according to the manufacturer’s procedure^25,104,113

.

Human cell-based COX-2 assay. The human cell-based COX-2

assay was carried out as previously described^25,104,114. Confluent

HUVECs (2 ꢁ 10⁴cells/well) in a 96-well plate were washed with

100 mL of RPMI-1640 containing 2% foetal calf serum (FCS), and

incubated with 300 U/mL of recombinant human IL-1b for 24 h at

37 ^ꢀC for induction of COX-2. After washing with Hanks buffer

containing 20 mM HEPES, pH 7.4 and 0.2% BSA, HUVECs were

pre-incubated in Hanks buffer containing 20 mM HEPES, pH 7.4

and 0.2% BSA, with or without 10 mL of test compound dissolved

in DMSO at final concentrations varying 1 nM to 1 mM (final

concentration, less than 0.1%) for 20 min before stimulation with

30 mM of A23187. After further 15 min incubation at 37 ^ꢀC with

A23187, the reaction medium was quantitated for 6-keto-PGF_1a,

that is, spontaneously degraded stable form of prostacyclin

(PGI₂), by a RIA kit (Amersham).

1

solid. Mp: 249–252 ^ꢀC. H NMR (270 MHz, DMSO-d₆) ꢀ 12.22

(1 H, br s), 11.98 (1 H, br s), 7.75 (1 H, d, J ¼ 8.72 Hz), 7.48 (1 H,

d, J ¼ 1.81 Hz), 7.11 (1 H, dd, J ¼ 8.72 Hz, J ¼ l.81 Hz), 4.08 (2 H,

s), 2.73 (1 H, quintet, J ¼ 6.24 Hz), 1.07 (4 H, d, J ¼ 6.24 Hz).

IR (KBr): 3304, 3013, 1709, 1624, 1566, 1443, 1414, 1387, 1340,

1286, 1248, 1217, 1200, 1157, 1057, 1045, 1022 cm^ꢂ1. MS

(EI direct) m/z: M^þ277. Anal. (C₁₄H₁₂ClNO₃) C, H, N.

In vivo studies

General. Animal experiment was conducted according to the

guideline of animal care and use, and its procedure was approved

by the Animal Ethics Committee in Pfizer Global Research &

Development, Nagoya Laboratories. The animal work was also

approved by the Pfizer Institutional Animal Care and Use

Committee (IACUC).

Biology

Materials

Human umbilical vein endothelial cells (HUVECs) were purchased

from Morinaga Institute of Biological Science (Yokohama,

Kanagawa, Japan). Recombinant human interleukin-1b (IL-1b)

was purchased from R&D Systems (Minneapolis, MN). A23187

(calcium ionophore) and indomethacin were purchased from Carrageenan-induced foot-oedema formation in rats. Specific

Sigma-Aldrich. Thromboxane B₂(TXB₂), 6-keto-prostaglandin pathogen free and virus antibody free (SPF/VAF) male Sprague–

F_1a(6-keto-PGF_1a) and prostaglandin E₂(PGE₂) were purchased Dawley (SD) rats [Crj:CD(SD), 5 weeks old, 110–130 g, Charles

from Cayman Chemical (Ann Arbor, MI). ꢁ-Carrageenan (Picnin- River Laboratories, Japan Inc., Hino, Tokyo, Japan] that were

A) was purchased from Zushikagaku (Zushi, Kanagawa, Japan). fasted overnight were injected intraplantarly with 0.1 mL of a 1%

{2-[(2-, 3- or 4-Substituted, or 2,4- or 3,4-disubstituted)-benzoyl, (w/v) ꢁ-carrageenan (Picnin-A) suspension in saline into the right

(bicyclic heterocycloalkanophenyl)carbonyl or cycloalkanecarbo- hind paw as previously reported^{25,104,115–117}. Either the vehicle

nyl]-(5- or 6-substituted)-1H-indol-3-yl}acetic acids 8a–f, 14, [5% (v/v) Tween 80 in distilled water] or a test compound was

22a–c, 29, 44a–j and 48a,b were used for pharmacological dosed orally in a volume of 1 mL/100 g body weight at 1 h before

evaluations as free acids, respectively. All other chemicals were carrageenan injection. Foot volume was measured by a water

obtained from Wako Pure Chemical Industries, Ltd. unless stated displacement plethysmometer (Unicom Co., Yachiyo, Chiba,

otherwise. All common chemicals were analytical or the highest Japan) before and 3 h after carrageenan injection. The increases

available grade.

in foot volume for 3 h were calculated. Foot oedema was

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors 11

compared with vehicle-control group, and the percent inhibition determine the percent inhibition of PGE₂production as the

was calculated taking the values in the control group as 0%.

following equation:

% Inhibition

PGE₂production in carrageenan-induced oedema site of

rats. Determination of PGE₂synthesised in the inflammatory

site was carried out essentially according to a previously

described method^25,118. Foot oedema in SPF/VAF male SD rats

[Crj:CD(SD), 5 weeks old, 110–130 g] was induced by intra-

plantar injection of a 1% (w/v) ꢁ-carrageenan suspension. The

measurement of inhibitory activity against carrageenan-induced

foot oedema for the test compound, that was administrated orally

at 1 h before carrageenan injection, was performed at 3 h after

carrageenan injection [refer ‘‘Carrageenan-induced foot-oedema

formation in rats’’ section]. And then immediately, the animals

were euthanised and sacrificed by cervical dislocation. The foot

was amputated, frozen in liquid nitrogen and stored at ꢂ80 ^ꢀC

until analysis. The frozen foot was crushed, mixed with 7 mL of

ethanol containing 10 mg/mL of indomethacin to inhibit PGE₂

production during further handling, pulverised in a Waring

blender and clarified by centrifugation at 2000 ꢁ g for 10 min at

PGE₂content_{vehicle control}ꢂ PGE₂content_{drug treatement}

¼

ꢁ 100:

PGE₂content_{vehicle control}ꢂ PGE₂content_basal

Physicochemistry

The lipophilicity values of COX-2 inhibitors were estimated as

values of ACD log D_7.4, the octanol–water distribution coefficient

for ionizable compounds at pH 7.4, calculated by ACD software,

ACD/Laboratories 9.0 (Advanced Chemistry Development, Inc.,

Ontario, Canada).

