Analogues of the C-terminal fragments of neurokinins with modifications at their C-terminal methionyl residue

Structure-activity studies

Receiied 8 April. accepted for publication 31 Jul) 1993

.4nalogues of SP+ I I have been synthesized in \$.liich the methionql residue is replaced successively by the Glu(OCH2CH3), Glu(0Bzl). Hse(CH3) and G I L I ( C O N H C H ~ ) residues. and analogues of NKA4-10 and NKB4-ro have been prepared in which the iiicthion~-l residue is rcplaccd by the Hse(Bz1) and Hse(CH3) residues, respectively. The SPI_I I analogues \\ere tested in three iri virro preparations representative of NK-I , NK-2 and NK-3 receptor types. Substitution of the SCH? group of the Met” side chain by the groups C O O C H I C H ~ and COOBzl has little affect on the agonist activity in NK-1 preparations, while in NK-2 the corresponding analogues are more potent than the parent octapeptide; that substituted with COOBzl being 8.2 times more potent than SP4-I l . In NK-3 preparations all analogues are weak agonists. The selectivity of all the analogues is reduced compared with the corresponding hexapeptide analogues. The sP4-1 I ana- logues, along with those of NK.A4_I,, and NKBJ-I, , . \\ere tested for their binding ability in the three recep- tor subtypes above. The SP+I I analogues show reduced affinit! for NK-1 receptors, while the NKA+lo and NKB.,-lo analogues have almost the same affinities as N K A and N K B for NK-2 and NK-3 receptors, re- spectively. The efl‘ect of the Iipophilicity of the Met” side chain, especially Ivhen a phenyl group is present in the side chain, at the NK-2 receptor is discussed. 0 hlunksgaard 1994.

Kej. wordy: analogues: binding assa? s: guinea pig ilc~itii: neurokinin .\: neurokinin B: trat colon; rat portal vein; sub- stance P

Tachykinins are a family of peptides which share the common C-terminal sequence Phe-X-Glg-Leu-Met- NH, (X = Phe, Val). It has been shown that the natu- ral mammalian tachykinins substance P (SP), neuro- kinin A (NKA) and neurokinin B (NKB) recognize at least three distinct receptor subtypes termed NK-1, NK-2 and NK-3, respectively (1). The above classifi- cation has been based upon the relative order of po- tency, in various bioassaqs, of the natural peptides. their fragments and synthetic selective agonists toivard one of the receptor subtypes (2, 3). The C-terminal fragments of SP (SPJ-II, SP6-11) retain substantial SP-

Abbreviations uscd arc in accordance nith the rules of the ICPAC- ILB Commission on Biochemical Nonienclature in Eiir. J . B k d m r . (1984) 138. 9-37:J. B i d Clww (1989) 264,663-673. Other abbre- viations arc: AcOEt. eth\I acet:ite: AcOH. acetic acid: Buc. [err- butjlosgcarbon>l. But. rerr-but>l: Brl. bcnrjl: Cpt-CI. l-o\o-l- chlorophospholane: DMF. .~..\-dimethjIfortiiamide: Fnioc. fluoren) lox) carbonyl: NMM..V-meth> Imorpholine: Ph. phcuhl: THF. tetralpdrofuran: T F 4 . trifluoroacettc acid: TLC. tlitti-1a~ cr chmi ia - tograph)

3 44

like activitj in most biological preparations (4-7), while the order of relative affinity in NK-1 preparations is SPLI I > sP6-1 I (2). Structure-activity studies using ei- ther the whole molecule of SP or its C-terminal frag- ments have revealed the importance of chain length and Met” residue for smooth muscle activity and that the common C-terminal part of tachykinins is critical for the activation of all receptor types (8- 12). Very recently several no\el agonists and antagonists have been syn- thesized which are either peptides resulting from ap- propriate modifications, including the Met residue, of the natural neurokinins or their C-terminal fragments (13- 18) or non-peptides (19) and are characterized by high potency and selectivity.

