CA2045494A1 - Reduced irreversible bombesin antagonists - Google Patents

Abstract

(57) Abstract A peptide of the formula (1) R-A B-C-Trp-Ala-Val-X-Y-T-W, wherein R represents a group or the formula 4-(ClCH2CH2)2N-C6H4-CH2CH(NHR1)CO-;3-(ClCH2CH2)2N-C6H4-CH2CH(NHR1)CO-;4-(ClCH2CH1)2N-C6H4-CO-: 3-(ClCH2CH2),N-C6H4-CO-; ClCH2CH2NHCO-; ClCH=CH-CO-, BrCH=CH-CO-, CH2=CCICO-, CH2=BrCO-(either cis or trans isomers); (a); CH=C-CO-; ClCH2CH2CH2N(NO)CO-; ClCH2CO-CH(R2)NHCO(CH2)2CO-; A repres-ents a valence bond, or a Gly, Leu-Gly, Arg-Leu-Gly, or Gln-Arg-Leu-Gly residue, B represents a valence bond or a Asn, Phe or Thr residue C represents a Gln or His residue, X represents a Gly or ala residue; Y represents a valence bond or a His(R3), his(R3). Phe, phe, Ser, ser. Ala or ala residue; T represents a valence bond, or a Leu, leu, Phe or phe residue; W
represents a group of the formula OR2, NH2, NH(CH2)4)CH3, NH(CH2)2C6H5, Met-R4, Leu-R4, Ile-R4 or Nle-R4; R1 re-presents a hydrogen atom, a Boc group or an acyl group, R2 represents a hydrogen atom, a linear or branched alyphatic chain having from 1 to 11 carbon atoms, a benzyl or a C1-C21 phenyl group, R3 represents a hydrogen atom or a Tos, Dnp or Bzl group, R4 represents NH2, NH-NH2 or OR2, one or more peptide bonds (CONH) are replaced by reduced peptide bonds (CH2, NH), and the pharmaceutically acceptable salts thereof and pharmaceutically acceptable salts are bombesin antagonists. Their preparation and pharmaceutical compositions containing them are also described.

Description

~'O91/06563 PCT/~90/~1836 R~DUCED I~RFVERSI~LE BOMBESIN ~NTAGON STS

The present invention rela~es to peptide derivat~ves, to pharmaceutical compositions containing them, to processes for their preparation, and to their application as therapeutic agents.
In this specification symbols and abbreviations are those commonly used in peptide chemistry (see Eur.J. Biochem. (1984~ 138, 9-37).
Consequently, the three~letter amino acid symbols dehote the L
configuration of chiral amino acids. D-amino acids are represented by small letters: e.g., ala = D~Ala. Other s~mbols and abbxevia-tions used are- AA, amino acid; Ac, acetyl; AcOEt, ethylacetate;
B~S, bombesin; Boc, t-butoxycarbonyl; BuO~, n-butyl alcohol; BOP, benzotriazolyloxy-tris[dimethylamino~phosphonium hexafluorophospha-te; Cab, [p-bisl2-chloroethyl)amino]b~nzoyl; dec., decomposition;
DCC, N,N'-dicyclohexylcarbodiimide; DCHA, dicyclohexylamine; DCI, N,N'-dicyclohexylurea; DMAP, 4-dimethylaminopyridine; DMF, freshly distilled dimethylformamide; DMSO, dimethylsulfoxide; Dnp, 2,4-di-nitrophenyl; EGF, epidermal grow~h factor; EtOH, ethyl alcohol; FA~
(or FD) MS, fast atom bombardment ~or ~ield desorption~ mass spectrometry; ECC, ethylchlorocarbonate; EI-MS, electron impact mass spectrometry; Et20, diethyl ether; 51p, L-pyro~lutamic acid;
h-GRP (or p-GR~), human (or porcine) gastrin releasing peptide;

HCl/AcOH, anhydrous HCl in glacial acetic acid; HOBt, l-hydroxyben~
zotriazole; I.D., internal diameter; HOSu, N-hydroxysuccinimide;

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2 PCT/EP~0/01836 Mel, [bis(2-chloroethyl)amino~- L-ph~nylalanine; MeOH, methyl ~lcohol; m.p., melting point; mod., mo~ification; n.d., not determined; Nle, L-norleucine; NMM, N-methylmorpholine; NMR, nuclear magnetic resonance; oSu~ N-hydroxysuccinLmidyl; Pd/C, palladium on charcoal; PE, petrole~ ether 40-70; RP-HPLC, reversed phase high performance liquid chromatography; SCLC, small cell lung carcinoma; TFA, trifluoroacetic acid; THF, tetrahydro-furan; TLC, thin layer chromatography; Tos, p-toluensulphonyl;
TsOH, p-toluensulphonic acid.
The capital letter psi - ~ - between two amino acids indicates an amide bond replacement by the fu~ction specified between the brackets.
The invention provides a peptide of the formula I:
R - A - B--C - Trp--Ala ~Val ~ X - Y ~ T--W
wherein R represents a group of the formula 4-IClCH~CH~)2N-C6H~-CH2CH~NHRl)CO-IpMel);

3-~ClCH2CH2)2N-C~H~-CH2CH(NHR1)CO-~mMel);

4-(ClCH2CH~)2N-C6H~-CO-(Cab); 3-(ClCH2C~12)~N-C6H4 CO-;
ClCH2CH~NHCO-; ClCH=CH-CO-, BrCH=CH-CO-, CH~=CClCO-, CH2=CBrCO- ~either cis or trans isomers);
CH2-CH-CH~-CO-; CH - C-CO-; ClCH2CH2CH~NtNO)CO-;
o ClCHzCO-CH(Rz)NHCO(CHz) 2C-;

,' .:, ~
,' ~ ':

