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Design, Synthesis and Biological Evaluation of Antagonists of LHRH by Criteria of Potency, Safety and SolubilityAnders Ljungqvista, Dong-Mei Feng3, Cyril Bowers*5, William A. Hookc, and Karl Folkers3 *a Institute for Biomedical Research, The University o f Texas at Austin, Texas b Tulane University School o f Medicine, New Orleans, Louisiana, U.S.A. c National Institute o f Dental Research, National Institutes o f Health, Bethesda,

Maryland 28092, USA

Z. Naturforsch. 46b, 1231 -1 2 3 6 (1991); received March 1, 1991LHRH-Antagonists, Antiovulatory Activity, Histamine Release, Solubility

Some analogs o f Antide and congeners with higher water solubility have been synthesized by substitutions in positions 1, 5 or 6 with hydrophilic residues. In position 1, D -3 -Q a l has been incorporated in four peptides and D -3 -P a l in one peptide. In positions 5 and 6, D and L -3-P al, PzAla and (DSer)Lys have been tried. In one peptide, D -(A cD Ser)L ys was substi­tuted in position 6.

Most o f the new analogs had lower AOA (antiovulatory activity) than the parent com­pounds but three potent analogs were identified. The first one,[N -A c-D -3 -Q a l',D p C lP h e2,D -3 -P a l3,c-PzACAla5,D -P icL y s6,ILys8,D -A la l0]-L H R H , had 55% AOA at 0.25 ng and 100% at 0.5 /ug. Its E D 50 for in vitro histamine release was 171 ± 17 ^g/ml which is an increase from 49± 4.8 >ug/ml for the parent compound with N - A c - D -2 -N a l [1],

The second analog,[N - A c-D -2 -N a l',D p C lP h e2,D -3 -P a l3,PicLys5,D -(D S er)L ys6,ILys8,D -A la 10]-L H R H , had 69% AOA at 0.25 /ug and 95% at 0.5 fig. This analog released somewhat more histamine than the parent analog featuring D -P icL y s6, the E D S0 being 18 //g/ml compared to 93± 11 for the parent analog.

The third analog is:[ N - A c - D -2 -N a t1, DpClPhe2,D -3 -P a l3,c-PzACAla5,D -P z A la 6,ILys8,D A la’]-L H R H . The AOA for this analog was 63% at 0.25 //g and the E D 50 for histamine release 88±6.4 ^g/ml.

histamine from mast cells [1], These features in­clude a group of hydrophobic amino acids at the N-terminal and strongly basic residues in positions6 and 8 , notably D - Arg6 ,Arg8. A prime example of this class of antagonists is [N -A c-D -2 -N a l',D -4 F -P h e 2 ,D -T rp 3 ,D -A rg6]- LHRH [2], It was recently shown that some antag­onists bind to rat peritoneal mast cells and mem­brane preparations and that the binding was relat­ed to the release of histamine [3].

We recently developed Antide (analog 1, Table III) which lacked strongly basic residues and which showed high potency and negligible histamine re­lease [4], Further evaluation of the anaphylactoid activity of Antide concluded that Antide “repre­sents a new generation of LHRH antagonists with an improved safety margin” [5].

Prolonged duration of inhibition of gonado­tropin secretion in overectomized monkeys using single [6 ] or multiple [7] doses of Antide has also been established.

The same group observed a long-term supres- sion of testosterone secretion in male monkeys after a single dose of Antide [8 ].

