J . Med. Chem. 1995,38, 934-941
934
Synthesis and Biological Evaluation of NK1 Antagonists Derived from L-Tryptophan Angus M. MacLeod,*,+Margaret A. Cascieri,* Kevin J. Merchant,' Sharon Sadowski,$Sarah Hardwicke,' Richard T. Lewis,' D. Euan MacIntyre,s Joseph M. Metzger,§ Tung Ming Fong,* Sara Shepheard," F. David Tattersall,' Richard Hargreaves," and Raymond Baker' Merck, Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K., and Merck Research Laboratories, Rahway, New Jersey 07065 Received September 7, 1994@
The 3,5-bis(trifluoromethyl)benzylester of N-acetyl-L-tryptophan (31,which was derived from the screening lead N-ethyl-L-tryptophan benzyl ester, has been used as a starting point to identify high-affinity substance P receptor antagonists with improved in vivo activity. Altering the ester moiety to a n amide or ether led to a substantial loss in binding affinity, but conversion to a ketone provided compounds with affinity comparable to the equivalent esters. A homochiral synthesis of the key intermediate amino ketone 15 was developed which allows its preparation on a large scale. From this intermediate a range of amine-containing acylamino derivatives were prepared with affinity optimized in the morpholinylbutyramide 161 which has a n IC50 of 0.17nM at the human NK1 receptor. In addition to improving affinity, the amino group also provided aqueous solubility for a number of these derivatives. When tested in vivo the quinuclidine derivative L-737,488(16i) was found to be a n orally active (ID50 = 1.8 mgkg) inhibitor of substance P-induced dermal extravasation in the guinea pig.
Introduction N-acetyl-L-tryptophan,l0 with high affinity at the human NK1 (hNK1) receptor, which we developed from a lead Substance P is an 11-amino acid peptide,l with the identified through screening the Merck sample collecsequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leution. The 3,5-bis(trifluoromethyl)benzylester 3 was Met-NH2, which has been implicated in a variety of shown to be a potent competitive substance P antagonist disease states including rheumatoid arthritis: migraine headache^,^ and asthma4 and is involved in the transwith an IC50 of 1.6 nM at the human NK1 receptor. It belongs to a class of mission of pain ~ i g n a l s . ~ Compound 3 was active in vivo, after intraperitoneal structurally related peptide neurotransmitters collecdosing, in suppression of substance P-induced dermal tively known as the tachykinins that bind to the extravasation in the guinea pig but was substantially neurokinin receptors NK1, NK2, and NK.3. Substance less potent when dosed orally. The poor oral activity of P binds t o all three of these receptors but with highest these compounds was attributed to the likely rapid affinity toward the NK1 subtype. The recent identificaenzymatic hydrolysis of the biologically labile ester of tion6 of CP-96,345(11,the first nonpeptide substance P 3,and our first approach to improving oral activity was antagonist, has made possible investigations into the to replace the ester with a stable bioisostere. In this pharmacology of the neurokinins that were not feasible paper we describe the synthesis and structure-activity with peptide antagonists and has confirmed the wide of amide, ether, and ketone analogues of these esters potential utility of this class of compounds. For exand demonstrate that the ketones have receptor binding ample, it has been demonstrated7 from studies pert o the esters, with oral activity in affinities equivalent formed in ferrets that some substance P antagonists are animal models of inflammation. In addition, we have highly effective antiemetics and may be useful in identified an auxiliary amine binding site which leads alleviating Cisplatin- or radiation-induced emesis in the to enhanced affinity and allows for improved aqueous treatment of cancer, as well as sickness caused by other emetogens. solubility in this class of compounds. In addition to its NK1 activity, CP-96,345 has been showns to bind with high affinity to L3H1diltiazem binding sites on the L-type calcium channel, and this may be responsible for some, but not all, of the functional effects seen with this compound. However, in an extensiong of the medicinal chemistry of 1, the piperidine-based compound CP-99,994(2) has been developed as another high-affinity NK1 ligand but with substan(1) CP-96,345 (2) CP-99,994 tially reduced calcium channel binding. Several other structurally diverse series of nonpeptide substance P antagonists have now been reported including esters of +
Department of Medicinal Chemistry, Neuroscience Research Cen-
tre. Department of Molecular Pharmacology and Biochemistry, Rahway, NJ. 8 Department of Cellular and Molecular Pharmacology, Rahway, NJ. 'I Pharmacology Department, Neuroscience Research Centre. @Abstractpublished in Advance ACS Abstracts, February 1, 1995.
0022-2623/95/1838-0934$09.00/0
(3)
0 1995 American Chemical Society
Evaluation of L-Tryptophan-DerivedNKI Antagonists
Scheme 1
IC (8)
(7)
=Reagents and conditions: (a) LiAlH4, THF, then BoczO, DMAF', CH3CN; (b) NaH, DMF, 3,5-dimethylbenzyl bromide; (c) HC1, MeOH; (d) AczO, pyridine.
Chemistry
Ether derivatives of L-tryptophan were prepared (Scheme 1)by reduction of N-Boc-L-tryptophan followed by protection of the amine groups t o give alcohol 4. Subsequent alkylation of 4 using sodium hydride and 3,5-dimethylbenzyl bromide gave ether 5 accompanied by substantial amounts of the oxazolidinone 6; no attempt was made to improve this synthesis because of the poor activity of the acetamide 8 (see below). Amide analogues 9 and 10 were made using conventional amide coupling reactions and are detailed in the Experimental Section. Synthesis of the equivalent ketones
Journal of Medicinal Chemistry, 1995, Vol. 38, No. 6 935
has been described in a preliminary communicationll and is detailed in Scheme 2. N-Boc-L-tryptophanwas activated using isobutyl chloroformate and coupled to N,O-dimethylhydroxylamineto give the Weinreb amide 11. This was reacted at low temperature12with lithium dimethyl methylphosphonate to give the keto phosphonate 12 which could be purified by chromatography but was more conveniently, after aqueous workup, condensed with 3,5-bis(trifluoromethyl)benzaldehyde to give the unsaturated ketone 13 as yellow crystals in 47% yield over the two steps. Reduction of the olefin was preferentially carried out under hydrogenation conditions using Wilkinson's catalyst and was followed by removal of the Boc protecting group with ethereal HC1 to give the amino ketone 15 in 96% yield from 13. Using this sequence, 50 g batches of 15 (hydrochloride salt) could be prepared in 36% overall yield from N-BOC-Ltryptophan with ee 90% as measured by 'H NMR of the diastereomeric camphanamide derivatives. The enantiomeric excess was routinely enhanced t o give homochiral material (within the limits of detection) by precipitation of the salt from hot methanol on addition of water. Acylation of amine 15 was variously carried out by carbodiimide-, carbonyldiimidazole-, or mixed anhydride-mediated couplings with acids or by reaction with acyl chlorides to give a range of amide derivatives, 16. Biology
A stable CHO cell line expressing the human NK1 receptor was used13 to determine binding affinities of compounds prepared in this series with [12511Tyr8substance P as radioligand. Inhibition of substance P-induced inositol phosphate accumulation in CHO cells expressing the human NK1 receptor was assayed as previously described.13 NK1 receptor binding assays with a Gln165Ala mutant were performed as previously described14and results compared with binding affinities for the wild-type receptor carried out under the same conditions. Substance P-induced plasma extravasation assays were performed in guinea pigs. Test compounds were administered by oral dosing 1h before a challenge with
Scheme 2
0
BocHN+l(OMe)2 0 0
=Reagents and conditions: (a) i-BuOCOC1, Et3N, DMF, then HNMeOMe, -15 "C; (b) LiCHZPO(OMe)z, THF, -78 "C; (c) 3,5bis(trifluoromethyl)benzaldehyde,KzC03, CH3CN; (d) Wilkinson's catalyst, EtOAc, Hz, 40 psi; (e) EtzO, HC1.
936 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 6
0.5 pmol of substance P injected into the dorsal skin of the animals. Leakage of plasma into the skin surrounding the sites of injection was determined by measuring levels of Evans blue dye which had been introduced intravenously prior to the agonist challenge. Cardiovascular effects of CP-99,994, CP-96,345, and 16g,i were measured in ferrets of either sex (approximate body weight, 1.5 kg). Anesthesia was induced with sodium pentobarbitone (40 mgkg ip) and maintained with sodium barbitone (300 mgkg ip). The trachea was cannulated, .and the ferrets were then artificially ventilated with room air. The femoral artery and vein were cannulated for the recording of blood pressure and the administration of test compounds, respectively.
MacLeod et al. Table 1. Human NK1 Receptor Binding
67 f 10 9 1467 f 450 22 f 3 10 1033 f 330 767 i 368 a Displacement of lz5I-labeled substance P from the cloned human NK1 receptor expressed in CHO cells. All binding data are reported as the mean f SD for n 23 determinations. 17a 1% 8
binding affinity. We have also shown18that the bicyclic ring system of 18 may be dismantled to give acyclic diphenylalaninyl ethers (19) which retain good binding affinity; the S-enantiomer of 19 has an IC50of 2.5 nM at the NKI receptor. Assuming that the benzyloxy
Results and Discussion In an earlier paper, we described a series of novel, nonpeptide substance P receptor antagonists based on esters of L-tryptophan. Optimal NK1 affinities within this class were achieved with benzyl esters substituted at both metu-positions with a lipophilic group, where the strongly electronegative trifluoromethyl was particularly preferred. Structural variations around the a-amino group generally had less of an influence on activity although acyl derivatives were superior to a basic amine at this position. In order to improve the in vivo activity of these compounds, we investigated two separate approaches toward replacing the ester group while attempting to maintain binding affinity. In the preceding paper we described analogues in which the ester and the a-acylamino group are incorporated into a single heterocyclic ring providing compounds with NK, activity somewhat lower (> 7-fold) in affinity than the related esters.15 In this paper we describe an acyclic approach to replacing the ester through relatively conservative modifications t o the reference N-acetyland N-benzoyltryptophanesters 3 and 17a,b (hNK1 IC50 = 1.6, 67, and 22 nM, respectively).
(17a) R = CH3 (17b) R = Ph
We have previously shown,16from binding studies on point mutants of the human NK1 receptor, that the benzyl group of the tryptophan ester antagonists interacts with the same residue on the receptor protein (His2651 as the benzyl group of a series of quinuclidine ether antagonists (18) developed in our laboratories.
This is consistent with the preference in both series for 3,5-disubstitution on the aryl ring to achieve optimal
CH3
Ph
(19)
groups of the three series of compounds represented by 3,18, and 19 can be superimposed in a model of receptor binding, our initial assessment of the tryptophan esters was that the ether type oxygen, rather than the carbonyl, would correspond t o the oxygen in compounds 18 and 19 and would be important for high-affinity binding. Surprisingly, the ether 8 turned out (Table 1)to be more than 10-fold lower in affinity than the corresponding ester 17a. Although there is a significant difference in the activity of these two compounds, it is not possible to rule out that this is due to the increased conformational flexibility of 8 introducing a larger entropy factor into the binding process. Amide analogues of the esters were prepared and proved t o be even less complementary to the receptor with affinities of both the secondary amide 9 and its N-methyl homologue 10 reduced more than 50-fold from the analogous ester l7b.
(9) R = H (10) R = CH3
In contrast to this, the N-acetylamino ketone 16a had binding affinity (Table 2) within 2-fold of the corresponding ester 3, suggesting that the ethereal ester oxygen plays no role in the binding of these compounds. The difference in activity of the amides, when compared with the esters and ketones, is quite striking and suggests either that the amides are unable to adopt the required binding conformation because of reduced freedom of bond rotation, or that the amide N-H or N-methyl adversely interacts with the r e ~ e p t 0 r . lIn ~ the preceding paper15 we described the activities of conformationally restricted hydantoin and diketopiperazine analogues of 10 which have binding affinities > 100-foldweaker than the corresponding ester 3. It is notable that two distinct cis and trans amide rotamers, in a ratio of 3:2, are observed in the DMSO-& lH NMR spectrum for 10 at 300 K. The signals for these rotamers coalesce between
Evaluation of L-Tryptophan-Derived NKI Antagonists Table 2. Human NK1 Receptor Binding
R
Compound
'Method of Preparation
CP 96,345 CP 99,994 3 16a 16b 16c 16d 16e
Table 3. Binding to Gln165Ala Mutant Receptor
hNKl lCsa(nMIb 0.45 0.5 1.6
-CH3 -CH2NMe2 -(CH2)2" '(CH2)3NMe2 -(CH2)4NMe2
16f
A B B B A
3.1 i 1.7 5.0 k 3.6 1.8 I 0.2 0.7 10.2 0.6 _+ 0.1
B
3.5 i 0.7
16g
< N
C
0.5 i 0.1
16h