~'091/06563 3 ~ PCT/EP90tO1836 A represents a valence bond, or a Gly, Leu-Gly, Arg-Leu-rJly~ or Gln-Arg-Leu-Gly residue, B represents a valence bond or a Asn, Thr or phe residue;
C represents a Gln or His residue, X represents a Gly or ala residue;
Y represents a valence bond, or a HiS ~ R3 ), his~R,), Phe, phe, Ser, ser, Ala or ala residue;
T represents a valence bond, or a Leu, leu, Phe or phe residue;
W represents a group of the formula OR2, NH2, NH(CH2)4C~,, NHICH2)2C6H~, Met-R4, Leu-R4, Ile-R4, or Nle-~;
R represents a hydrogen atom, a ~oc group or an acyl group having from l to ll carbon atoms.
R~ represents a hydrogen atom, a linear or bra~ched alyphatic chaln having from l to l1 carbon atoms, a benzyl or a phenyl group.
Preferred alyphatic chains which Rz may represent incl~de methyl, ethyl, n-propyl-, iso-propyl, n-butyl and iso-butyl groups.
R3 represents a hydrogen atom or a Tos, Dnp or Bzl group, and R4 represents NH2, NH-NH2 or ~2 In addition, one or more peptide bonds ICO~) are replaced by reduced peptide bonds ~CH2NH).
Preferred acyl group which Rl may represent are aliphatic acyl group such as acetyl, formyl, propionyl and butirryl or aromatic such as benzoyl optionally substituted by nitro, metho~y, amino group or halogen atoms.
Preferably in the formula I R represents pMel or Cab, Rl represents hydroqen atom, Boc or acetyl group, A represents a valence bond, B
represents a valence bond or phe residue, C represents a Gln residue, X represents a His(Dnp), His or Gly residue most preferably Gly, Y represents a valence bond, T represents a Leu residue, W represents a group of the formula Leu-NH~ or Nle-NH, and t~e r~ r~ e ~ r~ ~u~ n ~ ~

O91/06563 4 ~ j f7`. ~ ~ PCT/EP90/OIB36 Salts of these peptides with pharmaceutically acceptable acids are within the scope o~ the invention. Such acid addition salts can be derived from a variely of inorganic and organic acids such as sulfuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulfamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trlfluoracetic, benzoic, salicylic, gluconic, ascorbic and related acids.

The synthesis of the peptides of the invention may be accomplished by classical solution methods. The syn~hesis consists essentially of appropriate successive condensations o~ protected amino acids or pep~ides. The condensations are carried out so that the resulting peptides have the desired sequence of amino acid residues.
The amino acids and peptides, which can be condensed according to methods known in peptide chemistry, have the amino and carboxyl groups, not involved in peptide bond formation, blocked by suitable protecting groups capable of being removed by acid or alkali treatment or by hydrogenolysis.
For the protection of the amino group the followiDg protective groups may, for example, be employed: benzyloxycarbonyl~
t-butoxycarbonyl, trityl, f ormyl, trif luoracetyl, o-nitrophenylsul-phenyl, 4-methyloxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 3,5-dimethoxy-a-a'-dimethylbenzyloxycarbonyl or methylsulphon~le-thoxycarbonyl.
For the protection of the carboxyl group the following protective groups may, for example, be employed: methyl, ethyl, t-butyl, benzyl, p-nitrobenzyl or fluorenylmethyl, amide, hydrazide, t-butoxycarbonyl hydrazide or benzyloxycarbonyl hydrazide.

, . ... ... ..

Wo 91/06563 ~ PCT/EP90/01836 The hydrcxy functions of h~drox; amino acids and the imino Cunction of histidine may be protected by suitable protecting groups (throughout all the synthesis or only during a few steps) or may be unprotected. For the protection of the hydroxy function the following protective groups may, for example, be employed; t-butyl, benzyl, acetyl. For the protection of the imidazole imino function the following groups may, for example, be used: Z,4-dinitrophenyl, tosyl, benzyl. De-protecting reactions are carried out according to methods known per se in peptide chemistry.
The condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptidic linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide or an aetivated ester, or by direct condensa-tion between a free ami~o group and a free car~oxyl group, in the presence of a condensing agent such as dicyclohexylcarbodiLmide, alone or together with a racemization preventing agent, such as N-hydroxysuccinimide or 1-hydro~ybenzotriazole, or togeSher with an activating agent such as 4-dimethylamino-pyridine. The condensation may ~e carried out in a solvent such as di~2~hylformamide, dimethylacetamide, pyridine, acetonitrile, tetrahydrofuran or --N-methyl-2-pyrrolidone.
The fonmation of a reduced peptide bo~d is accomplished by condensation of an N-protected amino acid alde~yde with a C-protected amino acid or peptide in the prese~ce o a reduci~g agent, such as NaBH~CN. The aldehyde, in turn, is usually obtained by condensing an N-protected amino acid with N,O-dLmathylhydroxyl-amine, and reducing the resulting amide with a suitable reducing agent, such as LiAlH~.

.. . . .

WO9l/06563 ~ CT/EP90/0183h The reac~ion temperature may be from -30C to room temperature. The xeaction time is generally from 1 to 120 hours.
The scheme of syn.hesis, the protecting groups and condensing agents are selected so as to avoid the risk o~ racemization.

Bioloaical act~vitv The peptides of the present inve~ntion are endowed with potent antagonism versus "in vitro'` and "in vivo~ effects induced by bombesin, such as contraction of smooth musculature, modification of behaviour of central origin and mitogenesis.
Bombesin ~BS) is a tetradecapeptide of formula Glp-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-~et-NH~, originally isolated from the skin of a frog. The bioloyical activity resides in the C-terminal part of th~ molecule: BBS~6-14)nonapeptide is as active as the parent compound. The human counterpart of bombesin is a 27 amino acid peptide, known as gastrin-releasing peptide (h-GRP).
Bombesin and bombesin-like peptides display a number of biological ~ -activities (J.H. Walsh (1983) in ~'9rain Peptides'l, D.T. Krieger, M.J. Brownstein and J.B. ~artin (edsj, Wiley Interscience Publ., pp. 941-960), including autocri~e growth-promoting ef~ects on human small cell Iung carcinoma ISCLC) (F. Cu~ti~ta et a~. (1985) Cancer Survey, 4, 707-727), auto¢rine and/or para~ri~ stimulation of human prostatic cancer cell proliferation (M. ~olog~a e~ al~, Cancer, in press ) and modulation of the E~F receptor ~I. Zachary and E. Rozengurt (1985~ Cancer Surveys, 4, 729-76~)o .... ..
-, .

