U.S. patent application number 14/528362 was filed with the patent office on 2015-02-26 for prodrug comprising a drug linker conjugate.
The applicant listed for this patent is ASCENDIS PHARMA A/S. Invention is credited to Felix Cleemann, Ulrich Hersel, Silvia Kaden, Harald Rau, Thomas Wegge.
Application Number | 20150057221 14/528362 |
Document ID | / |
Family ID | 40823379 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150057221 |
Kind Code |
A1 |
Cleemann; Felix ; et
al. |
February 26, 2015 |
PRODRUG COMPRISING A DRUG LINKER CONJUGATE
Abstract
The present invention relates to a prodrug or a pharmaceutically
acceptable salt thereof comprising a drug linker conjugate D-L,
wherein -D is an amine containing biologically active moiety; and
-L is a non-biologically active linker moiety -L.sup.1 represented
by formula (I): ##STR00001## wherein the dashed line indicates the
attachment to the amine of the biologically active moiety and
wherein R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, X,
X.sup.1, X.sup.2, X.sup.3 have the meaning as indicated in the
description and the claims and wherein L.sup.1 is substituted with
one to four groups L.sup.2-Z and optionally further substituted,
provided that the hydrogen marked with the asterisk in formula (I)
is not replaced by a substituent; wherein L.sup.2 is a single
chemical bond or a spacer; and Z is a carrier group. The invention
also relates to A-L, wherein A is a leaving group, pharmaceutical
composition comprising said prodrugs and their use as
medicaments.
Inventors: |
Cleemann; Felix;
(Heidelberg, DE) ; Hersel; Ulrich; (Heidelberg,
DE) ; Kaden; Silvia; (Heidelberg, DE) ; Rau;
Harald; (Heidelberg, DE) ; Wegge; Thomas;
(Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASCENDIS PHARMA A/S |
Hellerup |
|
DK |
|
|
Family ID: |
40823379 |
Appl. No.: |
14/528362 |
Filed: |
October 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12865693 |
Oct 8, 2010 |
8906847 |
|
|
PCT/EP09/51079 |
Jan 30, 2009 |
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14528362 |
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Current U.S.
Class: |
514/7.6 ;
514/11.7; 514/21.3; 514/211.13; 530/300; 530/308; 530/324;
540/551 |
Current CPC
Class: |
A61K 47/542 20170801;
A61K 47/545 20170801; A61K 31/553 20130101; A61K 47/65 20170801;
A61K 38/26 20130101; A61K 47/60 20170801; A61K 38/1709 20130101;
A61K 47/54 20170801; A61P 43/00 20180101; A61K 38/18 20130101 |
Class at
Publication: |
514/7.6 ;
530/308; 514/11.7; 540/551; 514/211.13; 530/300; 530/324;
514/21.3 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 38/17 20060101 A61K038/17; A61K 38/18 20060101
A61K038/18; A61K 38/26 20060101 A61K038/26; A61K 31/553 20060101
A61K031/553 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2008 |
EP |
08150973.9 |
Dec 5, 2008 |
EP |
08170872.9 |
Claims
1. A prodrug or a pharmaceutically acceptable salt thereof
comprising: a drug-linker conjugate D-L; wherein: -D is a nitrogen
containing biologically active moiety; -L is a non-biologically
active linker moiety -L.sup.1; and -L.sup.1 comprises an
amine-containing nucleophile, and is represented by formula (I):
##STR00088## wherein: the dashed line indicates the attachment to
the nitrogen of the biologically active moiety by forming an amide
bond; X is C(R.sup.4R.sup.4a), N(R.sup.4), O,
C(R.sup.4R.sup.4a)--C(R.sup.5R.sup.5a),
C(R.sup.5R.sup.5a)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--N(R.sup.6), N(R.sup.6)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--O, or O--C(R.sup.4R.sup.4a); X.sup.1 is C, or
S(O); X.sup.2 is C(R.sup.7R.sup.7a, or
C(R.sup.7R.sup.7a)--C(R.sup.8R.sup.8a); X.sup.3 is O, S, or N--CN;
R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4,
R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8,
R.sup.8a are independently selected from the group consisting of H,
and C.sub.1-4 alkyl; optionally, one or more of the pairs
R.sup.1a/R.sup.4a, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, and
R.sup.7a/R.sup.8a form a chemical bond; optionally, one or more of
the pairs R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a,
R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, R.sup.8/R.sup.8a are joined
together with the atom to which they are attached to form a
C.sub.3-7 cycloalkyl; or 4 to 7 membered heterocyclyl; optionally,
one or more of the pairs R.sup.1/R.sup.4, R.sup.1/R.sup.5,
R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.4/R.sup.6, R.sup.7/R.sup.8,
R.sup.2/R.sup.3 are joined together with the atoms to which they
are attached to form a ring A; optionally, R.sup.3/R.sup.3a are
joined together with the nitrogen atom to which they are attached
to form a 4 to 7 membered heterocycle; A is selected from the group
consisting of: phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 4 to 7 membered heterocyclyl, and 9 to 11
membered heterobicyclyl; and --N(R.sup.3R.sup.3a) is the
amine-containing nucleophile; wherein L.sup.1 is substituted with
one to four groups L.sup.2-Z and optionally further substituted,
provided that the hydrogen marked with the asterisk in formula (I)
is not replaced by a substituent; wherein L.sup.2 is a single
chemical bond or a spacer; and wherein Z is a carrier group.
2. The prodrug of claim 1; wherein X.sup.3 is O.
3. The prodrug of claim 1; wherein: X is N(R.sup.4); X.sup.1is C;
and X.sup.3 is O.
4. The prodrug of claim 1; wherein X.sup.2 is
C(R.sup.7R.sup.7a).
5. The prodrug of claim 1; wherein L.sup.1 is selected from the
group consisting of: ##STR00089## ##STR00090## ##STR00091##
##STR00092## wherein: R is H, or C.sub.1-4 alkyl; and Y is NH, O,
or S.
6. The prodrug of claim 1; wherein L.sup.1 is selected from the
group consisting of: ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## wherein R is H, or C.sub.1-4
alkyl.
7. The prodrug of claim 1; wherein: L.sup.2 is a single chemical
bond; or L.sup.2-Z is selected from the group consisting of:
COOR.sup.9, OR.sup.9, C(O)R.sup.9, C(O)N(R.sup.9R.sup.9a),
S(O).sub.2N(R.sup.9R.sup.9a), S(O)N(R.sup.9R.sup.9a),
S(O).sub.2R.sup.9, S(O)R.sup.9,
N(R.sup.9)S(O).sub.2N(R.sup.9aR.sup.9b), SR.sup.9,
N(R.sup.9R.sup.9a), OC(O)R.sup.9, N(R.sup.9)C(O)R.sup.9a,
N(R.sup.9)S(O).sub.2R.sup.9a, N(R.sup.9)S(O)R.sup.9a,
N(R.sup.9)C(O)OR.sup.9a, N(R.sup.9)C(O)N(R.sup.9aR.sup.9b,
OC(O)N(R.sup.9R.sup.9a), T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl,
and C.sub.2-50 alkynyl; wherein T, C.sub.1-50 alkyl, C.sub.2-50
alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one
or more R.sup.10, which are the same or different; and wherein
C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.11)--, --S(O).sub.2N(R.sup.11)--,
--S(O)N(R.sup.11)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--, --S--, --N(R.sup.11)--,
--OC(O)R.sup.11, --N(R.sup.11)C(O)--, --N(R.sup.11)S(O).sub.2--,
--N(R.sup.11)S(O)--, --N(R.sup.11)C(O)O--,
--N(R.sup.11)C(O)N(R.sup.11a)--, and --OC(O)N(R.sup.11R.sup.11a);
and wherein: R.sup.9, R.sup.9a, and R.sup.9b are independently
selected from the group consisting of H, Z, T, C.sub.1-50 alkyl,
C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein T, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
substituted with one or more R.sup.10, which are the same or
different; and wherein C.sub.1-50, alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of: T, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.11)--, --S(O).sub.2N(R.sup.11)--,
--S(O)N(R.sup.11)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.11)S(O)N(R.sup.11)--, --S--, --N(R.sup.11)--,
--OC(O)R.sup.11, --N(R.sup.11)C(O)--, --N(R.sup.11)S(O).sub.2--,
--N(R.sup.11)S(O)--, --N(R.sup.11)C(O)O--,
--N(R.sup.11)C(O)N(R.sup.11a)--, and --OC(O)N(R.sup.11R.sup.11a); T
is selected from the group consisting of: phenyl, naphthyl,
indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4 to 7
membered heterocyclyl, and 9 to 11 membered heterobicyclyl; wherein
T is optionally substituted with one or more R.sup.10, which are
the same or different; R.sup.10 is selected from the group
consisting of: Z, halogen, CN, oxo (.dbd.O), COOR.sup.12,
OR.sup.12, C(O)R.sup.12, C(O)N(R.sup.12R.sup.12a),
S(O).sub.2N(R.sup.12R.sup.12a), S(O)N(R.sup.12R.sup.12a),
S(O).sub.2R.sup.12, S(O)R.sup.12,
N(R.sup.12)S(O).sub.2N(R.sup.12R.sup.12b), SR.sup.12,
N(R.sup.12R.sup.12a), NO.sub.2; OC(O)R.sup.12,
N(R.sup.12)C(O)R.sup.12a, N(R.sup.12)S(O).sub.2R.sup.12a,
N(R.sup.12)S(O)R.sup.12a, N(R.sup.12)C(O)OR.sup.12a,
N(R.sup.12)C(O)N(R.sup.12aR.sup.12b), OC(O)N(R.sup.12R.sup.12a),
and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally
substituted with one or more halogen, which are the same or
different; and R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, and
R.sup.12b are independently selected from the group consisting of:
H, Z, or C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally
substituted with one or more halogen, which are the same or
different; provided that one of R.sup.9, R.sup.9a, R.sup.9b,
R.sup.10, R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, and R.sup.12b
is Z.
8. The prodrug of claim 1; wherein L.sup.2 is a C.sub.1-20 alkyl
chain, which is: optionally interrupted by one or more groups
independently selected from --O-- and C(O)N(R.sup.3aa); and
optionally substituted with one or more groups independently
selected from OH and C(O)N(R.sup.3aaR.sup.3aaa); and wherein
R.sup.3aa and R.sup.3aaa are independently selected from the group
consisting of H, and C.sub.1-4 alkyl.
9. The prodrug of claim 1; wherein L.sup.2 has a molecular weight
in the range of from 14 g/mol to 750 g/mol.
10. The prodrug of claim 1; wherein L.sup.2 is attached to Z via a
terminal group selected from the group consisting of:
##STR00104##
11. The prodrug of claim 1; wherein L is represented by formula
(Ia): ##STR00105## wherein R.sup.3aa and R.sup.3aaa: are
independently selected from the group consisting of H and C.sub.1-4
alkyl; or are joined together with the nitrogen atom to which they
are attached to form a 4 to 7 membered heterocycle.
12. The prodrug of claim 1; wherein L is represented by formula
(Ib): ##STR00106## wherein R.sup.3aa is H or C.sub.1-4 alkyl.
13. The prodrug of claim 1; wherein R.sup.1 in formula (I) is
L.sup.2-Z.
14. The prodrug of claim 1; wherein R.sup.3 in formula (I) is
L.sup.2-Z.
15. The prodrug of claim 1; wherein R.sup.3 and R.sup.3a in formula
(I) are joined together with the nitrogen atom to which they are
attached to form a 4 to 7 membered heterocycle which is substituted
with L.sup.2-Z.
16. The prodrug of claim 1; wherein D-H is a small molecule
bioactive agent or a biopolymer.
17. The prodrug of claim 1; wherein D-H is a biopolymer selected
from the group of biopolymers consisting of proteins, polypeptides,
oligonucleotides, and peptide nucleic acids.
18. The prodrug of claim 1; wherein D-H is a polypeptide selected
from the group of polypeptides consisting of: ACTH, adenosine
deaminase, agalsidase, alfa-1 antitrypsin (AAT), alfa-1 proteinase
inhibitor (API), alteplase, amylins (amylin, symlin), anistreplase,
anerod serine protease, antibodies (monoclonal or polyclonal, and
fragments or fusions), antithrombin III, antitrypsins, aprotinin,
asparaginases, atosiban, biphalin, bivalirudin, bone-morphogenic
proteins, bovine pancreatic trypsin inhibitor (BPTI), cadherin
fragments, calcitonin (salmon), collagenase, complement C1 esterase
inhibitor, conotoxins, cytokine receptor fragments, DNase,
dynorphine A, endorphins, enfuvirtide, enkephalins,
erythropoietins, exendins, factor VII, factor VIIa, factor VIII,
factor VIIIa, factor IX, fibrinolysin, fibroblast growth factor
(FGF), growth hormone releasing peptide 2 (GHRP2), fusion proteins,
follicle-stimulating hormones, gramicidin, ghrelin,
desacyl-ghrelin, granulocyte colony stimulating factor (G-CSF),
galactosidase, glucagon, glucagon-like peptides,
glucocerebrosidase, granulocyte macrophage colony stimulating
factor (GM-CSF), human heat shock proteins (HSP),
phospholipase-activating protein (PLAP), gonadotropin chorionic
(hCG), hemoglobins, hepatitis B vaccines, hirudin, human serine
protease inhibitor, hyaluronidases, idurnonidase, immune globulins,
influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10,
11, 12, 13, 21), IL-1 receptor antagonist (rhIL-1ra), insulins,
insulin like growth factors, insulin-like growth factor binding
protein (rhIGFBP), interferons (alfa 2a, alfa 2b, alfa 2c, beta 1a,
beta 1b, gamma 1a, gamma 1b), intracellular adhesion molecule,
keratinocyte growth factor (KGF), P-selectin glycoprotein ligand
(PSGL), transforming growth factors, lactase, leptin, leuprolide,
levothyroxine, luteinizing hormone, lyme vaccine, natriuretic
peptides (ANP, BNP, CNP and fragments), neuropeptide Y,
pancrelipase, pancreatic polypeptide, papain, parathyroid hormone,
PDGF, pepsin, peptide YY, platelet activating factor
acetylhydrolase (PAF-AH), prolactin, protein C, thymalfasin,
octreotide, secretin, sermorelin, soluble tumor necorsis factor
receptor (TNFR), superoxide dismutase (SOD), somatropins (growth
hormone), somatoprim, somatostatin, streptokinase, sucrase,
terlipressin, tetanus toxin fragment, tilactase, thrombins,
thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis
factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator
(tPA), TSH, urodilatin, urate oxidase, urokinase, vaccines,
vascular endothelial growth factor (VEGF), vasoactive intestinal
peptide, vasopressin, ziconotide, lectin, and ricin.
19. The prodrug of claim 1; wherein D-H is a protein prepared by
recombinant DNA technologies.
20. The prodrug of claim 1; wherein D-H is a protein selected from
the group of proteins consisting of: antibody fragments, single
chain antigen binding proteins, catalytic antibodies, and fusion
proteins.
21. The prodrug of claim 1; wherein D-H is a small molecule
bioactive agent selected from the group of agents consisting of:
central nervous system-active agents, anti-infective,
anti-allergic, immunomodulating, anti-obesity, anticoagulants,
antidiabetic, anti-neoplastic, antibacterial, anti-fungal,
analgesic, contraceptive, anti-inflammatory, steroidal,
vasodilating, vasoconstricting, and cardiovascular agents with at
least one primary or secondary amino group.
22. The prodrug of claim 1; wherein D-H is a small molecule
bioactive agent selected from the group of agents consisting of:
acarbose, alaproclate, alendronate, amantadine, amikacin,
amineptine, aminoglutethimide, amisulpride, amlodipine, amotosalen,
amoxapine, amoxicillin, amphetamine, amphotericin B, ampicillin,
amprenavir, amrinone, anileridine, apraclonidine, apramycin,
articaine, atenolol, atomoxetine, avizafone, baclofen, benazepril,
benserazide, benzocaine, betaxolol, bleomycin, bromfenac,
brofaromine, carvedilol, cathine, cathinone, carbutamid,
cefalexine, clinafloxacin, ciprofloxacin, deferoxamine,
delavirdine, desipramine, daunorubicin, dexmethylphenidate,
dexmethylphenidate, diaphenylsulfon, dizocilpine, dopamin,
dobutamin, dorzolamide, doxorubicin, duloxetine, eflornithine,
enalapril, epinephrine, epirubicin, ergoline, ertapenem, esmolol,
enoxacin, ethambutol, fenfluramine, fenoldopam, fenoterol,
fingolimod, flecaimide, fluvoxamine, fosamprenavir, frovatriptan,
furosemide, fluoexetine, gabapentin, gatifloxacin, gemiflocacin,
gentamicin, grepafloxacin, hexylcaine, hydralazine,
hydrochlorothiazide, icofungipen, idarubicin, imiquimod, inversine,
isoproterenol, isradipine, kanamycin A, ketamin, labetalol,
lamivudine, levobunolol, levodopa, levothyroxine, lisinopril,
lomefloxacin, loracarbef, maprotiline, mefloquine, melphalan,
memantine, meropenem, mesalazine, mescaline, methyldopa,
methylenedioxymethamphetamine, metoprolol, milnacipran,
mitoxantron, moxifloxacin, norepinephrine, norfloxacin,
nortriptyline, neomycin B, nystatin, oseltamivir, pamidronic acid,
paroxetine, pazufloxacin, pemetrexed, perindopril, phenmetrazine,
phenelzine, pregabalin, procaine, pseudoephedrine, protriptyline,
reboxetine, ritodrine, sabarubicin, salbutamol, serotonin,
sertraline, sitagliptin, sotalol, spectinomycin, sulfadiazin,
sulfamerazin, sertraline, sprectinomycin, sulfalen,
sulfamethoxazol, tacrine, tarnsulosin, terbutaline, timolol,
tirofiban, tobramycin, tocainide, tosufloxacin, trandolapril,
tranexamic acid, tranylcypromine, trimerexate, trovafloxacin,
valaciclovir, valganciclovir, vancomycin, viomycin, viloxazine, and
zalcitabine.
23. The prodrug of claim 1; wherein Z is a polymer of at least 500
Da or a C.sub.8-18 alkyl group.
