CA2179889A1 - Inhibitors of serine proteases, bearing a chelating group - Google Patents
Inhibitors of serine proteases, bearing a chelating groupInfo
- Publication number
- CA2179889A1 CA2179889A1 CA002179889A CA2179889A CA2179889A1 CA 2179889 A1 CA2179889 A1 CA 2179889A1 CA 002179889 A CA002179889 A CA 002179889A CA 2179889 A CA2179889 A CA 2179889A CA 2179889 A1 CA2179889 A1 CA 2179889A1
- Authority
- CA
- Canada
- Prior art keywords
- group
- peptide
- formula
- acid
- chelating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Optics & Photonics (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
Abstract
A trypsin-like serine protease inhibiting peptide or pseudo-peptide bearing a chelating group capable of complexing a detectable element, the chelating group being linked to an amino group of the peptide or pseudo-peptide such that the chelating group does not interfere with or prevent the binding of the peptide or pseudo-peptide to thrombin, preferably a compound of formula (I) or (II) in free form, in salt form or in the form of a complex, e.g. a complex with a detectable element, its preparation, its use as a medicament or, when complexed with a -emitting radionuclide, as a radiopharmaceutical, e.g. for thrombus imaging, and pharmaceutical compositions containing such a compound.
Description
WO 95/20603 P~ ~
217988g .~
INHIBITORS OF SERINE PROTEASES, BEARING A CHELATING GROUP
This invention relates to inhibitors of serine proteases, e.g. thrombin, and conjugates of such inhibitors used as thrombus imaging agents.
5 Thrombin and other serine proteases and inhibitors therefor are discussed in WO 94t20526, the disclosure o~
which is incorporated herein by reference.
It has now been found that peptidic and pseudo-peptidic trypsin-like serine protease inhibitors which are 10 transition state analogues and which comprise an amino group which can be modified with a chelating group without interfering with or preventing their binding to thrombin, provide novel compounds having interesting properties .
15 By transition state analogues are meant compounds as defined by A.J. Barrett et al. in Proteinase Inhibitors, Ed. Elsevier Amsterdam, 1986, p. 57.
The term "pseudo-peptide" or "pseudo-peptidic" as used herein refers to a peptide isostere in which the peptide 20 bond between 2 amino acid residues, whether natural or unnatural, is replaced with any isoste-ic group, e.g.
-CH2-NH-. The pseudo-peptide may comp~ e one or more such isosteric bonds.
.
Accordinçr to the invention, there is provided a 25 trypsin-like serine protease inhibiting peptide or 2 ~ 7 ~ 8 8 9 r~ 9 pseudo-peptide bearing a chelating group capable of complexing a detectable element, the rhf.l~t;nr group being linked to an amino group of the peptide or pseudo-peptide such that the rhPl~t;nJ group ao~es not , 5 interf ere with or prevent the binding of the peptide or pseudo-peptide to thrombin. ~, These ~ ~U~ are referred to thereafter as compounds of the invention.
The rhelAtinr group is linked by a covalent bond to the 10 amino group of the peptide or pseudo-peptide. The chelating group is preferably attached to the N-terminal amino group of the peptide or pseudo-peptide.
The chelating group may be attached either directly or indirectly, e.g. by means of a spacer group, to the amino 15 group of the peptide or pseudo-peptide. Preferably the chelating group is coupled to the peptide or pseudo-peptide or to the peptide-spacer or (pseudo-peptide)-spacer moiety by an amide bond.
Preferred _ ~u--ds of the invention are those comprising 20 a C-t~r~n;niql boronic acid derivative residue, particularly a C-t~rm;n~l boronic acid derivative of lysine, ornithine, arginine or an ~l ;rh~t;c amino alcohol. More preferred are those comprising additionally at least one unnatural -amino acid having 25 a hydrophobic side chain.
In one ~ t the present invention provides a compound of formula I
W~ I ~Z~[ (A3)q(A2)p(Al)o]r-NH - ~-B\ I ~
WO 9~l20603 % 1 ~ 9 8 8 9 P~
wherein Y1 and Y2, independently, are OH or F or, taken together, form a moiety derived from a dihydroxy compound having at least two hydroxy groups separated by at least two 5 connecting atoms in a chain or ring, said chain or ring comprising 1 to about 20 carbon atoms and, optionally, a heteroatom which can be N, S, or O;
R1 is a substituted alkyl selected from the group consisting of - (CH,) z-X, -CH (CH3) - (CH2) 2-X, -CH2-CH(CH3)-CH2-X, -(CH2)2-CH(CH3)-X, and -(CH2)2-C(CH3)2-X, where X is -NH2, -NH-C (NH) -NH2 or -S-C (NH) -NH2, and z is 3 to 5;
m is 1 or 2;
r, o, p and g are, independently, either 1 or 0;
15 Al, A2 and A3, ;nfl~rPn~lPnt1y, are amino acids of L- or D-configuration selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, ~et, Phe, Pro, Ser, Thr, Trp, Tyr and Val;
W is a chelating group attached to the N-terminal amino 20 group of the compound, and Z is a direct bond or a spacer in free form or in salt form.
Preferred significances for A~, A2, A3, Rl, Yl, Y2, P, ~1, r and o are as disclosed in EP EP-A2-293,881, the 25 disclosure of which is incorporated herein by reference.
Wo 95/20603 2 1 ~ 9 8 8 9 ` P~
A particularly preferred compound of formula I is /OH
w--z--(DJ-Phe-Pro--NEI C--B\
OH
(C~2) 3 ..
NH C ' NH2 NH
In a further or alternative embodiment the invention provides a compound o~ formula II
.
W--Z--Y--N--CE~-B II
I I `Q
R~ Rs wherein W, Z and m are as defined above, 5 Y is a se~auence of n amino acids selected such that the n+1 amino acid peptide Y-Lys or Y-Arg has an affinity for the active site of a trypsin-like protease, where n is an integer from 1 to 10 preferably 1 to 4, and in which at least one amino acid is an unnatural amino acid having a 10 hydrophobic side chain;
Ql and Q2, which may be the same or different, are selected from -OH, COR2, -CONR2R3, -NR2R3, and -OR6, or and Q2 taken together form a diol residue, and wherein R2, Rl and R6, which may be the same or different, are C1 10alkyl, C6 l0aryl, C6 l0aralkyl, or phenyl substituted by up to three qroups selected from C1 ~alkyl, halogen and Cl ~alkoxy, 095/20603 2~98189 r~ .s is R~ is hydrogen or C1 10alkyl;
Rs is a group -Z1-X1, where in Z 1 i s - ( CH2 ) -; - CH ( CH3 ) - ( Cx2 ) 2 - ' -CH2 -CH ( CHl ) -CH2-, - ( CH2 ) 2 -CH ( CH3 ) -, - ( CH2 ) 2-C ( CH3 ) 2 - ' -CH ( CH3 ) - ( CH2 ) 3 ~ ~ -CH2-CH ( CH3 ) - ( CH2 ) 2- ' -(CH2)2-CH(CX3)-CH2-, --(cx2)3-c(cH3)2-l -(CX2)2-C(CH3)2-CH2-, (CH2)3-CH(CH3)-, -(CH2)3-CH(CH3)-CH2-, C6 10aryl, or C6 10aralkyl wherein s is 2, 3, 4 or 5;
and wherein X1 is -NH2, -NH-C(NH)-NH2, -S-C(NH)-NH2, -N3, Cl ~alkoxy, C1 4alkylthio, -Si ~CH3) 3, OH, SH, NR7R8, or phenyl, optionally substituted by up to three groups selected from halogen, OH and C1 ~alkoxy, or C (NH) R1oi wherein R7 is H or C1 10alkyl; Ra is Cl 10alkyl, -CO-R9 or CS-Rg; R1o is C1 3alkyl or N(CH3)2, or N
wherein R9 is H or C1 10alkyl, Cl l0alkoxy, C6 lOaryl, C6 10aralkyl and Rll and R12 (which may be the same or different) are H or C1 10alkyl;
or R4 and Rs together form a trimethylene group, and the asymmetric carbon atom marked ~ may have the D-or L-configuration, or represent any mixture of these.
Preferred significances 3for Y, R~, Rsl Ql and Q2 are as indicated below and also as disclosed in EP-A-471,651 and WO 94/20526, the disclosure of which is incu, uo, ~ted herein by re3ference.
By an unnatural amino acid is meant any amino acid other than D- or L- Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, ~et, Phe, Pro, Ser, Thr, ~rp, Tyr, or Val.
Wo sS/20603 ` ~ r.~
2~ 79~89 Preferred unnatural amino acic~s are o~ formu~a III
R~8 --NH--CH--C ~
O . .
in which R13 is a hydrophobic group. Preferred hydrophobic groups consist of a methylene group linked to an aromatic group optionally substituted by a polar 5 group, to an alicyc~ic group having at least two rings and no polar substituents, or to a tert.butyl or trimethylsilyl group. Pre~erably R13 is R13 ' where Rl8 ' is a group of formulae c), d), e), f), g), h) or i) C) ~ --CH~--si (C~3 ) 3 ~CH3~ d) --CH2--C (CE13 ) 3 ~\1 f' ~cH2--e) h~
r - ~ r C ( CH3 ) 3 uDnatur~l ~mino ~1dr of fonm la III may oe i~ D- or ,~ Wo 9sl20603 2 ~ 7 9 8 8 9 F~l~;, c~
L-form or any mixture of these, but are preferably in D-form.
Particularly preferred compounds of formula II are those compounds in which Ql and Q2 taken together represent the . 5 group OPin of formula a).
--O
-O I ~
a) The amino acids constituting Y are -amino acids which may be selected from the L-amino acids naturally occurring in proteins, their -orresponding PnAnt i nmPric D-amino acids or chemically ~ iPA alpha-amino acids 10 such as glutamic acid gam.ma-piperidide:
Glu--N ~
or pipecolic acid (Plp), provided that at least one amino acid is an unnatural amino acid having a hydrophobic side chain .