~ . .

U'O 91/06563 ~ PCT/EP90/01836 A bombesin a~tagonis~, by competins with the natuxal ligand for the receptor(s), would inhibit the triggering of the cascade of events leading to abnormal ceil proliferation.
Different approaches in this direction have been followed ~y different research groups. A series of C-terminal bombesin nona-and decapeptides, characterized by amino acid deletion, inversion or substitution, has been the object of a previous patent application by our side ~EP Patent Application n 89102283.2).
These peptides, however, like other ~S antagonists, usually show : -moderate a_finity for the ~BS recep~or.
The compouncs Oc the present invention, due to the ~lkylating moiety, display grea~er receptor zffinity than the parent peptides, and behave as receptor antagonists either when given in combination with bombesin or when administered 24 hours before kom~esin challenge. In addition, owing to the presence of reduced peptide bonds, water solubility and, in many cases, also antagonistic properties are increased.

Biolo~ l test results The binding affinity of the compounds o~ the present invention for the bombesin receptors was determined on mouse Swiss 3T3 :
fibroblasts (I. Zachary and E. Rozengurt ~l9851 Proc. ~atl. Acad.
Sci. USA, 82, 7616-7620) (Table 1).
The effect on mitogenesis was determined in quiescent and confluent Swiss 3T3 cells maintained in serum free m~dium (~.N.Corps et al ' " ~ ' , ' .~
.

:. - . . , :
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W091t06563 ~",~ PCTtEP90tO1836 (1985) Biochem J. 231, 781-785). In a first set of experiments, analogues were given alone or in combination with bombesin. In a second set of experiments, cells were pre-treated with the alkyla-ting peptides, washed, left at 37C for 24 hours and then challen-ged with bombesin. In both cases, DNA synthesis was evaluated as [H3]thymidine incorporation ~Table 2).
In addition, exposure to these peptides in the 0.1-50 ~M range was associated with significant reduction in the growth of SCLC cell lines (such as NCI-H345, NCI-N592, NCI-H69, NCI-H128), as well as of prostatic carcinoma cell lines (such as DU145 and~PC3) ~Table 3).
Parenteral administration of these peptides at doses ranging between 1 ng/kg - 100 mg/kg to nude mice was associated with significant growth reduction of the above mentioned transplanted human SCLC and prostatic carcinoma cell lines.

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- ~'091/06563 PCT/EP90/01836 ABLE l BINDING AE'FINITY OF BOMEESIN
AL~YLATING ANALOG~FS ON MOUSF SWISS 3T3 FIBROBLASTS

....
COMPOUND IC50 tnM)*

, . . . . _ .

I 839 + 178 II 28 + l III 2340 + 291 IV 2.3 + l.0 V 0.9 + 0.5 Ref erence peptides:

BBS 12.6 + 0.65 Spantide ll~00 ~pro2]Spantide l4000 [Leul3~tCH2-NH)Leul4~BB5 214 + 30 * mea~ value ~ S.E.M. ~

.
, ~ , - . , . ~ ~ , .
~-, . . .
.

~'0 91/06563 PCI/EP90/01836 1 0 ~ g ~; D ,~ ~

~3 ]THYMIDINE INCORPORATION IN MOUS~ SWISS 3T3 FIBRO~LASTS

COMPOUND FOLD INCREASE OVER BASAL VALUE ~ INHIBITION IN THE PRESENCE OF
25nM BBS
A
SnM 50nM 0.5~M 5 ~M 0.5~M S ~H 0.5~M S ~M
. . _ _ , . _ . .

l n.d. n.d. 1.8 1.7 54 ~ 5 75 ; 4 69 ~ 2 84 ~ 3 II n.d. n.d. 0.8 0.8 86 ~ 3 90 l 3 S5 l 5 86 ~ 6 III n.d. n.d. 0.8 0.7 34 ~ 21 86 ~ 1 46 l 14 61 _ 16 IV n.d. n.d. 1.1 1.3 79 ~ 7 S5 ~ 8 0 a5 ~ 7 V n.d. n.d. 1.1 0.9 83 ~ 8 85 ~ 7 0 39 ~ 7 Reference peptides:
BBS 3.0;1 Leu~(CH,-NH)Leu'^)BBS 1 1 ~ 29~10 ~ 56~4 0 0 A~ analogues are gi~en in combin~tion with ~BS
B- cells are pre-treated wi~h analogue~, washed, left at 37-C for 24 h and then challenged with BBS

: ' .
.. , : , , , :
, :.
'' ~, , 2 ~ é

~LE 3 "IN VITRO" ACTIYITY OF ALRYLATING ANALOGUE ON SCLC C~LL LIN~S

. _ .. .. ... .. . .... _ _ . . _ _ . _ COMPOUND I ~ 3o ( nM J
~CI-N592 NCI-H69 . _ . . .. _ _ .
~ .

Reference peptide :
Le~ (CH,-NH)Leu'~]BBS ~20 l,660 'O 91/06563 PCr/EP90/01836 1 2 ~ L~
The peptides of the formula 1, .herefore, find application in the therapy of human neoplasms which are modulated in their growth and progression by peptides of the GRP family, either directly or in concert with other growth factors.
In addition, these alkylating analogues can be used in the management of the clinical s~mptoms associates with these deseases and due to hypersecretion of GRP-like peptides.
The compounds of the invention can be administered by the usual routes, for example, parenterally, e.g. by intravenous injection or infusion, or by intramuscular, subcutaneous, intracavity and ~ntranasal administration.
The dosage depends on the age, weight and condition of the patient and on the administration route.
On the basis of the "in vitro" and "in vivo" data in mice it can be estimated that the therapeutic doses in humans will be in the range of 1 ng~Xg - 100 mg/kg, once to 6 times daily.
Moreover, the toxicity of the peptides o~ the present invention is quite negligible.
The invention also provides p~armaceutical compocitions containing a compound of formula ~I~ as the active substa~ce, in association with one or more pharmaceutically acceptable excipients.
The pha~maceutical compositions o~ the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form.
For instance, solutions for intravenous injection or infusion may contain as carrier, for example, sterile water or, preferably, they may be in the form of sterile aqueous isotonic saline solutions.