Results and discussion

Chemistry

A synthetic method for {6-chloro-2-[(2-, 3- or 4-substituted or

2,4-disubstituted)-benzoyl]-1H-indol-3-yl}acetic acid derivatives

4 ^ꢀC. PGE₂was extracted by a Sep-Pak C18 cartridge (Waters using indole derivatives and aroyl chlorides as starting materials

Corporation), and dried in vacuum. Samples were diluted to a

final volume of 0.5 mL with assay buffer [50 mM Tris/HCl,

pH 7.4 containing 0.9% NaCl, 0.01% Triton X-100 and 0.1% (phenylsulfonyl)-1H-indole 2 with benzenesulfonyl chloride,

is depicted in Scheme 1 (Method A). Thus, commercially

available 6-chloroindole 1 was converted into 6-chloro-1-

(w/v) bactogelatin] and the levels of PGE₂were determined by

RIA according to the manufacture’s direction (RIA kit,

Amersham). Either the vehicle [5% (v/v) Tween 80 in distilled

water] or the test compound was dosed orally in a volume of

1 mL/100 g body weight 1 h before carrageenan injection as

already described in ‘‘Carrageenan-induced foot-oedema forma-

then arylcarbonylated with various (2-, 3- or 4-substituted or

2,4-disubstituted)-benzoyl chlorides 3a–f via lithiation with tert-

BuLi at 2-position of the indole core, that was attached with the

2-carbanion-stabilising (phenylsulfonyl)amino-group, to afford

aryl[6-chloro-1-(phenylsulfonyl)-1H-indol-2-yl]methanones 4a–f,

respectively¹⁰⁵, followed by hydrolysis of the phenylsulfonyl

tion in rats’’ section. The mean values of compound-treated group portion to afford aryl(6-chloro-1H-indol-2-yl)methanones

were compared to that of vehicle-treated control group to

5a–f, respectively. Next, 3-position of indole core, which

Scheme 1. Synthesis of {6-chloro-2-[(2-, 3- or 4-substituted or 2,4-disubstituted)-benzoyl]-1H-indol-3-yl}acetic acids 8a–f. Reagents and conditions:

(a) n-Bu₄NHSO₄, 50% aqueous KOH, benzene, room temp to 0 ^ꢀC; then benzenesulfonyl chloride, 0 ^ꢀC to room temp; (b) tert-BuLi/pentane, THF,

ꢂ78 ^ꢀC to 0 ^ꢀC, or ꢂ78 ^ꢀC; (c) K₂CO₃/THF–MeOH–H₂O or 2 N NaOH/EtOH–THF, up to reflux; (d) diethyl malonate, AcONa, Mn(OAc)₃ꢃ 2H₂O,

AcOH, room temp to 80 ^ꢀC; (e) TFA, Et₃SiH, CH₂Cl₂, room temp to reflux; (f) 2 N NaOH, EtOH, reflux; then acidified by 2 N HCl, room temp.

12 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

was adjacent to the electron-withdrawing aroyl group on

In case 1-bromo-(3 or 4-substituted)-benzenes were available

the core, was acetoxymalonylated by oxidative radical condi- as starting materials, {6-chloro-2-[(3- or 4-substituted)-benzoyl]-

tion with diethyl malonate and Mn(III) in AcOH^106,107to 1H-indol-3-yl}acetic acid derivatives were prepared as depicted

afford diethyl 2-acetoxy-2-[2-(arylcarbonyl)-6-chloro-1H-indol- in Scheme 3 (Method B). Thus, after introduction of carboxylic

3-yl]malonates 6a–f, respectively, followed by reductive deace- acid group onto 2-position of the above compound 2 with

toxylation with Et₃SiH in TFA¹⁰⁷to give diethyl lithium diisopropylamide (LDA) and carbon dioxide (CO₂) gas,

indolylmalonates 7a–f, respectively. Finally, hydrolysis and the resulting 6-chloro-1-(phenylsulfonyl)-1H-indole-2-carboxylic

decarboxylation of the diethyl-malonate moiety^106,107afforded acid 15 was converted into 6-chloro-1H-indole-2-carboxylic

the requisite {6-chloro-2-[(2-, 3- or 4-substituted or 2,4-disub- acid 16 by hydrolysis of the phenylsulfonyl moiety, and then

stituted)-benzoyl]-1H-indol-3-yl}acetic acid derivatives 8a–f as the resulting acid was led to 6-chloro-N-methoxy-N-methyl-

sole products in good yields, respectively.

1H-indole-2-carboxamide 17 via the corresponding acid chloride.

As well, [6-chloro-2-(cyclohexanecarbonyl)-1H-indol-3-yl]a- The N-methoxy-N-methylamide moiety was substituted by (3- or

cetic acid 14 was prepared as a sole product in good yield by way 4-substituted)-benzenes with (substituted phenyl)lithium reagents

of cyclohexanecarbonylation at 2-position of compound 2 with that were prepared from 1-bromo-(3- or 4-substituted)-benzenes

cyclohexanecarbonyl chloride 9 under similar conditions as used 18a–c and n-BuLi in situ by bromo-group–metal exchange^108–110

.

for the synthesis of compounds 4a–f, and subsequent process The resulting (6-chloro-1H-indol-2-yl)[(3- or 4-substituted)-phe-

in the same way as described in method A (Scheme 2). nyl]methanones 19a–c were converted into the final products in

Scheme 2. Synthesis of [6-chloro-2-(cyclohexanecarbonyl)-1H-indol-3-yl]acetic acid 14. Reagents and conditions: (a) tert-BuLi/pentane, THF,

ꢂ78 ^ꢀC; (b) 2 N NaOH, EtOH–THF, up to reflux; (c) diethyl malonate, AcONa, Mn(OAc)₃ꢃ 2H₂O, AcOH, room temp to 80 ^ꢀC; (d) TFA, Et₃SiH,

CH₂Cl₂, room temp to reflux; (e) 2 N NaOH, EtOH, reflux; then acidified by 2 N HCl, room temp.

Scheme 3. Synthesis of {6-chloro-2-[(3- or 4-substituted)-benzoyl]-1H-indol-3-yl}acetic acids 22a–c. Reagents and conditions: (a) LDA, THF,

ꢂ78 ^ꢀC; then CO₂(gas), ꢂ78 ^ꢀC; (b) 2 N NaOH, EtOH, reflux; (c) i) SOCl₂, DMF, heating several times; then concentrated in vacuo. ii) N,O-

dimethylhydroxylamine hydrochloride, CH₂Cl₂, room temp; then pyridine, 0 ^ꢀC; (d) n-BuLi/hexane, THF, ꢂ78 ^ꢀC; (e) diethyl malonate, AcONa,

Mn(OAc)₃ꢃ 2H₂O, AcOH, room temp to 80 ^ꢀC; (f) TFA, Et₃SiH, CH₂Cl₂, room temp to reflux; (g) 2 N NaOH, EtOH, reflux; then acidified by 2 N HCl,

room temp.