~ ore preferred ~ u~-ds of formula II are those in which 15 Y is a sequence of two amino acids, of which the N-tPrminAl amino acid is the unnatural amino acid and the other amino acid is L-proline ( Pro ), e . g . a r ~--ld of f ormula IIa w--Z ~ I ~ ~--T ~ B~ ~
~, R, ~ 5 7 IIa WO 95/20603 2 ~ 7 9 ~ ~ 9 P~
in which W, Z, R~ and R~8 are as defined above, and R'~ is - ( CH, ) ,. -X1 , in which X1' is -NH2, -NH-C(NH)-NH~, -N3, OH -Si(CH3)3, phenyl, monochloro-phenyl, dimethylamino-phenyl, -NH-C(O)-CH3, -NHC(O)H, -NHC(S)-NHCE3, -NH-C(O)-C~CH3)~, -NH-C (O) -CH~-OE[3, -NH-C (O) -CH2-C1 or -NH-C (O) -OCH3 and s' is 2,3 or 4 Suitable chelating groups e . g . as W are physiologically acceptable chelating groups capable of complexing a detectable element.
Preferably the rhf~ t;n~ group has a substantially hydrophilic character. Examples cf r71Pl~;nr groups include iminodicarboxylic groups or polyaminopoly-carboxylic groups, e.g. those derived from non cyclic ligands such as ethylene diaminetetraacetic acid (EDTA), diethylene triamine p~n~re~; c acid (DTPA), ethylene glycol -O, O ' -bis ( 2 -aminoethyl ) -N, N, N ', N ' -tetraacetic acid (EGTA), N,N'-bis(hydroxybenzyl)ethylPnP~ min~-N,N'-diacetic acid (HBED) and triethylenetetramine hexaacetic acid (TTHA); those derived from substituted-EDTA or -DTPA, such as p-isothiocyanato-benzyl-EDTA or -DTPA, p-a}nino-benzyl-DTPA or 7~-amino-Cl ~alkylene-DTPAi those derived from macrocyclic ligands, e.g. 1,4,7,10-tetra azacyclo-~ r~n~-N,N' ,N" ,N' ' ' -tetraacetic acid (DOTA), 1, 4, 7,10-tetra-azacyclotridecane-1, 4, 7,10-tetraacetic acid (TITRA) and 1,4,8,11-tetraazacyclotetradecane -N,N',N",N' ' '-terr~ lcer; r acid (TETA); those derived from N-substituted or C-substituted macrocyclic amines as mentioned above including also cyclames, such as 1,4,8,11-terr~7~ryclott3L~c.de~cu.e (TETRA), and as disclosed in EP 304,780 Al and in WO 89tO1476-A.
Chelating groups particularly suitable for Tc li3h~ll;ng may be any of the known chelating groups capable of WO 95/20603 2 ~ 7 g 8 8 g r~
complexing 39=Tc, e.g. as disclosed in EP-A-279,41~
DOS 41 28 183 or WO 92/05154, the disclosures of which are incorporated herein by reference. These complexing groups may be linked to the peptide or pseudo-peptide via a group of formula -(R20)~,-R21 wherein n~ is 0 or 1, R20 is C~ 6alkylene, -O-Cl 6alkylene or -~H-Cl 6alkylene and R2l is -NCS, a carboxy group or a functional derivative thereof, e.g. acid halide, anhydride or hydrazide, optionally through a spacer group.
Preferred chelating groups are radicals of formula ~j) W ' -CH2 -XA-NH- ( j ) wherein ~ ~
W ~ is a chelating group derived f rom a non-cyclic or cyclic polyamino-polyacetic acid or anhydride, and XA is phenylene or C1 ~alkylene.
Groups derived from DTPA or substituted DTPA, e.g.
OH ~NEI
0~
I~` ~
O ~0 OqJ O
OH o~
are preferred as r~ At;ng groups.
Suitable spacer groups, e.g. as Z, include groups of 20 formula ~k) -Rz2-R23-CO- (k) Wo 95l20603 - ~ r~ 7S~'~ 'S
217g889' '~
wherein R22 is a divalent residue derived from a functional moiety capable of - covalently reacting with the chelating agent, and R23 is C~ alkylene optionally interrupted by one or more heteroatoms or residues selected from O, S, CO, -NH-, -NHCO-, N(Cl ~alkyl)-CO- and -N~Cl ~alkyl) -, C2 llalkenylene or -CX(R24) - wherein R24 is a residue attached to a natural or unnatural ~-amino acid, e.g. hydrogen, Cl 1,alkyl, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl -methyl .
In the compounds of formulae I and II, m~is rr~fl~r~hly 1.
The compounds of the invention may exist in free or salt form. Salts include acid addition salts, e.g. with organic acids, polymeric acids or inorganic acids; for example, hydrochlorides and acetates, and salt forms obtA;n~h~e with the carboxylic acid groups present in the rl~ t; ng group, e . g . alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts .
The present invention also includes a process for the production of the ~ ds of the invention. They may be produced by analogy to known methods.
The ~ ' of the invention may be produced for example as follows:
a) removing at least one protecting group which is present in a trypsin-like serine protease inhibiting peptide of the invention bearing a protecting group;
~ WO 95l20603 2 ~ ~ ~ 8 8 ~ r~ c - ~
b1 linking togeth r by an amide bond two peptide fragments each of them containing at least one amino acid in protected or unprotected form and , one of them containing the chelating group, wherein the amide bond is such that the desired amino acid se~uence is obtained, and then optionally effecting step a) ahove;
c~ linking together a chelating agent and the desired peptide in protected or unprotected form in such a way that the chelating group is attached to the desired amino group of the peptide, and then optionally effecting step a) above; or d) removing or converting a functional group of an unprotected or a protected peptide bearing a chelating group so that the desired unprotected or protected peptide bearing a chelating group -; s obtained and if appropriate carrying out step 5 j above, 20 and :-2covering the compound of the invention thus obt. .ed in free form or in salt form.
The above reactions may be effected analogously to known methods, e . g . as described in the following examples, in particular process steps a) and c) above.
25 When the chelating group is attached by an amide bond, this may be carried out analogously to the methods used for amide formation. Where desired, in these reactions, protecting groups which are s~ Ah~ e for use in peptides or for the desired chelating groups may be 30 used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.
WO 95/20603 r~ r ~
~17~88~ --~hen it is desired to attach the chelating group to the N-trrm;n~l amino group of a peptide or peptide fragment used as starting material, and which comprises one or more side chain amino groups, these side chain amino 5 groups are conveniently protected with a protecting group, e.g. as used in peptide chemistry.
The peptide fragment bearing the chelating group and used in step b) above may be prepared by reacting the peptide fragment comprising at least one amino acid in lO protected or unprotected form with the chelating agent.
The reaction may be performed by analogy with step c).
The chelating agent used ln process step c) may be known or prepared analogously to known ~L~,~eduLes. The ~ ULld used is such that it allows the introduction 15 of the desired rh~l~t;nrJ group on the peptide, e.g. a polyaminopolycarboxylic acid as disclosed, a salt or anhydride thereof.
In the above process, amino-acids, peptide fragments or peptides used as starting materials, which have the 20 chelating group attached through a spacer group to the peptide, e.g. a radical of formula (j) as defined above, may be prepared by reacting the corrPC~nn~;nr amino-acids or peptides free from spacer group in conventional manner with a corresponding 25 sp2cer-yielding ,- uL-d, such as an acid or reactive acid derivative comprising the spacer group. For example, an acid of formula R'z2-R23-COO~ or a reactive acid derivative thereof such as an active ester, wherein R ' 22 iS a functional moiety capaole of 3 0 covalently reacting with the chelating agent may be used. F ~ c of active ester groups or carboxy activating çlroups are e . g . 4 -nitrophenyl, pentachlorophenyl, pentafluorophenyl, 5llrr~n;m;~yl or 1 -hydroxy-benzotriazolyl .
Alternatively the chelating agent may be reacted first with a spacer group-yielding compound, in order to bear the spacer group and then be reacted in conventional 5 manner with the peptide, peptide fragment or amino -acid .
The peptides or peptide fragments used as starting materials in process steps b), c) and d) may be p~epared e.g. as disclosed in EP-A2-293,881, EP-A2-471, 561, or WO 94/20526.
The compounds of the invention may be purified in convPntirn~l manner, e.g. by chromatography. Preferably the compounds of the invention contain less than 596 by weight of peptides free of chelating groups.
15 In accordance with the present invention it has now been found that the CU ~I~UU11dX of the invention exhibit rh~-^ological activity and are therefore ^PI~tically useful.
The CULII~L~U11d::~ of the invention have trypsin-like serine 20 protease inhibiting properties, as indicated in the test methods ~lP~r;hP~l in WO 9~/20526 The compounds of the invention are useful as inhibitors of trypsin-like serine proteases and may be employed and administered as described in WO 94/20526.
25 The compounds of the invention may be administered in free form or in phArr~Aputically acceptable salt form.
Such salts may be prepared in conventional manner and exhibit the same order of activity as the free .u!,ds. 13 WO 95/20603 ` r~ 1 r'c '~
2~7~8~8~ --In accordance with the foregoing, the present invention further provides:
a) a compound of the invention or a~ ph;ll-mqr~ tically acceptable salt thereof for use as a rhprrsrellt; rAl;
b) a method o~ preventing or- treating disorders caused by an excess of one or more trypsin-like serine protease in a subj ect in need of such a treatment, which method comprises administering to said subject an effective amount o~ a compound of the invention or a rhs~-reutically acceptable salt thereof i c) a rhsrr~~~utical composition comprising a compound of the invention in free form or in rhq~-reutically acceptable salt form in association with a ph~ e~ltically acceptable diluent or carrier.
According to a further embodiment of the invention, the c~ ~u--d~ of the invention can be complexed with a 2 0 detectable ~lement .
Accordingly, the present invention also provides the compounds of the invention as def ined above which are complexed with a detectable element ~hereinafter referred to as chelates of the invention), in free form 25 or in salt form, their preparation and their use as diagnostic agents.
By detectable element is meant any element, preferably a metal ion which exhibits a L)LU~ L Ly detectable in diagnostic t~rhn;r~ R, e.g. a metal ion which emits a 3 0 detectable radiation or a metal ion which is capable of .
wo 95/20603 r~
2179~9 inf luencing NMR relaxation properties .
Suitable detectable metal ions include for example pArAmArnrtic ions, e.g. Gd3~, Fe3, Mn2~ and Cr2 fluorescent metal ions, e.g. Eu3', and radinn-~rl;~71~c, 5 e.g. r-elr!itting rA~l;rn~lr7 i.7PC, positron-emitting radinn--rl ;~.oc e.g. 6~Ga.