24. The prodrug of claim 1; wherein Z is selected from the group of
optionally crosslinked polymers consisting of: poly(propylene
glycol), poly(ethylene glycol), dextran, chitosan, hyaluronic acid,
alginate, xylan, mannan, carrageenan, agarose, cellulose, starch,
hydroxyalkyl starch (HAS), poly(vinyl alcohols), poly(oxazolines),
poly(anhydrides), poly(ortho esters), poly(carbonates),
poly(urethanes), poly(acrylic acids), poly(acrylamides),
poly(acrylates), poly(methacrylates), poly(organophosphazenes),
polyoxazoline, poly(siloxanes), poly(amides),
poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters),
poly(iminocarbonates), poly(amino acids), collagen, gelatin,
hydrogel, a blood plasma protein, and copolymers thereof.
25. The prodrug of claim 1; wherein Z is a protein.
26. The prodrug of claim 1; wherein Z is a protein selected from
the group consisting of albumin, transferrin, and
immunoglobulin.
27. The prodrug of claim 1; wherein Z is a linear or branched
poly(ethylene glycol) with a molecular weight from 2,000 Da to
150,000 Da.
28. The prodrug of claim 1; wherein: D-H is a GLP-1 receptor
agonist; and Z is a hydrogel.
29. The prodrug of claim 28; wherein the GLP-1 receptor agonist is
Exendin-4.
30. The prodrug of claim 28; wherein in formula (I): X is
N(R.sup.4); X.sup.1 is C; and X.sup.3 is O.
31. The prodrug of claim 1; wherein D-H is a GLP-1 receptor
agonist; and wherein: L is represented by formula (Ia):
##STR00107## wherein R.sup.3aa and R.sup.3aaa: are independently
selected from the group consisting of H and C.sub.1-4 alkyl; or are
joined together with the nitrogen atom to which they are attached
to form a 4 to 7 membered heterocycle; and wherein Z is a hydrogel;
or L is represented by formula (Ib): ##STR00108## wherein R.sup.3aa
is H or C.sub.1-4 alkyl.
32. A prodrug precursor comprising: a compound of formula Act-L;
wherein: Act is a leaving group; and L is a non-biologically active
linker moiety L.sup.1 which comprises an amine-containing
nucleophile, and which is represented by formula (I): ##STR00109##
wherein: the dashed line indicates the attachment to the nitrogen
of the biologically active moiety by forming an amide bond; X is
C(R.sup.4R.sup.4a), N(R.sup.4), O,
C(R.sup.4R.sup.4a)--C(R.sup.5R.sup.5a),
C(R.sup.5R.sup.5a)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--N(R.sup.6), N(R.sup.6)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--O, or O--C(R.sup.4R.sup.4a); X.sup.1 is C; or
S(O); X.sup.2 is C(R.sup.7R.sup.7a; or
C(R.sup.7R.sup.7a)--C(R.sup.8R.sup.8a); X.sup.3 is O; S; or N--CN;
R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4,
R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8,
R.sup.8a are independently selected from the group consisting of H,
and C.sub.1-4 alkyl; optionally, one or more of the pairs
R.sup.1a/R.sup.4a, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, and
R.sup.7a/R.sup.8a form a chemical bond; optionally, one or more of
the pairs R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a,
R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, and R.sup.8/R.sup.8a are joined
together with the atom to which they are attached to form a
C.sub.3-7 cycloalkyl, or 4 to 7 membered heterocyclyl; optionally,
one or more of the pairs R.sup.1/R.sup.4, R.sup.1/R.sup.5,
R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.4/R.sup.6, R.sup.7/R.sup.8,
and R.sup.2/R.sup.3 are joined together with the atoms to which
they are attached to form a ring A; optionally, R.sup.3/R.sup.3a
are joined together with the nitrogen atom to which they are
attached to form a 4 to 7 membered heterocycle; A is selected from
the group consisting of: phenyl, naphthyl, indenyl, indanyl,
tetralinyl, C.sub.3-10 cycloalkyl, 4 to 7 membered heterocyclyl,
and 9 to 11 membered heterobicyclyl; and --N(R.sup.3R.sup.3a) is
the amine-containing nucleophile; wherein L.sup.1 is substituted
with one to four groups L.sup.2-Z and optionally further
substituted, provided that the hydrogen marked with the asterisk in
formula (I) is not replaced by a substituent; wherein L.sup.2 is a
single chemical bond or a spacer, and wherein Z is a carrier
group.
33. The prodrug precursor of claim 32; wherein Act is chloride,
bromide, fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl,
N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl,
pentafluorophenoxy, 2-thiooxo-thiazolidinyl, or
N-hydroxysulfosuccinimidyl.
34. A pharmaceutical composition comprising: a prodrug of claim 1
or a pharmaceutical salt thereof; and a pharmaceutically acceptable
excipient.
35. A method comprising: administering the prodrug of claim 1.
36. A method comprising: administering the pharmaceutical
composition of claim 34.
37. The prodrug of claim 1; wherein D-H is a GLP-1 receptor
agonist; and wherein L is represented by formula (Ib): ##STR00110##
wherein: R.sup.3aa is H or C.sub.1-4 alkyl; and Z is a hydrogel.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/865,693, filed on Jul. 30, 2012, which is a
national phase of and thus claims priority from, PCT Patent
Application No. PCT/EP2009/051079 filed on Jan. 30, 2009--the
disclosures of which are incorporated herein by reference in their
entirety--which claims priority from EP 08170872.9 filed on Dec. 5,
2008, and from EP 08150973.9 filed on Feb. 1, 2008.
[0002] The present invention relates to a prodrug or a
pharmaceutically acceptable salt thereof comprising a drug linker
conjugate D-L. The invention also relates to pharmaceutical
compositions comprising said prodrugs and their use as
medicaments.
[0003] To enhance physicochemical or pharmacokinetic properties of
a drug in vivo such drug can be conjugated with a carrier.
[0004] Typically, carriers in drug delivery are either used in a
non-covalent fashion, with the drug physicochemically formulated
into a solvent-carrier mixture, or by covalent attachment of a
carrier reagent to one of the drug's functional groups.
[0005] However the non-covalent approach requires a highly
efficient drug encapsulation to prevent uncontrolled, burst-type
release of the drug. Restraining the diffusion of an unbound, water
soluble drug molecule requires strong van der Waals contacts,
frequently mediated through hydrophobic moieties. Many
conformationally sensitive drugs, such as proteins or peptides, are
rendered dysfunctional during the encapsulation process and/or
during subsequent storage of the encapsulated drug. In addition,
such amino-containing drugs readily undergo side reactions with
carrier degradation products (see, for example, D. H. Lee et al.,
J. Contr. Rel., 2003, 92, 291-299). Furthermore, dependence of the
release mechanism of the drug upon biodegradation may cause
interpatient variability.
[0006] Alternatively, the drugs may be conjugated to a carrier
through covalent bonds. This approach is applied to various classes
of molecules, from so-called small molecules, through natural
products up to larger proteins. Covalent drug carrier conjugates
can be divided into two groups. Firstly, conjugates, where the
covalent bond between carrier and drug is mostly present during the
action of the drug ("permanent covalent bond"), i.e. a derivative
of the drug exhibits its pharmacological effects as it is known for
the drug as such. Secondly, the covalent bond is mostly previously
cleaved to release the drug as such, which can exhibit its known
pharmacological effects. In the latter case the covalent drug
carrier conjugate is called carrier linked prodrug or carrier
prodrug.
[0007] In order to ensure cleavage of the covalent bond between
carrier and drug easy removal of said bond in vivo is required to
release the drug (prodrug activation).
[0008] Prodrug activation may occur by enzymatic or non-enzymatic
cleavage of the bond between the carrier and the drug molecule, or
a sequential combination of both, i.e. an enzymatic step followed
by a non-enzymatic rearrangement.
[0009] Enzymatically induced prodrug activation is characterized in
that the cleavage in enzyme-free in-vitro environment such as an
aqueous buffer solution, of e.g., an ester or amide may occur, but
the corresponding rate of hydrolysis may be much too slow and not
therapeutically useful. In an in-vivo environment, esterases or
amidases are typically present and the esterases and amidases may
cause significant catalytic acceleration of the kinetics of
hydrolysis from twofold up to several orders of magnitude.
Therefore, the cleavage is predominantly controlled by the
enzymatic reaction.
[0010] A major drawback of predominantly enzymatic cleavage is
interpatient variability. Enzyme levels may differ significantly
between individuals resulting in biological variation of prodrug
activation by the enzymatic cleavage. The enzyme levels may also
vary depending on the site of administration. For instance it is
known that in the case of subcutaneous injection, certain areas of
the body yield more predictable therapeutic effects than others. To
reduce this unpredictable effect, non-enzymatic cleavage or
intramolecular catalysis is of particular interest.
[0011] Therefore, enzyme-independent autocatalytic cleavage of
carrier and biologically active moiety is preferred. In most cases
this is achieved by an appropriately designed linker moiety between
the carrier and the biologically active moiety, which is directly
attached to the functional group of a biologically active moiety
via covalent bond.
[0012] Specific linker types are known in the art.
[0013] Y. Sohma et al., J. Med. Chem. 46 (2003), 4124-4135 describe
ester based prodrugs, where the carrier is water-soluble and the
biologically active moiety is derived from HIV-1 protease inhibitor
KNI-727. The linker used is attached to the biologically active
moiety via ester group. The mechanism of this prodrug system is
cyclization-activation by cyclic imide formation for the cleavage
of ester bonds.
[0014] However this is disadvantageous because of the instability
of the ester functional group. Furthermore, ester groups may be
less chemoselectively addressable for the conjugation of the
carrier or linker and the drug.
[0015] A. J. Garman et al. (A. J. Garman, S. B. Kalindjan, FEBS
Lett. 1987, 223 (2), 361-365 1987) use PEG5000-maleic anhydride for
the reversible modification of amino groups in tissue-type
plasminogen activator and urokinase. Regeneration of functional
enzyme from PEG-uPA conjugate upon incubation at pH 7.4 buffer by
cleavage of the maleamic acid linkage follows first order kinetics
with a half-life of 6.1 h. A disadvantage of the maleamic acid
linkage is the lack of stability of the conjugate at lower pH
values. This limits the applicability of the maleamic acid linkage
to biologically active agents which are stable at basic (high) pH
values, as purification of the biologically active agent polymer
conjugate has to be performed under basic (high pH) conditions to
prevent premature prodrug cleavage.
[0016] In WO-A 2004/108070 prodrug system based on
N,N-bis-(2-hydroxyethyl)glycine amide (bicine) linker is described.
In this system two PEG carrier molecules are linked to a bicine
molecule coupled to an amino group of the drug molecule. The first
two steps in prodrug activation is the enzymatic cleavage of the
first linkages connecting both PEG carrier molecules with the
hydroxy groups of the bicine activating group. Different linkages
between PEG and bicine are described resulting in different prodrug
activation kinetics. The second step in prodrug activation is the
cleavage of the second linkage connecting the bicine activating
group to the amino group of the drug molecule. The main
disadvantage of this system is the connection of the polymer to the
bicine linker resulting in a slow hydrolysis rate of this second
bicine amide linkage (t1/2>3 h in phosphate buffer).
Consequently the release of a bicine-modified prodrug intermediate
may show different pharmacokinetic, immunogenic, toxicological, and
pharmacodynamic properties as compared to the parent native drug
molecule.
[0017] Another bicine-based system is described in WO-A
2006/136586.
[0018] Accordingly, there is a need for alternative carrier-linked
prodrugs, where the linker allows an autocatalytic cleavage to
release a drug in an unmodified form without remaining residues
originating from the linker.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows an example of the cleavage resulting in a
cyclic imide.
[0020] FIGS. 2A-2G, collectively referred to as FIG. 2, depict
further details concerning compound numerals, starting materials,
synthesis method, molecular weight (MW), and MS data.
[0021] FIG. 3 shows in vivo and in vitro linker cleavage data of
13b, wherein in vivo (triangles) and in vitro (diamonds) cleavage
kinetics are shown by semilogarithmic representation.
DETAILED DESCRIPTION OF EMBODIMENTS
[0022] Thus, an object of the present invention is to provide such
drug linker conjugates, where the linker is covalently attached via
a cleavable bond to a biologically active moiety (representing the
drug after release), and where the linker is further covalently
attached via a permanent bond to a carrier directly or via a spacer
to form the carrier-linked prodrug.
[0023] This object is achieved by a prodrug or a pharmaceutically
acceptable salt thereof comprising a drug linker conjugate D-L,
wherein:
[0024] -D is a nitrogen containing biologically active moiety;
and
[0025] -L is a non-biologically active linker moiety -L.sup.1
represented by formula (I):
##STR00002##
[0026] wherein the dashed line indicates the attachment to the
nitrogen of the biologically active moiety by forming an amide
bond;
[0027] X is C(R.sup.4R.sup.4a), N(R.sup.4), O,
C(R.sup.4R.sup.4a)--C(R.sup.5R.sup.5a),
C(R.sup.5R.sup.5a)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--N(R.sup.6), N(R.sup.6)--C(R.sup.4R.sup.4a),
C(R.sup.4R.sup.4a)--O, or O--C(R.sup.4R.sup.4a);
[0028] X.sup.1 is C; or S(O);
[0029] X.sup.2 is C(R.sup.7,R.sup.7a; or
C(R.sup.7,R.sup.7a)--C(R.sup.8, R.sup.8a);
[0030] X.sup.3 is O; S; or N--CN;
[0031] R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a,
R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a,
R.sup.8, R.sup.8a are independently selected from the group
consisting of H; and C.sub.1-4 alkyl;
[0032] Optionally, one or more of the pairs R.sup.1a/R.sup.4a,
R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.7a/R.sup.8a form a
chemical bond;
[0033] Optionally, one or more of the pairs R.sup.1/R.sup.1a,
R.sup.2/R.sup.2a, R.sup.4/R.sup.4a, R.sup.5/R.sup.5a,
R.sup.7/R.sup.7a, R.sup.8/R.sup.8a are joined together with the
atom to which they are attached to form a C.sub.3-7 cycloalkyl; or
4 to 7 membered heterocyclyl;
[0034] Optionally, R.sup.4/R.sup.6 are joined together with the
atoms to which they are attached to form a saturated 4 to 7
membered heterocyclyl;
[0035] Optionally, one or more of the pairs R.sup.1/R.sup.4,
R.sup.1/R.sup.5, R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.4/R.sup.6,
R.sup.7/R.sup.8, R.sup.2/R.sup.3 are joined together with the atoms
to which they are attached to form a ring A;
[0036] Optionally, R.sup.3/R.sup.3a are joined together with the
nitrogen atom to which they are attached to form a 4 to 7 membered
heterocycle;
[0037] A is selected from the group consisting of phenyl; naphthyl;
indenyl; indanyl; tetralinyl, C.sub.3-10 cycloalkyl; 4 to 7
membered heterocyclyl; and 9 to 11 membered heterobicyclyl; and
[0038] wherein L.sup.1 is substituted with one to four groups
L.sup.2-Z and optionally further substituted, provided that the
hydrogen marked with the asterisk in formula (I) is not replaced by
a substituent; wherein
[0039] L.sup.2 is a single chemical bond or a spacer; and
[0040] Z is a carrier group.
[0041] It was surprisingly found, that the scope of
cyclization-activation by cyclic imide formation can be extended
from ester to even carrier-linked amide prodrugs, despite the much
greater stability of the amide bond under aqueous conditions. It
was observed that N,N'-biscarboxamides linked to a nucleophile
carrying moiety through one amide bond and to the drug molecule
through the second amide bond exhibit autohydrolysis in a range
that is useful for prodrug applications. In addition, it was
discovered that linkers can be designed that include a carrier
permanently attached to the N,N' biscarboxamide motif in such a
fashion that cyclic imide formation can be employed as a
self-activation principle in carrier-linked amide prodrug
design.
[0042] Examples for such preferred cyclic cleavage products are
substituted succinimide or glutarimide ring structures.
Prerequisite for such cyclization activation is the presence of an
amine-containing nucleophile in the linker structure and another
amide bond which is not the amide prodrug bond but an amide bond
substituted with a hydrogen atom.
[0043] In case of succinimide- or a glutarimide-activated prodrug
cleavage, the amine-containing nucleophile serves as a neighbouring
group to enhance the nucleophilicity of the nitrogen contained in
the permanent amide bond which in turn attacks the prodrug amide
carbonyl group and consequently induces intramolecular acylation of
the permanent amide bond generating the cyclic imide ring.
[0044] Therefore preferred linker structures comprise a permanent
linkage to a carrier, an amine-containing nucleophile, and a
permanent amide bond with a hydrogen attached to the nitrogen of
the amide bond. Corresponding carrier-linked prodrugs comprise a
linker containing a permanent linkage to a carrier, an
amine-containing nucleophile and said permanent amide bond, and a
nitrogen containing biologically active moiety derived from the
drug conjugated to the linker by means of a cleavable amide
bond.
[0045] FIG. 1 shows an example of the cleavage resulting in a
cyclic imide. The nitrogen of the biologically active moiety is
shown as hydrogen containing amine, which results in a drug having
a primary amine functional group. However also, e.g., a secondary
amine may be part of the drug. For simplification reasons the one
to four mandatory substituents L.sup.2-Z including the carrier are
not shown.
[0046] Preferred properties of the prodrug are given by a half-life
of hydrolysis in aqueous buffer at pH 7.4 and 37.degree. C. between
1 h and 3 months; similar rates of hydrolysis under physiological
conditions in buffer and plasma.
[0047] The prodrug according to the present invention may show
excellent in vivo/in vitro correlation of linker cleavage, a high
degree of enzyme independence and can be stored at lower pH (pH
dependent cleavage).
[0048] Within the meaning of the present invention the terms are
used as follows.
[0049] "Biologically active moiety D" means the part of the drug
linker conjugate, which results after cleavage in a drug D-H of
known biological activity.
[0050] "Non-active linker" means a linker which does not show the
pharmacological effects of the drug derived from the biologically
active agent.
[0051] "Alkyl" means a straight-chain or branched carbon chain.
Each hydrogen of an alkyl carbon may be replaced by a
substituent.
