synthesis of a potent agonist of substance p by modifying the methionyl and glutaminyl residues of...

hi. J. Peptide Protein Res. 38, 1991, 350-356

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of

substance P Structure-activity relationships

KOSTAS KARAGIANNIS' , AMALIA MANOLOPOULOU' , GEORGE STAVROPOULOS' , CONSTANTINE POULOS' CHRISTOPHER C. JORDAN* and RUSSELL M. HAGAN'

'Department of Chemistry. University of Patras, Patras, Greece; 2Department of Neuropharmacology, Glaxo Group Research Ltd., Hertfordshire, UK

Received I 1 January, accepted for publication 10 May 1991

Analogues of [Om6]-SP,,, have been synthesized in which the Met'' residue is replaced by glutamate y-alkylesters. These analogues were tested in three in vitro preparations representative of NK- 1, NK-2, and NK-3 receptor types. Substitution of the SCH, group of the Met" side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonsists in NK-I and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH, group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH, at NK-I receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by y-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Om6]-SP,,, . Other y-alkyl esters of the glutamic acid reduce its biological activity.

Key words: analogues; y-alkyl esters; guinea pig ileum; rat colon; rat portal vein; substance P

The undecapeptide substance P (SP) is a mammalian tachykinin which evokes responses in a variety of pharmacological systems. It can be divided into two domains according to physical and chemical properties; the N-terminal portion which carries polar groups and the remainder of the molecule which is hydrophobic. From the latter, the C-terminal hexa-

Abbreviations used are in accordance with rules of IUPAC-IUB Commission on Biochemical Nomenclature in European J. Biochem. (1984) 138, 9-37; J. Biol. Chem. (1989) 264, 663-673. Other abbreviations are: AcOH, acetic acid; AcOEt, ethylacetate; Boc, tert.-butyloxycarbonyl; Bzl, benzyl; Cpt-C1, l-oxo-l-chlorophospholane; DCC, dicyclohexylcarbodiimide; DCHA, dicyclo- hexylamine; DCM, dichloromethane; DCU, dicyclohexylurea; DEAD, diethyl azodicarboxylate; DMF, N,N-dimethylforrnamide; HOBt, I-hydroxybenzothiazole; NMM, N-methylmorpholine; MeOH, methanol; THF, tetrahydrofuran; TLC, thin-layer chromatography; Z, benzyloxycarbonyl; (CH,O)Z, p-methoxybenzyl- oxycarbonyl.

peptide amide (SPCl1. H-Gln-Phe-Phe-Gly-Leu-Met- NH,) is the minimal peptide fragments that retains substantial SP-like agonist activity in most pharmacological tests (1). This applies to contraction of gastro- intestinal smooth muscle (1 ,2), promotion of salivaqy secretion (3) and to the hypotensive effects of this group of peptides (4). In neuronal preparations (rat spinal cord and rat superior cervical ganglion, in vitro), the C-terminal hexapeptide is somewhat more active than the parent undecapeptide (5,6). However, the N-terminal residues should not be considered redundant since they may contribute to the potency of the compound in some test systems, whilst in others (e.g. release of histamine from mast cells), there is an absolute requirement for the N-terminal basic residues (7,8). In view of the importance of the C-terminal hexapeptide for biological activity, this sequence should provide a basis for examining some aspects of the structure-activity relationships for tachykinin agonists.

350

Potent agonist of substance P peptides either with HCl in acetic acid or with catalytic hydrogenation.

Structural modifications in the model hexapeptide H-Om-Phe-Phe-Gly-Leu-Met-NH, (4) involved replacement of the methionyl residue by y-alkyl esters of glutamic acid Glu(OR), (R = CH,, C2H5, n-C,H,, n-C,H,, cC6H,, , t-C,H,). The resulting analogues were tested in the guinea pig ileum longitudinal smooth muscle preparation (GPI), rat colon muscularis mucosae (RC) and rat portal vein (RPV). Equi- potent molar ratios (EPMR), were expressed as the ratio EC,, Test Compound/ECw, Standard. The standards used were substance P methyl ester (SP-OCH,) in GPI, neurokinin A (NKA) in RC, and neurokinin B (NKB) in RPV. The results are summarized in Table I.