Suitable r-emitting rA~7; nnllrl; rl~c include ~hose which are useful in diagnostic technislues. The r-emitting rAr7; nnllrli~c advantageously have a half-life of from 1 10 hour to 40 days, preferably from 5 hours to 4 days, more pref erably f rom 5 hours to 3 days and pref erably should exhibit a single r-~m; cs;rn, preferably in the range of 100-200 KeV. Examples are radirn--rl ;tll,q derived from Gallium, Indium, Technetium, Ytterbium, Rhenium and ~11Al 1 ;llm e.g. 67Ga, ~ n 99=Tc l69yb The chelates of the invention may be ~L~5Lt:.l by reacting the ~ ~oul~d with a cc~LL~ ;nr ~7~t~rtAhle element yielding compound, e.g. a metal salt, pref erably a water-soluble salt . The reaction may be 20 carried out by analogy with known methods, e.g. as disclosed in Perrin, Organic Ligand, Chemical Data Series 22. NY Pergamon Press (1982); in Krejcarit and Tucker, Biophys. Biochem. Res. Com. 77: 581 ~1977) and in Wagner and Welch, J. Nucl. Med. 2Q: 428 (1979).
25 Preferably the complexing of a c~ ulld of the invention is effected at a pH at which it is stable.
Alternatively the detectable element may also be provided to the solution as a complex with an ;nt~ -';Ate chelating agent, e.g. a chelating agent 30 which forms a chelate complex that renders the element soluble at the physiological pH of the complex but is Wo 95~20603 217 9 8 8 9 ~ r~ s less the~modynamically stable than the chelate. ExamPle of such an ;nt~ tf~ chelating agent is 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron). In such a process, the detectable element exchanges the 5 ligand.
The above mentioned reactions may be effected analogously to known methods. The above mentioned reactions are conveniently effected under conditions avoiding trace metal ~ontArn;n~tion Preferably dis-10 tilled de-ionized water, ultrapure reagents, no carrier-added radioactivity etc. are used to reduce the effects of trace metal.
The chelates of the invention may exist e.g. in free or salt form. Salts include acid addition salts with e.g.
15 organic acids, polymeric acids or tn-~rg~n;c acids, for example hydrochlorides and acetates, and salt forms ~ht~;n:-hl,~ with the carboxylic acid groups present in the molecule which do not participate to the chelate f ormatioII .
20 The chelates of the invention and their pharmaceutical acceptable salts exhibit diagnostic activity and are therefore useful as imaging agents, e.g. v; c~l; c~tion of thrombi. They exhibit significant serum stability and binding affinity to thrombin, as indicated in 25 standard i n y~ tests . The compound of Example 4 has a half life of 210 hours in human serum, when tested ~n Yi~Q .
Accordingly, the present invention also provides a method for i Y~S~ detection of a thrombus in a subject 30 which comprises a~ administering a chelate of the invention to said subject and b) recording the lo~l;c~t;,,n of the thrombus targeted by said chelate.
~ Wo gs/20603 ~ 1 ~ 9 8 8 g r.~
The chelates of the invention for use as an imaging agent in the above method may be administered parPnt~r~1 ly, preferably intravenously, e.g. in the ~e - form of injectable solutions or suspensions, preferably 5 in a single injectLon. The appropriate dosage will of course vary ri~r~n~;n5 upon, for example, the compound and the type of detectable element used. A suitable dose to be inj ected is in the range to enable imaging by photnsr~nn;n~ procedures known in the art. A
lO chelate of the invention may advantageously be administered in a dose comprising 0.1 to lO mCi of a stably chelated radinn-1r1; ~lr .
In animals an indicated dosage range may be of from 0.1 to 1 ug/kg of a rr~ro1~nrl of the invention labeled with 0 . 02 to 0 . 5 mCi r-emitting r;-~; onl1r1; ~,~ . In larger animals, f or example humans, an indicated dosage range may be of from 1 to 20 ug compound labeled e.g. with 3 to 10 mCi r-emitting r~ n-~r1 i~1~, preferably 2 to 5 mCi.
20 The localisation of a throm.bus with the chelates may be followed by the ~,Jr,~ lr7;nrJ imaging technir~ues, e.g.
using nuclear medicine imaging instrumentation, for example a scanner, a conventional gam.ma camera to obtain planar images, a rotating r-camera to perform 25 Si~gle Photon ~m; RRion Computer T~ ,hy (SPECT) or suitable instrumentation to perform Photon Emission T, ~ (PET), each preferably computer assisted.
The chelates of the invention may be administered in free form or in ~h~rr~~~lltically acceptable salt form.
3 0 Such salts may be prepared in conventional manner and exhibit the same order of activity as the free wo 95~20603 ;~ 1 7 ~ 8 8 ~ Pcr/EPss/00269 The chelates_of the invention are particularly useful in detecting thrombi formation resulting from vascular thrombotic disorders caused by myocardial infarction, artheriosclerosis, stroke, tl;RspTn;n:~ted intravascular S coagulation, peri- and post-operative to surgery, vascular recanalisations, immobility, atrial f ibril lation and heart ; n ~1l f f i r i ~n~y .
The chelates of the invention for use-in the method of the present invention may pre_erably be prepared 10 shortly before administration to a subject, i.e. the radiolabeling with the desired detectable metal ion, may be performed shortly before administration.
According to a further aspect of the invention, there is provided a ~hArr-rF~lltical composition comprisin~ a 15 chelate according to the invention in free or in rhAnr--Plltically acceptable salt form, together with one or more phArr~ ltically acceptable carriers or diluents theref or .
Such a composition may be manufactured in conventional 2 0 manner .
A composition according to the invention may also be presented in separate package with instructions for mixing the compound with the metal ion and for the administration of the resulting chelate. It may also be 25 presented in twin-pack ~orm, that is, as a sin,r,le package crmtAin;nrJ separate unit dosages of the compound and the detectable metal ion with instructions for mixing them and for administration of the chelate.
A diluent or carrier may be present in the unit dosage 3 0 f orms .
In the f ollowing examples, all temperatures are in C
Wo ss/2o6o3 r~
~17g889 and [a]D20- values are uncorrected.
The following abbreviations are employed:
Bzl = benzyl Z = benzyloxycarbonyl Boc = t-butyloxycarbonyl Ac = acetyl MeOH = methyl alcohol EtOH = ethyl alcohol EtOAc = ethyl acetate DCC = dicyclohexylr~rhod;; m; ~1 HONSu = N-hydroxy-sllrr;n;m;~?
OPin = p;nAned; Ql TFA = trif luoroacetic acid THF = tetrahydrofuran DMF = dimethyl formamide Np = p-nitrophenyl TLC = thin layer chromatography Baa = -NX-CH- (CH2CH2CH2Br) B-TMSa 1 = trime thyl s ilyl A 1 A n; n ~
BoroLys = -NH-CH-(CX2-CH2-CH2-CH2-NH2)B-DTPA- (p-NX-Bzl) = OH NH~
, 1. ~
o'~
OH~ ~N ~ ~OH
0 ~0 0~ 0 OH OH
(t-Bu) -DTPA- (p-NH Bzl) = t-butyl ester (4x) of DTPA- (p-NH-Bz l ) WO9~/20603 2~79889 F~~ 'C~ ~ ~
D~P.7~-(D~ 1-Pro-BoroLv~-OPi~) 2 ~NH, COOH ~51 N
HOOC ~ ~COOH ,3L
O
~NH2 ~) D~PA-(D-q~ l-P~., .. _~v(6-N-Fmo~)Lvl3-OP
H-D-TMSal-Pro-Boro (e-N-Fmoc) Lys-OPin (tri~luoroacetate~
(428 . 4 mg O . 5 mMol~ ia dissolved in dioxane and is treated with 5 . 0 ml saturated aqueous NaHCO3 and DTPA-anhydride (0.268 g 0.75 mMol~. The mixture is stirred ~or a . 5 h at room temperature. Acetic acid ( 3 5 . 0 ml ~ is added and the mixture is concentrated in vacuo at 40. The resulting residue is treated with 50 ml EtOAc and H20. The aS~ueous layer is extracted with EtOAc and the c n~d organic layers are washed with H2O. A3ter drying over Na2SO and concentration in vacuo the residue is chromato~rr~rh~f~ on a 4. 0 x 25 cm ~PI C-column RP18 (10 um) H2O/MeOH/acetic acid. The desired` product is obtained as a white foam MS: 1842 (N + H) .
b) ~rp~ l-P ~ ,Lvn-OP~
DTPA- (D-TMSal-Pro-Boro ~e -N-Fmoc) Lys-OPin) 2 (1 . 92 g, 1. 042 mMol) o~ step al is treated with 5 0 ml piperidine in DMF (20 9~ solution) at room temperature ~ WO 95/20603 2 ~ 7 9 8 8 9 r~
for 15 min. The resulting suspension is diluted with 50 ml of ether and the desired product precipitates as a crude material. This material is purified over Dowex (l-X4, 200-40D meshj, EtOH/H2O (1:1) and 10 96 AcOH.
5 Af ter removal of the solvent the desired product is . obtained as a white foam and is crystallized from EtOH/ether to give the title - ~ Qllnr~ as an amorphous white solid. MS: 1398 (M + H) '; [a]D = -72 .0 ( c = 0 . 3 5 in MeOH ) .
10 H-D-TMSal-Pro-Boro(~-N-Fmoc)Lys-OPin(trifluoroacetate), used as starting material, may be prepared as follows:
A) soc-D-TMQ- 1 -Pro-soroLv~-oPin may be prepared as disclosed in Example 5 of EP-A-471, 651.
s) soc-D-TM~-l-pro-soro(~-N-Fmoc)LvD-op~
Boc-D-TMSal-Pro-BoroLys-OPin(620.7 mg,1.0 mMol) and Fmoc-OSu (337.3 mg, 1.0 mMol) in dioxane are treated at room temperature with saturated aqueous NaHCO3 solution. After 45 min, the reaction mixture is 20 diluted with EtOAc and is washed with water and brine.
Af ter drying over Na2SO~ and concentration in vacuo, the title compound is used without further purification in the next step.
