Efficient synthesis of apricoxib, CS-706, a selective cyclooxygenase-2 inhibitor, and evaluation of inhibition of prostaglandin E2 production in inflammatory breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2011.08.050

An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using copper catalyzed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis to an aldehyde, and condensation with sulfanilamide.

Full text of DOI:10.1016/j.bmcl.2011.08.050

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6071–6073
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of apricoxib, CS-706, a selective cyclooxygenase-2 inhibitor,
and evaluation of inhibition of prostaglandin E2 production
in inflammatory breast cancer cells
⇑
Pijus K. Mandal, Eric M. Freiter, Allison L. Bagsby, Fredika M. Robertson, John S. McMurray
Department of Experimental Therapeutics, M.D. Anderson Cancer Center, 1862 East Road, Houston, TX 77054-3005, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using
copper catalyzed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis
to an aldehyde, and condensation with sulfanilamide. This method provided multi-gram access of aproc-
oxib in good yield. Apricoxib exhibited potency equal to celecoxib at inhibition of prostaglandin E2
synthesis in two inflammatory breast cancer cell lines.
Received 13 July 2011
Revised 10 August 2011
Accepted 11 August 2011
Available online 19 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Apricoxib
CS-706
Cox-2 inhibitor
Cyclooxygenase 2
Homoallylic ketone
Cyclooxygenase (Cox) enzymes, also known as prostaglandin H
synthases (PGHS), catalyze the rate limiting step in the formation
of inflammatory prostaglandins, most notably the inflammatory
mediator, prostaglandin E2 (PGE2).1,2 Cox-1 is constitutively ex-
pressed and PGE2 derived from this isoform is associated with sur-
vival of specific populations of epithelial cells such as crypt stem
cells in the gastrointestinal tract.3 Cox-2 is transcribed from an
inducible immediate early gene primarily responsible for the pro-
duction of PGE2. Since this isoform was first cloned and sequenced
in 1992,4 numerous studies have documented the association be-
tween elevated gene expression and proliferation, invasion, angio-
genesis and metastasis in human tumors from different organ sites,
thus validating Cox-2 as a useful therapeutic target.5,6 In invasive
breast cancer, Cox-2 mRNA and protein are elevated regardless of
hormone receptor or Her-2 neu status.7–9
clinically as anti-tumor, anti-angiogenesis and chemopreventive
agents10,11 continue.
Apricoxib, (CS-706, 1) 2-(4-ethoxyphenyl)-4-methyl-1-(4-sulfa-
moylphenyl)-pyrrole, a small-molecule, orally active, selective
Cox-2 inhibitor was discovered by investigators at Daiichi Sankyo
in 1996.14,15 Clinical studies demonstrated potent analgesic activ-
ity16,17 and preclinical studies demonstrated good pharmacokinet-
ics, pharmacodynamics and gastrointestinal tolerability.18 As an
anticancer agent, preclinical studies demonstrated efficacy in bili-
ary tract cancer models19 and colorectal carcinoma,20 and Recamp
et al.21 recently reported a Phase I trial of 1 in combination with
erlotinib in lung cancer.
Our goal is to evaluate the clinical potential of apricoxib in
inflammatory breast cancer models, which requires multi-gram
quantities. Daichi Sankyo reported the synthesis of 1 in a patent14
and appears to have provided the compound for the clinical and
preclinical studies mentioned above. The original synthetic route
is outlined in Scheme 1. Though the initial two steps were accom-
plished with decent yields, the final step of pyrrolidine formation
followed by dehydration and dehydrocyanation produced only 3%
of 1 as a brown powder. The yield in the last step of the synthesis
of the 2-(4-methoxyphenyl) analog, 2-(4-methoxyphenyl)-4-
methyl-1-(4-sulfamoylphenyl)-pyrrole, was 6%,14 indicating that
this synthesis route is problematic.
Selective Cox-2 inhibitors, such as celecoxib (CelebrexÒ, Pfizer,
Inc), in addition to treating pain and inflammation, showed great
promise as inhibitors of tumor growth and angiogenesis.10,11
Unfortunately, due to the observation of unacceptably high rate
of cardiovascular side effects,12,13 the Cox-2 selective inhibitor
rofexocib (VioxxÒ, Merck, Inc) was removed from the market on
September 30, 2004, with
a black box warning issued for
CelebrexÒ. Consequently, efforts to develop next generation
Cox-2 inhibitors with different toxicity profiles that can be used
The route used to prepare the 4-ethyl analog, 2-(4-ethoxyphenyl)
-4-ethyl-1-(4-sulfamoylphenyl)-pyrrole, in the same patent em-
ployed the well known Paal–Knorr condensation of the intermediate
⇑
Corresponding author.
E-mail address: jsmcmur@mdanderson.org (J.S. McMurray).
c
-ketoaldehyde 7 and sulfanilamide (Scheme 2).14 The process
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.050

6072
P. K. Mandal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6071–6073
Scheme 3. Efficient synthesis of apricoxib (1).
We envisioned that 7 could be prepared by ozonolysis of homo-
allylic ketone (8) (Route B). A recent development in the synthesis
of homoallylic ketones by Dorr and Lubell via copper-catalyzed
cascade addition of alkenylmagnesium bromide to an ester24 was
examined. Treatment of commercially available methyl 4-eth-
oxybenzoate with 1-propenylmagnesium bromide (4.0 equiv) in
presence of CuCN (0.6 equiv) resulted in 95% yield of desired ke-
tone 8 after silica gel chromatography, along with a minor amount
of unreacted ester (Scheme 3).25
Scheme 1. Original synthetic route to apricoxib (1).14
The product was a mixture of cis/trans R/S stereoisomers, as de-
tected in the 1H NMR spectrum, and was used directly in the next
step without separation. Ozone was bubbled through a solution of
8 in MeOH/CH2Cl2 at À78 °C, until all starting materials were
consumed. The ozonide was then reduced to aldehyde 7 by treat-
ment with Me2S overnight. Removal of volatiles and subsequent
addition and evaporation of toluene gave the crude 1,4-dicarbonyl
compound 7 which was sufficiently pure for the following conden-
sation step. The 1H NMR signal at 9.78 ppm of the crude product
confirmed the formation of the aldehyde. No attempt was made
to characterize the enantiomeric ratio of 7 since the dehydration/
aromatization reaction of the next step removes the chirality of
the product. Treatment of 7 with sulfanilamide in 40% acetic
acid-acetonitrile at 70 °C for 3 h resulted in a brown product. Puri-
fication by silica gel flash chromatography yielded 71% of pure 1 as
a white solid.26
Scheme 2. Retro synthetic route to 1.
Our lots of apricoxib were assayed for the ability to inhibit pro-
duction of PGE2 in two inflammatory breast cancer lines, SUM149
and SUM190.27 As shown in Table 1, after stimulation with arachi-
donic acid, SUM149 cells produced 3 pg/mL/1000 cells of PGE2.
Apricoxib at 100 nM resulted in 70% inhibition (1 pg/mL/1000
involved conventional enamine alkylation with the appropriate
phenacyl bromide in an inert solvent followed by acid hydrolysis
leading to the desired dicarbonyl compound. In our hands, coupling
of 4-ethoxyphenacyl bromide (5)22 and 1-(N,N-diisopropylamino)-
1-propene (6) in solvents such as toluene, CH2Cl2, dry THF at 0 °C,
room temperature, or reflux produced at best 18% yield of 7. Purifi-
cation of 6 through distillation was fruitless so crude enamine was
used, which likely contributed to the low yields. The corresponding
1-piperidino-1-propene also did not give high yields of 7. In a later
cells). Concentrations of 1 and 10 lM resulted in 91% and 97% inhi-
bition, respectively. Although SUM190 cells produced double the
amount of PGE2, the degree of inhibition with apricoxib was the
same. The well known Cox-2 inhibitor, celecoxib was equally po-
tent as 1.
In summary, we present a highly efficient synthesis of the
promising Cox-2 inhibitor, apricoxib, in only three steps from com-
mercially available starting materials. The key is the improved
patent researchers at Daichi Sankyo used c-ketoaldehyde protected
as a ketal23 and condensed this with sulfanilamide under acid condi-
tions.22 Note that 5 and 6 also had to be synthesized which add fur-
ther steps to the preparation of 1.
Table 1
Apricoxib inhibits PGE2 in inflammatory breast cancer cells
a
a
SUM149
DMSO
PGE2 (pg/mL/1000 cells)
SUM190
DMSO
PGE2 (pg/mL/1000 cells)
3.15 0.19
0.97 0.24
0.28 0.07
0.11 0.01
1.19 0.13
0.43 0.08
0.13 0.04
1.00
0.31
0.09
0.03
0.38
0.14
0.04
6.43 1.87
2.10 0.81
0.50 0.15
0.22 0.05
2.87 0.74
0.81 0.13
0.18 0.01
1.00
0.33
0.08
0.03
0.45
0.13
0.03
0.1
l
M Apricoxib
M Apricoxib
M Apricoxib
M Celecoxib
M Celecoxib
M Celecoxib
0.1
l
M Apricoxib
M Apricoxib
M Apricoxib
M Celecoxib
M Celecoxib
M Celecoxib
1
l
1 l
10
l
10 l
0.1
l
0.1 l
1
l
1 l
10
l
10 l
a
PGE2 = average PGE2 production of triplicate samples standard deviation.

P. K. Mandal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6071–6073
6073
21. Reckamp, K.; Gitlitz, B.; Chen, L. C.; Patel, R.; Milne, G.; Syto, M.; Jezior, D.;
Zaknoen, S. Cancer 2011, 117, 809.
22. Kojima, S.; Ooyama, J. WO2008020617A, 2008.
23. Okazaki, R; Kojima, S., US20050148780. 2005.
24. Dorr, A. A.; Lubell, W. D. Can. J. Chem. 2007, 85, 1006.
synthesis of the c-ketoaldehyde intermediate, 7. Multi-gram quan-
tities have been prepared for use in preclinical studies. Our lots of
apricoxib potently inhibit Cox-2 activity in inflammatory breast
cancer cells. Details of further biological evaluation will be pub-
lished under separate cover.
25. Synthesis of 1-(4-ethoxy-phenyl)-3-methyl-hex-4-en-1-one (8): To
a stirred
suspension of CuCN (1.8 g, 20.0 mmol) in 50 mL of dry THF at À78 °C under
argon, a solution of 1-propenylmagnesium bromide (133.2 mmol, 265 mL of
0.5 M solution in THF) was added dropwise. The slurry was stirred for an
additional 30 min and then a solution of methyl 4-ethoxybenzoate (6.0 g,
33.3 mmol) in 60 mL of dry THF was added slowly. The stirred reaction mixture
was allowed to warm to room temperature overnight. The reaction was
quenched with ice cold saturated aqueous NaH2PO4 (100 mL) and the mixture
was extracted with ether (4 Â 100 mL). The combined ether extracts were
washed with brine (2 Â 100 mL), dried (MgSO4), filtered, and evaporated to
dryness. The crude homoallylic ketone was purified by silica gel flash
chromatography using a gradient of ethyl acetate in hexane as the eluent to
give 8 (7.4 g, 95%) as a colorless oil. 1H NMR (CDCl3, 300.0 MHz) d 1.04–1.07 (m,
3H), 1.44 (t, J = 6.9 Hz, 3H), 1.6–1.64 (m, 3H), 2.8–2.96 (m, 2.5H), 3.2 (m, 0.5H),
4.1 (q, J = 6.9 Hz, 2H), 5.25 (m, 0.5H), 5.34–5.46 (m, 1.5H), 6.92 (d, J = 9.0 Hz,
2H), 7.92 (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3, 75.0 MHz) d 12.9, 14.6, 17.9, 20.4,
21.0, 28.4, 33.0, 45.4, 45.5, 63.7, 114.1, 123.1, 123.4, 130.2, 130.3, 135.5, 136.0,
141.9, 162.7, 198.1. M+H calcd 233.154, found 233.2482.
Acknowledgments
We are grateful to the National Cancer Institute for support
(P.K.M., J.S.M.) CA096652, the Cancer Center Support Grant
CA016672 for support of both our NMR facility and the Transla-
tional Chemistry Core facility which provided HRMS, the American
Airlines-Komen For the Cure Foundation Promise Grant KGO81287
(F.M.R., E.M.F. and A.L.B.) and The State of Texas Fund for Rare and
Aggressive Breast Tumors (FMR, EMF, and ALB).
Supplementary data
26. Synthesis of Apricoxib (1): Homoallylic ketone (8) (5.0 g, 21.53 mmol) in 180 mL
of CH2Cl2/MeOH (1:5) was treated with ozone bubbles at À78 °C until a blue
coloration persisted. The solution was purged with argon, 8.0 mL of
dimethylsulfide (21.5 mmol) was added, and the reaction mixture then
warmed slowly to rt overnight. The solvent was evaporated under vacuum
to give 7 which was then diluted with 100 mL of 40% acetic acid in acetonitrile,
(v/v) and sulfanilamide (4.0 g, 23.2 mmol) was added. The mixture was
refluxed until complete consumption of 1,4-dicarbonyl compound was
detected by TLC (ca. 3 h). After cooling to room temperature, the product
was concentrated under vacuum and diluted with 250 mL of ethyl acetate. The
organic layer then washed with saturated Na2CO3 solution (3 Â 50 mL)
followed by brine (1 Â 50 mL), dried (MgSO4), and evaporated to dryness.
The crude brown material was purified by silica gel flash chromatography
using a gradient of EtOAc in hexane to give apricoxib as white solid (5.5 g,
15.43 mmol, 71%). Mp 161–163 °C (lit. 135–139 °C14). 1H NMR (CDCl3,
300.0 MHz) d 1.32 (t, J = 6.9 Hz, 3H), 2.1 (s, 3H), 3.92 (q, J = 6.9 Hz, 2H), 4.95
(s, 2H), 6.14 (m, 1H), 6.63 (m, 1H), 6.69 (d, J = 6.6 Hz, 2H), 6.94 (d, J = 6.6 Hz,
2H), 7.13 (d, J = 6.6 Hz, 2H), 7.74 (d, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75.0 Hz) d
11.7, 14.8, 63.4, 82.4, 113.2, 114.4, 121.0, 121.1, 124.9, 125.2, 127.4, 129.7,
133.6, 138.7, 144.2, 158.0. M+H calcd 357.1273, found 357.1252.
Supplementary data (1H, 13C, and COSY NMR spectra of com-
pounds 1 and 8) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.08.050.
References and notes
1. Rouzer, C. A.; Marnett, L. J. J. Lipid Res. 2009, 50, S29.
2. Hla, T.; Bishop-Bailey, D.; Liu, C. H.; Schaefers, H. J.; Trifan, O. C. Int. J. Biochem.
Cell Biol. 1999, 31, 551.
3. Cohn, S. M. Z.; Schloemann, S.; Tessner, T.; Seibert, K.; Stenson, W. F. J. Clin.
Invest. 1997, 99, 1367.
4. Hla, T.; Neilson, K. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7384.
5. Wang, D.; Dubois, R. N. Semin. Oncol. 2004, 31, 64.
6. Menter, D. G.; Schilsky, R. L.; DuBois, R. N. Clin. Cancer Res. 2010, 16, 1384.
7. Parrett, M.; Harris, R.; Joarder, F.; Ross, M.; Clausen, K.; Robertson, F. Int. J.
Oncol. 1997, 10, 503.
8. Hwang, D.; Scollard, D.; Byrne, J.; Levine, E. J. Natl. Cancer Inst. 1998, 90, 455.
9. Soslow, R. A.; Dannenberg, A. J.; Rush, D.; Woerner, B. M.; Khan, K. N.;
Masferrer, J.; Koki, A. T. Cancer 2000, 89, 2637.
