U.S. patent application number 10/659178 was filed with the patent office on 2005-12-29 for boronic acid salts.
This patent application is currently assigned to Trigen Limited. Invention is credited to Combe-Marzelle, Sophie Marie, Deadman, John Joseph, Dolman, Mark, Kakkar, Sanjay Kumar, Kennedy, Anthony James, Madge, David Jonathan.
Application Number | 20050288253 10/659178 |
Document ID | / |
Family ID | 32719549 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050288253 |
Kind Code |
A1 |
Madge, David Jonathan ; et
al. |
December 29, 2005 |
Boronic acid salts
Abstract
Salts of a peptide boronic acid drug, for example of
Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2. The counter-ion to the
boronate may be an alkali metal or derived from a strongly basic
organic nitrogen-containing compound.
Inventors: |
Madge, David Jonathan;
(London, GB) ; Dolman, Mark; (London, GB) ;
Deadman, John Joseph; (Melbourne, AU) ; Kennedy,
Anthony James; (London, GB) ; Combe-Marzelle, Sophie
Marie; (London, GB) ; Kakkar, Sanjay Kumar;
(London, GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
Trigen Limited
|
Family ID: |
32719549 |
Appl. No.: |
10/659178 |
Filed: |
September 9, 2003 |
Current U.S.
Class: |
514/64 ;
562/7 |
Current CPC
Class: |
C07K 5/06191 20130101;
A61P 9/00 20180101; A61P 7/00 20180101; C07K 5/06078 20130101; A61K
38/00 20130101; A61P 7/02 20180101 |
Class at
Publication: |
514/064 ;
562/007 |
International
Class: |
C07F 005/02; A61K
031/69 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 2002 |
GB |
GB 0220764.5 |
Sep 9, 2002 |
GB |
GB 0220822.1 |
Apr 4, 2003 |
GB |
GB 0307817.7 |
May 16, 2003 |
GB |
GB 0311237.2 |
Jul 4, 2003 |
GB |
GB 0315691.6 |
Claims
1. A pharmaceutically acceptable base addition salt of a boronic
acid of formula (I): 31wherein Y comprises a hydrophobic moiety
which, together with the aminoboronic acid residue
--NHCH(R.sup.9)--B(OH).sub.2, has affinity for the substrate
binding site of thrombin; and R.sup.9 is a straight chain alkyl
group interrupted by one or more ether linkages and in which the
total number of oxygen and carbon atoms is 3, 4, 5 or 6 or R.sup.9
is --(CH.sub.2).sub.m--W where m is from 2, 3, 4 or 5 and W is --OH
or halogen.
2. The salt of claim 1 wherein R.sup.9 is an alkoxyalkyl group.
3. The salt of claim 1 wherein YCO-- comprises an amino acid
residue which binds to the S2 subsite of thrombin, the amino acid
residue being N-terminally linked to a moiety which binds the S3
subsite of thrombin.
4. The salt of claim 1 wherein Y comprises a dipeptide which binds
to the S3 and S2 binding sites of thrombin.
5. The salt of claim 4 wherein the S3-binding amino acid residue is
of (R)-configuration, the S2-binding residue is of
(S)-configuration, and the fragment --NHCH(R.sup.9)--B(OH) is of
(R)-configuration.
6. The salt of claim 5 wherein R.sup.9 is an alkoxyalkyl group.
7. The salt of claim 1 wherein the boronic acid has a Ki for
thrombin of about 100 nM or less.
8. The salt of claim 1 wherein the salt comprises a salt of the
boronic acid with metal or a strongly basic organic
nitrogen-containing compound.
9. The salt of claim 1 wherein the salt comprises a salt of the
boronic acid with an alkali metal, an aminosugar, a guanidine or an
amine of formula (XI): 32where n is from 1 to 6, R.sup.2 is H,
carboxylate or derivatised carboxylate, R.sup.3 is H,
C.sub.1-C.sub.4 alkyl or a residue of a natural or unnatural amino
acid.
10. The salt of claim 4 wherein the Y dipeptide is N-terminally
protected or N-terminally unprotected, and the peptide linkages in
the dipeptide are unsubstituted or independently N-substituted by a
C.sub.1-C.sub.13 hydrocarbyl, wherein the C.sub.1-C.sub.13
hydrocarbyl contains no heteratoms or at least one in-chain or
in-ring nitrogen, oxygen or sulfur atom, and the C.sub.1-C.sub.13
hydrocarbyl is unsubstituted or substituted by a substituent
selected from halo, hydroxy and trifluoromethyl.
11. The salt of claim 1 which consists essentially of an acid salt
in which one B--OH group of formula (I), when trigonally
represented, remains protonated.
12. The salt of claim 5 which comprises boronate ions derived from
the peptide boronic acid and has a stoichiometry consistent with
the boronate ions carrying a single negative charge.
13. The salt of claim 6 which consists essentially of a monosodium
or monolithium salt of the boronic acid.
14. A pharmaceutically acceptable base addition salt of a boronic
acid of formula (II): 33where: X is H or an amino-protecting group;
aa.sup.1 is an amino acid residue having a hydrocarbyl side chain
containing no more than 20 carbon atoms and comprising at least one
cyclic group having up to 13 carbon atoms; aa.sup.2 is an imino
acid residue having from 4 to 6 ring members; R.sup.1 is a group of
the formula --(CH.sub.2).sub.s-Z, where s is 2, 3 or 4 and Z is
--OH, --OMe, --OEt or halogen.
15. The salt of claim 14 wherein aa.sup.1 is selected from Phe, Dpa
and wholly or partially hydrogenated analogues thereof.
16. The salt of claim 15 wherein aa.sup.1 is of
R-configuration.
17. The salt of claim 14 wherein aa.sup.2 is a residue of an imino
acid of formula (IV) 34where R.sup.11 is --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --S--CH.sub.2--, --S--C(CH.sub.3).sub.2--
or --CH.sub.2--CH.sub.2--CH.sub- .2--, and, when the formula (IV)
ring is 5- or 6-membered, the formula (IV) ring is unsubstituted or
is substituted at one or more --CH.sub.2-- groups by from 1 to 3
C.sub.1-C.sub.3 alkyl groups.
18. The salt of claim 17 wherein aa.sup.2 is of
S-configuration.
19. The salt of claim 14, wherein aa.sup.1-aa.sup.2 is
(R)-Phe-(S)-Pro and the fragment --NH--CH(R.sup.1)--B(OH).sub.2 is
of (R)-configuration.
20. The salt of claim 15 wherein the boronic acid is of formula
(VIII): X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2 (VIII), wherein X is
R.sup.6--(CH.sub.2).sub.p--C(O)--,
R.sup.6--(CH.sub.2).sub.p--S(O).sub.2-- -,
R.sup.6--(CH.sub.2).sub.p--NH--C(O)-- or
R.sup.6--(CH.sub.2).sub.p--O--- C(O)-- wherein p is 0, 1, 2, 3, 4,
5 or 6 and R.sup.6 is H or a 5 to 13-membered cyclic group which is
unsubstituted or substituted by 1, 2 or 3 substituents selected
from halogen; amino; nitro; hydroxy; a C.sub.5-C.sub.6 cyclic
group; C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 alkyl containing,
or linked to the cyclic group through, an in-chain O atom, the
aforesaid alkyl groups optionally being substituted by a
substituent selected from halogen, amino, nitro, hydroxy and a
C.sub.5-C.sub.6 cyclic group.
21. The salt of claim 15 wherein the salt comprises a salt of the
boronic acid with an alkali metal, an aminosugar or an amine of
formula (XI): 35where n is from 1 to 6, R.sup.2 is H, carboxylate
or derivatised carboxylate, R.sup.3 is H, C.sub.1-C.sub.4 alkyl or
a residue of a natural or unnatural amino acid.
22. A pharmaceutical product comprising a therapeutically effective
amount of a boronate salt which consists essentially of a single
base addition salt of a boronic acid formula (XX): 36where: X is H
or an amino-protecting group; aa.sup.1 is an amino acid residue of
R-configuration having a hydrocarbyl side chain containing no more
than 20 carbon atoms and comprising at least one cyclic group
having up to 13 carbon atoms; aa.sup.2 is an imino acid residue of
S-configuration having from 4 to 6 ring members; C* is a chiral
centre of R-configuration; and R.sup.1 is a group of the formula
--(CH.sub.2).sub.s-Z, where s is 2, 3 or 4 and Z is --OH, --OMe,
--OEt or halogen.
23. A pharmaceutical formulation adapted for oral administration,
comprising a) a first species selected from a boronic acid of
formula (I), and boronate ions of said boronic acid and equilibrium
forms of said boronic acid and said boronate ions: 37wherein Y
comprises a hydrophobic moiety which, together with the
aminoboronic acid residue --NHCH(R.sup.9)--B(OH).sub.2, has
affinity for the substrate binding site of thrombin; and R.sup.9 is
a straight chain alkyl group interrupted by one or more ether
linkages and in which the total number of oxygen and carbon atoms
is 3, 4, 5 or 6 or R.sup.9 is --CH.sub.2).sub.m--W where m is from
2, 3, 4 or 5 and W is --OH or halogen; and (b) a second species
selected from pharmaceutically acceptable metal ions, said metal
ions having a valency of n, lysine, arginine and aminosugars,
wherein the formulation has an observed stoichiometry of first to
second species essentially consistent with a notional stoichiometry
of 1:1 when the second species is a metal ion with a valency of 1
or is lysine, arginine or an aminosugar, or an observed
stoichiometry of n:1 when the second species is a metal ion with a
valency of greater than 1.
24. A method of inhibiting thrombin in the prophylaxis or therapy
of disease, comprising orally administering to a mammal suffering
from, or at risk of suffering from, thrombosis a therapeutically
effective amount of the salt defined in claim 1.
25. A method for preventing thrombosis in a haemodialysis circuit
of a patient, for preventing a cardiovascular event in a patient
with end stage renal disease, for preventing venous thromboembolic
events in a patient receiving chemotherapy through an indwelling
catheter, for preventing thromboembolic events in a patient
undergoing a lower limb arterial reconstructive procedure, or for
treating by way of therapy or prophylaxis an arterial disease
selected from acute coronary syndromes, cerebrovascular thrombosis,
peripheral arterial occlusion and arterial thrombosis resulting
from atrial fibrillation, valvular heart disease, arterio-venous
shunts, indwelling catheters or coronary stents, the method
comprising orally administering to a mammal a therapeutically
effective amount of the salt defined in claim 16.
26. An oral pharmaceutical formulation, comprising a
therapeutically effective amount of the salt of claim 1.
27. A medicament adapted for oral administration and comprising a
therapeutically effective amount of a pharmaceutically acceptable
base addition salt of a boronic acid which is a selective thrombin
inhibitor and has a neutral aminoboronic acid residue capable of
binding to the thrombin S1 subsite linked through a peptide linkage
to a hydrophobic moiety capable of binding to the thrombin S2 and
S3 subsites, the salt comprising a cation having a valency n and
having an observed stoichiometry consistent with a notional
stoichiometry (boronic acid:cation) of n:1.
28. A medicament of claim 27 wherein the boronic acid has a Ki for
thrombin of about 100 nM or less.
29. A method for making a salt of claim 1, comprising: combining in
a solvent diethanolamine and an ester of a boronic acid as defined
in claim 1; allowing or causing a precipitate to form and
recovering the precipitate; converting the precipitated material
into the free organoboronic acid by contacting the precipitated
material with an aqueous acid or base; and reacting the
organoboronic acid with a base of a pharmaceutically acceptable
multivalent metal to form to a salt as defined in claim 1.
Description
BACKGROUND
[0001] The present disclosure relates to pharmaceutically useful
products obtainable from organoboronic acids. The disclosure also
relates to the use of members of the aforesaid class of products,
to their formulation, their preparation, their synthetic
intermediates and to other subject matter.
[0002] The disclosure further relates to oral pharmaceutical
formulations containing the described products.
[0003] Boronic Acid Compounds
[0004] It has been known for some years that boronic acid compounds
and their derivatives, e.g. esters, have biological activities,
notably as inhibitors or substrates of proteases. For example,
Koehler et al. Biochemistry 10:2477, 1971 report that
2-phenylethane boronic acid inhibits the serine protease
chymotrypsin at millimolar levels. The inhibition of chymotrypsin
and subtilisin by arylboronic acids (phenylboronic acid,
m-nitro-phenylboronic acid, m-aminophenylboronic acid,
m-bromophenylboronic acid) is reported by Phillip et al, Proc. Nat
Acad. Sci. USA 68:478-480, 1971. A study of the inhibition of
subtilisin Carlsberg by a variety of boronic acids, especially
phenyl boronic acids substituted by Cl, Br, CH.sub.3, H.sub.2N, MeO
and others, is described by Seufer-Wasserthal et al, Biorg. Med.
Chem. 2(1):35-48, 1994.
[0005] In describing inhibitors or substrates of proteases, P1, P2,
P3, etc. designate substrate or inhibitor residues which are
amino-terminal to the scissile peptide bond, and S1, S2, S3, etc.,
designate the corresponding subsites of the cognate protease in
accordance with: Schechter, I. and Berger, A. On the Size of the
Active Site in Proteases, Biochem. Biophys. Res. Comm., 27:157-162,
1967. In thrombin, the S1 binding site or "specificity pocket" is a
well defined slit in the enzyme, whilst the S2 and S3 binding
subsites (also respectively called the proximal and distal
hydrophobic pockets) are hydrophobic and interact strongly with,
respectively, Pro and (R)-Phe, amongst others.
[0006] Pharmaceutical research into serine protease inhibitors has
moved from the simple arylboronic acids to boropeptides, i.e.
peptides containing a boronic acid analogue of an .alpha.-amino
carboxylic acid. The boronic acid may be derivatised, often to form
an ester. Shenvi (EP-A-145441 and U.S. Pat. No. 4,499,082)
disclosed that peptides containing an .alpha.-aminoboronic acid
with a neutral side chain were effective inhibitors of elastase and
has been followed by numerous patent publications relating to
boropeptide inhibitors of serine proteases. Specific, tight binding
boronic acid inhibitors have been reported for elastase (K.sub.i,
0.25 nM), chymotrypsin (K.sub.i, 0.25 nM), cathepsin G (K.sub.i, 21
nM), .alpha.-lytic protease (K.sub.i, 0.25 nM), dipeptidyl
aminopeptidase type IV (K.sub.i, 16 pM) and more recently thrombin
(Ac-D-Phe-Pro-boroArg-OH (DuP 714 initial K.sub.i 1.2 nM).
[0007] Claeson et al (U.S. Pat. No. 5,574,014 and others) and
Kakkar et al (WO 92/07869 and family members including U.S. Pat.
No. 5,648,338) disclose thrombin inhibitors having a neutral
C-terminal side chain, for example an alkyl or alkoxyalkyl side
chain.
[0008] Modifications of the compounds described by Kakkar et al are
included in WO 96/25427, directed to peptidyl serine protease
inhibitors in which the P2-P1 natural peptide linkage is replaced
by another linkage. As examples of non-natural peptide linkages may
be mentioned --CO.sub.2--, --CH.sub.2O--, --NHCO--,
--CHYCH.sub.2--, --CH.dbd.CH--, --CO(CH.sub.2).sub.pCO-- where p is
1, 2 or 3, --COCHY--, --CO.sub.2--CH.sub.2NH--, --CHY--NX--,
--N(X)CH.sub.2--N(X)CO--, --CH.dbd.C(CN)CO--, --CH(OH)--NH--,
--CH(CN)--NH--, --CH(OH)--CH.sub.2-- or --NH--CHOH--, where X is H
or an amino protecting group and Y is H or halogen, especially F.
Particular non-natural peptide linkages are --CO.sub.2-- or
--CH.sub.2O--.
[0009] Metternich (EP 471651 and U.S. Pat. No. 5,288,707, the
latter being assigned to Trigen Limited) discloses variants of
Phe-Pro-BoroArg boropeptides in which the P3 Phe is replaced by an
unnatural hydrophobic amino acid such as trimethylsilylalanine,
p-tert.butyl-diphenyl-silyloxym- ethylphenylalanine or
p-hydroxymethylphenylalanine and the P1 side chain may be neutral
(alkoxyalkyl, alkylthioalkyl or trimethylsilylalkyl).
[0010] The replacement of the P2 Pro residue of borotripeptide
thrombin inhibitors by an N-substituted glycine is described in
Fevig J M et al Bioorg. Med. Chem. 8: 301-306 and Rupin A et al
Thromb. Haemost. 78(4): 1221-1227, 1997. See also U.S. Pat. No.
5,585,360 (de Nanteuil et al).
[0011] Amparo (WO 96/20698 and family members including U.S. Pat.
No. 5,698,538) discloses peptidomimetics of the structure
Aryl-linker-Boro(Aa), where Boro(Aa) may be an aminoboronate
residue with a non-basic side chain, for example BoroMpg. The
linker is of the formula --(CH.sub.2).sub.mCONR-- (where m is 0 to
8 and R is H or certain organic groups) or analogues thereof in
which the peptide linkage --CONR-- is replaced by --CSNR--,
--SO.sub.2NR--, --CO.sub.2--, --C(S)O-- or --SO.sub.2O--. Aryl is
phenyl, naphthyl or biphenyl substituted by one, two or three
moieties selected from a specified group. Most typically these
compounds are of the structure
Aryl-(CH.sub.2).sub.n--CONH--CHR.sup- .2--BY.sup.1.gamma.2, where
R.sup.2 is for example a neutral side chain as described above and
n is 0 or 1.
[0012] Non-peptide boronates have been proposed as inhibitors of
proteolytic enzymes in detergent compositions. WO 92/19707 and WO
95/12655 report that arylboronates can be used as inhibitors of
proteolytic enzymes in detergent compositions. WO 92/19707
discloses compounds substituted meta to the boronate group by a
hydrogen bonding group, especially acetamido (--NHCOCH.sub.3),
sufonamido (--NHSO.sub.2CH.sub.3) and alkylamino. WO 95/12655
teaches that ortho-substituted compounds are superior.
[0013] Boronate enzyme inhibitors have wide application, from
detergents to bacterial sporulation inhibitors to pharmaceuticals.
In the pharmaceutical field, there is patent literature describing
boronate inhibitors of serine proteases, for example thrombin,
factor Xa, kallikrein, elastase, plasmin as well as other serine
proteases like prolyl endopeptidase and Ig AI Protease. Thrombin is
the last protease in the coagulation pathway and acts to hydrolyse
four small peptides form each molecule of fibrinogen, thus
deprotecting its polymerisation sites. Once formed, the linear
fibrin polymers may be cross-linked by factor XIIIa, which is
itself activated by thrombin. In addition, thrombin is a potent
activator of platelets, upon which it acts at specific receptors.
Thrombin also potentiates its own production by the activation of
factors V and VIII.
[0014] Other aminoboronate or peptidoboronate inhibitors or
substrates of serine proteases are described in:
[0015] U.S. Pat. No. 4,935,493
[0016] EP 341661
[0017] WO 94/25049
[0018] WO 95/09859
[0019] WO 96/12499
[0020] WO 96/20689
[0021] Lee S-L et al, Biochemistry 36:13180-13186, 1997
[0022] Dominguez C et al, Bioorg. Med. Chem. Lett. 7:79-84,
1997
[0023] EP 471651
[0024] WO 94/20526
[0025] WO 95/20603
[0026] WO97/05161
[0027] U.S. Pat. No. 4,450,105
[0028] U.S. Pat. No. 5,106,948
[0029] U.S. Pat. No. 5,169,841.
[0030] Peptide boronic acid inhibitors of hepatic C virus protease
are described in WO 01/02424.
[0031] Matteson D S Chem. Rev. 89: 1535-1551, 1989 reviews the use
of .alpha.-halo boronic esters as intermediates for the synthesis
of inter alia amino boronic acids and their derivatives. Matteson
describes the use of pinacol boronic esters in non-chiral synthesis
and the use of pinanediol boronic esters for chiral control,
including in the synthesis of amino and amido boronate esters.
[0032] Contreras et al J. Organomet Chem. 246: 213-217, 1983
describe how intramolecular N.fwdarw.B coordination was
demonstrated by spectroscopic studies on cyclic boronic esters
prepared by reacting Me.sub.2CHCMe.sub.2--BH.sub.2 with
diethanolamines.
[0033] Boronic acid and ester compounds have displayed promise as
inhibitors of the proteasome, a multicatalytic protease responsible
for the majority of intracellular protein turnover. Ciechanover,
Cell, 79:13-21, 1994, teaches that the proteasome is the
proteolytic component of the ubiquitin-proteasome pathway, in which
proteins are targeted for degradation by conjugation to multiple
molecules of ubiquitin. Ciechanover also teaches that the
ubiquitin-proteasome pathway plays a key role in a variety of
important physiological processes.
[0034] Adams et al, U.S. Pat. No. 5,780,454 (1998), U.S. Pat. No.
6,066,730 (2000), U.S. Pat. No. 6,083,903 (2000) and equivalent WO
96/13266, and U.S. Pat. No. 6,297,217 (2001) describe peptide
boronic ester and acid compounds useful as proteasome inhibitors.
These documents also describe the use of boronic ester and acid
compounds to reduce the rate of muscle protein degradation, to
reduce the activity of NF-.kappa.B in a cell, to reduce the rate of
degradation of p53 protein in a cell, to inhibit cyclin degradation
in a cell, to inhibit the growth of a cancer cell, to inhibit
antigen presentation in a cell, to inhibit NF-.kappa.B dependent
cell adhesion, and to inhibit HIV replication. Brand et al, WO
98/35691, teaches that proteasome inhibitors, including boronic
acid compounds, are useful for treating infarcts such as occur
during stroke or myocardial infarction. Elliott et al, WO 99/15183,
teaches that proteasome inhibitors are useful for treating
inflammatory and autoimmune diseases.
[0035] Unfortunately, organoboronic acids can be relatively
difficult to obtain in analytically pure form. Thus, alkylboronic
acids and their boroxines are often air-sensitive. Korcek et al, J.
Chem. Soc. Perkin Trans. 2:242, 1972, teaches that butylboronic
acid is readily oxidized by air to generate 1-butanol and boric
acid.
[0036] It is known that derivatisation of boronic acids as cyclic
esters provides oxidation resistance. For example, Martichonok V et
al J. Am. Chem. Soc. 118: 950-958, 1996 state that diethanolamine
derivatisation provides protection against possible boronic add
oxidation. U.S. Pat. No. 5,681,978 (Matteson D S et al) teaches
that 1,2-diols and 1,3 diols, for example pinacol, form stable
cyclic boronic esters that are not easily oxidised.
[0037] Wu et al, J. Pharm. Sci., 89:758-765, 2000, discuss the
stability of the compound N-(2-pyrazine)
carbonyl-phenylalanine-leucine boronic add (LDP-341, also known as
bortezomib), an anti-cancer agent. It is described how "during an
effort to formulate [LDP-341] for parenteral administration, the
compound showed erratic stability behaviour". The degradation
pathways were investigated and it was concluded that the
degradation was oxidative, the initial oxidation being attributed
to peroxides or molecular oxygen and its radicals.
[0038] WO 02/059131 discloses boronic acid products which are
described as stable. In particular, these products are certain
boropeptides and/or boropeptidomimetics in which the boronic acid
group has been derivatised with a sugar. The disclosed sugar
derivatives, which have hydrophobic amino acid side chains, are of
the formula 1
[0039] wherein:
[0040] P is hydrogen or an amino-group protecting moiety;
[0041] R is hydrogen or alkyl;
[0042] A is 0, 1 or 2;
[0043] R.sup.1, R.sup.2 and R.sup.3 are independently hydrogen,
alkyl, cycloalkyl, aryl or --CH.sub.2--R.sup.5;
[0044] R.sup.5, in each instance, is one of aryl, aralkyl, alkaryl,
cycloalkyl, heterocyclyl, heteroaryl, or --W--R.sup.6, where W is a
chalcogen and R.sup.6 is alkyl;
[0045] where the ring portion of any of said aryl, aralkyl,
alkaryl, cycloalkyl, heterocyclyl, or heteroaryl in R.sup.1,
R.sup.2, R.sup.3 or R.sup.5 can be optionally substituted; and
[0046] Z.sup.1 and Z.sup.2 together form a moiety derived from a
sugar, wherein the atom attached to boron in each case is an oxygen
atom.
[0047] Some of the disclosed compounds are sugar derivatives of
LDP-341 (see above).
[0048] Many drugs comprise an active moiety which is a carboxylic
acid. There are a number of differences between carboxylic acids
and boronic acids, whose effects on drug delivery, stability and
transport (amongst others) have not been investigated. One feature
of trivalent boron compounds is that the boron atom is sp.sup.2
hybridised, which leaves an empty 2p.sub.z orbital on the boron
atom. A molecule of the type BX.sub.3 can therefore act as an
electron-pair acceptor, or Lewis acid. It can use the empty
2p.sub.z orbital to pick up a pair of nonbonding electrons from a
Lewis base to form a covalent bond. BF.sub.3 therefore reacts with
Lewis bases such as NH.sub.3 to form acid-base complexes in which
all of the atoms have a filled shell of valence electrons.