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors 13

a similar way for compounds 8a–f from compounds 5a–f in phenyl}acrylates, (ii) the synthesis of 2-bromo-1-(aryl, bicyclic

Method A. Thus, radical acetoxymalonylation for the aryl(1 H- heterocycloalkanophenyl or cycloalkyl)ethanones and (iii) the

indol-2-yl)methanones was performed to afford diethyl 2-acetoxy- synthesis of the desired {2-[(4-substituted or 3,4-disubstituted)-

2-[2-(arylcarbonyl)-6-chloro-1H-indol-3-yl]malonates

20a–c, benzoyl or (bicyclic heterocycloalkanophenyl)carbonyl]-(5-

followed by reductive deacetoxylation to form respective diethyl or 6-substituted)-1H-indol-3-yl}acetic acid derivatives via

indolylmalonates 21a–c, respectively. Subsequent hydrolysis indole-skeleton construction (Method C), similar to the already-

and decarboxylation of the diethyl malonates afforded requisite described synthesis of [2-{[(4-substituted or 4,5-disubstituted)-

{6-chloro-2-[(3- or 4-substituted)-benzoyl]-1H-indol-3-yl}acetic pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-

acids 22a–c as sole products in good yields, respectively. Utilising 1H-indol-3-yl]acetic acid derivatives by Hayashi et al.^25,104

the same manner, [2-(4-chlorobenzoyl)-5-fluoro-1H-indol-3-yl]a-

cetic acid 29 was also prepared from commercially available amino]phenyl}acrylates 30a–g were synthesised via Heck–

5-fluoro-1H-indole-2-carboxylic acid 23 as shown in Scheme 4.

Mizoroki reaction¹¹⁹or via known procedure¹²⁰, respectively, as

Alternatively, {2-[(4-substituted or 3,4-disubstituted)-benzoyl described already by Hayashi et al.^25,104

First, various methyl 3-{(4- or 5-substituted)-2-[(arylsulfonyl)

or (bicyclic heterocycloalkanophenyl)carbonyl]-(5- or 6-substi-

Second, except commercially available 4-chlorophenacyl

tuted)-1H-indol-3-yl}acetic acid derivatives were prepared bromide 31, various 2-bromo-1-[(4-substituted or 3,4-disubsti-

by convergent synthesis consisted of three independent parts, tuted)phenyl or bicyclic heterocycloalkanophenyl]ethanones

that is, (i) the synthesis of methyl 3-{2-[(arylsulfonyl)amino] were synthesised as depicted in Scheme 5. In the case of

Scheme 4. Synthesis of [2-(4-chlorobenzoyl)-5-fluoro-1H-indol-3-yl]acetic acid 29. Reagents and conditions: (a): (i) SOCl₂, DMF, heating several

times; then concentrated in vacuo. (ii) N,O-dimethylhydroxylamine hydrochloride, CH₂Cl₂, room temp; then pyridine, 0 ^ꢀC; (b) n-BuLi/hexane, THF,

ꢂ78 ^ꢀC; (c) diethyl malonate, AcONa, Mn(OAc)₃ꢃ 2H₂O, AcOH, room temp to 80 ^ꢀC; (d) TFA, Et₃SiH, CH₂Cl₂, room temp to reflux; (e) 2 N NaOH,

EtOH, reflux; then acidified by 2 N HCl, room temp.

Scheme 5. Synthesis of 2-bromo-1-[(4-substituted or 3,4-disubstituted)-phenyl or bicyclic-heterocycloalkanophenyl]ethanones 35, 38, 40 and 42.

Reagents and conditions: (a) i) n-BuLi/hexane, Et₂O, ꢂ78 ^ꢀC to ꢂ20 ^ꢀC; then DMA/Et₂O, ꢂ20 ^ꢀC to room temp; (b) tetrabutylammonium tribromide,

CH₂Cl₂–MeOH; (c) methanesulfonyl chloride, pyridine, CH₂Cl₂, 0 ^ꢀC to room temp; (d) Br₂, HBr/AcOH, CHCl₃, 0 ^ꢀC to room temp.

14 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

Scheme 6. Synthesis of {2-[(4-substituted or 3,4-disubstituted)-benzoyl or (bicyclic heterocycloalkanophenyl)carbonyl]-(5- or 6-substituted)-

1H-indol-3-yl}acetic acids 44a–j. Reagents and conditions: (a) K₂CO₃, acetone; then DBU; (b) 2 N NaOH, MeOH–THF or MeOH or EtOH, reflux;

then acidified by 2 N HCl, room temp. Note: Refer text in this article and Refs. 25 and 104 by Hayashi et al. for the preparation of methyl 3-{(4- or

5-substituted)-2-[(arylsulfonyl)amino]phenyl}acrylates 30a–g.

dihydrobenzofuran-5-yl)ethanone 42 were also synthesised from

the corresponding (bicyclic heterocycloalkanophenyl)ethanones

39 and 41 by bromination with bromine in HBr/AcOH^25,104

respectively.

,

Third, the above methyl 3-{(4- or 5-substituted)-2-

[(arylsulfonyl)amino]phenyl}acrylates 30a–g and the above

bromides 31, 35, 38, 40 and 42 were converted into indoline

skeletons by Michael addition reaction with K₂CO₃, and subse-

quent one-pot dephenylsulfonation assisted-aromatisation with

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) afforded methyl

{2-[(4-substituted or 3,4-disubstituted)-benzoyl or (bicyclic het-

erocycloalkanophenyl)carbonyl]-(5- or 6-substituted)-1H-indol-3-

yl}acetates 43a–j, respectively. Then hydroxylation of the methyl

acetates gave requisite {2-[(4-substituted or 3,4-disubstituted)-

benzoyl or (bicyclic heterocycloalkanophenyl)carbonyl]-(5- or

6-substituted)-1H-indol-3-yl}acetic acid derivatives 44a–j,

respectively (Scheme 6).