~'O ~1t06563 1 3 ~f!J~ PCT/EPg~/01836 Suspensions or solutions for lntramuscular injections may co~ain, together with the active compound, a pharmaceu~ically acceptable carrier, e.g., steriIe water, olive oil, ethyl oleate, glycols (e.g., propylene glycol) and, if desired, a suitable amount of lidocaine hydrochloride.
Furthermore, according to the invention, there is provided a method of treating neuroendocrine neoplasms (such as small cell lung carcinoma and prostatic carcinoma) or the clinical symptoms associate~ with these diseases in patients in need of it, comprising administering to the said patients a composition of the invention.

Chemistry Methods:

a) TLC was performed on pre-coated plates of silica gel 60 F_5~
(Merck~, layer thickness 0.2S mm, length 20 cm, wi~h the ' following eluents:
System A: n-butanol~acetic acid~water = 600/lS0/lS0 by volume System B: chlorofor~methanol~ 9~/l by volume System C: chloroform/methanol = ~0/lO by volume Syst = D: t~luene/ethyl acetate/acetic acidiw~ter =

lO0/lO/20/lO by volume.

.... . . ..

, ~O 91/06563 ~ ~ J~ r~ PCr/EP90/01836 b) Analy~ical RP-~PL. was performed on a Hewlett ~ackard Mod.
1084 apparatus on a LiChrosorb Hibar RP-18 column (Merck) 250 x 4 mm I.D., par.icle diameter 5 ~. The following eluents were used:
A= KH2PO~ 20 mM, pH 3.5/acetonitrile 9/l by volume;
~= KH2PO~ 20 mM, pH 3.5/acetonitrile 3/7 by volume.

The elution was programmed with a linear gradient from 60% to 90~ B over a period of 20 min tSystem A) or from 30 to 70% B
over a period of lS min ~System B), and then isocratically for lS min, with a flow rate of 1 ml~min.
The peptides were characterized by their retention time (RT).

c) Preparative RP-HPLC was performed using a Delta Prep 3000 apparatus (Waters) on a Deltapa~ column ~Waters), 300 x 19 mm I.D., particle diameter, 10 ~. The ~ollowing eluents were used:
A= 0.05% TFA in water;
B= 0.05% TFA in acetonitrile/water 7/3 by volume.
Flow rate= 24 ml/min; detection wav~length= 220 nm.
Elution methods are reported in the single examples.
In each case, fractions were checked by analytical RP-~PLC aDd those showing a purity greater than 98~ were pooled. After remoYal of acetonitrile, the solutions uere lyophilized.

d~ Amino acid analysis was carried out on acid hydrolysates ~either at 110C for 22 h in h N ~Cl + 0.1% phenol or at 100C
, .

i ,: ' .: ' .

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91/06~;63 1 5 ~ n; ~ ~ PCr~EP90/01836 for 15 h in 3 N ~ercaptoethansulfonic acid, botn under N~).
Only natural æmino ~cid residues were determinea. Due to partial decomposi~ion in normal hydrolysis conditions, Trp was determined only in hydrolysates with mercaptoethansulfonic acid.

EXamP1e 1 Pr~paration of pMel-Gln-Trp-Ala-Val-Gly-H~slDnp~-Leu~(CH2NH)Met-NH. ~I).

Step 1 ~oc ~ (Ia) 43.45 g (200 mmol) of Boc-Val-OH were dissolved in 500 ml of anhydrous THF, cooled at -25C and treated with 22.48 ml (200 ~mol) of NMM, followed by 19.80 ml ~200 mmol) of ~CC. After stirring for 2 min at -12C, a pre-coo~ed solution o~ 67.47 g ~200 mmol~ of H-Gly-OBzl . TsO~ and 22~48 ml (200 ml) of NMM i~ 500 ml of anhydrous DMF was added. ~he reaction mixture was stirred for Z
hours at - 12-Co then filtered fr~m salts and the solutio~
evaporated u~der reduced pressure. The oily residue was dissol~ed in 1200 ml of AcOEt and the solutiol~ washed successively with 10%
citric a~::id (5 x 100 ml), brine, 596 Na~O, (5 x 100 ml) and brine to neutrality. ~fter dr~ing over Na2$0~, ~he solvent was evaporated and the residue purified by flash-chromatographY on silica gel, eluting with AcOE~/MeO~ 95~5. ~6.68 g t78% yield) of compound Ia were obta m ed from PE: m.p. 76-78-C; [~ 2~.0~ tC
1, MeOH); FD-MS: ~/z 365 (100, M~); RfD 0.70s RT~ ll.B.

, , 1~ :' ' "

:

~'O91/06563 6dr., ~ /EP9~/01836 ~ 6 Step 2 H-Val-Gly-O~zl . HCl_(~ b) 56.40 g (154.75 mmol~ of ~oc-Val-Gly-O~zl tIa) were made to react for 30 min at room temperature with 570 ml of 1.33 N HCl/AcOH. The solvent was removed under reduced pressure and the oily residue evapor2ted twice from DMF and washed with EtzO. 44.2 g (95% yield) of compound Ib were obtained as an oil: FD-MS: m/z 265 (100, MH-) as f ree base Rf ,~ O . 5 4; RT~ 6 . 7 .

~ .
Step 3 Boc-Ala-Val-Gly-O~ Ic) Starting from 27.81 g (147 mmol) of Boc-Ala-OH a~d 44.2 g (147 mmol) of ~-Val-Gly-OBzl . HCl (Ib), and operating as described for the preparation of Ia, but replacing AcOEt with CH2Cl2 in the washings, 54.82 g (68~ yield) of compound Ic were obtained from CH~Cl2/PE: m.p. 142-146~C; FD-MS: m/æ 436 ~100, ~;); R~ 0.26; RT~

9.4.

