Bioorganic & Medicinal Chemistry Letters 24 (2014) 4794–4797

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Enhanced water-soluble derivative of PC407 as a novel potentialCOX-2 inhibitor injectable formulation

http://dx.doi.org/10.1016/j.bmcl.2014.09.0070960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel./fax: +86 2984776783.E-mail address: blezhang@fmmu.edu.cn (B.-L. Zhang).

Hong-Fei Liu, Ning Wan, Meng-Lei Huan, Yi-Yang Jia, Xiao-Feng Yuan, Si-Yuan Zhou, Bang-Le Zhang ⇑Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China

a r t i c l e i n f o

Article history:Received 25 July 2014Revised 26 August 2014Accepted 2 September 2014Available online 15 September 2014

Keywords:COX-2 inhibitorProdrugSolubilityInjectable formulation

a b s t r a c t

PC407 is an effective COX-2 inhibitor in non-steroidal anti-inflammatory drug development but the poorsolubility limits their usefulness. The aim of the study was to prepare and evaluate 4-oxo-4-[4-(5-(naph-thalen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamido]butyrate disodium, a derivativeof PC407 with enhanced water solubility for injectable formulation. The prepared derivative displayedinteresting high aqueous solubility (20.3 mg/mL, much superior to the parent compound PC407,1.6 lg/mL) with confirmed in vivo analgesic activity. This derivative represents the profiles of prodrugand potential candidate of PC407 for the development of injectable COX-2 inhibitor due to extraordinarywater solubility, low toxicity, and impressive analgesic activity.

� 2014 Elsevier Ltd. All rights reserved.

The prostaglandin (PG) synthase (cyclooxygenase), a keyenzyme involved in the metabolism of arachidonic acid to prosta-glandins, is the main target for non-steroidal anti-inflammatorydrugs (NSAIDs). Two important isoforms of cyclooxygenase withentirely different roles have been identified, namely, the constitu-tive cyclooxygenase-1 (COX-1) and the inducible cyclooxygenase-2(COX-2). COX-2 can be induced at injury and causes inflammation,fever, and pain.1,2 Specifically inhibiting COX-2 over COX-1 is ben-eficial and can avoid the undesirable gastrointestinal and renal sideeffects associated with classical nonselective NSAIDs.3 Celecoxib(4-[5-(4-methylphenyl)-3-trifluoromethyl-1H-pyrazoyl-1-yl]ben-zenesulfonamide, 1, Fig. 1), a 1,5-diarylpyrazole class of compound,is considered as selective COX-2 inhibitor with fewer gastrointesti-nal side effects at therapeutic concentrations,4–6 Celecoxib is animportant drug for treatment of pain or inflammation caused bymany conditions such as rheumatoid arthritis, osteoarthritis, andfamilial adenomatous polyposis.5,7–10 4-(5-(Naphthalen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (PC407,3a, Fig. 1) is a promising celecoxib analog developed by our groupwith potent pharmacological activities in the treatment of pain,inflammation and antiproliferation based on the role of COX-2 inthe etiopathology of those diseases.11–15

Recent surveys have shown that many patients underwent sur-gery experience with severe or moderately severe operative andpostoperative pain which were poorly managed in clinical trials.COX-2 inhibitors have been proved to reduce acute to moderate

pain by targeting the COX-2 enzyme expressed at sites of injuryincluding surgical incisions recently.16–19 However, celecoxib andits derivatives often show high apolar characteristics, practicallyinsoluble at physiological pH, and is classified as class II of biophar-maceutics classification system, which restricts the options of for-mulations available, along with high variability in absorption andlow bioavailability after oral administration.6 In addition, the onlyavailable injectable COX-2 inhibitor to treat pain is parecoxibsodium (2b, Fig. 1), while the parent drug valdecoxib was with-drawn from the market in 2005 by FDA because of its safety.8

Therefore, design and synthesis of new injectable COX-2 inhibitorsfor better management of pain is much required. As a part of ourongoing program to design novel effective selective COX-2 inhibi-tors and unveil the potential of COX-2 inhibitor for parenteraladministration, we conceived the idea to convert PC407 to suitablederivative using its amenable sulfonamide (SO2NH2) group. Hereinwe describe the approach for preparation of PC407 derivative toachieve high water solubility for development of injectable formu-lation. The physicochemical characteristics, water solubility, stabil-ity in aqueous solution, cleavage to the parent drug and in vivoanalgesic property of the derivative were also detected.