Furthermore,

(6-chloro-2-cycloalkanecarbonyl-1H-indol-3-

yl)acetic acid derivatives were prepared using the above conver-

gent synthesis as depicted in Scheme 7. Thus, cyclobutyl methyl

ketone 45a and cyclopropyl methyl ketone 45b were brominated

by tetrabutylammonium tribromide to afford the corresponding

2-bromo-1-cyclobutylethanone 46a and 2-bromo-1-cyclopropy-

lethanone 46b, respectively. Subsequent Michael addition reaction

of methyl 3-{4-chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate

30f and the above bromides 46a,b with K₂CO₃, followed by

aromatisation in situ with DBU gave methyl (6-chloro-2-

cycloalkanecarbonyl-1H-indol-3-yl)acetates 47a,b, respectively,

which were hydrolysed to afford requisite (6-chloro-2-cyclobuta-

Scheme 7. Synthesis of [2-cycloalkanecarbonyl-(5- or 6-substituted)-1H-

indol-3-yl]acetic acids 48a,b. Reagents and conditions: (a) tetrabutyl-

ammonium tribromide, CH₂Cl₂–MeOH; (b) K₂CO₃, acetone; then

DBU; (c) 2 N NaOH, EtOH, reflux; then acidified by 2 N HCl, room

temp.

4-chloro-3-fluorophenyl derivative, N,N-dimethylacetamide 32 necarbonyl-1H-indol-3-yl)acetic acid 48a and (6-chloro-

was coupled with (4-chloro-3-fluorophenyl)lithium, that was 2-cyclopropanecarbonyl-1H-indol-3-yl)acetic

generated from 1-bromo-4-chloro-3-fluorobenzene 33 in situ respectively.

acid

48b,

with n-BuLi, to form 1-(4-chloro-3-fluorophenyl)ethanone 34.

The detailed procedures and obtained data for the above

For 4-[(methylsulfonyl)amino]phenyl derivative, 4⁰-aminoaceto- respective compounds are described in the ‘‘Experimental’’

phenone 36 was converted into N-(4-acetylphenyl)methanesulfo- section and in the Appendix of the Supplementary Material.

namide 37 with methanesulfonyl chloride. Then the acetyl

All of these final test compounds {2-[(2-, 3- or 4-substituted,

moieties of the above 1-arylethanones 34 and 37 were brominated or 2,4- or 3,4-disubstituted)-benzoyl, (bicyclic hetero-

with tetrabutylammonium tribromide^111,112to afford 2-bromo-1- cycloalkanophenyl)carbonyl or cycloalkanecarbonyl]-(5- or

(4-chloro-3-fluorophenyl)ethanone 35 and N-[4-(2-bromoacetyl) 6-substituted)-1H-indol-3-yl}acetic acid derivatives 8a–f, 14,

phenyl]methanesulfonamide 38, respectively. For bicyclic-hetero- 22a–c, 29, 44a–j and 48a,b were used for SAR studies with

cycloalkanophenyl derivatives, 2-bromo-1-(2,3-dihydrobenzo[b] pharmacological evaluations as free acids, respectively.

[1,4]dioxin-6-yl)ethanone

40

and

2-bromo-1-(2,3-

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors 15

Structure–activity relationships of COX-2 inhibitors

IC₅₀against COX-2 over IC₅₀against COX-1 in the human

cellular studies for the compounds. As described already²⁵, the

above 6-keto-PGF_1aproduction in HUVEC in our study condition

In vitro activities and selectivities against COX isoforms

Actually, design, synthesis and SAR of various novel {2-[(2-, 3- was established as a COX-2 induction-dependent response.

or 4-substituted, or 2,4- or 3,4-disubstituted)-benzoyl, (bicyclic In this in vitro design and SAR study using human cellular

heterocycloalkanophenyl)carbonyl or cycloalkanecarbonyl]-(5- or assays, the acid-type novel analogues gave significant findings

6-substituted)-1H-indol-3-yl}acetic acid analogues were investi- regarding potency and selectivity of their COX-2 inhibition

gated to seek and identify various chemotypes of orally potent dependent on the 5- or 6-substituent of the indole core and on the

and selective COX-2 inhibitors as potential agents for the 2-[(2-, 3- or 4-substituted, or 2,4- or 3,4-disubstituted)-benzoyl,

treatment of inflammatory diseases. (bicyclic heterocycloalkanophenyl)carbonyl or cycloalkanecarbo-

At first, the in vitro inhibitory activities and selectivities for the nyl] portion, indeed. The results of the in vitro SAR study for the

compounds against enzymatic activities of COX isozymes, analogues are summarised in Tables 1 and 2.

inducible COX-2 and constitutional COX-1, in human cells

First, substituted-phenyl group effects at 2-benzoyl portion of

were evaluated with our reported procedures^25,104. In brief, {6-chloro-2-[(2-, 3- or 4-substituted, or 2,4- or 3,4-disubstituted)-

the activities of the COX isozymes were determined (i) by 6-keto- benzoyl]-1H-indol-3-yl}acetic acid analogues were studied

prostaglandin F_1a(6-keto-PGF_1a, spontaneously degraded stable- in vitro. Thus, meta- or para-toluoyl analogues 8b and 8c,

form of PGI₂) production in human umbilical vein endothelial meta- or para-trifluoromethylphenyl analogues 22c and 8d, and

cells (HUVECs) that were expressing COX-2 induced by meta- or para-fluorophenyl analogues 22a and 22b exhibited

interleukin-1b (IL-1b) for COX-2, and (ii) by thromboxane B₂potent COX-2 inhibitions, with showing respective patterns of

(TXB₂, spontaneously degraded stable-form of TXA₂) production regioselectivities by their substituents for the inhibitory activities.

in human washed platelets (HWPs) for COX-1, respectively^113,114

.

For the selectivity indices of human COX-2 inhibitors in this on the phenyl core of the analogues gave more potent activities

Specifically, para-methyl and -trifluoromethyl groups (8c and 8d)

study in vitro, COX-2-inhibition selectivities were estimated as in vitro (i.e. the IC₅₀values were 0.192 mM and 0.209 mM for

Table 1. In vitro structureꢂactivity relationships of inhibitory effects against COX-2/COX-1 activities in human cellular assays for {2-[(2-, 3- or

4-substituted, or 2,4- or 3,4-disubstituted)-benzoyl]-(5- or 6-substituted)-1H-indol-3-yl}acetic acid analogues 8a–f, 22a–c, 29 and 44a–g.