C) ~-D-Tr~8~1 -Pro-Boro ( c -N-Fmoc ) I,v~ -OPin (t--if 1'''~''~-- ' ~) The product of step B) (2.36 g, 2.8 mMol) is treated with 8.5 ml TFA/H2O (95:5) and 0.85 ml anisol. After 1 h at room temperature, the reaction mixture is concentrated in vacuo and the resulting oil is chromatographed on a 3.2 x 25 cm HPLC column RP18 ( 10 um), H2O/MeOH/TFA. The desired product is obtained as a white foam, MS: 743 (M + H) WO 95/20603 2 ~ 7 9 8 8 9 r~
".upr.w 2: 11 T~ ~ ~a O~ EX~D1~ 1 1 mg of compound of Example 1 is dissolved in 5 ml O . 01 M acetic acid. The resulting solution is passed through a 0.22 u Millex-GV filter and dispensed in 5 0.1 ml pQrtions and stored at -20C. IllIncl3 (Amersham, 1 mCi/100 ul) is prediluted in an equal volume of 0 . 5 M sodium acetate and labeling is carried out by mixing the ligand with the InCl3 solution and gentle homogenisation at room temperature.
HEPES buffer, pX 7.4, is then added to make a 10-6M
solution .
~y~llUPT.l/! 3 D~PA--(D-~-~71 )--CO(CH~) ~CO--~_~/UQ-l--P. ~, 3~ ~.L~r~-OP~ n O --~ ~
~f ~ ~N ~ ~N~
O ~0 OqJ O ..
~ a~
a ) ~OOC ( c~, ) ,Co-D-T~Q- 1 -Pro-B~-o ( ~ -N-F ) L~ -OPi~
15 X-D-TMSal -Pro -Boro ~ ~ -N-Fmoc ) Lys -OPin ( tri f luoro-acetate of Example 1, step C) (0.857 g,l.0 mMol) is dissolved in dioxane and is treated at room temperature with saturated aS~ueous NaXCO3 solution and succinic anhydride (0.11 g, 1.10 mMol). After 45 min, the 20 reaction mixture is diluted with EtOAc and is washed ~ Wo 95/20603 217 9 8 ~ 9 r~
with aqueous NaHSO,-601ution (1 M) and brine. The organic layer is dried over Na2SO~ and concentrated in ~~
vacuo. The resulting crude product is chromatographed on a 3.2 x 25 cm HPLC-column RP18 (10 l~m) H20/MeOH.
5 The desired product is obtained as a white foam, MS:
,- 843 (M + H) .
}:~) (t.BU)-~q!pl~-(D-N~-Bzl)-co(c~)~co-D-~rM-Boro ( e -N-lr ) -LVEl-OP~ r~
The product of step a) (623 mg, 0.739 mMol) in THF is 10 cooled to 0 and is treated with N-methyl morpholine (0.081 ml, 0.739 mMol and pivaloyl chloride (0.095 ml, 0.776 mMol) to form the sLixed anhydride. After stirri~g for 1 h at 0, the reaction mixture is treated with ('Bu)-DTPA-(p-NH2-Bzl) (575 mg, 0.739 mMol) (as described in Bioconjugate Chem. 1991, 2(3), 180-186) and additional N-methyl morpholine (0.081 ml, 0.739 mMol). The reaction mixture is stirred for 15 h at room temperature, and then diluted with EtOAc and washed with aqueous citric acid solution (1 M), aqueous 20 saturated NaHCO3-solution and brine. The organic layer is dried over Na2SO~ and concentrated in vacuo. The resulting oil is chromatographed on a 3.2 x 25 cm HPLC-column RP18 (10 um), H2O/MeOH/TFA. The desired product is obtained as a white foam, MS: 1603 (M + H) .
25 c) lYrV.p_(D_~-B'~l )-co(~ co-D-Tr~ l-pro-Boro(~-N-F
) I.~--OP ~ ~
The product of step b) (576 mg, 0.296 mMol) is s~r~n;fied by treatment with TFA/H20 (95:5) in the presence of anisol/et~n-~flithiol (1:1) for 7 h at room 3 0 temperature . Excess TFA is removed in vacuo and the resultir~g residue is treated with ether to precipitate the desired product. After filtration and drying under reduced pressure, the title ~-, uL-d is obtained as an amorphous solid foam of the trifluoroacetate MS: 1323 W0 95/20603 P ~
217g~89 (M + H) -~-s~1l-co~c~,),CO-D-TM~Al-3?~0-sorol.~s-OPirL
The product of step c) ~326 mg, 0.2463 n~ol) is treated 5 with 2 . O ml piperidine in DMF (20 96 solution) at room temperature for 15 min. The resulting mixture is diluted with ether and the desired product precipitates as a white solid . This material is purif ied over Dowex (1-X4, 200-400 mesh), ~tOH/H2O (1:1) and 10 96 AcOH.
10 After removal of the solvent the title compound is obtained as a white lyophilisate (from H2O) ~S: 1101 (M + H) ', [a]D = -31.8 (c = 0.27 in MeOH) .
~P~r.T~ 4 ~ n 1~ o r r 1 ~ 3 The lllIn labeled c _-uLld is prepared by using the same 15 procedure as disclosed in Example 2.
217988g .~
INHIBITORS OF SERINE PROTEASES, BEARING A CHELATING GROUP
This invention relates to inhibitors of serine proteases, e.g. thrombin, and conjugates of such inhibitors used as thrombus imaging agents.
5 Thrombin and other serine proteases and inhibitors therefor are discussed in WO 94t20526, the disclosure o~
which is incorporated herein by reference.
It has now been found that peptidic and pseudo-peptidic trypsin-like serine protease inhibitors which are 10 transition state analogues and which comprise an amino group which can be modified with a chelating group without interfering with or preventing their binding to thrombin, provide novel compounds having interesting properties .
15 By transition state analogues are meant compounds as defined by A.J. Barrett et al. in Proteinase Inhibitors, Ed. Elsevier Amsterdam, 1986, p. 57.
The term "pseudo-peptide" or "pseudo-peptidic" as used herein refers to a peptide isostere in which the peptide 20 bond between 2 amino acid residues, whether natural or unnatural, is replaced with any isoste-ic group, e.g.
-CH2-NH-. The pseudo-peptide may comp~ e one or more such isosteric bonds.
.
Accordinçr to the invention, there is provided a 25 trypsin-like serine protease inhibiting peptide or 2 ~ 7 ~ 8 8 9 r~ 9 pseudo-peptide bearing a chelating group capable of complexing a detectable element, the rhf.l~t;nr group being linked to an amino group of the peptide or pseudo-peptide such that the rhPl~t;nJ group ao~es not , 5 interf ere with or prevent the binding of the peptide or pseudo-peptide to thrombin. ~, These ~ ~U~ are referred to thereafter as compounds of the invention.
The rhelAtinr group is linked by a covalent bond to the 10 amino group of the peptide or pseudo-peptide. The chelating group is preferably attached to the N-terminal amino group of the peptide or pseudo-peptide.
The chelating group may be attached either directly or indirectly, e.g. by means of a spacer group, to the amino 15 group of the peptide or pseudo-peptide. Preferably the chelating group is coupled to the peptide or pseudo-peptide or to the peptide-spacer or (pseudo-peptide)-spacer moiety by an amide bond.
Preferred _ ~u--ds of the invention are those comprising 20 a C-t~r~n;niql boronic acid derivative residue, particularly a C-t~rm;n~l boronic acid derivative of lysine, ornithine, arginine or an ~l ;rh~t;c amino alcohol. More preferred are those comprising additionally at least one unnatural -amino acid having 25 a hydrophobic side chain.
In one ~ t the present invention provides a compound of formula I
W~ I ~Z~[ (A3)q(A2)p(Al)o]r-NH - ~-B\ I ~
WO 9~l20603 % 1 ~ 9 8 8 9 P~
wherein Y1 and Y2, independently, are OH or F or, taken together, form a moiety derived from a dihydroxy compound having at least two hydroxy groups separated by at least two 5 connecting atoms in a chain or ring, said chain or ring comprising 1 to about 20 carbon atoms and, optionally, a heteroatom which can be N, S, or O;
R1 is a substituted alkyl selected from the group consisting of - (CH,) z-X, -CH (CH3) - (CH2) 2-X, -CH2-CH(CH3)-CH2-X, -(CH2)2-CH(CH3)-X, and -(CH2)2-C(CH3)2-X, where X is -NH2, -NH-C (NH) -NH2 or -S-C (NH) -NH2, and z is 3 to 5;
m is 1 or 2;
r, o, p and g are, independently, either 1 or 0;
15 Al, A2 and A3, ;nfl~rPn~lPnt1y, are amino acids of L- or D-configuration selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, ~et, Phe, Pro, Ser, Thr, Trp, Tyr and Val;
W is a chelating group attached to the N-terminal amino 20 group of the compound, and Z is a direct bond or a spacer in free form or in salt form.
Preferred significances for A~, A2, A3, Rl, Yl, Y2, P, ~1, r and o are as disclosed in EP EP-A2-293,881, the 25 disclosure of which is incorporated herein by reference.
Wo 95/20603 2 1 ~ 9 8 8 9 ` P~
A particularly preferred compound of formula I is /OH
w--z--(DJ-Phe-Pro--NEI C--B\
OH
(C~2) 3 ..
NH C ' NH2 NH
In a further or alternative embodiment the invention provides a compound o~ formula II
.
W--Z--Y--N--CE~-B II
I I `Q
R~ Rs wherein W, Z and m are as defined above, 5 Y is a se~auence of n amino acids selected such that the n+1 amino acid peptide Y-Lys or Y-Arg has an affinity for the active site of a trypsin-like protease, where n is an integer from 1 to 10 preferably 1 to 4, and in which at least one amino acid is an unnatural amino acid having a 10 hydrophobic side chain;
Ql and Q2, which may be the same or different, are selected from -OH, COR2, -CONR2R3, -NR2R3, and -OR6, or and Q2 taken together form a diol residue, and wherein R2, Rl and R6, which may be the same or different, are C1 10alkyl, C6 l0aryl, C6 l0aralkyl, or phenyl substituted by up to three qroups selected from C1 ~alkyl, halogen and Cl ~alkoxy, 095/20603 2~98189 r~ .s is R~ is hydrogen or C1 10alkyl;
Rs is a group -Z1-X1, where in Z 1 i s - ( CH2 ) -; - CH ( CH3 ) - ( Cx2 ) 2 - ' -CH2 -CH ( CHl ) -CH2-, - ( CH2 ) 2 -CH ( CH3 ) -, - ( CH2 ) 2-C ( CH3 ) 2 - ' -CH ( CH3 ) - ( CH2 ) 3 ~ ~ -CH2-CH ( CH3 ) - ( CH2 ) 2- ' -(CH2)2-CH(CX3)-CH2-, --(cx2)3-c(cH3)2-l -(CX2)2-C(CH3)2-CH2-, (CH2)3-CH(CH3)-, -(CH2)3-CH(CH3)-CH2-, C6 10aryl, or C6 10aralkyl wherein s is 2, 3, 4 or 5;
and wherein X1 is -NH2, -NH-C(NH)-NH2, -S-C(NH)-NH2, -N3, Cl ~alkoxy, C1 4alkylthio, -Si ~CH3) 3, OH, SH, NR7R8, or phenyl, optionally substituted by up to three groups selected from halogen, OH and C1 ~alkoxy, or C (NH) R1oi wherein R7 is H or C1 10alkyl; Ra is Cl 10alkyl, -CO-R9 or CS-Rg; R1o is C1 3alkyl or N(CH3)2, or N
wherein R9 is H or C1 10alkyl, Cl l0alkoxy, C6 lOaryl, C6 10aralkyl and Rll and R12 (which may be the same or different) are H or C1 10alkyl;
or R4 and Rs together form a trimethylene group, and the asymmetric carbon atom marked ~ may have the D-or L-configuration, or represent any mixture of these.