Step 4 H- ~ Cl_~Id) Starting from 27 g (62 mmol) of Boc-~la-Val-Gly-OBzl ~Ic), and oper~ting as descri~ed for the preparation of Ib, 22.65 g (98 yield) of compound ~d were obtained from Meo~JAcoE~/pE: m.p 178-181C; FD-MS m/z 3~6 (100, M~) as free base; R~ 0.53; RT
7Ø

, '0 9l/06563 PCT/EP90/0183b steD ~

~oc-Trp-Ala-Val-Glv-03zl (Ie) The condensation was carried out as described for Ia, starting from 18.41 g ~60.5 mmol) of Boc-Trp-OH and 22.50 g 160.5 mmol) of H-~la-Val-Gly-O~zl (Id). The crude product was then dissolved in DMF and precipitated by dropping the solution with stirring at 0C
into a 10% citric acid acqueous solution. The precipitate was filtered and washed with water to neutrality, then dried at 40%
over PzO5~ 35.70 9 (95% yield) of compound Ie were obtàined: m.p.
154-177C (dec.); FD-MS: m/z 621 (100, M -); Rf~ 0.10; RT~ 13,2.

S~ep 6 H-Trp-Ala-Val-Gly-O~ HCl (If~

33.75 g (54.28 mmol) of Boc-Trp-AIa-Val-Gly-O~zl (Ie) were ma~e to react for 30 min at room temperature wi~h 340 ml of 1.3~ N ~Cl/
AcOH, 34 ml of anisole and 17 ml of 2-mercaptoethanol. ~he solvents were removed under reduced pressuLe and the oily residue evaporated twice from DMF. The product was precipitated ~rom MeO~/PE and washed several times with PE a~d then with Et2O. 26.~5 g (88%

yield) of compound If were obtained: m.p. llB 122C; FD-MS: m/z ~21 (100, M~-) as free base; Rf~ 0.66; RT~ 5.8.

' ~O9l/06~63 PCT/EP90/01835 Step ~ t 3 fioc-~ln-TrD-Ala-'~al-Giv-OBzl (Ia) Starting from 11.73 g ~47.66 mmolJ o~ Boc-Gln-OH and 26.6 g (47.66 mmol) of H-Trp-Ala-Val-Gly-O~zl . HCl ~I~), and operating as described for Ie, 29.86 g t83~ yield~ of compound VII were obtained from ~JeOH/CH~C1~/Et,o/PE: m.p. 208-211C (dec.); FD-MS: m/z 749 (100, M- ): Rfc 0.51; RT~ 7.6.

Step 8 Boc-Gln-TrD-Ala-Val-Gly-OH_(Ih) 53 ml of a solution composed by 12 ml (318 mmol) of 99~ fcrmic acid and 33 ml ~300 mmol) of NMM in 1 1 of MeOH were added with stirring to a suspension of 3 g (4 mmol) of goc-Gln-Trp-Ala-val~Gly-oB
tIg) and 1.86 g of 10% Pd/C in 80 ml of DMF.
The reaction mixture was s~irred for 1 h at room temperature, the catalyst was fil~ered off and the solvent evaporated in vacuo. The residue was ground with AcOEt, giving 2.2 g (84% yield) of co~pound Ih: FD-~5: m/z 682 (l00, ~Na~~, 659 (40, M-~) Rf~ 0.52; R5~ 9Ø

,:,. : . . . . ; :
, . . ...
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, Y~'O 91/06563 PCI /EP90/01836 ., 19 Step 9 ~ s Boc-Leu-N~CHl?OCH~ (Ti) 24.93 g (100 mmol) of Boc-Leu-OH . H20 were dehydrated by evaporation from 200 ml of DMF, and dissolved in 350 ml of CHzCl~.
9.95 9 (102 mmol) of HCl . ~(CH,)OCH, and 3.05 g (2 mmol) of DMAP
were added with stirring at ~0C, followed by a few drops of DMF to obtain an almost clear solution. A solution of 20.65 g ~100 mmol) of DCC in 130 ml of CH2C12 and a solution of 11.24 ml (100 mmol) of NMM in 130 ml of CH2C12 were then dropped separately in~30 min, keeping the reaction temperature at 0C. After an additional hour at room temperature, the reaction mixture was filtered ~rom salts and DCU, and evaporated. The residue was d~ssolved in AcOEt, filtered from other DCU, and washed successively with 10~ citric acid (5 x 100 ml), 5% NaRCO3 (15 x 100 ml) and brine to neutrality.
After evaporation of the solvent, the oily residue was purified ~y flash-chromatography on silica gel, eluting first with PE~Et2O
85/15 (to remove a faster moving impurity), and ~hen wi~h PE/Et20 1/1. 17.38 g (57% yield) of pure compund Ii were recovered as an oil:EI- MS: m/z 201 (4, M-OtBu~, 173 t2, ~-~oc); R~ 0.44; RT~
10.4; RTD 19.1.
:

.

, ; :

:

~'O9l/06563 PCT/EP90/0lg36 Step l0 f~ ,~ "7 soc-Leu-H (Ii) 8.4 g (30.58 mmol) of Boc-Leu-N(CH3)0CH3 (Ii) were dissolved in 350 ml of anhydrous Et.O and made to react at 0C with 3.48 9 (91.74 rnmol~ of LiAl~ added portionwise in lS mi~. The ~eaction mixture was stirred for 15 min at 0C, ~hen 175 ml of AcOEt, followed by 700 ml of 10~ citric acid, were added, keeping the reaction temperature at 0C. After 30 min stirring the reaction mixture was extracted with AcOEt ~5 x 300 ml~, the combined organic layers were washed with 10% citric acid, then with brine to neutrality, and dried over Na25O~. Evaporation of the solvent gave 6.26 g t95%
yield) of crude oily compound Ij: EI-MS: m~z lB6 ~7, M-CHO); Rf~
0.38; RT~ 7.7; RTn 15.