The above results, along with the results from our recent studies using the model hexapeptide [Omh]- SPO-l1 modified in the Met” residue (10, 12, 19, 20), prompted us to investigate the effect on activity of cer- tain of thc above modifications when applied to the SPi-1 I , NKAJ-Io or NKBl-lo fragments. Thus we have sjnthesized a series of analogues of sp4-11 (1) where the SCH? group of Meti’ has been replaced by

Neurokinin agonists

carboxylic-phosphinic anhydride method (22, 23). This method involves formation of the corresponding mixed phosphinic anhydride by reacting the Fmoc-amino acid derivative with 1-0x0- 1-chlorophospholane (Cpt-CI) (24) in the presence of NMM followed by aminolysis. N"-Amino protection was performed either with the Boc-group, which was then removed with HCl in ace- tic acid, or the Fmoc-group, which was rcmoved M ' i t h 20% piperidine in DMF. For the side chain protection the tert-butyl group was used and was rcmoved Lvitli HCI in acetic acid.

Structural modifications in the octapeptide H-Pro- Gin-Gin-Phe-Phe-Gly-Leu-Met-NH:! 1 involved re- placement of the SCH3 group of methionine by COOC~HS, COOBzl, OCH3 and CONHCH3, while in thc hcptapcptidcs H-Asp-Ser-Phe-Val-GIy-Leu-Met- NH2 and H-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 the SCH3 group of methionine was replaced by OBzl and OCH3, respectively. The resulting octapeptide ana- logues were tested in guinea pig ileum longitudinal smooth muscle preparation (GPI, NK- l), rat colon muscularis niucosae (RC, NK-2) and rat portal vein (RPV, NK-3). Equipotent molar ratios (EPMR) were expressed as the ratio ECso(test compound)! ECso(standard). The standards used were substance P methyl ester (SP-OCH3) in GPI, neurokinin A (NKA) in RC and neurokinin B (NKB) in RPV. Results are summarized in Table 1. All analogues, except 8, were tested for their abilities to compete for binding of [3H]-

COOCHXH3, COOBzl, OCH3 and CONHCH3, and analogues of NKA4-l" and NKB4-1" where the SCH3 group of Met'" has been replaced by OCH3 and OBzl, respectively. The synthetic analogues were tested in three different preparations representative of the pro- posed NK-I, NK-2 and NK-3 tachykinin receptors (1). Activities of SP4-11 analogues were determined in both functional and radioligand binding assays, while activi- ties of NKA4-10 and NKB4-10 analogues were mea- sured in binding assays only. Structure-activity corre- lations are reported.

RESULTS AND DISCUSSION

The analogues of the C-terminal octapeptide of sub- stance P were synthesized in solution by coupling the protected N-terminal hexapeptide acid Boc-Pro-Gln- Gln-Phe-Phe-Gly-OH (2) to the C-terminal dipeptides H-Leu-X-NH2 [ X = Glu(OC2Hs), Glu(OBzl), Hse(CH3) and Glu(NHCH3)I with the DCC/HOBt method. The analogues [Hse(Bzl)'"]-NKA4-1o (16) and [ Hs~(CH~)" ' ] -NKB~-~" (19) were synthesized ac- cording to the procedure described in Figs. 1 and 2. The fragment couplings were performed in solution with thc DCC/HOBt method, and the peptidcs used in frag- ment couplings were synthesizcd in solution stepwise by the REMA method (21). The Finoc-Scr(But)-OH and Fmoc-Asp(0Bu')-OH amino acid derivatives wcre coupled to the amino component by the mixed

Gly

I H

i REMA, i i HCl/AcOH followed by NMH, iii H2/Pd-C, iv DCC/HOBt, v Cpt-Cl/NMM, vi 20% piperidine in DYF, vii HCl/AcOH.

e(Bz1)

-OH

-NH2

-NH2

-NH2

-NH2

-NH 2

-NH2

-NH

-NH2

-NH2

-NH2

FIGURE 1

Sqnthcsis of [ Hsc(Bzl)"']-NKA4_,,,.