Intravenous administration is the preferred dosing route foracute pain, so we sought to identify an effective derivative ofPC407 suitable for intravenous administration. Benzenesulfon-amide group as a pharmacophore in PC407 is a good choice forderivative approach to explore the possibility to develop an inject-able formulation suitable for management of pain. A water solubleform was accessible by N-acylation of the sulfonamide (SO2NH2)followed by preparation of its sodium salt, which would serve as

Figure 1. Selective COX-2 inhibitors and injectable derivatives.

H.-F. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4794–4797 4795

possible prodrug for the parent compound PC407. Upon in vivoadministration, the derivative would cleave to regenerate the sul-fonamide pharmacophore and become active.

The synthesis of water soluble derivative is outlined inScheme 1. The parent drug PC407 required for the synthesis ofprodrug, was prepared according to the procedures reported byPenning group4 or our group.11–14 Briefly, reacting 1-(naphtha-len-2-yl)ethanone 4 with ethyl trifluoroacetate in the presence ofNaOMe provided the expected 1,3-dicarbonyl adduct 5, whichwas carried out by the condensation with 4-hydrazinobenzenesul-fonsmide hydrochloride in anhydrous EtOH under refluxing condi-tion to give the desired product. The pure PC407 (3a) was readilyisolated by flash silica chromatography in 44% yield. With the com-pound 3a in hand, the N-acylation of the sulfonamide (SO2NH2) inPC407 using 5 equiv of succinic anhydride was performed inrefluxing anhydrous CH2Cl2 for 20 h in the presence of triethyl-amine, leading to the formation of 6 in 83% yield. When treated6 with NaHCO3 in methanol at 0–25 �C, the corresponding diso-dium salt 3b was obtained in 87% yield, with abnormally highmelting point compared with 6. The products in above steps werecharacterized by spectroscopic method and the purity of the finalcompound was 99.5%, as determined by HPLC with UV detectionat 250 nm (see Supplementary material).

Our criteria to evaluate the injectable derivative for intravenousadministration were based on water solubility, stability in aqueous

Scheme 1. Synthesis of injectable derivative of PC407. Reagents and conditions:hydrazinobenzenesulfonsmide hydrochloride, EtOH, refluxing, 20 h; (iii) 5 equiv (CH2CO

solution, cleavage to the parent drug in vivo and analgesic efficacy.As indicated above, the aqueous solubility was of critical impor-tance in order to develop an intravenous dosage form. A validatedHPLC method was employed for the simultaneous measurement ofPC407 (3a) and the prodrug 3b. HPLC analyses were performed atroom temperature by C-18 reverse phase HPLC column. The mobilephase was 20% of 0.01 M NaOAc–HOAc (320:1) buffer and 80% ofMeOH mixture. The flow rate was fixed at 1.0 mL/min and theUV detection wavelength used was 250 nm. The retention timesof parent drug PC407 (3a) and 3b in HPLC are 10.61 and8.16 min, respectively (Fig. 2), which meets the requirement inanalysis of the prodrug and PC407.

When measured the solubility of prodrug 3b in water, the pro-drug was found to be highly soluble, provided a solubility of20.3 mg/mL in water for injection, whereas PC407 is practicallyinsoluble in water (1.6 lg/mL), which confirmed the solubilitywas highly improved. The stability of 3b in aqueous solution wasalso measured after 12 h at room temperature by HPLC, % releaseof PC407 from prodrug 3b is negligible (approximately 1.2%, seeSupplementary material Fig. 1S). On the other hand, when prodrug3b was administered intravenously at a dose of 5 mg/kg to the rats(three males and three females), the plasma concentrations of pro-drug 3b and PC407 (3a) were determined at the time of 0 (pre-dosebaseline), 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 and 300 minafter the start of the drug taking and shown in Figures 3 and 4,

(i) 1 equiv CF3CO2Et, 1.1 equiv NaOMe, ether, refluxing, 10 h; (ii) 1 equiv 4-)2O, TEA, CH2Cl2; refluxing, 20 h; (iv) 2.1 equiv NaHCO3, MeOH, 0–25 �C.

Figure 2. HPLC chromatogram of PC407 (3a) and prodrug 3b.

Figure 3. Plasma concentrations of prodrug 3b after intravenous administration ofprodrug 3b in rats. Date were shown as mean values ± SD, n = 6.