O

HO

R¹

5

O

2

R³

2

R²

N

6

3

H

R⁴

4

R⁵

In vitro

Human cell assay

Compounds

No

COX-2^a

COX-1^b

R¹

R²

R³

R⁴

R⁵

IC₅₀(mM)

Selectivity of COX-1/COX-2 ^c

8a

8b

8c

8d

8e

8f

22a

22b

22c

29

44a

44b

44c

44d

44e

44f

44g

H

Cl

H

F

H

MeO

H

Me

H

Cl

H

Me

H

F

H

CF₃

H

F

H

Me

CF₃

H

Cl

H

F

3.31

0.248

0.192

0.209

9.54

NT^d

1.92

0.613

410.0

NT

–

7.75

3.19

447.6

–

410.0

0.229

0.228

0.340

0.264

0.0156

41.00

0.493

0.904

410.0

NT

–

1.87

–

0.431

NT

6.39

7.94

H

–

F

H

Cl

24.2

509

–

14.0

4.49

–

tert-Bu

H

MeO

F₃C-O

H

410.0

6.92

4.06

410.0

Cl

H

NHMs

^aIC₅₀values of compounds against COX-2 activity were determined as inhibitory activity against 6-keto-prostaglandin F_1asynthesis in human

umbilical vein endothelial cells that were expressing COX-2 by interleukin-1b-stimulation.

^bIC₅₀values of compounds against COX-1 activity were determined as inhibitory activity against thromboxane B₂production in human washed

platelets.

^cThe COX-2-inhibition selectivities of the compounds were calculated as the ratios of the IC₅₀values against COX-1 to the IC₅₀values against COX-2

in the above human cellular assays, respectively.

^dNT: not tested.

16 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

Table 2. In vitro structureꢂactivity relationships of inhibitory effects against COX-2/COX-1 activities in human cellular assays for

{2-[cycloalkanecarbonyl or (bicyclic heterocycloalkanophenyl)carbonyl]-(5- or 6-substituted)-1H-indol-3-yl}acetic acid analogues 14, 44h–j and

48a,b.

O

HO

R¹

5

O

2

R³

R²

N

6

H

In vitro

Human cell assay

Compounds

COX-2^a

IC₅₀(mM)

0.0730

COX-1^b

No

R¹

H

R²

Cl

R³

IC₅₀(mM)

Selectivity of COX-1/COX-2^c

15.9

14

1.16

44h

44i

44j

H

F₃C

H

Cl

H

0.148

0.389

0.0566

410.0

2.97

467.6

425.7

52.4

O

Cl

O

48a

48b

H

Cl

2.84

410.0

43.52

410.0

–

^aIC₅₀values of compounds against COX-2 activity were determined as inhibitory activity against 6-keto-prostaglandin F_1asynthesis in human

umbilical vein endothelial cells that were expressing COX-2 by interleukin-1b-stimulation.

^bIC₅₀values of compounds against COX-1 activity were determined as inhibitory activity against thromboxane B₂production in human washed

platelets.

^cThe COX-2-inhibition selectivities of the compounds were calculated as the ratios of the IC₅₀values against COX-1 to the IC₅₀values against COX-2

in the above human cellular assays, respectively.

compounds 8c and 8d, respectively) than the corresponding meta- their COX-2 inhibition over COX-1 inhibition in the human

methyl and -trifluoromethyl groups (8b and 22c) (i.e. the IC₅₀cellular assays demonstrated the significance of electron-density

values were 0.248 mM and 0.340 mM for compounds 8b and 22c, distribution and bulkiness for the substituted-phenyl portion

respectively); while meta- and para-fluoro groups gave almost the within some ranges. Notably, COX-2 selectivity of para-

same activities each other (i.e. the IC₅₀values were 0.229 mM and trifluoromethylphenyl analogue 8d was greater than 47.6-fold,

0.228 mM for compounds 22a and 22b, respectively). On the other which was considerably higher than that of meta- and para-

hand, compared with the meta- and para-toluoyl analogues 8b and toluoyl analogues 8b and 8c, meta-fluorophenyl analogue 22a,

8c, ortho-toluoyl analogue 8a reduced activity (IC₅₀¼ 3.31 mM). and meta-fluoro-para-chlorophenyl analogue 44f, that is, 7.75-,

Furthermore, meta-fluoro-para-chlorophenyl analogue 44f 3.19-, 1.87- and 4.49-fold for compounds 8b, 8c, 22a and 44f,

retained potent activity (IC₅₀¼ 0.904 mM), whereas ortho-chloro- respectively. In detail, as substituent effects on the phenyl core,

phenyl analogue 8e and ortho,para-dichlorophenyl analogue 8f (i) more electron-negative para-trifluoromethyl group (8d) was

much reduced or lacked activity. Overall, compared with the much prefer to less electron-negative (rather electron-donating)

ortho-substituent, the meta- and para-substituents were favour- para-methyl group (8c), (ii) larger meta-methyl group (8b) was

able for COX-2 inhibition. Besides, para-methanesulfonylamido- better than smaller meta-fluoro group (22a) and (iii) a pair of

phenyl analogue 44g was inactive, which would show limitation meta-fluoro and para-chloro groups (44f), whose para-group is

of the size or volume for the substituent effect at this position larger and more electron-negative, was more favourable compared

owing to the steric hindrance against binding-site of the protein. with a pair of meta-fluoro and para-hydrogen groups (22a),

Also, the selectivity study of the [6-chloro-2-(2-, 3- or whose para-group is smaller and less electron-negative,

4-substituted)-benzoyl-1H-indol-3-yl]acetic acid analogues for respectively.

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors 17

Second, substituent effects at 5- or 6-position on the indole ring-size. Indeed, the rank of the orders of COX-2 inhibitory

core of [2-(4-chlorobenzoyl)-(5- or 6-substituted)-1H-indol-3- activities for the cycloalkyl analogues in vitro was cyclohexyl

yl]acetic acid analogues were studied in vitro. In this study, 5- or analogue 14 (IC₅₀¼ 0.0730 mM)4cyclobutyl analogue 48a

6-fluoro group (29 or 44a) and 5-trifluoromethoxy group (44e), (IC₅₀¼ 2.84 mM)4cyclopropyl analogue 48b (IC₅₀¼ greater

thus (relatively) electron-negative groups, were effective to gain than 10.0 mM). Overall, as especially indicated by the lacking-

potent or highly potent COX-2 inhibitory activities in vitro, that or reducing-aromaticity effect on six-membered ring, the aroma-

is, the IC₅₀values were 0.264, 0.0156 and 0.493 mM for ticity of this portion attached with 2-carbonyl group of the indole

compounds 29, 44a and 44e, respectively. But, replacement by skeleton was unnecessary or not so important for potent and

5- or 6-methoxy group (44d or 44c) or 5-tert-butyl group (44b), selective COX-2 inhibition on the corresponding interaction-site

thus rather electron donating and/or bulky group, led to loss or in COX-2 protein as far as the size of this portion was appropriate

reduction of the activity. Importantly, the above potent or highly or sufficient for the potent and selective interaction between the

potent 5- or 6-substituted indolyl analogues 29, 44a and 44e compound and COX-2, which shows a potential of further

exhibited much high or high selectivities of COX-2 inhibition analogue-design around them.