315

M. Antoniou and C. Poulos

i REH.4, i i H C 1 / . 4 c O H followed by NHY, i i i H Z / P d - C , iv D C C / H O B t .

v C p t - C l / H H H , v i 2 0 % p i p e r i d i n e i n DHF, vi i H C l / A c O H , v i i i N H 3 / C H 3 0 H .

FIGURE 2 Synthesis of [ H ~ ~ ( C H ~ ) " ' ] - N K B , _ I ~ , .

substance P to rabbit cerebral cortical membranes (NK- l), binding of the NK-2 antagonist ligand [3H]- GR100679 to rat colon membranes (NK-2) and binding of the NK-3 agonist ligand [ 3H j-senktide to guinea pig cerebral cortical membranes (NK-3). Affinity constants (pK, values) were determined. and the results are sum- marized in Table 2.

The above modifications in the C-terminal octapep- tide of SP were selected based on the effect that the same modifications had on the activitj, !!,hen they ap-

plied to the model hexapeptide [Orn6]-SPs-~ I (20) (10, 12. 19. 20). Thus the [Orn6,Glu(OCHKH3)11]-SP~-~ I

(21) analogue showed increased selectivity at NK-1 receptors, the [ Orn6,Glu(OBzl)1 '1-sp0-1 I (22) in- creased activity at NK-2 receptors, the [Ornh,Hse(CHJ)11]-SP6-1 1 (23) increased activity at NK-3 receptors and the [Orn6,Glu(NHCH3)I ']-SP6- 11 (24) increased selectivity for NK-1 receptors when compared mith the parent hexapeptide (Table 1).

All the analogues were full agonists at the NK-1

No. Pcptidc Equipotent rnolar ratios (EPMR)"

Guinea pig ileum Rat colon Rat portal vein NK- 1 NK-2 NK-3

1 7 8 9 10 20 21

23 24

7 7 _ _

SP,~Il [ Glu(OCH,CH)' 1-S PA. i 1

[Glu(OBzl)"]-SP,., I

[Hs~(CHI)"]-SP, . I I [Glu(NHCH,)"]-SPA I j

[ Orn"]-SP,,., [Om". Glu(OCH-CH ?)'I]-SPe., I [Orn". Glu(OBzi)llJ-SP,.., [Orn". Hse(CHx)ll]-SPc, [Orn". Clu( N H C H I]-SP,,~I I

7 9

2.9

13.1 19.2 2 6 6.1 7.1

15.2 26.1

- ii ._

94 13 11.4

I20 2950

148.1 1096

79 2 834

3073'

64.5b 3178 1417 293

> 3810' 2000

> 300W

692 > 494 I t

_ _

~ ~~~~

EPMR valuez for compounds 20-24 ha \c hccn takcn from ref\. 12. 19 and 20. and t h e numhcr of cxpcrimcnts is I I =4

I O " , , E ,,,.,, of NKR at 1 0 O p ~ . ? O " , E',,,,, of NKB at 100 phi.

3", E,,,,,, of NKB at 10 { I M .

I' Calculated from ref. 2.

cPartial agonist x F = 0.5.

346

Neurokinin agonists

TABLE 2 Biiidiizg affirzities of tuchykiiiin uiiulog~ies

No. Peptide pK, values,' ~ ~

NK- I NK-2 NK-3

1 [CIU(OCH~CH,)"]-SP~.I I 7.00 4.96 5.47 9 [Hse(CHT)" ] -SP4~] 6.42 5.04 6.85 10 [ Glu(NHCH3)' l]-SP+ I I 5.41 5.36 5.1 1 16 [ H s ~ ( B z I ) ~ " ] - N K A ~ ~ 5.62 6.89 5.85 19 [ H s ~ ( C H ~ ) " ' ] - N K B ~ . , ~ ~ < 5.68 5.68 6.90 26 Substance P 9.32 4.57 6.77 27 NKA 6.66 6.97 5.92 28 NKB 6.45 5.17 7.67 29 Physalaemin 8.82 - 6.34 30 Eledoisin 6.35 5.75 7.83 31 GR94800 - 9.00 6.00 32 G R 100679 5.80 9.45 6.10