Preferred significances 3for Y, R~, Rsl Ql and Q2 are as indicated below and also as disclosed in EP-A-471,651 and WO 94/20526, the disclosure of which is incu, uo, ~ted herein by re3ference.
By an unnatural amino acid is meant any amino acid other than D- or L- Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, ~et, Phe, Pro, Ser, Thr, ~rp, Tyr, or Val.
Wo sS/20603 ` ~ r.~
2~ 79~89 Preferred unnatural amino acic~s are o~ formu~a III
R~8 --NH--CH--C ~
O . .
in which R13 is a hydrophobic group. Preferred hydrophobic groups consist of a methylene group linked to an aromatic group optionally substituted by a polar 5 group, to an alicyc~ic group having at least two rings and no polar substituents, or to a tert.butyl or trimethylsilyl group. Pre~erably R13 is R13 ' where Rl8 ' is a group of formulae c), d), e), f), g), h) or i) C) ~ --CH~--si (C~3 ) 3 ~CH3~ d) --CH2--C (CE13 ) 3 ~\1 f' ~cH2--e) h~
r - ~ r C ( CH3 ) 3 uDnatur~l ~mino ~1dr of fonm la III may oe i~ D- or ,~ Wo 9sl20603 2 ~ 7 9 8 8 9 F~l~;, c~
L-form or any mixture of these, but are preferably in D-form.
Particularly preferred compounds of formula II are those compounds in which Ql and Q2 taken together represent the . 5 group OPin of formula a).
--O
-O I ~
a) The amino acids constituting Y are -amino acids which may be selected from the L-amino acids naturally occurring in proteins, their -orresponding PnAnt i nmPric D-amino acids or chemically ~ iPA alpha-amino acids 10 such as glutamic acid gam.ma-piperidide:
Glu--N ~
or pipecolic acid (Plp), provided that at least one amino acid is an unnatural amino acid having a hydrophobic side chain .
~ ore preferred ~ u~-ds of formula II are those in which 15 Y is a sequence of two amino acids, of which the N-tPrminAl amino acid is the unnatural amino acid and the other amino acid is L-proline ( Pro ), e . g . a r ~--ld of f ormula IIa w--Z ~ I ~ ~--T ~ B~ ~
~, R, ~ 5 7 IIa WO 95/20603 2 ~ 7 9 ~ ~ 9 P~
in which W, Z, R~ and R~8 are as defined above, and R'~ is - ( CH, ) ,. -X1 , in which X1' is -NH2, -NH-C(NH)-NH~, -N3, OH -Si(CH3)3, phenyl, monochloro-phenyl, dimethylamino-phenyl, -NH-C(O)-CH3, -NHC(O)H, -NHC(S)-NHCE3, -NH-C(O)-C~CH3)~, -NH-C (O) -CH~-OE[3, -NH-C (O) -CH2-C1 or -NH-C (O) -OCH3 and s' is 2,3 or 4 Suitable chelating groups e . g . as W are physiologically acceptable chelating groups capable of complexing a detectable element.
Preferably the rhf~ t;n~ group has a substantially hydrophilic character. Examples cf r71Pl~;nr groups include iminodicarboxylic groups or polyaminopoly-carboxylic groups, e.g. those derived from non cyclic ligands such as ethylene diaminetetraacetic acid (EDTA), diethylene triamine p~n~re~; c acid (DTPA), ethylene glycol -O, O ' -bis ( 2 -aminoethyl ) -N, N, N ', N ' -tetraacetic acid (EGTA), N,N'-bis(hydroxybenzyl)ethylPnP~ min~-N,N'-diacetic acid (HBED) and triethylenetetramine hexaacetic acid (TTHA); those derived from substituted-EDTA or -DTPA, such as p-isothiocyanato-benzyl-EDTA or -DTPA, p-a}nino-benzyl-DTPA or 7~-amino-Cl ~alkylene-DTPAi those derived from macrocyclic ligands, e.g. 1,4,7,10-tetra azacyclo-~ r~n~-N,N' ,N" ,N' ' ' -tetraacetic acid (DOTA), 1, 4, 7,10-tetra-azacyclotridecane-1, 4, 7,10-tetraacetic acid (TITRA) and 1,4,8,11-tetraazacyclotetradecane -N,N',N",N' ' '-terr~ lcer; r acid (TETA); those derived from N-substituted or C-substituted macrocyclic amines as mentioned above including also cyclames, such as 1,4,8,11-terr~7~ryclott3L~c.de~cu.e (TETRA), and as disclosed in EP 304,780 Al and in WO 89tO1476-A.
Chelating groups particularly suitable for Tc li3h~ll;ng may be any of the known chelating groups capable of WO 95/20603 2 ~ 7 g 8 8 g r~
complexing 39=Tc, e.g. as disclosed in EP-A-279,41~
DOS 41 28 183 or WO 92/05154, the disclosures of which are incorporated herein by reference. These complexing groups may be linked to the peptide or pseudo-peptide via a group of formula -(R20)~,-R21 wherein n~ is 0 or 1, R20 is C~ 6alkylene, -O-Cl 6alkylene or -~H-Cl 6alkylene and R2l is -NCS, a carboxy group or a functional derivative thereof, e.g. acid halide, anhydride or hydrazide, optionally through a spacer group.
Preferred chelating groups are radicals of formula ~j) W ' -CH2 -XA-NH- ( j ) wherein ~ ~
W ~ is a chelating group derived f rom a non-cyclic or cyclic polyamino-polyacetic acid or anhydride, and XA is phenylene or C1 ~alkylene.
Groups derived from DTPA or substituted DTPA, e.g.
OH ~NEI
0~
I~` ~
O ~0 OqJ O
OH o~
are preferred as r~ At;ng groups.
Suitable spacer groups, e.g. as Z, include groups of 20 formula ~k) -Rz2-R23-CO- (k) Wo 95l20603 - ~ r~ 7S~'~ 'S
217g889' '~
wherein R22 is a divalent residue derived from a functional moiety capable of - covalently reacting with the chelating agent, and R23 is C~ alkylene optionally interrupted by one or more heteroatoms or residues selected from O, S, CO, -NH-, -NHCO-, N(Cl ~alkyl)-CO- and -N~Cl ~alkyl) -, C2 llalkenylene or -CX(R24) - wherein R24 is a residue attached to a natural or unnatural ~-amino acid, e.g. hydrogen, Cl 1,alkyl, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl -methyl .
In the compounds of formulae I and II, m~is rr~fl~r~hly 1.
The compounds of the invention may exist in free or salt form. Salts include acid addition salts, e.g. with organic acids, polymeric acids or inorganic acids; for example, hydrochlorides and acetates, and salt forms obtA;n~h~e with the carboxylic acid groups present in the rl~ t; ng group, e . g . alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts .
The present invention also includes a process for the production of the ~ ds of the invention. They may be produced by analogy to known methods.
The ~ ' of the invention may be produced for example as follows:
a) removing at least one protecting group which is present in a trypsin-like serine protease inhibiting peptide of the invention bearing a protecting group;
~ WO 95l20603 2 ~ ~ ~ 8 8 ~ r~ c - ~
b1 linking togeth r by an amide bond two peptide fragments each of them containing at least one amino acid in protected or unprotected form and , one of them containing the chelating group, wherein the amide bond is such that the desired amino acid se~uence is obtained, and then optionally effecting step a) ahove;
c~ linking together a chelating agent and the desired peptide in protected or unprotected form in such a way that the chelating group is attached to the desired amino group of the peptide, and then optionally effecting step a) above; or d) removing or converting a functional group of an unprotected or a protected peptide bearing a chelating group so that the desired unprotected or protected peptide bearing a chelating group -; s obtained and if appropriate carrying out step 5 j above, 20 and :-2covering the compound of the invention thus obt. .ed in free form or in salt form.
The above reactions may be effected analogously to known methods, e . g . as described in the following examples, in particular process steps a) and c) above.
25 When the chelating group is attached by an amide bond, this may be carried out analogously to the methods used for amide formation. Where desired, in these reactions, protecting groups which are s~ Ah~ e for use in peptides or for the desired chelating groups may be 30 used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.
WO 95/20603 r~ r ~
~17~88~ --~hen it is desired to attach the chelating group to the N-trrm;n~l amino group of a peptide or peptide fragment used as starting material, and which comprises one or more side chain amino groups, these side chain amino 5 groups are conveniently protected with a protecting group, e.g. as used in peptide chemistry.
The peptide fragment bearing the chelating group and used in step b) above may be prepared by reacting the peptide fragment comprising at least one amino acid in lO protected or unprotected form with the chelating agent.
The reaction may be performed by analogy with step c).
The chelating agent used ln process step c) may be known or prepared analogously to known ~L~,~eduLes. The ~ ULld used is such that it allows the introduction 15 of the desired rh~l~t;nrJ group on the peptide, e.g. a polyaminopolycarboxylic acid as disclosed, a salt or anhydride thereof.
In the above process, amino-acids, peptide fragments or peptides used as starting materials, which have the 20 chelating group attached through a spacer group to the peptide, e.g. a radical of formula (j) as defined above, may be prepared by reacting the corrPC~nn~;nr amino-acids or peptides free from spacer group in conventional manner with a corresponding 25 sp2cer-yielding ,- uL-d, such as an acid or reactive acid derivative comprising the spacer group. For example, an acid of formula R'z2-R23-COO~ or a reactive acid derivative thereof such as an active ester, wherein R ' 22 iS a functional moiety capaole of 3 0 covalently reacting with the chelating agent may be used. F ~ c of active ester groups or carboxy activating çlroups are e . g . 4 -nitrophenyl, pentachlorophenyl, pentafluorophenyl, 5llrr~n;m;~yl or 1 -hydroxy-benzotriazolyl .