[0049] Boric acid, accordingly, can act as a Lewis add, accepting
OH.sup.-:
B(OH).sub.3+H.sub.2O.fwdarw.B(OH).sub.4.sup.-+H.sup.+
[0050] Further, boronic acids of the type RB(OH).sub.2 are dibasic
and have two pKa's. Another point of distinction about boron
compounds is the unusually short length of bonds to boron, for
which three factors may be responsible:
[0051] 1. Formation of p.pi.-p.pi. bonds;
[0052] 2. Ionic-covalent resonance;
[0053] 3. Reduced repulsions between non-bonding electrons.
[0054] The presumed equilibria of boronic and carboxylic acids in
aqueous KOH are shown below (excluding formation of
RBO.sub.2.sup.2-): 2
[0055] Thrombosis
[0056] Hemostasis is the normal physiological condition of blood in
which its components exist in dynamic equilibrium. When the
equilibrium is disturbed, for instance following injury to a blood
vessel, certain biochemical pathways are triggered leading, in this
example, to arrest of bleeding via clot formation (coagulation).
Coagulation is a dynamic and complex process in which proteolytic
enzymes such as thrombin play a key role. Blood coagulation may
occur through either of two cascades of zymogen activations, the
extrinsic and intrinsic pathways of the coagulation cascade. Factor
VIIa in the extrinsic pathway, and Factor IXa in the intrinsic
pathway are important determinants of the activation of factor X to
factor Xa, which itself catalyzes the activation of prothrombin to
thrombin, whilst thrombin in turn catalyses the polymerization of
fibrinogen monomers to fibrin polymer. The last protease in each
pathway is therefore thrombin, which acts to hydrolyze four small
peptides (two FpA and two FpB) from each molecule of fibrinogen,
thus deprotecting its polymerization sites. Once formed, the linear
fibrin polymers may be cross-linked by factor XIIIa, which is
itself activated by thrombin. In addition, thrombin is a potent
activator of platelets, upon which it acts at specific receptors.
Thrombin activation of platelets leads to aggregation of the cells
and secretion of additional factors that further accelerate the
creation of a hemostatic plug. Thrombin also potentiates its own
production by the activation of factors V and VIII (see Hemker and
Beguin in: Jolles, et. al., "Biology and Pathology of Platelet
Vessel Wall Interactions," pp. 219-26 (1986), Crawford and Scrutton
in: Bloom and Thomas, "Haemostasis and Thrombosis," pp. 47-77,
(1987), Bevers, et. al., Eur. J. Biochem. 122:429-36, 1982, Mann,
Trends Biochem. Sci. 12:229-33, 1987).
[0057] Proteases are enzymes which cleave proteins at specific
peptide bonds. Cuypers et al., J. Biol. Chem. 257:7086, 1982, and
the references cited therein, classify proteases on a mechanistic
basis into five classes: serine, cysteinyl or thiol, acid or
aspartyl, threonine and metalloproteases. Members of each class
catalyse the hydrolysis of peptide bonds by a similar mechanism,
have similar active site amino acid residues and are susceptible to
class-specific inhibitors. For example, all serine proteases that
have been characterised have an active site serine residue.
[0058] The coagulation proteases thrombin, factor Xa, factor VIIa,
and factor IXa are serine proteases having trypsin-like specificity
for the cleavage of sequence-specific Arg-Xxx peptide bonds. As
with other serine proteases, the cleavage event begins with an
attack of the active site serine on the scissile bond of the
substrate, resulting in the formation of a tetrahedral
intermediate. This is followed by collapse of the tetrahedral
intermediate to form an acyl enzyme and release of the amino
terminus of the cleaved sequence. Hydrolysis of the acyl enzyme
then releases the carboxy terminus.
[0059] As indicated above, platelets play two important roles in
normal hemostasis. First, by aggregating, they constitute the
initial hemostatic plug which immediately curtails bleeding from
broken blood vessels. Secondly, the platelet surface can become
activated and potentiate blood clotting, a property referred to as
platelet procoagulant activity. This may be observed as an increase
in the rate of activation of prothrombin by factor Xa in the
presence of factor Va and Ca.sup.2+, referred to as the
prothrombinase reaction. Normally, there are few (if any) clotting
factors on the surface of unstimulated platelets but, when
platelets are activated, negatively charged phospholipids
(phosphatidylserine and phospatidylinositol) that are normally on
the cytoplasmic side of the membrane become available and provide a
surface on which two steps of the coagulation sequence occur. The
phospholipid on the surface of activated platelets profoundly
accelerates the reactions leading to the formation of thrombin, so
that thrombin can be generated at a rate faster than its
neutralisation by antithrombin III. The reactions that occur on the
platelet surfaces are not easily inhibited by the natural
anticoagulants in blood such as antithrombin III, either with or
without heparin. (See Kelton and Hirsch in: Bloom and Thomas,
"Haemostasis and Thrombosis," pp. 737-760, (1981); Mustard et al
in: Bloom and Thomas, "Haemostasis and Thrombosis," pp. 503526,
(1981); Goodwin et al; Biochem. J 308:15-21, 1995).
[0060] A thrombus can be considered as an abnormal product of a
normal mechanism and can be defined as a mass or deposit formed
from blood constituents on a surface of the cardiovascular system,
for example of the heart or a blood vessel. Thrombosis can be
regarded as the pathological condition wherein improper activity of
the hemostatic mechanism results in intravascular thrombus
formation. Three basic types of thrombi are recognised:
[0061] the white thrombus which is usually seen in arteries and
consists chiefly of platelets;
[0062] the red thrombus which is found in veins and is composed
predominantly of fibrin and red cells;
[0063] the mixed thrombus which is composed of components of both
white and red thrombi.
[0064] The composition of thrombi is influenced by the velocity of
blood flow at their sites of formation. In general white
platelet-rich thrombi form in high flow systems, while red
coagulation thrombi form in regions of stasis. The high shear rate
in arteries prevents the accumulation of coagulation intermediates
on the arterial side of the circulation: only platelets have the
capacity to form thrombi binding to the area of damage via von
Willebrand factor. Such thrombi composed only of platelets are not
stable and disperse. If the stimulus is strong then the thrombi
will form again and then disperse continually until the stimulus
has diminished. For the thrombus to stabilise, fibrin must form. In
this respect, small amounts of thrombin can accumulate within the
platelet thrombus and activate factor Va and stimulate the platelet
procoagulant activity. These two events lead to an overall increase
in the rate of activation of prothrombin by factor Xa of 300,000
fold. Fibrin deposition stabilises the platelet thrombus. Indirect
thrombin inhibitors, for example heparin, are not clinically
effective at inhibiting stimulation of platelet procoagulant
activity. Accordingly, a therapeutic agent which inhibits platelet
procoagulant activity would be useful for treating or preventing
arterial thrombotic conditions.
[0065] On the venous side of circulation, the thrombus is comprised
of fibrin: thrombin can accumulate because of the slower flow on
the venous side and platelets play only a minor role.
[0066] Thrombosis is thus not considered to be a single indication
but, rather, is a class of indications embracing distinct
sub-classes for which differing therapeutic agents and/or protocols
may be appropriate. Thus, regulatory authorities treat disorders
such as, for example, deep vein thrombosis, cerebrovascular
arterial thrombosis and pulmonary embolism as distinct indications
for the purposes of licensing medicines. Two main sub-classes of
thrombosis are arterial thrombosis and venous thrombosis. Arterial
thrombosis includes such specific disorders as acute coronary
syndromes [for example acute myocardial infarction (heart attack,
caused by thrombosis in a coronary artery)], cerebrovascular
arterial thrombosis (stroke, caused by thrombosis in the
cerebrovascular arterial system) and peripheral arterial
thrombosis. Examples of conditions caused by venous thrombosis are
deep vein thrombosis and pulmonary embolism.
[0067] The management of thrombosis commonly involves the use of
antiplatelet drugs (inhibitors of platelet aggregation) to control
future thrombogenesis and thrombolytic agents to lyse the newly
formed clot, either or both such agents being used in conjunction
or combination with anticoagulants. Anticoagulants are used also
preventatively (prophylactically) in the treatment of patients
thought susceptible to thrombosis.
[0068] Currently, two of the most effective classes of drugs in
clinical use as anticoagulants are the heparins and the vitamin K
antagonists. The heparins are ill-defined mixtures of sulfated
polysaccharides that bind to, and thus potentiate, the action of
antithrombin III. Antithrombin III is a naturally occurring
inhibitor of the activated clotting factors IXa, Xa, XIa, thrombin
and probably XIIa (see Jaques, Pharmacol. Rev 31:99-166, 1980). The
vitamin K antagonists, of which warfarin is the most well-known
example, act indirectly by inhibiting the post-ribosomal
carboxylations of the vitamin K dependent coagulation factors II,
VII, 1.times. and X (see Hirsch, Semin. Thromb. Hemostasis 12:1-11,
1986). While effective therapies for the treatment of thrombosis,
heparins and vitamin K antagonists have the unfortunate side
effects of bleeding, heparin-induced thrombocytopenia (in the case
of heparin) and marked interpatient variability, resulting in a
small and unpredictable therapeutic safety margin.
[0069] The use of direct acting inhibitors of thrombin and other
serine protease enzymes of the coagulation system is expected to
alleviate these problems. To that end, a wide variety of serine
protease inhibitors have been tested, including boropeptides, i.e.
peptides containing a boronic acid analogue of an .alpha.-amino
acid. Whilst direct acting boronic acid thrombin inhibitors have
been discussed earlier in this specification, they are further
described in the following section.
[0070] Neutral P1 Residue Boropeptide Thrombin Inhibitors
[0071] Claeson et al (U.S. Pat. No. 5,574,014 and others) and
Kakkar et al (WO 92/07869 and family members including U.S. Pat.
No. 5,648,338) disclose lipophilic thrombin inhibitors having a
neutral (uncharged) C-terminal (P1) side chain, for example an
alkoxyalkyl side chain.
[0072] The Claeson et al and Kakkar et al patent families disclose
boronate esters containing the amino acid sequence
D-Phe-Pro-BoroMpg [(R)-Phe-Pro-BoroMpg], which are highly specific
inhibitors of thrombin. Of these compounds may be mentioned in
particular Cbz-(R)-Phe-Pro-BoroMpg- -OPinacol (also known as TRI
50b). The corresponding free boronic acid is known as TRI 50c. For
further information relating to TRI 50b and related compounds, the
reader is referred to the following documents:
[0073] Elgendy S et al., in The Design of Synthetic Inhibitors of
Thrombin, Claeson G et al Eds, Advances in Experimental Medicine,
340:173-178, 1993.
[0074] Claeson G et al, Biochem J. 290:309-312, 1993
[0075] Tapparelli C et al, J Biol Chem, 268:4734-4741, 1993
[0076] Claeson G, in The Design of Synthetic Inhibitors of
Thrombin, Claeson G et al Eds, Advances in Experimental Medicine,
340:83-91, 1993
[0077] Phillip et al, in The Design of Synthetic Inhibitors of
Thrombin, Claeson G et al Eds, Advances in Experimental Medicine,
340:67-77, 1993
[0078] Tapparelli C et al, Trends Pharmacol. Sci. 14:366-376,
1993
[0079] Claeson G, Blood Coagulation and Fibrinolysis 5:411-436,
1994
[0080] Elgendy et al, Tetrahedron 50:3803-3812, 1994
[0081] Deadman J et al, J. Enzyme Inhibition 9:29-41, 1995
[0082] Deadman J et al, J. Medicinal Chemisty 38:1511-1522,
1995.
[0083] The tripeptide sequence of TRI 50b has three chiral centres.
The Phe residue is considered to be of (R)-configuration and the
Pro residue of natural (S)-configuration, at least in compounds
with commercially useful inhibitor activity; the Mpg residue is
believed to be of (R)-configuration in isomers with commercially
useful inhibitor activity. Thus, the active, or most active, TRI
50b stereoisomer is considered to be of R,S,R configuration and may
be represented as: 3
[0084] (RSR)-TRI 50b: Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg Pinacol
[0085] Whilst indirect acting thrombin inhibitors have been found
useful for the treatment of patients susceptible to or suffering
from venous thrombosis, the same is not true of arterial
thrombosis, because it would be necessary to raise the dosage used
in the treatment of venous thrombosis by many times in order to
treat (prevent) arterial thrombosis. Such raised dosages typically
cause bleeding, which makes indirect acting thrombin inhibitors
unsuitable or less preferable for treating arterial thrombosis.
Heparin and its low molecular weight derivatives are indirect
thrombin inhibitors, and so are unsuitable to treat arterial
thrombosis. Oral direct thrombin inhibitors are in development for
arterial indications but may have lower than desirable therapeutic
indices, i.e. may have higher than desirable levels of bleeding at
therapeutic doses.
[0086] Many organoboronic acid compounds may be classified as
lipophilic or hydrophobic. Typically, such compounds include
amongst others:
[0087] boropeptides of which all or a majority of the amino acids
are hydrophobic
[0088] boropeptides of which at least half of the amino acids are
hydrophobic and which have a hydrophobic N-terminal substituent
(amino protecting group)
[0089] non-peptides based on hydrophobic moieties.
[0090] Oral Absorption
[0091] Absorption in the gastrointestinal tract can be by an active
or a passive route. Active absorption by transport mechanisms tends
to be variable between individuals and with intestinal content
(Gustafsson et al, Thrombosis Research, 2001, 101, 171-181). The
upper intestine has been identified as the principal site of oral
drug absorption. In particular, the duodenum is the customary
target site for absorption of orally administered drugs because of
its large surface area. The intestinal mucosa acts as a barrier
that controls passive transcellular absorption: the absorption of
ionic species is blocked whilst the transcellular absorption of
lipophilic molecules is favoured (Palm K et al., J. Pharmacol and
Exp. Therapeutics, 1999, 291, 435-443).
[0092] Orally administered drugs are required to be consistently
and adequately absorbed. Variability of absorption between
individuals or between different occasions in the same individual
is unwelcome. Similarly, drugs which have a low level of
bioavailability (only a small portion of the administered active
agent is absorbed) are generally unacceptable.
[0093] Non-ionised compounds are favoured for passive absorption, a
route associated with invariability, and are therefore preferred
for consistent absorption. Lipophilic species are particularly
favoured by passive absorption mechanisms and, accordingly,
non-ionic, lipophilic drugs are indicated to be most favoured for
consistent and high oral absorption.
[0094] Typical functionalities required for interaction of drugs
with their physiological targets are functional groups such as
carboxylic and sulphonic acids. These groups exist as the
protonated form in the stomach (at pH 2-3), but will be ionised to
some extent at the higher pH of the intestinal fluid. One strategy
that has been used to avoid the ionisation of the carboxylates or
sulphonates is to present them as ester forms, which are cleaved
once absorbed into the vascular lumen.
[0095] For example, the direct acting thrombin inhibitor
melagatran, which has sub-optimal gastrointestinal absorption, has
terminal carboxy and amidino groups and is a pure zwitterion at pH
8-10 when the carboxylic acid and amidino groups are both charged.
A prodrug H 376/95 was therefore developed which has protecting
groups for the carboxylic acid and for the amidine and is a more
lipophilic molecule than melagatran. The prodrug has a permeability
coefficient across cultured epithelial Caco-2 cells 80 times higher
than that of melagatran and oral bioavailability 2.7-5.5 times
higher than that of melagatran as well as much smaller variability
in the area under the drug plasma concentration vs. time curve
(Gustafsson et al, Thrombosis Research, 2001, 101, 171-181).
[0096] Oral Absorption of Boropeptides, Boropeptidomimetics and
other Organoboronates
[0097] The boronate ester group of TRI 50b is rapidly cleaved in
the conditions of the plasma to form the corresponding boronic acid
group, which is considered to be the active moiety which inhibits
the catalytic site of thrombin.
[0098] Boronic acids are divalent functional groups, with
boron-oxygen bond lengths (1.6 .ANG.) more typical of single bonds,
unlike superficially comparable C--O and S--O bonds in carboxylic
and sulphonic acids. Consequently the boronic acid group has two
ionisation potentials. The boronic acid group will be partly
ionised at pH's of the duodenal fluid and not suited to the desired
passive duodenal uptake. Thus, a charged boronate inhibitor
H-D-PheProBoroArg is absorbed by a predominantly active transport
mechanism (Saitoh, H. and Aungst, B. J., Pharm. Res., 1999, 16,
1786-1789).
[0099] The peptide boronic acid formed by such cleavage of TRI 50b
(the acid is designated TRI 50c) is relatively insoluble in water,
especially at acidic or neutral pH, and tends to be poorly absorbed
in the stomach and duodenum. The acid has the structure
Cbz-Phe-Pro-BoroMpg-OH.
[0100] Whereas the peptide boronic acid Cbz-Phe-Pro-BoroMpg-OH is
partly ionised under duodenal conditions and, to that extent,
unfavoured for passive transport, esters of the acid are designed
for a high rate of passive (thus consistent) transport. The
tripeptide sequence Phe-Pro-Mpg has a non-basic P1 side chain
(specifically, methoxypropyl), such that the tripeptide consists of
three non-polar amino acids. The esters of the peptide boronic acid
are non-ionisable and the ester-forming species further impart
lipophilic properties, so encouraging a high rate of passive
transport.
[0101] Computational techniques have confirmed that TRI 50b and
other diol esters of Cbz-Phe-Pro-BoroMpg-OH can be predicted to
have good bioavailability. Thus, polar surface area (PSAd) is a
parameter predictive of bioavailability and PSAd values of greater
than 60 .ANG. correlate well with passive transcellular transport
and with bioavailability of known drugs (Kelder, J. Pharm. Res.,
1999, 16, 1514-1519). Measurements for diol esters of the above
peptide boronic acid, including the pinacol ester TRI 50b, show
that the diol esters have PSAd values well above 60 .ANG.,
predictive of passive transport and good bioavailability as shown
in Table 1:
1TABLE 1 PSAd values of selected diol esters of
Cbz-Phe-Pro-BoroMpg-OH Diol PSAd Value Pinacol 98.74 Pinanediol
90.64
[0102] The corresponding monohydroxy alcohol (e.g. alkanol) esters
were considered too unstable, spontaneously cleaving to liberate
the acid in-vitro. Esters of diols such as pinanediol and pinacol
have enhanced kinetic stability over esters of monohydroxy
alcohols, in that after partial hydrolysis to the mono-ester
derivative they will tend to reassociate by a facile
intra-molecular reaction.
BRIEF SUMMARY OF THE DISCLOSURE
[0103] To counterbalance these highly desirable features of TRI
50b, it has been discovered that TRI 50b tends to hydrolyse. Thus
in the acid conditions of an HPLC assay, TRI 50b is converted to
the acid form with a short half life, which implies potential
intraduodenal hydrolysis in the duodenum and elsewhere in the
gastro-intestinal tract into ionic species which would resist
passive transport and, if anything, be absorbed by active
transport, indicative at best of variable bioavailability.
[0104] The instability of TRI 50b to hydrolysis also presents
potential disadvantages in preparation of the compound and its
formulation, as well as in the storage of pharmaceutical
formulations containing it.
[0105] Another challenging difficulty which has been posed by TRI
50b is that the data show significant variation in bioavailability
between subjects. Such variability can make a drug candidate
unacceptable and it would therefore be desirable to reduce the
observed variability.
[0106] An ideal solution to the instability of TRI 50b would be
development of a diol ester more stable to hydrolysis, as such a
diol ester like TRI 50b can be predicted to be oxidation resistant
as compared with TRI 50c. In this regard, it is known that ring
size can affect boronate stability and glycolato boron has been
shown to have enhanced aqueous stability compared to pinacol (D. S.
Matteson, Stereodirected Synthesis with Organoboranes,
Springer-Verlag, 1995, ch. 1). Similarly, the pinanediol ester is
more stable than the pinacol; this is believed to be because the
pinanediol group is highly sterically hindered and disfavours
nucleophilic attack on the boron. In fact transesterification from
pinacol to pinanediol has been reported (Brosz, C S, Tet Assym,
8:1435-1440, 1997) whereas the reverse process is unfavourable. The
pinanediol ester however is considered too slow to cleave in plasma
and there remains a need to provide an improved diol ester.
[0107] Another solution to the instability of TRI 50b would be to
administer in its place TRI 50c. However, TRI 50c data suggest that
TRI 50c too suffers from variability in bioavailability.
[0108] TRI 50c suffers further from instability, in that there is a
problematic tendency for the boropeptide moiety itself to degrade
via de-boronation (carbon-boron bond cleavage), by a pathway
previously considered to be oxidative as taught by the literature
(e.g. Wu et al, discussed above). The level of degradation can be
remarkably high.
[0109] The properties discussed above of TRI 50b and TRI 50c will
not be restricted to such compounds but will be shared by other
boropeptide esters and acids, even if the properties of such other
boropeptides differ quantitatively.
[0110] The present disclosure is predicated on, amongst other
things, the finding that certain organoboronic acid products are
indicated to be of enhanced stability.
[0111] The benefits of the present disclosure include a solution to
the problem of boronate diol ester and especially TRI 50b
instability, that is to say the presently disclosed products
provide inter alia pharmacologically active compounds which are
more stable than TRI 50b and other comparable esters in the sense
of stability to hydrolysis. The disclosure further includes a
solution to the problem of organoboronic acid instability, that is
to say the presently disclosed products provide inter alia
pharmacologically active compounds which are more stable to
deboronation than TRI 50c. The stability provided within the
framework of the disclosure is not absolute but is improved
relative to the comparator compounds. The benefits offered by the
disclosure further include the provision of unexpected products
which have usefulness in oral formulations.
[0112] There is disclosed an amino boronic acid derivative which
avoids the disadvantages of pinacol esters. The disclosure further
includes a peptide boronic acid derivative which is indicated to be
of enhanced stability. In particular, the disclosure includes
amongst other subject a pharmaceutical formulation which comprises
an amino boronic acid derivative and which can provide in the
duodenum an ionic boropeptide species whilst avoiding the
disadvantages of pinacol esters, and can provide bioavailability.
Further included are boronic acid derivatives which are of relative
stability to hydrolysis and deboronation and are useful in oral
formulations for inhibiting thrombin.
[0113] The disclosure concerns a pharmaceutically acceptable base
addition salt of certain organoboronic acid drugs, specifically
hydrophobic boropeptides (e.g di- or tri-peptides), and more
specifically thrombin inhibitors having a non-basic P1 group. As a
class, such salts are not only contrary to the direction of the
prior art but additionally have an improved level of stability
which cannot be explained or predicted on the basis of known
chemistry.
[0114] In one aspect, the disclosure relates to pharmaceutically
acceptable base addition salts of boronic acids which have a
neutral aminoboronic acid residue capable of binding to the
thrombin S1 subsite linked through a peptide linkage to a
hydrophobic moiety capable of binding to the thrombin S2 and S3
subsites. In a first embodiment, there is disclosed a
pharmaceutically acceptable base addition salt of a boronic acid
of, for example, formula (I): 4
[0115] wherein
[0116] Y comprises a hydrophobic moiety which, together with the
aminoboronic acid residue --NHCH(R.sup.9)--B(OH).sub.2, has
affinity for the substrate binding site of thrombin; and
[0117] R.sup.9 is a straight chain alkyl group interrupted by one
or more ether linkages (e.g. 1 or 2) and in which the total number
of oxygen and carbon atoms is 3, 4, 5 or 6 (e.g. 5) or R.sup.9 is
--(CH.sub.2).sub.m--W where m is 2, 3, 4 or 5 (e.g. 4) and W is
--OH or halogen (F, Cl, Br or I). R.sup.9 is an alkoxyalkyl group
in one subset of compounds, e.g. alkoxyalkyl containing 4 carbon
atoms.
[0118] The disclosure comprises pharmaceutically acceptable base
addition salts of hydrophobic boronic acid inhibitors of thrombin,
and therefore includes such salts of peptide boronic acids which
have a partition coefficient between 1-n-octanol and water
expressed as log P of greater than 1.0 at physiological pH and
25.degree. C. Some peptide boronic acids useful in the disclosure
have a partition coefficient of at least 1.5. A class of
hydrophobic peptide boronic acids useful in the disclosure has a
partition coefficient of no more than 5.
[0119] Disclosed as certain examples are pharmaceutically
acceptable base addition salts of hydrophobic boronic acid
inhibitors of thrombin. Such inhibitors may contain hydrophobic
amino acids, and this class of amino acids includes those whose
side chain is hydrocarbyl, hydrocarbyl containing an in-chain
oxygen and/or linked to the remainder of the molecule by an
in-chain oxygen or heteroaryl, or any of the aforesaid groups when
substituted by hydroxy, halogen or trifluoromethyl. Representative
hydrophobic side chains include alkyl, alkoxyalkyl, either of the
aforesaid when substituted by at least one aryl or heteroaryl,
aryl, heteroaryl, aryl substituted by at least one alkyl and
heteroaryl substituted by at least one alkyl. Proline and other
imino acids which are ring-substituted by nothing or by one of the
moieties listed in the previous sentence are also hydrophobic.
[0120] Some hydrophobic side chains contain from 1 to 20 carbon
atoms, e.g. non-cyclic moieties having 1, 2, 3 or 4 carbon atoms.
Side chains comprising a cyclic group typically but not necessarily
contain from 5 to 13 ring members and in many cases are phenyl or
alkyl substituted by one or two phenyls.
[0121] Hydrophobic non-peptides are typically based on moieties
which may form a side chain of a hydrophobic amino add, as
described above.