In GPI (NK-1 receptor type) all the analogues showed decreased activity to the parent hexapeptide but all were full agonists (maximum response not significantly different from that of SP-OCH,). This was in contrast to the t-butyl ester analogue, which was 2.7 times more active than the parent hexapeptide and approximately equiactive to SP-OCH:, . In the series of analogues 33-38, there is no obvious correlation between activity and lipophilicity of the position 11 side-chain. However the changes in potency observed within this series (e.g. in GPI) demonstrates that biological activity is sensitive to modifications at this site. Yet surprisingly, the group of compounds 34,36, 37, 39 which have rather similar activities in GPI (EMPR 6.4-9.5) contains the greatest range of structural variations of the ester group.

When the above analogues were tested in rat colon (NK-2 receptor type) the loss of potency relative to the parent hexapeptide was of similar magnitude to that observed compared on the GPI while decreased selec- tivity for all the analogues was observed compared to NKA.

The exception is the t-butyl ester analogue 38, which is more active than the parent hexapeptide. It is also worth noting that the potency of the analogues 33-38 is improved as the lipophilicity of the ester group increases leading finally to analogue 38, which is more potent than the parent hexapeptide. This i s in agreement with the behaviour of the y-benzyl ester analogue 39, which is even more potent that the t-butyl ester analogue 38, perhaps because of the influence of the benzene ring, which is able to contribute more strongly to binding than the neutral aliphatic groups.

From the above results it is evident that the structural requirements of NK-1 and NK-2 receptors for the methionine side chain are different and might be stereochemical. All the analogues may be characterized as SP-like rather than NKA-like peptides but their behaviour also shows that the lipophilicity of the side chain at position 11 may be an additional factor for successful interaction with the NK-2 receptor.

In rat portal vein (NK-3 receptor type) all the analogues showed very low agonist activity and lower

In previous studies (9), it has been shown that replacement of the SCH, group of the Met in the C-terminal hexapeptide of substance P by charged groups reduces the biological activity of SP,,I at NK-1 receptors in guinea pig ileum and at the NK-2 receptors in rat colon, while replacement by a benzyl ester group had little effect on the activity of SP6-ll in the first preparation but, in the second preparation, the activity was increased. The behaviour of the glutamate benzyl ester analogue prompted us to investigate other glutamate esters as replacements for Met" in the model hexapeptide analogue [Om6]-SP,,, (10). The synthesized analogues were tested in the guinea pig ileum, rat colon muscularis mucosae and rat portal vein, representative of the proposed NK-1, NK-2, and NK-3 subtypes of neurokinin receptor (1 1) respectively. Structure-activity relations are reported.

RESULTS AND DISCUSSION

The analogues of the C-terminal hexapeptide of substance P were synthesized in solution by coupling the protected N-terminal tetrapeptide acid Boc-Om(Boc)- Phe-Phe-Gly-OH (1 0) or Z-Orn(Z)-Phe-Phe-Gly-OH to the C-terminal dipeptides H-Leu-X-NH, [x =

nC, H,), Glu(O-nC,H,), Glu(O-t-C,H,)] either with the DCC/HOBt or the mixed carboxylic-phosphinic anhydride method (12,13). The latter method involves formation of the corresponding mixed phosphinic anhydride by reacting the peptide acid with l-oxo-l- chlorophospholane (Cpt-C1) (14) in the presence of NMM followed by aminolysis. We applied the above organophosphorus methodology as further examples of our work on the application of mixed carboxylic- phosphinic anhydrides in fragment couplings (1 3,15). As expected, it proved to be in our case more con- venient than the DCC/HOBt method because the crude products are obtained in high yield and purity and can be used further without any purification. These are the results of regiospecificity that the mixed phosphinic anhydrides show during aminolysis, while the sole byproduct is the phosphinic acid, l-oxo-l- hydroxyphospholane, which is soluble in water and easily removed during the work-up procedure. For the esterification of the y-carboxyl group of glutamic acid the SOCl,/ROH method (16) was used and was successful with methanol and ethanol, while for l-propanol and 1-butanol the DEAD method (17) gave much better results. For the conversion of the a- carboxyl group of glutamic acid derivates to the corresponding amides the mixed carboxylic-carbonic anhydride (1 8) and the DCC/+ NH; OBt (19) methods were used. The latter method, which was applied in the synthesis of 13, 14, and 17, gave much better yields (ca. 75%) while with the former method yields of 15 and 16 were much lower (ca. 50%). Final products were obtained by deprotection of the hexa-

Glu(OCH,), Glu(OC,H,), GlU(OCC6H,,), Glu(0-

35 1

K. Karagiannis et al. TABLE 1

Biological activity of SP,,, analogues

No. Peptide Equipotent molar ratios (EPMR)

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

1 SPI-II OCH, I

4 [o~61-sp6-ll 2.6 148.1 2000 33 [Om6 ,Glu(OCH,)" 1-SP,, I 44.0 1688 > 3000" 34 [Om6 ,Glu(OC, H,)"]-SP,, I 6.4 1096 > 3000" 35 [Orn6,Glu(OC3H,-n)"]-SP,, I 64.7 567 > 333' 36 [Om6 ,Glu(OC, H,-n)"]-SP,, I 5.9 212 >w 37 [Om6 ,Glu(OC6H, I -c)"]-SP,, I 9.5 1027 > 3000"

39 [Om6,Glu(OBzl)"]-SP,ll 7.1 79.2 -

2 NKA 1 3 NKB 1

38 [Om6,Glu(OC, H,-t)"]-SP,, I 0.95 138 1071

'Maximum effect compared to that of NKB: 33 28% at lOop~ , 34 20% at l O o p ~ , 35 12% at 3 0 p ~ , 3640% at 3 0 p ~ , 37 10% at 1 0 0 ~ ~ .

maximum effect compared to the parent hexapeptide, the exception being again the t-butylester analogue 38, which is a full agonist and I .9 times more active than the parent hexapeptide. The increased activity of the latter analogue is rather surprising, since the steric properties of the t-butyl group differ substantially from those of the SCH, group.

In conclusion, methionine in position 11 of substance P can be replaced by a glutaminyl y-t-butyl ester residue thus resulting in a very potent agonist when compared to either the parent hexapeptide or SP-OCH, in guinea pig ileum. This is the first example in the literature where replacement of methionine in a substance P fragment by an amino acid that does not contain sulphur at the &position in its side chain leads to an analogue with higher potency than the parent fragment at the NK-1 receptor sub-type. It appears that the role of the sulphur containing Met" side chain for activation of the NK-I receptor can also be ful- filled by the t-butyl ester group. At the other receptor sub-types the same groups also increase the potency of the parent hexapeptide but the analogue is consider- ably less potent than IWA and NKB.

EXPERIMENTAL PROCEDURES

Chemistry Capillary melting points were determined on a Buchi SPM-20 apparatus and are reported uncorrected. Optical rotations were measured with a Carl Zeiss precision polarimeter ( 0 . 0 0 5 O ) . Analysis by TLC was on precoated plates of silica gel 60 F254 (Merck) with the following solvent systems: Rf, chloroform- methanol (6 : I), Rfi I-butanol-acetic acid-water (4 : 1 : I), Rf, I-butanol-acetic acid-water-pyridine (30 : 6 : 24 : 20), Rf4 toluene-n-hexane-ethylacetate (7 : 3 : 20). The products on TLC plates were detected

by UV light and either chlorination followed by a solution of 1% starch - 1% KI (1 : 1) or ninhydrin. For column chromatography silica gel 60 (20-230 mesh ASTM, Merck) was used. For gel filtration Sephadex LH-20 with methanol as eluent and Sepha- dex G-15 with 0 . 5 ~ acetic acid were used. Partition chromatography was performed on Sephadex G-25F packed with the water phase and equilibrated with the organic phase in the following systems:

(A) I-butanol-acetic acid-water (4 : 1 : 5). (B) 1 -butanol-acetic acid-pyridine-water

(60: 1: 10:85). Retention times (tR) of peptides were measured by RP-HPLC with Lichrosorb RP-18, column 250 x 4mm 5pm with the following solvent system: A 0.06% TFA in CH3CN, B 0.06% TFA in H20, 8%- 92% (A : B) isocratic elution for 5 min and then linear gradient 8%-92% (A:B) to 52%-48% (A:B) for 20 min, UV detection at 218 nm, flow rate 2 mL/min. The elemental analyses were within f 0.40% of the calculated values. Amino acid analyses of the final products were performed on an LKB 4400 amino acid analyzer. Samples were hydrolyzed by boiling in 6 N HCI containing 0.1% phenol at 110' for 18h in evacuated sealed ampules. Methodology for FAB mass spectra analysis has been previously reported (9). DCM and DMF were distilled immediately before use over CaH, . Boc-Glu(OcC, H, , )-OH DCHA and HCI H-Glu(0Bu-t)-NH, were purchased from Bachem.