Introduction

Many potent antagonists of LHRH (the luteinizing hormone releasing hormone, /?Glu,His,Trp,Ser,Tyr,Gly,Leu,Arg,Pro,Gly-NH2) have structural features which cause release of

Abbreviations fo r the unnatural amino acidsAbu = 2-aminobutyric acid(AcDSer)Lys = N £-(N-acetyl-D-seryl)lysineAOPP = 2-(3'-amino-2'-oxo-l'-pyrrolidino)-

4-methylpentanoic acidc-PzACAla = c/s-3-(4-pyrazinylcarbonylaminocyclo-

hexyl)alanine(DSer)Lys = N £-(D-seryl)lysineILys = NMsopropyllysine2-Nal = 3-(2-naphthyl)alanine NicLys = N e-nicotinoyllysine3-Pal = 3-(3-pyridyl)alanine />ClPhe = 3-(4-chlorophenyl)alanine PicLys = N £-picolinoyllysine PzAla = 3-pyrazinylalanine 3-Qal = 3-(3-quinolyl)alanine* Reprint requests to Prof. K. Folkers.

Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen0932-0776/91/0900- 1231/S01.00/0

1232 A. Ljungqvist et al. ■ Design, Synthesis and Biological Evaluation o f A ntagonists o f LH RH

The mechanism(s) for this long-term action of Antide seems to include binding to serum proteins for a peripheral depot effect, and structural stabili­ty to enzymic cleavage. Binding to proteins result­ed from studies using a radioreceptor assay [9],

If a depot effect, regardless of mechanism, is at least partially responsible for the long duration of action of Antide, the relatively poor water solubili­ty at physiological pH may actually be an advan­tage. There are, however, reports that low water solubility has caused problems [ 1 0 , 1 1 ].

It was thus considered of importance to modify Antide and some potent analogs [4, 12] in order to increase their water solubility and to study how this would effect the biological activities of the re­sulting analogs.

We describe the synthesis and bioassays of some new analogs with improved water solubility.

Experimental

MaterialsBOC-Ser(OBzl), B O C -Pro, B O C -Leu and

B O C -D -A la were obtained from Peninsula Laboratories, Belmont, CA. Abu was obtained from Sigma Chemical Company, St. Louis, MO and it was converted to the BOC-derivative using standard procedures [13].

B O C -D -2 -N al, B O C -D -pC lP he, B O C -D - and L -3-Pal, BOC-ILys(Z) dicyclohexylamine salt, B O C -D - and L-NicLys and B O C -D -3-Q al were all provided by Dr. Narasimha Rao of the Southwest Foundation for Biomedical Research, San Antonio, TX. B O C -D - and L -PzA la were synthesized by literature methods [14].

B O C -A O PP was kindly provided by Dr. R. M. Freidinger, Merck, Sharp & Dohme, West Point, PA [15, 16], a -BOC - cis- D — and L-4-am ino- cyclohexylalanine were provided by Dr. Narasim­ha Rao, and were converted to the corresponding B O C -cw -D - and L-PzA C A la derivatives by acylation with the /»-nitrophenylester [17] of pyra- zinecarboxylic acid from the Aldrich Chemical Company, Milwaukee, WI, in DMF.

B O C -D - and L-PicLys were similarly pre­pared from « -B O C -D - and L -L y s and picolinic acid /^-nitrophenylester [18] in DMF. a -B O C -D - and L-(DSer)Lys, protected at the D -S er moiety by the Z group at the amino group and by the benzyl group at the hydroxyl function, were synthesized by acylation of B O C -D - and L -L y s in DM F by Z -D -S e r(O B z l)-O N p in the presence of 1-hydroxybenzotriazole. The

B O C -D -(A cD Ser)Lys, protected as the benzyl derivative at the D -S e r hydroxyl, was similarly prepared by the reaction between A c-D -Ser(O B zl)O N p and B O C -D -L y s .

All synthesized amino acids and intermediates were characterized by NM R and were homogen­ous on TLC.

The benzhydrylamine hydrochloride resin was purchased from Beckman Bioproducts, Palo Alto, CA. The dicyclohexylcarbodiimide was from Aid- rich and was distilled in vacuo before use. The dichloromethane was distilled from sodium car­bonate. All other solvents and reagents were reagent grade.

SynthesisThe peptides were synthesized by the solid-

phase method using a Beckman automated 990 peptide synthesizer. The protocol used was essen­tially as described [19], The peptide was cleaved from the resin with concomitant removal of all protecting groups by treatment with doubly dis­tilled HF( 1) at 0 °C for 1 h in the presence of about 10% anisole of/?-cresol. The HF was then evapor­ated, in vacuo, first by a water aspirator and then by pump vacuum overnight. The residue was then extracted 2 -3 times with ether in order to remove non-peptidic material. The crude peptide was sub­sequently extracted with aqueous acetic acid and the extract was lyophilized.

Purification and CharacterizationPurification was achieved by chromatography

on S i0 2 (EM, 230-400 mesh) with the solvent sys­tem «-butanol:acetic acid:water 4:1:2 or 4:1:5 (upper phase) followed by gel filtration on Se- phadex G 25 with 6 % aqueous acetic acid as the eluant. An alternative purification method was gel filtration as above followed by chromatography on Sephadex LH 20 with the solvent system water:«-butanol:acetic acid:methanol 90:10:10:8.

The purity was checked by TLC, amino acid analysis and HPLC.

The peptides gave single spots on TLC (EM, 0.25 mm SiO^) in four different solvent systems (Table I).

Amino acid analyses were carried out on a Beck­man 118 CL amino acid analyzer after hydrolysis in constant boiling HC1 for 24 h using standard procedures [19]. The unnatural amino acids were qualitatively determined with the exception of3-Pal which was quantified. The data are in Table II. The purity was further checked by HPLC using a Waters instrument with a 660 solvent pro-

A. Ljungqvist et al. • Design, Synthesis and Biological Evaluation o f A ntagonists o f LH R H 1233

grammer and a Vydac C 18 column. The flow rate was 1.5 ml/min and the absorbance was recorded at 210 nm. Different gradients of increasing con­centration of acetonitrile in 0.01 M K H 2P 0 4, ad­justed to pH 3 with H 3 P O 4 , were employed. All peptides were estimated to be 97 -99% pure in this system (data not shown).

Table I. TLC data.

# Rf \ Rf 2 Rf 3 RfA R( 5

2. 0.68 0.32 0.48 0.543. 0.70 0.35 0.74 0.844. 0.55 0.38 0.43 0.736. 0.62 0.38 0.47 0.777. 0.58 0.33 0.45 0.758. 0.62 0.35 0.53 0.809. 0.69 0.34 0.78 0.85

11. 0.74 0.35 0.48 0.6212. 0.70 0.37 0.39 0.5313. 0.68 0.32 0.76 0.8414. 0.67 0.31 0.76 0.8315. 0.61 0.39 0.42 0.7517. 0.68 0.35 0.71 0.7818. 0.69 0.38 0.73 0.6820. 0.64 0.33 0.50 0.8121. 0.71 0.41 0.55 0.8222. 0.66 0.37 0.53 0.81

Solvent systems:1. /j-BuOH : p y : H O A c: H 20 = 4:1:1:2;2. h-BuOH :HOAc: H20 = 4:1:2;3. /7-BuO H :p y :H O A c:H 20 = 40:1:10:20;4. n -B u 0 H :p y :H 0 A c:H 20 = 30:10:3:12;5. E tO A c: p y : H O A c: H 20 = 5:5:1:3.

Biological assaysAOA in rats was determined as reported [20].

The wheal area/10 jug of analog was calculated as described [4], The histamine release was assayed in rat mast cells as reported [21, 22], The ED 50 value reported is the concentration in //g/ml that releases 50% of total releasable histamine. The biological data are in Table III.

Results and Discussion

The strategy for design was to make single-resi- due changes in analogs with good potency in order to increase water solubility. Positions 1, 5 and 6

were chosen for these changes. Position 1 has D -2 -N al in five analogs selected for this study. D —2-Nal has been a dominant substituent in posi­tion 1 for some time [23], and its very lipophilic character made it a suitable candidate for substitu­tion. As a replacement, we chose D -3-Q al which isosteric with D -2-N al but much more hydrophilic. In one peptide, D -3-Pal was placed in position 1.

Positions 5 and 6 were selected because, in An- tide and its congeners, they are occupied by a new class of amino acids, acylated lysines and amino- cyclohexylalanines. Since in earlier analogs, these positions were often occupied by basic, hydrophil­ic residues [23], the relatively poor water solubility of Antide and its analogs may be, at least partly, attributed to these residues.

Table II. Amino acid analyses.

# Ser Pro Ala Leu Lys 3-Pal I Lys 2-Nal /jClPhe others

2. 0.90 0.98 1.03 1.00 1.88 0.90 + + 3-Qal +3. 1.00 0.94 1.04 1.00 1.05 0.96 + + + PzAla+4. 2.05 1.10 1.05 0.99 1.90 0.90 + + +6. 1.94 1.00 0.90 1.00 1.98 1.00 + + +7. 1.92 1.00 1.06 0.98 2.01 1.02 + + +8. 1.96 0.96 1.02 1.01 2.03 1.00 + + +9. 1.04 0.91 1.07 1.01 0.92 0.99 + + + PzAla +

11. 0.91 0.96 1.11 0.97 1.00 1.03 + + ACAla* + 3-Qal +12. 0.97 0.87 1.16 1.04 0.96 1.99 + + ACAla +13. 1.00 0.94 1.04 1.00 1.00 + + + PzAla + ACAla +14. 0.91 0.92 0.94 0.97 2.01 + + + ACAla+15. 1.93 1.09 1.07 1.00 1.01 0.90 + + + ACAla +16. 0.81 1.11 1.13 1.01 1.92 + + + ACAla +18. 0.98 0.98 1.00 1.02 1.01 + + + ACAla + PzAla +20. 0.92 0.96 1.10 1.94 1.06 + + 3-Qal +A bu +21. 0.89 0.97 1.12 1.00 1.02 + + + AOPP** +22. 0.94 1.01 1.05 0.95 1.02 + + AOPP + , 3-Qal +

* ACAla = 4-aminocyclohexylalanine; ** AOPP is hydrolyzed to yield two peaks, one o f which may be lactam acidwith a free amino group and other may be the acid with the lactam ring hydrolyzed.

1234 A. Ljungqvist et al. • Design. Synthesis and Biological Evaluation o f A ntagonists o f LH RH

Table III. Biological data for LHRH antagonists of the general sequence:N - A c - X aa1 .DpClPhe2,D - 3-Pal\Ser4,Yaa5.Zaa6,Waa7,I Lys8,Pro9,D - A la10- N H2.

# Xaa Yaa Zaa WaaAOA0.125

%/7/g0.25 0.5

E D S0 for hist, release ^g/ml

Wheal area mm2/ 10 //g

| a* D -2 -N a l NicLys D -N icL y s Leu _ 0 36 >300 132.7 ± 02. D -3-Q a l NicLys D -N icL y s Leu - - 0 88.8±4.03. D -2 -N a l NicLys D -P zA la Leu - 0 88 83.0±7.54. D -2 -N a l NicLys D -(D Ser)L ys Leu - 0 30 26±3.4 81.2± 2.75.a D -2 -N a l PicLys D -P icL ys Leu - 40 100 93 ±11 123.0 ± 06. D -2 -N a l (DSer)Lys D -P icL ys Leu - 0 13 22±4.7 106.9 ± 3.17. D -2 -N a l PicLys D -(D Ser)L ys Leu 20 69 95 18 103.9±5.28. D -2 -N a l PicLys D -(A cD Ser)L ys Leu - 0 - 53 ±11 97.2±2.29. D -2 -N a l PicLys D -P zA la Leu - 9 - 115.4±2.4

10.b D -2 -N a l c-PzACAla D -P icL ys Leu - 67 90 49±4.8 99.5±4.511. D -3 -Q a l c-PzACAla D -P icL ys Leu 17 55 100 171 ± 17 82.9±7.012. D -3 -P a l c-PzACAla D -P icL ys Leu - 20 - 11813. D -2 -N a l c-PzACAla D -P zA la Leu - 63 - 88±6.4 120.6±7.314. D -2 -N a l c-PzACAla D -3 -P a l Leu - 44 68 ± 1 .8 113.1 ±3.915. D -2 -N a l c-PzACAla D -(D Ser)L ys Leu - 11 117.8 ± 2.816.b D -2 -N a l PicLys c-D -P zA C A la Leu 29 73 100 28±7.5 122.8 ±5.717. D -2 -N a l 3-Pal r-D -P zA C A la Leu - 33 - 16 ± 2.0 101,7±4.318. D -2 -N a l PzAla c-D -P zA C A la Leu - 44 - 28±4.7 101,6± 2.219.b D -2 -N a l PicLys D -P icL ys Abu - 36 100 273±27 91.0± 5.420. D -3 -Q a l PicLys D -P icL ys Abu - 0 - 103.9± 3.721. D -2 -N a l PicLys AOPP - - 44 71 99.5±4.522. D -3 -Q a l PicLys AOPP — - 0 - 89.5±5.5

a From reference 4; b from reference 12; * Antide.

In positions 5 and 6 , the more hydrophilic ami­no acids, D - and L -3-Pal, PzAla, (DSer)Lys and D -(A cD Ser)Lys were used.

Substitutions in position 1

D -2-N al has been replaced by D -3-Q al in four peptides. In Antide (analog 1) the AOA decreased from 36 to 0% at 0.5 /ig (analog 2). The wheal area decreased considerably, from 132.7 to 8 8 . 8 mm [2],

When D -3-Q al was incorporated into analog10 [12] the AOA potency remained high, 55% at 0.25 /.ig and 17% at 0.125 /.ig. Even more interest­ing, however, may be the observation that the ED 50 for histamine release increased from 49 to 171 f.igjm\ and the wheal area is small, only82.9 mm [2], These data support the concept that a very lipophilic N-terminal promotes histamine re­lease. Analog 11 may have a high margin of safety.

Analog 19 with Abu in position 7 [12] and analog with AOPP [15, 16] in positions 6 -7 have also been substituted with D -3-Q al in position 1. The resulting analogs 2 0 and 2 2 were both inactive at

0.25 jug as compared to AOA values of 36 and 44% for the parent compounds.

In summary, four peptides have been substitut­ed with D -3-Q al in position 1. One retained its potency, and the other three showed no activity at the level tested. The analog that retained its potency had the largest lipophilicity o f the group, featuring c-PzACAla5 ,D -P icL ys 6 ,Leu7. Antide has NicLys5 ,D -N icL ys 6 ,Leu7. Analog 19 has PicLys5 ,D -P icL ys 6 ,Abu 7 and analog 21 has PicLys5, AOPP6 -7 which lacks the long acylated side chain normally present in position 6 . These three latter peptides lost their AOA upon replacement of D -2 -N al by D -3-Q al. Consequently, it seems that in order for the hydrophilic quinolylalanine to be effective, its hydrophilicity should be balanced by increased lipophilicity elsewhere in the mole­cule.

Analog 12 has the considerably smaller D -3-Pal instead of D -2-N al in position 1. The AOA de­creased to 20% at 0.25 //g, but taking into account the structural difference between D -2-N al and D-3-Pal, the value of 20% is noteworthy.

A. Ljungqvist et al. • Design, Synthesis and Biological E valuation o f A ntagonists o f L H R H 1235

Substitutions in position 5

Analog 6 has PicLys5 in the parent analog (5) [4] replaced by (DSer)Lys. This change caused a large decrease in potency, and the AOA was 13 and 0% at 0.5 and 0.25 jig, respectively, as compared to 100 and 40% for analog 5. The ED 50 for histamine release was decreased fourfold, from 93 to 22 jug/ ml. This result is not surprising since (DSer)Lys contains a basic amino group and PicLys does not.

The very potent analog 16 [12], with the c-D -PzA C A la moiety in position 6 has been mod­ified in position 5 with 3-Pal and PzAla (analogs 17 and 18). Both substitution reduced the potency to ca. one-half or from 73% to 33 and 44%, re­spectively, at 0.25 jig. The analogs did, however, retain considerable activity. The ED 50 value was reduced from 28 to 16 for the 3-Pal5 analog, but the corresponding value for the analog with the much less basic [24] PzAla remained unchanged.

Substitutions in position 6

D - PzAla has been incorporated in position 6 in Antide (analog 1), analog 5 and analog 10. In the case of Antide, a considerable increase in potency was noted at 0.5 jig , 8 8 vs. 36% (analog 3). At 0.25 jug, like Antide, analog 3 was however inac­tive. Its wheal area was notably small, 83 mm2.

In analog 5, the same substitution caused a sub­stantial decrease in potency, 9 vs. 40% at 0.25 jug (analog 9), but analog 13 was equipotent with its parent compound 10, 63 vs. 67% at the same dose level. It is evident, as has been often noted, that the same amino acid residue can cause widely different effects on AOA depending on the sequence in which it is introduced.

Of importance is the lowering of histamine re­lease by D -P zA la 6 introduction into analog 13; the ED 50 increased from 49 to 8 8 jug/m\.

In analog 14, D - 3-Pal6 replaced D - PicLys6 in the sequence of analog 10. This change lowered AOA moderately, from 67 to 44% at 0.25 jug, but the ED 50 for histamine release increased from 49 to

6 8 jig/mX. D -P zA la may be slightly better then D -3 -P al in position 6 .

D -(D Ser)Lys has been incorporated in position6 of the three peptides 1, 5 and 10. Analog 4, based on Antide, was equipotent, 30% AOA at 0.5 jug. The histamine release increased by a factor 10; the ED 50 being 26 jug/m\ compared with >300 jug/m\ for Antide. Compound 7, based on analog 5, showed increased potency, 69 vs. 40% at 0.25 jug. It had 20% AOA at 0.125 jug. The ED 50 value was about 18, that is it released more histamine than be parent compound, analog 5 with D - PicLys6. Analog7 with D -(D Ser)Lys6 had high potency. As a com­parison, analog 15, featuring the usually very ef­fective c-PzACAla5 had only 11% AOA at 0.25 jug. It seems that the (DSer)Lys moiety is effective only if very specific structural features are present in the rest of the chain [25]. The high potency of analog 7 is also remarkable in comparison with analog 6

which has (DSer)Lys in position 5 and was in­active at 0.25 jug. The latter analog was designed toward reducing histamine release by moving the basic residue from position 6 to position 5 accord­ing to Rivier et al. [26], The ED 50 value for analog 6 was, however, 22 compared to 18 for analog 7, that is they are very similar.

Analog 8 has D -(A cD Ser)Lys in position 6 . This peptide was designed in order to learn if his­tamine release would be affected upon acetylation of the serine amino group. This was the case. The ED 50 for 8 was 53, an increase from the value of 18 for analog 7. The wheal areas are also very similar,97.2 vs. 103.9 mm2. Analog 8 was inactive at 0.25 jig which indicates that the basicity may be necessary in this residue for good AOA potency.

Appreciation is expressed to Dr. Marvin Karten and the Contraceptive Development Branch of the National Institute of Health (Contract NOl- H D -1-3101) for the support of this research. We also grateful for the generous gift of B O C -A O PP from Dr. Roger M. Freidinger of Merck, Sharp & Dohme.

1236 A. Ljungqvist et al. • Design. Synthesis and Biological Evaluation o f A ntagonists o f L H R H

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