[0122] Hydrophobic compounds may contain, for example, one amino
group and/or one acid group (e.g. --COOH, --B(OH).sub.2).
Generally, they do not contain multiple polar groups of any one
type.
[0123] One class of hydrophobic organoboronic acids have a
partition coefficient between 1-n-octanol and water expressed as
log P of greater than 1 at physiological pH and 25.degree. C. For
example, TRI 50c has a partition coefficient of approximately
2.
[0124] Some sub-classes of hydrophobic organoboronic acids are
those described by Formulae (I) and (III)) below, under the heading
"Detailed Description of Several Examples".
[0125] Also disclosed as another embodiment is a pharmaceutically
acceptable base addition salt of a 5
[0126] peptide boronic acid of formula (II): where:
[0127] X is a moiety bonded to the N-terminal amino group and may
be H to form NH.sub.2. The identity of X is not critical but may be
a particular X moiety described above. In one example there may be
mentioned benzyloxycarbonyl.
[0128] aa.sup.1 is an amino acid having a hydrocarbyl side chain
containing no more than 20 carbon atoms (e.g. up to 15 and
optionally up to 13 C atoms) and comprising at least one cyclic
group having up to 13 carbon atoms. In certain examples, the cyclic
group(s) of aa.sup.1 have/has 5 or 6 ring members. For instance,
the cyclic group(s) of aa.sup.1 may be aryl groups, particularly
phenyl. Typically, there are one or two cyclic groups in the
aa.sup.1 side chain. Certain side chains comprise, or consist of,
methyl substituted by one or two 5- or 6-membered rings.
[0129] More particularly, aa.sup.1 is Phe, Dpa or a wholly or
partially hydrogenated analogue thereof. The wholly hydrogenated
analogues are Cha and The disclosure therefore includes medicaments
comprising salts, e.g. metal salts, of organoboronic acids which
are thrombin inhibitors, particularly selective thrombin
inhibitors, having a neutral P1 (S1-binding) moiety. For more
information about moieties which bind to the S3, S2 and S1 sites of
thrombin, see for example Tapparelli C et al, Trends Pharmacol.
Sci. 14: 366-376, 1993; Sanderson P et al, Current Medicinal
Chemistry, 5: 289-304, 1998; Rewinkel 3 et al, Current
Pharmaceutical Design, 5:1043-1075, 1999; and Coburn C Exp. Opin.
Ther. Patents 11(5): 721-738, 2001. The thrombin inhibitory salts
of the disclosure are not limited to those having S3, S2 and S1
affinity groups described in the publications listed in the
preceding sentence.
[0130] Dcha.
[0131] aa.sup.2 is an imino acid having from 4 to 6 ring members.
Alternatively, aa.sup.2 is Gly N-substituted by a C.sub.3-C.sub.13
hydrocarbyl group, e.g. a C.sub.3-C.sub.8 hydrocarbyl group
comprising a C.sub.3-C.sub.6 hydrocarbyl ring; the hydrocarbyl
group may be saturated, for example exemplary N-substituents are
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. As a
hydrocarbyl group containing one or more unsaturated bonds may be
mentioned phenyl and methyl or ethyl substituted by phenyl, e.g.
2-phenylethyl, as well as .beta.,.beta.-dialkylphenylethy- l.
[0132] There is a debate in the literature as to whether boronates
in aqueous solution form the `trigonal` B(OH).sub.2 or
`tetrahedral` B(OH).sub.3.sup.- boron species, but NMR evidence
seems to indicate that at a pH below the first pKa of the boronic
acid the main boron species is the neutral B(OH).sub.2. In the
duodenum the pH is likely to be between 6 and 7, so the trigonal
species is likely to be predominant here. In any event, the symbol
--B(OH).sub.2 includes tetrahedral as well as trigonal boron
species, and throughout this specification symbols indicating
trigonal boron species embrace also tetrahedral species. The symbol
further may further include boronic groups in anhydride form.
[0133] The salts may be in the form of solvates, particularly
hydrates.
[0134] The salts may comprise, or consist essentially of, acid
salts in which the boronic acid is singly deprotonated. The
disclosure therefore includes products having a metal/boronate
stoichiometry consistent with the boronate groups in the product
predominantly (more than 50 mol %) carrying a single negative
charge.
[0135] Oral formulations of the salts are also provided herein. In
particular, there are provided oral formulations comprising the
salts in the solid phase, for example particulate salts formulated
as compressed tablets or as capsules.
[0136] According to a further aspect of the present disclosure,
there is provided a method of treatment of a condition where
anti-thrombotic activity is required which method comprises oral
administration of a therapeutically effective amount of a salt of a
boronic add of formula (I) to a person suffering from, or at risk
of suffering from, such a condition.
[0137] As described further hereinafter, there are provided also
haemodialysis solutions comprising a salt of the disclosure.
[0138] The salts described herein include products obtainable by
(having the characteristics of a product obtained by) reaction of
the boronic acid with a strong base and the term "salt" herein is
to be understood accordingly. The term "salt" in relation to the
disclosed products, therefore, does not necessarily imply that the
products contain discrete cations and anions and is to be
understood as embracing products which are obtainable using a
reaction of a boronic acid and a base. The disclosure embraces
products which, to a greater or lesser extent, are in the form of a
coordination compound. The disclosure thus provides also products
obtainable by (having the characteristics of a product obtained by)
reaction of a boronic acid (I) with a strong base a well as the
therapeutic, including prophylactic, use of such products.
[0139] The present disclosure is not limited as to the method of
preparation of the salts, provided that they contain a boronate
species derived from boronic acid (I) and a counter-ion. Such
boronate species may be boronate anions in any equilibrium form
thereof. The term "equilibrium form" refers to differing forms of
the same compounds which may be represented in an equilibrium
equation (e.g. boronic acid in equilibrium with a boronic anhydride
and in equilibrium with different boronate ions). Boronates in the
solid phase may form anhydrides and the disclosed boronate salts
when in the solid phase may comprise boronate anhydrides, as a
boronic equilibrium species. It is not required that the salts be
prepared by reaction of a base containing the counter-ion and the
boronic acid (I). Further, the disclosure includes salt products
which might be regarded as indirectly prepared by such an acid/base
reaction as well as salts obtainable by (having the characteristics
of products obtained by) such indirect preparation. As examples of
possibly indirect preparation may be mentioned processes in which,
after initial recovery of the salt, it is purified and/or treated
to modify its physicochemical properties, for example to modify
solid form or hydrate form, or both.
[0140] In some embodiments, the cations of the salts are
monovalent.
[0141] In some embodiments the salts comprise anhydride species; in
others they are essentially free of anhydride species.
[0142] The salts may be in isolated form. The salts may have a
purity, e.g. as determined by the method of Example 28, of at least
about 90%, e.g. of greater than or equal to about 95%. In the case
of pharmaceutical formulations, such salt forms may be combined
with pharmaceutically acceptable diluents, excipients or
carriers.
[0143] The disclosure includes a method for preparing the salts
from the corresponding boronic acid as an intermediate, as well as
the intermediate boronic acid of Formula (I) and a method for
preparing it.
[0144] Further aspects and embodiments of the disclosure are set
forth in the following description and claims.
[0145] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of the words, for
example "comprising" and "comprises", mean "including but not
limited to", and are not intended to (and do not) exclude other
moieties, additives, components, integers or steps.
[0146] This patent application contains data indicating that the
stability (resistance to deboronation) of organoboronic acids may
be increased by providing them in the form of salts, e.g. metal
salts. In single experiments, the ammonium salt of TRI 50c appeared
to decompose on drying to yield ammonia, whilst the choline salt
demonstrated rapid decomposition to a deboronated impurity.
Although experiments have not been conducted to reproduce these
unrepeated observations, there is provided a sub-class in which the
ammonium and choline salts are excluded. The salt may be an acid
salt. In any event, this stabilisation technique forms part of the
disclosure and is applicable, inter alia, to organoboronic acids
described under the heading "BACKGROUND" and to organoboronic acids
described in publications mentioned under that heading.
BRIEF DESCRIPTION OF THE DRAWINGS
[0147] FIG. 1 is a plot referred to in Example 32, showing oral
phase clearance and kinetics following p.o. dosing with TRI 50b or
TRI 50c.
[0148] FIG. 2 is a second plot referred to in Example 32, showing
oral phase clearance and kinetics following intraduodenal dosing
with TRI 50b or TRI 50c.
DETAILED DESCRIPTION OF SEVERAL EXAMPLES
[0149] Glossary
[0150] The following terms and abbreviations are used in this
specification:
[0151] The expression "acid salt" as applied to a salt of a boronic
acid refers to salts of which a single --OH group of the
trigonally-represented acid group --B(OH).sub.2 is deprotonated.
Thus salts wherein the boronate group carries a single negative
charge and may be represented as --B(OH)(O.sup.-) or as
[--B(OH).sub.3].sup.- are add salts. The expression encompasses
salts of a cation having a valency n wherein the molar ratio of
boronic acid to cation is approximately n to 1. In practical terms,
the observed stoichiometry is unlikely to be exactly n:1 but will
be consistent with a notional n:1 stoichiometry. For example, the
observed mass of the cation might vary from the calculated mass for
a n:1 stoichiometry by no more than about 10%, e.g. no more than
about 7.5%; in some cases an observed mass of a cation might vary
from the calculated mass by no more than about 1%. Calculated
masses are suitably based on the trigonal form of the boronate. (At
an atomic level, a salt stoichiometrically consistent with being an
acid salt might contain boronates in a mix of protonation states,
whose average approximates to single deprotonation and such "mixed"
salts are included in the term "acid salt"). Examples of acid salts
are monosodium salts and hemizinc salts.
[0152] .alpha.-Aminoboronic acid or Boro(aa) refers to an amino
acid in which the CO.sub.2 group has been replaced by BO.sub.2.
[0153] The term "amino-group protecting moiety" refers to any group
used to derivatise an amino group, especially an N-terminal amino
group of a peptide or amino acid. Such groups include, without
limitation, alkyl, acyl, alkoxycarbonyl, aminocarbonyl, and
sulfonyl moieties. However, the term "amino-group protecting
moiety" is not intended to be limited to those particular
protecting groups that are commonly employed in organic synthesis,
nor is it intended to be limited to groups that are readily
cleavable.
[0154] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings or
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0155] The expression "thrombin inhibitor" refers to a product
which, within the scope of sound pharmacological judgement, is
potentially or actually pharmaceutically useful as an inhibitor of
thrombin, and includes reference to substance which comprises a
pharmaceutically active species and is described, promoted or
authorised as a thrombin inhibitor. Such thrombin inhibitors may be
selective, that is they are regarded, within the scope of sound
pharmacological judgement, as selective towards thrombin in
contrast to other proteases; the term "selective thrombin
inhibitor" includes reference to substance which comprises a
pharmaceutically active species and is described, promoted or
authorised as a selective thrombin inhibitor.
[0156] The term "heteroaryl" refers to a ring system which has at
least one (e.g. 1, 2 or 3) in-ring heteroatoms and has a conjugated
in-ring double bond system. The term "heteroatom" includes oxygen,
sulfur and nitrogen, of which sulfur is sometimes less
preferred.
[0157] "Natural amino acid" means an L-amino acid (or residue
thereof) selected from the following group of neutral (hydrophobic
or polar), positively charged and negatively charged amino
acids:
[0158] Hydrophobic Amino Acids
[0159] A=Ala=alanine
[0160] V=Val=valine
[0161] I=Ile=isoleucine
[0162] L=Leu=leucine
[0163] M=Met=methionine
[0164] F=Phe=phenylalanine
[0165] P=Pro=proline
[0166] W=Trp=tryptophan
[0167] Polar (Neutral or Uncharged) Amino Acids
[0168] N=Asn=asparagine
[0169] C=Cys=cysteine
[0170] Q=Gln=glutamine
[0171] G=Gly=glycine
[0172] S=Ser=serine
[0173] T=Thr=threonine
[0174] Y=Tyr=tyrosine
[0175] Positively Charged (Basic) Amino Acids
[0176] R=Arg=arginine
[0177] H=His=histidine
[0178] K=Lys=lysine
[0179] Negatively Charged Amino Acids
[0180] D=Asp=aspartic acid
[0181] E=Glu=glutamic acid.
[0182] ACN=acetonitrile
[0183] Amino acid=.alpha.-amino acid
[0184] Base addition salt=a salt which is prepared from addition of
an inorganic base or an organic base to a free acid (in this case
the boronic acid).
[0185] Cbz=benzyloxycarbonyl
[0186] Cha=cyclohexylalanine (a hydrophobic unnatural amino
add)
[0187] Charged (as applied to drugs or fragments of drug molecules,
e.g. amino acid residues)=carrying a charge at physiological pH, as
in the case of an amino, amidino or carboxy group
[0188] Dcha=dicyclohexylalanine (a hydrophobic unnatural amino
acid)
[0189] Dpa=diphenylalanine (a hydrophobic unnatural amino acid)
[0190] Drug=a pharmaceutically useful substance, whether the active
in vivo principle or a prodrug
[0191] i.v.=intravenous
[0192] Mpg=3-methoxypropylglycine (a hydrophobic unnatural amino
acid)
[0193] Multivalent=valency of at least two, for example two or
three
[0194] Neutral (as applied to drugs or fragments of drug molecules,
e.g. amino acid residues)=uncharged=not carrying a charge at
physiological pH
[0195] Pinac=Pinacol=2,3-dimethyl-2,3-butanediol
[0196] Pinanediol=2,3-pinanediol=2,6,6-trimethylbicyclo
[3.1.1]heptane-2,3-diol
[0197] Pip=pipecolinic acid
[0198] p.o.=per os=by way of the mouth (thus an oral formulation is
administered p.o.)
[0199] s.c.=subcutaneous
[0200] Strong base=a base having a sufficiently high pKb to react
with a boronic acid. Suitably such bases have a pKb of 7 or more,
e.g. 7.5 or more, for example about 8 or more
[0201] THF=tetrahydrofuran
[0202] Thr=thrombin
[0203] The Compounds
[0204] The products of the disclosure comprise salts of boronic
acids which have a neutral aminoboronic acid residue capable of
binding to the thrombin S1 subsite linked through a peptide linkage
to a hydrophobic moiety capable of binding to the thrombin S2 and
S3 subsites. The disclosure includes salts of acids of formula (I):
6
[0205] wherein
[0206] Y comprises a hydrophobic moiety which, together with the
aminoboronic acid residue --NHCH(R.sup.9)--B(OH).sub.2, has
affinity for the substrate binding site of thrombin; and
[0207] R.sup.9 is a straight chain alkyl group interrupted by one
or more ether linkages and in which the total number of oxygen and
carbon atoms is 3, 4, 5 or 6 (e.g. 5) or R.sup.9 is
--(CH.sub.2).sub.m--W where m is from 2, 3, 4 or 5 (e.g. 4) and W
is --OH or halogen (F, Cl, Br or I). As examples of straight chain
alkyl interrupted by one or more ether linkages (--O--) may be
mentioned alkoxyalkyl (one interruption) and alkoxyalkoxyalkyl (two
interruptions). R.sup.9 is an alkoxyalkyl group in one subset of
compounds, e.g. alkoxyalkyl containing 4 carbon atoms.
[0208] Typically, YCO-- comprises an amino acid residue (whether
natural or unnatural) which binds to the S2 subsite of thrombin,
the amino acid residue being N-terminally linked to a moiety which
binds the S3 subsite of thrombin.
[0209] In one class of Formula (I) acids, YCO-- is an optionally
N-terminally protected dipeptide residue which binds to the S3 and
S2 binding sites of thrombin and the peptide linkages in the acid
are optionally and independently N-substituted by a
C.sub.1-C.sub.13 hydrocarbyl group optionally containing in-chain
and/or in-ring nitrogen, oxygen or sulfur and optionally
substituted by a substituent selected from halo, hydroxy and
trifluoromethyl. The N-terminal protecting group, when present, may
be a group X as defined above (other than hydrogen). Normally, the
acid contains no N-substituted peptide linkages; where there is an
N-substituted peptide linkage, the substituent is often 1C to 6C
hydrocarbyl, e.g. saturated hydrocarbyl; the N-substituent
comprises a ring in some embodiments, e.g. cycloalkyl, and may be
cyclopentyl, for example. One class of acids has an N-terminal
protecting group (e.g. an X group) and unsubstituted peptide
linkages.
[0210] Where YCO-- is a dipeptide residue (whether or not
N-terminally protected), the S3-binding amino acid residue may be
of R configuration and/or the S2-binding residue may of S
configuration. The fragment --NHCH(R.sup.9)--B(OH) may of R
configuration. The disclosure is not restricted to chiral centres
of these conformations, however.
[0211] In one class of compounds, the side chain of P3 (S3-binding)
amino acid and/or the P2 (S2-binding) amino acid is a moiety other
than hydrogen selected from a group of formula A or B:
--(CO).sub.a--(CH.sub.2).sub.b-D.sub.c-(CH.sub.2).sub.d-E (A)
--(CO).sub.a--(CH.sub.2).sub.b-D.sub.c-C.sub.e(E.sup.1)(E.sup.2)(E.sup.3)
(B)
[0212] wherein
[0213] a is 0 or 1;
[0214] e is 1;
[0215] b and d are independently 0 or an integer such that (b+d) is
from 0 to 4 or, as the case may be, (b+e) is from 1 to 4;
[0216] c is 0 or 1;
[0217] D is O or S;
[0218] E is H, C.sub.1-C.sub.6 alkyl, or a saturated or unsaturated
cyclic group which normally contains up to 14 members and
particularly is a 5-6 membered ring (e.g. phenyl) or an 8-14
membered fused ring system (e.g. naphthyl), which alkyl or cyclic
group is optionally substituted by up to 3 groups (e.g. 1 group)
independently selected from C.sub.1-C.sub.6 trialkylsilyl, --CN,
--R.sup.13, --R.sup.12OR.sup.13, --R.sup.12COR.sup.13,
--R.sup.12CO.sub.2R.sup.13 and --R.sup.12O.sub.2CR.sup.13, wherein
R.sup.12 is --(CH.sub.2).sub.f-- and R.sup.13 is
--(CH.sub.2).sub.gH or by a moiety whose non-hydrogen atoms consist
of carbon atoms and in-ring heteroatoms and number from 5 to 14 and
which contains a ring system (e.g. an aryl group) and optionally an
alkyl and/or alkylene group, wherein f and g are each independently
from 0 to 10, g particularly being at least 1 (although --OH may
also be mentioned as a substituent), provided that (f+g) does not
exceed 10, more particularly does not exceed 6 and most
particularly is 1, 2, 3 or 4, and provided that there is only a
single substituent if the substituent is a said moiety containing a
ring system, or E is C.sub.1-C.sub.6 trialkylsilyl; and E.sup.1,
E.sup.2 and E.sup.3 are each independently selected from --R.sup.15
and -J-R.sup.15, where J is a 5-6 membered ring and R.sup.15 is
selected from C.sub.1-C.sub.6 trialkylsilyl, --CN, --R.sup.13,
--R.sup.12OR.sup.13, --R.sup.12COR.sup.13,
--R.sup.12CO.sub.2R.sup.13, --R.sup.12O.sub.2CR.sup.13, and one or
two halogens (e.g. in the latter case to form a -J-R.sup.15 moiety
which is dichlorophenyl), where R.sup.12 and R.sup.13 are,
respectively, an R.sup.12 moiety and an R.sup.13 moiety as defined
above (in some acids where E.sup.1, E.sup.2 and E.sup.3 contain an
R.sup.13 group, g is 0 or 1);
[0219] in which moiety of Formula (A) or (B) any ring is
carbocyclic or aromatic, or both, and any one or more hydrogen
atoms bonded to a carbon atom is optionally replaced by halogen,
especially F.
[0220] In certain examples, a is 0. If a is 1, c may be 0. In
particular examples, (a+b+c+d) and (a+b+c+e) are no more than 4 and
are more especially 1, 2 or 3. (a+b+c+d) may be 0.
[0221] Exemplary groups for E, E.sup.1, E.sup.2 and E.sup.3 include
aromatic rings such as phenyl, naphthyl, pyridyl, quinolinyl and
furanyl, for example; non-aromatic unsaturated rings, for example
cyclohexenyl; saturated rings such as cyclohexyl, for example. E
may be a fused ring system containing both aromatic and
non-aromatic rings, for example fluorenyl. One class of E, E.sup.1,
E.sup.2 and E.sup.3 groups are aromatic (including heteroaromatic)
rings, especially 6-membered aromatic rings. In some compounds,
E.sup.1 is H whilst E.sup.2 and E.sup.3 are not H; in those
compounds, examples of E.sup.2 and E.sup.3 groups are phenyl
(substituted or unsubstituted) and C.sub.1-C.sub.4 alkyl, e.g.
methyl.
[0222] In one class of embodiments, E contains a substituent which
is C.sub.1-C.sub.6 alkyl, (C.sub.1-C.sub.5 alkyl)carbonyl, carboxy
C.sub.1-C.sub.5 alkyl, aryl (including heteroaryl), especially
5-membered or preferably 6-membered aryl (e.g. phenyl or pyridyl),
or arylalkyl (e.g. arylmethyl or arylethyl where aryl may be
heterocyclic and is preferably 6-membered).
[0223] In another class of embodiments, E contains a substituent
which is OR.sup.13, wherein R.sup.13 can be a 6-membered ring,
which may be aromatic (e.g. phenyl) or is alkyl (e.g. methyl or
ethyl) substituted by such a 6-membered ring.
[0224] A class of moieties of formula A or B are those in which E
is a 6-membered aromatic ring optionally substituted, particularly
at the 2-position or 4-position, by --R.sup.13 or --OR.sup.13.
[0225] The disclosure includes salts in which the P3 and/or P2 side
chain comprises a cyclic group in which 1 or 2 hydrogens have been
replaced by halogen, e.g. F or Cl.
[0226] The disclosure includes a class of salts in which the side
chains of formula (A) or (B) are of the following formulae (C), (D)
or (E): 7
[0227] wherein q is from 0 to 5, e.g. is 0, 1 or 2, and each T is
independently hydrogen, halogen (e.g. F or Cl), --SiMe.sub.3,
--R.sup.13, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13 or
--O.sub.2CR.sup.13. In some embodiments of structures (D) and (E),
T is at the 4-position of the phenyl group(s) and is --R.sup.13,
--OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13 or
--O.sub.2CR.sup.13, and R.sup.13 is C.sub.1-C.sub.10 alkyl and more
particularly C.sub.1-C.sub.6 alkyl. In one sub-class, T is
--R.sup.13 or --OR.sup.13, for example in which f and g are each
independently 0, 1, 2 or 3; in some side chains groups of this
sub-class, T is --R.sup.12OR.sup.13 and R.sup.13 is H.
[0228] In one class of the moieties, the side chain is of formula
(C) and each T is independently R.sup.13 or OR.sup.13 and R.sup.13
is C.sub.1-C.sub.4 alkyl. In some of these compounds, R.sup.13 is
branched alkyl and in others it is straight chain. In some
moieties, the number of carbon atoms is from 1 to 4.
[0229] In many dipeptide fragments YCO-- (which dipeptides may be
N-terminally protected or not), the P3 amino acid has a side chain
of formula (A) or (B) as described above and the P2 residue is of
an imino acid.
[0230] The disclosure therefore includes medicaments comprising
salts, e.g. metal salts, of organoboronic acids which are thrombin
inhibitors, particularly selective thrombin inhibitors, having a
neutral P1 (S-binding) moiety. For more information about moieties
which bind to the S3, S2 and S1 sites of thrombin, see for example
Tapparelli C et al, Trends Pharmacol. Sci. 14: 366-376, 1993;
Sanderson P et al, Current Medicinal Chemistry, 5: 289-304, 1998;
Rewinkel J et al, Current Pharmaceutical Design, 5:1043-1075, 1999;
and Coburn C Exp. Opin. Ther. Patents 11(5): 721-738, 2001. The
thrombin inhibitory salts of the disclosure are not limited to
those having S3, S2 and S1 affinity groups described in the
publications listed in the preceding sentence.
[0231] The boronic acids may have a Ki for thrombin of about 100 nM
or less, e.g. about 20 nM or less.
[0232] A subset of the Formula (I) acids comprises the acids of
Formula (III): 8
[0233] X is a moiety bonded to the N-terminal amino group and may
be H to form NH.sub.2. The identity of X is not critical but may be
a particular X moiety described above. In one example there may be
mentioned benzyloxycarbonyl.
[0234] In certain examples X is R.sup.6--(CH.sub.2).sub.p--C(O)--,
R.sup.6--(CH.sub.2).sub.p--S(O).sub.2--,
R.sup.6--(CH.sub.2).sub.p--NH--C- (O)-- or
R.sup.6--(CH.sub.2).sub.p--O--C(O)-- wherein p is 0, 1, 2, 3, 4, 5
or 6 (of which 0 and 1 are preferred) and R.sup.6 is H or a 5 to
13-membered cyclic group optionally substituted by 1, 2 or 3
substituents selected from halogen, amino, nitro, hydroxy, a
C.sub.5-C.sub.6 cyclic group, C.sub.1-C.sub.4 alkyl and
C.sub.1-C.sub.4 alkyl containing, and/or linked to the 5 to
13-membered cyclic group through, an in-chain 0, the aforesaid
alkyl groups optionally being substituted by a substituent selected
from halogen, amino, nitro, hydroxy and a C.sub.5-C.sub.6 cyclic
group. More particularly X is R.sup.6--(CH.sub.2).sub.p--C(O)-- or
R.sup.6--(CH.sub.2).sub.p--O--C(O)-- and p is 0 or 1. Said 5 to
13-membered cyclic group is often aromatic or heteroaromatic, for
example is a 6-membered aromatic or heteroaromatic group. In many
cases, the group is not substituted.
[0235] Exemplary X groups are (2-pyrazine) carbonyl, (2-pyrazine)
sulfonyl and particularly benzyloxycarbonyl.
[0236] aa.sup.1 is an amino acid residue having a hydrocarbyl side
chain containing no more than 20 carbon atoms (e.g. up to 15 and
optionally up to 13 C atoms) and comprising at least one cyclic
group having up to 13 carbon atoms. In certain examples, the cyclic
group(s) of aa.sup.1 have/has 5 or 6 ring members. For instance,
the cyclic group(s) of aa.sup.1 may be aryl groups, particularly
phenyl. Typically, there are one or two cyclic groups in the
aa.sup.1 side chain. Certain side chains comprise, or consist of,
methyl substituted by one or two 5- or 6-membered rings.
[0237] More particularly, aa.sup.1 is Phe, Dpa or a wholly or
partially hydrogenated analogue thereof. The wholly hydrogenated
analogues are Cha and Dcha.
[0238] aa.sup.2 is an imino acid residue having from 4 to 6 ring
members.
[0239] An exemplary class of products comprises those in which
aa.sup.2 is a residue of an imino acid of formula 9
[0240] where R.sup.11 is --CH.sub.2--, CH.sub.2--CH.sub.2--,
--S--CH.sub.2-- or --CH.sub.2--CH.sub.2--CH.sub.2--, which group
when the ring is 5 or 6-membered is optionally substituted at one
or more --CH.sub.2-- groups by from 1 to 3 C.sub.1-C.sub.3 alkyl
groups, for example to form the R.sup.11 group
--S--C(CH.sub.3).sub.2--. Of these imino acids,
azetidine-2-carboxylic acid, especially (s)-azetidine-2-carboxylic
acid, and more particularly proline are illustrative.
[0241] It will be appreciated from the above that a very preferred
class of products consists of those in which aa.sup.1-aa.sup.2 is
Phe-Pro. In another preferred class, aa.sup.1-aa.sup.2 is Dpa-Pro.
In other products, aa.sup.1-aa.sup.2 is Cha-Pro or Dcha-Pro. Of
course, also included are corresponding product classes in which
Pro is replaced by (s)-azetidine-2-carboxylic acid.
[0242] R.sup.9 is as defined previously and may be a moiety R.sup.1
of the formula --(CH.sub.2).sub.5-Z. Integer s is 2, 3 or 4 and W
is --OH, --OMe, --OEt or halogen (F, Cl, I or, preferably, Br).
Particularly illustrative Z groups are --OMe and --OEt, especially
--OMe. In certain examples s is 3 for all Z groups and, indeed, for
all compounds of the disclosure. Particular R.sup.1 groups are
2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 4-bromobutyl,
4-chlorobutyl, 4-methoxybutyl and, especially, 3-bromopropyl,
3-chloropropyl and 3-methoxypropyl. Most preferably, R.sup.1 is
3-methoxypropyl. 2-Ethoxyethyl is another preferred R.sup.1
group.
[0243] Accordingly, a very preferred class of salts consists of
those of acids of the formula X-Phe-Pro-Mpg-B(OH).sub.2, especially
Cbz-Phe-Pro-Mpg-B(OH).sub.2; also preferred are analogues of these
compounds in which Mpg is replaced by a residue with another of the
particularly preferred R.sup.1 groups and/or Phe is replaced by Dpa
or another aa.sup.1 residue.
[0244] The aa.sup.1 moiety of the salt is preferably of R
configuration. The aa.sup.2 moiety is preferably of
(S)-configuration. Particularly preferred salts have aa.sup.1 of
(R)-configuration and aa.sup.2 of (S)-configuration. The chiral
centre --NH--CH(R.sup.1)--B-- is preferably of (R)-configuration.
It is considered that commercial formulations will have the chiral
centres in (R,S,R) arrangement, as for example in the case of salts
of Cbz-Phe-Pro-BoroMpg-OH: 10
[0245] The disclosure includes salts of
Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg-OH (and of other compounds of the
formula X-(R)-Phe-(S)-Pro-(R)-boroMpg-OH) which are at least 90%
pure, e.g. at least 95% pure.
[0246] In broad terms, the salts described herein may be considered
to correspond to reaction products of an organoboronic acid as
described above with a strong base, e.g. a basic metal compound;
the salts are however not limited to products resulting from such a
reaction and may be obtained by alternative routes.
[0247] The salts are therefore obtainable by contacting an acid of
formula (I) with a strong base. The disclosure thus contemplates
products (compositions of matter) having the characteristics of a
reaction product of an acid of formula (I) and a strong base. The
base is pharmaceutically acceptable.
[0248] As suitable salts may be mentioned salts of metals, e.g. of
monovalent or divalent metals, and stronger organic bases, for
example:
[0249] 1. Alkali metal salts;
[0250] 2. Divalent, e.g. alkaline earth metal, salts;
[0251] 3. Group III metals;
[0252] 4. Salts of strongly basic organic nitrogen-containing
compounds, including:
[0253] 4A. Salts of guanidines and their analogues;
[0254] 4B. Salts of strongly basic amine, examples of which include
(i) aminosugars and (ii) other amines.
[0255] Of the above salts, particularly illustrative are alkali
metals, especially Na and Li. Also illustrative are
aminosugars.
[0256] Specific salts are of the acid boronate though in practice
the acid salts may contain a very small proportion of the doubly
deprotonated boronate. The term "acid boronate" refers to trigonal
--B(OH).sub.2 groups in which one of the B--OH groups is
deprotonated as well as to corresponding tetrahedral groups in
equilibrium therewith. Acid boronates have a stoichiometry
consistent with single deprotonation.
[0257] The disclosure includes therefore products (compositions of
matter) which comprise salts which may be represented by formula
(V): 11
[0258] where Y.sup.n+ is a pharmaceutically acceptable cation
obtainable from a strong base, and aa.sup.1, aa.sup.2, X and
R.sup.1 are as defined above. Also included are products in which
R.sup.1 is replaced by another R.sup.9 group.
[0259] One class of salts have a solubility of about 10 mM or more,
e.g. of at least about 20 mM, when their solubility is determined
as described in the examples at a dissolution of 25 mg/ml. More
particularly yet they have a solubility of least 50 mM when their
solubility is determined as described in the examples at a
dissolution of 50 mg/ml.
[0260] The disclosure includes salts of boronic acids (I) having an
observed stoichiometry consistent with the salt being of (being
representable by) the formula "(boronate.sup.-).sub.n
cation.sup.n+". One class of such salts are represented by the
formula:
[Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)(O.sup.-)]M.sup.+
[0261] where M.sup.+ represents a monovalent cation, especially an
alkali metal cation. It will be understood that the above
representation is a notional representation of a product whose
observed stoichiometry is unlikely to be literally and exactly 1:1.
In any event, a particular salt is
Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2 monosodium salt (TGN 255).
In the above formula, the trigonally-represented boronate
represents, as always, boronates which are trigonal, tetrahedral or
mixed trigonal/tetrahedral.
[0262] Particularly exemplary are products which comprise:
[0263] (i) species selected from (a) acids of formula (VIII):
X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2 where X is H or an
amino-protecting group, especially Cbz, (b) boronate anions
thereof, and (c) any equilibrium form of the aforegoing (e.g. an
anhydride); and
[0264] (ii) ions having a valency n in combination with said
species, the species and said ions having an observed stoichiometry
consistent with a notional species:ion stoichiometry of n:1. In one
class of salts, n is 1.
[0265] Considering the counter-ions in turn:
[0266] 1. Monovalent Metal, Especially Alkali Metal Salts
[0267] Suitable alkali metals include lithium, sodium and
potassium. All of these are remarkably soluble. Lithium and sodium
are illustrative because of their high solubility. The lithium and
particularly sodium salts are of surprisingly high solubility in
relation to potassium amongst others. Sodium is most used in many
instances. Salts containing mixtures of alkali metals are
contemplated by the disclosure.
[0268] The disclosure includes products comprising salts of the
formula (VI) 12
[0269] where M.sup.+ is an alkali metal ion and aa.sup.1, aa.sup.2,
X and R.sup.1 are as defined above, as well as salts in which both
hydroxy groups of the boronate group are in salt form (preferably
with another identical M.sup.+ group) and mixtures of such salts.
Included also are products wherein R.sup.1 is replaced by another
R.sup.9 group.
[0270] 2. Divalent. E.g. Alkaline Earth Metal (Group II Metal)
Salts
[0271] One example of a divalent metal is calcium. Another suitable
divalent metal is magnesium. Also contemplated is zinc. The
divalent metals are usually used in a boronic acid:metal ratio of
substantially 2:1, in order to achieve the preferred monovalent
boronate moiety. Salts containing mixtures of divalent metals, e.g.
mixtures of alkaline earth metals, are also contemplated.
[0272] Further disclosed are products (compositions of matter)
which comprise salts which may be represented by the formula (VII):
13
[0273] where M.sup.2+ is a divalent metal cation, e.g. an alkaline
earth metal or zinc cation, and aa.sup.1, aa.sup.2', X and R.sup.9
are as defined above, as well as salts in which both hydroxy groups
of the boronate group are deprotonated and mixtures of such salts.
As previously indicated, the boronate may comprise a tetrahedral
species.
[0274] Thus the disclosure includes salts of the formula
[Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)(O.sup.-)].sub.2M.sup.2+, for
example oral formulations containing
[Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)(O.sup.-)]- .sub.2Ca.sup.2+,
suitably adapted for the salt to enter the duodenum without
suffering gastric hydrolysis, e.g. by enteric coating.
[0275] 3. Group III Metals
[0276] Suitable Group III metals include aluminium and gallium.
Salts containing mixtures of Group III metals are also
contemplated.
[0277] The disclosure includes products comprising salts of the
formula (VIII): 14
[0278] where M.sup.3+ is a Group III metal ion and aa.sup.1,
aa.sup.2', X and R.sup.9 are as defined above, as well as salts in
which both hydroxy groups of the boronate group are in salt form
and mixtures of such salts. As previously indicated, the boronate
may comprise a tetrahedral species.
[0279] 4. Strongly Basic Organic Nitrogen-Containing Compounds
[0280] The disclosure includes products obtainable by (having the
characteristics of a product obtained by) reaction of a peptide
boronic acid as defined above and a strong organic base. Two
illustrative classes of organic base are described in sections 4A
and 4B below. Particularly preferred are acid salts (in which one
of the two boronic --OH groups is deprotonated). Most commonly, the
salts contain a single type of organic counter-ion (disregarding
trace contaminants) but the disclosure contemplates salts
containing mixtures of organic counter-ions; in one sub-class, the
different counter-ions all fall within the section 4A family
described below or, as the case may be, in the section 4B family
below; in another subclass, the salts comprise a mixture of organic
counter-ions which are not all from the same family (4A or 4B).
[0281] Suitable organic bases include those with a pKb of 7 or
more, e.g. 7.5 or more, for example in the region of 8 or more.
Bases which are less lipophilic [e.g. have at least one polar
functional group (e.g. 1, 2 or 3 such groups) for example hydroxy]
are favoured; thus aminosugars are one favoured class of base.
[0282] 4A. Guanidines and their Analogues
[0283] The guanidino compound (guanidine) may in principle be any
soluble and pharmaceutically acceptable compound having a guanidino
or a substituted guanidino group, or a substituted or unsubstituted
guanidine analogue. Suitable substituents include aryl (e.g.
phenyl), alkyl or alkyl interrupted by an ether or thioether
linkage and, in any event, typically contain from 1 to 6 and
especially 1, 2, 3, or 4 carbon atoms, as in the case of methyl or
ethyl. The guanidino group may have 1, 2, 3 or 4 substituent groups
but more usually has 1 or 2 substituent groups, for instance on a
terminal nitrogen. One class of guanidines is monoalkylated;
another class is dialkylated. As guanidine analogues may be
mentioned thioguanidines and 2-amino pyridines. Compounds having
unsubstituted guanidino groups, for example guanidine and arginine,
form one particular class.
[0284] Salts containing mixtures of guanidines are contemplated by
the disclosure.
[0285] A particular guanidino compound is L-arginine or an
L-arginine analogue, for example D-arginine, or the D- or,
preferably, L-isomers of homoarginine or agmatine [(4-aminobutyl)
guanidine]. Less preferred arginine analogues are
NG-nitro-L-arginine methyl ester, for example, and constrained
guanidine analogues, particularly 2-amino pyrimidines, for example
2,6-quinazolinediamines such as 5,6,7,8-tetrahydro-2,6-quinazolin-
ediamine, for example. The guanidino compound may also be a
peptide, for example a dipeptide, containing arginine; one such
dipeptide is L-tyrosyl-L-arginine.
[0286] Some particular guanidino compounds are compounds of formula
(VII): 15
[0287] where n is from 1 to 6 and for example at least 2, e.g. 3 or
more, and in many instances no more than 5. Most particularly, n is
3, 4 or 5. R.sup.2 is H or carboxylate or derivatised carboxylate,
for example to form an ester (e.g. a C.sub.1-C.sub.4 alkyl ester)
or amide. R.sup.3 is H, C.sub.1-C.sub.4 alkyl or a residue of a
natural or unnatural amino acid (e.g. tyrosine). The compounds of
formula (IV) are usually of L-configuration. The compounds of
formula (IV) are arginine (n=3; R.sup.2=carboxyl; R.sup.3.dbd.H)
and arginine derivatives or analogues.
[0288] The disclosure includes products comprising salts of the
formula (IX) 16
[0289] where aa.sup.1, aa.sup.2, X and R.sup.1 are as defined
previously and G.sup.+ is the protonated form of a pharmaceutically
acceptable organic compound comprising a guanidino group or an
analogue thereof, as well as salts in which both hydroxy groups of
the boronate group are in salt form (preferably with another
identical G.sup.+ group) and mixtures of such salts. Also included
are products wherein R.sup.1 is replaced by another R.sup.9
group.
[0290] 4B. Strongly Basic Amines
[0291] The disclosure includes products obtainable by (having the
characteristics of a product obtained by) reaction of a peptide
boronic acid as defined above and a strong organic base which is an
amine. The amine may in principle be any soluble and
pharmaceutically acceptable amine.
[0292] It is envisaged that a desirable class of amine includes
those having polar functional groups in addition to a single amine
group, as such compounds will be more hydrophilic and thus more
soluble than others. In certain salts, the or each additional
functional group is hydroxy. Some amines have 1, 2, 3, 4, 5 or 6
additional functional groups, especially hydroxy groups. In one
illustrative class of amines the ratio of (amino plus hydroxy
groups):carbon atoms is from 1:2 to 1:1, the latter ratio being
particularly preferred. These amines with one or more additional
polar functional groups may be a hydrocarbon, especially an alkane,
substituted by the amino group and the additional polar group(s).
The amino group may be substituted or unsubstituted and, excluding
amino substituents, the polar base may contain, for example, up to
10 carbon atoms; usually there are no less than three such carbon
atoms, e.g. 4, 5 or 6. Aminosugars are included in this category of
polar bases.
[0293] The disclosure includes products comprising salts of the
formula (X) 17
[0294] where aa.sup.1, aa.sup.2, X and R.sup.1 are as defined
previously and A.sup.+ is the protonated form of a pharmaceutically
acceptable amine, as well as salts in which both hydroxy groups of
the boronate group are in salt form (preferably with another
identical A.sup.+ group) and mixtures of such salts. In one class
of such products, A.sup.+ is the protonated form of an amine
described in section 4B(i) below; in another class A.sup.+ is the
protonated form of an amine described in 4B(ii) below. Also
included are products in which R.sup.1 is replaced by another
R.sup.9 group.
[0295] Two illustrative classes of amine base are described in
sections 4B(i) and 4B(ii) below. Particularly preferred are acid
salts (in which one of the two boronic --OH groups is
deprotonated). Most commonly, the salts contain a single type of
amine counter-ion (disregarding trace contaminants) but the
disclosure contemplates salts containing mixtures of amine
counter-ions; in one sub-class, the different counter-ions all fall
within the subsection 4B(i) family described below or, as the case
may be, in the sub-section 4B(ii) family below; in another
subclass, the salts comprise a mixture of organic counter-ions
which are not all from the same family (4B(i) or 4B(ii)).
[0296] 4B(i) Aminosugars
[0297] The identity of the aminosugar is not critical. Preferred
aminosugars include ring-opened sugars, especially glucamines.
Cyclic aminosugars are also envisaged as useful. One class of the
aminosugars is N-unsubstituted and another, preferred, class is
N-substituted by one or two N-substituents (e.g. one). Suitable
substituents are hydrocarbyl groups, for example and without
limitation containing from 1 to 12 carbon atoms; the substituents
may comprise alkyl or aryl moieties or both. Exemplary substituents
are C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7
and C.sub.8 alkyl groups, in particular methyl and ethyl, of which
methyl is illustrative. Data indicate that aminosugars, especially
N-methyl-D-glucamine, are of surprisingly high solubility.
[0298] A most preferred aminosugar is N-methyl-D-glucamine: 18
[0299] 4B(ii) Other Amines
[0300] Other suitable amines include amino acids (whether naturally
occurring or not) whose side chain is substituted by an amino
group, especially lysine.
[0301] Some amines are compounds of formula (XI): 19
[0302] where n, R.sup.2 and R.sup.3 are as defined in relation to
formula (IV). The compounds of formula (VI) are usually of
L-configuration. The compounds of formula (VI) are lysine (n=4;
R.sup.2=carboxyl; R.sup.3=H) and lysine derivatives or analogues. A
most preferred amine is L-lysine.
[0303] Other suitable amines are nitrogen-containing heterocycles.
At least usually, such heterocyclic compounds are alicyclic; one
class of the heterocyclic compounds is N-substituted and another,
preferred, class is N-unsubstituted. The heterocycles may contain 6
ring-forming atoms, as in the cases of piperidine, piperazine and
morpholine. One class of amines includes N-containing heterocycles
substituted by polar substituents, espedally hydroxy, e.g. 1, 2 or
3 times.
[0304] The disclosure therefore includes amines other than
aminosugars which have one or more (e.g. 1, 2, 3, 4, 5 or 6) polar
substituents, especially hydroxy, in addition to one amine group.
Such compounds may have a ratio of (amino plus hydroxy
groups):carbon atoms of 1:2 to 1:1, the latter ratio being
particularly preferred.
[0305] The disclosure includes mixed salts, i.e. salts containing a
mixture of boropeptide moieties and/or counterions but single salts
are preferred.
[0306] The salts in solid form may contain a solvent, e.g. water.
There are included a class of products in which the salts are
essentially anhydrous. Also included is a class in which the salts
are hydrates.
[0307] Use of the Products of the Disclosure
[0308] The salts of the disclosure are thrombin inhibitors. They
are therefore useful for inhibiting thrombin. There are therefore
provided compounds which have potential for controlling haemostasis
and especially for inhibiting coagulation, for example in the
treatment or prevention of secondary events after myocardial
infarction. The medical use of the compounds may be prophylactic
(including to treat thrombosis as well as to prevent occurrence of
thrombosis) as well as therapeutic (including to prevent
re-occurrence of thrombosis or secondary thrombotic events).
[0309] The salts may be employed when an anti-thrombogenic agent is
needed. Further, it has been found that the salts, including those
of boronic acids of Formula (III), are beneficial in that the class
is useful for treating arterial thrombosis by therapy or
prophylaxis. The disclosed salts are thus indicated in the
treatment or prophylaxis of thrombosis and hypercoagulability in
blood and tissues of animals including man. The term "thrombosis"
includes inter alla atrophic thrombosis, arterial thrombosis,
cardiac thrombosis, coronary thrombosis, creeping thrombosis,
infective thrombosis, mesenteric thrombosis, placental thrombosis,
propagating thrombosis, traumatic thrombosis and venous
thrombosis.
[0310] It is known that hypercoagulability may lead to
thromboembolic diseases.
[0311] Examples of venous thromboembolism which may be treated or
prevented with compounds of the disclosure include obstruction of a
vein, obstruction of a lung artery (pulmonary embolism), deep vein
thrombosis, thrombosis associated with cancer and cancer
chemotherapy, thrombosis inherited with thrombophilic diseases such
as Protein C deficiency, Protein S deficiency, antithrombin III
deficiency, and Factor V Leiden, and thrombosis resulting from
acquired thrombophilic disorders such as systemic lupus
erythematosus (inflammatory connective tissue disease). Also with
regard to venous thromboembolism, compounds of the disclosure are
useful for maintaining patency of indwelling catheters.
[0312] Examples of cardiogenic thromboembolism which may be treated
or prevented with compounds of the disclosure include
thromboembolic stroke (detached thrombus causing neurological
affliction related to impaired cerebral blood supply), cardiogenic
thromboembolism associated with atrial fibrillation (rapid,
irregular twitching of upper heart chamber muscular fibrils),
cardiogenic thromboembolism associated with prosthetic heart valves
such as mechanical heart valves, and cardiogenic thromboembolism
associated with heart disease.
[0313] Examples of conditions involving arterial thrombosis include
unstable angina (severe constrictive pain in chest of coronary
origin), myocardial infarction (heart muscle cell death resulting
from insufficient blood supply), ischemic heart disease (local
ischemia due to obstruction (such as by arterial narrowing) of
blood supply), reocclusion during or after percutaneous
transluminal coronary angioplasty, restenosis after percutaneous
transluminal coronary angioplasty, occlusion of coronary artery
bypass grafts, and occlusive cerebrovascular disease. Also with
regard to arterio-venous (mixed) thrombosis, anti-thrombotic
compounds of the disclosure are useful for maintaining patency in
arteriovenous shunts.
[0314] Other conditions associated with hypercoagulability and
thromboembolic diseases which may be mentioned inherited or
acquired deficiencies in heparin cofactor II, circulating
antiphospholipid antibodies (Lupus anticoagulant), homocysteinemia,
heparin induced thrombocytopenia and defects in fibrinolysis.
[0315] Particular uses which may be mentioned include the
therapeutic and/or prophylactic treatment of venous thrombosis and
pulmonary embolism. Preferred indications envisaged for the
products of the disclosure (notably the salts of TRI 50c)
include:
[0316] Prevention of venous thromboembolic events (e.g. deep vein
thrombosis and/or pulmonary embolism). Examples include patients
undergoing orthopaedic surgery such as total hip replacement, total
knee replacement, major hip or knee surgery; patients undergoing
general surgery at high risk for thrombosis, such as abdominal or
pelvic surgery for cancer; and in patients bedridden for more than
3 days and with acute cardiac failure, acute respiratory failure,
infection.
[0317] Prevention of thrombosis in the haemodialysis circuit in
patients, in patients with end stage renal disease.
[0318] Prevention of cardiovascular events (death, myocardial
infarction, etc) in patients with end stage renal disease, whether
or not requiring haemodialysis sessions.
[0319] Prevention of venous thrombo-embolic events in patients
receiving chemotherapy through an indwelling catheter.
[0320] Prevention of thromboembolic events in patients undergoing
lower limb arterial reconstructive procedures (bypass,
endarteriectomy, transluminal angioplasty, etc).
[0321] Treatment of venous thromboembolic events.
[0322] Prevention of cardiovascular events in acute coronary
syndromes (e.g. unstable angina, non Q wave myocardial
ischaemia/infarction), in combination with another cardiovascular
agent, for example aspirin (acetylsalicylic acid; aspirin is a
registered trade mark in Germany), thrombolytics (see below for
examples), antiplatelet agents (see below for examples).
[0323] Treatment of patients with acute myocardial infarction in
combination with acetylsalicylic acid, thrombolytics (see below for
examples).
[0324] The thrombin inhibitors of the disclosure are thus indicated
both in the therapeutic and/or prophylactic treatment of all the
aforesaid disorders.
[0325] In one method, the products of the disclosure are used for
the treatment of patients by haemodialysis, by providing the
product in the dialysis solution, as described in relation to other
thrombin inhibitors in WO 00/41715. The disclosure therefore
includes dialysing solutions and dialysing concentrates which
comprise a product of the disclosure, as well as a method of
treatment by dialysis of a patient in need of such treatment, which
method comprises the use of a dialysing solution including a low
molecular weight thrombin inhibitor. Also included is the use of an
anti-thrombotic product of the disclosure for the manufacture of a
medicament for the treatment by dialysis of a patient, in which the
anti-thrombotic product of the disclosure is provided in the
dialysing solution.
[0326] In another method, the products of the disclosure are used
to combat undesirable cell proliferation, as described in relation
to other thrombin inhibitors in WO 01/41796. The undesirable cell
proliferation is typically undesirable hyperplastic cell
proliferation, for example proliferation of smooth muscle cells,
especially vascular smooth muscle cells. The products of the
disclosure particularly find application in the treatment of
intimal hyperplasia, one component of which is proliferation of
smooth muscle cells. Restenosis can be considered to be due to
neointimal hyperplasia; accordingly intimal hyperplasia in the
context of the disclosure includes restenosis.
[0327] The products of the disclosure are also contemplated for the
treatment of ischemic disorders. More particularly, they may be
used in the treatment (whether therapeutic or prophylactic) of an
ischemic disorder in a patient having, or at risk of, non-valvular
atrial fibrillation (NVAF) as described in relation to other
thrombin inhibitors in WO 02/36157. Ischemic disorders are
conditions whose results include a restriction in blood flow to a
part of the body. The term will be understood to include thrombosis
and hypercoagulability in blood, tissues and/or organs. Particular
uses that may be mentioned include the prevention and/or treatment
of ischemic heart disease, myocardial infarction, systemic embolic
events in e.g. the kidneys or spleen, and more particularly of
cerebral ischemia, including cerebral thrombosis, cerebral embolism
and/or cerebral ischemia associated with non-cerebral thrombosis or
embolism (in other words the treatment (whether therapeutic or
prophylactic) of thrombotic or ischemic stroke and of transient
ischemic attack), particularly in patients with, or at risk of,
NVAF.
[0328] The products of the disclosure are also contemplated for the
treatment of rheumatic/arthritic disorders, as described in
relation to other thrombin inhibitors in WO 03/007984. Thus, the
products of the disclosure may be used in the treatment of chronic
arthritis, rheumatoid arthritis, osteoarthritis or ankylosing
spondylitis
[0329] Moreover, the products of the disclosure are expected to
have utility in prophylaxis of re-occlusion (i.e. thrombosis) after
thrombolysis, percutaneous trans-luminal angioplasty (PTA) and
coronary bypass operations; the prevention of re-thrombosis after
microsurgery and vascular surgery in general. Further indications
include the therapeutic and/or prophylactic treatment of
disseminated intravascular coagulation caused by bacteria, multiple
trauma, intoxication or any other mechanism; anticoagulant
treatment when blood is in contact with foreign surfaces in the
body such as vascular grafts, vascular stents, vascular catheters,
mechanical and biological prosthetic valves or any other medical
device; and anticoagulant treatment when blood is in contact with
medical devices outside the body such as during cardiovascular
surgery using a heart-lung machine or in haemodialysis.
[0330] The products of the disclosure are further indicated in the
treatment of conditions where there is an undesirable excess of
thrombin without signs of hypercoagulability, for example in
neurodegenerative diseases such as Alzheimer's disease. In addition
to its effects on the coagulation process, thrombin is known to
activate a large number of cells (such as neutrophils, fibroblasts,
endothelial cells and smooth muscle cells). Therefore, the
compounds of the disclosure may also be useful for the therapeutic
and/or prophylactic treatment of idiopathic and adult respiratory
distress syndrome, pulmonary fibrosis following treatment with
radiation or chemotherapy, septic shock, septicaemia, inflammatory
responses, which include, but are not limited to, edema, acute or
chronic atherosclerosis such as coronary arterial disease, cerebral
arterial disease, peripheral arterial disease, reperfusion damage,
and restenosis after percutaneous trans-luminal angioplasty
(PTA).
[0331] The salts may also be useful in the treatment of
pancreatitis.
[0332] The salts described herein are further considered to be
useful for inhibiting platelet procoagulant activity. The
disclosure provides a method for inhibiting platelet pro-coagulant
activity by administering a salt of a boronic acid described herein
to a mammal at risk of, or suffering from, arterial thrombosis,
particularly a human patient. Also provided is the use of such
salts for the manufacture of medicaments for inhibiting platelet
procoagulant activity.
[0333] The use of products of the disclosure as inhibitors of
platelet pro-coagulant activity is predicated on the observation
that the boronic acids described herein are indicated to be
effective at inhibiting arterial thrombosis as well as venous
thrombosis.
[0334] Indications involving arterial thrombosis include acute
coronary syndromes (especially myocardial infarction and unstable
angina), cerebrovascular thrombosis and peripheral arterial
occlusion and arterial thrombosis occurring as a result of atrial
fibrillation, valvular heart disease, arterio-venous shunts,
indwelling catheters or coronary stents. Accordingly, in another
aspect there is provided a method of treating a disease or
condition selected from this group of indications, comprising
administering to a mammal, especially a human patient, a salt of
the disclosure. The disclosure includes products for use in an
arterial environment, e.g. a coronary stent or other arterial
implant, having a coating which comprises a salt according to the
disclosure.
[0335] The salts of the disclosure may be used prophylactically to
treat an individual believed to be at risk of suffering from
arterial thrombosis or a condition or disease involving arterial
thrombosis or therapeutically (including to prevent re-occurrence
of thrombosis or secondary thrombotic events).
[0336] There is therefore included the use of selective thrombin
inhibitors (organoboronic add salts) described herein for treatment
of the above disorders by prophylaxis or therapy as well as their
use in pharmaceutical formulations and the manufacture of
pharmaceutical formulations.
[0337] Administration and Pharmaceutical Formulations
[0338] The salts may be administered to a host, for example, in the
case where the drug has anti-thrombogenic activity, to obtain an
anti-thrombogenic effect. In the case of larger animals, such as
humans, the compounds may be administered alone or in combination
with pharmaceutically acceptable diluents, excipients or carriers.
The term "pharmaceutically acceptable" includes acceptability for
both human and veterinary purposes, of which acceptability for
human pharmaceutical use is preferred. In the case of oral
administration, the compounds are preferably administered in a form
which prevents the salt of the disclosure from contact with the
acidic gastric juice, such as enterically coated formulations,
which thus prevent release of the salt of the disclosure until it
reaches the duodenum.
[0339] The enteric coating is suitably made of carbohydrate
polymers or polyvinyl polymers, for example. Examples of enteric
coating materials include, but are not limited to, cellulose
acetate phthalate, cellulose acetate succinate, cellulose hydrogen
phthalate, cellulose acetate trimellitate, ethyl cellulose,
hydroxypropyl-methylcellulose phthalate,
hydroxypropylmethylcellulose acetate succinate, carboxymethyl
ethylcellulose, starch acetate phthalate, amylose acetate
phthalate, polyvinyl acetate phthalate, polyvinyl butyrate
phthalate, styrene-maleic acid copolymer,
methyl-acrylate-methacrylic acid copolymer (MPM-05),
methylacrylate-methacrylic acid-methylmethacrylate copolymer
(MPM-06), and methylmethacrylate-methacrylic acid co-polymer
(Eudragit.RTM. L & S). Optionally, the enteric coating contains
a plasticiser. Examples of the plasticiser include, but are not
limited to, triethyl citrate, triacetin, and diethyl phthalate.
[0340] The salts of the disclosure may be combined and/or
co-administered with any cardiovascular treatment agent. There are
large numbers of cardiovascular treatment agents available in
commercial use, in clinical evaluation and in pre-clinical
development, which could be selected for use with a product of the
disclosure for the prevention of cardiovascular disorders by
combination drug therapy. Such agent can be one or more agents
selected from, but not limited to several major categories, namely,
a lipid-lowering drug, including an IBAT (ileal Na.sup.+/bile acid
cotransporter) inhibitor, a fibrate, niacin, a statin, a CETP
(cholesteryl ester transfer protein) inhibitor, and a bile acid
sequestrant, an anti-oxidant, including vitamin E and probucol, a
IIb/IIIa antagonist (e.g. abciximab, eptifibatide, tirofiban), an
aldosterone inhibitor (e.g. spirolactone and epoxymexrenone), an
adenosine A2 receptor antagonist (e.g. losartan), an adenosine A3
receptor agonist, a beta-blocker, acetylsalicylic acid, a loop
diuretic and an ACE (angiotensin converting enzyme) inhibitor.
[0341] The salts of the disclosure may be combined and/or
co-administered with any antithrombotic agent with a different
mechanism of action, such as the antiplatelet agents
acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane
receptor and/or synthetase inhibitors, prostacyclin mimetics and
phosphodiesterase inhibitors and ADP-receptor (P.sub.2 T)
antagonists.
[0342] The products of the disclosure may further be combined
and/or co-administered with thrombolytics such as tissue
plasminogen activator (natural, recombinant or modified),
streptokinase, urokinase, prourokinase, anisoylated
plasminogen-streptokinase activator complex (APSAC), animal
salivary gland plasminogen activators, and the like, in the
treatment of thrombotic diseases, in particular myocardial
infarction.
[0343] The salts of the disclosure may be combined and/or
co-administered with a cardioprotectant, for example an adenosine
A1 or A3 receptor agonist.
[0344] There is also provided a method for treating an inflammatory
disease in a patient that comprises treating the patient with a
product of the disclosure and an NSAID, e.g., a COX-2 inhibitor.
Such diseases include but are not limited to nephritis, systemic
lupus, erythematosus, rheumatoid arthritis, glomerulonephritis,
vasculitis and sarcoidosis. Accordingly, the anti-thrombotic salts
of the disclosure may be combined and/or co-administered with an
NSAID.
[0345] Typically, therefore, the salts described herein may be
administered to a host to obtain a thrombin-inhibitory effect, or
in any other thrombin-inhibitory or anti-thrombotic context
mentioned herein.
[0346] Actual dosage levels of active ingredients in the
pharmaceutical compositions of this disclosure may be varied so as
to obtain an amount of the active compound(s) that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration (referred to herein as a
"therapeutically effective amount"). The selected dosage level will
depend upon the activity of the particular compound, the severity
of the condition being treated and the condition and prior medical
history of the patient being treated. However, it is within the
skill of the art to start doses of the compound at levels lower
than required for to achieve the desired therapeutic effect and to
gradually increase the dosage until the desired effect is
achieved.
[0347] For example, it is currently contemplated that, in the case
of oral administration of salts of TRI 50c, the salts might for
instance be administered in an amount of from 0.5 to 2.5 mg/Kg
twice daily, calculated as TRI 50c. Other salts might be
administered in equivalent molar amounts. The disclosure is not
limited to administration in such quantities or regimens and
includes dosages and regimens outside those described in the
previous sentence.
[0348] According to a further aspect of the disclosure there is
provided an oral pharmaceutical formulation including a product of
the disclosure, in admixture with a pharmaceutically acceptable
adjuvant, diluent or carrier.
[0349] Solid dosage forms for oral administration include capsules,
tablets (also called pills), powders and granules. In such solid
dosage forms, the active compound is typically mixed with at least
one inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or one or more: a)
fillers or extenders such as starches, lactose, sucrose, glucose,
mannitol and silicic acid; b) binders such as
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,
sucrose and acacia; c) humectants such as glycerol; d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates and sodium
carbonate; e) solution retarding agents such as paraffin; f)
absorption accelerators such as quaternary ammonium compounds; g)
wetting agents such as cetyl alcohol and glycerol monostearate; h)
absorbents such as kaolin and bentonite clay and i) lubricants such
as talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate and mixtures thereof. In the case of
capsules and tablets, the dosage form may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycol, for example.
[0350] Suitably, the oral formulations may contain a dissolution
aid. The dissolution aid is not limited as to its identity so long
as it is pharmaceutically acceptable. Examples include nonionic
surface active agents, such as sucrose fatty acid esters, glycerol
fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan
trioleate), polyethylene glycol, polyoxyethylene hydrogenated
castor oil, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers,
polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid
esters, polyoxyethylene alkylamines, polyoxyethylene alkyl
thioethers, polyoxyethylene polyoxypropylene copolymers,
polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty
acid esters, propylene glycol monofatty acid esters,
polyoxyethylene propylene glycol monofatty acid esters,
polyoxyethylene sorbitol fatty acid esters, fatty acid
alkylolamides, and alkylamine oxides; bile acid and salts thereof
(e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid,
dehydrocholic acid and salts thereof, and glycine or taurine
conjugate thereof); ionic surface active agents, such as sodium
laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates,
ether phosphates, fatty acid salts of basic amino acids;
triethanolamine soap, and alkyl quaternary ammonium salts; and
amphoteric surface active agents, such as betaines and
aminocarboxylic acid salts.
[0351] The active compounds may also be in micro-encapsulated form,
if appropriate, with one or more of the above-mentioned
excipients.
[0352] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compounds, the liquid
dosage forms may contain inert diluents commonly used in the art
such as water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan and mixtures thereof.
Besides inert diluents, the oral compositions may also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavouring and perfuming agents. Suspensions,
in addition to the active compounds, may contain suspending agents
such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol
and sorbitan esters, microcrystalline cellulose, aluminium
metahydroxide, bentonite, agar-agar, and tragacanth and mixtures
thereof.
[0353] The product of the disclosure may be presented as solids in
finely divided solid form, for example they may be micronised.
Powders or finely divided solids may be encapsulated.
[0354] The active compound may be given as a single dose, in
multiple doses or as a sustained release formulation.
[0355] It will be understood from the aforegoing that there are
provided pharmaceutical products comprising a metal salt, e.g. an
alkali metal salt, of a boronic acid of Formula (I) in dry fine
particle form, suitable for administration. The metal salt may be
in a form appropriate for oral administration. The metal salt is
suitably an acid salt.
[0356] Synthesis
[0357] 1. Peptide/Peptidomimetic Synthesis
[0358] The synthesis of boropeptides, including, for example,
Cbz-D-Phe-Pro-BoroMpg-OPinacol is familiar to those skilled in the
art and described in the prior art mentioned above, including
Claeson et al (U.S. Pat. No. 5,574,014 and others) and Kakkar et al
(WO 92/07869 and family members including U.S. Pat. No. 5,648,338).
It is described also by Elgendy et al Adv. Exp. Med. Biol. (USA)
340:173-178, 1993; Claeson, G. et al Biochem. J. 290:309-312, 1993;
Deadman et al J. Enzyme Inhibition 9:29-41, 1995, and by Deadman et
al J. Med. Chem. 38:1511-1522, 1995.
[0359] Stereoselective synthesis with S or R configuration at the
chiral B-terminal carbon may be conducted using established
methodology (Elgendy et al Tetrahedron. Lett. 33:4209-4212, 1992;
WO 92/07869 and family members including U.S. Pat. No. 5,648,338)
using (+) or (-)-pinanediol as the chiral director (Matteson et al
J. Am. Chem. Soc. 108:810-819, 1986; Matteson et al
Organometallics. 3:1284-1288, 1984). Another approach is to resolve
the requisite aminoboronate intermediate (e.g. Mpg-BOPinacol) to
selectively obtain the desired (R)-isomer and couple it to the
dipeptide moiety (e.g. Cbz-(R)-Phe-(S)-Pro, which is the same as
Cbz-D-Phe-L-Pro) which will form the remainder of the molecule.
[0360] The boropeptides may be synthesised initially in the form of
boronic acid esters, particularly esters with diols. Such diol
esters may be converted to the peptide boronic acid as described
next.
[0361] 2. Ester to Acid Conversion
[0362] A peptide boronate ester such as
Cbz-(R)-Phe-Pro-BoroMpg-OPinacol may be hydrolysed to form the
corresponding acid.
[0363] A novel technique for converting a diol ester of a peptide
boronic acid of formula (I) into the acid comprises dissolving the
diol ester in an ether and particularly a dialkyl ether, reacting
the thus-dissolved diol with a diolamine, for example a
dialkanolamine, to form a product precipitate, recovering the
precipitate, dissolving it in a polar organic solvent and reacting
the thus-dissolved product with an aqueous medium, e.g. an aqueous
add, to form the peptide boronic acid. The boronic add may be
recovered from the organic layer of the mixture resulting from the
reaction, for example by removing the solvent, e.g. by evaporation
under vacuum or distillation. The reaction between the diol ester
and the diolamine may be carried out under reflux, for example.
[0364] The identity of the diol is not critical. As suitable diols
may be mentioned aliphatic and aromatic compounds having hydroxy
groups that are substituted on adjacent carbon atoms or on carbon
atoms substituted by another carbon. That is to say, suitable diols
include compounds having at least two hydroxy groups separated by
at least two connecting carbon atoms in a chain or ring. One class
of diols comprises hydrocarbons substituted by exactly two hydroxy
groups. One such diol is pinacol and another is pinanediol; there
may also be mentioned neopentylglycol, 1,2-ethanediol,
1,2-propanediol, 1,3-propanediol, 2,3-butanediol,
1,2-diisopropylethanediol, 5,6-decanediol and
1,2-dicyclohexylethanediol.
[0365] The alkyl groups of the dialkyl ether preferably have 1, 2,
3 or 4 carbon atoms and the alkyl groups may be the same or
different. An exemplary ether is diethyl ether.
[0366] The alkyl groups of the dialkanolamine preferably have 1, 2,
3 or 4 carbon atoms and the alkyl groups may be the same or
different. An exemplary dialkanolamine is diethanolamine. The
diethanolamine/boronic acid reaction product hydrolyses in water at
room temperature and the rate of hydrolysis may be accelerated by
adding acid or base.
[0367] The polar organic solvent is preferably CHCl.sub.3. Other
examples are polyhalogenated alkanes generally and ethyl acetate.
In principle, any polar organic solvent is acceptable other than
alcohols.
[0368] The aqueous acid is suitably a strong inorganic acid at a pH
in the region of 1 such as hydrochloric acid, for example.
[0369] After reaction with the acid, the reaction mixture is
suitably washed with, for example, NH.sub.4Cl or another mild
base.
[0370] An example of a specific procedure is as follows
[0371] 1. The pinacol or pinanediol ester of the selected peptide
boronic acid is dissolved in diethylether.
[0372] 2. Diethanolamine is added and the mixture is refluxed at
40.degree. C.
[0373] 3. The precipitated product is removed (filtered), washed
(usually several times) with diethyl ether or another polar organic
solvent other than an alcohol, and dried (e.g. by evaporation under
vacuum).
[0374] 4. The dry product is dissolved in a polar organic solvent
other than an alcohol, e.g. CHCl.sub.3. Aqueous acid or base is
added, e.g hydrochloric acid (pH 1), and the mixture is stirred for
e.g. approximately 1 h at room temperature.
[0375] 5. The organic layer is removed and washed with NH.sub.4Cl
solution.
[0376] 6. The organic solvent is distilled off and the residual
solid product is dried.
[0377] The above process results in the formation of what may
conveniently be referred to as a "diolamine adduct" of the peptide
boronic adds of formula (I), especially such adducts with
diethanolamine, and such adducts are themselves included in the
disclosure. The molecular structure of such adducts is not known:
they might comprise a compound in which the two oxygens and the
nitrogen of the diolamine are all coordinated to the boron; they
might comprise ions. The adducts are however considered to be
esters. A particular novel product included in the disclosure is
that obtainable by reacting a pinacol or pinanediol ester of a
compound of Formula VIII, particularly (R,S,R)-TRI 50c, and
diethanolamine, i.e. the novel product is an (R,S,R)-TRI
50c/diethanolamine "adduct" where the acid is (R,S,R)-TRI 50c.
[0378] The diolamine materials of the disclosure may be defined as
a composition of matter comprising:
[0379] (i) a species of formula (XII) 20
[0380] wherein X is H or an amino protecting group, the boron atom
is optionally coordinated additionally with a nitrogen atom, and
the valency status of the terminal oxygens is open (they may be
attached to a second covalent bond, be ionised as --O.sup.-, or
have some other, for example intermediate, status); and, in bonding
association therewith
[0381] (ii) a species of formula (XIII) 21
[0382] wherein the valency status of the nitrogen atom and the two
oxygen atoms is open. It will be appreciated that the terminal
oxygen atoms of the species of formula (IX) and the oxygen atoms of
the species of formula (X) may be the same oxygen atoms, in which
case the species of formula (X) forms a diol ester with the species
of formula (IX).
[0383] It will be appreciated that the aforegoing technique
comprises an example of a method for recovering an organoboronic
acid product, the method comprising providing in a solvent a
dissolved mixture comprising the organoboronic acid in a soluble
form and a compound having two hydroxy groups and an amino group
(i.e. a diolamine), causing or allowing the organoboronic acid and
the diolamine to react to form a precipitate, and recovering the
precipitate. The soluble form of the organoboronic acid may be a
diol ester, as discussed above. The solvent may be an ether, as
discussed above. The organoboronic acid may be one of the
organoboronic acids referred to in this specification, for example
it may be of Formula (I), (II) or (III). The method described in
this paragraph is novel and forms an aspect of the disclosure. A
recovery method is filtration.
[0384] The reaction between the diolamine and the soluble form of
the organoboronic acid is suitable carried out at an elevated
temperature, for example under reflux.
[0385] Another aspect of the disclosure is a method for recovering
an organoboron species, comprising
[0386] providing, in a form soluble in an ether, an organoboronic
acid, for example a drug such as, e.g., a compound of formula
(III);
[0387] forming a solution of the soluble form in the ether;
[0388] combining the solution with a dialkanolamine and allowing or
causing the dialkanolamine to react with the soluble form of the
organoboronic acid to form an insoluble precipitate; and
[0389] recovering the precipitate.
[0390] The term "soluble" in the preceding paragraph refers to
species which are substantially more soluble in the reaction medium
than is the precipitated product. In variants of the method, the
ether is replaced by toluene or another aromatic solvent.
[0391] The diethanolamine precipitation technique described above
is an example of another novel method, which is a method for
recovering from ether solution a pinacol or pinanediol ester of a
peptide boronic acid, comprising dissolving diethanolamine in the
solution, allowing or causing a precipitate to form and recovering
the precipitate. The disclosure encompasses variants of this
methods in which another diol than pinacol or pinanediol is
used.
[0392] The precipitated material, i.e. the "adduct", may be
converted into the free organoboronic acid, for example by
contacting it with an acid. The acid may be an aqueous acid, for
example an aqueous inorganic acid, e.g. as described above. The
precipitate may be dissolved, for example in an organic solvent,
prior to being contacted with the acid.
[0393] The disclosure therefore provides a method for making an
organoboronic acid, comprising converting its diolamine reaction
product to the acid.
[0394] The acid resulting from the methods described in the
previous two paragraphs may be converted to a salt of the acid with
a multivalent metal, which salt may in turn be formulated into a
pharmaceutical composition in oral dosage form.
[0395] 3. Salt Synthesis
[0396] In general, the salts may be prepared by contacting the
relevant peptide boronic acid with a strong base appropriate to
form the desired salt. In the case of metal salts, the metal
hydroxides are suitable bases (alternatively, metal carbonates
might be used, for example), whilst sometimes it is more convenient
to contact the acid with a relevant metal alkoxide (eg methoxide),
for which purpose the corresponding alkanol is a suitable solvent.
Salts with organic bases may be prepared by contacting the peptide
boronic acid with the organic base itself. Illustrative salts are
acid salts (one --BOH proton replaced) and, to make add salts with
a monovalent cation, the add and the base are suitably reacted in
substantially equimolar quantities. Generally stated, therefore,
the usual acid:base molar ratio is substantially n:1, where n is
the valency of the cation of the base.
[0397] In one procedure, a solution of the peptide boronic acid in
a water-miscible organic solvent, for example acetonitrile or an
alcohol (e.g. ethanol, methanol, a propanol, for example
iso-propanol, or another alkanol), is combined with an aqueous
solution of the base. The acid and the base are allowed to react
and the salt is recovered. The reaction is typically carried out at
ambient temperature (e.g. at a temperature of from 15 to 30.degree.
C., e.g. 15 to 25.degree. C.), but an elevated temperature may be
used, for example up to the boiling point of the reaction mixture
but more usually lower, e.g. a temperature of up to 40.degree. C.
or 50.degree. C. The reaction mixture may be allowed to stand or be
agitated (usually stirred).
[0398] The time during which the acid and the base are allowed to
react is not critical but it has been found desirable to maintain
the reaction mixture for at least one hour. A period of from one to
two hours is usually suitable but longer reaction times may be
employed.
[0399] The salt may be recovered from the reaction mixture by any
suitable method, for example evaporation or precipitation.
Precipitation may be carried out by adding an excess of a miscible
solvent in which the salt has limited solubility. In one preferred
technique, the salt is recovered by evacuating the reaction mixture
to dryness. The salt is preferably thereafter purified, for example
by redissolving the salt before filtering the resulting solution
and drying it, for example by evacuating it to dryness. The
redissolution may be performed using water, e.g. distilled water.
The salt may then be further purified, for example in order to
remove residual water by further redissolution in a suitable
solvent, which is advantageously ethyl acetate or THF followed by
evaporating to dryness. The purification procedure may be carried
out at ambient temperature (say, 15 to 30.degree. C., e.g. 15 to
25.degree. C.), or at a modestly elevated temperature, such as e.g.
a temperature not exceeding 40.degree. C. or 50.degree. C.; for
example the salt may be dissolved in water and/or solvent by
agitating with or without warming to, for example, 37.degree.
C.
[0400] Also included is a method for drying the salts of the
disclosure and other peptide boronic acid salts, comprising
dissolving them in an organic solvent, e.g. ethyl acetate or THF,
and then evaporating to dryness, e.g. by evacuation.
[0401] Generally, preferred solvents for use in purifying the salts
are ethyl acetate or THF, or perhaps another organic solvent.
[0402] A general procedure for synthesising salts of
Cbz-Phe-Pro-BoroMpg-OH is as follows: Cbz-Phe-Pro-BoroMpg-OH
(20.009, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added the
requisite base in solution in distilled water (190 ml); the base is
added as a 0.2M solution for a monovalent cation. The resultant
clear solution is allowed to react for example by being left to
stand or being agitated, for a usual period, in either case, of
from one to two hours. The reaction is typically carried out at
ambient temperature (e.g. 15-30.degree. C., e.g. 15 to 25.degree.
C.) but alternatively the temperature may be elevated (e.g. up to
30.degree. C., 40.degree. C. or 50.degree. C.). The reaction
mixture is then evacuated to dryness under vacuum with its
temperature not exceeding 37.degree. C., typically to yield a white
brittle solid or an oil/tacky liquid. The oil/tacky liquid is
redissolved in the minimum amount of distilled water necessary (200
ml to 4L), typically with warming (e.g. to 30-40.degree. C.),
usually for up to 2 hours. The solution is filtered, suitably
through filter paper, and evacuated to dryness, again with the
temperature of the solution not exceeding 37.degree. C., or freeze
dried. The resultant product is dried under vacuum overnight to
normally yield a white brittle solid. If the product is present as
an oil or tacky solid then it is dissolved in ethyl acetate and
evacuated to dryness to produce the product as a white solid. The
white solid is typically a coarse, amorphous powder.
[0403] In variations of the aforegoing general procedure, the
acetonitrile is replaced by another water-miscible organic solvent,
notably an alcohol, as discussed above, especially ethanol,
methanol, iso-propanol or another propanol.
[0404] Where a boronic acid salt is less soluble in a selected
reaction medium for salt formation such that its direct preparation
from the corresponding acid and base is inconvenient, the less
soluble salt may be prepared from a salt more soluble in the
reaction medium.
[0405] There is provided also the use of a boronic acid to make a
salt of the disclosure. Included also is a method of preparing a
product of the disclosure, comprising contacting a boronic acid,
e.g. of formula (I), (II) or (III), with a base capable of making
such a salt.
[0406] The peptide boronic acid of formula (I) used to prepare the
pharmaceutical preparations is typically of GLP or GMP quality, or
in compliance with GLP (good laboratory practice) or GMP (good
manufacturing practice); such acids are included in the
disclosure.
[0407] Similarly the acids are usually sterile and/or acceptable
for pharmaceutical use, and one aspect of the disclosure reside in
a composition of matter which is sterile or acceptable for
pharmaceutical use, or both, and comprises a peptide boronic acid
of formula (I). Such a composition of matter may be in particulate
form or in the form of a liquid solution or dispersion.
[0408] The intermediate add may be in isolated form and such
isolated acids are included in the disclosure, especially isolated
acids which are a peptide boronic acid of formula (VIII):
X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2 (VIII)
[0409] wherein X is H (to form NH.sub.2) or an amino-protecting
group.
[0410] One typical way of providing the intermediate acids is as a
particulate composition consisting predominantly of such a peptide
boronic acid, and these compositions are included in the
disclosure. The peptide boronic acid often forms at least 75% by
weight of the composition and typically at least 85% by weight of
the composition, e.g. at least 95% by weight of the
composition.
[0411] Another typical way of providing the intermediate acids is
as a liquid composition consisting of, or consisting essentially
of, a peptide boronic acid of formula (II) and a liquid vehicle in
which it is dissolved or suspended. The liquid vehicle may be an
aqueous medium, e.g. water, or an alcohol, for example methanol,
ethanol, isopropanol, or another propanol, another alkanol or a
mixture of the aforegoing.
[0412] The compositions of the intermediate acids are generally
sterile. The compositions may contain the peptide boronic acid in
finely divided form, to facilitate further processing.
[0413] Separation of Stereoisomers
[0414] The stereoisomers of a peptide boronic ester or a synthetic
intermediate aminoboronate may be resolved in, for example, any
known way. In particular, stereoisomers of boronic esters may be
resolved by HPLC.
EXAMPLES
Examples 1 to 3 and 33
Introductory Remarks
[0415] Apparatus
[0416] Throughout the following procedures of Examples 1 to 3 and
33, standard laboratory glassware and, where appropriate,
specialised apparatus for handling and transferring of air
sensitive reagents are used.
[0417] All glassware is heated at 140-160.degree. C. for at least 4
hours before use and then cooled either in a desiccator or by
assembling hot and purging with a stream of dry nitrogen.
[0418] Solvents
[0419] The organic solvents used in the procedures of Examples 1 to
3 and 33 are all dry. Suitably, they are dried over sodium wire
before use.
[0420] Dryness
[0421] In the drying procedures of Example 1 to 3 and 33, products
are tested for dryness (including dryness in terms of organic
solvent) by observing weight loss on drying. The following
procedure was followed to determine loss on drying: a sample was
placed in a vacuum drier and dried at 40.degree. C. at 100 mbar for
2 hours. Products are considered dry when the decrease in weight
upon drying is less than 0.5% of the total weight of the starting
material.
[0422] Examples 1 to 3 and 33 describe performance of the following
reaction scheme and conversion of the resultant TRI 50c to sodium
and calcium salts thereof: 2223
Example 1
Synthesis of TRI 50D
[0423] Step 1: Z-DIPIN B
[0424] Procedure A
[0425] 17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole)
iodine and 127 ml dry tetrahydrofuran are charged and heated to
reflux. Then 15 ml of a solution of 66 g (608 mmole)
1-chloro-3-methoxypropane in 185 ml dry tetrahydrofuran are added
and stirred under reflux until the vigorous reaction starts. After
the initial exotherm ceases, the solution of
1-chloro-3-methoxypropane is added slowly to maintain gentle reflux
until all the magnesium is consumed. After the reaction is
finished, the reaction mixture is cooled to ambient temperature and
slowly added to a solution of 64.4 g (620 mmole) trimethylborate in
95 ml dry tetrahydrofuran; the latter solution is cooled to below
0.degree. C. and, if it warms up during the course of the reaction,
the reaction mixture must be added to it sufficiently slowly to
maintain the temperature of this solution below 65.degree. C. Upon
complete addition, the reaction mixture is allowed to warm to about
0.degree. C. and stirred for another 60 minutes. Then a solution of
22.4 ml sulfuric acid in 400 ml water is added slowly so as to
maintain the temperature below 20.degree. C. The layers are allowed
to settle and the phases are separated. The aqueous layer is
rewashed three times with 200 ml tert.-butylmethylether. The
combined organic layers are allowed to settle and additional water
separated from this solution is removed. The organic layer is dried
over magnesium sulfate, filtered and evaporated to dryness. The
evaporation residue is filtered from the precipitated solid and the
filtrate dissolved in 175 ml toluene. 34.8 g (292 mmole) pinacol is
charged to the solution followed by stirring at ambient temperature
for not less than 10 hours. The solution is evaporated to dryness,
dissolved in 475 ml n-heptane and washed three times with 290 ml
saturated aqueous solution of sodium hydrogen carbonate. The
n-heptane solution is evaporated to dryness and the evaporation
residue distilled and the fraction with Bp 40-50.degree. C. at
0.1-0.5 mbar recovered.
[0426] Boiling point: 40-50.degree. C./0.1-0.5 mbar
[0427] Yield: 40.9 g (70%) Z-DIPIN B (oil)
[0428] Procedure B
[0429] 17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole)
iodine and 127 ml dry tetrahydrofuran are charged and heated to
reflux. Then 15 ml of a solution of 66 g (608 mmole)
1-chloro-3-methoxypropane in 185 ml dry tetrahydrofuran are added
and stirred under reflux until the vigorous reaction starts. After
the initial exotherm ceases, the solution of
1-chloro-3-methoxypropane is added slowly to maintain gentle
reflux. After the reaction is finished, the reaction mixture is
cooled to ambient temperature and slowly added to a solution of
64.4 g (620 mmole) trimethylborate in 95 ml dry tetrahydrofuran,
maintaining the temperature of this solution below minus 65.degree.
C. Upon complete addition, the reaction mixture is allowed to warm
to about 0.degree. C. and stirred for another 60 minutes. Then a
solution of 22.4 ml sulfuric acid in 400 ml water is added slowly
so as to maintain the temperature below 20.degree. C. The organic
solvent is removed by distillation under vacuum. 300 ml n-heptane
is charged to the aqueous solution of the evaporation residue
followed by addition of 34.8 g (292 mmole) pinacol. The
two-phase-mixture is stirred at ambient temperature for not less
than 2 hours. After allowing the layers to settle, the aqueous
phase is separated. 300 ml n-heptane is charged to the aqueous
solution and the two-phase-mixture is stirred at ambient
temperature for not less than 2 hours. After allowing the layers to
settle, the aqueous phase is separated. The organic layers are
combined and washed once with 200 ml water, followed by 200 ml
saturated sodium hydrogen carbonate solution and two further washes
with 200 ml water each. The n-heptane solution is evaporated to
dryness and the evaporation residue distilled and the fraction with
Bp 40-50.degree. C. at 0.1-0.5 mbar recovered.
[0430] Boiling point: 40-50.degree. C./0.1-0.5 mbar
[0431] Yield: 40.9 g (70-85%) Z-DIPIN B (oil)
[0432] Step 2: Z-DIPIN C
[0433] 16.6 g (164 mmole) diisopropylamine and 220 ml
tetrahydrofuran are charged and cooled to -30 to -40.degree. C. To
this solution 41.8 g (163 mmole) n-butyl lithium, 25% in n-heptane
is added, followed by stirring at 0 to -5.degree. C. for one hour.
This freshly prepared solution of lithium diisopropylamide is
cooled to -30.degree. C. and then added to a solution of 27.9 g
(139 mmole) Z-DIPIN B in 120 ml tetrahydrofuran and 35.5 g (418
mmole) dichloromethane at a temperature between -60 and -75.degree.
C. The solution is stirred at that temperature for half an hour
followed by addition of 480 ml (240 mmole) 0.5N anhydrous
Zinc(II)-chloride in tetrahydrofuran or 32.5 g (240 mmole)
anhydrous solid Zinc(II)-chloride. After stirring at -65.degree. C.
for one hour, the reaction mixture is allowed to warm to ambient
temperature and stirred for another 16-18 hours. The reaction
mixture is evaporated to dryness (i.e. until solvent is removed)
and followed by addition of 385 ml n-heptane. The reaction mixture
is washed with 150 ml 5% sulfuric acid, with 190 ml saturated
sodium hydrogen carbonate solution, and 180 ml saturated sodium
chloride solution. The organic layer is dried over magnesium
sulfate, filtered and evaporated to dryness (i.e. until solvent is
removed). The oily residue is transferred into the next step
without further purification.
[0434] Yield: 19 g (55%) Z-DIPIN C
[0435] Step 3: Z-DIPIN D
[0436] To a solution of 23.8 g (148 mmole) hexamethyldisilazane in
400 ml tetrahydrofuran at -15.degree. C. is added 34.7 g (136
mmole) n-butyl lithium, 25% in n-heptane and stirred for one hour.
The solution is cooled to -55.degree. C. followed by the addition
of 30.6 g (123 mmole) Z-DIPIN C dissolved in 290 ml tetrahydrofuran
and 35 ml tetrahydrofuran to this freshly prepared solution of
LiHMDS. The solution is allowed to warm to ambient temperature and
stirred for 12 hours. The reaction mixture is evaporated to
dryness, the evaporation residue dissolved in 174 ml n-heptane,
washed with 170 ml water and 75 ml saturated sodium chloride
solution. The organic phase is dried over magnesium sulfate,
filtered and evaporated to complete dryness (i.e. until solvent is
removed). The oily residue is dissolved in 100 g n-heptane. This
solution is carried over into the next step without further
purification.
[0437] Yield: 32.2 g (70%) Z-DIPIN D
[0438] Step 4: Z-DIPIN (TRI 50b, Crude)
[0439] A solution of 26.6 g (71 mmole) Z-DIPIN D in 82.6 g
n-heptane is diluted with 60 ml n-heptane and cooled to -60.degree.
C. followed by introduction of 10.5 g (285 mmole) hydrogen
chloride. The reaction mixture is subsequently evacuated and
flushed with nitrogen, while the temperature is increased in
increments of about 20.degree. C. to ambient temperature. The
solvent is removed from the oily precipitate and replaced several
times by 60 ml fresh n-heptane. The oily residue is dissolved in 60
ml tetrahydrofuran (Solution A).
[0440] To a different flask 130 ml tetrahydrofuran, 24.5 g (61.5
mmole) Z-D-Phe-Pro-OH and 6.22 g (61.5 mmole) N-methylmorpholine
are charged and cooled to -20.degree. C. To this solution a
solution of 8.4 g (61.5 mmole) isobutylchloroformate in 20 ml
tetrahydrofuran is added and stirred for 30 minutes, followed by
addition of Solution A at -25.degree. C. Upon complete addition, up
to 16 ml (115 mmole) triethylamine is added to adjust the pH to
9-10, measured using a pH stick. The reaction mixture is allowed to
warm to ambient temperature and stirred for 3 hours, still under
nitrogen. The solvent is evaporated to dryness and the evaporation
residue dissolved in 340 ml tert.-butylmethylether (t-BME). The
solution of Z-DIPIN in t-BME is washed twice with 175 ml 1.5%
hydrochloric acid. The combined acidic washes are given a rewash
with 175 ml t-BME. The combined organic layers are washed with 175
ml water, with 175 ml saturated sodium hydrogen carbonate solution,
with 175 ml 25% sodium chloride solution, dried over magnesium
sulfate and filtered. This solution is carried over into the next
step without further purification.
[0441] Yield: 29.9 g (80%) Z-DIPIN
Example 2
Synthesis of TRI 50D (Diethanolamine Adduct of TRI 50C)
[0442] The starting material used in this Example is the solution
of TRI 50b ("Z-DIPIN") obtained in Example 1. The solution is
carried forward to the synthesis of TRI 50d without further
purification. The solution of Z-DIPIN in t-BME (containing 7.0 g
(11.5 mmole) (R,S,R) TRI 50b, calculated based on HPLC results of
Z-DIPIN) is evaporated to dryness and the evaporation residue
dissolved in 80 ml diethylether. 1.51 g (14.4 mmole) diethanolamine
is added and the mixture heated at reflux for at least 10 hours,
during which process the product precipitates. The suspension is
cooled to 5-10.degree. C., filtered and the filter residue washed
with diethylether.
[0443] To improve chiral and chemical purity the wet filter cake (7
g) is dissolved in 7 ml dichloromethane, cooled to 0-5.degree. C.
and the product precipitated by addition of 42 ml diethylether and
filtered. The isolated wet product is dried at 35.degree. C. in
vacuum or at least 4 hours, until day.
[0444] Yield: 5.5 g (80%) Tri50d
[0445] Melting Point: 140-145.degree. C.
Example 3
Preparation of Sodium Salt of TRI 50C
[0446] 1.5 kg (2.5 mole) TRI 50d Example 2 is dissolved in 10.5 L
dichloromethane. 11 L 2% hydrochloric acid is added and the mixture
is stirred for at most 30 minutes (optimally about 20 minutes) at
room temperature. A precipitate forms in the organic phase. After
stirring, the layers are allowed to settle and separated. The
aqueous layer is rewashed twice with 2.2 L dichloromethane. The
combined organic layers are washed with a solution of 625 g
ammonium chloride in 2.25 L water. (The ammonium chloride buffers
the pH of the aqueous extractions to be within a range of from
about pH 1-2 to about pH 4-5, as strongly acidic conditions might
cleave peptide bonds). The organic phase is dried over magnesium
sulfate, filtered and the filtrate evaporated to dryness. An assay
of the free boronic acid is performed (by the RP HPLC method of
Example 28 for at most 30 mins (optionally about 20 min) at room
temperature) and the amounts of the solvents and base for
conversion of the acid to the salt are calculated. If 2.5 mol of
the free acid is obtained, the evaporation residue is dissolved in
5 L acetonitrile followed by addition of a solution of 100 g (2.5
mole) sodium hydroxide as a 5% solution in 2.2 L water. The
solution is stirred for two hours at ambient temperature (e.g.
15-30.degree. C., optimally room temperature) and then evaporated
in vacuum (of ca. 10 mmHg) at a temperature not exceeding
35.degree. C. The evaporation residue is repeatedly dissolved in
3.5 L fresh acetonitrile and evaporated to dryness to remove traces
of water. If the evaporation residue is dry, it is dissolved in 3 L
acetonitrile (or alternatively in 6 L THF) and slowly added to a
mixture of 32 L n-heptane and 32 L diethylether. The addition is
performed slowly enough to avoid lumping or sticking of the product
and is carried out over a period of not less than 30 minutes. The
precipitated product is filtered off, washed with n-heptane and
dried under vacuum at a temperature initially of about 10.degree.
C. and then increasing to a limit of about 35.degree. C., until
dry.
[0447] Yield: 1.0 kg (70%) Tri50c sodium salt.
Example 4
Alternative Conversion of TRI 50B to TRI 50C
[0448] The synthetic procedures described in this and subsequent
synthetic examples were generally performed under nitrogen and
using dry solvents as supplied from commercial sources.
[0449] 1. Approximately 300 g of TRI 50b, obtained by the HPLC
purification of racemic TRI 50b, were dissolved in approximately
2.5 L diethylether. It is estimated that different batches of TRI
50b had isomeric purities ranging from 85% R,S,R to in excess of
95% R,S,R.
[0450] 2. Approximately 54 ml diethanolamine were added (1:1
stoichiometry with total TRI 50b content), and the mixture was
refluxed at 40.degree. C.
[0451] 3. The precipitated product was removed, washed several
times with diethylether and dried.
[0452] 4. The dry product was dissolved in CHCl.sub.3. Hydrochloric
acid (pH 1) was added and the mixture was stirred approximately 1 h
at room temperature.
[0453] 5. The organic layer was removed and washed with NH.sub.4Cl
solution.
[0454] 6. The organic solvent was distilled off and the residual
solid product was dried.
[0455] Typical yield: Approximately 230 g
Example 5
Preparation of Lithium Salt of TRI 50C
[0456] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added LiOH as a
0.2M solution in distilled water (190 ml). The resultant clear
solution is stirred for 2 hours at room temperature and then
evacuated to dryness under vacuum with its temperature not
exceeding 37.degree. C. The resultant oil/tacky liquid is
redissolved in 500 ml distilled water necessary with light warming
for about 20 minutes. The solution is filtered through filter paper
and evacuated to dryness, again with the temperature of the
solution not exceeding 37.degree. C. The resultant product is dried
under vacuum overnight to normally yield a white brittle solid.
[0457] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0458] Yield 17.89 g.
[0459] Microanalysis:
2 C % Found H % Found N % Found B % Found Metal % Found (Calc.)
(Calc.) (Calc.) (Calc.) (Calc.) 57.14 6.60 7.34 2.07 Li 1.26
(61.03) (6.64) (7.90) (2.03) (1.31)
Example 6
UV/Visible Spectra of Lithium Salt of TRI 50C
[0460] UV/Visible spectra of the salt resulting from the procedure
of Example 5 were recorded in distilled water at 20.degree. C. from
190 nm to 400 nm. The salt gave .lambda..sub.max at 210 and 258 nm.
The weight of the dried salt was then measured for the purposes of
calculating the extinction coefficient. The .lambda..sub.max at 258
nm was used. The extinction coefficient was calculated using the
formula:--
A=.epsilon.cl where
[0461] A is the absorbance
[0462] C is the concentration
[0463] l the path length of the UV cell
[0464] and .epsilon. is the extinction coefficient.
[0465] Extinction coefficient: 451
Example 7
Aqueous Solubility of Lithium Salt of TRI 50C
[0466] The salt used in this Example was made using a modification
of the process described in Example 5. The modified process differs
from that described in that 100 mg of TRI 50c was used as starting
material, the product of the redissolution in water was dried by
freeze drying and the filtration was carried out through a 0.2
.mu.m filter. The salt is believed to contain about 85% of R,S,R
isomer.
[0467] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 370C, the sample filtered and the UV
spectrum measured. The salt left a white residue of undissolved
material. The lithium salt was comparatively soluble and so was
redissolved at 50 mg/ml in the same manner previously
described.
[0468] Solubility when dissolved at 25 mg/ml: 43 mM (23 mg/ml).
[0469] Solubility when dissolved at 50 mg/ml: 81 mM (43 mg/ml).
Example 8
Preparation of Sodium Salt of TRI 50C (TGN 255)
[0470] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added NaOH as a
0.2M solution in distilled water (190 ml). The resultant clear
solution is stirred for 2 hours at room temperature and then
evacuated to dryness under vacuum with its temperature not
exceeding 37.degree. C. The resultant oil/tacky liquid is
redissolved in 500 ml distilled water with light warming for about
15-20 minutes. The solution is filtered through filter paper and
evacuated to dryness, again with the temperature of the solution
not exceeding 37.degree. C. The resultant product is dried under
vacuum overnight to normally yield a white brittle solid. The
product may be present as an oil or tacky solid due to residual
water, in which case it is dissolved in ethyl acetate and evacuated
to dryness to produce the product as a white solid.
[0471] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0472] Yield: Over 50%.
[0473] Microanalysis:
3 C % Found H % Found N % Found B % Found Metal % Found (Calc.)
(Calc.) (Calc.) (Calc.) (Calc.) 59.93 6.47 7.31 1.91 Na 3.81
(59.24) (6.44) (7.67) (1.98) (4.20)
Example 9
UV/Visible Spectra of Sodium Salt of TRI 50C
[0474] UV/Visible spectra of the sodium salt resulting from the
procedure of Example 8 were recorded in distilled water at
20.degree. C. from 190 nm to 400 nm. The salt gave .lambda..sub.max
at 210 and 258 nm. The weight of the dried salt was then measured
for the purposes of calculating the extinction coefficient. The
.lambda..sub.max at 258 nm was used. The extinction coefficient was
calculated using the formula:--
A=.epsilon.cl where
[0475] A is the absorbance
[0476] C is the concentration
[0477] l the path length of the UV cell
[0478] and .epsilon. is the extinction coefficient.
[0479] Extinction coefficient: 415.
Example 10
Aqueous Solubility of Sodium Salt of TRI 50C
[0480] The salt used in this Example was made using a modification
of the process described in Example 8. The modified process differs
from that described in that 100 mg of TRI 50c was used as starting
material, the product of the redissolution in water was dried by
freeze drying and the filtration was carried out through a 0.2
.mu.m filter. The salt is believed to contain about 85% of R,S,R
isomer.
[0481] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 37.degree. C., the sample filtered and
the UV spectrum measured. The salt left a white residue of
undissolved material. The sodium salt was comparatively soluble and
so was redissolved at 50 mg/ml in the same manner previously
described.
[0482] Solubility when dissolved at 25 mg/ml: 44 mM (25 mg/ml).
[0483] Solubility when dissolved at 50 mg/ml: 90 mM (50 mg/ml).
Example 11
Preparation of Potassium Salt of TRI 50C
[0484] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added KOH as a
0.2M solution in distilled water (190 ml). The resultant clear
solution is stirred for 2 hours at room temperature and then
evacuated to dryness under vacuum with its temperature not
exceeding 37.degree. C. The resultant oil/tacky liquid is
redissolved in 1L distilled water with warming to 37.degree. C. for
about 2 hours. The solution is filtered through filter paper and
evacuated to dryness, again with the temperature of the solution
not exceeding 37.degree. C. The resultant product is dried under
vacuum overnight to normally yield a white brittle solid.
[0485] Yield: 14.45 mg.
[0486] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0487] Microanalysis:
4 C % Found H % Found N % Found B % Found Metal % Found (Calc.)
(Calc.) (Calc.) (Calc.) (Calc.) 54.84 6.25 7.02 2.01 K 4.29 (57.55)
(6.26) (7.45) (1.92) (6.94)
Example 12
UV/Visible Spectra of Potassium Salt of TRI 50C
[0488] UV/Visible spectra of the potassium salt resulting from the
procedure of Example 11 were recorded in distilled water at
20.degree. C. from 190 nm to 400 nm. TRI 50C and the salt gave
.lambda..sub.max at 210 and 258 nm. The weight of the dried salt
was then measured for the purposes of calculating the extinction
coefficient. The .lambda..sub.max at 258 nm was used. The
extinction coefficient was calculated using the formula:--
A=.epsilon.cl where
[0489] A is the absorbance
[0490] C is the concentration
[0491] l the path length of the UV cell
[0492] and .epsilon. is the extinction coefficient.
[0493] Extinction coefficient: 438.
Example 13
Aqueous Solubility of Potassium Salt of TRI 50C
[0494] The salt used in this Example was made using a modification
of the process described in Example 11. The modified process
differs from that described in that 100 mg of TRI 50c was used as
starting material, the product of the redissolution in water was
dried by freeze drying and the filtration was carried out through a
0.2 .mu.m filter. The salt is believed to contain about 85% of
R,S,R isomer.
[0495] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 37.degree. C., the sample filtered and
the UV spectrum measured. The salt left a white residue of
undissolved material.
[0496] Solubility when dissolved at 25 mg/ml: 29 mM (16 mg/ml).
Example 14
Preparation of Zinc Salt of TRI 50C
[0497] The relative solubility of zinc hydroxide is such that, if
the hydroxide had been used to prepare the corresponding TRI 50c
salt using the procedure of Example 5, they would not have resulted
in homogeneous salt formation. A new method was therefore developed
to prepare the zinc salt, as described in this and the next
examples.
[0498] TRI 50c sodium salt (2.24 g, 4.10 mM) was dissolved in
distilled water (100 ml) at room temperature and zinc chloride in
THF (4.27 ml, 0.5M) was carefully added with stirring. A white
precipitate that immediately formed was filtered off and washed
with distilled water. This solid was dissolved in ethyl acetate and
washed with distilled water (2.times.50 ml). The organic solution
was evacuated to dryness and the white solid produced dried over
silica in a desiccator for 3 days before microanalysis. Yield 1.20
g.
[0499] .sup.1H NMR 400 MHz, .delta..sub.H(CD.sub.3OD) 7.23-7.33
(20H, m, ArH), 5.14 (4H, m, PhCH.sub.2O), 4.52 (4H, m, aCH), 3.65
(2H, m), 3.31 (12H, m), 3.23 (6H, s, OCH.sub.3), 2.96 (4H, d, J 7.8
Hz), 2.78 (2H, m), 2.58 (2H, m), 1.86 (6H, m), 1.40 (10H, m).
[0500] .sup.13C NMR 75 MHz .delta..sub.C(CD.sub.3OD) 178.50,
159.00, 138.05, 137.66, 130.54, 129.62, 129.50, 129.07, 128.79,
128.22, 73.90, 67.90, 58.64, 58.18, 56.02, 38.81, 30.06, 28.57,
28.36, 25.29.
[0501] FTIR (KBr disc) .nu..sub.max (cm.sup.-1) 3291.1, 3062.7,
3031.1, 2932.9, 2875.7, 2346.0, 1956.2, 1711.8, 1647.6, 1536.0,
1498.2, 1452.1, 1392.4, 1343.1, 1253.8, 1116.8, 1084.3, 1027.7,
916.0, 887.6, 748.6, 699.4, 595.5, 506.5.
Example 15
Preparation of Arginine Salt of TRI50C
[0502] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added arginine as
a 0.2M solution in distilled water (190 ml). The resultant clear
solution is stirred for 2 hours at room temperature and then
evacuated to dryness under vacuum with its temperature not
exceeding 37.degree. C. The resultant oil/tacky liquid is
redissolved in 2L distilled water with warming to 37.degree. C. for
2 hours. The solution is filtered through filter paper and
evacuated to dryness, again with the temperature of the solution
not exceeding 37.degree. C. The resultant product is dried under
vacuum overnight to normally yield a white brittle solid.
[0503] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0504] Yield: 10.54 g.
[0505] Microanalysis:
5 C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.)
(Calc.) 52.47 7.12 15.25 1.52 (56.65) (7.20) (14.01) (1.54)
Example 16
UV/Visible Spectra of Arginine Salt of TRI50C
[0506] UV/Visible spectra of the salt resulting from the procedure
of Example 15 were recorded in distilled water at 20.degree. C.
from 190 nm to 400 nm. TRI 50C and the salt gave .lambda..sub.max
at 210 and 258 nm. The weight of the dried salt was then measured
for the purposes of calculating the extinction coefficient. The
.lambda..sub.max at 258 nm was used. The extinction coefficient was
calculated using the formula:--
A=.epsilon.cl where
[0507] A is the absorbance
[0508] C is the concentration
[0509] l the path length of the UV cell
[0510] and .epsilon. is the extinction coefficient.
[0511] Extinction coefficient: 406.
Example 17
Aqueous Solubility of Arginine Salt of TRI50C
[0512] The salt used in this Example was made using a modification
of the process described in Example 15. The modified process
differs from that described in that 100 mg of TRI 50c was used as
starting material, the product of the redissolution in water was
dried by freeze drying and the filtration was carried out through a
0.2 .mu.m filter. The salt is believed to contain about 85% of
R,S,R isomer.
[0513] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 37.degree. C., the sample filtered and
the UV spectrum measured. The salt left a white residue of
undissolved material.
[0514] Solubility when dissolved at 25 mg/ml: 14 mM (10 mg/ml).
Example 18
Preparation of Lysine Salt of TRI50C
[0515] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added L-lysine as
a 0.2M solution in distilled water (190 ml). The resultant clear
solution is stirred for 2 hours at room temperature and then
evacuated to dryness under vacuum with its temperature not
exceeding 37.degree. C. The resultant oil/tacky liquid is
redissolved in 3L distilled water with warming to 37.degree. C. for
2 hours. The solution is filtered through filter paper and
evacuated to dryness, again with the temperature of the solution
not exceeding 37.degree. C. The resultant product is dried under
vacuum overnight to normally yield a white brittle solid. The
product may be present as an oil or tacky solid (due to residual
water), in which case it is then dissolved in ethyl acetate and
evacuated to dryness to produce the product as a white solid.
[0516] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0517] Yield: 17.89.
[0518] Microanalysis:
6 C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.)
(Calc.) 57.03 7.43 10.50 1.72 (59.11) (7.36) (10.44) (1.61)
Example 19
UV/Visible Spectra of Lysine Salt of TRI50C
[0519] UV/Visible spectra of the salt resulting from the procedure
of Example 18 were recorded in distilled water at 20.degree. C.
from 190 nm to 400 nm. TRI 50C and the salt gave .lambda..sub.max
at 210 and 258 nm. The weight of the dried salt was then measured
for the purposes of calculating the extinction coefficient. The
.lambda..sub.max at 258 nm was used. The extinction coefficient was
calculated using the formula:--
A=.epsilon.cl where
[0520] A is the absorbance
[0521] C is the concentration
[0522] l the path length of the UV cell
[0523] and .epsilon. is the extinction coefficient.
[0524] Extinction coefficient: 437.
Example 20
Aqueous Solubility of Lysine Salt of TRI50C
[0525] The salt used in this Example was made using a modification
of the process described in Example 18. The modified process
differs from that described in that 100 mg of TRI 50c was used as
starting material, the product of the redissolution in water was
dried by freeze drying and the filtration was carried out through a
0.2 .mu.m filter. The salt is believed to contain about 85% of
R,S,R isomer.
[0526] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 37.degree. C., the sample filtered and
the UV spectrum measured. The salt left a white residue of
undissolved material.
[0527] Solubility when dissolved at 25 mg/ml: 13 mM (8.6
mg/ml).
Example 21
Preparation of N-METHYL-D-GLUCAMINE Salt of TRI50C
[0528] Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 4
(20.00 g, 38.1 mM) is dissolved in acetonitrile (200 ml) with
stirring at room temperature. To this solution is added
N-methyl-D-glucamine as a 0.2M solution in distilled water (190
ml). The resultant clear solution is stirred for 2 hours at room
temperature and then evacuated to dryness under vacuum with its
temperature not exceeding 37.degree. C. The resultant oil/tacky
liquid is redissolved in 500 ml distilled water with light warming
for about 20 minutes. The solution is filtered through filer paper
and evacuated to dryness, again with the temperature of the
solution not exceeding 37.degree. C., or freeze dried. The
resultant product is dried under vacuum overnight to normally yield
a white brittle solid.
[0529] The salt was then dried under vacuum over silica to constant
weight (72 h).
[0530] Yield: 21.31 g.
[0531] Microanalysis:
7 C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.)
(Calc.) 56.67 7.28 7.74 1.63 (56.67) (7.41) (7.77) (1.50)
Example 22
UV/Visible Spectra of N-METHYL-D-GLUCAMINE Salt of TRI50C
[0532] UV/Visible spectra of the salt resulting from the procedure
of Example 21 were recorded in distilled water at 20.degree. C.
from 190 nm to 400 nm. TRI 50C and the salt gave .lambda..sub.max
at 210 and 258 nm. The weight of the dried salt was then measured
for the purposes of calculating the extinction coefficient. The
.lambda..sub.max at 258 nm was used. The extinction coefficient was
calculated using the formula:--
A=.epsilon.cl where
[0533] A is the absorbance
[0534] C is the concentration
[0535] l the path length of the UV cell
[0536] and .epsilon. is the extinction coefficient.
[0537] Extinction coefficient: 433.
Example 23
Aqueous Solubility of N-METHYL-D-GLUCAMINE Salt of TRI50C
[0538] The salt used in this Example was made using a modification
of the process described in Example 21. The modified process
differs from that described in that 100 mg of TRI 50c was used as
starting material, the product of the redissolution in water was
dried by freeze drying and the filtration was carried out through a
0.2 .mu.m filter. The salt is believed to contain about 85% of
R,S,R isomer.
[0539] To determine maximum aqueous solubility 25 mg of the dried
salt were shaken in water at 37.degree. C., the sample filtered and
the UV spectrum measured. The salt was observed to fully dissolve.
The salt was comparatively soluble and so was redissolved at 50
mg/ml in the same manner previously described.
[0540] Solubility when dissolved at 25 mg/ml: 35 mM (25 mg/ml).
[0541] Solubility when dissolved at 50 mg/ml: 70 mM (50 mg/ml).
Example 24
Alternative Preparation of Arginine Salt of TRI50C
[0542] The arginine salt is formed simply by adding a slight molar
excess of L-arginine to a solution of 0.2-0.3 mmol of TRI 50c in 10
ml of ethyl acetate. The solvent is evaporated after one hour, and
the residue is triturated twice with hexane to remove excess
arginine.
Example 25
Solubility of TRI50C
[0543] The UV/visible spectra of TRI 50c resulting from the
procedure of Example 5 and its solubility were obtained as
described above in relation to the salts. The solubility of TRI 50c
when dissolved at 50 mg/ml was 8 mM (4 mg/ml).
[0544] .sup.13C NMR 75 MHz .delta..sub.C(CD.sub.3C(O)CD.sub.3)
206.56, 138.30, 130.76, 129.64, 129.31, 129.19, 129.09, 128.20,
128.04, 74.23, 73.55, 67.78, 58.76, 56.37, 56.03, 48.38, 47.87,
39.00, 25.42, 25.29.
[0545] FTIR (KBr disc) .nu..sub.max (cm.sup.-1) 3331.3, 3031.4,
2935.3, 2876.9, 2341.9, 1956.1, 1711.6, 1639.9, 1534.3, 1498.1,
1453.0, 1255.3, 1115.3, 1084.6, 1027.6, 917.3, 748.9, 699.6, 594.9,
504.5, 467.8.
Example 26
Analysis of Sodium and Zinc Salts of (R,S,R) TRI 50C
[0546] The following salts were prepared using a boronate:metal
stoichiometry of n:1, where n is the valency of the metal, using
(R,S,R) TRI 50c of higher chiral purity than that used to prepare
the salt described in Example 10.
[0547] A. Sodium Salt
8 Analytical data HPLC or LC/MS: HPLC betabasic C18 Column,
CH.sub.3CN, Water Estimated Purity: >95% by UV
(.lambda..sub.215nm) Micro analysis: Calcd. Found. C: 59.24 59.93
H: 6.44 6.47 N: 7.67 7.31 Other: B: 1.98 1.91 Na: 4.20 3.81
Physical Properties Form: Amorphous solid Colour: White Melting
Point: N/A Solubility: Soluble in aqueous media ca.about.50 mg/ml
M.sub.w: 547.40
[0548] B. Zinc Salt
9 Analytical data HPLC or LC/MS: HPLC betabasic C18 Column,
CH.sub.3CN, Water Estimated Purity: >95% by UV
(.lambda..sub.215nm) Micro analysis: Calcd. Found. C: 58.21 56.20
H: 6.33 6.33 N: 7.54 7.18 Other: B: 1.94 1.84 Zn: 5.87 7.26
Physical Properties Form: Amorphous solid Colour: White Melting
Point: N/A Solubility: Soluble in aqueous media ca.about.2 mg/ml
M.sub.w: 1114.18
[0549] Notes: The trigonal formula of the acid boronate is used in
the calculated microanalyses. It is believed that a lower sodium
salt solubility is reported in example 11 because the salt tested
in example 11 had lower chiral purity.
CONCLUSION
[0550] The sodium and zinc salts have all been prepared with a
stoichiometry of, respectively, one metal ion to one molecule of
TRI 50c and one metal ion to two molecules of TRI 50c. The value
found for the sodium salt is close to and thus consistent with that
calculated for this 1:1 stoichiometry. For the zinc salt an excess
of zinc was found; nonetheless, the zinc salt comprises a
significant proportion of acid boronate.
Example 27
Stability
[0551] An assay of TRI 50c and its sodium and lysine salts before
and after drying.
[0552] Method
[0553] TRI 50c and its Na, Ca and Lys salts were weighed into HPLC
vials and stored in a desiccator over phosphorus pentoxide for 1
week. For sample analysis, 5 mg of dried and non-dried material was
weighed in a 5 mL volumetric flask and dissolved in 1 mL
acetonitrile and filled up with water to 5 mL.
[0554] The compounds were investigated by HPLC. For the impurity
profiles, an HPLC peak area percentage was calculated. The results
are shown in Table 1.
10 TABLE 1 Amount Purity Compound [.mu.g/mL] (% area) TRI 50c dry
1000.0 82.00 TRI 50c non-dried 947.3 85.54 TRI 50c Na salt dry 1024
98.81 TRI 50c Na salt non-dried 1005.8 98.61 TRI 50c Lys salt dry
813.3 90.17 TRI 50c Lys salt non-dried 809.8 92.25
[0555] The purity of the acid was lowered by the drying process but
the purity of the salts was less affected; the purity of the sodium
salt was not significantly reduced. Large differences in response
factors will reduce the actual impurity levels, however.
[0556] This example indicates that the salts of the disclosure,
particularly the metal salts, e.g. alkali metal salts, are more
stable than the acids, notably TRI 50c.
Example 28
Stability
[0557] This Example compares the stability of TRI 50c and TRI 50c
lysine salt when filled into enteric-coated hard gelatin
capsules.
11 1. Tabulated Results Climatic Purity Purity conditions (HPLC %
(HPLC % Area).sup.3 Compound Packing 1.5 month.sup.0) Area) T0 T1
TRI50c capsules 25.degree. C./60% 99 73.9 in blister .sup.
r.h..sup.4 TRI50c capsules 40.degree. C./75% 99 73.9 in blister r.h
TRI50c capsules.sup.1 40.degree. C./75% 99 75.3 r.h TRI50c capsules
25.degree. C./60% 90.2.sup.2) 90.5 Lysine Salt in blister r.h.
TRI50c capsules 40.degree. C./75% 90.2.sup.2 91.8 Lysine Salt in
blister r.h TRI50c capsules.sup.1 40.degree. C./75% 90.2.sup.2 90.6
Lysine Salt r.h Notes: .sup.0)1.5 month storage at given
conditions, samples were then stored at room temperature until
analytical testing. .sup.1capsules stored at the respective
climatic conditions without blister. .sup.2)purity of the batch
before storage. .sup.3purity of the stored batch (capsules were
poured out, the contents of the capsules were then analyzed).
.sup.4r.h. = relative humidity
CONCLUSION
[0558] There was no significant difference in the purities of the
lysine salt at T.sub.0 and T.sub.1.
[0559] 2. Analytical Procedure
[0560] 2.1 Sample Preparation
[0561] 2.1.1 Assay of TRI 50c and Salts
[0562] TRI 50c-standard (free acid) was stored in a desiccator over
phosphorus pentoxide for 2 days for drying. Afterwards, the
reference standard was weighed in a volumetric flask and dissolved
in a mixture of acetonitrile and water (25/75 v/v %). Aliquots of
the resulting solution (ST 1A) were diluted successively with water
as shown in the dilution scheme of table 4.
12 Stock- and Calibration solutions of Tri 50c Net weight Purity
Dissolved Conc. Calibr. mg % Salt-Factor in ml Solvent [.mu.g/ml]
[.mu.g/ml] ST 1 A 40.8 98.23 1 10 ACN/water 4007.8 C4000 25/75 (v/v
%) ml ST [.mu.g/ml] ad ml Solvent [.mu.g/ml] ST 2 A 5 1 A 4007.8 10
water 2003.9 C2000 ST 3 A 5 2 A 2003.9 10 water 1001.9 C1000 ST 4 A
5 3 A 1001.9 10 water 501.0 C500 ST 5 A 5 4 A 500.9 10 water 250.5
C250 ST 6 A 1 3 A 1001.9 10 water 100.2 C100 ST 7 A 1 6 A 100.2 10
water 10.0 C10
[0563] 2.1.2 Impurity Profile of the Stored Capsules
[0564] The stored capsules of every batch at corresponding climatic
condition were removed and 10 mg of the content was weighed in a 10
ml volumetric flask and dissolved in 10 ml of a mixture of
acetonitrile/water (25/75 v/v %). These solutions were injected for
impurity profile analysis and for quantification respectively.
[0565] 3. Data Evaluation
[0566] The quantitative evaluation and the impurity profile
analysis were performed using an HPLC-PDA method. The processing
wavelength was set as 258 nm.
[0567] 4. Analytical Parameters
[0568] 4.1 Equipment and Software
13 Autosampler Waters Alliance 2795 Pump Waters Alliance 2795
Column oven Waters Alliance 2795 Detection Waters 996 diode array,
extracted wavelength 258 nm Software version Waters Millennium
Release 4.0
[0569] 4.2 Stationary Phase
14 Analytical Column ID S71 Material X-Terra .TM. MS C.sub.18, 5
.mu.m Supplier Waters, Eschborn, Germany Dimensions 150 mm .times.
2.1 mm (length, internal diameter)
[0570] 4.3 Mobile Phase
[0571] Aqueous phase: A: 0.1% HCOOH in water
[0572] Organic phase: C: ACN
[0573] Gradient conditions:
15 Time Flow % A % C 0.00 0.5 90 10 27.0 0.5 10 90 27.1 0.5 90 10
30.0 0.5 90 10
Example 29
TRI 50B Inhibition of Platelet Procoagulant Activity
[0574] Platelet pro-coagulant activity may be observed as the
increase, in rate of activation of prothrombin by factor Xa in the
presence of factor Va upon the addition of platelets pretreated
with thrombin, caused by thrombin alone, collagen alone or a
mixture of thrombin and collagen. This property is due to an
increase in anionic phospholipid on the surface of the platelet
with concomitant release of microvesicle from the surface. This is
an essential physiological reaction and people whose platelets have
reduced ability to generate procoagulant activity (Scott syndrome)
show an increased tendency for bleeding.
[0575] Method:
[0576] Washed platelets were treated with either 1.15 nM thrombin,
231 g/ml collagen or a mixture of both at the same concentration at
37.degree. C. TRI 50b was added either for 1 minute prior to the
addition of activator or immediately after the incubation with
activator. Platelet procoagulant activity was determined as
described previously (Goodwin C A et al, Biochem J. 1995 8, 308:
15-21).
[0577] TRI 50b proved to be a potent inhibitor of platelet
procoagulant activity with IC.sub.50's as summarised below:
[0578] Table 2: Influence of TRI 50b on the induction of platelet
procoagulant activity by various agonists:
16TABLE 2 IC50 plus pre- IC50 without Fold acceleration incubation
incubation Agonist without TRI 50b (nM) (nM) Thrombin 30 8 3000
Collagen 45 200 300 Thrombin/Collagen 110 3 80
[0579] Table 2 records, for example, that when platelets were
treated with thrombin they caused a 30-fold acceleration of the
rate of activation of prothrombin in comparison with control
platelets. Treatment with TRI 50 reduced such acceleration by half
at the various TRI 50 concentration levels given. The significant
potency of TRI 50 is evidenced by the fact that the IC.sub.50
values are in the nanomolar range.
[0580] TRI 50b does not have an effect on ADP, collagen or
epinephrine induced aggregation of washed platelets.
Example 30
Rabbit Extracorporeal Shunt Model
[0581] Introduction
[0582] The technique describes an animal model in which a platelet
rich thrombus is produced. The activity of TRI 50b and heparin are
compared.
[0583] The carotid artery and jugular vein of anaesthetised rabbits
were used to create an extracorporeal circuit containing a
suspended foreign surface (silk thread). Thrombus deposition is
initiated by creation of high sheer stress turbulent arterial blood
flow, platelet activation, followed by coagulation in the presence
of thrombogenic surfaces. Histopathological studies have shown that
the thrombus is platelet rich.
[0584] Materials and Methods
[0585] Animals:
[0586] NZW rabbits (males 2.5-3.5 kg) were used. The animals were
allowed food and water up to the induction of anaesthesia.
[0587] Anaesthesia:
[0588] Animals were premedicated with fontanel/fluanisone (Hypnorm)
0.15 ml total by intramuscular injection. General anaesthesia was
induced with methohexitone (10 mg/ml) to effect, followed by
endotracheal intubation. Anaesthesia was maintained with isoflurane
(1-2.0%) carried in oxygen/nitrous oxide.
[0589] Surgical Preparation:
[0590] The animals were placed in dorsal recumbency and the ventral
cervical region prepared for surgery. The left carotid artery and
right jugular vein were exposed. The artery was cannulated with a
large Portex.RTM. catheter (yellow gauge), cut to a suitable
length. The vein was cannulated with a Silastic.RTM. catheter. The
shunt comprised of a 5 cm length of `auto analyser` line
(purple/white gauge). Joins to the shunt on the arterial side were
made with intermediate size Silastic.RTM. tubing. The shunt was
filled with saline before exposure to the circulation. The right
femoral artery was cannulated for the measurement of blood
pressure.
[0591] Thread Preparation and insertion:
[0592] The central section of the shunt contained a thread 3
centimetres in length. This consisted of 000 gauge Gutterman sewing
silk so as to give four strands with a single knot at the end. (The
knot section was outside the shunt).
[0593] Blood Flow
[0594] Blood flow velocity was determined by use of `Doppler`
probes (Crystal Biotech). A silastic probe was positioned over the
carotid artery at the point of insertion of the arterial catheter.
Flow was recorded on a chart recorder using heat sensitive
paper.
17TABLE 3 RESULTS ANTI- TREAT- THROMBUS WEIGHT THROMBOTIC MENT DOSE
AFTER 20 minute run ACTIVITY Control N/A 22.4 .+-. 2.2 mg (n = 5)
TRI 50b 10 mg/kg iv 9.78 .+-. 1.9 mg (n = 5) Active 3.0 mg/kg iv
15.3 .+-. 2.2 mg (n = 5) Active HEPARIN 100 u/kg iv 22.9 .+-. 1.65
mg (n = 4) Inactive 300 u/kg iv 10.5 .+-. 1.4 mg (n = 4) Active
(Severe bleeding)
[0595] Discussion
[0596] Table 3 shows that, under high arterial shear conditions, a
TRI 50b dose of 3 mg/kg to 10 mg/kg iv significantly inhibits
thrombus formation without bleeding, whereas a heparin dose within
the normal clinical range for treating venous thrombosis (100 u/kg
iv heparin) was ineffective. The higher dose of heparin, though
active, caused severe bleeding. These results, which show TRI 50b
effectively inhibiting arterial thrombosis without causing
bleeding, are consistent with TRI 50b inhibiting platelet
procoagulant activity. In contrast, the thrombin inhibitor heparin,
when administered at an approximately equi-effective dose (in terms
of inhibition of arterial thrombosis), produced the severe bleeding
normal when thrombin inhibitors are used to treat arterial
thrombosis.
Example 31
Comparison of Bleeding Times
[0597] The aim of the study was to compare the bleeding times of
heparin with TRI 50b in a suitable model. It is accepted that
heparin is a poor inhibitor of platelet procoagulant activity (J.
Biol. Chem. 1978 Oct. 10; 253(19):6908-16; Miletich 3P, Jackson C
M, Majerus P W1: J. Clin. Invest 1983 May; 71(5): 1383-91).
[0598] Bleeding times were determined in a rat tail bleeding model
following intravenous administration of heparin and TRI 50b. The
doses employed were chosen on the basis of their efficacy in the
rat Wessler and dynamic models and were as follows:
[0599] TRI 50b: 5 and 10 mg/kg
[0600] Heparin: 100 units/kg
[0601] Materials and Methods
[0602] Anaesthesia
[0603] Rats were anaesthetised with sodium pentabarbitone at 60
mg/kg (2.0 ml/kg of 30 mg/ml solution by ip. injection).
Supplemental anaesthetic was given ip. as required.
[0604] Surgical Preparation
[0605] A jugular vein was cannulated for the administration of test
compound. The trachea was also cannulated with a suitable cannula
and the animals allowed to breathe `room air` spontaneously.
[0606] Compound Administration
[0607] These were given in the appropriate vehicle at 1.0 ml/kg
intravenously. Heparin was administered in saline, whilst TRI 50b
was dissolved in ethanol, and then the resultant solution added to
water for injection (1 part ethanol to 5 parts water).
[0608] Technique
[0609] Two minutes following compound administration the distal 2
mm of the animal's tail was sectioned with a new scalpel blade and
the tail immersed in warm saline (37.degree. C.) contained in a
standard `universal` container, so that the blood stream was
clearly visible. The bleeding time recording was started
immediately following transection until the cessation of blood flow
from the tip of the tail. A period of 30 seconds was allowed after
the blood flow from the tail had stopped to ensure that bleeding
did not re-commence, if bleeding did start again the recording time
was continued for up to a maximum of 45 minutes.
[0610] Results
[0611] Table 4 gives a summary of the bleeding results and shows
the increases above base line values.
18 TABLE 4 Bleeding time min Treatment (.+-.SEM.sup..dagger.)
Saline 5.1 .+-. 0.6 Heparin 100 u/kg iv >40* TRI 50b 5 mg/kg iv
11.3 .+-. 1.2 TRI 50b 10 mg/kg iv 30.4 .+-. 5.2 *Severe bleeding in
all animals, with no cessation after 40 minutes. .sup..dagger.SEM =
standard error of the mean
[0612] Discussion
[0613] The results show that TRI 50b was superior to heparin
(produced less bleeding) at all doses. It should be noted that when
100 u/kg heparin is compared with 5 mg/kg TRI 50b, heparin-treated
animals bled more extensively than those receiving TRI 50b; it was
previously established (Example 25) that heparin at a dose of 100
u/kg is a less effective inhibitor of arterial thrombosis than TRI
50b at a dose of 3.0 mg/kg. Heparin is primarily a thrombin
inhibitor and a poor inhibitor of platelet procoagulant activity;
the results are therefore consistent with TRI 50b exerting
anti-coagulant activity by inhibition of platelet coagulant
activity in addition to thrombin inhibiting activity.
Example 32
TRI 50B as a Prodrug for TRI 50C: Pharmacokinetics and
Absorption
[0614] Materials and Methods
[0615] Animals
[0616] Rats, body weight circa 250-300 g were used. The animals
were fasted only on the day of use for the iv stage. Animals were
fasted on the night prior to study for the oral and intraduodenal
studies, water was allowed up to the time of anaesthesia.
19TABLE 5 oral phase Treatment Dose mg/kg po n TRI 50b 20 mg/kg 2
TRI 50c 20 mg/kg 2
[0617]
20TABLE 6 intraduodenal phase Treatment Dose mg/kg po n TRI 50b 20
mg/kg 3 TRI 50c 20 mg/kg 3
[0618] Dose
[0619] Formulation (TRI 50b/TRI 50c) These were dosed in a
formulation prepared as follows: 48 mg/ml of TRI 50b is dissolved
in ethanol: PEG 300 (2:3 vol: vol). Just before administration, 5
volumes of this solution is mixed with 3 volumes of 5% kollidon 17
8F.
[0620] 1) Both compounds were dosed by oral gavage, or directly
into the duodenum, at 20 mg/kg.
[0621] The compounds were dosed in a PEG/ethanol/kollidon
formulation which was prepared immediately before, as described
immediately under the heading "Dose": Stock 15.0 mg/ml. This was
dosed at 1.33 ml/kg (equivalent to 30 mg/kg).
[0622] Methods
[0623] Oral Gavage
[0624] Rats were dosed at 20 mg/kg. Approximately 30 minutes
following dosing the rats were anaesthetised.
[0625] Intraduodenal Administration
[0626] The compounds were instilled directly into the duodenum
after anaesthesia and surgical procedures had been completed.
[0627] Blood sampling
[0628] Oral Phase
[0629] Blood (0.81 ml) was taken from the carotid cannula into
(0.09 ml) of 3.8% w/v tri sodium citrate following anaesthesia and
surgery. The first samples were taken one-hour post dose. Then at,
1.5, 2, 4 hours post dose.
[0630] Intraduodenal Phase
[0631] Blood samples were taken: Pre dose, then at 0.25, 0.5, 0.75,
1.0, 2, 3 and 4 hours post dosing.
[0632] Plasma
[0633] This was obtained by centrifugation (3000 RPM for 10 min)
and stored at -20.degree. C. prior to analysis.
[0634] Results
[0635] Pharmacokinetic Analysis
[0636] FIG. 1: oral phase clearance and kinetics following dosing
with TRI 50b or its free acid (TRI 50c).
[0637] FIG. 2: oral phase clearance and kinetics following
intraduodenal dosing with TRI 50b or its free acid (TRI 50c).
CONCLUSION
[0638] When given by the intraduodenal route TRI 50b achieved a
higher bioavailability (peak plasma concentration) than the free
acid. The data are consistent with TRI 50b being rapidly hydrolysed
in plasma to TRI 50c and with TRI 50c being the active
principle.
[0639] The results of examples 29 to 32 indicate that
administration of TRI 50c as a salt will provide a way to treat
arterial thrombosis and/or venous thrombosis.
Example 33
Preparation of Calcium Salt of TRI 50C (TGN 167)
[0640] 1.5 kg (2.5 mole) TRI 50d from Example 2 is dissolved in
10.5 L dichloromethane. 11 L 2% hydrochloric acid is added and the
mixture is stirred for at most 30 minutes (optimally about 20
minutes) at room temperature. After stirring the layers are allowed
to settle and separated. The aqueous layer is given a rewashed
twice with 2.2 L dichloromethane. The combined organic layers are
washed with a solution of 625 g ammonium chloride in 2.25 L water.
The organic phase is dried over magnesium sulfate, filtered and the
filtrate evaporated to dryness. An assay of the free boronic acid
is performed and the amounts of the solvents and base for
conversion of the acid to the salt are calculated. If 2.5 mol of
the free acid is obtained, the evaporation residue is dissolved in
5 L acetonitrile followed by addition of a suspension of 93 g (1.25
mole) calcium hydroxide in 1 L water. The solution is stirred for
two hours at ambient temperature (e.g. 15-30.degree. C., optimally
room temperature) and then evaporated under vacuum (of ca. 10 mmHg)
at a temperature initially of about 10.degree. C. and then
increasing to a limit of about 35.degree. C. The evaporation
residue is repeatedly dissolved in 3.5 L fresh acetonitrile and
evaporated to dryness to remove traces of water. If the evaporation
residue is dry, it is dissolved in 6 L tetrahydrofuran and slowly
added to a mixture of 32 L n-heptane and 32 L diethylether. The
addition is performed slowly enough to avoid lumping or sticking of
the product and is carried out over a period of not less than 30
minutes. The precipitated product is filtered off, washed with
n-heptane and dried under vacuum (of ca. 10 mmHg) at a temperature
below 35.degree. C. until dry.
[0641] Yield: 0.98 kg (70%) Tri50c calcium salt.
[0642] The procedures of Examples 1 to 3 and 33 may be scaled up
and, if operated carefully, will produce highly pure salts. In the
diethanolamine precipitation step it is important to use 1.25
equivalents of diethanolamine per equivalent of (R,S,R) TRI 50b. In
the hydrolysis of the diethanolamine ester, it is important to
avoid excessively long contact with the aqueous acid. Likewise the
TRI 50b should be synthesised via the Grignard reaction to Z-DIPIN
A.
Example 34
Human Clinical Studies
[0643] In human clinical volunteer studies with doses of up to 2.5
mg/kg i.v. (dosages which significantly prolong the thrombin
clotting time), TRI 50b had no effect on Simplate bleeding time
(i.e. bleeding time measured using a Simplate.RTM. bleeding time
device).
[0644] It will be appreciated from the foregoing that the
disclosure provides boronic acid salts useful for pharmaceutical
purposes and which feature one or more of the following attributes:
(1) improved amount of oral bioavailability; (2) improved
consistency of oral bioavailability; (3) improved stability; and
(4), in any event, not suggested by the prior art.
[0645] The selection of active ingredient for a pharmaceutical
composition is a complex task, which requires consideration not
only of biological properties (including bioavailability) but also
of physicochemical properties desirable for processing, formulation
and storage. Bioavailability itself is dependent on various
factors, often including in vivo stability, solvation properties
and absorption properties, each in turn potentially dependent on
multiple physical, chemical and/or biological behaviours.
[0646] Advantageously, at least preferred products of the
disclosure have adequate absorption and bioavailability. For
commercial utility, a product having less good solubility may be
selected by virtue of a superior overall combination of
properties.
[0647] The present disclosure includes the subject matter of the
following paragraphs:
[0648] 1. A pharmaceutically acceptable base addition salt of a
boronic acid of formula (I): 24
[0649] wherein
[0650] Y comprises a hydrophobic moiety which, together with the
aminoboronic acid residue --NHCH(R.sup.9)--B(OH).sub.2, has
affinity for the substrate binding site of thrombin; and
[0651] R.sup.9 is a straight chain alkyl group interrupted by one
or more ether linkages and in which the total number of oxygen and
carbon atoms is 3, 4, 5 or 6 or R.sup.9 is --(CH.sub.2).sub.m--W
where m is 2, 3, 4 or 5 and W is --OH or halogen (F, Cl, Br or
I).
[0652] 2. A salt of paragraph 1 wherein R.sup.9 is an alkoxyalkyl
group.
[0653] 3. A salt of paragraph 1 or paragraph 2 wherein YCO--
comprises an amino acid which binds to the S2 subsite of thrombin,
the amino acid being N-terminally linked to a moiety which binds
the S3 subsite of thrombin.
[0654] 4. A salt of paragraph 1 or paragraph 2 wherein Y is an
optionally N-terminally protected dipeptide which binds to the S3
and S2 binding sites of thrombin and the peptide linkages in the
acid are optionally and independently N-substituted by a
C.sub.1-C.sub.13 hydrocarbyl optionally containing in-chain or
in-ring nitrogen, oxygen or sulfur and optionally substituted by a
substituent selected from halo, hydroxy and trifluoromethyl.
[0655] 5. A salt of paragraph 4 wherein said dipeptide is
N-terminally protected.
[0656] 6. A salt of paragraph 4 or paragraph 5 wherein all the
peptide linkages in the acid are unsubstituted.
[0657] 7. A salt of any of paragraphs 1 to 6 wherein S3-binding
amino acid residue is of R configuration, the S2-binding residue is
of S configuration, and the fragment --NHCH(R.sup.9)--B(OH) is of R
configuration.
[0658] 8. A salt of any of paragraphs 1 to 7 wherein the boronic
acid has a Ki for thrombin of about 100 nM or less.
[0659] 9. A salt of paragraph 8 wherein the boronic acid has a Ki
for thrombin of about 20 nM or less.
[0660] 10. A pharmaceutically acceptable base addition salt of a
boronic acid of formula (II): 25
[0661] where:
[0662] X is H (to form NH.sub.2) or an amino-protecting group;
[0663] aa.sup.1 is an amino acid having a hydrocarbyl side chain
containing no more than 20 carbon atoms and comprising at least one
cyclic group having up to 13 carbon atoms;
[0664] aa.sup.2 is an imino acid having from 4 to 6 ring
members;
[0665] R.sup.1 is a group of the formula --(CH.sub.2).sub.s-Z,
where s is 2, 3 or 4 and Z is --OH, --OMe, --OEt or halogen (F, Cl,
Br or I).
[0666] 11. A salt of paragraph 10 wherein aa.sup.1 is selected from
Phe, Dpa and wholly or partially hydrogenated analogues
thereof.
[0667] 12. A salt of paragraph 10 wherein aa.sup.1 is selected from
Dpa, Phe, Dcha and Cha.
[0668] 13. A salt of any of paragraphs 10 to 12 wherein aa.sup.1 is
of R-configuration.
[0669] 14. A salt of paragraph 10 wherein aa.sup.1 is (R)-Phe or
(R)-Dpa.
[0670] 15. A salt of paragraph 10 wherein aa.sup.1 is (R)-Phe.
[0671] 16. A salt of any of paragraphs 10 to 15 wherein aa.sup.2 is
a residue of an imino acid of formula (IV) 26
[0672] where R.sup.11 is --CH.sub.2--, --CH.sub.2--CH.sub.2--,
--S--CH.sub.2--, --S--C(CH.sub.3).sub.2-- or
--CH.sub.2--CH.sub.2--CH.sub- .2--, which group, when the ring is
5- or 6-membered, is optionally substituted at one or more
--CH.sub.2-- groups by from 1 to 3 C.sub.1-C.sub.3 alkyl
groups.
[0673] 17. A salt of paragraph 16 wherein aa.sup.2 is of
S-configuration.
[0674] 18. A salt of paragraph 16 wherein aa.sup.2 is an
(S)-proline residue.
[0675] 19. A salt of paragraph 10, wherein aa.sup.1-aa.sup.2 is
(R)-Phe-(S)-Pro.
[0676] 20. A salt of any of paragraphs 10 to 19 wherein the
fragment --NH--CH(R.sup.1)--B(OH).sub.2 is of R configuration.
[0677] 21. A salt of any of paragraphs 10 to 20 wherein R.sup.1 is
2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 3-bromopropyl,
3-chloropropyl or 3-methoxypropyl.
[0678] 22. A salt of any of paragraphs 10 to 20 wherein R.sup.1 is
3-methoxypropyl.
[0679] 23. A salt of paragraph 10 which is a salt of a compound of
formula (VIII):
X-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2 (VIII).
[0680] 24. A salt of any of paragraphs 10 to 23 where X is
R.sup.6--(CH.sub.2).sub.p--C(O)--,
R.sup.6--(CH.sub.2).sub.p--S(O).sub.2-- -,
R.sup.6--(CH.sub.2).sub.p--NH--C(O)-- or
R.sup.6--(CH.sub.2).sub.p--O--- C(O)-- wherein p is 0, 1, 2, 3, 4,
5 or 6 and R.sup.6 is H or a 5 to 13membered cyclic group
optionally substituted by 1, 2 or 3 substituents selected from
halogen, amino, nitro, hydroxy, a C.sub.5-C.sub.6 cyclic group,
C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 alkyl containing, and/or
linked to the cyclic group through, an in-chain 0, the aforesaid
alkyl groups optionally being substituted by a substituent selected
from halogen, amino, nitro, hydroxy and a C.sub.5-C.sub.6 cyclic
group.
[0681] 25. A salt of paragraph 24 wherein said 5 to 13-membered
cyclic group is aromatic or heteroaromatic.
[0682] 26. A salt of paragraph 25 wherein said 5 to 13-membered
cyclic group is phenyl or a 6-membered heteroaromatic group.
[0683] 27. A salt of any of paragraphs 24 to 26 wherein X is
R.sup.6--(CH.sub.2).sub.p--C(O)-- or
R.sup.6--(CH.sub.2).sub.p--O--C(O)-- and p is 0 or 1.
[0684] 28. A salt of any of paragraphs 10 to 22 wherein X is
benzyloxycarbonyl.
[0685] 29. A salt of any of paragraphs 1 to 28 which does not
comprise a choline or ammonium salt.
[0686] 30. A salt of any of paragraphs 1 to 29 which comprises
boronate ions derived from the boronic acid and monovalent
counter-ions.
[0687] 31. A salt of any of paragraphs 1 to 29 which is a salt of
the peptide boronic acid with an alkali metal or a strongly basic
organic nitrogen-containing compound.
[0688] 32. A salt of paragraph 21 wherein the strongly basic
organic nitrogen compound has a pKb of about 7 or more, e.g. about
7.5 or more.
[0689] 33. A salt of paragraph 31 or paragraph 32 wherein the
strongly basic organic nitrogen-containing compound is a guanidine,
a guanidine analogue or an amine.
[0690] 34. A salt of any of paragraphs 1 to 33 which is a salt of
the boronic acid with a metal.
[0691] 35. A salt of any of paragraphs 1 to 28 which is a salt of
the boronic acid with an alkali metal, an aminosugar, a guanidine
or an amine of formula (XI): 27
[0692] where n is from 1 to 6, R.sup.2 is H, carboxylate or
derivatised carboxylate, R.sup.3 is H, C.sub.1-C.sub.4 alkyl or a
residue of a natural or unnatural amino acid.
[0693] 36. A salt of any of paragraphs 1 to 28 which is a salt of
the boronic acid with a guanidine or with an amine of formula (IX):
28
[0694] where n is from 1 to 6, R.sup.2 is H, carboxylate or
derivatised carboxylate, R.sup.3 is H, C.sub.1-C.sub.4 alkyl or a
residue of a natural or unnatural amino acid.
[0695] 37. A salt of paragraph 36 which is a guanidine salt of the
boronic acid.
[0696] 38. A salt of paragraph 37 which is a salt of the boronic
acid with L-arginine or an L-arginine analogue.
[0697] 39. A salt of paragraph 38 wherein the L-arginine analogue
is D-arginine, or the D- or L-isomers of homoarginine, agmatine
[(4-aminobutyl) guanidine], NG-nitro-L-arginine methyl ester, or a
2-amino pyrimidines.
[0698] 40. A salt of paragraph 37 which is a salt of the boronic
acid with a guanidine of formula (VII) 29
[0699] where n is from 1 to 6, R.sup.2 is H, carboxylate or
derivatised carboxylate, R.sup.3 is H, C.sub.1-C.sub.4 alkyl or a
residue of a natural or unnatural amino acid.
[0700] 41. A salt of paragraph 40 where the derivatised carboxylate
forms a C.sub.1-C.sub.4 alkyl ester or amide.
[0701] 42. A salt of paragraph 40 or paragraph 41 wherein the
compound of formula (VII) is of L-configuration.
[0702] 43. A salt of paragraph 37 which is an L-arginine salt of
the peptide boronic acid.
[0703] 44. A salt of paragraph 36 which is a salt of the boronic
acid with an amine of formula (IX).
[0704] 45. A salt of paragraph 41 where the derivatised carboxylate
forms a C.sub.1-C.sub.4 alkyl ester or amide.
[0705] 46. A salt of paragraph 44 or paragraph 45 wherein the amine
of formula (IX) is of L-configuration.
[0706] 47. A salt of paragraph 44 which is an L-lysine salt of the
boronic acid.
[0707] 48. A salt of any of paragraphs 1 to 28 which is a potassium
salt.
[0708] 49. A salt of any of paragraphs 1 to 28 which is a sodium
salt.
[0709] 50. A salt of any of paragraphs 1 to 28 which is a lithium
salt.
[0710] 51. A salt of any of paragraphs 1 to 28 which is an
aminosugar salt.
[0711] 52. A salt of paragraph 51 wherein the aminosugar is a
ring-opened sugar.
[0712] 53. A salt of paragraph 52 wherein the aminosugar is a
glucamine.
[0713] 54. A salt of paragraph 51 wherein the aminosugar is a
cyclic aminosugar.
[0714] 55. A salt of any of paragraphs 51 to 54 wherein the
aminosugar is N-unsubstituted.
[0715] 56. A salt of any of paragraphs 51 to 54 wherein the
aminosugar is N-substituted by one or two substituents.
[0716] 57. A salt of paragraph 56 wherein the or each substituent
is a hydrocarbyl group.
[0717] 58. A salt of paragraph 57 wherein the or each substituent
is selected from the group consisting of C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7 and C.sub.8 alkyl groups
[0718] 59. A salt of any of paragraphs 56 to 58 wherein there is a
single N-substituent.
[0719] 60. A salt of paragraph 51 wherein the glucamine is
N-methyl-D-glucamine.
[0720] 61. A salt of any of paragraphs 1 to 60 which comprises
boronate ions derived from the peptide boronic acid and has a
stoichiometry consistent with the boronate ions carrying a single
negative charge.
[0721] 62. A salt of any of paragraphs 1 to 60 wherein the salt
consists essentially of acid salt in which one B--OH group of the
boronate group, when trigonally represented, remains
protonated.
[0722] 63. A salt of any of paragraphs 1 to 62 wherein the salt
comprises a boronate ion derived from the peptide boronic acid and
a counter-ion and wherein the salt consists essentially of a salt
having a single type of counter-ion.
[0723] 64. A salt of any of paragraphs 1 to 28 which is a
monolithium or monosodium salt.
[0724] 65. A salt of paragraph 23 which is a monolithium or
monosodium salt.
[0725] 66. A solid phase monosodium or monolithium salt, containing
no more than 1% water by weight, of
Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH).sub.2.
[0726] 67. A salt of paragraph 66 which contains no more than 0.5%
water by weight.
[0727] 68. A salt of paragraph 66 which contains no more than 0.1%
water by weight.
[0728] 69. A salt of any of paragraphs 66 to 68 which is the
monosodium salt.
[0729] 70. A salt of any of paragraphs 1 to 65 which is in the
solid phase.
[0730] 71. A salt of paragraph 70 which is substantially dry.
[0731] 72. A product for use as a pharmaceutical, comprising a salt
of any of paragraphs 1 to 71.
[0732] 73. A pharmaceutical formulation in oral dosage form
comprising a salt of any of paragraphs 1 to 71 and a
pharmaceutically acceptable diluent, excipient or carrier.
[0733] 74. A formulation of paragraph 73 which is a solid
formulation.
[0734] 75. A pharmaceutical formulation of paragraph 74 which is
adapted to release the salt in the duodenum.
[0735] 76. A pharmaceutical formulation of paragraph 75 which is
enterically coated.
[0736] 77. The use of a salt of any of paragraphs 1 to 71 for the
manufacture of a medicament for treating thrombosis.
[0737] 78. The use of paragraph 77 wherein the disease is an acute
coronary syndrome.
[0738] 79. The use of paragraph 77 wherein the disease is acute
myocardial infarction.
[0739] 80. The use of paragraph 77 wherein the disease is a venous
thromboembolic event, selected from the group consisting of deep
vein thrombosis and pulmonary embolism.
[0740] 81. The use of a salt of any of paragraphs 1 to 71 for the
manufacture of an oral medicament for preventing thrombosis in a
haemodialysis circuit of a patient, for preventing a cardiovascular
event in a patient with end stage renal disease, for preventing
venous thromboembolic events in a patient receiving chemotherapy
through an indwelling catheter, or for preventing thromboembolic
events in a patient undergoing a lower limb arterial reconstructive
procedure.
[0741] 82. The use of a salt of any of paragraphs 1 to 71 for the
manufacture of an oral medicament for treating by way of therapy or
prophylaxis an arterial disease selected from acute coronary
syndromes, cerebrovascular thrombosis, peripheral arterial
occlusion and arterial thrombosis resulting from atrial
fibrillation, valvular heart disease, arterio-venous shunts,
indwelling catheters or coronary stents.
[0742] 83. An oral pharmaceutical formulation comprising a
combination of (i) a salt of any of paragraphs 1 to 71 and (ii) a
further pharmaceutically active agent.
[0743] 84. An oral pharmaceutical formulation comprising a
combination of (i) a salt of any of paragraphs 1 to 71 and (ii)
another cardiovascular treatment agent.
[0744] 85. A formulation of paragraph 84 wherein the other
cardiovascular treatment agent comprises a lipid-lowering drug, a
fibrate, niacin, a statin, a CETP inhibitor, a bile acid
sequestrant, an anti-oxidant, a IIb/IIIa antagonist, an aldosterone
inhibitor, an A2 antagonist, an A3 agonist, a beta-blocker,
acetylsalicylic acid, a loop diuretic, an ace inhibitor, an
antithrombotic agent with a different mechanism of action, an
antiplatelet agent, a thromboxane receptor and/or synthetase
inhibitor, a fibrinogen receptor antagonist, a prostacyclin
mimetic, a phosphodiesterase inhibitor, an ADP-receptor (P2 T)
antagonist, a thrombolytic, a cardioprotectant or a COX-2
inhibitor.
[0745] 86. A formulation of any of paragraphs 83 to 85 which is
substantially dry.
[0746] 87. The use of a salt of any of paragraphs 1 to 71 for the
manufacture of a medicament for treating, for example preventing, a
cardiovascular disorder in co-administration with another
cardiovascular treatment agent.
[0747] 88. A oral medicament comprising a salt of a boronic acid
which is a selective thrombin inhibitor and has a neutral
aminoboronic acid residue capable of binding to the thrombin S1
subsite linked through a peptide linkage to a hydrophobic moiety
capable of binding to the thrombin S2 and S3 subsites, the salt
comprising a cation having a valency n and having an observed
stoichiometry consistent with a notional stoichiometry (boronic
acid:cation) of n:1.
[0748] 89. A medicament of paragraph 88 wherein the boronic acid
has a Ki for thrombin of about 100 nM or less, and optionally of
about 20 nM or less.
[0749] 90. A pharmaceutical formulation adapted for oral
administration and comprising in the solid phase a compound which
is a source of boronate species corresponding to the acid
Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(- OH).sub.2 and a source of
pharmaceutically acceptable cations other than choline and
ammonium.
[0750] 91. A method for forming a boronic acid of formula (I) or a
product capable of being a source of such a boronic acid in vivo:
30
[0751] wherein
[0752] Y comprises a hydrophobic moiety which, together with the
aminoboronic acid residue --NHCH(R.sup.9)--B(OH).sub.2, has
affinity for the substrate binding site of thrombin; and
[0753] R.sup.9 is a straight chain alkyl group interrupted by one
or more ether linkages and in which the total number of oxygen and
carbon atoms is 3, 4, 5 or 6 or R.sup.9 is --(CH.sub.2).sub.m--W
where m is from 2, 3, 4 or 5 and W is --OH or halogen (F, Cl, Br or
I),
[0754] the method comprising:
[0755] providing a diethylether solution of an ester of the
acid;
[0756] dissolving diethanolamine in the solution;
[0757] allowing or causing a precipitate to form and recovering the
precipitate, and converting the precipitated material into an end
product selected from the free organoboronic acid or a product
capable when in vivo of being a source of such a boronic acid.
[0758] 92. The method of paragraph 91, which further comprises
formulating said end product into a pharmaceutical composition.
[0759] 93. A method for forming a salt of any of paragraphs 1 to
71, comprising: combining an acetonitrile solution of the
corresponding boronic acid with a pharmaceutically acceptable base
to form the salt.
[0760] 94. A method of stabilising an organoboronic acid,
comprising providing it in the form of a salt thereof.
[0761] 95. A method of presenting an organoboronic acid drug in
stabilised form for pharmaceutical use, comprising providing the
organoboronic acid drug in the form of a pharmaceutically
acceptable base addition salt thereof in the solid phase.
* * * * *