CA2080227A1 - Phosphonopeptides with collagenase inhibiting activity - Google Patents
Phosphonopeptides with collagenase inhibiting activityInfo
- Publication number
- CA2080227A1 CA2080227A1 CA002080227A CA2080227A CA2080227A1 CA 2080227 A1 CA2080227 A1 CA 2080227A1 CA 002080227 A CA002080227 A CA 002080227A CA 2080227 A CA2080227 A CA 2080227A CA 2080227 A1 CA2080227 A1 CA 2080227A1
- Authority
- CA
- Canada
- Prior art keywords
- alpha
- leucyl
- methylamide
- hydrogen
- phosphonopropyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108060005980 Collagenase Proteins 0.000 title description 15
- 102000029816 Collagenase Human genes 0.000 title description 15
- 229960002424 collagenase Drugs 0.000 title description 8
- 230000002401 inhibitory effect Effects 0.000 title description 3
- 238000000034 method Methods 0.000 claims abstract description 39
- 230000008569 process Effects 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims description 171
- MGJXBDMLVWIYOQ-UHFFFAOYSA-N methylazanide Chemical compound [NH-]C MGJXBDMLVWIYOQ-UHFFFAOYSA-N 0.000 claims description 63
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 51
- 229910052739 hydrogen Inorganic materials 0.000 claims description 48
- 239000001257 hydrogen Substances 0.000 claims description 48
- 125000000217 alkyl group Chemical group 0.000 claims description 37
- -1 2-imidazolinyl group Chemical group 0.000 claims description 27
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 26
- 150000003839 salts Chemical class 0.000 claims description 25
- MHDVGSVTJDSBDK-UHFFFAOYSA-N dibenzyl ether Chemical compound C=1C=CC=CC=1COCC1=CC=CC=C1 MHDVGSVTJDSBDK-UHFFFAOYSA-N 0.000 claims description 20
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 18
- 150000001408 amides Chemical class 0.000 claims description 16
- 238000011282 treatment Methods 0.000 claims description 14
- 150000002431 hydrogen Chemical group 0.000 claims description 13
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 13
- 239000012453 solvate Substances 0.000 claims description 13
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- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 9
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- CKQYFZPCICOPMQ-VKHMYHEASA-N (2s)-2-amino-n-methylpropanamide Chemical compound CNC(=O)[C@H](C)N CKQYFZPCICOPMQ-VKHMYHEASA-N 0.000 claims description 8
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 7
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- 125000005936 piperidyl group Chemical group 0.000 claims description 7
- 230000015556 catabolic process Effects 0.000 claims description 6
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 claims description 3
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- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical group C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 125000000623 heterocyclic group Chemical group 0.000 claims description 2
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- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims 3
- XTPRSWPAZJPVMR-UHFFFAOYSA-N 2-hydroxyethylazanide Chemical compound [NH-]CCO XTPRSWPAZJPVMR-UHFFFAOYSA-N 0.000 claims 2
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- VFRSADQPWYCXDG-LEUCUCNGSA-N ethyl (2s,5s)-5-methylpyrrolidine-2-carboxylate;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.CCOC(=O)[C@@H]1CC[C@H](C)N1 VFRSADQPWYCXDG-LEUCUCNGSA-N 0.000 description 18
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- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 16
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- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
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- 239000011368 organic material Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
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- 208000028169 periodontal disease Diseases 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 150000003017 phosphorus Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229910003446 platinum oxide Inorganic materials 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 150000004492 retinoid derivatives Chemical class 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 102200066678 rs1554618767 Human genes 0.000 description 1
- 102200021964 rs786205555 Human genes 0.000 description 1
- 102220086214 rs864622379 Human genes 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 230000036555 skin type Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000001593 sorbitan monooleate Substances 0.000 description 1
- 235000011069 sorbitan monooleate Nutrition 0.000 description 1
- 229940035049 sorbitan monooleate Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 201000004415 tendinitis Diseases 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
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- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
Phosphorous derivatives having structure (I), processes for their preparation and their use as collagenase inhibitors.
Description
VVO 91tl5507 PCT/GB91/00~3g - 1 - -- 'i' "'' '~7 PHOSPHONOPEPTIDES ~ITH COLLAGENASE INHIBITING ACTIYITY
The present invention relates to novel ?hosphorus derivatives, processes for their preparation and ~he - us-5 in medicine. In particular, the preser.~ invention -ela-es to thei- use as inhibitors of enzymes o- ths col_agen2^--family o- neutral metallo?roteases, fc- _re-~-n~ 2--"-' _:^
and othe- diseases.
10 The mammalian collagenase familv c~ er.zvmes com-~-ises c numbe- c- proteases, exem?lified Dy in_e-s-i~i2: (-Y?-- --collagenase itsel r, the stromelysins (a so know. 2s proteoglycanases or transins), fibroblas. and polymorphonuclear leucocyte gelat nases (also know-. zc 15 collagen-IV-ases), and 'pump-1' (putative me.allo?ro~e2s_ 1, uterine metalloprotease). Membership of the mammalian collagenase family of proteases is evident by possessior.
of a number of highly characteristic and experimentally verifiable properties. [Goldberc et a'., v. Bic'. Chem.
20 26iC, 6600, 1986; Whitham et al., Biocherr. ~. 2~^, 5'3, 1986, Breathnach e~ 2'., Nucleic Acids Res., ~ c, 198?~ ller et al., Biochem. v., 253, 1~ " ' 98~; C~~
e- al., J. Biol. Chem., 263, 657v, 198&; Mu-phy ~- c ., ~iochem. J., 258, 463, 198C; Quar.-~n e~ a ., ~iocne. .