4796 H.-F. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4794–4797

respectively. After intravenous administration of prodrug 3b, theplasma concentration of 3b dropped rapidly as it was convertedinto PC407. Compound 3b was undetectable in plasma after 1.5 hin rats (Fig. 3). The peak of PC407 concentration was reached afew min (about 10) after which it declined for up to 4 h. Theresults demonstrate that PC407 can be released from 3b and theconversion to the active parent drug occurs in vivo throughbiotransformation.

Thus, the pharmacological profiles of prodrug 3b were evalu-ated both in vitro and in vivo. The in vitro inhibition studies withCOX-1 and COX-2 enzymes, in vivo analgesic activity and toxicityin mice are detected. When tested the inhibitory activity ofPC407 and 3b against recombinant isoforms of cyclooxygenasesCOX-1 and COX-2 by the protocols described in the literature,11

3b showed negligible inhibition even at 10 lM both on COX-1and COX-2, showing the profile of prodrug. On the contrary,reference drug PC407 exhibited 100% inhibition of COX-2 isoform,

without any inhibition of COX-1 isoform at 0.1 lM concentration,which was consistent with the known facts.4,11 As expected, pro-drug 3b showed relatively less potency in COX-2 inhibitionin vitro than PC407 due to the block of sulfonamide, which is themain pharmacophore group in PC407.11

In vivo pharmacological evaluation of the water soluble prodrug3b was carried out to assess their potential analgesic activity in theacetic acid-induced writhing assay.20–22 The writhing test is a noci-ceptive model characterized by repeated contractions of theabdominal muscles. A single writhe is defined as a wave of con-strictions and elongations passing caudally along the abdominalwall, accompanied by a twisting of the trunk and followed byextension of the hind limbs. Pain responses were scored by count-ing the number of abdominal contractions during 15 min or 30 minperiod after intraperitoneal injection of acetic acid. As summarizedin Table 1, when the analgesic activities were conducted for PC407and its water soluble prodrug 3b at equimolar dose, 0.1 mmol/kg(56 mg/kg of the disodium salts 5, equivalent to 42 mg/kg ofPC407 on a molar basis), it was found that the prodrug 3b showedexcellent analgesic activity in mice pain model. When the cumula-tive number of writhing responses was recorded for 15 min, PC407was found to be better than its prodrug 3b, reducing visceral painby 83% (PC407) and 77% (prodrug), respectively. When recordedfor 30 min, the salt 3b showed a slightly higher efficacy to thatof PC407 in vivo, reducing visceral pain by 83% (prodrug) and81% (PC407), respectively. The differences of the in vivo activitiesbetween prodrug 3b and PC407 during 15 min and 30 min period,could be the reason for drug-releasing properties of prodrug, whichcan be explained by the results of in vivo biotransformation to theactive parent drug. Further in vivo toxicity studies of prodrug 3b inmice indicated that the maximum tolerable dose is over 1500 mg/kg and therefore represents a potential prodrug candidate of PC407for the development of injectable COX-2 specific inhibitor.

In summary, a water soluble prodrug of PC407 has been synthe-sized based on sulfonamide (SO2NH2) group as potent injectableCOX-2 inhibitor. Studies revealed that the designed prodrug 3bhas good water solubility, aqueous stability, and in vivo analgesicactivity in intravenous administration. This novel strategy offersa formulation perspective based on the significantly improved

Figure 4. Plasma concentrations of PC407 (3a) after intravenous administration of prodrug 3b in rats. Date were shown as mean values ± SD, n = 6.

Table 1Pharmacological evaluation of prodrug 3b and PC407 in acetic acid-induced writhingassaya

Entry Group Pain responsesb,c (15 min) Pain responsesb,d (30 min)

1 Model 37.2 ± 7.3 57.1 ± 7.62 PC407 (3a) 6.2 ± 1.5* (83%) 10.3 ± 2.2* (81%)3 3b 8.5 ± 2.2* (77%) 9.7 ± 3.4* (83%)

a Carried out using 10 mice per group at the dose of 0.1 mmol/kg test compound.b Date shown as mean values ± SD.c Scored by counting the number of abdominal contractions during 15 min period

after ip injection of acetic acid.d Number of abdominal contractions during 30 min period after ip injection of

acetic acid.* P < 0.01, compared with model group.