Step ll Boc-Leu~(CH,NH)Met~NH~ (Ik) .
To a solu~ion of 6.14 g (28.52 mmol) of Boc-Leu-H ~ in 100 ml of 1% AcOH in anhydrous MeOH, 4.24 g (28.52 mmol) of HCl ~-Met-NH~ -were added, followed by 4.21 g (57 mmol) of Naa~C~ added portion-wise in 30 min at room tempera~ure. After 40 min additional !
~ "' `' `' " ' ` ~

2 1 PCT/EP90/()1~36 stirring the solut~on WaS ~veporated, ~he residue taken up in 300 ml of 5~ NaHCO, and the product extracted with AcOEt (5 x 100 ml).
The organic phase waS washed with brin~! to neutrality, dried over Na2so~ and concentrated. 5.30 g t53% yield~ of pure compound Ik were obtained: m.p. 124-126C; FD-MS: m/z 347 (100, M'~); Rf~ 0.16;
RT~ 6.2; RTD 12.4.

Step 12 H-Leu~(CH~NH)Met-NH, . 2 HCl ~Il) A solution of 1.04 g (3 mmol) of Boc-Leu~(C~NH)Met-NH2 (I~) in 10 ml of 1.33 N ~CllAcOH, containing 1 ml of anisole and 0.5 ml of 2-mercaptoethanOl~ was stirred or 20 min at rGom t~mperature.
Solvents were removed at reduced pressure and the oily residue was evaporated three times fro~ DMF and once from ~eOH, then it was triturated with AcOEt and Et~O. 1.44 9 (98.~ yield) of compound Il were obtained in two crops: EI-MS: m/z 247 (1, M~-), 203 (6, M-CONH2) as free base; Rf~ 0.58; RT~ 3.6~

}XO 91/06563 PCr/EP90/01R36 Step 13 Boc-His(Dnp)-Leu~(cH2NH)Met-NH? ~Im) 1.29 g (2.68 mmol) of Boc-His[Dnp)-OH iPrOH were evaporated three times from DMF to remove the isopropyl alcohol of crystallization, then were dissolved in 15 ml of DMF, cooled at -25C, and made to react with 0.30 ~1 (2.68 mmol) of NMM, followed by 0.27 ml ~2.68 mmol) of ECC. After 2 min stirring at -12C, a cold solution of 0.858 g ~2.68 mmol) of H-LeuY~CH2NH)Met-NH2 . 2 HCl ~ and 0.60 ml (5.36 mmol) of NMM in 15 ml of DMF were added. Th~ reaction mixture was kept for ~0 min at -12C, then ~or 30 min at 0C. The solvent was remove~ in vacuo and the residue was dissolved in AcOEt, washed with 5~ NaHCO~ an~ then brine to neutrality. After drying over Na~SO~ the solvent was evaporated and the oily xesidue purified by flash-chromatography on silica gel, eluting with AcOEt containing increasing amoun~ of MeOH ~from 0.5% to 10%). The product was recovered by evaporation of the solven~s and tritura-tion with Et20: 1.23 g (70.7% yield) of compound Im were obtained:
m.p. 70C (mod.) - 90C (dec.); ~D-MS: m/z 651 ~100, MH ); Rf~
0.57; RT~ 12Ø

- , ..

: .
: :

~'0 91/06563 2 3 PCT/EP90/01836 Ste? 14 H-His(DnD)-Leu~(CH?NH)Met-NH7 ._2 HCl (In) Starting from 1.16 g ~1.78 m~ol) of Boc-HistDnp)-Leu~(CH.NH)Met-NH2 (Im), and operating as described in step 12, 1.09g (98% yield) o' compound In were obtained from AcOEt: 110C ~mod.) - 200C (dec.~;
FD-MS: m/z 551 ~100, MH-) as free base; Rf~ 0.41; RT~ 4.2; RTn 7-1-Step 15 Boc-Gln-Trp-Ala-Val-Gly-~is(Dnp)-Leu~(CR2N~Met-N~2 L~L

156 mg (1.16 mmol~ of ~OBt, 239 mg ~1.16 mmol) of DCC, 660 mg ~1.06 mmol) of H-His(Dnp)-Leu~(CH2NH)Met-NH2 ~ 2 ~Cl (In) and 0.23 ml (2.12 mmol) of NMM were successively added to a solution of 700 mg (1.06 mmol) of Boc-Gln-Trp-Ala-Val-Gly-OH ~Ih) in 8 ml of DM~. The reaction mixture was stirred at 0C for 1 h and at room temperature overnight, then it was filtered and evapQrated in vacuo. The oily residue was dissolved in DMF and poured with stirring i~to a 5%
NaHCO, aqueous solution. The suspensio~ was filtered and the produc~ washed wit~ water to neu~rali~y. The crude ~aterial was purified by flash-chromatography in the eluen~ system composed by , - .

:~ .

~'091/06563 2 4 ~ /EP90/01836 AcOEt/MeOH 8/2. 820 mg (65% yielc) o~ compound Io were obtained from MeoH/AcoEt/Et2o 128C (mod.) - 145C (dec.); FA~-MS: m/z 1192 (23, ~H ); Rf~ 0.10; R~ 10.6.

Step 16 H-Gln-TrP-Ala-v-al-Glv-His(DA ~ ~ 2 HCl~(Ip) .
800 mg (0.67 mmol) of Boc-Gln-~rp-Ala-val-Gly-His~Dnp)-Leu~tcH2NH) Met-NH2 (Io) were dissolved in a mixture of 8 ml of 1. 33 N HCl/
AcOH, 0.8 ml of anisole and 0.4 ml of 2-mercaptoethanol, and made to react for gO min at room temperature. The solvent were removed ln vacuo and the residue was ground with Et~O, giving 0.765 mg (98%
yield) of compound Ip: m.p. 165C (mod.) - 220C (dec.); F~3-MS:
m/z 1092 (6, MH~) as free base; Rf~ 0.20; RT~ 4.5 RT8 12.1.

.

' :

: . . . . . . .

~, .

.
. .

