Note: Descriptions are shown in the official language in which they were submitted.
~'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;
'
.
.
WO91/06563 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
'
" ~ ' , ' .~
.
:. - . . , :
.
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.
.
. .
- ~'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 ,~ ~
TA3LE 2
~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
: '
.
.. , : , , , :
, :.
'' ~,
,
W 0 91/06563 PCT/EP90/01836
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
. _ . . .. _ _ .
~ .
II 110940
IV 700783
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
1 4
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
,: ' .: ' .
;
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
1 7
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
1 8
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
,:,. : . . . . ; :
, . . ...
-. .
,
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.
:
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~'O9l/06563 PCT/EP90/0lg36
2 0
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
2 2
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
- ,
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~'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.
.
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WO 91/06~S3 PCT~EP90/01836
2 5
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
2 6
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.
~ ,
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WO 9l~06563 PCT/EP~0/01836
2 7
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
' ~
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.
- '~'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~
,' ; ' ' ~ ' : - ,
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~'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
, ~ .: . ": ~ . . . .
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