L' Results are single experiments with data for each test concentration determined in triplicate.

receptor but less potent than the parent octapeptide. The rank order of potency was SP4-1 I > 7 > 8 > 9 > 10, which is similar to that of the corresponding hexapep- tide analogues. The only difference is that the isosteric analogues [Hse(CHs)"]-SP4-1 I (9) was more potent than the [Glu(NHCH3)"]-SP4_11 (lo), while in the hexapeptide series the opposite was found.

The most potent octapeptide analogue at the NK-2 receptor was 8, where the SCH3 group of Met" has been replaced by a benzyl ester group and it was 8.2 times more potent than the parent octapeptide. Com- paring the order of potency of the octapeptide ana- logues, 8> 7 > S P ~ - I ~ > 9 > 10, with that of the corre- sponding hexapeptide analogues, 22> [ Orn6]-SP6-l I

> 23> 21 > 24, we may assume that the analogues of the octapeptide and hexapeptide series had similar be- haviour. The only difference, in the above order of po- tencies, is that the ethyl ester analogue (7) is more potent than the parent octapeptide, while the corresponding hexapeptide analogue 21 was less potent than the par- ent hexapeptide.

In the NK-3 receptor type, all the analogues were very weak agonists with order of potency sp4-11 > 9 > 8 > 7 > 1 0 . It is interesting to note that the isos- teric analogue [Hse(CH3)"]-SP4-11 (9) was less potent than the parent octapeptide, in contrast to the corre- sponding hexapeptide analogue (23), which was more potent than the parent hexapeptide (Table 1).

These results further support previous observations that SP fragments have different sensitivities to modi- fications compared to SP and that modifications at the Met" residue have variable effects on activity and/or selectivity. It is also interesting to note that the selec- tivity of the SP4-11 analogues for NK-1 receptors with respect to NK-2 receptors was reduced compared to the selectivity of the corresponding hexapeptide ana- logues.

Comparing the binding affinities of the above ana- logues it is interesting to note that 7 showed selectiv- ity for the NK-1 receptor type. The isosteric analoguc [Hse(CH3)11]-SP4-~ I (9) maintained good affinity for the NK-3 receptor (less than 1 log unit less active than NKB), with low affinity for the NK-1 receptor. These results prompted us to investigate the effect on binding activity of the replacement of the SCH3 group of Metlo in NKB4-10 by OCH3 and in NKA4-10 by OBzl. The latter was selected, as the presence of the phcnyl ring in the side chain of methionine increased its apparent affinity for the NK-2 receptor. From Table 2 we can see that the analogue [ Hse(CH3)1°]-NKB~-1,, (19) has the same NK-3 binding affinity as the corresponding SP octapeptide (9) but also has very low activity at NK- 1 and NK-2 receptors, and it is almost equipotent to NKB (with a difference in pK, values less than 1 log unit). On the other hand, [Hse(Bzl)"']-NKA1-1o (16) was equipotent to NKA at the NK-2 receptor.

In conclusion, the chain length of the SP fragments and Met" side chain are important factors for activity at NK-1 receptors. As the chain length grows, the lipophilic character of the side chain of Met1' in SP and of Metlo in NKA seems to be an important factor for activity and affinity at the NK-2 receptor. This is more important when a phenyl group is present. The Met" side chain is not as important for activity at the NK-3 receptor as it is for the NK-1 receptor. Activity of NKB analogues at NK-3 receptors can be maintained or in- creased by replacement of sulfur by oxygen.