Preparation of Boc-Glu(0R)-OH * DCHA

Boc-Glu(OCH,)-OH * DCHA (5). Glutamic acid was converted to the HCI salt of H-Glu(OCH3)-OH by the SOCl,/MeOH method [ 161. The hydrochloride y-methyl ester of glutamic acid was converted to Boc-Glu(OCH,)-OH by the S-Boc-2-mercapto-4,6-

352

Potent agonist of substance P DCM. The reaction mixture was then left to stand for 24 h at room temperature. The precipitated DCU was filtered and the solvent was removed in vucuo. The remaining residue was taken up in AcOEt, washed with 5% NaHCO,, water, 10% NaCl and dried (Na, SO,). The solvent was evaporated and the residue was solidified by the addition of petroleum ether 60-80 yielding the desired product. This method was applied for the synthesis of the amides 13, 14, and 17 (Table 2). Compound 15 shows low solubility in DCM and AcOEt, and thus its preparation was carried out in DMF according to the above procedure, while the work-up procedure was as follows: At the end of the reaction, the solvent was removed in vacuo and the residue was solidified upon the addition of 5% NaHCO,. The solid was filtered and washed on the filter with 5% NaHCO,, water and dried over P,05 in vacuo.

b. To a solution of Boc-Glu(0R)-OH (3mmol) in THF (10mL) cooled to - 15" was added NMM (3 mmol) followed by isobutylchloroformate (3mmol). After 2min a solution of 58% NH40H (0.6 mL, 3 mmol) precooled to - 15" was added and the reaction mixture left to stand at the above temperature for 1 h. At the end saturated NaCl was added and the mixture was extracted with AcOEt which was then washed with 5% NaHCO,, water and dried (Na,SO,). The solvent was evaporated in vucuo and the residue was crystallized with the addition of petroleum ether 60-80" to yield the corresponding amides which were recrystallized from the appropriate solvent. This procedure was applied for amides 15 and 16 (Table 2).

Preparation of dipeptides Y-Glu(0R)-NH, A portion of Boc-Glu(0R)-NH, (5 mmol) was deprotected with 1 N HC1 in AcOH for 1 h at room temperature. After evaporation of the acetic acid the residue was solidified by the addition of dry ether. The precipitated hydrochloride salt (HCl, H-Glu(0R)- NH,) was filtered, washed with ether, dried in vucuo over KOH pellets and was used further without any purification. The above hydrochloride salt was dissolved in DMF (3mL), cooled to - 15", neutralized with NMM and allowed to react with a sample of Ncr-protected leucine (8 mmol) dissolved in THF (15 mL) and preactivated at - 15" for 2min with isobutylchloroformate (8 mmol) and NMM (8 mmol). After 4 h the solvent was evaporated and the residue was partitioned in AcOEtlwater. The organic phase was washed with 5% NaHC03, water, 10% citric acid, water and dried (Na,SO,). Evaporation of the solvent yielded the desired product (Table 2).

dimethylpyrimidine method (20) and characterized as DCHA salt 5. Yield 37% (based on glutamic acid), m.p. 150-153", [a]: + 11.7" (c 2, MeOH), W2 0.82, Rf, 0.75.

Boc-Glu(OC,H,)-OH DCHA (6). This was prepared following exactly the procedure applied in the synthesis of 5. Yield 34% (based on glutamic acid), m.p. 124-126", [a]: + 6.5" (cl, MeOH), Rf, 0.75, Rf, 0.68.

Boc-Glu(OC,H,-n)-OH- DCHA (9) . A sample of Boc-Glu-OBzl (2.19 g, 6.5 mmol) was dissolved in anhydrous THF (10 mL) followed by the addition of 1-propanol (4.95 mL, 65 mmol). After cooling the solution to - 10' triphenylphosphine (1.7g, 6.5mmol) and DEAD (1.01 mL, 6.5 mmol) were added. Stirring was continued for 10min at - 10" and for 20min at room temperature. The above procedure for the addition of triphenylphosphine and DEAD was repeated twice adding a total of 19.5mmol triphenylphosphine and 19.5 mmol DEAD. The reaction mixture was stirred at room temperature for 18 h, then diluted with 30 mL etherlpetroleum-ether 60-80" (1 : 1) and left at 0" for 12 h. The precipitated triphenylphosphine oxide was filtered, the solvent was evaporated and the residue was dissolved in ether which was washed successively with 5% NaHCO,, water and dried (Na,SO,). The ether was evaporated and the oil residue was submitted to column flash chromatography on silica gel 60 (230-400 mesh ASTM Merck) using toluene as eluant. Fractions containing a compound with Rf, 0.31 were collected and the solvent was evaporated to yield 1.95 g (79%) of an oil which failed to crystallize and corresponded to the esterification product. Boc-Glu(OC, H,-n)-OBzl (7) as shown by comparing its IR spectrum with that of Boc-Glu-OBzl. The diester 7 (1.7 g, 4.48 mmol) was dissolved in 60 mL MeOH/H20 (14 : 1) and was hydro- genated over 10% Pd/C (200mg) for 3th while the progress of the reaction was followed by TLC. At the end the solvent was removed in vucuo yielding 1.22g (94%) of Boc-Glu(OC,H,-n)-OH (8) as an oil which failed to crystallize and was converted to its DCHA salt 9. Yield 70%, m.p. 124-126", [a]g + 8.98" (c4, DMF), Rf, 0.77, Rf3 0.68.

Boc-Glu(OC,H,-n)-OH - DCHA (12). The deriva- tive 12 was prepared according to the procedure described for 9 through the intermediates Boc- Glu(OC,H, -n)-OBzl (10) and Boc-Glu(OC,H, -n)- OH (11) which were obtained as oils. Yield 41% (based on Boc-Glu-OBzl) m.p. 132-133", [a]:: + 10.53" (c4, DMF), Rf, 0.80, Rf, 0.66.

Preparation of Boc-Glu(0R)-NH, a. To a solution of BooGlu(0R)-OH (3mmol) in DCM cooled to 0" was added m + + - O B t (3mmol) followed by a precooled solution of DCC (3 mmol) in

Synthesis of Z- Orn ( Z ) -Phe- Phe-Gly-0 H (26) Starting from HCl * H-Gly-OEt and using the mixed anhydride method described for the synthesis of the dipeptide Y-Leu-Glu(0R)-NH, we synthesized in a

353

K. Karagiannis et al. TABLE 2

Physical constants of amino acid and dipeptide derivatives Y-Glu(0R)-NH,

No. Y R m.p., recryst. solvent Yield, [a]:: Rfl Rf* "C (%.) (deg)

13 BOC CH, 126-127 AcOEt/petroleum 72 - 6.Y 0.51 0.67

14 BOC C2HS 128-129 AcOEt 71 - 2.2b 0.52 0.73 15 BOC n-C, H, 121-125 AcOEt/petroleum 50 - 1.1' 0.50 0.80

16 BOC n-C, H, 116-117 AcOEt/petroleum 48 - 2.T 0.56 0.81

17 BOC cyclo- 142-143 AcOEt 76 + 14.8' 0.59 0.73

18 Boc-L~u CH3 160-162 AcOEt 74 - 26.7' 0.53 0.70 19 (CH,O)Z-Leu C2 HS 170-172 AcOEtjpetroleum 77 -11.8' 0.55 0.76

20 Bw-L~u n-C3 H, 105-106 AcOEt/petroleum 64 - 24.W 0.58 0.72

21 Bm-Leu n-C, H, 125- 126 AcOEt/petroleum 61 - 24.8' 0.59 0.80

22 B O C - ~ U cyclo 133- 135 AcOEt/petroleum 71 - 24.2' 0.60 0.67

23 z-Leu t-C,Hg 136-137 AcOEt/petroleum 71 - I 1.8' 0.56 0.85

ether 60-80'

ether 60-80"

ether 60-80'

C6Hll

ether 60-80'

ether 60-80'

ether 60-80'

C6Hll ether 60-80'

' e l DMF. b ~ 2 CH,OH 'c2 DMF. d~ 3 CH,OH.

stepwise manner the tetrapeptide Z-Orn(Z)-Phe-Phe- Gly-OEt (25), m.p. 195-197", [a]: + 19.20" (cl , DMF), Rf, 0.77, Rf, 0.84. A portion of 25 (3.90g, 5mmol) was dissolved in DMF (30mL) and 1 N NaOH (SSmL, 5.5mmol) was added under stirring over a period of 15min at room temperature. The reaction was followed by TLC and was completed in 2 h. At the end the solvent was evaporated in vacuo and the residue was dissolved in H,O (50mL) and acifidied with 5% HC1 to pH 2 at 0". The precipitated acid 26 was filtered, washed with water, dried in vacuo over P,05 and recrystallized from MeOH/ether. Yield 3.05g (81%), m.p. 185-189", [a]; - 26.01" (cl ,

Anal. calc. for C4,Hq5N20,: C 65.50, H 6.03, N 9.31. Found: C 65.62, H 5.91, N 9.10.

DMF), Rf, 0.73, Rf3 0.66.

Preparation of protected hexapeptide analogues a. To a solution of Boc-Orn(Boc)-Phe-Phe-Gly-OH, 24, (1 mmol) in DMF (10 mL) cooled to 0" were added NMM (1 mmol) and 1 -oxo- 1 -chlorophospholane (1 mmol). After an activation time of 30min the hydrochloride salt [HCl - Leu-Glu(0R)-NH, ] (1 mmol) was added followed by NMM (1 mmol). The reaction mixture was left for 24 h at 0" and the pH was kept at 7.5-8 by adding NMM. At the end the solvent was evaporated and the remaining residue was solidified by the addition of saturated NaHCO, . The solid was filtered, washed on the filter with water, 10% citric acid, water and dried in vacuo over P205. This procedure was applied in the preparation of peptides 27, 28, and 31 (Table 3).

b. A portion (1 mmol) of HCl * H-Leu-Glu(0R)- NH, (R = n-C,H,, n-C4H9) was dissolved in DMF (SmL), neutralized with NMM and allowed to react with a sample of Boc-Orn(Boc)-Phe-Phe-Gly-OH (1 mmol) dissolved in DMF (10 mL) and preactivated at 0" for O.5h with HOBt (1.6mmol) and DCC (1 mmol). The reaction mixture was left to stand for 2 h at 0" and then for 24 h at room temperature, while the pH of the reaction mixture was adjusted to 7.5-8 with NMM. The precipitated DCU was filtered and the solvent was evaporated in vacuo. The remaining residue was solidified by trituration with saturated NaHCO,, filtered, washed on the filter with water, 10% citric acid, water and dried in vacuo over P,05. This procedure was applied for peptides 29 and 30 (Table 3), which were purified by column chromatography on silica gel 60 using as eluent CHCl,/MeOH (6 : 1). The above coupling procedure was also applied in the synthesis of 32 (Table 3), but the carboxyl component was the tetrapeptide acid 26 and deprotection of the dipeptide Z-Leu-Glu(OC,H,-t)-NH, (23) was carried out with hydrogenolysis over 10% Pd/C in DMF/H,O (9 : 1). The product 32 was purified by recrystallization from DMF/ether.

Preparation of ff-Om-Phe-Phe-Gly-LX-NH, A sample Boc-Orn(Boc)-Phe-Phe-Gly-Leu-X-NH, (200-400mg) was deprotected with 1 N HCl in acetic acid for 1 h at room temperature. The solvent was removed in vacuo and the residue was solidified by the addition of dry ether. The solid was filtered, washed several times with dry ether and dried over KOH

354

Potent agonist of substance P TABLE 3

Physical constants of protected hexapeptides Y-Om( Y)-Phe-Phe-Gly-Leu-Glu(0R)-NH,

No. Y R m.p., Yield, % bl'd ,deg Rf, Rf2 Rf, "C c l DMF

21 BOC CH, 206-208 74 - 24.2 0.72 0.77 0.81 2a Boc CZH5 214217 73 - 28.5 0.56 0.80 0.78 29 BOC n-C, H, 211-212 52 - 19.2 0.67 0.83 0.86

31 BOC cyclo 212-21 5 71 - 30.9 0.60 0.76 0.82

32 2 t-CdH, 222-225 dw. 74 - 35.3 0.65 0.70 0.79

30 BOC n-C4H, 2 w 2 0 1 62 -21.4 0.68 0.86 0.90

C6Hll

pellets in vacuo. The deprotected hexapeptides were dissolved in water, filtered through a Millipore filter, and lyophilized. The products were purified by gel filtration on Sephadex G-15 (2 x 85 cm) using 0.5 M acetic acid as eluent, followed by partition chromatography on Sephadex G-25F (2 x 85cm) with 1- butanol-acetic acid-water (4 : 1 : 5 v/v, upper phase). Deprotection of the hexapeptide 32 was achieved by hydrogenolysis over 10% Pd/C in DMF/H,O (9: 1) for 53 h. The catalyst was removed by filtration and the filtrate was evaporated in vacuo. The resulting residue was diluted with water, filtered through a Millipore filter and lyophilized. This product was further purified by partition chromatography on Sephadex G-25F (2 x 85cm) with I-butanol-acetic acid-pyridine-water (60 : 1 : 10 : 85 by vol. upper phase) to yield 38. For physical constants see Table 4.

Bioassays Agonist activity at NK-1 receptors was determined from contractile responses of guinea-pig longitudinal smooth muscle (G.P.1) recorded under isotonic con- ditions, at 37", in the presence of atropine (1 p ~ ) , mepyramine (1 p ~ ) , methysergide (1 p ~ ) , and indo- methacin (1 p ~ ) . Agonist activity at NK-2 receptors was determined from contractile responses of the rat colon muscularis mucosae preparation (RC) in the presence of antagonists as described for the GPI.

Tissues were mounted in 2 mL organ baths and doses of agonist were added in volumes of < 220 pL (expo- sure time 20 s for GPI: 45 s for RC). Preparations were washed thoroughly between doses and the inter dose interval was 7 min (GPI) or 15 min (RC).

Experiments were conducted as 3 + 3 (GPI) or 2 + 2 (RC) assays against SP-OCH, or NKA respectively as standards using a randomized block design. Each concentration of agonist was tested four times. Data were analyzed by analysis of variance. Only those assays demonstrating significant regression of response against concentration, no significant deviation from parallelism and no significant deviation from linearity were used to calculate equipotent molar rations (EPMR).

Agonist activity at NK-3 receptors was determined from contractile activity in the everted rat portal vein preparation [21] (RPV) at 25". Isometric contractions (resting tension 0.5 g) were recorded in response to serially applied doses of agonist administered at inter- vals of 15 min. Concentration-response curves were established for standard (neurokinin B) and test compound and ECa values were determined.

Guinea-pig ileum and rat colon preparations were bathed in Tyrode solution of the following composition: (mM) Na+ 149.1, K+2.8, Ca2+1.8, M$+2.1, C1- 147.5, H2PO;0.3, HCO; 11.9, glucose 5.6, bubbled with 95% oxygen/5% carbon dioxide. Rat

TABLE 4 Physical constants of peptides H-Om- Phe- Phe-Gly-Leu-Glu(0R) -NH,

No. R Formula m.p. Yield [a]; TLC HPLC FAB-MS Amino acid analysisb "C (%) (deg) tR mlz

c I DMF Rf, Rf, (M + HI+ L ~ U Gly Phe -Glu

33 CH, C3,H,N80, 125-127 75 -34.8 0.27 0.57 19.0 740 1.01 1.00 1.98 0.96 34 C2H5 C28HSN808 129-131 76 -35.9 0.32 0.55 21.2 754 1.00 1.01 1.98 0.98 35 n-C,H, C,,H,,N,O, 220-225" 63 -26.3 0.36 0.59 22.5 768 1.01 1.00 1.94 0.98 36 n-C,H, C,H,N,08 126128 72 -30.2 0.39 0.65 23.6 782 1.02 1.01 1.96 0.99 37 c~cIoC,HII C4,H6zN,O, 145-148 71 -38.3 0.38 0.24 23.8 808 1.04 1.01 1.96 0.99 38 t-C4H, C,H,N,O, 128-130 69 -31.0 0.44 0.62 23.2 782 1.04 1.00 1.99 0.98

"Decomposition. present but not measured.

355

K. Karagiannis et al.

portal vein preparations were bathed in Krebs- Henseleit solution of the following composition: (m) Na+143, K+5.9, Ca2+1.25, M$+0.6, C1- 125.2, HCO; 25, H2P041.2, SO:-0.6, glucose 11.1 and bubbled with 95% oxygen/5% carbon dioxide.

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Dr. Constantine Poulos Department of Chemistry University of Patras Patras Greece

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synthesis of a potent agonist of substance p by modifying the methionyl and glutaminyl residues of...

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