H.-F. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4794–4797 4797

pharmacological profiles suitable for intravenous administration.Thus, this prodrug was identified as a potential candidate ofCOX-2 inhibitor for injectable formulation and further consideredfor detailed pharmacokinetic and pharmacodynamic evaluationin view of its potential clinic advantages.

Acknowledgments

Financial support of this research by National Science &Technology Major Project ‘Significant New Drug Creation andManufacture Program’ of China (No. 2011ZXJ09106-04C) andNational Natural Science Foundation of China (No. 21342017) isgratefully acknowledged. We also would like to thank the financialsupport by ‘Excellent Young Talents Support Program’ in FourthMilitary Medical University.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.007.

References and notes

1. Vane, J. R.; Botting, R. M. Inflamm. Res. 1998, 47, S78.2. Needleman, P.; Isakson, P. C. J. Rheumatol. 1997, 24, 6.3. Zeidan, A. Z.; AlSayed, B.; Bargaoui, N.; Djebbar, M.; Djennane, M.; Donald, R.;

ElDeeb, K.; Joudeh, R. A.; Nabhan, A.; Schug, S. A. Pain Pract. 2013, 13, 316.4. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,

S.; Graneto, M. I.; Lee, L. R.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;Isakson, P. C. J. Med. Chem. 1997, 40, 1347.

5. Frampton, J. E.; Keating, G. M. Drugs 2007, 67, 2433.6. Paulson, S.; Vaughn, M.; Jessen, S.; Lawal, Y.; Gresk, C.; Yan, B.; Maziasz, T.;

Cook, C.; Karim, A. J. Pharmacol. Exp. Ther. 2001, 297, 638.7. Scheen, A. J. Rev. Med. Liege 2004, 59, 565.8. Dogne, J. M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251.9. Steinbach, G.; Lynch, P. M.; Phillips, R. K.; Wallace, M. H.; Hawk, E.; Gordon, G.

B.; Wakabayashi, N.; Saunders, B.; Shen, Y.; Fujimura, T.; Su, L. K.; Levin, B. N.Eng. J. Med. 2000, 342, 1946.

10. Kalgutkar, A. S.; Zhao, Z. Curr. Drug Targets 2001, 2, 79.11. Lu, Z.; Xiong, X.; Zhang, B.; Lin, G.; Shi, Y.; Liu, Z.; Meng, J.; Zhou, Y.; Mei, Q. Acta

Pharmacol. Sin. 2005, 26, 1505.12. Hao, L.; Mei, Q.; Zhang, B.; Jia, M.; Li, X.; Zhang, F. Acta Pharmacol. Sin. 2004, 25,

1509.13. Li, Y.; Niu, Y.; Wu, H.; Zhang, B.; Sun, Y.; Huang, H.; Li, Q.; Fan, L.; Liu, L.; Mei, Q.

Cancer Sci. 2009, 100, 2451.14. Zhang, B.; Mei, Q.; He, W.; Zhou, S. Chin. J. New Drugs 2002, 11, 859.15. Li, Y.; Niu, Y.; Sun, Y.; Mei, L.; Zhang, B.; Li, Q.; Liu, L.; Zhang, R.; Chen, J.; Mei, Q.

Nutr. Cancer 2014, 66, 29.16. Phillips, E.; Hinck, B.; Pedro, R.; Makhlouf, A.; Kriedberg, C.; Hendlin, K.; Monga,

M. Urology 2009, 74, 994.17. Sun, T.; Sacan, O.; White, P. F.; Coleman, J.; Rohrich, R. J.; Kenkel, J. M. Pain Med.

2008, 106, 950.18. Huang, Y. M.; Wang, C. M.; Wang, C. T.; Lin, W. P.; Horng, L. C.; Jiang, C. C. BMC

Musculoskelet. Disord. 2008, 9, 77.19. Matsota, P.; Nakou, M.; Kalimeris, K.; Batistaki, C.; Pandazi, A.;

Kostopanagiotou, G. Arch. Med. Sci. 2013, 9, 877.20. Katz, N. Semin. Arthritis Rheum. 2002, 32, 15.21. Collier, H. O.; Dinneen, L. C.; Johnson, C. A.; Schneider, C. Br. J. Pharmacol. 1968,

32, 295.22. Hargreaves, K.; Dubner, R.; Brown, F.; Flores, C.; Joris, J. Pain 1988, 32, 77.