EXPERIMENTAL Chemistry Capillary melting points were determined on a Buchi SMP-20 apparatus and are reported uncorrected. Op- tical rotations were measured with a Carl Zeiss preci- sion polarimeter (0.005 "). Analysis by TLC was on

341

M. Antoniou and C. Poulos

precoated plates of silica gel F254 (Merck) mith the following solvent systems: Rfl chloroform-methanol (6: I), Rfi 1-butanol-acetic acid-water (4: 1: I) , Rfi 1-butanol-acetic acid-water-pyridine (30:6:24:20) and Rf4 chloroform-methanol-acetic acid (85: 10:5). The products on TLC plates were detected by UV light and either chlorination followed by a solution of 1"" starch-] ob KI (1: 1 v/v) or ninhydrin. Retention times ( t R ) of peptides were measured by RP-HPLC with a Supercosil LC-18 column (250 x 4.6 mm 5 pin) with the following solvent systems: (A) 0.1 O 0 TFA in water, (B)0.1o0 TFA in CH3CN, 904,,-10", (A:B) isocratic elution for 5 min and then a linear gradient 70-30", (A:B) to 10-90",, (A:B) for 25 min (for compounds 7-10), 70-30",, (A:B) isocratic elution for 5 min and then a linear gradient 70-30"" (A:B) to 40-60"" (A:B) for 20 min (for compounds 16 and 19), UV detection at 257 nm, flow rate 1 mL.'min. The elemental analyses of amino acid derivatives and dipeptides were within

0.40", of the calculated values. Methodology for amino acid analyses of the final products and for FAB mass spectral analysis has been previously reported (17). D M F was distilled immediately before use over CaH2.

Deprotectioii of the tert-butj,lo.~j,~Nrboii!,lgroiip. A sample (2 mmol) of the N"-Boc protected peptide was dissolved in 1 N HCI 1 N acetic acid (10 mL). After 1 h at room temperature the solvent was removed iii v m i o at 25 ' C, and the residue was solidified by the addition of drq ether. The resultant hydrochloride salt was filtered, washed with dry ether, dried ii? iucuo over KOH pellets and was then used in the coupling without further pu- rification.

Deprotection of 9TPuoreii~~lo.u~~crrrb~)iij~/ group. A portion of the N"-Fmoc protected peptide was dissolved in 20% piperidine solution in DMF. After 30 min at room temperature the solvent was removed iti wciio and the residue solidified with the addition of dry ether. The solid was filtered, washed with ether, dried and \vas used without any purification.

Geriernl procedures f i r coupliiigs

Procedure A . To a solution of N1-Boc protected amino acid (4.8 mmol) in T H F (8 mL) cooled to - 15 C was added N M M (4.8 mmol) followed by isobutylchioro- formate (4.8 mmol). After 2 min a solution of the hy- drochloride salt of the amino component (3 nimol) in D M F ( 5 mL) precooled to - 15 'C and neutralized nith NMM was added to the reaction mixture. Lvhich was left to stand at the above temperature for 3 h and then allowed to warm to room temperature. At the end the solvent was evaporated iii vnc~io, and the residue was dissolved in AcOEt which was then washed with 5", NaHCO3, water, lo", citric acid, bvater and dried (Na2SO4). The solvent was removed iii wcuo and the

348

residue was solidified with the addition of petroleum ether 60-80 C to yield the desired product.

Procedure B. A portion ( 1 mmol) the hydrochloride salt of the amino component was dissolved in D M F ( 5 mL), neutralized with N M M and allowed to react with a sample of Boc-Pro-Gln-Gln-Phe-Phe-Gly-OH (1 mmol) dissolved in D M F (10 mL) and preactivated at 0 'C for 0.5 h with HOBt (1.6 mmol) and DCC ( 1 mmol). The reaction mixture was left to stand for 2 h at 0 ' C and then for 24 h at room temperature, while the pH of the reaction mixture was adjusted to 7.5-8 nith NMM. The precipitated DCU was filtered, and the solvent was evaporated i~7 ~wcuo. Thc remaining residue was solidified by trituration with saturated NaHCOi, filtered, washed on the filter with water, lo"; citric acid, Lvater and then dried in L ~ U O over P ~ 0 5 .