' WO 91/06~S3 PCT~EP90/01836 Step l7 Boc-}~Mel-Gln-T~p-Ala-Val-C~ly-HistDnD)-Leur'(CH,NH)Met-NH._(I) 321 mg (O.64 m~ol) of Boc-pMel-OSu [prepared extemporaneously from 259 mg (O.64 mmol) of ~oe-pMel-OH (see our UK Pat. Appl. N~
8906000.9), 77 mg (0.67 mmol) of HOSu and 132 mg (0.64 ~mol) of DCC
in 5 ml of DMF) were added dropwise to a cooled solution tQC) of 500 mg (0.43 mmol) of H-Gln-Trp-Ala-Val-~ly-His(Dnp)-Leu~(CH2NH~
Met-NH2 2 HCl (Ip) and 0.096 ml ~0.87 mmol) of NM~ in l0 ml of DMF. The reaction mixture was stirred overnight at room tempera-ture, then it was poured dropwise into a 5% NaHCO, aqueous solu-tion. The suspension was stirred for l0 min at room temperature, then filtered and washed with water to neutrality. The crude product (520 mg, 78~ yield) was puri.fied by preparative ~P-XPLC, running a gradient from ~0% to 100% of eluent ~ in eluent A over 20 min, with a flow rate of 30 ml/min. 286 mg (45~ yield) of compou~d I were obtained: m.p. 140C (mod.) - 170C ~dec.~; AA ratios: Glu l, Gly 0.93 tl), Ala 0.98 tl), Val l.00 ll) (pNel, Trp, His(Dnp) and Leu~(CH2NH)Me~-N~ n.d.); FAB-MS: m/2 1478 (l0, MH~j; Rf~ 0.50;
RT ~ 2 7 . O .

~0 91/06563 PCT/EP9~/01836 Exam~le 2 _ _ .

Preparation of Boc-PMel-Gln-Trp-Ala-val-Gl~-His-Leu~(CH2NH)Met-NH2 (II) 180 mg (0.12 mmol) of Boc-pMel-Gln-Trp-Ala-Val-Gly-His~Dnp)-Leu ~[CH2NH)Met-NH2 (I) were suspended in 7.2 ml of 0.02 M RH2PO~
(brought to pH 8 with lN NaOH), then 7.2 ml of 2-mercaptoethanol were added. The resulting solution was stirred for _~ min at room temperature, then i~ was concen~rated in vacuo and poured dropwise into Et~O. The crude pxoduct was filtered and purified first by flash-chromatography on silica gel, in the solvent system AcOEt/
MeOH 7/3 v/v; then by preparative RP-HPLC, running a gradi~nt fro~
30~ to 90% of elunt B in eluent A over 20 m~, wlth a flow ra~c of 24 ml/min. 82 mg (52% yield) of compound I~ were obtained: m.p.
75C ~mod.) - 120 (dec.); ~A ratios: Glu 1, Gly 0.97 (1~, Ala 0.99 (1), Val 1.02 (1), His 0.94 (1) (p~el, Trp and Leu ~ (C~2NK)Met-NH2 n.d.); FAB-MS: m/z 1312 (7, MH ~; Rf~ 0.14: RT~ 18.1.

~ , ~; :
. :. , ; , WO 9l~06563 PCT/EP~0/01836 Example 3 Preparation of Ac-pMel-Gln-Trp-Ala-Val-Gly-His~Dnp)~L ~ Met-NHz_~III) Step 1 Ac-~Mel-OH (IIIa) A solution of 0.991 mg ~9 mmol) of a~etyl imidazole in i~ ml of DMF was dropped with stirxing into a solution of 500 mg ~ mmol) of H~pMel~OH (SIGMA) in 10 ml of DMF. The reaction mixture was stirred for S h at room temperature, ~hen the solvent was evapora-ted in vacuo. The crude material was purif ied through its DC~A
salt. 312 mg ~60~ yield) o~ compound III a were obtained from AcOEt/Et~O: m.p. 52-54C; EI-MS: m~Z 346 ~2, m~ ); Rfn 0.33; RT~
12.8.

St~p 2 Ac-pMel-Gln-Tr~-Ala-Val-Gly-His~D ~

70 mg (O.2 mmol) of Ac-pNel-OH wexe dissolved in 5 ml of DMF, then 233 mg (0.2 mmol) of H-~ln-Trp-Ala-Val-Gly-HistDnp~-Leu ICH2NH) Met-NHz 2 HCl IIP) were added, followed, at 5DC, by 0.0~6 ml l0.6 mmol) of NMM and 88.5 mg (O.2 mmol) of BOP. The reaction mixture was stirred at room temperature for 4 . 5 h, then it was poured ' ~
.

.

- '~'O 9l/06563 2 8 2~ 1PCT/EP90/~l836 dropwise into AcOEt. Tne crude product was fil~ered, ~ashed with AcO~t and purified by preparative R?-HPLC, running a gradient 'rom 60% to 90% of eluent B in eluent A over 40 min, with a flow rate of 24 ml/min. 128 mg of compound III (45~ yield) were obtained: m.p.
124-150C (dec.); AA ratios: Glu 1, Gly o.ag ~1), Ala 0.98 (1), Val 0.94 (1), (Trp, His(Dnp) and Leu~(CH2NH)Met-NH~ n.d.); FA~-MS:
m/z 1420 (16, M~); Rf~ 0.57; RTA 18.15.

ExamDle 4 Preparation of Cab-Gln-TrP-Ala-Val-Glv-His(DnP)-Leu~(C~NH)Met -NH7 tIV) Starting ~rom 0.20 g ~0.172 mmol) of H-Gln~Trp-Ala-Val-Gly-His(DnP) Leu~(CH2NH)Met-N~2 ~ 2 HCl ~Ip), 0.068 g (0.258 mmol) of [p-bis(2-chloroethyl)amino]benzoic acid (Cab-OH), 0.115 g (0.25~ mmol) of BOP and 0.057 ml (O.516 mmol) of NMN, and operating as described for the preparation of compound III, a crude m~terial was obtained, which was purified by preparative ~P HPLC, running ~ gradient from 30~ to 90% of eluent B in eluent A over 20 min, wi~h a flow rate Qf 24 ml/min. 0.138 g t60% yield) of compound III were ob~ained: m.p.
128-150C (dec.); AA ratios: Glu 1.02 (1), Gly 1, Ala 1.00 (1~, Val 0.95 (1) (Trp, His(DnpJ and Leu~(CH2N~Met-NH2 n.d.); FAB- MS: m/z 1336 (13, MH~ ~ 0.47; RT~ 19~9~

,' ; ' ' ~ ' : - , .

~'09l/06563 PCT/~P90/01~36 2 9 ~s~
Examvle 5 Preparation of Cab-Gln-Trp-Ala-Val-GlY-His-Leu~CH~NH)M_t-NH7 ~V) 0.20 g (0.15 mmol) of Cab-Gln-Trp-Ala-Val-Gly-His(Dnp)-Leu ~ CH2NH) Met-NH2 ~IV) were suspended in 10.5 rnl of 0.1 M KH2PO4 ~brought to pH 8.1 with lN KOH), then 10.5 ml of 2-mercaptoethanol were added.
The resulting solution was stirred for 30 min at room temperature, then it was concentxated in vacuo. The product was extracted with BuOH, and the organic layer was washed twice with water and evaporated. The residue was dissolved in MeOH and precipitated with EtzO. The crude product ~as purified ~y preparative RP-HPLC, running a gradient from 50% to 90~ of eluent ~ in eluent A ovèr 30 min, with a flow rate o~ 24 ml/min: 96 mg (55% yield) of compound V
were ob~ained: m.p. 128-150C (dec.); AA ratios: Glu 1.08 (1), Gly 1, Ala 0.90 ~1), Val 0.91 (1), Trp 1.10 ~1), His 1.09 (1) (Leu (CH2NH) Met NH2 n.d.); FAB-MS: m/z 1170 ~23, MH'); Rf~ 0.39; RT~
14.1.

Operating as described in ~he previous examples, th~ following peptides were also prepared:
H-pMel-~ln-Trp-Ala-Val-~ly-Leu~C~zNH)Leu-NH2 H-pMel-5ln-Trp-Ala-val-Gly-Leu~(cH2NH~Nle -NH2 Ac-pMel-Gln-Trp-Ala-Val-Gly-LeU~(cH2NH)Nle-NH2 Ac-pMel-phe-Gln-Trp-Ala-val-Gly-Leu~(cH2~)Nle-NH2 Boc-pMel-phe-Gln-Trp-Ala-Val-Gly-Leu~(CH2NH)Nle-NH2 Boc-pMel-Gln-Trp-Ala-Val-Gly-LeUy~ CH2NH)Nle-N~2 H-pMel-phe-Gln-Trp-Ala-val-Gly-Leu~(cH2NH)Nle-NHz ,, ~O 91/06563 3 ~ PCT/EP90/01836 Cab-Gln-Trp-Ala-Va1-Gly-Leu ~CH2NH)Met~NHz ~ ~ A~ rJ
Cab-Gln-Trp-Ala-Val-Gly-Leu ~(CH2NH)Leu-NH2 Cab-Gln-Trp-Ala-Val-Gly-Leu ~lCH2NH)Nle-NH2 Cab-phe-Gln-Trp-Ala-Val-Gly-LeU r (cH2NH,Met-NH2 Cab-phe-Gln-Trp-Ala-Val-Gly-LeU ~(CH2NH)Leu-NH2 Cab-phe-Gln-Trp-Ala-Val-Gly-Leu ~(CHzNH)Nle-NHz , ~ .: . ": ~ . . . .
, ~
,~ : ~ , ..

, :
', ' '

Claims (5)

-

1. A peptide of the formula I:
I
wherein R represents a group of the formula 4-(ClCH2CH2)2N-C6H4-CH2CH(NHR1)CO-;
3-(ClCH2CH2)2N-C6H4-CH2CH(NHR1)CO-;
4-(ClCH2CH2)2N-C6H4-CO-; 3-(ClCH2CH2)2N-C6H4-CO-;
ClCH2CH2NHCO-; ClCH=CH-CO-, BrCH-CH-CO-, CH2=CClCO-, CH2=CBrCO- (either cis or trans isomers);
; CH = C-CO-; ClCH2CH2CH2N(NO)CO-;
ClCH2CO-CH(R2)NHCO(CH2)2CO-;
A represents a valence bond, or a Gly, Leu-Gly, Arg-Leu-Gly, or Gln-Arg-Leu-Gly residue, B represents a valence bond or a Asn, phe or Thr residue;
C represents a Gln or His residue, X represents a Gly or ala residue;
Y represents a valence bond, or a His(R3), his(R3), Phe, phe, Ser, ser, Ala or ala residue;
T represents a valence bond, or a Leu, leu, Phe or phe residue;
W represents a group of the formula OR,, NH2, NH(CH2)4CH3, NH(CH2)2C6H5, Met-R4, Leu-R4, Ile-R4, or Nle-R4;
R1 represents a hydrogen atom, a Boc group or a C1-C12 acyl group, R2 represents a hydrogen atom, a linear or branched alyphatic chain having from 1 to 11 carbon atoms, a benzyl or a phenyl group, R3 represents a hydrogen atom or a Tos, Dnp or Bzl group, R4 represents NH2, NH-NH2 or OR2, one or more peptide bonds (CONH) are replaced by reduced peptide bonds (CH2NH), and the pharmaceutically acceptable salts thereof.

2. A peptide of the formula I according to claim 1 wherein R represents pMel or Cab, R1 represents hydrogen atom, Boc or acetyl group, A and Y represent valence bonds, B represents a valence bond or a phe residue, C represents a Gln residue, X
represents a His (Dnp), His or Gly residue, T represents a Leu residue, W represents a group of the formula Leu-NH2 or Nle-NH2 and the reduced peptide bond (CH2NH) is that between T
and W.

3. A pharmaceutical composition comprising a peptide according to claim 1 or 2 or a pharmaceutically acceptable salt of such a peptide in admixture with a pharmaceutically acceptable diluent or carrier.

4. A process for the preparation of a peptide according to claim 1 or 2, the process comprising condensing amino acids and/or amino acid derivatives in the desired sequence and/or peptide fragments containing these amino acids or their derivatives in the desired sequence to give the desired peptide, the end carboxylic acid group being activated for the peptide linkage and the remaining groups being protected and deprotecting the resultant compound and/or converting the resultant peptide into a pharmaceutically acceptable salt thereof.

5. The use of peptides according to claims 1 and 2 for the preparation of a pharmaceutical suitable for therapy of human neoplasms.