over COX-1 inhibition, that is, 509-fold for 6-fluoro analogue

COX-2 has approximately 60% similarity to COX-1 in coding-

44a, 24.2-fold for 5-fluoro analogue 29 and 14.0-fold for 5- sequence comparison between the isozymes, also as a structural

trifluoromethoxy analogue 44e, respectively. As described above, difference between COX-2 and COX-1 originated from the amino

the structural and physicochemical properties of the substituents acid sequence regarding shape and size of the cyclooxygenase

are significant for the present potent and selective COX-2 inhibi- active-sites, COX-2 has valine 523 and COX-1 has isoleucine 523

tors; and the strong effects of 5- and/or 6-substituents located on in the corresponding active-sites, hence the active site of COX-2

the indole core in vitro were also demonstrated in the design and is larger than that of COX-1^{4,18,99–103,121}. Actually, the present

SAR studies of various novel [2-{[(4-substituted or 4,5-disubsti- in vitro SAR study of the diverse novel acid-types of analogues

tuted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubsti- clarified the variety of effects of 5- or 6-substituent on the indole

tuted)-1H-indol-3-yl]acetic acid analogues by Hayashi et al.^25,104

,

core and of 2-[(2-, 3- or 4-substituted, or 2,4- or 3,4-disubsti-

which achieved highly potent and selective COX-2 inhibitory tuted)-benzoyl, (bicyclic heterocycloalkanophenyl)carbonyl or

activities for the analogues with respective manners as reported. cycloalkanecarbonyl] portion, owing to the respective chemotypes

Third, SAR of {2-(bicyclic heterocycloalkanophenyl)carbonyl- and structural features that include shapes, sizes/volumes,

(6-chloro or 5-trifluoromethyl)-1H-indol-3-yl}acetic acid positions and electronic states, for their potent and selective

analogues was studied in vitro. Indeed, (6-chloro-1H-indol-3- COX-2 inhibition processes in vitro as critical and key factors of

yl)acetic acid analogue 44h and (5-trifluoromethyl-1H-indol-3- highly organised multisite interaction/coordination with pharma-

yl)acetic acid analogue 44i as 2-(2,3-dihydrobenzo[b][1,4] cophores in the enzyme protein, which determine dynamic

dioxine-6-carbonyl) derivatives and [6-chloro-2-(2,3-dihydroben- molecular interaction-mechanisms, -strengths and -modes (pos-

zofuran-5-carbonyl)-1H-indol-3-yl]acetic acid analogue 44j ition and orientation) of the ligands. Interestingly, bulkier size/

exhibited potent or highly potent COX-2 inhibitory activities substituent effects for COX-2/COX-1 selectivities, such as

in vitro, that is, the IC₅₀values were 0.148, 0.389 and 0.0566 mM profitablities of bicyclic group, bulkier mono- or disubstituent(s)

for compounds 44h, 44i and 44j, respectively. Furthermore, these bearing-(hetero)aryl group and relatively large-ring group at

2-(bicyclic heterocycloalkanophenyl)carbonyl analogues exhib- 2-carbonyl portion on the indole core of the analogues, have been

ited high selectivities of COX-2 inhibition over COX-1 inhibition, demonstrated as results of this study and the previous design and

that is, greater than 67.6-fold, greater than 25.7-fold, and 52.4- SAR studies by Hayashi et al.^25,104for the respective series of

fold for compounds 44h, 44i and 44j, respectively. In comparison COX-2 inhibitors within the appropriate ranges of positions,

of compounds 44h and 44i, the 6- or 5-substituent apparently shapes and sizes for whole/local molecular structures as

affected the respective in vitro activities and selectivities, similar described, which would be associated with the differences of

to the cases of 6- or 5-substituents on indole core of the above shape, size and interaction-/coordination-mode of the ligand-

2-(monocyclic aroyl) analogues as described. Besides, the bicyc- interaction/-coordination sites between COX-2 and COX-1 pro-

lic-group effects for their potent and selective COX-2 inhibitions teins; whereas appropriate electronic effects/states for the

were similar to that of our previously reported study by Hayashi substituents and for ꢂ-electron densities on the 2-carbonyl

et al. for 2-[(bicyclic cycloalkanopyridinyl)carbonyl] analogue, (hetero)aryl portion have been also important for the COX-2

that is, [6-chloro-2-(5,6,7,8-tetrahydroisoquinolin-3-ylcarbonyl)- selective inhibitions in the present study and in the previous SAR

1H-indol-3-yl]acetic acid whose IC₅₀value of COX-2 inhibition studies in vitro by Hayashi et al.¹⁰⁴, respectively. All together,

was 0.0192 mM and selectivity of the COX-2 inhibition over the present and the previously reported SAR studies in vitro and

COX-1 inhibition was greater than 521-fold in the human cellular the respective diverse analogues per se have provided unique and

assays¹⁰⁴. These studies demonstrates the profitability of the significant information to recognise/understand potent and select-

present and previous analogues that have bicyclic-group portion ive interaction between COX-2 and the artificial inhibitors

with some degree of bulkiness and volume at 2-carbonyl group, in vitro for further COX-2 inhibitor study regarding molecular

for potent and selective interactions between COX-2 and the mechanisms of actions, and have demonstrated various useful

molecules in the enzyme protein.

directions for further design of potent and selective COX-2

Fourth, SAR of (6-chloro-2-cycloalkanecarbonyl-1H-indo- inhibitors, which can be exploited to elucidate further detailed

l-3-yl)acetic acid analogues was studied in vitro. Notably, underlying the mechanisms and to discover/develop further

2-cyclohexanecarbonyl analogue 14 exhibited highly potent COX-2 inhibitors.

COX-2 inhibitory activity with COX-2 inhibition selectivity

over COX-1 inhibition in vitro, that is, the IC₅₀value was

In vivo oral activities in the rats and physicochemical

0.0730 mM and the selectivity was 15.9-fold. Hence, the incorp-

properties

oration of 2-cyclohexanecarbonyl group instead of the above

2-aroyl groups was well effective in potent and selective COX-2 Encouraged by the potent and selective COX-2 inhibitory

inhibition as well. Besides, compared with compound 14, other 2- activities of the novel acid-types of analogues in the

cycloalkanecarbonyl analogues with smaller rings showed weaker above study in vitro, SAR of representative novel {2-[(3- or

activities dependent on the degree of reduction of the respective 4-substituted)-benzoyl

or

cycloalkanecarbonyl]-(5-

or

18 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

6-substituted)-1H-indol-3-yl}acetic acid analogues for oral anti- activities for the analogues. For example, lipophilicity values for

inflammatory efficacies in peripheral-inflammation model were the whole structures of the compounds were calculated as ACD

investigated in vivo as the next step of this study. Thus, in vivo log D at pH 7.4 (ACD log D_7.4). The results of the SAR studies of

inhibitory effects against oedema-swelling stimulated by intra- oral activities in vivo and physicochemical properties for COX-2

plantarly injected-carrageenan in the hind paw of specific inhibitors as potential anti-inflammatory drugs are summarised

pathogen free and virus antibody free (SPF/VAF) male in Table 3.

Sprague–Dawley (SD) rats, and in vivo suppressive effects against

Actually, in this in vivo SAR study with the analogues for oral

PGE₂production stimulated by carrageenan in the foot of the inhibitory effects against carrageenan-induced oedema-swelling

SPF/VAF SD rats, that was the oedema-formation site in the in the foot of SPF/VAF SD rats, 6-chloro-2-[4-(trifluoromethyl)

above peripheral-inflammation model, were evaluated for the benzoyl]-1H-indolyl analogue 8d, 6-chloro-2-(cyclohexanecarbo-

compounds per os (po)^{25,104,115–118}. As well, physicochemical nyl)-1H-indolyl analogue 14, 6-chloro-2-(4-fluorobenzoyl)-1H-

properties of the compounds were estimated to consider key indolyl analogue 22b and 2-(4-chlorobenzoyl)-5-fluoro-

factors of the in vivo oral activities, together with in vitro 1H-indolyl analogue 29 exhibited 25.6–43.0% inhibitions at

Table 3. In vivo structureꢂactivity relationships of oral inhibitory effects against oedema formation and prostaglandin E₂production in peripheral-

inflammation model rats and physicochemical properties for COX-2 inhibitors 8d, 14, 22b, 22c and 29.

O

HO

R¹

5

O

2

R³

R²

N

6

H

In vivo

Physicochemical properties

Oral anti-inflammatory

effect^a

Oral suppressive effect

on PGE₂production^b

Compounds

MW^c

Da

TPSA^dLipophilicity^e

HBD^f

HBA^g

2

No

R¹R²

R³

% Inhibition (10 mg/kg) % Inhibition (10 mg/kg)

ACD log D_7.4Number Number

˚

A

8d

H

Cl

32.9

56.1

381.73

70.16

0.37

2

3

CF₃

14

H

Cl

31.2

25.6

53.6

66.6

319.78

331.73

70.16

0.01

2

3

22b

ꢂ0.36

F

22c

29

H

F

Cl

H

7.40

33.3

91.1

381.73

331.73

70.16

0.47

2

3

CF₃

43.0

–0.29

Cl

^aOral inhibitory effects of COX-2 inhibitors against foot-oedema formation by carrageenan in SPF/VAF male SD rats.

^bOral inhibitory effects of COX-2 inhibitors against prostaglandin E₂(PGE₂) production in the carrageenan-stimulated foot of SPF/VAF male SD rats.

^cMW, molecular weight.

^dTPSA, topological polar surface area.

^eLipophilicity values calculated as ACD log D at pH 7.4, predicted by ACD/Laboratories 9.0.

^fHBD, hydrogen bond donor.

^gHBA, hydrogen bond acceptor.

DOI: 10.3109/14756366.2013.864650

Design, synthesis and SAR of novel COX-2 inhibitors 19

10 mg/kg per os. Especially, compound 29 demonstrated the most 6-substituted)-1H-indol-3-yl}acetic acid analogues would be

potent inhibitory efficacy in the model among these compounds explicable by their inhibitory effects against PGE₂and PGI₂

(10 mg/kg, po). These orally potent anti-oedematous efficacies of productions in the peripheral oedema-sites, directly depended

the compounds would be due to their potent COX-2 inhibitions on their intrinsic inhibitory potencies against COX-2, their

in vitro (IC₅₀¼ 0.0730–0.264 mM), which are in accordance with physicochemical properties and their structural features, which

our previous reports by Hayashi et al.^25,104for [2-{[(4-substituted is in accordance with the detailed mechanism-study for the

or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted previously reported analogues by Hayashi et al.^25,104

or 5,6-disubstituted)-1H-indol-3-yl]acetic acid analogues that Taken together, the present study demonstrates that (i) the

.

exhibited highly potent and selective COX-2 inhibitions in vitro novel acid-types of COX-2 inhibitors 8d, 14, 22b and 29 were

and orally potent anti-oedematous efficacies in the peripheral- orally potent potential anti-inflammatory drugs in vivo, and

inflammation model. In contrast, 6-chloro-2-[3-(trifluoromethyl) (ii) the SARs of the analogues regarding in vitro and in vivo

benzoyl]-1H-indol-3-yl analogue 22c exhibited less potent inhibitory activities against inflammatory-mediator productions

in vivo anti-oedematous efficacy (7.40% at 10 mg/kg, po) owing derived from COX-2 with the physicochemical properties

to or contributed by its weaker COX-2 inhibition in vitro and structural-features of the analogues clarified the essential

(IC₅₀¼ 0.340 mM) and less appropriate physicochemical property and key factors to achieve the orally potent anti-oedematous

compared with the above compounds as discussed later.

efficacies in the peripheral-inflammation model of rats in vivo,

Furthermore, SAR of these COX-2 inhibitors for in vivo oral which led to the in vivo mechanisms of actions for the analogues,

suppressive effects against carrageenan-induced PGE₂synthesis as unique and significant findings. These results also show

in the foot of the SPF/VAF SD rats was studied. Thus, the in vivo possibility of the present series of various novel acid-types of

oral suppressive effects of the above compounds 8d, 14, 22b, 22c analogues, as well as other/further compounds around the

and 29 against PGE₂production in the peripheral-inflammation analogues that have potent and selective COX-2 inhibitory

site were 33.3–91.1% inhibitions at 10 mg/kg per os. Significantly, activities and appropriate physicochemical-properties/structural-

compound 29 exhibited the most potent inhibitory activity in features, as orally potent anti-inflammatory drugs in the in vivo

the model among them (10 mg/kg, po). As well, the tendency of model. Moreover, the present in vitro and in vivo SARs and the

the degree of the in vivo oral suppressive effects among most diverse COX-2 inhibitors per se, together with our previously-

of the above compounds against PGE₂production in the site reported COX-2 inhibitor studies and the diverse COX-2 inhibi-

(10 mg/kg, po) were similar to that of the degree of in vivo oral tors therein^25,104, might be helpful/useful to investigate further

anti-oedematous effects among the compounds in the peripheral- COX-2 inhibitor study in vivo for the treatment of inflammatory

inflammation model rats (10 mg/kg, po), although the respective diseases/syndromes, injuries/traumas, infections, and/or chronic

values of the percent inhibitions for the oral suppressive effects inflammation-related disorders such as RA and OA, and/or for

on PGE₂production and for the oral anti-oedematous effects chemotherapy or chemoprevention of cancers/tumours, and to

were influenced by the respective in vivo endpoints-dependent elucidate underlying further molecular mechanisms of actions for

sensitivities per os for the analogues.

the orally potent agents in the in vitro and in vivo environments.

In the viewpoints of molecular characteristics, the above orally

potent compounds 8d, 14, 22b and 29 in the peripheral-

inflammation model rats possess favourable physicochemical

properties and appropriate structural features to achieve orally

potent anti-inflammatory activities and high plasma-to-brain

selectivities at the oral (systemic)-administration conditions in

the SD rats as our previously described COX-2 inhibitor studies

by Hayashi et al.^25,104. Indeed, the above present compounds 8d,

14, 22b and 29 have far low lipophilicities for the respective

whole structures under physiological plasma condition (ACD log

D_7.4¼ –0.36 to 0.37), appropriate ranges or values of other

properties such as molecular weight (MW; 319.78–381.73 Da),

Conclusions

Through this drug-discovery study, various novel {2-[(2-, 3- or 4-

substituted, or 2,4- or 3,4-disubstituted)-benzoyl, (bicyclic

heterocycloalkanophenyl)carbonyl or cycloalkanecarbonyl]-(5-

or 6-substituted)-1H-indol-3-yl}acetic acid analogues were

designed, synthesised and biologically evaluated to seek and

identify various chemotypes of potent and selective COX-2

inhibitors for the treatment of inflammatory diseases. In fact,

diverse acid-types of novel potent and selective COX-2 inhibitors

were discovered in vitro, showing a variety of indispensable

structural features/factors that are responsible for the potencies

and the selectivities in vitro. Furthermore, novel COX-2 inhibitors

8d, 14, 22b and 29 exhibited orally potent anti-oedematous

efficacies in the carrageenan-induced peripheral-inflammation

model of SPF/VAF male SD rats with orally potent inhibitory

activities against PGE₂production in the peripheral-inflammation

sites of the rats, which were dependent on the intrinsic COX-2

inhibitory activities, physicochemical properties and structural

features of the respective molecules. These unique and significant

findings that include the in vitro and in vivo SAR results

demonstrate that the representative compounds 8d, 14, 22b and 29

per se are orally potent potential anti-inflammatory drugs in vivo

with unique and promising potential in or around the present acid-

types of analogues, which would be quite useful to elucidate or

understand underlying potent and selective interaction between

COX-2 and COX-2 inhibitors in vitro. Moreover, the present study

and the findings might be helpful to investigate further COX-2

inhibitor study in vivo for the treatment of inflammatory diseases/

syndromes, injuries/traumas, infections, OA and RA, and/or for

chemotherapy or chemoprevention of cancers/tumours, and to

clarify underlying further molecular mechanisms of actions for

2

˚

topological polar surface area (TPSA; 70.16 A ), the numbers

of hydrogen bond donor (HBD; two) and hydrogen bond acceptor

(HBA; three), and hydrophilic functionalities such as HBDs at

plural sites of the respective molecules. In contrast, compared

with these compounds, 6-chloro-2-[3-(trifluoromethyl)benzoyl]-

1H-indol-3-yl analogue 22c, that had weaker potency in vitro as

mentioned and higher lipophilicity (ACD log D_7.4¼ 0.47),

showed lower anti-inflammatory effect and lower suppressive

effect on PGE₂production in vivo (po), although other

physicochemical properties and structural features (e.g. MW,

TPSA and multisite HBD functionalities) that related to the oral

activities are within the above appropriate-ranges in common with

all these compounds.

As reported already^{4,5,122–126}, mechanisms of peripheral-

inflammatory oedema-formation by carrageenan comprise multi-

phasic processes in vivo that include the production of PGE₂and

PGI₂(as inflammatory mediators) owing to COX-2 induction/

upregulation and COX-2 activity in the peripheral-inflammatory

sites. Therefore, the mechanisms of actions of the oral anti-

inflammatory effects for the present series of novel {2-[(3-

or 4-substituted)-benzoyl or cycloalkanecarbonyl]-(5- or

20 S. Hayashi et al.

J Enzyme Inhib Med Chem, Early Online: 1–22

´

22. Duque J, Dıaz-Mun˜oz MD, Fresno M, In˜iguez MA. Up-regulation of

cyclooxygenase-2 by interleukin-1b in colon carcinoma cells. Cell

Signal 2006;18:1262–9.

the orally potent agents in vivo, together with our previously

described unique and significant studies of orally potent novel

[2-{[(4-substituted or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-

(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic

acid analogues as highly potent and selective COX-2 inhibitors

by Hayashi et al.^25,104, and with the highly organised physico-

chemical properties and structural features of the molecules for

dynamic enzyme-inhibition processes in vitro and in vivo

as described in this and the previous studies.

23. Kelley DJ, Mestre JR, Subbaramaiah K, et al. Benzo[a]pyrene

up-regulates cyclooxygenase-2 gene expression in oral epithelial

cells. Carcinogenesis 1997;18:795–9.

24. Kiritoshi S, Nishikawa T, Sonoda K, et al. Reactive oxygen species

from mitochondria induce cyclooxygenase-2 gene expression in

human mesangial cells. Potential role in diabetic nephropathy.

Diabetes 2003;52:2570–7.

25. Hayashi S, Sumi Y, Ueno N, et al. Discovery of a novel COX-2

inhibitor as an orally potent anti-pyretic and anti-inflammatory drug:

design, synthesis, and structure–activity relationship. Biochem

Pharmacol 2011;82:755–68.

Declaration of interest

We report no declarations of interest.

26. Kowalski ML, Makowska J. Use of nonsteroidal anti-inflammatory

drugs in patients with aspirin hypersensitivity. Safety of cyclo-

oxygenase-2 inhibitors. Treat Respir Med 2006;5:399–406.

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Appendix. Supplementary Material

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Design, synthesis and structure-activity relationship studies of novel and diverse cyclooxygenase-2 inhibitors as anti-inflammatory drugs

DOI: 10.3109/14756366.2013.864650

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