Procedure C. A sample of NX-Fmoc protected amino acid derivative (1.5 equiv.) was dissolved in the mini- mum amount of D M F followed by the addition of N M M (1.5 equiv.) and Cpt-CI (1.5 equiv.) at -10 "C. After an activation time of 10 min, a solution of the hqdrochloride salt of the amino component (1 equiv.) in D M F precooled to - 10 ' C and neutralized with N M M \vas added to the reaction mixture, which was left to stand at the above temperature for 3 h and then allowed to bvarm up to room temperature. At the end the sol- vent cvas removed in ~ J N C U O and the residue was solidi- fied by the addition of ethanol. The solid was filtered, ivashed on the filter with water, 10'; citric acid, water and dried iii i w u o over PzOS. Reprecipitation from DMF-ether gave the desired product.

S! titketis of Boc-Pro-Gb2-Glii-Phe-Phe-Gly-OH (2). Ac- cording to procedure A and starting from Boc-Gln- Gln-Phe-Phe-Gly-OBzl (22), the corresponding hexa- peptide benzylester was synthesized, and upon hqdrogenolqsis over lo", PdjC in DMFIHrO (9:l) it yielded the acid 2. Overall yield 70"/,, m.p. 226-228 " C (decomp.), [XI? -33.4' (c 1, DMF), Rfi 0.62, Rf3 0.71, Rf4 0.1 1.

Sjwtheris of protected octnpeptide nrinlogues 3-6. A por- tion of the HCI.H-Leu-X-NH2 [X = Glu(OCH2CH3), Glu(0Bzl). Hse(CH3), Glu(CONHCH3)I was coupled to the acid 2 according to procedure B. The isolated product was reprecipitatcd from DMF/ether.

C H J ) - N H ~ (3), yield 7OoO, m.p. 222-224 "C, [ r ] ?

Boc-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(0Bzl)- N H i (4), yield 72",, m.p. 231-233 "C , [ r ] g -39.7 (c

Boc-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse(CH3)-

Boc-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(OCH2

-17.1 ' ( C 1, DMF), Rf, 0.79, Rf3 0.69, Rfj 0.71.

1. DMF), Rfi 0.75, Rf3 0.79, Rfj 0.74.

NH2 (5), yield 82",, m.p. 227-229 "C, [x ]E -27.9' (c

Boc-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(NHCH3)- 1. DMF), Rfl 0.10, Rf3 0.27, Rfj 0.56.

Neurokinin agonists

according to procedure C. Yield 85 y o , 1n.p. 209-2 1 1 ' C from DMF/ether, [XI? -25.3 ' (c 1, DMF), Rfl 0.59, Rf2 0.70, Rf3 0.75.

NH? (6), yield 74%, n1.p. 215-217 "C, [x]g -22.7 (c 1 , DMF), Rfi 0.18, Rf3 0.31, Rf4 0.33.

Preparation of H-Pro-Gln-Gln-Phe-Phe-Gly-Leu-X-NH2. A sample of Boc-Pro-Gln-Gln-Phe-Phe-Gly-Leu-X- NH2 (200-300 rng) was deprotected according to the general procedure described above. The deprotected octapeptides were dissolved in 1 M AcOH, filtered through a Millipore filter and lyophilized. They were purified by partition chromatography on Sephadex G25F (2 x 85 cm) with 1-butanol-acetic acid-water (4:1:5 vjv, upper phase). H-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(OCH2CH3)-

NH2 (7), yield 729;, m.p. 209-21 1 "C, [a12 -25.6' (c

nzjz 993 ( M + H) + ; amino acid analysis: Proo.97, GIu2.98, Phel .99, G l y l . ~ ~ , Leuo.98. H-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(OBzl)-NH2

(8), yield 7 6 2 , m.p. 218-220 "C, [r]? -45.1 O (c 1, AcOH), Rfi 0.23, Rf3 0.58, f R 23.0 min; FAB-MS rn/z 1055 ( M + H) + ; amino acid analysis: Pro0.98, GIu2.99, Phe 1.97, Gly I .OO, Leuo.98. H-Pro-Cln-Gln-Phe-Phe-Gly-Leu-Hse(CH3)-NH2

(9), yield 73"/,, m.p. 227-229 "C, [ z ] g -34.5 " (c 1, AcOH), Rf2 0.12, Rf3 0.47, t~ 20.0 min; FAB-MS m / z 952 ( M + H) + ; amino acid analysis: Proo.98, Glu1.99, Phei.ol, GlyI.oo, Leuo.97, Hse present but not measured.

H-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Glu(CONHCH3)- NH? (lo), yield 6 9 2 , m.p. 215-217 "C, [XI? -22.4 O

m,'z 977 ( M + H) + ; amino acid analysis: Pro0.96, Glu2.97, Phe1.99, Gly~.(~o, Leuo.99.

1, DMF), Rf2 0.35, Rf3 0.41, t K 19.0 min; FAB-MS:

(C 1, DMF), Rf2 0.31, Rf3 0.33, t R 23.5 min; FAB-MS

Sjwthesis of Boc-Phe- Val-Gb-OH ( I I ) . According to procedure A and starting from TosOH . H-Gly-OBzl the corresponding tripeptide benzylester was synthe- sized which upon hydrogenolysis over 10% PdjC in DMF/H20(9:l)yielded the acid 11. Overall yield 30"/,, m.p. 159-161 "C, [r]? -19.0" (c 1, DMF), Rf2 0.58, Rf3 0.59, Rf4 0.64.

Sjxtliesis of Boc-Phe-Phe- Val-Gly-OH (12). According to procedure A and starting from Boc-Phe-Val-Gly- OBzl the corresponding tetrapeptide benzylester was synthesized which upon hydrogenolysis over 10% PdjC in DMF/HiO (9:l) yielded the acid 12. Overall yield 73",, m.p. 139-141 'C, [a]:: -19.8' (c 1, DMF), Rf2 0.53, Rf7 0.51, Rf4 0.71.

Sjwthesis of Boc-Plie- Val-GIy-Leu-Hse(Bzl)-NH2 (13). Compound 13 was synthesized by coupling the acid 11 to the HCl.H-Leu-Hse(Bzl)-NH* (20) according to procedure B. Yield 81 yo, m.p. 198-199 "C from DMF/ ether, [ x ] E -32.4 (c 1, DMF), Rfl 0.43, Rf2 0.85, Rf3 0.84.

Synthesis of Fmoc-SetfBu-t)-Phe- Val- Gb-Leu-Hse(Bzl)- NH2 (14). Compound 14 was synthesized from 13

S.ynthesis of Fn7oc-Asp(OBu-t)-Se$Bu-t)-Phe- V d - G(i.- Leu-Hse(BzO-NH2 (15). Compound 15 was synthesized from 14 according to procedure C. Yield 81",, m.p. 215-217 " C from DMF/ether, [z ]S -23.8 ' (c 1, DMF), Rfi 0.73, Rf2 0.60, Rf3 0.65.

Synthesis of H-A sp-Se r- Ph e- Val- Glv- L eu- H ye(Bzl)- NH? (16). A sample of the heptapeptide 15 (200-300nig) was deprotected with 20% piperidine in DMF, accord- ing to the general procedure described above, followed by deprotection of the tert-butyl group with HCIlAcOH. The deprotected heptapeptide was dissolved in 1 M AcOH, filtered through a Millipore filter and lyo- philized. It was purified by partition chromatography on Sephadex G25F (2 x 85 cm) with 1-butanol-acetic acid-water (4:1:5 v/v, upper phase). Yield 56",, m.p. 201-203 "C, [ r ] c -18.8 O (c 1, DMF), Rf? 0.34,

amino acid analysis: Aspo 97, Sero 94, Phel (11, Val0 ~ ) h ,

Glyloo, Leu0 97, Hse present but not measured.