Alternatively the chelating agent may be reacted first with a spacer group-yielding compound, in order to bear the spacer group and then be reacted in conventional 5 manner with the peptide, peptide fragment or amino -acid .
The peptides or peptide fragments used as starting materials in process steps b), c) and d) may be p~epared e.g. as disclosed in EP-A2-293,881, EP-A2-471, 561, or WO 94/20526.
The compounds of the invention may be purified in convPntirn~l manner, e.g. by chromatography. Preferably the compounds of the invention contain less than 596 by weight of peptides free of chelating groups.
15 In accordance with the present invention it has now been found that the CU ~I~UU11dX of the invention exhibit rh~-^ological activity and are therefore ^PI~tically useful.
The CULII~L~U11d::~ of the invention have trypsin-like serine 20 protease inhibiting properties, as indicated in the test methods ~lP~r;hP~l in WO 9~/20526 The compounds of the invention are useful as inhibitors of trypsin-like serine proteases and may be employed and administered as described in WO 94/20526.
25 The compounds of the invention may be administered in free form or in phArr~Aputically acceptable salt form.
Such salts may be prepared in conventional manner and exhibit the same order of activity as the free .u!,ds. 13 WO 95/20603 ` r~ 1 r'c '~
2~7~8~8~ --In accordance with the foregoing, the present invention further provides:
a) a compound of the invention or a~ ph;ll-mqr~ tically acceptable salt thereof for use as a rhprrsrellt; rAl;
b) a method o~ preventing or- treating disorders caused by an excess of one or more trypsin-like serine protease in a subj ect in need of such a treatment, which method comprises administering to said subject an effective amount o~ a compound of the invention or a rhs~-reutically acceptable salt thereof i c) a rhsrr~~~utical composition comprising a compound of the invention in free form or in rhq~-reutically acceptable salt form in association with a ph~ e~ltically acceptable diluent or carrier.
According to a further embodiment of the invention, the c~ ~u--d~ of the invention can be complexed with a 2 0 detectable ~lement .
Accordingly, the present invention also provides the compounds of the invention as def ined above which are complexed with a detectable element ~hereinafter referred to as chelates of the invention), in free form 25 or in salt form, their preparation and their use as diagnostic agents.
By detectable element is meant any element, preferably a metal ion which exhibits a L)LU~ L Ly detectable in diagnostic t~rhn;r~ R, e.g. a metal ion which emits a 3 0 detectable radiation or a metal ion which is capable of .
wo 95/20603 r~
2179~9 inf luencing NMR relaxation properties .
Suitable detectable metal ions include for example pArAmArnrtic ions, e.g. Gd3~, Fe3, Mn2~ and Cr2 fluorescent metal ions, e.g. Eu3', and radinn-~rl;~71~c, 5 e.g. r-elr!itting rA~l;rn~lr7 i.7PC, positron-emitting radinn--rl ;~.oc e.g. 6~Ga.
Suitable r-emitting rA~7; nnllrl; rl~c include ~hose which are useful in diagnostic technislues. The r-emitting rAr7; nnllrli~c advantageously have a half-life of from 1 10 hour to 40 days, preferably from 5 hours to 4 days, more pref erably f rom 5 hours to 3 days and pref erably should exhibit a single r-~m; cs;rn, preferably in the range of 100-200 KeV. Examples are radirn--rl ;tll,q derived from Gallium, Indium, Technetium, Ytterbium, Rhenium and ~11Al 1 ;llm e.g. 67Ga, ~ n 99=Tc l69yb The chelates of the invention may be ~L~5Lt:.l by reacting the ~ ~oul~d with a cc~LL~ ;nr ~7~t~rtAhle element yielding compound, e.g. a metal salt, pref erably a water-soluble salt . The reaction may be 20 carried out by analogy with known methods, e.g. as disclosed in Perrin, Organic Ligand, Chemical Data Series 22. NY Pergamon Press (1982); in Krejcarit and Tucker, Biophys. Biochem. Res. Com. 77: 581 ~1977) and in Wagner and Welch, J. Nucl. Med. 2Q: 428 (1979).
25 Preferably the complexing of a c~ ulld of the invention is effected at a pH at which it is stable.
Alternatively the detectable element may also be provided to the solution as a complex with an ;nt~ -';Ate chelating agent, e.g. a chelating agent 30 which forms a chelate complex that renders the element soluble at the physiological pH of the complex but is Wo 95~20603 217 9 8 8 9 ~ r~ s less the~modynamically stable than the chelate. ExamPle of such an ;nt~ tf~ chelating agent is 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron). In such a process, the detectable element exchanges the 5 ligand.
The above mentioned reactions may be effected analogously to known methods. The above mentioned reactions are conveniently effected under conditions avoiding trace metal ~ontArn;n~tion Preferably dis-10 tilled de-ionized water, ultrapure reagents, no carrier-added radioactivity etc. are used to reduce the effects of trace metal.
The chelates of the invention may exist e.g. in free or salt form. Salts include acid addition salts with e.g.
15 organic acids, polymeric acids or tn-~rg~n;c acids, for example hydrochlorides and acetates, and salt forms ~ht~;n:-hl,~ with the carboxylic acid groups present in the molecule which do not participate to the chelate f ormatioII .
20 The chelates of the invention and their pharmaceutical acceptable salts exhibit diagnostic activity and are therefore useful as imaging agents, e.g. v; c~l; c~tion of thrombi. They exhibit significant serum stability and binding affinity to thrombin, as indicated in 25 standard i n y~ tests . The compound of Example 4 has a half life of 210 hours in human serum, when tested ~n Yi~Q .
Accordingly, the present invention also provides a method for i Y~S~ detection of a thrombus in a subject 30 which comprises a~ administering a chelate of the invention to said subject and b) recording the lo~l;c~t;,,n of the thrombus targeted by said chelate.
~ Wo gs/20603 ~ 1 ~ 9 8 8 g r.~
The chelates of the invention for use as an imaging agent in the above method may be administered parPnt~r~1 ly, preferably intravenously, e.g. in the ~e - form of injectable solutions or suspensions, preferably 5 in a single injectLon. The appropriate dosage will of course vary ri~r~n~;n5 upon, for example, the compound and the type of detectable element used. A suitable dose to be inj ected is in the range to enable imaging by photnsr~nn;n~ procedures known in the art. A
lO chelate of the invention may advantageously be administered in a dose comprising 0.1 to lO mCi of a stably chelated radinn-1r1; ~lr .
In animals an indicated dosage range may be of from 0.1 to 1 ug/kg of a rr~ro1~nrl of the invention labeled with 0 . 02 to 0 . 5 mCi r-emitting r;-~; onl1r1; ~,~ . In larger animals, f or example humans, an indicated dosage range may be of from 1 to 20 ug compound labeled e.g. with 3 to 10 mCi r-emitting r~ n-~r1 i~1~, preferably 2 to 5 mCi.
20 The localisation of a throm.bus with the chelates may be followed by the ~,Jr,~ lr7;nrJ imaging technir~ues, e.g.
using nuclear medicine imaging instrumentation, for example a scanner, a conventional gam.ma camera to obtain planar images, a rotating r-camera to perform 25 Si~gle Photon ~m; RRion Computer T~ ,hy (SPECT) or suitable instrumentation to perform Photon Emission T, ~ (PET), each preferably computer assisted.
The chelates of the invention may be administered in free form or in ~h~rr~~~lltically acceptable salt form.
3 0 Such salts may be prepared in conventional manner and exhibit the same order of activity as the free wo 95~20603 ;~ 1 7 ~ 8 8 ~ Pcr/EPss/00269 The chelates_of the invention are particularly useful in detecting thrombi formation resulting from vascular thrombotic disorders caused by myocardial infarction, artheriosclerosis, stroke, tl;RspTn;n:~ted intravascular S coagulation, peri- and post-operative to surgery, vascular recanalisations, immobility, atrial f ibril lation and heart ; n ~1l f f i r i ~n~y .
The chelates of the invention for use-in the method of the present invention may pre_erably be prepared 10 shortly before administration to a subject, i.e. the radiolabeling with the desired detectable metal ion, may be performed shortly before administration.
According to a further aspect of the invention, there is provided a ~hArr-rF~lltical composition comprisin~ a 15 chelate according to the invention in free or in rhAnr--Plltically acceptable salt form, together with one or more phArr~ ltically acceptable carriers or diluents theref or .
Such a composition may be manufactured in conventional 2 0 manner .
A composition according to the invention may also be presented in separate package with instructions for mixing the compound with the metal ion and for the administration of the resulting chelate. It may also be 25 presented in twin-pack ~orm, that is, as a sin,r,le package crmtAin;nrJ separate unit dosages of the compound and the detectable metal ion with instructions for mixing them and for administration of the chelate.
A diluent or carrier may be present in the unit dosage 3 0 f orms .
In the f ollowing examples, all temperatures are in C
Wo ss/2o6o3 r~
~17g889 and [a]D20- values are uncorrected.
The following abbreviations are employed:
Bzl = benzyl Z = benzyloxycarbonyl Boc = t-butyloxycarbonyl Ac = acetyl MeOH = methyl alcohol EtOH = ethyl alcohol EtOAc = ethyl acetate DCC = dicyclohexylr~rhod;; m; ~1 HONSu = N-hydroxy-sllrr;n;m;~?