[0052] "C.sub.1-4 alkyl" means an alkyl chain having 1-4 carbon
atoms, e.g. if present at the end of a molecule: methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl tert-butyl, or
e.g.--CH.sub.2--, --CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH(C.sub.2H.sub.5)--,
--C(CH.sub.3).sub.2--, when two moieties of a molecule are linked
by the alkyl group. Each hydrogen of a C.sub.1-4 alkyl carbon may
be replaced by a substituent.
[0053] "C.sub.1-6 alkyl" means an alkyl chain having 1-6 carbon
atoms, e.g. if present at the end of a molecule: C.sub.1-4 alkyl,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl;
tert-butyl, n-pentyl, n-hexyl, or e.g. --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH.sub.2CH.sub.2--CH.sub.2--, --CH(C.sub.2H.sub.5)--,
--C(CH.sub.3).sub.2--, when two moieties of a molecule are linked
by the alkyl group. Each hydrogen of a C.sub.1-6 alkyl carbon may
be replaced by a substituent.
[0054] Accordingly, "C.sub.1-18 alkyl" means an alkyl chain having
1 to 18 carbon atoms and "C.sub.8-18 alkyl" means an alkyl chain
having 8 to 18 carbon atoms. Accordingly, "C.sub.1-50 alkyl" means
an alkyl chain having 1 to 50 carbon atoms.
[0055] "C.sub.2-50 alkenyl" means a branched or unbranched alkenyl
chain having 2 to 50 carbon atoms, e.g. if present at the end of a
molecule: --CH.dbd.CH.sub.2, --CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH.sub.2, --CH.dbd.CH--CH.sub.2--CH.sub.3,
--CH.dbd.CH--CH.dbd.CH.sub.2, or e.g. --CH.dbd.CH--, when two
moieties of a molecule are linked by the alkenyl group. Each
hydrogen of a C.sub.2-50 alkenyl carbon may be replaced by a
substituent as further specified. Accordingly, the term "alkenyl"
relates to a carbon chain with at least one carbon carbon double
bond. Optionally, one or more triple bonds may occur.
[0056] "C.sub.2-50 alkynyl" means a branched or unbranched alkynyl
chain having 2 to 50 carbon atoms, e.g. if present at the end of a
molecule: --C.ident.CH, --CH.sub.2--C.ident.CH,
CH.sub.2--CH.sub.2--C.ident.CH, CH.sub.2--C.ident.C--CH.sub.3, or
e.g. --C.ident.C-- when two moieties of a molecule are linked by
the alkynyl group. Each hydrogen of a C.sub.2-50 alkynyl carbon may
be replaced by a substituent as further specified. Accordingly, the
term "alkynyl" relates to a carbon chain with at lest one carbon
carbon triple bond. Optionally, one or more double bonds may
occur.
[0057] "C.sub.3-7 cycloalkyl" or "C.sub.3-7 cycloalkyl ring" means
a cyclic alkyl chain having 3 to 7 carbon atoms, which may have
carbon-carbon double bonds being at least partially saturated, e.g.
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,
cycloheptyl. Each hydrogen of a cycloalkyl carbon may be replaced
by a substituent. The term "C.sub.3-7 cycloalkyl" or "C.sub.3-7
cycloalkyl ring" also includes bridged bicycles like norbonane or
norbonene. Accordingly, "C.sub.3-5 cycloalkyl" means a cycloalkyl
having 3 to 5 carbon atoms.
[0058] Accordingly, "C.sub.3-10 cycloalkyl" means a cyclic alkyl
having 3 to 10 carbon atoms, e.g. C.sub.3-7 cycloalkyl;
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. The term
"C.sub.3-10 cycloalkyl" also includes at least partially saturated
carbomono- and -bicycles.
[0059] "Halogen" means fluoro, chloro, bromo or iodo. It is
generally preferred that halogen is fluoro or chloro.
[0060] "4 to 7 membered heterocyclyl" or "4 to 7 membered
heterocycle" means a ring with 4, 5, 6 or 7 ring atoms that may
contain up to the maximum number of double bonds (aromatic or
non-aromatic ring which is fully, partially or un-saturated)
wherein at least one ring atom up to 4 ring atoms are replaced by a
heteroatom selected from the group consisting of sulfur (including
--S(O)--, --S(O).sub.2--), oxygen and nitrogen (including
.dbd.N(O)--) and wherein the ring is linked to the rest of the
molecule via a carbon or nitrogen atom. Examples for a 4 to 7
membered heterocycles are azetidine, oxetane, thietane, furan,
thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole,
pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole,
thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline,
tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine,
pyrazolidine, oxazolidine, isoxazolidine, thiazolidine,
isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran,
tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine,
pyrimidine, piperazine, piperidine, morpholine, tetrazole,
triazole, triazolidine, tetrazolidine, diazepane, azepine or
homopiperazine.
[0061] "9 to 11 membered heterobicyclyl" or "9 to 11 membered
heterobicycle" means a heterocyclic system of two rings with 9 to
11 ring atoms, where at least one ring atom is shared by both rings
and that may contain up to the maximum number of double bonds
(aromatic or non-aromatic ring which is fully, partially or
un-saturated) wherein at least one ring atom up to 6 ring atoms are
replaced by a heteroatom selected from the group consisting of
sulfur (including --S(O)--, --S(O).sub.2--), oxygen and nitrogen
(including .dbd.N(O)--) and wherein the ring is linked to the rest
of the molecule via a carbon or nitrogen atom. Examples for a 9 to
11 membered heterobicycle are indole, indoline, benzofuran,
benzothiophene, benzoxazole, benzisoxazole, benzothiazole,
benzisothiazole, benzimidazole, benzimidazoline, quinoline,
quinazoline, dihydroquinazoline, quinoline, dihydroquinoline,
tetrahydroquinoline, decahydroquinoline, isoquinoline,
decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline,
benzazepine, purine or pteridine. The term 9 to 11 membered
heterobicycle also includes spiro structures of two rings like
1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles like
8-aza-bicyclo[3.2.1]octane.
[0062] In case the compounds according to formula (I) contain one
or more acidic or basic groups, the invention also comprises their
corresponding pharmaceutically or toxicologically acceptable salts,
in particular their pharmaceutically utilizable salts. Thus, the
compounds of the formula (I) which contain acidic groups can be
used according to the invention, for example, as alkali metal
salts, alkaline earth metal salts or as ammonium salts. More
precise examples of such salts include sodium salts, potassium
salts, calcium salts, magnesium salts or salts with ammonia or
organic amines such as, for example, ethylamine, ethanolamine,
triethanolamine or amino acids. Compounds of the formula (I) which
contain one or more basic groups, i.e. groups which can be
protonated, can be present and can be used according to the
invention in the form of their addition salts with inorganic or
organic acids. Examples for suitable acids include hydrogen
chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric
acid, lactic acid, salicylic acid, benzoic acid, formic acid,
propionic acid, pivalic acid, diethylacetic acid, malonic acid,
succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,
sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic
acid, isonicotinic acid, citric acid, adipic acid, and other acids
known to the person skilled in the art. If the compounds of the
formula (I) simultaneously contain acidic and basic groups in the
molecule, the invention also includes, in addition to the salt
forms mentioned, inner salts or betaines (zwitterions). The
respective salts according to the formula (I) can be obtained by
customary methods which are known to the person skilled in the art
like, for example by contacting these with an organic or inorganic
acid or base in a solvent or dispersant, or by anion exchange or
cation exchange with other salts. The present invention also
includes all salts of the compounds of the formula (I) which, owing
to low physiological compatibility, are not directly suitable for
use in pharmaceuticals but which can be used, for example, as
intermediates for chemical reactions or for the preparation of
pharmaceutically acceptable salts.
[0063] The term "pharmaceutically acceptable" means approved by a
regulatory agency such as the EMEA (Europe) and/or the FDA (US)
and/or any other national regulatory agency for use in animals,
preferably in humans.
[0064] "Pharmaceutical composition" means one or more active
ingredients, and one or more inert ingredients, as well as any
product which results, directly or indirectly, from combination,
complexation or aggregation of any two or more of the ingredients,
or from dissociation of one or more of the ingredients, or from
other types of reactions or interactions of one or more of the
ingredients. Accordingly, the pharmaceutical compositions of the
present invention encompass any composition made by admixing a
compound of the present invention and a pharmaceutically acceptable
excipient (pharmaceutically acceptable carrier).
[0065] The term "excipient" refers to a diluent, adjuvant, or
vehicle with which the therapeutic is administered. Such
pharmaceutical excipient can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, including but not limited to peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred
excipient when the pharmaceutical composition is administered
orally. Saline and aqueous dextrose are preferred excipients when
the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as liquid excipients for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsions, tablets, pills, capsules, powders, sustained-release
formulations and the like. The composition can be formulated as a
suppository, with traditional binders and excipients such as
triglycerides. Oral formulation can include standard excipients
such as pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical excipients are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the therapeutic, preferably in purified form, together with a
suitable amount of excipient so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0066] Preferably, X.sup.3 is O.
[0067] Preferably, X is N(R.sup.4), X.sup.1 is C and X.sup.3 is
O.
[0068] Preferably, X.sup.2 is C(R.sup.7R.sup.7a).
[0069] Preferably, L.sup.1 is selected from the group consisting
of
##STR00003## ##STR00004## ##STR00005## ##STR00006##
[0070] wherein R is H; or C.sub.1-4 alkyl; Y is NH; O; or S; and
R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4,
X, X.sup.1, X.sup.2 have the meaning as indicated above.
[0071] Even more preferred, L.sup.1 is selected form the group
consisting of
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017##
[0072] wherein R has the meaning as indicated above.
[0073] At least one (up to four) hydrogen is replaced by a group
L.sup.2-Z. In case more than one group L.sup.2-Z is present each
L.sup.2 and each Z can be selected independently. Preferably, only
one group L.sup.2-Z is present resulting in the formula
D-L.sup.1-L.sup.2-Z.
[0074] In general, L.sup.2 can be attached to L.sup.1 at any
position apart from the replacement of the hydrogen marked with an
asterisk in formula (I). Preferably, one to four of the hydrogen
given by R, R.sup.1 to R.sup.8 directly or as hydrogen of the
C.sub.1-4 alkyl or further groups and rings given by the definition
of R and R.sup.1 to R.sup.8 are replaced by L.sup.2-Z.
[0075] Furthermore, L.sup.1 may be optionally further substituted.
In general, any substituent may be used as far as the cleavage
principle is not affected.
[0076] Preferably, one or more further optional substituents are
independently selected from the group consisting of halogen; CN;
COOR.sup.9; OR.sup.9; C(O)R.sup.9; C(O)N(R.sup.9R.sup.9a);
S(O).sub.2N(R.sup.9R.sup.9a); S(O)N(R.sup.9R.sup.9a);
S(O).sub.2R.sup.9; S(O)R.sup.9;
N(R.sup.9)S(O).sub.2N(R.sup.9aR.sup.9b); SR.sup.9;
N(R.sup.9R.sup.9a); NO.sub.2; OC(O)R.sup.9; N(R.sup.9)C(O)R.sup.9a;
N(R.sup.9)S(O).sub.2R.sup.9a; N(R.sup.9)S(O)R.sup.9a;
N(R.sup.9)C(O)OR.sup.9a; N(R.sup.9)C(O)N(R.sup.9aR.sup.9b;
OC(O)N(R.sup.9R.sup.9a); T; C.sub.1-50 alkyl; C.sub.2-50 alkenyl;
or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl; C.sub.2-50
alkenyl; and C.sub.2-50 alkynyl are optionally substituted with one
or more R.sup.10, which are the same or different and wherein
C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of T, --C(O)O--; --O--; --C(O)--;
--C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.11a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0077] R.sup.9, R.sup.9a, R.sup.9b are independently selected from
the group consisting of H; T; and C.sub.1-50 alkyl; C.sub.2-50
alkenyl; or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl;
C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are optionally
substituted with one or more R.sup.10, which are the same or
different and wherein C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of T, --C(O)O--; --O--;
--C(O)--; --C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.11a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0078] T is selected from the group consisting of phenyl; naphthyl;
indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 4 to 7
membered heterocyclyl; or 9 to 11 membered heterobicyclyl, wherein
T is optionally substituted with one or more R.sup.10, which are
the same or different;
[0079] R.sup.10 is halogen; CN; oxo (.dbd.O); COOR.sup.12;
OR.sup.12; C(O)R.sup.12; C(O)N(R.sup.12R.sup.12a);
S(O).sub.2N(R.sup.12R.sup.12a); S(O)N(R.sup.12R.sup.12a);
S(O).sub.2R.sup.12; S(O)R.sup.12;
N(R.sup.12)S(O).sub.2N(R.sup.12aR.sup.12b); SR.sup.12;
N(R.sup.12R.sup.12a); NO.sub.2; OC(O)R.sup.12;
N(R.sup.12)C(O)R.sup.12a; N(R.sup.12)S(O).sub.2R.sup.12a;
N(R.sup.12)S(O)R.sup.12a; N(R.sup.12)C(O)OR.sup.12a;
N(R.sup.12)C(O)N(R.sup.12aR.sup.12b); OC(O)N(R.sup.12R.sup.12a); or
C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different;
[0080] R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, R.sup.12b are
independently selected from the group consisting of H; or C.sub.1-6
alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0081] The term "interrupted" means that between two carbons a
group is inserted or at the end of the carbon chain between the
carbon and hydrogen.
[0082] L.sup.2 is a single chemical bond or a spacer. In case
L.sup.2 is a spacer, it is preferably defined as the one or more
optional substituents defined above, provided that L.sup.2 is
substituted with Z.
[0083] Accordingly, when L.sup.2 is other than a single chemical
bond, L-Z is COOR.sup.9; OR.sup.9; C(O)R.sup.9;
C(O)N(R.sup.9R.sup.9a); S(O).sub.2N(R.sup.9R.sup.9a);
S(O)N(R.sup.9R.sup.9a); S(O).sub.2R.sup.9; S(O)R.sup.9;
N(R.sup.9)S(O).sub.2N(R.sup.9aR.sup.9b); SR.sup.9;
N(R.sup.9R.sup.9a); OC(O)R.sup.9; N(R.sup.9)C(O)R.sup.9a;
N(R.sup.9)S(O).sub.2R.sup.9a; N(R.sup.9)S(O)R.sup.9a;
N(R.sup.9)C(O)OR.sup.9a; N(R.sup.9)C(O)N(R.sup.9aR.sup.9b);
OC(O)N(R.sup.9R.sup.9a); T; C.sub.1-50 alkyl; C.sub.2-50 alkenyl;
or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl; C.sub.2-50
alkenyl; and C.sub.2-50 alkynyl are optionally substituted with one
or more R.sup.10, which are the same or different and wherein
C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of -T-, --C(O)O--; --O--; --C(O)--;
--C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.11a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0084] R.sup.9, R.sup.9a, R.sup.9b are independently selected from
the group consisting of H; Z; T; and C.sub.1-50 alkyl; C.sub.2-50
alkenyl; or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl;
C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are optionally
substituted with one or more R.sup.10, which are the same or
different and wherein C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of T, --C(O)O--; --O--;
--C(O)--; --C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.11a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0085] T is selected from the group consisting of phenyl; naphthyl;
indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 4 to 7
membered heterocyclyl; or 9 to 11 membered heterobicyclyl, wherein
t is optionally substituted with one or more R.sup.10, which are
the same or different;
[0086] R.sup.10 is Z; halogen; CN; oxo (.dbd.O); COOR.sup.12;
OR.sup.12; C(O)R.sup.12; C(O)N(R.sup.12R.sup.12a);
S(O).sub.2N(R.sup.12R.sup.12a); S(O)N(R.sup.12R.sup.12a);
S(O).sub.2R.sup.12; S(O)R.sup.12;
N(R.sup.12)S(O).sub.2N(R.sup.12R.sup.12b); SR.sup.12;
N(R.sup.12R.sup.12a); NO.sub.2; OC(O)R.sup.12;
N(R.sup.12)C(O)R.sup.12a; N(R.sup.12)S(O).sub.2R.sup.12a;
N(R.sup.12)S(O)R.sup.12a; N(R.sup.12)C(O)OR.sup.12a;
N(R.sup.12)C(O)N(R.sup.12aR.sup.12b); OC(O)N(R.sup.12R.sup.12a); or
C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different;
[0087] R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, R.sup.12b are
independently selected from the group consisting of H; Z; or
C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different;
[0088] provided that one of R.sup.9, R.sup.9a, R.sup.9b, R.sup.10,
R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, R.sup.12b is Z.
[0089] More preferably, L.sup.2 is a C.sub.1-20 alkyl chain, which
is optionally interrupted by one or more groups independently
selected from --O--; and C(O)N(R.sup.3aa); optionally substituted
with one or more groups independently selected from OH; and
C(O)N(R.sup.3aaR.sup.3aaa); and wherein R.sup.3aa, R.sup.3aaa are
independently selected from the group consisting of H; and
C.sub.1-4 alkyl.
[0090] Preferably, L.sup.2 has a molecular weight in the range of
from 14 g/mol to 750 g/mol.
[0091] Preferably, L.sup.2 is attached to Z via a terminal group
selected from
##STR00018##
[0092] In case L.sup.2 has such terminal group it is furthermore
preferred that L.sup.2 has a molecular weight in the range of from
14 g/mol to 500 g/mol calculated without such terminal group.
[0093] Preferably, L is represented by formula (Ia)
##STR00019##
[0094] wherein R.sup.4, L.sup.2, and Z have the meaning as
indicated above, and wherein R.sup.3aa, R.sup.3aaa are
independently selected from the group consisting of H; and
C.sub.1-4 alkyl; or are joined together with the nitrogen atom to
which they are attached to form a 4 to 7 membered heterocycle.
[0095] Preferably, R.sup.4 is H; or methyl.
[0096] Preferably, L is represented by formula (Ib)
##STR00020##
[0097] wherein R.sup.1, R.sup.1a, R.sup.4, L.sup.2 and Z have the
meaning as indicated above, and wherein R.sup.3aa is H; or
C.sub.1-4 alkyl. Preferably, R.sup.4 is H; or methyl
[0098] Preferably, R.sup.1 in formula (I) is L.sup.2-Z.
[0099] Preferably, R.sup.3 in formula (I) is L.sup.2-Z.