10. Masferrer, J. L.; Leahy, K. M.; Koki, A. T.; Zweifel, B. S.; Settle, S. L.; Woerner, B.
M.; Edwards, D. A.; Flickinger, A. G.; Moore, R. J.; Seibert, K. Cancer Res. 2000,
60, 1306.
11. Abou-Issa, H. M.; Alshafie, G. A.; Seibert, K.; Koki, A. T.; Masferrer, J. L.; Harris, R.
E. Anticancer Res. 2001, 21, 3425.
12. Psaty, B. M.; Furberg, C. D. N. Eng. J. Med. 2005, 352, 1133.
13. Graham, D. J.; Campen, D.; Hui, R.; Spence, M.; Cheetham, C.; Levy, G.; Shoor, S.;
Ray, W. A. Lancet 2005, 365, 475.
14. Kimura, T; Noguchi, Y; Nakao, A; Suzuki, K; Ushiyama, S; Kawara, A; Miyamoto,
M. EP 799823 A1 1997.
15. Rao, P. P.; Grover, R. K. IDrugs 2009, 12, 711.
16. Moberly, J. B.; Xu, J.; Desjardins, P. J.; Daniels, S. E.; Bandy, D. P.; Lawson, J. E.;
Link, A. J.; Truitt, K. E. Clin. Ther. 2007, 29, 399.
17. Rohatagi, S.; Kastrissios, H.; Sasahara, K.; Truitt, K.; Moberly, J. B.; Wada, R.;
Salazar, D. E. Br J. Clin. Pharmacol. 2008, 66, 60.
18. Ushiyama, S.; Yamada, T.; Murakami, Y.; Kumakura, S.; Inoue, S.; Suzuki, K.;
Nakao, A.; Kawara, A.; Kimura, T. Eur. J. Pharmacol. 2008, 578, 76.
19. Kiguchi, K.; Ruffino, L.; Kawamoto, T.; Franco, E.; Kurakata, S.; Fujiwara, K.;
Hanai, M.; Rumi, M.; DiGiovanni, J. Mol. Cancer Ther. 2007, 6, 1709.
20. Senzaki, M.; Ishida, S.; Yada, A.; Hanai, M.; Fujiwara, K.; Inoue, S.; Kimura, T.;
Kurakata, S. Int. J. Cancer 2008, 122, 1384.
27. PGE2 production in inflammatory breast cancer cells: SUM149 or SUM190 cells
were cultured in F12 media (Invitrogen) supplemented with 10% fetal bovine
serum, 5 lg/mL insulin (Sigma–Aldrich, St. Louis, MO), 1 lg/mL hydrocortisone
(Sigma–Aldrich), and antibiotic/antimycotic (Invitrogen/Life Technologies,
Carlsbad, CA). Cells were seeded in triplicate in six-well tissue culture plates
in a humidified cell culture incubator at 37 °C with 5% CO2. When cells reached
80% confluence, culture media was removed by aspiration, cells were washed
once with sterile PBS and fresh medium containing the indicated Cox-2
inhibitor, either Apricoxib, or Celecoxib at concentrations of 0.1, 1, and 10 lM,
or an equivalent amount of DMSO used as the vehicle control was added. Cells
were pre-incubated for 2 h in the presence of either the Cox-2 inhibitor or
DMSO at 37 °C. Following the 2 h pre-incubation time period, PGE2 production
was stimulated by the addition of sodium arachidonate (Cayman Chemical,
Ann Arbor, MI), added directly to growth medium, with gentle agitation, to a
final concentration of 10 lM, and incubated for an additional 2 h. Conditioned
supernatant was then isolated from cells and stored at À80 °C until further
analysis. Following removal of supernatants, cells were immediately
trypsinized and cell number was assessed. PGE2 was assayed from
conditioned supernatant using a competitive enzyme immunoassay specific
to PGE2 (Cayman Chemical), as recommended by the manufacturer. The
concentration of PGE2 was normalized to total cell number per well, expressed
as pg PGE2/mL conditioned supernatant/1000 cells. Assays were run three
times and are expressed and mean standard deviation.