OPin = p;nAned; Ql TFA = trif luoroacetic acid THF = tetrahydrofuran DMF = dimethyl formamide Np = p-nitrophenyl TLC = thin layer chromatography Baa = -NX-CH- (CH2CH2CH2Br) B-TMSa 1 = trime thyl s ilyl A 1 A n; n ~
BoroLys = -NH-CH-(CX2-CH2-CH2-CH2-NH2)B-DTPA- (p-NX-Bzl) = OH NH~
, 1. ~
o'~
OH~ ~N ~ ~OH
0 ~0 0~ 0 OH OH
(t-Bu) -DTPA- (p-NH Bzl) = t-butyl ester (4x) of DTPA- (p-NH-Bz l ) WO9~/20603 2~79889 F~~ 'C~ ~ ~
D~P.7~-(D~ 1-Pro-BoroLv~-OPi~) 2 ~NH, COOH ~51 N
HOOC ~ ~COOH ,3L
O
~NH2 ~) D~PA-(D-q~ l-P~., .. _~v(6-N-Fmo~)Lvl3-OP
H-D-TMSal-Pro-Boro (e-N-Fmoc) Lys-OPin (tri~luoroacetate~
(428 . 4 mg O . 5 mMol~ ia dissolved in dioxane and is treated with 5 . 0 ml saturated aqueous NaHCO3 and DTPA-anhydride (0.268 g 0.75 mMol~. The mixture is stirred ~or a . 5 h at room temperature. Acetic acid ( 3 5 . 0 ml ~ is added and the mixture is concentrated in vacuo at 40. The resulting residue is treated with 50 ml EtOAc and H20. The aS~ueous layer is extracted with EtOAc and the c n~d organic layers are washed with H2O. A3ter drying over Na2SO and concentration in vacuo the residue is chromato~rr~rh~f~ on a 4. 0 x 25 cm ~PI C-column RP18 (10 um) H2O/MeOH/acetic acid. The desired` product is obtained as a white foam MS: 1842 (N + H) .
b) ~rp~ l-P ~ ,Lvn-OP~
DTPA- (D-TMSal-Pro-Boro ~e -N-Fmoc) Lys-OPin) 2 (1 . 92 g, 1. 042 mMol) o~ step al is treated with 5 0 ml piperidine in DMF (20 9~ solution) at room temperature ~ WO 95/20603 2 ~ 7 9 8 8 9 r~
for 15 min. The resulting suspension is diluted with 50 ml of ether and the desired product precipitates as a crude material. This material is purified over Dowex (l-X4, 200-40D meshj, EtOH/H2O (1:1) and 10 96 AcOH.
5 Af ter removal of the solvent the desired product is . obtained as a white foam and is crystallized from EtOH/ether to give the title - ~ Qllnr~ as an amorphous white solid. MS: 1398 (M + H) '; [a]D = -72 .0 ( c = 0 . 3 5 in MeOH ) .
10 H-D-TMSal-Pro-Boro(~-N-Fmoc)Lys-OPin(trifluoroacetate), used as starting material, may be prepared as follows:
A) soc-D-TMQ- 1 -Pro-soroLv~-oPin may be prepared as disclosed in Example 5 of EP-A-471, 651.
s) soc-D-TM~-l-pro-soro(~-N-Fmoc)LvD-op~
Boc-D-TMSal-Pro-BoroLys-OPin(620.7 mg,1.0 mMol) and Fmoc-OSu (337.3 mg, 1.0 mMol) in dioxane are treated at room temperature with saturated aqueous NaHCO3 solution. After 45 min, the reaction mixture is 20 diluted with EtOAc and is washed with water and brine.
Af ter drying over Na2SO~ and concentration in vacuo, the title compound is used without further purification in the next step.
C) ~-D-Tr~8~1 -Pro-Boro ( c -N-Fmoc ) I,v~ -OPin (t--if 1'''~''~-- ' ~) The product of step B) (2.36 g, 2.8 mMol) is treated with 8.5 ml TFA/H2O (95:5) and 0.85 ml anisol. After 1 h at room temperature, the reaction mixture is concentrated in vacuo and the resulting oil is chromatographed on a 3.2 x 25 cm HPLC column RP18 ( 10 um), H2O/MeOH/TFA. The desired product is obtained as a white foam, MS: 743 (M + H) WO 95/20603 2 ~ 7 9 8 8 9 r~
".upr.w 2: 11 T~ ~ ~a O~ EX~D1~ 1 1 mg of compound of Example 1 is dissolved in 5 ml O . 01 M acetic acid. The resulting solution is passed through a 0.22 u Millex-GV filter and dispensed in 5 0.1 ml pQrtions and stored at -20C. IllIncl3 (Amersham, 1 mCi/100 ul) is prediluted in an equal volume of 0 . 5 M sodium acetate and labeling is carried out by mixing the ligand with the InCl3 solution and gentle homogenisation at room temperature.
HEPES buffer, pX 7.4, is then added to make a 10-6M
solution .
~y~llUPT.l/! 3 D~PA--(D-~-~71 )--CO(CH~) ~CO--~_~/UQ-l--P. ~, 3~ ~.L~r~-OP~ n O --~ ~
~f ~ ~N ~ ~N~
O ~0 OqJ O ..
~ a~
a ) ~OOC ( c~, ) ,Co-D-T~Q- 1 -Pro-B~-o ( ~ -N-F ) L~ -OPi~
15 X-D-TMSal -Pro -Boro ~ ~ -N-Fmoc ) Lys -OPin ( tri f luoro-acetate of Example 1, step C) (0.857 g,l.0 mMol) is dissolved in dioxane and is treated at room temperature with saturated aS~ueous NaXCO3 solution and succinic anhydride (0.11 g, 1.10 mMol). After 45 min, the 20 reaction mixture is diluted with EtOAc and is washed ~ Wo 95/20603 217 9 8 ~ 9 r~
with aqueous NaHSO,-601ution (1 M) and brine. The organic layer is dried over Na2SO~ and concentrated in ~~
vacuo. The resulting crude product is chromatographed on a 3.2 x 25 cm HPLC-column RP18 (10 l~m) H20/MeOH.
5 The desired product is obtained as a white foam, MS:
,- 843 (M + H) .
}:~) (t.BU)-~q!pl~-(D-N~-Bzl)-co(c~)~co-D-~rM-Boro ( e -N-lr ) -LVEl-OP~ r~
The product of step a) (623 mg, 0.739 mMol) in THF is 10 cooled to 0 and is treated with N-methyl morpholine (0.081 ml, 0.739 mMol and pivaloyl chloride (0.095 ml, 0.776 mMol) to form the sLixed anhydride. After stirri~g for 1 h at 0, the reaction mixture is treated with ('Bu)-DTPA-(p-NH2-Bzl) (575 mg, 0.739 mMol) (as described in Bioconjugate Chem. 1991, 2(3), 180-186) and additional N-methyl morpholine (0.081 ml, 0.739 mMol). The reaction mixture is stirred for 15 h at room temperature, and then diluted with EtOAc and washed with aqueous citric acid solution (1 M), aqueous 20 saturated NaHCO3-solution and brine. The organic layer is dried over Na2SO~ and concentrated in vacuo. The resulting oil is chromatographed on a 3.2 x 25 cm HPLC-column RP18 (10 um), H2O/MeOH/TFA. The desired product is obtained as a white foam, MS: 1603 (M + H) .
25 c) lYrV.p_(D_~-B'~l )-co(~ co-D-Tr~ l-pro-Boro(~-N-F
) I.~--OP ~ ~
The product of step b) (576 mg, 0.296 mMol) is s~r~n;fied by treatment with TFA/H20 (95:5) in the presence of anisol/et~n-~flithiol (1:1) for 7 h at room 3 0 temperature . Excess TFA is removed in vacuo and the resultir~g residue is treated with ether to precipitate the desired product. After filtration and drying under reduced pressure, the title ~-, uL-d is obtained as an amorphous solid foam of the trifluoroacetate MS: 1323 W0 95/20603 P ~
217g~89 (M + H) -~-s~1l-co~c~,),CO-D-TM~Al-3?~0-sorol.~s-OPirL
The product of step c) ~326 mg, 0.2463 n~ol) is treated 5 with 2 . O ml piperidine in DMF (20 96 solution) at room temperature for 15 min. The resulting mixture is diluted with ether and the desired product precipitates as a white solid . This material is purif ied over Dowex (1-X4, 200-400 mesh), ~tOH/H2O (1:1) and 10 96 AcOH.
10 After removal of the solvent the title compound is obtained as a white lyophilisate (from H2O) ~S: 1101 (M + H) ', [a]D = -31.8 (c = 0.27 in MeOH) .
~P~r.T~ 4 ~ n 1~ o r r 1 ~ 3 The lllIn labeled c _-uLld is prepared by using the same 15 procedure as disclosed in Example 2.
Claims (9)
1. A compound of formula II in free or salt form II
wherein W is a chelating group attached to the N-terminal amino group of the compound;
Z is a direct bond or a spacer;
Y is a sequence of 2 amino acids of which the N-terminal amino acid is an unnatural amino acid of formula III
III
wherein R18 is a group of formula (c), (d), (e), (f), (g), (h) or (i) c) ?CH2?Si(CH3)3 d) -CH2-C(CH3)3 f) e) h) g) i) and the other amino acid is L-proline;
Q1 and Q2 are both -OH or together represent a group of formula (a) a) R4 is hydrogen or C1-10alkyl;
R5 is a group -Z1-X1, wherein Z1 is -(CH2)?-; -CH(CH3)-(CH2)2-, -CH2-CH(CH3)-CH2-, -(CH2)2-CH(CH3)-, -(CH2)2-C(CH3)2-, -CH(CH3)-(CH2)3-, -CH2-CH(CH3)-(CH2)2-, -(CH2)2-CH(CH3)-CH2-, -(CH2)3-C(CH3)2-, -(CH2)2-C(CH3)2-CH2-, (CH2)3-CH(CH3)-, -(CH2)3-CH(CH3)-CH2-, C5-10aryl, or C6-10aralkyl wherein s is 2, 3, 4 or 5;
and wherein X1 is -NH2, -NH-C(NH)-NH2, -S-C(NH)-NH2, -N3, C1-4alkoxy, C1-4alkylthio, -Si(CH3)3, OH, SH, NR7R8, or phenyl, optionally substituted by up to three groups selected from halogen, OH and C1-4alkoxy, or C(NH)R10;
wherein R7 is H or C1-10alkyl; R8 is C1-10alkyl, -CO-R9 or CS-R9; R10 is C1-3alkyl or N(CH3)2, or NR11R12;
wherein R9 is H or C1-10alkyl, C1-10alkoxy, C6-10aryl, C6-10aralkyl and R11 and R12 (which may be the same or different) are X or C1-10alkyl;
or R4 and R5 together form a trimethylene group;
m is 1 or 2, and the asymmetric carbon atom marked * may have the D- or L-configuration, or represent any mixture of these.