[0100] Preferably, R, R.sup.3a in formula (I) are joined together
with the nitrogen atom to which they are attached to form a 4 to 7
membered heterocycle, wherein the heterocycle is substituted with
L.sup.2-Z.
[0101] Preferably, D-H is a small molecule bioactive agent or a
biopolymer.
[0102] Preferably, D-H is a biopolymer selected from the group of
biopolymers consisting of proteins, polypeptides, oligonucleotides,
and peptide nucleic acids.
[0103] "Oligonucleotides" means either DNA, RNA, single-stranded or
double-stranded, siRNA, miRNA, aptamers, and any chemical
modifications thereof with preferably 2 to 1000 nucleotides.
Modifications include, but are not limited to, those which provide
other chemical groups that incorporate additional charge,
polarizability, hydrogen bonding, electrostatic interaction, and
fluxionality to the nucleic acid ligand bases or to the nucleic
acid ligand as a whole. Such modifications include, but are not
limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of
5-bromo or 5-iodo-uracil; backbone modifications, methylations,
unusual base-pairing combinations such as the isobases isocytidine
and isoguanidine and the like. Modifications can also include 3'
and 5' modifications such as capping and change of
stereochemistry.
[0104] Preferably, D-H is a polypeptide selected from the group of
polypeptides consisting of ACTH, adenosine deaminase, agalsidase,
alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API),
alteplase, amylins (amylin, symlin), anistreplase, anerod serine
protease, antibodies (monoclonal or polyclonal, and fragments or
fusions), antithrombin III, antitrypsins, aprotinin, asparaginases,
atosiban, biphalin, bivalirudin, bone-morphogenic proteins, bovine
pancreatic trypsin inhibitor (BPTI), cadherin fragments, calcitonin
(salmon), collagenase, complement C1 esterase inhibitor,
conotoxins, cytokine receptor fragments, DNase, dynorphine A,
endorphins, enfuvirtide, enkephalins, erythropoietins, exendins,
factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX,
fibrinolysin, fibroblast growth factor (FGF), growth hormone
releasing peptide 2 (GHRP2), fusion proteins, follicle-stimulating
hormones, gramicidin, ghrelin, desacyl-ghrelin, granulocyte colony
stimulating factor (G-CSF), galactosidase, glucagon, glucagon-like
peptides, glucocerebrosidase, granulocyte macrophage colony
stimulating factor (GM-CSF), human heat shock proteins (HSP),
phospholipase-activating protein (PLAP), gonadotropin chorionic
(hCG), hemoglobins, hepatitis B vaccines, hirudin, human serine
protease inhibitor, hyaluronidases, idurnonidase, immune globulins,
influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10,
11, 12, 13, 21), IL-1 receptor antagonist (rhIL-1ra), insulins,
insulin like growth factors, insulin-like growth factor binding
protein (rhIGFBP), interferons (alfa 2a, alfa 2b, alfa 2c, beta 1a,
beta 1b, gamma 1a, gamma 1b), intracellular adhesion molecule,
keratinocyte growth factor (KGP), P-selectin glycoprotein ligand
(PSGL), transforming growth factors, lactase, leptin, leuprolide,
levothyroxine, luteinizing hormone, lyme vaccine, natriuretic
peptides (ANP, BNP, CNP and fragments), neuropeptide Y,
pancrelipase, pancreatic polypeptide, papain, parathyroid hormone,
PDGF, pepsin, peptide YY, platelet activating factor
acetylhydrolase (PAF-AH), prolactin, protein C, thymalfasin,
octreotide, secretin, sermorelin, soluble tumor necorsis factor
receptor (TNFR), superoxide dismutase (SOD), somatropins (growth
hormone), somatoprim, somatostatin, streptokinase, sucrase,
terlipressin, tetanus toxin fragment, tilactase, thrombins,
thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis
factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator
(tPA), TSH, urodilatin, urate oxidase, urokinase, vaccines,
vascular endothelial growth factor (VEOF), vasoactive intestinal
peptide, vasopressain, ziconotide, lectin and ricin.
[0105] Preferably, D-H is a protein prepared by recombinant DNA
technologies.
[0106] Preferably, D-H is a protein selected from the group of
proteins consisting of antibody fragments, single chain antigen
binding proteins, catalytic antibodies and fusion proteins.
[0107] Preferably, D-H is a small molecule bioactive agent selected
from the group of agents consisting of central nervous
system-active agents, anti-infective, anti-allergic,
immunomodulating, anti-obesity, anticoagulants, antidiabetic,
anti-neoplastic, antibacterial, anti-fungal, analgesic,
contraceptive, anti-inflammatory, steroidal, vasodilating,
vasoconstricting, and cardiovascular agents with at least one
primary or secondary amino group.
[0108] Preferably, D-H is a small molecule bioactive agent selected
from the group of agents consisting of acarbose, alaproclate,
alendronate, amantadine, amikacin, amineptine, aminoglutethimide,
amisulpride, amlodipine, amotosalen, amoxapine, amoxicillin,
amphetamine, amphotericin B, ampicillin, amprenavir, amrinone,
anileridine, apraclonidine, apramycin, articaine, atenolol,
atomoxetine, avizafone, baclofen, benazepril, benserazide,
benzocaine, betaxolol, bleomycin, bromfenac, brofaromine,
carvedilol, cathine, cathinone, carbutamid, cefalexine,
clinafloxacin, ciprofloxacin, deferoxamine, delavirdine,
desipramine, daunorubicin, dexmethylphenidate, dexmethylphenidate,
diaphenylsulfon, dizocilpine, dopamin, dobutamin, dorzolamide,
doxorubicin, duloxetine, eflornithine, enalapril, epinephrine,
epirubicin, ergoline, ertapenem, esmolol, enoxacin, ethambutol,
fenfluramine, fenoldopam, fenoterol, fingolimod, flecainide,
fluvoxamine, fosamprenavir, frovatriptan, furosemide, fluoexetine,
gabapentin, gatifloxacin, gemiflocacin, gentamicin, grepafloxacin,
hexylcaine, hydralazine, hydrochlorothiazide, icofungipen,
idarubicin, imiquimod, inversine, isoproterenol, isradipine,
kanamycin A, ketamin, labetalol, lamivudine, levobunolol, levodopa,
levothyroxine, lisinopril, lomefloxacin, loracarbef, maprotiline,
mefloquine, melphalan, memantine, meropenem, mesalazine, mescaline,
methyldopa, methylenedioxymethamphetamine, metoprolol, milnacipran,
mitoxantron, moxifloxacin, norepinephrine, norfloxacin,
nortriptyline, neomycin B, nystatin, oseltamivir, pamidronic acid,
paroxetine, pazufloxacin, pemetrexed, perindopril, phenmetrazine,
phenelzine, pregabalin, procaine, pseudoephedrine, protriptyline,
reboxetine, ritodrine, sabarubicin, salbutamol, serotonin,
sertraline, sitagliptin, sotalol, spectinomycin, sulfadiazin,
sulfamerazin, sertraline, sprectinomycin, sulfalen,
sulfamethoxazol, tacrine, tamaulosin, terbutaline, timolol,
tirofiban, tobramycin, tocainide, tosufloxacin, trandolapril,
tranexamic acid, tranylcypromine, trimerexate, trovafloxacin,
valaciclovir, valganciclovir, vancomycin, viomycin, viloxazine, and
zalcitabine.
[0109] Preferably, Z is a polymer of at least 500 Da or a
C.sub.8-18 alkyl group.
[0110] Preferably, Z is selected from the group of optionally
crosslinked polymers consisting of poly(propylene glycol),
poly(ethylene glycol), dextran, chitosan, hyaluronic acid,
alginate, xylan, mannan, carrageenan, agarose, cellulose, starch,
hydroxyalkyl starch (HAS), poly(vinyl alcohols), poly(oxazolines),
poly(anhydrides), poly(ortho esters), poly(carbonates),
poly(urethanes), poly(acrylic acids), poly(acrylamides),
poly(acrylates), poly(methacrylates), poly(organophosphazenes),
polyoxazoline, poly(siloxanes), poly(amides),
poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters),
poly(iminocarbonates), poly(amino acids), collagen, gelatin,
hydrogel or a blood plasma protein, and copolymers thereof.
[0111] Preferably, Z is a protein.
[0112] Preferably, Z is a protein selected from the group
consisting of albumin, transferrin, immunoglobulin.
[0113] Preferably, Z is a linear or branched poly(ethylene glycol)
with a molecular weight from 2,000 Da to 150,000 Da.
[0114] Even more preferred is a prodrug of the present invention,
wherein D-H is a GLP-1 receptor agonist; L is L.sup.1 represented
by formula (I) as indicated above; and Z is a hydrogel. Even more
preferably, in formula (I) X is N(R.sup.4), X.sup.1 is C and
X.sup.3 is O. Even more preferably, L is represented by formula
(Ia) as indicated above.
[0115] GLP-1 is one of the intestinal peptide hormones that are
released into the circulatory system after food intake. It augments
the postprandial release of insulin, when nutritions (especially
carbohydrates) are absorbed and their level postprandially
elevated. GLP-1 associates with GLP-1 receptor sites located on
pancreatic .beta.-cells and elevates endogenous cAMP levels in a
dose dependent manner. In isolated rat islets in the presence of
above normoglycemic glucose levels, GLP-1 stimulates the release of
insulin. A therapeutic potential for GLP-1 in type 2 diabetes
patients was suggested before, owing to the profound efficacy of
this insulinotropic peptide to stimulate secretion of insulin when
glucose levels are elevated and to cease doing so upon return to
normoglycemia. The antidiabetogenic effect of glucagon-like
peptide-1 (7-36) amide in normal subjects and patients with
diabetes mellitus is described e.g. in N. Engl. J. Med.
326(20):1316-1322. In vitro studies and animal experiments suggest
that GLP-1 improves insulin sensitivity and has an anabolic effect
on pancreatic cells. In humans, GLP-1 was also reported to suppress
glucagon secretion, decelerate gastric emptying, and induce
satiety, leading to weight loss if administered for weeks and
months.
[0116] Exendin-4 is reported to associate with GLP-1 receptors
located on pancreatic beta-cells with 2.5 times higher affinity
than GLP-1. In isolated rat islets and beta-cells in presence of
glucose, exendin enhances secretion of insulin in a dose-dependent
fashion. Exendin-4 is a high potency agonist and truncated
exendin-(9-39)-amide an antagonist at the glucagon-like peptide
1-(7-36)-amide receptor of insulin-secreting beta-cells (see J.
Biol. Chem. 268(26):19650-19655). Studies in type 2 diabetic
rodents revealed that exendin-4 is 5530-fold more potent than GLP-1
in lowering blood glucosc levels. Also, the duration of
glucose-lowering action after a single administration of exendin-4
is significantly longer compared to GLP-1 (see e.g. Diabetes
48(5):1026-1034). Plasma half-life of exendin-4 in humans was
described to be only 26 minutes. Exendin-4 reduces fasting and
postprandial glucose and decreases energy intake in healthy
volunteers (sec e.g. Am. J. Physiol. Endocrinol. Metab.
281(1):E155-61).
[0117] Accordingly in an even more preferred embodiment the GLP-1
receptor agonist is Exendin-4.
[0118] Hydrogels to be used are known in the art. Suitable
hydrogels may be used which are described in WO-A 2006/003014.
Accordingly, a hydrogel may be defined as a three-dimensional,
hydrophilic or amphiphilic polymeric network capable of taking up
large quantities of water. The networks are composed of
homopolymers or copolymers, are insoluble due to the presence of
covalent chemical or physical (ionic, hydrophobic interactions,
entanglements) crosslinks. The crosslinks provide the network
structure and physical integrity. Hydrogels exhibit a thermodynamic
compatibility with water which allow them to swell in aqueous
media. The chains of the network are connected in such a fashion
that pores exist and that a substantial fraction of these pores are
of dimensions between 1 nm and 1000 nm.
[0119] Another object of the present invention is a pharmaceutical
composition comprising a prodrug of the present invention or a
pharmaceutical salt thereof together with a pharmaceutically
acceptable excipient.
[0120] Yet another object of the present invention is a prodrug of
the present invention or a pharmaceutical composition of the
present invention for use as a medicament.
[0121] Yet another object of the present invention is a method of
treating, controlling, delaying or preventing in a mammalian
patient in need of the treatment of one or more conditions
comprising administering to said patient a therapeutically
effective amount of a prodrug of the present invention or a
pharmaceutical composition of the present invention or a
pharmaceutically acceptable salt thereof.
[0122] Another object of the present invention is a prodrug
precursor of formula Act-L, wherein L has the meaning as indicated
above and Act is a leaving group.
[0123] Preferably, Act is chloride, bromide, fluoride,
nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl,
N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl,
pentafluorophenoxy, 2-thiooxo-thiazolidinyl, or
N-hydroxysulfosuccinimidyl.
EXAMPLES
Materials and Methods
[0124] Materials: Side chain protected Exendin-4 (J. Eng et al., J.
Biol. Chem. 1992, 267, 11, 7402-7405) on Rink amide resin, side
chain protected BNP-32a (human, Cys10 and Cys26 exchanged for Ala)
on chlorotrityl resin, side chain protected BNP-32b with ivDde
side-chain protecting group on Lys14 (human, Cys10 and Cys26
exchanged for Ala) on chlorotrityl resin, and side chain protected
human growth hormone releasing factor fragment 1-29 amide
(GRF(1-29)) on Rink amide (each synthesized by Fmoc-strategy) were
obtained from Peptide Specialty Laboratories GmbH, Heidelberg,
Germany. Standard side chain protecting groups were used except for
Lys27 of Exendin-4 and Lys21 of ORF(1-29) where Mmt side-chain
protecting groups were used.
[0125] 40 kDa methoxy poly(ethylene glycol) maleimido-propionamide
(PEG40 kDa-maleimide) was obtained from Chirotech Technology Ltd,
Cambridge, UK.
[0126] 2-Chlorotrityl chloride resin, Sieber amide resin and amino
acids were from Merck Biosciences GmbH, Schwalbach/Ts, Germany, if
not stated otherwise. Fmoc-D-Homocysteine(Trt)-OH and
S-Trityl-3-mercaptopropionic acid (Trt-MPA) were obtained from
Bachem AG, Bubendorf, Switzerland.
O--(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol
(Fmoc-Pop-OH) was obtained from Polypure AS, Oslo, Norway.
Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine (Fmoc-Acp-OH) was
purchased from NeoMPS SA, Strasbourg, France.
cis-Cyclohexane-1,2-dicarboxylic anhydride was obtained from Alfa
Aesar GmbH & Co KG, Karlsruhe, Germany.
[0127] All other chemicals were from Sigma-ALDRICH Chemie GmbH,
Taufkirchen, Germany.
[0128] Solid phase synthesis was performed on 2-Chlorotrityl
chloride resin with a loading of 1.3 mmol/g or Sieber amide resin
with a loading of 0.55 mmol/g. Syringes equipped with polypropylene
frits were used as reaction vessels.
[0129] Loading of the first amino acid to resins was performed
according to manufacturer's instructions.
[0130] Fmoc Deprotection:
[0131] For Fmoc protecting-group removal, the resin was agitated
with 2/2/96 (v/v/v) piperidine/DBU/DMF (two times, 10 min each) and
washed with DMF (ten times).
[0132] ivDde Deprotection:
[0133] For ivDde protecting-group removal, the resin was agitated
with 98/2 (v/v) DMF/hydrazine hydrate (3 times, 10 min each) and
washed with DMF (ten times).
[0134] Boc Protection:
[0135] The N-terminus of a peptide was boc-protected by agitating
the resin with 30 eq (boc).sub.2O and 60 eq pyridine in DCM. After
1 h the resin was washed with DCM (10 times).
[0136] Standard Coupling Condition for Acids:
[0137] Coupling of acids (aliphatic acids, Fmoc-amino acids) to
free amino groups on resin was achieved by agitating resin with 3
eq of acid, 3 eq PyBOP and 6 eq DIEA in relation to free amino
groups on resin (calculated based on theoretical loading of the
resin) in DMF at room temperature. After 1 hour resin was washed
with DMP (10 times).
[0138] 3-Maleimido Propionic Acid Coupling:
[0139] Coupling of 3-maleimido propionic acid to free amino groups
on resin was achieved by agitating resin with 2 eq of acid, 2 eq
DIC and 2 eq HOBt in relation to free amino groups in DMF at room
temperature. After 30 min, resin was washed with DMF (10
times).
[0140] Standard Protocol for Synthesis of Ureas on Resin:
[0141] Synthesis of ureas on resin was achieved by agitating resin
with 2.5 eq of bis(pentafluorophenyl) carbonate, 5 eq DIEA, and
0.25 eq DMAP in relation to free amino groups in DCM/ACN 1/1 at
room temperature. After 15 min resin was washed with DMF (10
times). 5 eq of amine was dissolved in DMF. Mixture was added to
resin and agitated for 60 min at room temperature. Resin was washed
with DMF (10 times).
[0142] Cleavage Protocol for Sieber Amide Resin:
[0143] Upon completed synthesis, the resin was washed with DCM (10
times), dried in vacuo and treated repeatedly (five times a 15
minutes) with 97/2/1 (v/v) DCM/TES/fFA. Eluates were combined,
volatiles were removed under a nitrogen stream and product was
purified by RP-HPLC. HPLC fractions containing product were
combined and lyophilized.
[0144] Cleavage Protocol for 2-Chlorotrityl Chloride Resin:
[0145] Upon completed synthesis, the resin was washed with DCM,
dried in vacuo and treated two times for 30 minutes with 6/4 (v/v)
DCM/HFIP. Eluates were combined, volatiles were removed under a
nitrogen stream and product was purified by RP-HPLC. HPLC fractions
containing product were combined and lyophilized.
[0146] Cleavage Protocol for Rink Amide Resin:
[0147] Upon completed synthesis, the resin was washed with DCM,
dried in vacuo and treated with 2 ml of TFA cleavage cocktail
(TFA/TES/Water/DTI 95/2/2/1) per 100 mg resin for 60 min at room
temperature. Volatiles were removed under a nitrogen stream.
Unpolar side products and protecting groups were removed by
precipitating peptide from diethyl ether. Precipitate was dried in
vacuo and dissolved in ACN/water 1/1 and purified by RP-HPLC.
[0148] Amine containing products obtained as TFA salts were
converted to the corresponding HCl salts using ion exchange resin
(Discovery DSC-SAX, Supelco, USA). This step was performed in case
the residual TFA was expected to interfere with e.g. a subsequent
coupling reactions.
[0149] RP-HPLC Purification:
[0150] RP-HPLC was done on a 100.times.20 or a 100.times.40 mm C18
ReproSil-Pur 300 ODS-3 5.mu. column (Dr. Maisch, Ammerbuch,
Germany) connected to a Waters 600 HPLC System and Waters 2487
Absorbance detector. Linear gradients of solution A (0.1% TFA in
H.sub.2O) and solution B (0.1% TFA in acetonitrile) were used. HPLC
fractions containing product were lyophilized.
[0151] Analytics: Electrospray ionization mass spectrometry
(ESI-MS) was performed on a Waters ZQ 4000 ESI instrument and
spectra were, if necessary, interpreted by Waters software
MaxEnt.
[0152] Size exclusion chromatography (SEC) was performed using an
Amersham Bioscience AEKTAbasic system equipped with a Superdex200
10/300 column (Amersham Bioscience/GE Healthcare), if not stated
otherwise. 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, 3 mM EDTA
was used as mobile phase
[0153] For Cation Exchange Chromatography, an Amersham Bioscience
AEKTAbasic system was equipped with a Source 15S filled HR16/10
column (Amersham Bioscience/GE Healthcare).
[0154] Desalting was performed using an Amersham Bioscience
AEKTAbasic system equipped with a HiPrep 26/10 Desalting column and
0.1% acetic acid in water as mobile phase.
[0155] In vitro linker hydrolysis and release of drug: Compounds
were dissolved in buffer A (10 mM sodium phosphate, 140 mM NaCl, pH
7.4, 3 mM EDTA) or buffer B (0.1 M Acetat 3 mM EDTA, pH 4.0), and
solution was filtered through a 0.2 m filter and incubated at
37.degree. C. Samples were taken at time intervals and analyzed by
RP-HPLC at 215 nm and ESI-MS. UV-signals correlating to liberated
drug molecule were integrated and plotted against incubation time.
In case of identical retention times of prodrug and drug, ratio of
mass signals was used to determine release kinetics.
[0156] For hydrogel conjugates, compounds were suspended in buffer
A and incubated at 37.degree. C. Samples were taken after
centrifugation of the suspension and analyzed by RP-HPLC at 215 nm.
UV-signals correlating to liberated drug molecule were integrated
and plotted against incubation time.
[0157] Curve-fitting software was applied to estimate the
corresponding halftime of release.
Example 1
Synthesis of Fatty Acid Carrier (1)
##STR00021## ##STR00022##
[0159] 1 was synthesized on sieber amide resin (477 mg, 0.262 mmol)
by coupling of Fmoc-Lys(ivDde)-OH, fmoc deprotection, coupling of
dodecanoic acid, ivDde deprotection, coupling of Fmoc-Pop-OH, fmoc
deprotection, coupling of 3-maleimido propionic acid, cleavage from
resin and purification as depicted above and described in
"Materials and Methods".
[0160] Yield: 128 mg (0.119 mmol).
[0161] MS: m/z 1101.0=[M+Na].sup.+ (MW calculated=1078.4
g/mol).
Example 2
Synthesis of Linker Reagent (2)
##STR00023##
[0163] Linker reagent 2 was synthesized on 3-chlorotrityl chloride
resin (300 mg, 0.39 mmol) by loading of resin with
Fmoc-Cys(Trt)-OH, fmoc deprotection, and on-resin urea formation
using N,N-dimethyl-ethylenediamine as amine, cleavage from resin as
depicted above and described in "Materials and Methods". For
RP-HPLC separation, 0.01% HCl in water was used as solution A and
0.01% HCl in acetonitrile was used as solution B.
[0164] Yield: 82 mg of HCl salt (0.16 mmol).
[0165] MS: m/z 478.2=[M+H].sup.+ (MW calculated=477.6 g/mol).
Example 3
Synthesis of Exendin-4 Linker Intermediate (3)
##STR00024##
[0167] 2 (14 mg, 0.027 mmol), PyBOP (14 mg, 0.027 mmol), and DIEA
(17 .mu.l, 0.10 mmol) were dissolved in 0.2 ml of dry DMF. Mixture
was added to 22 mg side-chain protected Exendin-4 on-resin (0.1
mmol/g, 2.2 .mu.mol) and agitated for 30 min at room temperature.
Resin was washed with DMF (10 times) and DCM (10 times). 3 was
cleaved from resin and purified by RP-HPLC as described in
"Materials and Methods".
[0168] Yield: 1.7 mg 3 as TFA salt (0.38 .mu.mol).
[0169] MS: m/z 1468.7=[M+3H].sup.3+ (MW calculated=4403 g/mol).
Example 4
Synthesis of Fatty Add-PEG-Linker-Exendin-4 Conjugate (4)
##STR00025##
[0171] 3 (1.7 mg, 0.38 .mu.mol) and 1 (0.6 mg, 0.58 mmol) were
dissolved in 500 .mu.l of acetonitrile/water 7/3 (v/v). 40 .mu.l of
0.5 M phosphate buffer (pH 7.4) were added and the mixture was
incubated at RT for 10 min. Conjugate 4 was purified by
RP-HPLC.
[0172] MS: m/z 1828.7=[M+3H].sup.3+ (MW calculated=5480 g/mol).
Example 5
Synthesis of Linker Intermediate (5a)
##STR00026##
[0174] Fmoc-Acp-OH.2HCl (100 mg, 0.21 mmol) was suspended in 400
.mu.l DMF/DMSO 1/1 (v/v). S-tritylcysteamine.HCl (75 mg, 0.21
mmol), PyBOP (109 mg, 0.21 mmol) and DIEA (146 .mu.l, 0.86 mmol)
were added and mixture was agitated for 60 min at RT. Fmoc group
was removed by adding 75 .mu.l piperidine and 25 .mu.l DBU. After
15 min mixture was hydrolyzed and acidified (AcOH) and compound was
purified by RP-HPLC. After lyophilization 98 mg (0.14 mmol, double
TFA salt) were obtained.
[0175] MS: m/z 511.6=[M+Na].sup.+ (MW calculated=488.7 g/mol).
[0176] Synthesis of cis-cyclohexane diacarboxylic acid amoxapine
monoamide (5b)
##STR00027##
[0177] Amoxapine (200 mg, 0.64 mmol) and
cis-cyclohexane-1,2-dicarboxylic anhydride (108 mg, 0.70 mmol) were
dissolved in 700 .mu.l of dry DMF. Pyridine (130 .mu.l, 1.6 mmol)
was added and mixture was stirred for 60 min at RT. Mixture was
quenched with 2 ml of acetonitrile/acetic acid/water (1/1/1) and
purified by RP-HPLC. After lyophilization 344 mg (0.49 mmol, double
TFA salt) of 5b were obtained.
[0178] MS: m/z 468.5=[M+H].sup.+ (MW calculated=468.0 g/mol).
[0179] Synthesis of Linker-Amoxapine Conjugate (5c)
##STR00028##
[0180] 5b (7 mg, 0.010 mmol) was preactivated by incubating with
PyBOP (12.5 mg, 0.024 mmol) and DIEA (5 .mu.l, 0.03 mmol) in 200
.mu.l of dry DMF for 45 min at RT. 5a (20 mg, 0.028 mmol) and DIEA
(15 .mu.l, 0.09 mmol) were added and mixture was incubated for
further 60 min. Mixture was quenched with 0.5 ml of
acetonitrile/acetic acid/water (1/1/1) and purified by RP-HPLC.
After lyophilization 3 mg (0.0026 mmol, double TFA salt) of 5c were
obtained.
[0181] MS: m/z 939.3=[M+H].sup.+ (MW calculated=938.6 g/mol).
[0182] For trityl deprotection, lyophilisate was incubated in 1 ml
HFIP and 3 .mu.l TES for 30 min. Mixture was evaporated and thiol
was purified by RP-HPLC. After lyophilization 2 mg (2.2 .mu.mol,
double TFA salt) of amoxapine-linker conjugate 5c were
obtained.
[0183] MS: m/z 697.1=[M+H].sup.+ (MW calculated=696.3 g/mol).
[0184] Synthesis of Fatty Acid-PEG-Amoxapine Conjugate (5)
##STR00029##
[0185] Amoxapine-linker conjugate 5c (2 mg, 2.2 .mu.mol) and 1 (3.5
mg, 3.2 .mu.mol) were dissolved in 900 .mu.l of acetonitrile/water
7/3 (v/v). 60 .mu.l of 0.5 M phosphate buffer (pH 7.4) were added
and the mixture was incubated at RT for 10 min. 5 was purified by
RP-HPLC.
[0186] MS: m/z 1774.9=[M+H].sup.+ (MW calculated=1774.7 g/mol).
Example 6
Synthesis of Linker Reagent (6)
##STR00030##
[0188] Fmoc-Asp(tBu)-OH (411 mg, 1 mmol), HOBt (153 mg, 1 mmol),
and DIC (160 .mu.l, 1 mmol) were dissolved in 2 ml of DMF and
incubated for 10 min at RT. N,N-dimethyl ethylenediamine (160
.mu.l, 1.5 mmol) was added and stirred at RT for 30 min. Acetic
acid (300 .mu.l) was added and
Fmoc-Asp(tBu)-NH--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 was purified
by RP-HPLC.
[0189] Yield: 220 mg (0.46 mmol)
[0190] MS Fmoc-Asp(tBu)-NH--(CH.sub.2).sub.2--N(CH.sub.3).sub.2:
m/z 504.6=[M+Na].sup.+ (MW calculated=481.6 g/mol).
[0191] Fmoc-Asp(tBu)-NH--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 (220
mg, 0.46 mmol) was dissolved in 3 ml of 98/2 (v/v) TFA/TES. After
30 min the solvent was removed under a nitrogen stream and 6 was
purified by RP-HPLC using 0.01% HCl in water as solvent A and 0.01%
HCl in acetonitril as solvent B.
[0192] Yield: 146 mg (0.32 mmol, HCl salt)
[0193] MS: m/z 426.5=[M+H].sup.+ (MW calculated=425.5 g/mol).
Example 7
Synthesis of Linker Reagents 7a and 7b
##STR00031##
[0195] Synthesis of 7a
[0196] Fmoc-Asp(tBu)-OH (300 mg, 0.73 mmol), HOBt (1112 mg, 0.73
mmol), and DIC (117 .mu.l, 0.73 mmol) were dissolved in 2 ml of DMF
and incubated for 10 min at RT. Boc-ethylenediamine (230 mg, 1.44
mmol) was added and stirred at RT for 30 min. Acetic acid (300
.mu.l) was added and Fmoc-Asp(tBu)-NH--(CH.sub.2).sub.2--NH-boc was
purified by RP-HPLC.
[0197] Yield: 205 mg (0.37 mmol)
[0198] MS intermediate: m/z 576.6=[M+Na].sup.+ (MW calculated=553.7
g/mol).
[0199] Fmoc-Asp(tBu)-NH--(CH.sub.2).sub.2--NH-boc (205 mg, 0.37
mmol) was dissolved in 3 ml of 98/2 (v/v) TFA/TES. After 30 min the
solvent was removed under a nitrogen stream and
Fmoc-Asp(H)--NH--(CH.sub.2).sub.2--NH.sub.2 was purified by
RP-HPLC.
[0200] Yield: 140 mg (0.27 mmol, TFA salt)
[0201] MS intermediate: m/z 398.8=[M+H].sup.+ (MW calculated=397.4
g/mol).
[0202] Fmoc-Asp(H)--NH--(CH.sub.2).sub.2--NH.sub.2 (140 mg, 0.27
mmol, TFA salt) was dissolved in 1 ml of DMF and DIEA (140 .mu.l,
0.81 mmol) and boc.sub.2O (100 mg, 0.46 mmol) added. The solution
was stirred at RT for 15 min and then acidified with acetic acid
(300 .mu.l). 7a was purified by RP-HPLC.
[0203] Yield 7a: 120 mg (0.24 mmol)
[0204] MS 7a: m/z 520.5=[M+Na].sup.+ (MW calculated=497.6
g/mol).
[0205] 7b was synthesized as described above except for the use of
H.sub.2N--(CH.sub.2).sub.2--N(CH.sub.3)-boc instead of
boc-ethylenediamine as amine in the first step.
[0206] Yield 7b: 115 mg
[0207] MS 7b: m/z 534.5=[M+Na].sup.+ (MW calculated=511.6
g/mol).
Example 8
Synthesis of Exendin-Linker Conjugates 8a, 8b and 8c
##STR00032##
[0209] Synthesis of 8a:
[0210] 7a (30 mg, 60 .mu.mol), HOBt (9 mg, 60 .mu.mol), DIEA (12
.mu.l, 70 .mu.mol), and DIC (10 .mu.l, 63 .mu.mol) were dissolved
in 200 .mu.l of DMF and immediately added to side-chain protected
Exendin-4 on resin (40 mg, 4 .mu.mol) and incubated for 1 h at room
temperature. Resin was washed ten times with DMF and then incubated
for 5 min with 500 .mu.l of 1/1/2 acetic anhydride/pyridine/DMF.
Resin was washed 10 times with DMF and fmoc group was removed.
Trt-mercaptopropionic acid was coupled and 8a was cleaved from
resin and purified by RP-HPLC.
[0211] Yield: 3.6 mg
[0212] MS 8a: m/z 1108.5=[M+4H].sup.4+; 1477.8=[M+3H].sup.3+ (MW
calculated=4432 g/mol).
[0213] 8b was synthesized as described above for 8a except for the
use of 7b instead of 7a.
[0214] Yield: 3.5 mg
[0215] MS 8b: m/z 1112.5=[M+4H].sup.4+; 1482.5=[M+3H].sup.3+ (MW
calculated=4446 g/mol).
[0216] 8c was synthesized as described above for 8a except for the
use of 6 instead of 7a.
[0217] Yield: 3.2 mg
[0218] MS 8c: m/z 1116.2=[M+4H].sup.4+; 1487.8=[M+3H].sup.3+ (MW
calculated=4460 g/mol).
Example 9
Synthesis of PEG40 kDa-Linker-Exendin Conjugates 9a, 9b, and 9c
##STR00033##
[0220] Synthesis of 9a:
[0221] 8a (3.6 mg) was dissolved in 300 .mu.l 2/1
water/acetonitrile and 50 mg PEG40 kDa-maleimide was added. 100
.mu.l 0.25 M sodium phosphate buffer pH 7 were added and after 5
min the solution was acidified with 50 .mu.l acetic acid.
[0222] 9a was purified by ion exchange chromatography using 10 mM
sodium citrate pH 3 as solvent A and 10 mM sodium citrate pH 3 and
1 M NaCl as solvent B and a step-gradient (0 to 40% B). Fractions
containing 9a were desalted and lyophilized:
[0223] Yield: 14 mg
[0224] 9b was synthesized as described above except for the use of
8b.
[0225] Yield 15 mg
[0226] 9c was synthesized as described above except for the use of
8c.
[0227] Yield: 13 mg
Example 10
Synthesis of Fatty Acid-Linker-Exendin Conjugate 10
##STR00034##
[0229] 8c (1 mg) was dissolved in 100 .mu.l 1/1 acetonitrile/water
and 1 (1 mg) in 100 .mu.l of 3/1 acetonitrile/water was added. 100
.mu.l of 0.25 M sodium phosphate buffer was added, the reaction was
stirred for 5 min, after which 10 was purified by RP-HPLC.
[0230] Yield: 1.3 mg
[0231] MS 10: m/z 1385.9=[M+4H].sup.4+; 1846.3=[M+3H].sup.3+ (MW
calculated=5528.3 g/mol).
Example 11
Synthesis of NHS-Activated Linker Reagent 11
##STR00035##
[0233] 7b (20 mg, 40 .mu.mol), N,N'-dicyclohexylcarbodiimide (10
mg, 48 .mu.mol), and NHS (8 mg, 70 .mu.mol) were dissolved in 300
.mu.l of dry DCM and stirred at RT for 1 h. Solvent was removed
under a nitrogen stream and 11 was purified by RP-HPLC and
lyophilized.
[0234] Yield: 22 mg (36 .mu.mol)
[0235] MS: m/z 631.5=[M+Na].sup.+ (MW calculated=608.7 g/mol).
Example 12
Synthesis of Linker-Exendin(Fluorescein) Conjugate (12a) and
Linker-GRF(1-29)(Fluorescein) Conjugate (12b)
##STR00036##
[0237] 6 (60 mg, 130 mmol HCl salt), HOBt (20 mg, 130 .mu.mol),
DIEA (40 .mu.l, 230 .mu.mol), and DIC (20 .mu.l, 126 .mu.mol) were
dissolved in 700 .mu.l of DMF and immediately added to side-chain
protected Exendin-4 on resin (120 mg, 12 .mu.mol) and incubated for
1 h at room temperature. Resin was washed ten times with DMF and
then incubated for 5 min with 1 ml of 1/1/2 (v/v/v) acetic
anhydride/pyridine/DMF. Resin was washed ten times with DMF and
fmoc group was removed. Trt-mercaptopropionic acid was coupled
according to standard coupling method and resin was washed five
times with DMF and ten times with DCM. Mmt protecting group of
Lys27 was removed by incubation of resin five times in 2 ml of 9/1
(v/v) DCM/HFIP for 5 min. Resin was washed five times with DCM and
five times with DMF and 5,6-carboxy-flourescein-NHS ester (20 mg,
42 .mu.mol) and DIEA (20 .mu.l, 115 .mu.l) in 300 .mu.l DMF were
added to resin and incubated for 30 min. 12a was cleaved from resin
and purified by RP-HPLC
[0238] Yield: 12 mg
[0239] MS 12a: m/z 1205.9=[M+4H].sup.4+; 1607.0=[M+3H].sup.3+ (MW
calculated=4818.3 g/mol). 12b was synthesized as described for 12a
except for the use of GRF(1-29) on resin (120 mg, 12 .mu.mol).
[0240] Yield: 11 mg
[0241] MS 12b: m/z 998.6=[M+4H].sup.4+; 1330.5=[M+3H].sup.3+ (MW
calculated=3989.6 g/mol).
[0242] Synthesis of Mercaptopropionyl-Exendin(Fluorescein) (12c)
and Mercaptopropionyl-GRF(1-29)Fluorescein) (12d)
##STR00037##
[0243] Trt-mercaptopropionic acid was coupled according to standard
coupling method to side-chain protected Exendin-4 on resin (120 mg,
12 .mu.mol). Mmt protecting group removal of Lys27 and
5,6-carboxy-flourescein-NHS ester coupling was performed as
described for 12a, 12c was cleaved from resin and purified by
RP-HPLC
[0244] Yield: 13 mg
[0245] MS 12c: m/z 1545.6=[M+3H].sup.3+ (MW calculated=4633
g/mol).
[0246] 12d was synthesized as described for 12c except for the use
of GRF(1-29) on resin (120 mg, 12 .mu.mol).
[0247] Yield: 11 mg
[0248] MS 12d: m/z 1269.1=[M+3H].sup.3+ (MW calculated=3804.3
g/mol).
Example 13
Synthesis of Reversible PEG40 kDa-Linker-Exendin(Fluorescein)
Conjugate (13a) and Reversible PEG40
kDa-Linker-GRF(1-29)(Fluorescein) Conjugate (13b)
##STR00038##
[0250] 12a (12 mg) was dissolved in 500 .mu.l of 1/I
acetonitrile/water and 120 mg PEG40 kDa-maleimide in 1 ml of 1/1
acetonitrile/water was added. 300 .mu.l of 0.25 M sodium phosphate
buffer pH 7.0 were added and solution was acidified after 10 min
with 300 .mu.l acetic acid. 13a was purified by cation exchange
chromatography, desalted, and then lyophilized.
[0251] Yield: 51 mg
[0252] 13b was synthesized as described for 13a except for the use
of 12b instead of 12a.
[0253] Yield: 46 mg
[0254] Synthesis of Permanent PEG40 kDa-Exendin(Fluorescein)
Conjugate (13c) and Permanent PEG40 kDa-GRF(1-29)(Fluorescein)
Conjugate (13d)
##STR00039##
[0255] 13c was synthesized as described for 13a except for the use
of 12c instead of 12a.
[0256] Yield: 55 mg
[0257] 13d was synthesized as described for 13a except for the use
of 12d instead of 12a.
[0258] Yield: 45 mg
Example 14
Synthesis of Linker-GRF(1-29) Conjugate 14
##STR00040##
[0260] 14 was synthesized as described for 8c except for the use of
side-chain protected GRF(1-29) resin.
[0261] Yield: 10 mg
[0262] MS 14: m/z 908.2=[M+4H].sup.4+; 12112=[M+3H].sup.3+ (MW
calculated=3631.3 g/mol).
Example 15
Synthesis of PEG40 kDa-Linker-GRF(1-29) Conjugate (15)
##STR00041##
[0264] 15 was synthesized as described for 9c except for the use of
14 and 10 mM sodium citrate pH 4 as solvent A and 10 mM sodium
citrate pH 4 and 1 M sodium chloride as solvent B for cation
exchange chromatography.
[0265] Yield: 11 mg
Example 16
Synthesis of Linker Intermediate 16
##STR00042##
[0267] N,N-dimethylethylenediamine (198 .mu.L, 1.8 mmol) and
NaCNBH.sub.3 (58 mg, 0.9 mmol) were dissolved in methanol (5 mL)
and brought to pH 5.5 by addition of AcOH (250 .mu.L). A suspension
of 2,4,6,-trimethoxybenzaldehyde (294 mg, 1.5 mmol) in EtOH (5 mL)
was added and the reaction was stirred at RT for 1 h. 5 N HCl (0.5
mL) was added and the mixture was stirred for further 12 h. The
solvent was removed under reduced pressure; the residue was
dissolved in sat. NaHCO.sub.3 and extracted 3.times. with DCM. The
combined organic phases were dried over NaSO.sub.4 and the solvent
was evaporated under reduced pressure.
[0268] Yield: 303 mg (1.13 mmol)
[0269] MS: m/z 269.3=[M+H].sup.+ (MW calculated=268.4 g/mol)
Example 17
Synthesis of Linker 17a and 17b
##STR00043##
[0271] Synthesis of 17a:
[0272] Fmoc-Asp(OtBu)-OH (322 mg, 0.78 mmol), Tmob-protected
diamine 16 (150 mg, 0.56 mmol), HATU (255 mg, 0.67 mmol) and DIEA
(290 .mu.L, 1.68 mmol) were dissolved in DMF (1.5 mL). The mixture
was stirred for 30 min, acidified with AcOH and purified by
RP-HPLC.
[0273] Yield: 463 mg (5.97 mmol, TFA salt, ca. 90% pure)
[0274] MS Fmoc-Asp(OtBu)-N(TMOB)CH.sub.2CH.sub.2N(CH.sub.3).sub.2:
m/z 662.5=[M+H].sup.+ (MW calculated 661.8 g/mol)
[0275]
Fmoc-Asp(OtBu)-N(Tmob)CH.sub.2C.sub.2CH.sub.2N(CH.sub.3).sub.2 (225
mg, 0.29 mmol) was dissolved in a solution of piperidine (50 .mu.L)
and DBU (15 .mu.L) in DMF (1.5 mL). The mixture was stirred at RT
for 1.5 h. AcOH was added and
H-Asp(OtBu)-N(TMOB)CH.sub.2CH.sub.2N(CH.sub.3).sub.2 was purified
by RP-HPLC.
[0276] Yield 114 mg (0.21 mmol, TFA salt)
[0277] MS H-Asp(OtBu)-N(Tmob)CH.sub.2CH.sub.2N(CH.sub.3).sub.2: m/z
462.4=[M+Na].sup.+ (MW calculated=439.6 g/mol)
[0278] The TFA salt of
H-Asp(OtBu)-N(Tmob)CH.sub.2CH.sub.2N(CH.sub.3).sub.2 (114 mg, 0.21
mmol) was dissolved in sat. NaHCO.sub.3 (10 mL) and extracted
3.times. with DCM (3.times.10 mL). The combined organic layers were
dried over NaSO.sub.4 and the solvent was removed under reduced
pressure. The residue was dissolved in DMF (1.0 mL),
6-tritylmeraptohexanoic acid (121 mg, 0.31 mmol), HATU (118 mg,
0.31 mmol) and DIEA (108 .mu.L, 0.62 mmol) were added. The mixture
was stirred for 30 min. AcOH was added (200 .mu.L) and
TrtS(CH.sub.2).sub.5CONH-Asp(OtBu)--N(Tmob)CH.sub.2CH.sub.2N(CH.sub.3).su-
b.2 was purified by RP-HPLC.
[0279] Yield: 95 mg (0.10 mmol, TFA salt)
[0280] MS
TrtS(CH.sub.2).sub.5CONH-Asp(OtBu)-N(Tmob)CH.sub.2CH.sub.2N(CH.s-
ub.3).sub.2: m/z 812.64=[M+H].sup.+ (MW calculated=812.1 g/mol)
[0281]
TrtS(CH.sub.2).sub.5CONH-Asp(OtBu)-N(Tmob)CH.sub.2CH.sub.2N(CH.sub.-
3).sub.2 (95 mg, 0.10 mmol) was dissolved in a 3:1 mixture of
MeOH/H.sub.2O (1.0 mL), LiOH (7.4 mg, 0.31 mmol) was added and the
mixture was stirred for 5 h at 60.degree. C. AcOH was added (100
.mu.L) and 17a was purified by RP-HPLC.
[0282] Yield: 64 mg (0.07 mmol, TFA salt)
[0283] MS 17a: m/z 756.5=[M+H].sup.+ (MW calculated=756.0
g/mol)
[0284] 17b was synthesized as described above except for the use of
Fmoc-NMe-Asp(OtBu)-OH instead of Fmoc-Asp(OtBu)-OH in the first
step.
[0285] Yield 17b: 16 mg (18 .mu.mol, TFA salt)
[0286] MS 17b: m/z 770.5=[M+H].sup.+ (MW calculated=770.0
g/mol)
Example 18
Synthesis of Linker-BNP Conjugates 18a and 18b
##STR00044##
[0288] Synthesis of 18a:
[0289] 17a (8.0 mg, 0.01 mmol), PyBOP (5.2 mg, 10 mmol) and DIEA (7
.mu.L, 40 .mu.mol) were dissolved in DMF (400 .mu.L) and
immediately added to resin bound, side chain protected BNP-32a (50
mg, 5 .mu.mol). After incubation for 2 h at RT, the resin was
washed with 10.times.DMF, 10.times.DCM and dried in vacuo. The
product was cleaved from the resin and purified by RP-HPLC.
[0290] Yield: 10.6 mg
[0291] MS 18a: m/z 930.4=[M+4H].sup.4+; 1240.1=[M+3H].sup.3+ (MW
calculated=3717.2 g/mol)
[0292] 18b was synthesized as described above except for the use of
17b instead of 17a.
[0293] Yield: 4.7 mg
[0294] MS 18b: m/z 933.9=[M+4H].sup.4+; 1244.7=[M+3H].sup.3+ (MW
calculated=3731.0 g/mol)
Example 19
Synthesis of PEG40 kDa-Linker-BNP Conjugates 19a and 19b
##STR00045##
[0296] 18a (5.2 mg) was dissolved in 1:1 H.sub.2O/acetonitrile
containing 0.1% TFA (200 .mu.L). A solution of PEG40 kDa-maleimide
(70 mg) in 1:1 H.sub.2O/acetonitrile (1.5 mL) and phosphate buffer
(30 .mu.L, pH 7.4, 0.5 M) was added. The solution was incubated at
RT, after 5 min AcOH (30 .mu.L) was added. 19a was purified by
cation exchange chromatography, desalted, and lyophilized.
[0297] Yield: 19.2 mg
[0298] 19b was synthesized as described for 19a except for the use
of 18b instead of 18a.
Example 20
Synthesis of Linker 20
##STR00046##
[0300] Fmoc-Asp(OH)OtBu (100 mg, 0.24 mmol),
H.sub.2N--(CH.sub.2).sub.2N(CH.sub.3)-boc (36 .mu.L, 0.20 mmol),
HATU (92 mg, 0.4 mmol) and DIEA (105 .mu.L, 0.60 mmol) were
dissolved in 1 mL DMF. The mixture was stirred for 1 h at RT,
acidified with AcOH (100 .mu.L) and purified by HPLC.
[0301] Yield: 91 mg (0.13 mmol)
[0302] MS Fmoc-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu:
590.3=[M+Na].sup.+ (MW calculated=567.7 g/mol)
[0303] Fmoc-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu (91 mg, 0.13
mmol) was dissolved in DMF (1.0 mL), piperidine (50 .mu.L) and DBU
(15 .mu.L) were added and the mixture was stirred for 45 min at RT.
AcOH (100 .mu.L ) was added and
NH.sub.2-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu was purified by
RP-HPLC.
[0304] Yield: 39 mg (0.09 mmol, TFA salt)
[0305] MS NH.sub.2-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu: m/z
368.1=[M+Na].sup.+ (MW calculated=345.4 g/mol)
[0306] NH.sub.2-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu (36 mg,
0.09 mmol) was dissolved in DMF (0.5 mL), 6-tritylmercaptohexanoic
acid (55 mg, 0.14 mmol), HATU (53 mg, 0.14 mmol) and DIEA (49
.mu.L, 0.28 mmol) were added. The mixture was stirred for 45 min.
AcOH was added (100 .mu.L) and
TrtS(CH.sub.2).sub.5CONH-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu
was purified by RP-HPLC.
[0307] Yield: 41 mg (0.06 mmol)
[0308] MS
TrtS(CH.sub.2).sub.5CONH-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)O-
tBu: m/z 740.6=[M+Na].sup.+ (MW calculated=718.0 g/mol)
[0309]
TrtS(CH.sub.2).sub.5CONH-Asp(NH(CH.sub.2).sub.2N(CH.sub.3)-boc)OtBu
(41 mg, 0.06 mmol) was dissolved in 1:1 dioxane/H.sub.2O (1.0 mL),
LiOH (4.1 mg, 0.17 mmol) was added and the mixture was stirred at
60.degree. C. for 1 h. AcOH (50 .mu.L) was added and 20 was
purified by RP-HPLC.
[0310] Yield: 31 mg (0.05 mmol)
[0311] MS 20: m/z 684.5=[M+Na].sup.+ (MW calculated=661.9
g/mol)
Example 21
Synthesis of Linker-Exendin Conjugate 21
##STR00047##
[0313] Resin bound, side chain protected exendin (50 mg, 5 .mu.mol)
with a Mmt protecting-group on Lys27 was first boc-protected at the
N-terminus (see Materials and Methods) and then incubated five
times (5 min) with 2 mL of 9/1 (v/v) DCM/HFIP to remove the Mmt
protecting group from Lys27. 20 (6.6 mg, 10 .mu.mol), PyBOP (5.2
mg, 10 .mu.mol) and DIEA (7 .mu.L, 40 .mu.mol) were dissolved in
DMF (400 .mu.L) and immediately added to the resin. Incubation for
3 h at RT, the resin was washed with 10.times.DMF, 10.times.DCM and
dried in vacuo. The product was cleaved from the resin and purified
by RP-HPLC.
[0314] Yield: 2.4 mg
[0315] MS 21: m/z 1497.2=[M+3H].sup.3+ (MW calculated=4488.0
g/mol)
Example 22
Synthesis of Fatty Acid-Linker-Exendin Conjugate 22
##STR00048##
[0317] 21 (2.6 mg) was dissolved in 200 .mu.l 1/1
acetonitrile/water and 1 (0.8 mg) in 400 .mu.l of 7/3
acetonitrile/water was added. 100 .mu.l of 0.25 M sodium phosphate
buffer was added, the reaction was stirred for 5 min after which 22
was purified by RP-HPLC.
[0318] Yield: 2.4 mg
[0319] MS 22: m/z 1388.3=[M+4H].sup.4+; 1857.1 [M+3H].sup.3+; (MW
calculated=5566.4 g/mol).
Example 23
Synthesis of Precursor 23
##STR00049##
[0321] 6-Tritylmercaptohexanoic acid (200 mg, 0.51 mmol),
(PfpO).sub.2CO (202 mg, 0.51 mmol) and collidine (340 .mu.L, 2.65
mmol) were dissolved in DMSO (1 mL) and stirred for 30 min at RT.
The mixture was added to a solution of Fmoc-Lys-OH (170 mg, 0.46
mmol) in H.sub.2O/pyridine/tBuOH (3:3:1, 6 mL). The reaction was
heated at 60.degree. C. for 2 h, diluted with EtOAc, extracted
2.times. with 0.1M H.sub.2SO.sub.4, 2.times. with brine and dried
over Na.sub.2SO.sub.4. The solvent was evaporated under reduced
pressure and the residue was purified by RP-HPLC.
[0322] Yield: 109 mg
[0323] MS 23: m/z 741.3 [M+H].sup.+ (MW calculated=741.0 g/mol)
Example 24
Synthesis of Linker 24a-24c
##STR00050##
[0325] 23 (186 mg, 0.25 mmol) and DIEA (160 .mu.L, 0.92 mmol) were
dissolved in DCM (2 mL), added to 2-chlorotrityl chloride resin
(312 mg, 1.3 mmol/g) and agitated for 45 min at RT. MeOH (0.6 mL)
was added and the resin was incubated for another 15 min. The resin
was washed with DCM (10.times.) and DMF (10.times.).
Fmoc-deprotection and urea formation was achieved according to
general procedures (see Materials and Methods) by reaction with
N-boc-ethylenediamine (57 .mu.L, 0.34 mmol), the product was
cleaved from the resin and purified by RP-HPLC.
[0326] Yield: 14 mg
[0327] MS 24a: m/z 705.4 [M+H].sup.+, 727.3 [M+Na].sup.+ (MW
calculated=704.9 g/mol)
[0328] 24b was synthesized as described for 24 except for the use
of N-boc-N-methylethylenediamine instead of instead of
N-boc-ethylenediamine.
[0329] Yield: 21 mg
[0330] MS 24b: m/z 719.3 [M+H].sup.+, 741.4 [M+Na].sup.+ (MW
calculated=719.0 g/mol)
[0331] 24c was synthesized as described for 24a except for the use
of N,N-dimethylethylenediamine instead of
N-boc-ethylenediamine.
[0332] Yield: 10 mg
[0333] MS 24c: m/z 633.2 [M+H].sup.+ (MW calculated=632.9
g/mol)
Example 25
Synthesis of Exendin-Linker Conjugates 25a-25c
##STR00051##
[0335] 24a (7.0 mg, 0.01 mmol), PyBOP (5.2 mg, 10 mmol) and DIEA (7
.mu.L, 40 .mu.mol) were dissolved in DMF (250 .mu.L), immediately
added to resin bound, side chain protected exendin (50 mg, 5
.mu.mol) and incubated for 2 h at RT. The resin was washed with DMF
(100.times.), DCM (10.times.) and dried in vacuo. The product was
cleaved from the resin and purified by RP-HPLC.
[0336] Yield: 1.6 mg
[0337] MS 25a: m/z 1511.8=[M+3H].sup.3+ (MW calculated=4530.8
g/mol)
[0338] 25b was synthesized as described for 25a except for the use
of 24b instead of 24a.
[0339] Yield: 4.3 mg
[0340] MS 25b: m/z 1516.3=[M+3H].sup.3+ (MW calculated=4544.8
g/mol)
[0341] 25c was synthesized as described for 25a except for the use
of 24c instead of 24a.
[0342] Yield: 1.3 mg
[0343] MS 25c: m/z 1520.4=[M+3H].sup.3+ (MW calculated=4558.8
g/mol)
Example 26
Synthesis of Fatty Acid-Linker Conjugates 26a-26c
##STR00052##
[0345] 25a (1.6 mg) was dissolved in 200 .mu.l 1/1
acetonitrile/water and 1 (0.11 mg) in 200 .mu.l of 7/3
acetonitrile/water was added. 30 .mu.l of 0.25 M sodium phosphate
buffer was added, the reaction was stirred for 5 min, after which
26a was purified by RP-HPLC.
[0346] MS 26a: m/z 1870.0=[M+3H].sup.3+ (MW calculated=5609.2
g/mol).
[0347] 26b was synthesized as described for 26a except for the use
of 25b instead of 25a.
[0348] MS 26b: m/z 1875.9=[M+3H].sup.3+, 1406.7=[M+4H].sup.4+ (MW
calculated=5623.2 g/mol)
[0349] 26c was synthesized as described for 26a except for the use
of 25e instead of 25a.
[0350] MS 26c: m/z 1879.4=[M+3H].sup.3+, 1410.5=[M+4H](MW
calculated=5637.2 g/mol)
Example 27
Synthesis of 20 KDa-PEG-Linker-Exendin Conjugates 27a-27c
##STR00053##
[0352] 25a (2.0 mg) was dissolved in 1:1 H.sub.2O/MeCN containing
0.1% TFA (200 .mu.l). A solution of PEG40 KDa-maleimide (18 mg) in
1:1 H.sub.2O/MeCN (1 ml) and phosphate buffer (15 .mu.l, pH 7.4,
0.5 M) was added. The solution was incubated at RT, after 5 min
AcOH (20 .mu.l) was added and 27a was purified by cation exchange
chromatography, desalted and lyophilized.
[0353] 27b was synthesized as described for 27a except for the use
of 25b instead of 25a.
[0354] 27c was synthesized as described for 27a except for the use
of 25c instead of 25a.
Example 28
Synthesis of Linker 28
##STR00054##
[0356] 6-Bromohexanoyl chloride (46 .mu.l, 0.31 mmol) was dissolved
in 0.2 ml CH.sub.2Cl.sub.2 and added to a solution of
H.sub.2N--CH.sub.2--CH.sub.2--STrt (100 mg, 0.28 mmol), DIEA (97
.mu.l, 0.56 mmol) in CH.sub.2Cl.sub.2 (0.8 ml). The mixture was
stirred for 2 h at RT. The reaction mixture was acidified with AcOH
(50 .mu.l) and the solvent was removed in vacuo. The residue was
purified on silica gel (heptane/EtOAc=1:1) to obtain
Br--(CH.sub.2).sub.5--CONH--(CH.sub.2).sub.2--STrt.
[0357] Yield: 137 mg (0.276 mmol, 98%)
[0358] MS Br--(CH.sub.2).sub.5--CONH--(CH.sub.2).sub.2--STrt:
518.9=[M+Na].sup.+, (MW calculated=496.5 g/mol)
[0359] N-Boc-ethylenediamine (81 .mu.l, 0.51 mmol) was added to a
solution of Br--(CH.sub.2).sub.5--CONH--(CH.sub.2).sub.2--STrt (230
mg, 0.46 mmol) and Na.sub.2CO.sub.3 (196 mg, 1.85 mmol) in DMF (0.8
ml). The reaction mixture was stirred for 10 h at 70.degree. C.
After cooling to RT the mixture was diluted with 4 ml
(MeCN/H.sub.2O=25:75, with 0.1% TFA) and purified by RP-HPLC to get
Boc-NH--(CH.sub.2).sub.2--NH--(CH.sub.2).sub.5--CONH--(CH.sub.2)--STrt.
[0360] Yield: 189 mg (0.27 mmol, 59%, TFA-salt)
[0361] MS
Boc-NH--(CH.sub.2)--NH--(CH.sub.2).sub.3--CONH--(CH.sub.2-STrt:
576.5=[M+H].sup.+, (MW calculated=575.5 g/mol)
[0362]
Boc-NH--(CH.sub.2)--NH--(CH.sub.2).sub.5--CONH--(CH.sub.2)--STrt
(189 mg, 0.27 mmol) and HCHO (35% aqueous, 113 .mu.l) were
dissolved in MeCN (1.5 ml) and NaCNBH.sub.3 (34 mg, 0.54 mmol) was
added. The reaction mixture was stirred for 5 h at RT. After
completion of the reaction (MS) the solution was diluted with
H.sub.2O (5 ml) and extracted with CH.sub.2Cl.sub.2 (3.times.5 ml).
The combined organic layers were dried over MgSO.sub.4, filtered
and the solvent was removed in vacuo. The residue was purified by
RP-HPLC to get
Boc-NH--(CH.sub.2).sub.2--N(CH)--(CH.sub.2).sub.5--CONH--(CH.sub.2).sub.2-
-STrt.
[0363] Yield: 62.8 mg (0.11 mmol, 39%)
[0364] MS
Boc-NH--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2)--CONH--(CH.sub-
.2).sub.2--STrt: 590.6=[M+H].sup.+, (MW calculated=589.0 g/mol)
[0365]
Boc-NH--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.5--CONH--(CH.-
sub.2h-STrt (62.8 mg, 0.11 mmol) was dissolved in THF (6 ml) and
HCl in dioxane (130 .mu.l, 4 M solution) was added. The reaction
mixture was stirred for 12 h at RT. 200 .mu.l HCl in dioxane was
added and the solvent was removed in vacuo. The residue was
purified by RP-HPLC to give
H.sub.2N--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.5--CONH--(CH.sub.-
2).sub.2--STrt and not consumed starting material
Boc-NH--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.5--CONH--(CH.sub.2)-
--STrt.
[0366] Yield: 32.8 mg (0.062 mmol, 44%, HCl-salt) 28 and 14.7 mg
(0.025 mmol, 23%, TFA-salt) starting material
[0367] MS 28: 490.5=[M+H].sup.+, (MW calculated=489.0 g/mol)
Example 29
[0368] General Procedure for the Synthesis Carboxylic Acid
Substituted BNP Precursors 29a and 29b
##STR00055##
[0369] Cis-cyclohexane-1,2-dicarboxylic anhydride (231 mg, 1.5
mmol) and pyridine (271 .mu.l, 2 mmol) were dissolved in DCM (2 ml)
and added to resin bound, side chain protected BNP-32a (300 mg).
Incubation for 1 h at RT, washed with 10.times.DCM and dried in
vacuo.
##STR00056##
[0370] Resin bound, side chain protected BNP-32b, carrying an ivDde
protecting group at Lys14, was first boc-protected at the
N-terminus, deprotected at Lys14 position (see Materials and
Methods) and then reacted with cis-cyclohexane-1,2-dicarboxylic
anhydride as described above for 29a.
Example 30
Synthesis of BNP-Linker-Thiols 30a and 30b
##STR00057##
[0372]
H.sub.2N--(CH.sub.2)--N(CH.sub.3)--(CH).sub.5--CONH--(CH.sub.2).sub-
.2--STrt 28 (5.2 mg, 0.01 mmol), PyBOP (5.2 mg, 0.01 mmol) and DIEA
(7.0 .mu.l, 0.04 mmol) were dissolved in DMF (300 .mu.l) and added
to resin bound, side chain protected BNP 29a (50 mg, 0.005 mmol).
Incubation for 2 h at RT, the resin was washed with DMF
(10.times.), DCM (10.times.) and dried in vacuo. The product was
cleaved from the resin and purified by RP-HPLC.
[0373] Yield: 9.8 mg
[0374] MS 30a: m/z 947.6=[M+4H].sup.4+, 1263.1=[M+3H].sup.3+ (MW
calculated=3786.3 g/mol)
##STR00058##
[0375] 30b was synthesized as described above except for the use of
resin bound BNP derivative 29b instead of 29a.
[0376] Yield: 7.4 mg
[0377] MS 30b: m/z 947.5=[M+4H].sup.4+, 1263.0=[M+3H].sup.3+ (MW
calculated=3786.3 g/mol)
Example 31
Synthesis of 40 KDa-PEG-Linker-BNP Conjugates 31a and 31b
##STR00059##
[0379] 30a (4 mg) was dissolved in 1:1 H.sub.2O/MeCN containing
0.1% TFA (200 .mu.l). A solution of PEG40 KDa-maleimide (42.2 mg)
in 1:1 H.sub.2O/MeCN (1 ml) and phosphate buffer (15 .mu.l, pH 7.4,
0.5 M) was added. The solution was incubated at RT, after 5 min
AcOH (20 .mu.l) was added and 31a was purified by cation exchange
chromatography, desalted and lyophilized.
[0380] Yield: 2.0 mg
##STR00060##
[0381] 31b was synthesized as described for 31a except for the use
of 30b instead of 30a.
[0382] Yield: 16.8 mg
Example 32
Synthesis of Linker 32
##STR00061##
[0384] 3-Bromopropionylchloride (62.5 .mu.l, 0.62 mmol) was
dissolved in 0.5 ml CH.sub.2Cl.sub.2 and added to a solution of
H.sub.2N--CH.sub.2--CH.sub.2-STrt (200 mg, 0.56 mmol), DIEA (196
.mu.l, 1.1 mmol) in CH.sub.2Cl.sub.2 (1 ml). The mixture was
stirred for 1 h at RT. The reaction mixture was acidified with AcOH
(100 .mu.l) and the solvent was removed in vacuo. The residue was
purified over silica gel (heptane/EtOAc=1:1) to obtain
Br--(CH.sub.2).sub.2CONH--(CH.sub.2).sub.2--STrt.
[0385] Yield: 223 mg (0.49 mmol, 87%)
[0386] MS Br--(CH.sub.2).sub.2CONH--(CH.sub.2).sub.2--STrt:
478.7=[M+Na], (MW calculated=454.7 g/mol)
[0387] N-Alloc-ethylenediaminc HC-salt (43.5 mg, 0.24 mmol) and
DIEA (38 .mu.l, 0.22 mmol) were added to a solution of
Br--(CH.sub.2).sub.2CONH--(CH.sub.2)--STrt (100 mg, 0.22 mmol) and
Na.sub.2CO.sub.3 (93 mg, 0.87 mmol) in DMF (1 ml). The reaction
mixture was stirred for 10 h at 70.degree. C. After cooling down to
RT the reaction mixture was diluted with 4 ml (MeCN/H.sub.2O=25:75,
with 0.1% TFA) and purified by HPLC to get
Alloc-NH--(CH.sub.2).sub.2--NH--(CH.sub.2).sub.2--CONH--(CH.sub.2).sub.2--
-STrt.
[0388] Yield: 61 mg (0.096 mmol, 44%, TFA salt)
[0389] MS
Alloc-NH--(CH.sub.2).sub.2--NH--(CH.sub.2).sub.2--CONH--(CH.sub.-
2).sub.2--STrt: 540.8=[M+Na].sup.+, (MW calculated=517.8,
g/mol)
[0390]
Alloc-NH--(CH.sub.2).sub.2--NH--(CH.sub.2).sub.2--CONH--(CH.sub.2).-
sub.2--STrt (60.9 mg, 0.096 mmol) was dissolved in CH.sub.2Cl.sub.2
and Boc.sub.2O (42 mg, 0.19 mmol) was added. The solution was
stirred for 20 h at RT. After completion the reaction was quenched
by addition of 70 .mu.l AcOH and the solvent was removed in vacuo.
The residue was diluted with 4 ml MeCN/H.sub.2O (25:75, with 0.1%
TFA) and purified by RP-HPLC to give
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-(CH.sub.2).sub.2--CONH--(CH.sub.2-
).sub.2--STrt.
[0391] Yield: 53.3 mg (0.086 mmol, 89%)
[0392] MS
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-(CH.sub.2).sub.2--CONH--(CH.s-
ub.2).sub.2--STrt: 640.6=[M+Na].sup.+, (MW calculated=617.9.
g/mol)
[0393]
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-(CH.sub.2).sub.2--CONH--(CH.sub.-
2).sub.2--STrt (48.3 mg, 0.078 mmol) was dissolved in THF,
triethylammonium format (62 .mu.l) and Pd(PPh.sub.3).sub.4 (16 mg)
were added. The solution was stirred for 12 h at RT and monitored
by MS. After completion the solvent was removed in vacuo. The
residue was dissolved in MeCN/H.sub.2O (50:50, with 0.1% TFA) and
purified by RP-HPLC to give
H.sub.2N--(CH.sub.2).sub.2--N(Boc)-(CH.sub.2).sub.2--CONH--(CH.sub.2).sub-
.2--STrt (31).
[0394] Yield: 20.1 mg (0.031 mmol, 40%, TPA-salt)
[0395] MS 31: 534.6=[M+H].sup.+, 556.6=[M+Na].sup.+, (MW
calculated=533.5 g/mol)
Example 33
Synthesis of BNP-Linker-thiols 33a and 33b
##STR00062##
[0397] 33a was synthesized as described for 30a except for the use
of 32 instead of 28.
[0398] Yield: 8.0 mg
[0399] MS 33a: m/z 933.5=[M+4H].sup.4+, 1244.3=[M+3H].sup.3+ (MW
calculated=3729.9 g/mol)
##STR00063##
[0400] 33b was synthesized as described for 30b except for the use
of 32 instead of 28.
[0401] Yield: 5.0 mg
[0402] MS 33b: m/z 933.5=[M+4H].sup.4+, 1244.3=[M+3H].sup.3+ (MW
calculated=3715.9 g/mol)
Example 34
Synthesis of 40 KDa-PEG-Linker-BNP Conjugates 34a and 34b
##STR00064##
[0404] 33a (4.3 mg) was dissolved in 1:1 H.sub.2O/MeCN containing
0.1% TFA (200 .mu.l). A solution of PEG40 KDa-maleimide (46.8 mg)
in 1:1 H.sub.2O/MeCN (1 ml) and phosphate buffer (20 .mu.l, pH 7.4,
0.5 M) was added. The solution was incubated at RT, after 5 min
AcOH (20 .mu.l) was added and 34a was purified by cation exchange
chromatography, desalted and lyophilized.
[0405] Yield: 9.7 mg
##STR00065##
[0406] 34b was synthesized as described for 34a except for the use
of 33b instead of 33a.
[0407] Yield: 11.5 mg
Example 35
Synthesis of Linker 35
##STR00066##
[0409] Bromoacetylbromide (54 .mu.l, 0.62 mmol) was dissolved in
0.5 ml CH.sub.2Cl.sub.2 and added to a solution of
H.sub.2N--CH.sub.2--CH.sub.2-STrt (200 mg, 0.56 mmol) and DIEA (196
.mu.l, 1.1 mmol) in CH.sub.2Cl.sub.2 (1 ml). The mixture was
stirred for 1 h at RT. The reaction mixture was acidified with AcOH
(100 .mu.l) and the solvent was removed in vacuo. The residue was
purified over silica gel (heptane/EtOAc=1:1) to obtain product
Br--CH.sub.2--CONH--(CH.sub.2).sub.2--STrt.
[0410] Yield: 245 mg (0.55 mmol, 99%)
[0411] MS Br--CH.sub.2--CONH--(CH.sub.2).sub.2--STrt:
462.4=[M+Na].sup.+, (MW calculated=440.4 g/mol)
[0412] N-Alloc-ethylenediamine HCl-salt (45 mg, 0.25 mmol) and DIEA
(79 .mu.l, 0.45 mmol) was added to a solution of
Br--CH.sub.2--CONH--(CH.sub.2).sub.2--STrt (100 mg, 0.23 mmol) in
DMF (1 ml). The reaction mixture was stirred for 10 h at 70.degree.
C. After cooling down to RT the reaction mixture was diluted with
H.sub.2O/Et.sub.2O (1:1, 40 ml) and the layers were separated. The
aqueous layer was extracted several times with Et.sub.2O. The
combined organic layers were dried with MgSO.sub.4, filtered and
the solvent was removed in vacuo. The residue was purified over
silica gel (DCM/MeOH=95:5) to give
Alloc-NH--(CH.sub.2).sub.2--NH--CH.sub.2--CONH--(CH.sub.2).sub.2--STrt.
[0413] Yield: 94 mg (0.186 mmol, 82%, contains residual DMF)
[0414] MS
Alloc-NH--(CH.sub.2).sub.2--NH--CH.sub.2--CONH--(CH.sub.2).sub.2-
--STrt: 526.8=[M+Na].sup.+, (MW calculated=503.8. g/mol)
[0415]
Alloc-NH--(CH.sub.2).sub.2--NH--CH.sub.2--CONH--(CH.sub.2).sub.2--S-
Trt (94 mg, 0.186 mmol, with DMF)) was dissolved in
CH.sub.2Cl.sub.2 and Boc.sub.2O (81 mg, 0.37 mmol) was added. The
solution was stirred for 20 h at RT. After completion the reaction
was quenched by addition of 100 .mu.l AcOH and the solvent was
removed in vacuo. The residue was diluted with 4 ml MeCN/H.sub.2O
(25:75, with 0.1% TFA) and purified by RP-HPLC to give
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-CH.sub.2--CONH--(CH.sub.2).sub.2--
-STrt.
[0416] Yield: 34.7 mg (0.057 mmol, 26%)
[0417] MS
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-CH.sub.2--CONH--(CH.sub.2).su-
b.2--STrt: 603.9=[M+Na].sup.+, (MW calculated=603.9. g/mol)
[0418]
Alloc-NH--(CH.sub.2).sub.2--N(Boc)-CH.sub.2--CONH--(CH.sub.2).sub.2-
--STrt (34.7 mg, 0.048 mmol) was dissolved in THF,
triethylammoniumformate (38 .mu.l) and Pd(PPh.sub.3).sub.4 (5 mg)
were added. The solution was stirred for 12 h at RT and monitored
by MS. After completion the solvent was removed in vacuo. The
residue was dissolved in MeCN/H.sub.2O (50:50, with 0.1% TFA) and
purified by RP-HPLC to give
H.sub.2N--(CH.sub.2).sub.2--N(Boc)-CH.sub.2--CONH--(CH.sub.2).sub.2--STrt
35.
[0419] Yield: 12.6 mg (0.019 mmol, 42%, TFA-salt)
[0420] MS 35: 520.1=[M+H].sup.+, 542.2=[M+Na].sup.+, (MW
calculated=519.2 g/mol)
Example 36
Synthesis of BNP-Linker-Thiols 36a and 36b
##STR00067##
[0422] 36a was synthesized as described for 30a except for the use
of 35 instead of 28.
[0423] Yield: 9.1 mg
[0424] MS 36a: m/z 930.0=[M+4H].sup.4+, 1239.6=[M+3H].sup.3+(MW
calculated=3715.9 g/mol)
##STR00068##
[0425] 36b was synthesized as described for 30b except for the use
of 35 instead of 28.
[0426] Yield: 8.0 mg
[0427] MS 36b: m/z 929.9=[M+4H].sup.4+, 1239.5=[M+3H].sup.3+ (MW
calculated=3715.9 g/mol)
Example 37
Synthesis of 40 KDa-PEG-Linker-BNP Conjugates 37a and 37b
##STR00069##
[0429] 36a (4.2 mg) was dissolved in 1:1 H.sub.2O/MeCN containing
0.1% TFA (200 .mu.l). A solution of PEG40 KDa-maleimide (68 mg) in
1:1 H.sub.2O/MeCN (1 ml) and phosphate buffer (20 .mu.l, pH 7.4,
0.5 M) was added. The solution was incubated at RT, after 5 min
AcOH (20 .mu.l) was added and 37a was purified by ion exchange
chromatography, desalted and lyophilized.
[0430] Yield: 16 mg
##STR00070##
[0431] 37b was synthesized as described for 37a except for the use
of 36b instead of 36a.
[0432] Yield: 18.5 mg
Example 38
Synthesis of Linker-Exendin Conjugates
[0433] Linker-exendin conjugates were synthesized according to
general synthesis method A, B, C, D, E or F.
##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075##
[0434] Method A
[0435] Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2
mmol) were dissolved in 0.3 ml of dry DMF. Mixture was added to
side chain protected exendin-4 on resin (2 .mu.mol) and agitated
for 30 min at room temperature. Resin was washed with DMF (10
times). PyBOP (0.1 mmol) and diamine (0.1 mmol) were dissolved in
0.3 ml of dry DMF. Mixture was added to resin and agitated for 30
min at room temperature. Resin was washed with DMF (10 times).
Exendin-linker conjugates were cleaved and purified by RP-HPLC as
described in "Materials and Methods".
[0436] Method B
[0437] Synthesis: As described for Method A except that diacid
anhydride and pyridine are replaced by diacid (0.2 mmol), HOBt (0.2
mmol), DIC (0.2 mmol), and collidine (0.4 mmol).
[0438] Method C
[0439] Synthesis. Diamine (0.6 mmol) was dissolved in 1 ml of dry
DCM and diacid anhydride (0.4 mmol) was added. Mixture was stirred
for 60 min at room temperature. DCM was removed, the residue was
dissolved in ACN/water/AcOH, and amino acid was purified by RP-HPLC
and lyophilized.
[0440] Amino acid (0.1 mmol), HOBt (0.1 mmol), DIC (0.1 mmol), and
collidine (0.2 mmol) were dissolved in 0.3 ml of dry DMF. Mixture
was added to exendin-4 on resin (2 .mu.mol) and agitated for 30 min
at room temperature. Resin was washed with DMF (10 times).
[0441] Exendin-linker conjugates were cleaved and purified by
RP-HPLC as described in "Materials and Methods".
[0442] Method D
[0443] Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2
mmol) were dissolved in 0.3 ml of dry DMF. Mixture was added to
exendin-4 on resin (2 .mu.mol) and agitated for 30 min at room
temperature. Resin was washed with DMF (10 times). PyBOP (0.1
mmol), HOBt (0.1 mmol), and collidine (0.4 mmol) were dissolved in
0.3 ml of dry DMF. Mixture was added to resin and agitated for 30
min at room temperature. Resin was washed with DMF (10 times).
Diamine (0.1 mmol) and DIEA (0.3 mmol) were dissolved in a mixture
of 0.4 ml of DMF and 0.4 ml of EtOH. Mixture was added to resin and
agitated for 30 min at room temperature.
[0444] Resin was washed with DMF (10 times).
[0445] Exendin-linker conjugates were cleaved and purified by
RP-HPLC as described in "Materials and Methods".
[0446] Method E
[0447] Synthesis: Fmoc amino acid (0.1 mmol), PyBOP (0.1 mmol) and
DIEA (0.2 mmol) were dissolved in 0.3 ml of dry DMF. Mixture was
added to exendin-4 on resin (2 .mu.mol) and agitated for 30 min at
room temperature. Fmoc protecting group was removed by incubating
resin in DMF/piperidine 4/1 (v/v) for 2.times.10 min. Resin was
washed with DMF (10 times) and DCM (10 times). p-Nitrophenyl
chloroformate (0.1 mmol) was dissolved in 0.3 ml of dry THF and
DIEA (0.2 mmol). Mixture was added to resin and agitated for 30 min
at room temperature. Resin was washed with DCM (10 times). Diamine
(0.1 mmol) was dissolved in 0.3 ml of DMF. Mixture was added to
resin and agitated for 30 min at room temperature.
[0448] Resin was washed with DMF (10 times).
[0449] Exendin-linker conjugates were cleaved and purified by
RP-HPLC as described in "Materials and Methods".
[0450] Method F
[0451] Synthesis: as described for Method A, followed by
fmoc-deprotection and acetylation: Fmoc protecting group was
removed as by incubating resin in DMF/piperidine 4/1 (v/v) for
2.times.10 min. Resin was washed with DMF (10 times). Acetylation
was performed by incubating resin with acetic
anhydride/pyridine/DMF 1/1/2 (v/v/v) for 30 min. Resin was washed
with DMF (10 times).
[0452] Exendin-linker conjugates were cleaved and purified by
RP-HPLC as described in "Materials and Methods".
[0453] Further details concerning compound numerals, starting
materials, synthesis method, molecular weight (MW) and MS data are
given in FIG. 2.
Example 39
Synthesis of Hydrogel-Linker-Exendin Conjugates 39
##STR00076##
[0455] Maleimide-functionalized hydrogel microparticles were
synthesized as described in EP 1 625 856 A1.
[0456] 30 mg of maleimide-derivatized hydrogel microparticles
(loading 40 .mu.mol/g, 1.2 .mu.mol) were reacted with 6 mg of
compound 25a (1.32 .mu.mol, 1.1 eq) in 600 .mu.l 20/80 (v/v)
acetonitrile/50 mM phosphate buffer (pH 7.4) for 10 min to give
exendin-linker loaded hydrogel microparticles 39. The loaded
hydrogel 39 was washed 5 times with 50/50 (v/v) acetonitrile/water
and three times with water.
Example 40
Synthesis of Linker 40
##STR00077##
[0458] Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 .mu.L, 1.0
mmol) were dissolved in DCM (2 mL), added to 2-chlorotrityl
chloride resin (312 mg, 1.3 mmol/g) and agitated for 45 min at RT.
Methanol (0.6 mL) was added and the resin was incubated for another
15 min. The resin was washed with DCM (10.times.) and DMF
(10.times.). Fmoc-deprotection and urea formation was achieved
according to general procedures (see Materials and Methods) by
reaction with linker intermediate 5a, the product was cleaved from
the resin and purified by RP-HPLC.
[0459] Yield: 53 mg
[0460] MS 40: m/z 604.4 [M+H].sup.+ (MW calculated=603.8 g/mol)
Example 41
Synthesis of Exendin-Linker Conjugate 41
##STR00078##
[0462] 40 (HCl salt, 14.0 mg, 0.02 mmol), PyBOP (10.2 mg, 0.02
mmol) and DIEA (17 .mu.L, 0.1 mmol) were dissolved in DMF (300
.mu.L), immediately added to resin bound, side chain protected
exendin (100 mg, 10 .mu.mol) and incubated for 4 h at RT. The resin
was washed with DMF (10.times.), DCM (10.times.) and dried in
vacuo. The product was cleaved from the resin and purified by
RP-HPLC.
[0463] Yield: 5.4 mg
[0464] MS 40: m/z 1510.9=[M+3H].sup.3+ (MW calculated=4530.1
g/mol)
Example 42
Synthesis of Fatty Acid-Linker Conjugate 42
##STR00079##
[0466] 41 (1.6 mg) was dissolved in 200 .mu.l 3/1
acetonitrile/water and 1 (0.11 mg) in 200 .mu.l of 3/1
acetonitrile/water was added. 30 .mu.l of 0.25 M sodium phosphate
buffer was added, the reaction was stirred for 5 min, after which
42 was purified by RP-HPLC. MS 42: m/z 1870.2=[M+3H].sup.3+ (MW
calculated=5608.4 g/mol).
Example 43
Synthesis of Hydrogel-Linker-Exendin Conjugate 43
##STR00080##
[0468] 43 was synthesized as described for 39 except for the use of
41 instead of 25a.
Example 44
Synthesis of Linker 44
##STR00081##
[0470] 44 was synthesized as described for 40 except for the use of
28 instead of 5a.
[0471] Yield: 74 mg
[0472] MS 44: m/z 605.4 [M+H].sup.+ (MW calculated=604.8 g/mol)
Example 45
Synthesis of Exendin-Linker Conjugate 45
##STR00082##
[0474] 45 was synthesized as described for 41 except for the use of
44 instead of 40.
[0475] Yield: 6.0 mg
[0476] MS 45: m/z 1511.3=[M+3H].sup.3+ (MW calculated=4531.1
g/mol)
Example 46
Synthesis of Fatty Add-Linker Conjugate 46
##STR00083##
[0478] 46 was synthesized as described for 42 except for the use of
45 instead of 41.
[0479] MS 46: m/z 1870.5=[M+3H].sup.3+ (MW calculated=5609.5
g/mol).
Example 47
Synthesis of Linker Intermediate 47
##STR00084##
[0481] Tritylsulfide (247 mg, 0.89 mmol) was suspended in 1 ml
DMSO. DBU (152 .mu.l, 1.02 mmol) and 6-bromohexan-1-ol (173 mg,
0.96) were added and mixture was stirred for 5 min at RT. Reaction
mixture was dissolved in 20 ml ethylacetate and washed with 1
NH.sub.2SO.sub.4 (2.times.) and brine (3.times.). Organic layer was
dried (Na.sub.2SO.sub.4) and volatiles were removed in vacuo.
Product was purified by flash chromatography on silica
(heptane/AcOEt 1/1).
[0482] Yield 283 mg (S-trityl)-6-mercaptohexan-1-ol
[0483] (S-Trityl)-6-mercaptohexan-1-ol (466 mg, 1.24 mmol) was
dissolved in 3.5 ml DCM, 0.5 ml DMSO and 0.6 ml NEt.sub.3, and
cooled in an ice bath. SO.sub.3-pyridine (408 mg, 2.57 mmol) was
suspended in 0.5 ml DMSO and added to reaction mixture. Ice bath
was removed and reaction was stirred for 60 min at RT. Reaction
mixture was dissolved in 20 ml Et.sub.2O and extracted with 1
NH.sub.2SO.sub.4 (2.times.) and brine (3.times.). Organic layer was
dried (Na.sub.2SO.sub.4) and volatiles were removed in vacuo.
Product was purified by flash chromatography on silica
(heptane/AcOEt 1/1).
[0484] Yield: 390 mg (S-trityl)-6-mercaptohexan-1-al 47
[0485] MS 47: m/z 243.1=[Trt].sup.+, 413.1=[M+K].sup.+ (MW
calculated=374.4 g/mol)
Example 48
Synthesis of Linker 48
##STR00085##
[0487] Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 .mu.L, 1.0
mmol) were dissolved in DCM (2 mL), added to 2-chlorotrityl
chloride resin (312 mg, 1.3 mmol/g) and agitated for 45 min at RT.
Methanol (0.6 mL) was added and the resin was incubated for another
15 min. The resin was washed with DCM (10.times.) and DMF
(10.times.). Fmoc-deprotection and urea formation was achieved
according to general procedures (see Materials and Methods) by
reaction with ethylene diamine. For reductive alkylation 47 (299
mg, 0.8 mmol) and Na(OAc).sub.3BH (340 mg, 1.6 mmol) were dissolved
in 0.5 mL DMF, 0.5 ml MeOH and 10 .mu.L AcOH, added to resin and
agitated for 2 h at RT. Resin was washed with DMF (10.times.) and
DCM (10.times.). Boc protection was performed by agitating resin in
a solution of boc anhydride (218 mg, 1.0 mmol) and DIEA (170 .mu.L,
1.0 mmol) in DCM. Resin was washed with DCM (10.times.) and product
was cleaved from the resin and purified by RP-HPLC.
[0488] Yield: 34 mg
[0489] MS 48: m/z 634.2 [M+H].sup.+ (MW calculated=633.9 g/mol)
Example 49
Synthesis of Exendin-Linker Conjugate 49
##STR00086##
[0491] 49 was synthesized as described for 41 except for the use of
48 instead of 40.
[0492] Yield: 4.8 mg
[0493] MS 49: m/z 1487.3=[M+3H].sup.3+ (MW calculated=4460.0
g/mol)
Example 50
Synthesis of Hydrogel-Linker-Exendin Conjugate 50
##STR00087##
[0495] 50 was synthesized as described for 39 except for the use of
49 instead of 25a.
Example 51
Release Kinetics In Vitro
[0496] Release of drug molecule from 38a to 38z, 38aa to 38ab, 4,
5, 9a, 9b, 9c, 10, 13a, 15, 19a, 19b, 22, 26a to 26c, 31a, 31b,
34a, 34b, 37a, 37b, 42, 43, 46, and 50 was effected by hydrolysis
in buffer at pH 7.4 and 37.degree. C. or pH 4 and 37.degree. C. as
described in "Materials and Methods".
TABLE-US-00001 Compound t.sub.1/2 buffer A (pH 7.4) t.sub.1/2
buffer B (pH 4.0) 38a <1 h 13 h 38b 20 h 72 d 38c >3 m >3
m 38d 58 d n.d. 38e 41 d n.d. 38f 23 h 114 d 38g 19 d none 38h 47 d
none 38i 69 h 108 d 38j 16 d n.d. 38k 40 min 6 d 38l 16 h n.d. 38m
17 h 66 d 38n 18 d n.d. 38o 11-12 h 22 d 38p 26 d 178 d 38q 26 d
210 d 38r 26 h 47 d 38s 80 min 80 h 38t 96 min 67 h 38u 51 d none
38v 47 d none 38w 8 d 3.2 a 38x 72 d n.d. 38y 11-14 h 105 d 38z 11
d 1.6 a 38aa 40 h 65 d 38ab 20 h 20 d 38ac 14 h n.d. 38ad 18 h n.d.
4 15 d n.d. 5 22 h n.d. 9a 340 h n.d 9b 360 h n.d 9c 120 h n.d 10
130 h n.d 13a 120 h n.d 13b 160 h n.d. 15 160 h n.d 19a 31 h n.d.
19b 18 h n.d. 22 40 d n.d. 26a 34 d n.d. 26b 40 d n.d. 26c 18 d
n.d. 31a 22 h n.d. 31b 95 h n.d. 34a 42 h n.d. 34b 205 h n.d. 37a
138 h n.d. 37b 639 h n.d. 42 10 d n.d. 43 17 d n.d. 46 13 d n.d. 50
35 d n.d.
Example 52
Release Kinetics In Vivo-In Vitro/In Vivo Correlation
[0497] Release kinetics in vivo were determined by comparing the
pharmacokinetics of 13a with the pharmacokinetics of 13c and 13b
with 13d, respectively, after intravenous injection into rat.
Animal studies were performed at Heidelberg Pharma AG, Heidelberg,
Germany. 13a (27 mg) was dissolved in 3.5 ml PBS and 500 .mu.l of
the resulting solution were injected intravenously into six rats
each. Male SD rats with approximately 270 g weight were used. Blood
samples were drawn at t=0, 2 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120
h, and 168 h, plasma was prepared, and plasma was analyzed for
fluorescein fluorescence using a Perkin-Elmer LS 50B
spektrometer.
[0498] Pharmacokinetics of 13c were determined as described for
13a. Pharmacokinetics of 13b and 13d were determined as described
for 13a, except for the use of 20 mg 13 b and 13d each in 2.5 ml
PBS and four rats.
[0499] Linker hydrolysis half-life was calculated from the ratio of
fluorescence of 13a compared to fluorescence of 13c and 13b
compared to 13d, respectively, at the respective time points.
[0500] Half-life of in vivo Linker hydrolysis was determined to be
115 h and 160 h for 13a and 13b, respectively, which is in
excellent correlation to the half-life of in vitro linker
hydrolysis of 120 h and 160 h for 13a and 13b, respectively.
[0501] FIG. 3 shows in vivo and in vitro linker cleavage data of
13b, wherein in vivo (triangles) and in vitro (diamonds) cleavage
kinetics are shown by semilogarithmic representation.
ABBREVIATIONS
[0502] Acp 4-(2-aminoethyl)-1-carboxymethyl-piperazine [0503] AcOH
acetic acid [0504] Boc t-butyloxycarbonyl [0505] Dab
2,4-diaminobutyric acid [0506] DBU 1,3-diazabicyclo[5.4.0]undecene
[0507] DCM dichloromethane [0508] Dda dodecanoic acid [0509] DIC
diisopropylcarbodiimide [0510] DIEA diisopropylethylamine [0511]
DMAP dimethylamino-pyridine [0512] DMF N,N-dimethylformamide [0513]
DMSO dimethylsulfoxide [0514] EDTA ethylenediaminetetraacetic acid
[0515] eq stoichiometric equivalent [0516] Fmoc
9-fluorenylmethoxycarbonyl [0517] HATU
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0518] HFIP hexafluoroisopropanol [0519] HEPES
N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) [0520]
HOBt N-hydroxybenzotriazole [0521] ivDde
1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methylbutyl [0522]
LCMS mass spectrometry-coupled liquid chromatography [0523] Mal
3-maleimido propionyl [0524] Mmnt 4-methoxytrityl [0525] MS mass
spectrum [0526] MW molecular mass [0527] n.d. not determined [0528]
PfpOH pentafluorophenol [0529] PyBOP
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate [0530] RP-HPLC reversed-phase high performance
liquid chromatography [0531] RT room temperature [0532] SEC size
exclusion chromatography [0533] Suc succinimidopropionyl [0534] TCP
2-chlorotrityl chloride resin [0535] TES triethylsilane [0536] TMOB
2,4,6-trimethoxybenzyl [0537] TFA trifluoroacetic acid [0538] THF
tetrahydrofurane [0539] UV ultraviolet [0540] VIS visual
* * * * *