Rf3 0.67, fR 13.5 min; FAB-MS / H / Z 827 ( M + H)';

Synthesis of Boc- Phe- Phe- Val- Glv- Leu- Hse/CH q)-NH? (17). Compound 17 was synthesized by coupling the acid 12 to the HCI.H-Leu-Hse(CH3)-NH? (20) accord- ing to procedure B. Yield 790/,, m.p. 151-153 "C from DMF/ether, [a]? -22.7" (c 1, DMF), Rfl 0.68, Rf. 0.75, Rf3 0.77.

Synthesis of Fnzoc-Asp(0Bu-ti-Phe-Pke- Vol- GIis-Leu- Hse(CHj)-NHz (18). Compound 18 was synthesized from 17 according to procedure C. Yield 88"". m.p. 178-180 " C from DMFjether, [x]g -20.5 ' (0 1, DMF), Rfl 0.65, Rfi 0.72, Rf3 0.77.

Sytzthesis of H-Asp-Phe-Phe- Val-GliT-Leu-Hse(CH 1 ) -

NH2 (19). Compound 19 was prepared according to the procedure described for 16. Yield 59",, m.p. 162-

t~ 11.5 min; FAB-MS m/z 794 ( (M + H) + ; amino acid analysis: Aspo 98, Phel 98, Valo ~ 6 , Glyl 00. Leu0 OO, Hse present but not measured.

164"C, [x]E -26.4" ( C 1, DMF), Rfi 0.31, Rf? 0.62,

Bioassay. Bioassays for determination of agonist ac- tivity have been previously described in detail (1 9).

NK-1 receptor binding assay. Potency at the tachykinin receptor was measured using a 13H]-SP binding assaj in rabbit cerebral cortical membranes (25). Thc assay was performed essentially as described by Dam & Quirion (26). Cerebral cortical membranes were incu- bated with [3H]-SP (0.5 nM) in the presence of various concentrations of competing drugs at 22 " C for 40 min. Nonspecific binding was defined as that remaining in the presence of physalaemin (1 PM). The reaction was

349

M. Antoniou and C. Poulos

terminated by rapid filtration, and radioactivity bound to filters was determined. Competition curves were analysed using the curve-fitting program ALLFIT, and pKi values were determined.

NK-2 receptor binding cissa~~. Potency at the tachykinin NK-2 receptor was determined by measuring the abil- ity of test compounds to inhibit binding of [3H]- GR100679, a tetrapeptide NK-2 antagonist (27), to rat colon membranes. The assay was performed essentially as described for the [ 'HI-SP binding assay. Rat colon membranes were incubated with [ j H 1-GR100679 (0.5 nM) in the presence of various concentrations of competing drugs at 22 C for 90 min. Nonspecific bind- ing was defined as that remaining in the presence of the heptapeptide NK-2 antagonist GR94800 (1 pM) (28). The reaction was terminated by rapid filtration, and radioactivity bound to filters was determined. Compe- tition curves were analysed using the curve-fitting pro- gram ALLFIT, and pK, values were determined.

NK-3 receptor binding assaj~. Potency at the tachykinin NK-3 receptor was measured using a ['HI-senktide binding assay in guinea pig cerebral cortical mem- branes. The assay was performed essentially as de- scribed by Guard er crl. (29). Guinea pig cerebral cor- tical membranes were incubated with [ 3H]-senktide ( 1 nM) and a range of concentrations of competing drugs at 22 " C for 60 min. Nonspecific binding was defined as that remaining in the presence of eledoisin (10 pM). The reaction was terminated by rapid filtra- tion, and radioactivity bound to filters was determined. Competition curves were analysed using the curve- fitting program ALLFIT, and pK, values \vere deter- mined.

ACKNOWLEDGEMENTS

We thank Dr. S.J. Ireland and Dr. I . Beresford. Department of Neuropharmacolog!, Glaxo Group Research Ltd.. CK. for biologi- cal testing, and Prokssor R. Raniage. Department of Cheniistr). Universit) of Edinburgh. L K . for elemental anal!sis and FAB-hlS spectra.

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.\fed. Chf'iJJ. 27. 949-954

.Address:

Dr. Cori.sruritii?e Poirlos Department of Chemistry Uniscrsit! of Patras PiitrLis Grcece

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