wherein W is a chelating group attached to the N-terminal amino group of the compound;
Z is a direct bond or a spacer;
Y is a sequence of 2 amino acids of which the N-terminal amino acid is an unnatural amino acid of formula III
III
wherein R18 is a group of formula (c), (d), (e), (f), (g), (h) or (i) c) ?CH2?Si(CH3)3 d) -CH2-C(CH3)3 f) e) h) g) i) and the other amino acid is L-proline;
Q1 and Q2 are both -OH or together represent a group of formula (a) a) R4 is hydrogen or C1-10alkyl;
R5 is a group -Z1-X1, wherein Z1 is -(CH2)?-; -CH(CH3)-(CH2)2-, -CH2-CH(CH3)-CH2-, -(CH2)2-CH(CH3)-, -(CH2)2-C(CH3)2-, -CH(CH3)-(CH2)3-, -CH2-CH(CH3)-(CH2)2-, -(CH2)2-CH(CH3)-CH2-, -(CH2)3-C(CH3)2-, -(CH2)2-C(CH3)2-CH2-, (CH2)3-CH(CH3)-, -(CH2)3-CH(CH3)-CH2-, C5-10aryl, or C6-10aralkyl wherein s is 2, 3, 4 or 5;
and wherein X1 is -NH2, -NH-C(NH)-NH2, -S-C(NH)-NH2, -N3, C1-4alkoxy, C1-4alkylthio, -Si(CH3)3, OH, SH, NR7R8, or phenyl, optionally substituted by up to three groups selected from halogen, OH and C1-4alkoxy, or C(NH)R10;
wherein R7 is H or C1-10alkyl; R8 is C1-10alkyl, -CO-R9 or CS-R9; R10 is C1-3alkyl or N(CH3)2, or NR11R12;
wherein R9 is H or C1-10alkyl, C1-10alkoxy, C6-10aryl, C6-10aralkyl and R11 and R12 (which may be the same or different) are X or C1-10alkyl;
or R4 and R5 together form a trimethylene group;
m is 1 or 2, and the asymmetric carbon atom marked * may have the D- or L-configuration, or represent any mixture of these.
2 A compound according to claim 1, in which X1 is -NH2, -NH-C(NH)-NH2, -N3, -OH, -Si(CH3)3, phenyl, monochloro-phenyl, dimethylamino-phenyl, -NH-C(O)-CH3, -NH-C(O)H, -NHC(S)-NHCH3, -NH-C(O)-CH(CH3)2, -NH-C (O) -CH2-CH3, -NH-C (O) -CX2-Cl or -NH-C (O) -OCH3 .
3. A compound according to claim 1 or 2 in which the chelatins group is an iminodicarboxylic acid group or a polyaminopolycarboxylic acid group.
4. A compound according to claim 1, 2 or 3, in which the chelating group is a radical of formula (j) W'-CH2-Xa-NH- (j) wherein W' is a chelating group derived rom a non-cyclic or cyclic polyamino-polyacetic acid or anhydride, and Xa is phenylene or C1-3alkylene.
5. A compound according to any one of claims 1-4 comprising a spacing group of ,formula (d) -R22-R23-Co- (d) wherein R22 is a divalent residue derived, from a functional moiety capable of covalently reacting with the chelating agent, and R23 is C1- 11alkylene optionally interrupted by one or more heteroatoms or residues selected from O, S, CO, -NH-, -NHCO-, N(C1-4alkyl)-CO- and -N(C1-4alkyl)-, C2-11alkenylene or -CH(R14)- wherein R14 is a residue attached to a natural or unnatural .alpha.-amino acid, e.g. hydrogen, C1-11alkyl, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl-methyl.
6. DTPA- (p-NH-Bzl) -CO (CH2)3CO-TMSal-Pro-BoroLys-OPin, in free or salt form.
7. A chelate comprising a compound according to any one of claims 1-6 complexed with a detectable element.
8. The diagnostic use of a chelate according to claim 7.
9. A diagnostic composition comprising a chelate according to claim 7, together with a pharmaceutically acceptable carrier or diluent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9401483A GB9401483D0 (en) | 1994-01-26 | 1994-01-26 | Organic compounds |
| GB9401483.4 | 1994-01-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2179889A1 true CA2179889A1 (en) | 1995-08-03 |
Family
ID=10749357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002179889A Abandoned CA2179889A1 (en) | 1994-01-26 | 1995-01-25 | Inhibitors of serine proteases, bearing a chelating group |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0741747A1 (en) |
| JP (1) | JPH09509653A (en) |
| AU (1) | AU1576495A (en) |
| CA (1) | CA2179889A1 (en) |
| GB (1) | GB9401483D0 (en) |
| IL (1) | IL112423A0 (en) |
| PE (1) | PE45295A1 (en) |
| SG (1) | SG47079A1 (en) |
| WO (1) | WO1995020603A1 (en) |
| ZA (1) | ZA95636B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9515489D0 (en) * | 1995-07-28 | 1995-09-27 | Sandoz Ltd | Organic compounds |
| DE10046514A1 (en) | 2000-09-15 | 2002-04-25 | Diagnostikforschung Inst | Process for imaging and diagnosis of thrombi using magnetic resonance imaging using particulate contrast media |
| KR20050057294A (en) | 2002-09-09 | 2005-06-16 | 트라이젠 리미티드 | Boronic acid salts and their use in the treatment of thrombosis |
| US7576206B2 (en) | 2003-08-14 | 2009-08-18 | Cephalon, Inc. | Proteasome inhibitors and methods of using the same |
| US7223745B2 (en) | 2003-08-14 | 2007-05-29 | Cephalon, Inc. | Proteasome inhibitors and methods of using the same |
| US7468383B2 (en) | 2005-02-11 | 2008-12-23 | Cephalon, Inc. | Proteasome inhibitors and methods of using the same |
| CN102725300B (en) | 2009-12-22 | 2015-03-11 | 赛福伦公司 | Proteasome inhibitors and processes for their preparation, purification and use |
| JP7164521B2 (en) | 2016-06-21 | 2022-11-01 | オリオン・オフサルモロジー・エルエルシー | carbocyclic prolinamide derivatives |
| KR102595723B1 (en) | 2016-06-21 | 2023-10-27 | 오리온 옵탈몰로지 엘엘씨 | Heterocyclic Prolinamide Derivatives |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4668503A (en) * | 1982-07-26 | 1987-05-26 | Trustees Of University Of Massachusetts | Process for labeling amines with 99m Tc |
| US5187157A (en) * | 1987-06-05 | 1993-02-16 | Du Pont Merck Pharmaceutical Company | Peptide boronic acid inhibitors of trypsin-like proteases |
| EP0403243B1 (en) * | 1989-06-16 | 1995-11-08 | Merck Frosst Canada Inc. | Chelate derivatives of atrial natriuretic factor (ANF) |
| GB9017694D0 (en) * | 1990-08-13 | 1990-09-26 | Sandoz Ltd | Improvements in or relating to organic chemistry |
| US5252721A (en) * | 1990-09-28 | 1993-10-12 | Neorx Corporation | S3 N chelating compounds |
| JPH06507174A (en) * | 1991-04-22 | 1994-08-11 | マリンクロッド・メディカル・インコーポレイテッド | Methods for detecting and localizing tissues with neurokinin 1 receptors |
-
1994
- 1994-01-26 GB GB9401483A patent/GB9401483D0/en active Pending
-
1995
- 1995-01-24 PE PE1995260194A patent/PE45295A1/en not_active Application Discontinuation
- 1995-01-24 IL IL11242395A patent/IL112423A0/en unknown
- 1995-01-25 JP JP7519884A patent/JPH09509653A/en active Pending
- 1995-01-25 AU AU15764/95A patent/AU1576495A/en not_active Abandoned
- 1995-01-25 CA CA002179889A patent/CA2179889A1/en not_active Abandoned
- 1995-01-25 EP EP95907611A patent/EP0741747A1/en not_active Ceased
- 1995-01-25 WO PCT/EP1995/000269 patent/WO1995020603A1/en not_active Application Discontinuation
- 1995-01-25 SG SG1996005775A patent/SG47079A1/en unknown
- 1995-01-26 ZA ZA95636A patent/ZA95636B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IL112423A0 (en) | 1995-03-30 |
| EP0741747A1 (en) | 1996-11-13 |
| JPH09509653A (en) | 1997-09-30 |
| AU1576495A (en) | 1995-08-15 |
| GB9401483D0 (en) | 1994-03-23 |
| ZA95636B (en) | 1996-07-26 |
| SG47079A1 (en) | 1998-03-20 |
| WO1995020603A1 (en) | 1995-08-03 |
| PE45295A1 (en) | 1995-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU633859B2 (en) | Improvements in or relating to organic compounds | |
| AU684069B2 (en) | Alpha-ketoamide derivatives as inhibitors of thrombosis | |
| CA2234610C (en) | New amino acid derivatives, processes for preparing them and pharmaceutical compositions containing these compounds | |
| EP0832068B1 (en) | Stable reagents for the preparation of radiopharmaceuticals | |
| KR20190126294A (en) | Peptide Ligands for Binding to Mt1-mmp | |
| HUT77137A (en) | Peptide derived radionuclide chelators | |
| CA2224153C (en) | Radiolabeled peptide compositions for site-specific targeting | |
| NZ242668A (en) | Amidinophenylalanine derivatives; preparatory processes and their use as anticoagulents | |
| ZA200502835B (en) | Conjugates of TC complexes and targeting moieties and their use in MRI diagnostic | |
| JPS62207251A (en) | Oligopeptidylnitrile derivative | |
| US5776894A (en) | Chelated somatostatin peptides and complexes thereof, pharmaceutical compositions containing them and their use in treating tumors | |
| JPH08508999A (en) | Radioisotope labeled compounds for thrombus imaging | |
| US7481993B2 (en) | Chelators for radioactively labeled conjugates comprising a stabilizing sidechain | |
| CA2183421A1 (en) | Labelled peptide compounds | |
| KR20070034064A (en) | Improved N4 Chelating Agent Complex | |
| US5753627A (en) | Use of certain complexed somatostatin peptides for the invivo imaging of somatostatin receptor-positive tumors and metastasis | |
| CA2179889A1 (en) | Inhibitors of serine proteases, bearing a chelating group | |
| NZ250653A (en) | Somatostatin derivatives and pharmaceutical or diagnostic imaging compositions | |
| EP1700608A1 (en) | Chelators for radioactively labeled conjugates comprising a stabilizing sidechain | |
| FI101967B (en) | Method for preparing a ligand complexed with a pharmaceutically acceptable radionuclide | |
| PL163432B1 (en) | Method for manufacturing peptide derivatives | |
| JPH07316190A (en) | Peptide compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |