Discovery and stereoselective synthesis of the novel isochroman
`neurokinin-1 receptor antagonist 'CJ-17,493'; (2008) 16 EBIOMC 15 7193-
`7205
`August 1, 2008
`Section: Pgs. 7193-7205 Vol. 16 No. 15 ISSN: 0968-0896
`Length: 9881 words
`History: Received: April 4, 2008; Revised: June 23, 2008; Accepted: June 24, 2008
`Author: Yuji Shishido (‡) yuji.shishido@raqualia.com; Hiroaki Wakabayashi (‡); Hiroki Koike; Naomi Ueno; Seiji
`Nukui (†); Tatsuya Yamagishi (‡); Yoshinori Murata; Fumiharu Naganeo; Mayumi Mizutani (‡); Kaoru Shimada (‡);
`Yoshiko Fujiwara; Ayano Sakakibara (‡); Osamu Suga; Rinko Kusano; Satoko Ueda; Yoshihito Kanai; Megumi
`Tsuchiya; Kunio Satake (‡)
`Pfizer Global Research & Development, Nagoya Laboratories, 5-2 Taketoyo, Aichi 470-2394, Japan
`(‡) Present address: RaQualia Pharma Inc. 5-2 Taketoyo, Aichi, 470-2341, Japan.
`(†) Present address: Pfizer Global Research & Development, La Jolla Laboratories, 10614 Science Center Drive,
`San Diego, CA 92121, USA.
`Body
`ABSTRACT
`A novel central nervous system (CNS) selective neurokinin-1 (NK1) receptor antagonist, (2S,3S)-3-[(1R)-6-methoxy-
`1-methyl-1-trifluoromethylisochroman-7-yl]-methylamino-2-phenylpiperidine 'CJ-17,493' (compound (+)-1), was
`synthesized stereoselectively using a kinetic resolution by lipase-PS as a key step. Compound (+)-1 displayed high
`and selective affinity (Ki=0.2nM) for the human NK1 receptor in IM-9 cells, potent activity in the [Sar9, Met(O2)11]SP-
`induced gerbil tapping model (ED50=0.04mg/kg, sc) and in the ferret cisplatin (10mg/kg, ip)-induced anti-emetic
`activity model (vomiting: ED90=0.07mg/kg, sc), all levels of activity comparable with those of CP-122,721. In
`addition, compound (+)-1 exhibited linear pharmacokinetics rather than the super dose-proportionality of CP-
`122,721 and this result provides a potential solution for the clinical issue observed with CP-122,721.
`FULL TEXT
`1 Introduction
`The neurokinin-1 (NK1) receptor is a member of the seven-transmembrane G-protein coupled family of receptors
`and is associated with sensory neurons in the peripheral and specific areas of the central nervous system. The
`neuropeptide 'Substance P' and its human neurokinin-1 (hNK1) receptor have been associated with various
`biological disorders such as anxiety, depression, emesis, asthma and inflammatory bowel disease (IBD). 1
`Recently, two selective NK1 receptor antagonists have been approved: aprepitant (Merck; Emend® ), 2 as part of a
`1 1. (a) J.D. Gale, B.T. O'Neill, J.M. Humphrey; Expert Opin. Ther. Patents; Vol. 11, (2001), p. 1837.
`(b) A. Lecci, C.A. Maggi; Expert Opin. Ther. Target; Vol. 7, (2003), p. 342.
`(c) J.S. Albert; Expert Opin. Ther. Target; Vol. 14, (2004), p. 1421.
`HELSINN EXHIBIT 2058
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00945
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`combination therapy with a corticosteroid and a 5-HT3 receptor antagonist for the prevention of acute and delayed
`chemotherapy-induced nausea and vomiting (CINV) in humans; and maropitant (Pfizer; Cerenia® ) 3 as a veterinary
`medication to prevent and treat acute vomiting in dogs (Fig. 1).
`Figure 1. NK1 receptor antagonists as anti-emetic drugs for humans and animals (dog).
`The Pfizer compounds CP-99,994 and CP-96,345 were the first non-peptide neurokinin-1 receptor antagonists to
`be disclosed (Fig. 1). 4 Using the piperidine derivatives CP-99,994 and GR-203040 (Glaxo), it was soon
`demonstrated that NK1 antagonism results in the inhibition of cisplatin-induced emesis in humans and animals and
`that the site of action is a part of the central nervous system. 5 Subsequently, CP-122,721, derived from CP-99,994
`by the introduction of a trifluoromethoxy group, demonstrated anti-emetic activity at one-tenth the effective dose of
`CP-99,994. However, CP-122,721 exhibited strong competitive inhibition (Ki=0.02μM against metabolic oxidation of
`bufuralol) of the CYP2D6 enzyme. Furthermore, a comparison of half-lives of CP-122,721 upon exposure to
`CYP2D6-deficient human liver microsomes (HLM) with or without addition of exogenous CYP2D6 enzyme
`(hereinafter called the CYP2D6 +/− assay) revealed that the half-life of the compound decreased more than 3-fold
`upon addition of exogenous CYP2D6. The first step of the major metabolic pathway for CP-122,721 was identified
`as the CYP2D6 catalyzed O-demethylation on the 2-methoxy moiety. 6
`Based on the prototype NK1 receptor antagonist 'CP-122,721', we initiated a program aimed at lowering the
`effective dose in the anti-emetic model and addressing the high affinity of CP-122,721 for the CYP2D6 enzyme. In
`order to address these major issues, our initial approach focused on the modification at the 5 (or 4, 5)-position(s) of
`the benzylamine moiety by introduction of fluorine atoms as in the 5-trifluoromethoxy group of CP-122,721, while
`retaining the 2-methoxy moiety which plays an important role in the antagonistic activity (Fig. 2). As a result, the
`highly potent and metabolically stable compound (+)-1 was found among a series of synthesized 4,5-fused
`derivatives containing an oxygen atom. (+)-1 showed anti-emetic activity comparable to that of CP-122,721 in the
`gerbil tapping assay, known to be connected to the central NK1 receptor site, and in the cisplatin-induced ferret
`emesis and vomiting model. In addition, compound 21 (5:1 diastereomer mixture of compound (+)-1) displayed a 6-
`fold lower affinity for the CYP2D6 enzyme and a 3-fold improvement in the CPY2D6 +/− assay, comparing favorably
`to the high affinity for CYP2D6 which had identified as a major issue for CP-122,721. This report summarizes our
`SAR pursuit from CP-122,721 to compound (+)-1, a compound with enhanced safety potential which may prove
`useful as an anti-emetic drug, and outlines the first synthetic method of optically active (+)-1 using a kinetic
`resolution by lipase-PS. 7
`2 2. J.J. Hale, S.G. Mills, M. MacCoss, P.E. Finke, M.A. Cascieri, S. Sadowski, E. Ber, G.G. Chicchi, M. Kurtz, J. Metzger, G.
`Eirmann, N.N. Tsou, F.D. Tattersall, N.M.J. Rupniak, A.R. Williams, W. Rycroft, R. Hargreaves, D.E. MacIntyre; J. Med. Chem.;
`Vol. 41, (1998), p. 4607.
`3 3. Ito, F.; Kondo, H.; Shimada, K.; Nakane, M.; Lowe III, J. A.; Rosen, T. J.; Yang, B. V. WO 9221677 A1.
`4 4. (a) R.M. Snider, J.W. Constantine, J.A. Lowe, K.P. Longo, W.S. Label, H.A. Woody, S.E. Drozda, M.C. Desai, F.J. Vinick,
`R.W. Spencer, H.-J. Hess; Science; Vol. 251, (1991), p. 435.
`(b) S. Mclean, A. Ganong, T. Seeger, D. Bryce, K. Pratt, L. Reynolds, C. Siok, J. Low, J. Heym; Science; Vol. 251, (1991), p.
`437.
`5 5. P. Ward, D.R. Armour, D.E. Bays, B. Evans, G.M. Giblin, N. Heron, T. Hubbard, K. Liang, D. Middlemiss, J. Mordaunt, A.
`Naylor, N.A. Pegg, P.M. Vinader, S.P. Watson, C. Bountra, D.C. Evans; J. Med. Chem.; Vol. 38, (1995), p. 4985.
`6 6. (a) K. Colizza, M. Awad, A. Kamel; Drug Metab. Dispos.; Vol. 35, (2007), p. 884.
`(b) R.S. Obach, J.M. Margolis, M.J. Logman; Drug Metab. Pharmacokinet; Vol. 22, (2007), p. 336.
`7 7. Process research of CJ-17493:. S. Caron, N.M. Do, J.E. Sieser, P. Arpin, E. Vazquez; Org. Process Res. Dev.; Vol. 11,
`(2007), p. 1015.
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`Figure 2. Approach to compound (+)-1 starting from CP-99,994 and CP-122,721.
`2 Discussion and results
`The results of our modifications around the 5 (or 4, 5)-position(s) of the benzylamine moiety are summarized in
`SAR Tables 1–3, focusing in turn on non-cyclic analogs (Table 1), 5-ring membered analogs (Table 2), and 6-ring
`membered analogs (Table 3). The conversion of the trifluoromethoxy group of CP-122,721 to a fluoroalkyl moiety
`led to an improvement in the affinity for IM-9 cells and in the specific interaction to CYP2D6, while conserving the
`hNK 1 receptor antagonistic activity in vitro (Table 1). In particular, the introduction of geminal-methyl groups or of
`fluorine groups (F, CF3) at the 5-benzylic position of CP-122,721 (compounds 4, 6 and 7) resulted in moderate
`inhibition of the metabolic oxidation of bufuralol at 5μM substrate, while retaining the intrinsic activity in the gerbil
`tapping assay as shown in Table 1. Furthermore, the 5- and 6-membered cyclized trifluoromethyl derivatives (10–
`13, 15–17, and 21) showed comparable to or improved activity over CP-122,721 in the gerbil tapping assay, while
`the introduction of an oxygen atom in the 5- and 6-membered derivatives (15 and 21) resulted in a significant
`increase in inhibitory activity (ED90<0.1mg/kg, sc) in the cisplatin-induced emesis assay, as shown in Tables 2 and
`3. Compound 21 was evaluated as a 5:1 R/S mixture at the 6-position. The lower activity in vivo of these in vitro
`potent compounds (in the binding assay) can be explained by a lower CNS penetration, as evidenced by the ratio of
`free fraction concentration in brain (or CSF) versus plasma.
`CP-122,721 demonstrated super dose-proportional kinetics among poor metabolizers (PMs) of CYP2D6 in a human
`PK study of healthy male subjects, 6 while the quinuclidine compound CJ-11,974 showed dose-proportional
`pharmacokinetics in clinical data. An in vitro assay of these compounds confirmed the difference in Km values for
`CP-122,721 and CJ-11,974 in human liver microsomes. 8 We therefore hypothesized that it is possible to predict
`the kinetic behavior of compounds in this series by their Ki values and the ratio of their half-life in the CYP2D6 +/−
`assay. Thus a new screening sequence was devised, consisting of an inhibitory assay of the 1′-hydroxylation of
`bufuralol at 5μM concentration of substrates, followed by a full inhibition assay to determine the inhibition constant
`(Ki) of the compound for the inhibition of bufuralol by CYP2D6 instead of the cumbersome measurement of Km
`values. In addition to the Ki value, the T1/2 ratio in HLM in the CPY2D6 +/− assay or HLM in the quinidine +/− was
`also determined. 9
`Compounds with potent anti-emetic efficacy were selected and compared for their effect on CYP2D6, as shown in
`Table 4. Compounds 2, 3, 15 and 21 showed high specific interaction (>60% inhibition at 5μM, Ki<1.0μM) with the
`CYP2D6 enzyme in the 50% inhibition assay using bufuralol at 5μM concentration of substrates, and low Ki values
`in the bufuralol metabolism assay by human CYP2D6, while tetrahydronaphthalene derivative 19 (a C-analog of
`compound 21) showed a definite improvement over 21. Among the compounds with inhibition constants more than
`25 times greater than the Ki value for CP-122,721 (Ki>0.5μM vs 0.02μM) in Table 4, compound 11 and 17
`demonstrated hypermetabolism in CYP2D6 +/− assay. A possible explanation for the 10-fold jump for 11 in the
`CYP2D6 +/− assay is a rapid metabolite formation despite its low affinity for the CYP2D6 enzyme.
`Finally, in order to study the relationship between pharmacological activity in vivo and affinity to CYP2D6 in vitro,
`the 5- and 6-membered derivatives (15 and 21) were selected. Since the respective Ki values of 0.66 and 0.12μM
`for 15 and 21 indicate a 6- to 30-fold lower affinity compared to the Ki value (0.02μM) for CP-122,721, we predicted
`that compound 21 would not demonstrate appreciable super dose-proportionality in humans at pharmacologically
`relevant doses. The half-life ratio in the CPY2D6 +/− of compound 21 is also 3-fold lower than the corresponding
`ratio (6.00) for CP-122,721. Furthermore, although the profile of compound 21 is comparable to that of the 5-
`membered-ring compound 15, the pharmacokinetics of compound 21 showed a 2- to 4-fold superiority in AUC, C max
`8 8. O-Demethylation of CP-122,71: the 'first step major metabolite' exhibits a Km value of 0.24 in human liver microsomes, while
`the two major metabolites of CJ-11974 exhibit Km values of 80 and 88μM in the metabolic pathway by human liver microsomes;
`detailed data of CP-122,721: Ref. 6(b); detailed data of CJ-11,974.
`(b) R.S. Obach; Drug Metab. Dispos.; Vol. 28, (2000), p. 1069.
`9 9. T. Takashima, S. Murase, K. Iwasaki, K. Shimada; Drug Metab. Pharmacokinet; Vol. 20, (2005), p. 177.
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`and half-life (dog, 5mg/kg, po). Thus our effort shifted to the development of an efficient synthesis of optically active
`(+)-1.
`Finally, the important Km value for synthesized compound (+)-1 was measured directly based on the results for the
`affinity to CYP2D6 enzyme for compound 21 (active-enriched 5:1 diastereomer mixture). While the major metabolite
`of CP-122,721 is O-demethylation in human liver microsomes, the metabolic routes for CJ-17,493 are 4-
`hydroxylation on the benzylamine moiety (not demethylation in 6-position as in the metabolism of CP-122,721) and
`N-dealkylation on the 3-carbon atom between the benzylic NH moiety and the piperidine skeleton. The Km values in
`human liver microsomes were 16.6 and 2.3μM for hydroxylation and N-dealkylation, that is, 10- to 70-fold higher
`than the Km of CP-122,721 in human liver microsomes. This Km ratio between CP-122,721 and CJ-17,493 was
`considered to be sufficient to predict a lack of super-proportional kinetics. In fact, the FIH study of CJ-17,493
`confirmed the dose-proportionality in a PK study at doses ranging from 30 to 380mg (individual subjects dose-
`adjusted AUC(0–inf)s were similar) and no differences between CYP2D6 EMs (n=20) and PMs (n=4).
`3 Chemistry
`Regioselective mono-bromination of the commercially available 23 was followed by chlorination of the hydroxyl
`group (Scheme 1). The conversion of 25 to 3,4-dihydro-1H-isochromen derivative 26 was then effected by selective
`lithiation at the 4-position using n-butyl lithium at −100°C followed by the addition of 1,1,1-trifluoroacetone, affording
`a 3.9:1 mixture of 26 and 3-methoxyphenetyl chloride. The by-product was converted to 1-methoxy-3-vinylbenzene
`using DBU, after which distillation of the crude product gave pure 26 in 77% yield. After conversion of 26 to 28,
`compound 28 was subjected to kinetic resolution using lipase-PS, leading to the optically active O-acetyl derivative
`29 with an enantiomeric excess of 94%. 10 Compound 29 was converted to the 1-methoxy derivative in two steps to
`furnish 32, which was formylated regioselectively at the 7-position using a combination of aluminum(III) chloride and
`dichloromethyl n-butyl ether (ratio in 5-/7-position=5.3:1, isolated yield 54%) (Table 7, entry 5). The reductive
`amination of 34 with (2S,3S)-3-amino-2-phenylpiperidine led to compound 1, which was treated with 10%
`hydrochloric methanol to give (+)-1·dihydrochloride. The optical purity of the obtained (+)-1·dihydrochloride was
`then increased to >99% de by two successive recrystallizations.
`Scheme 1. Synthesis of compound (+)-1 (CJ-17,493). Reagents and conditions: (a) bromine, pyridine (1.1 equiv),
`CH 2Cl2 (100%); (b) CCl4, triphenylphosphine, 85°C, 75%; (c) n-butyllithium, THF/hexane (3:1), −100°C then 1,1,1-
`trifluoroacetone, −70°C–rt; (d) DBU, toluene, reflux, 77% from 25; (e) 48% HBr aq AcOH, reflux, 100%; (f) AcCl,
`triethylamine, THF, rt, 89%; (g) lipase-PS, 10% sec-butanol/hexane, rt, 24h, 45%; (h) K2CO 3, methanol/H2O (2.5:1),
`rt, 93%, 94% ee; (i) MeI, 60% NaH, DMF, rt, 98%; (j) dichloromethyl n-butyl ether, AlCl3, CH2Cl2, 74%; (k) (2S,3S)-
`3-amino-2-phenylpiperidine, NaBH(OAc)3, CH2Cl2, rt, 24h, 100% (crude yield); (l) 10% HCl/methanol, methanol,
`78%.
`The following Table 5 summarizes the screening of lipases for the kinetic resolution of racemic compound 28. 11
`The lipase PS series of standard and immobilized reagents produced the optically active acetate 29 in more than
`60% enantiomer excess (ee). Thus the inexpensive standard lipase PS was selected, and we proceeded to study
`10 10. Enzymes were kindly supplied by Amano enzyme. Lipase AY (Candida rugosa), CHE (Cholesterol esterase), lipase AH
`(Pseudomonas, sp.), lipase PS (Pseudomonas sp.).
`It was difficult to obtain the exact enantiomeric excess of compound 29. After deacetylation, the enantiomeric excess of optical
`phenol 31 was analyzed by HPLC using a DICEL CHIRALCEL OJ column (4.6×250mm), No. 45-04-90412, eluent;
`hexane/isopropanol (95:5), flow rate: 1mL/min, temperature; 40°C, injection; 10μL, retention time; acetate=7.55, 8.73min,
`phenol=15.88, 24.35min.
`11 11. E. Mizuguchi, M. Takemoto, K. Achiwa; Tetrahedron: Asymmetry; Vol. 4, (1993), p. 1961.
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`the limited solvent choices, as shown in Table 6. Although the combinations of 10% alcohols with hexane only
`moderately increased the enantiomeric excess (ee) of compound 29, this combination of solvents allowed a better
`control of the rate of the kinetic resolution than the combination of alcohols and buffer solution. The conversion rate
`(%) at ambient temperature was carefully investigated on a 15g scale for 28 and the 50% conversion time was
`determined to be ca. 20–23h. Finally, a combination of lipase-PS using 10% sec-butanol/hexane as the solvent
`gave the optical acetate 29 in 94% ee (51% conversion) on multigram scale: 28 (38.4g)/lipase PS (35g)/10% sec-
`BuOH/hexane (1.3L)/rt 23h (Table 6, entry 5).
`Another issue in the synthesis of compound (+)-1 was the need to improve the regioselectivity in the formylation of
`compound 32 (Table 7). A 34/33 ratio of 1.5:1 was obtained using titanium chloride and dichloromethyl methyl
`ether, 12 compared with a 10–15:1 ratio when this combination of reagents was used to produce the 5-membered
`analog 40 in scheme 2. After testing various reagent combinations, we found that the combination of aluminum
`chloride (2.3 equiv) and dichloromethyl n-butyl ether (2.0 equiv) in dichloromethane with dry ice/methanol cooling
`increased the regioselectivity of the formylation to a ratio of 5–6: 1 (Table 7, entry 5).
`Scheme 2. Synthesis of compound 15. Reagents and conditions: (a) bromine, pyridine (1.2 equiv)/CH2Cl2, 0°C–rt,
`18h; (b) n-butyllithium, THF/hexane (3:1), −100°C then 1,1,1-trifluoroacetone, −70 to −30°C (39/37=13.2:1); (c)
`glycine, KOH, EtOH/H2O (3:2), reflux, 2h then distillation (bp 94–96°C/1.5mm Hg), 69% from 37; (d) dichloromethyl
`methyl ether, TiCl4, CH2Cl2, 82% (40/41=15:1); (e) (2S,3S)-3-amino-2-phenylpiperidine, NaBH(OAc)3, CH2Cl2, rt,
`24h, 100% (crude yield); (f) 10% HCl/methanol, ethyl acetate, 21% after three times recrystallization.
`Compound 15 was also synthesized according to the same sequence as compound 1 (Scheme 2). Cyclization of 38
`to 39 afforded a 13:1 mixture of 38 and 37, which was treated with glycine 13 to give a single compound 38 in 69%
`yield. The subsequent formylation of 39 using titanium chloride/dichloromethyl methyl ether gave 6-formyl derivative
`40 (82% yield) in a ratio of 15:1, as compared to the 5.3:1 ratio obtained in the formylation of compound 32 under
`the same conditions. The reductive amination of 40 with (2S,3S)-3-amino-2-phenylpiperidine led to compound 15 as
`a 1:1 diastereomer mixture, which was treated with 10% hydrochloric methanol to give 15·dihydrochloride. Finally,
`triple recrystallization of 15·dihydrochloride allowed the isolation of the enriched 1R diastereomer as a 49:1 mixture
`without the need for any kinetic resolution of an intermediate.
`For the synthesis of the carbocyclic analogs 12 and 19, the following general protocol was used (Scheme 3). The
`ketones (42 and 43) were transformed into the trifluoromethyl tertiary alcohols (44 and 45) using Ruppert's reagent
`(TMS-CF 3) followed by acidic hydrolysis. 14 Next, chlorination of the cyclic tertiary alcohols using titanium(IV)
`chloride (2 equiv) at −78°C followed by addition of dimethylzinc (2 equiv), all in one pot, afforded the desired
`products (48 and 49) in high yield. 15 These were converted to the desired formyl derivatives 52 and 53 using
`titanium(IV) chloride and dichloromethyl methyl ether as the first-line choice, the two compounds giving different
`regioselectivity results. The reductive amination of these formyl derivatives with (2S,3S)-tert-butyl 3-amino-2-
`phenylpiperidine-1-carboxylate led to compounds 12 and 19, which were treated with 10% hydrochloric methanol to
`give 12·dihydrochloride and 19·dihydrochloride, respectively.
`12 12. H. Meier, H. Kretzschmann, H. Kolshorn; J. Org. Chem.; Vol. 57, (1992), p. 6847.
`13 13. G.L. Rowley, G.L. Kenyon; J. Heterocycl. Chem.; Vol. 9, (1972), p. 203.
`14 14. (a) G.K.S. Prakash, R. Krishnamurti, G.A. Olah; J. Am. Chem. Soc; Vol. 111, (1989), p. 393.
`(b) T. Mukaiyama, Y. Kawano, H. Fujisawa; Chem. Lett.; Vol. 34, (2005), p. 1.
`15 15. H. Tanaka, Y. Shishido; Bioorg. Med. Chem. Lett.; Vol. 17, (2007), p. 6067.
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`Scheme 3. Synthesis of compound 12, 19 and 20 (indane and tetraline derivatives). Reagents and conditons: (a)
`TMSCF 3, TBAF (cat.), THF, rt, then dil HCl aq; (b) TiCl4 (2.2 equiv), CH2Cl2, −78°C; (c) 1.0M Me2Zn in hexane
`solution (2.0 equiv) (n=1:87%; n=2:92%); (d) TiCl4 (2.2 equiv), Cl2CHOMe (2.2 equiv), CH2Cl2, 0°C (n=1:85%; n=2:
`40%); (e) (2S,3S)-tert-butyl 3-amino-2-phenylpiperidine-1-carboxylate, NaBH(OAc)3, CH2Cl2, rt, 24h; (f) 2M HCl
`aq/ethyl acetate (n=1:86%; n=2:94% from 52 and 53); (g) 10% HCl/methanol.
`4 Evaluation of compound(+)-1 (CJ-17,493)
`4.1 In vitro data of compound (+)-1 (CJ-17,493)
`Compound (+)-1 binds with high affinity (Ki=0.2nM) to the NK1 receptor labeled by [3H]SP in the IM-9 human
`lymphoblastoma cell line. (The IC50 values in human IM-9 cell were <0.1, 0.34 and 0.79nM for compound (+)-1, the
`1S epimer of compound (+)-1 and substance P, respectively). On the other hand, it possesses moderate affinity to
`the verapamil binding site at the L-type Ca2+ channel labeled by [3H]desmethoxyverapamil (IC50=164nM) and the
`sodium channel site 2 labeled by [3H]batrachotoxinin (IC50=48nM). The functional activity of compound (+)-1 against
`the sodium channel was examined by whole-cell patch clamp using CHO-CNaIIA cells stably transfected with the
`rat Na+-channel type IIA-subunit. The compound had no significant activity against the sodium channel at up to 1μM
`concentration (13% inh.), and 88% inhibition was observed at a concentration of 10μM.
`4.2 Pharmacology of compound (+)-1 (CJ-17,493)
`The activity of optically active compound (+)-1 in the CNS was assessed by the intracelebroventricular injection of a
`stable NK1 receptor agonist, that is, the gerbil [Sar9, Met(O2)11]SP-induced tapping model. Compound (+)-1, dosed
`sc 30min before the agonist injection, inhibited the tapping response in a dose-dependent manner. The ED50 was
`0.043±0.003 mg/kg, sc (mean±SEM, n=3). Capsaicin elicits plasma extravasation in a variety of tissues through the
`release of endogenous SP from sensory afferents. In the guinea pig ureter, intraperitoneally injected capsaicin
`(30μM) produces a plasma protein leakage which is likely to be mediated by activation of the peripheral NK1
`receptor. In this model, (+)-1 showed dose-dependent inhibition with an ED50 of 0.005mg/kg, po. NK1 receptor
`antagonists have been shown to block retching and vomiting responses induced by a wide range of centrally and
`peripherally acting emetogens in the ferret. Intraperitoneal injection of cisplatin (10mg/kg ip), a chemotherapeutic
`agent, elicits acute emetic episodes such as retching, vomiting and gagging in the ferret. (+)-1, given sc 30min
`before cisplatin, inhibited the emesis response in a dose-dependent manner with ED90 values of 0.03 and
`0.07mg/kg, sc for retching and vomiting/gagging, respectively (Fig. 3).
`Figure 3. Activity of optically active (+)-1 on retching and vomiting/gagging induced by cisplatin (10mg/kg, ip) in
`ferrets. ∗P<0.05 and ∗∗P<0.01 versus control by Student's-test. Data are mean±SEM, n=4–5.
`4.3 PK profile of compound (+)-1 (CJ-17,493)
`In rats, the systemic plasma clearance (CL), volume of distribution at steady-state (Vdss), and half-life (T1/2) of
`optically active (+)-1 (0.3mg/kg, iv) were 0.66L/h/kg, 7L/kg, and 8.8h, respectively. After oral administration (1–
`30mg/kg), exposure to compound (+)-1 increased with dose and maximum drug concentrations (Cmax ) occurred
`within 0.5h post dose. Oral bioavailability of optically active (+)-1 administered as the hydrochloride salt dissolved in
`water was lower than 1% at the tested doses. In dogs, optically active (+)-1 at 1mg/kg, iv gave CL, Vdss, and T1/2 of
`1.9L/h/kg, 11L/kg, and 3.8h, respectively. Exposure in dogs after oral administration was significantly higher than
`that in rats and the oral bioavailability at 5mg/kg, administered p.o. as a water solution was 9.8%. Finally, the FIH
`study of compound (+)-1 confirmed the dose-proportionality in PK from 30 to 380mg doses (individual subjects
`dose-adjusted AUC(0–inf)s were similar) and no differences between CYP2D6 EMs (n=20) and PMs (n=4).
`5 Conclusion
`Page 6 of 20
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`As a result of our search for a new anti-emetic drug based on prototype 'CP-122,721', we have found a compound
`with high potency and selectivity (Ki=0.2nM) for the NK1 receptor in IM-9 cells and potent anti-emetic activity in the
`cisplatin (10mg/kg, ip)-induced anti-emetic ferret assay (vomiting: ED90=0.07mg/kg, sc) comparable with CP-
`122,721, while lowering the affinity for the CYP2D6 enzyme. Thus compound (+)-1′ CJ-17,493′ was identified as an
`anti-emetic candidate that addresses the PK issue of CP-122,721 in poor metabolizers (PMs).
`6 Experimental
`6.1 General
`Proton nuclear magnetic resonance (1H NMR) spectra were measured on a JEOL FX270 by using
`deuterochloroform or dimethylsulfoxide-d6, and proton chemical shifts are reported as δ values in parts per million
`(ppm) relative to tetramethylsilane as an internal standard. Spin multiplicities are given as s (singlet), d (doublet), t
`(triplet), q (quartet), dd (doublet of doublet), m (multiplet), and br s (broad singlet). Gas chromatography analysis
`was determined using Column: J&DB-1 30m×0.25mm (Film thickness 0.25mm), Carrier gas: He, 20psi (138kPa),
`Injector: split (ca. 100:1), T=250°C, Detector: FID: T=250°C, Oven 170°C (10min). The electron spray ionization
`mass spectra (ESI-MS) were obtained with Quattro-II triplequadrupole mass spectrometer (micromass, UK). Melting
`points were determined on a Yanagimoto micro-melting-point apparatus and were uncorrected. The results of
`elemental analyses were within ±0.4% of the theoretical values and reported only with symbols. Reactions were
`followed by TLC on Silica-gel 60 F254 precoated TLC plates (E. Merck) or NH2 HPTLC F 254s plates (E. Merck).
`Chromatographic separations were carried out on silica-gel (E. Merck, 70–230 mesh) using the indicated eluents.
`The color fixing agent of TLC plates was used Dragendorf's reagent in the case of the basic compounds.
`6.1.1 (2S,3S)-3-[(1R)-6-Methoxy-1-methyl-1-trifluoromethylisochroman-7-yl]methylamino-2-phenylpiperidine
`((+)-1)·dihydrochloride
`6.1.1.1 2-Bromo-5-methoxyphenethyl alcohol (24)
`To a stirred mixture of 3-methoxyphenethyl alcohol (50.0g, 0.33mol) and pyridine (29mL, 0.36mol) in anhydrous
`dichloromethane (560mL) was added bromine (20mL, 0.39mol) dropwise under nitrogen at 0°C. The organic
`solution was stirred at ambient temperature for 2h. To the mixture was added additional bromine (0.85mL,
`17.0mmol) at the same temperature to complete the reaction and the solution was stirred for 1.5h. The resultant
`mixture was quenched by addition of 10% aqueous sodium bisulfite (100mL), and extracted with dichloromethane.
`The organic extracts were washed with brine (100mL), dried over magnesium sulfate, and concentrated to give
`crude product 24 (76.0g, quant.).
`1H NMR (CDCl 3): δ 7.43 (d, J=8.8Hz, 1H), 6.83 (d, J=3.3Hz, 1H), 6.67 (dd, J=8.8, 3.3Hz, 1H), 3.91–3.81 (m, 2H),
`3.78 (s, 3H), 2.99 (t, J=6.6Hz, 2H). ∗OH (1 proton, not observed) GC retention time: 5.98min (compound 24),
`3.05min (3-methoxyphenethyl alcohol).
`6.1.1.2 2-Bromo-5-methoxyphenethyl chloride (25)
`To a stirred solution of compound 24 (150.0g, 0.66mol) in carbon tetrachloride (940mL) was added
`triphenylphosphine (190.0g, 0.72mol) portionwise at ambient temperature and the organic solution was stirred at
`84°C for 4h. After cooling the mixture to room temperature, additional triphenylphosphine (8.5g, 32.0mmol) was
`added to the solution and the reaction mixture was stirred at 84°C for 10h. The reaction mixture was filtered through
`Celite, and the filter cake was washed with carbon tetrachloride (5× 50mL). The combined filtrate and washings
`were washed with saturated aqueous sodium bicarbonate (100mL), brine (100mL), dried over magnesium sulfate,
`and concentrated in vacuo. The crude product was purified by flash chromatography (Silica-gel, 12×9cm, 2–3%
`ethyl acetate in hexane) to afford the compound 25 (281.0g, 85%), which was distilled (bp 133°C/1.9mmHg), to
`provide the compound 25 (247.0g, 75%).
`1H NMR (CDCl 3): δ 7.43 (d, J=8.7Hz, 1H), 6.83 (d, J=3.0Hz, 1H), 6.69 (dd, J=8.7, 3.0Hz, 1H), 3.79 (s, 3H), 3.73 (t,
`J=7.1Hz, 2H), 3.16 (t, J=7.1Hz, 2H).
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`Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'
`GC retention time: 5.64min (compound 25).
`6.1.1.3 6-Methoxy-1-methyl-1-trifluoromethylisochroman (26)
`To a stirred solution of compound 25 (80.0g, 0.32mol) in anhydrous tetrahydrofuran (1300mL)/hexane (450mL) was
`added n-butyllithium (230mL, 0.35mol) dropwise under nitrogen at −100°C (internal temperature). The reaction
`mixture was stirred at −93°C for 2h. To the reaction mixture was added a solution of 1,1,1-trifluoroacetone (37mL,
`0.42mol) in anhydrous tetrahydrofuran (120mL)/hexane (40mL) dropwise at −105°C to −70°C (internal temperature)
`for 40min using a dropping funnel and stirred overnight at −70°C to room temperature (The temperature was
`gradually raised to room temperature overnight). This was quenched by water (100mL), and extracted with
`dichloromethane. The combined organic extracts were dried over magnesium sulfate, and concentrated in vacuo to
`give a crude product. The ratio of desired compound and 3-methoxyphenethyl chloride was 3.9:1 as determined by
`1H NMR and GC. To remove the by-product (3-methoxyphenethyl chloride), the residue was diluted with toluene
`(530mL) and to the mixture was added 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) (37mL, 0.24mol). After the
`solution was stirred at 110°C for 16 h, aqueous 2M hydrochloric acid (200mL) was added to the stirred solution
`mixture at 0°C. The organic layer was extracted with ethyl acetate (500mL), washed with aqueous 2M hydrochloric
`acid (100mL), saturated aqueous sodium bicarbonate (100mL), brine (100mL), dried over magnesium sulfate, and
`concentrated in vacuo. The residue was passed through a short column (Silica-gel, 12×9cm, 5% ethyl acetate in
`hexane) to remove the baseline compound. The mixture was distilled to provide compound 26 (61.0g, 77%) (bp
`106°C/0.9mmHg) as a colorless oil.
`1H NMR (CDCl 3): δ 7.26 (d, J=8.9Hz, 1H), 6.81 (dd, J=8.9, 2.6Hz, 1H), 6.68 (d, J=2.6Hz, 1H), 4.14 (dt, J=11, 5.8Hz,
`1H), 3.90 (dt, J=11, 5.8Hz, 1H), 3.81 (s, 3H), 2.87-2.81 (m, 2H), 1.64 (s, 3H).
`GC retention time: 3.71min (compound 26), 3.07min (3-methoxyphenethyl chloride), 2.21min (3-vinylanisole).
`6.1.1.4 6-Hydroxy-1-methyl-1-trifluoromethylisochroman (27)
`To a stirred solution of compound 26 (71.0g, 0.29mol) in acetic acid (600mL) was added aqueous 48% HBr
`(300mL) and the mixture was stirred at 130°C for 13h. After the removal of solvent in vacuo, the reaction mixture
`was treated with aqueous 8M sodium hydroxide until the pH became 5 to 6. The resultant solution was extracted
`with ethyl acetate (2× 400mL) and the combined extracts were washed with brine (100mL), dried over magnesium
`sulfate, and concentrated in vacuo, followed by flash chromatography (Silica-gel, 15×20cm, 17% ethyl acetate in
`hexane) to afford the compound 27 (67.0g, quant.) as a colorless oil.
`1H NMR (CDCl 3): δ 7.22 (d, J=9.1Hz, 1H), 6.73 (dd, J=9.1, 2.6Hz, 1H), 6.63 (d, J=2.6Hz, 1H), 5.00 (s, 1H), 4.17–
`4.07 (m, 1H), 3.90 (dt, J=11, 5.8Hz, 1H), 2.84–2.74 (m, 2H), 1.64 (s, 3H).
`GC retention time: 4.26min (compound 27).
`6.1.1.5 6-Acetoxy-1-methyl-1-trifluoromethylisochroman (28)
`To a stirred solution of compound 27 (79.0g, 0.34mol) and triethylamine (