(N.Y.), 2~, 5327, 198g; ~ir3ced21-.~.anser., ~. Or2' ?a_~
'7, 445, 1988~.
The range o- therapeuti- a??lica- ions o- the inver.~io-described hereinafte- reflects th- fundamer._21 -~ie ^-c^liage-. and othe- p~o~einaceous subst-2res c~ tr~2 collagenase family or enzymes ir. ~he conne-_ive _-ssu-ma~~ _hroughou. the body. A? :-cat ons exter.d _^
c'irica' interven.ions in many d_seases an~ ?henomer.-lnvolvi:sg the dest-uc.ior. o_ col_agen ar.~ o~he- conne^-: :-~issùe comporen~s, and also normr o- ciso-aere^ _:ssu-remodel ing.
SUBSTITUTE SH~ET
WO9ltlSS07 PCT/GB9l/00~38 ~7 ~ 2 --Inhibitors of the collagenase family of enzymes are considered to provide useful treatments for:
arthritic diseases, such as rheumatoid and osteo-arthritis, soft tissue rheumatism, polychond-itis and tendonitis; bone resorption diseases, such as osteoporosis, Paget's disease, hyperpara-hyroidism and cholesteatoma; the enhanced collagen des~ruc~ion tha-occurs in association with diabetes; the recessive classes of dystrophic epidermolysis bullosa; pe-iodontal dlsease and related consequences of sinsival ?rodu_~ion cS
collagenase, or of PMNL collagenase -elease f^l'owln~
cellular infiltration to inflamed gir.siva, _ncluding by combating the greater susceptibili.y of diabetes ?atients to periodontal disease; corneal ulce-ati3r., e.g. that induced by alkali or other burns, by radiation, by v tamin E or retinoid deficiency; ulceration of the skin and gastro-intestinal tract, and abnormal wound healing;
post-operative conditions, including colonic anastomosis, in which collagenase levels are raised; cancer, where members of the collagenase family of enzymes have been implicated in the neo~ascularizat on required tO support tumour growth and survival [-. 3asse~ e. a ~ . ~ Nature, 3a8, 699, 1990] in the tissue remodelling required to accommoda.e the growing primary a-.d secor.d2ry tumours, and in the penetration of tumour cells througr. the basemen_ membrane of the vascular walls du- n me~as~asis; and demyelinating diseases of the cen.-a` and pe-ipheral nervous systems, including syndromes ln whi-h myelin loss is the p-imary pathological even~ an^ .h3se ir. whic:r.
demyelination follows axonal a~-o-;.y. ~he degrad2tion of myelin in these diseases, exem?ii ed by ~ultiple sclerosis, is mediated by membe-s ~- .he c~llagenase family cc enzymes.
SUBSTITU~E S~E~T
~091/15507 PCT/GB91/0053 - 3 - ~ .,7 As a particular example of the therapeutic value of inhibitors of the collagenase family of enzymes such as are disclosed in the present invention, chronic arthritic diseases leading to extensive loss of the collagen, proteoglycan and elactin components of the cartilage, bone and tendons within the joints, should be amenable to treatment with inhibitors of the collagenases, proteoglycanases (stromelysins) and gelatinases cu--en;ly thought to be the major enzymes involved.
These enzymes have been detected in extracts of synovia and cartilage tissue, and have also been extensively studied in tissue cultures of a wide range of connective tissues. Apzrt from control of the biosynthesis, secretion and activation of the enzymes, the most important natural regulation of these enzymes in normal and diseased states, is considered to be the endogenous production of inhibitors such as the family of Tissue Inhibitor of Metalloproteases (TIMPS), and alpha-2 macroglobulin. An imbalance between the local levels of the proteolytic enzymes and natural inhibitors wlll allow destruction of connective tissue components to occur.
The compounds described in the present invention, being synthetic and low molecular weight inhibitors of this family of enzymes, offer a therapeutically useful way -:
which a more normal or non-pathological balance between inhib~tion and enzymic activity can be restored: they thus act to complement and supplement the endogenous enzyme inhibitors. Indeed, because these enzymes usually act only within restricted pericellular environments, before being inactivated by inhibitors c -culating in the blood and present in most inflammatory exudates, the low molecular weight inhibitors disclosed here may be more SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/~53g ~-. . ,~. .~7 effective than endogenous proteinaceous inhibitors tha~
are excluded by their size from the localized regions of connective tissue destruction.
European Patent Application 88310492.9 (Beecham Group) discloses a class of phosphorus derivatives having activity as inhibitors of collagenase and utility ln the treatment of rheumatoid arthritis and related diseases ir, which collagenolytic activity is a contributing facto~.
Novel structurally related compounds have now been discovered, which are collagenase inhibitors and thus ~~
potential utility in the treatment of diseases in which collagenolytic activity and tissue remodelling is implicated.
According to the present invention there is provided a compound of general formula (I), or a salt, solvate or hydrate thereof:
OR N ~ H ~ N
(I) in which, 2 is hydrogen, Cl_6 alkyl or optionally substituted benzyl;
Rl is hydrogen or Cl_6 alkyl;
R2 is C3-6 alkyl;
(CH ) NR R6 ~(CH2)nNHcoR7~ -(CH2)ncON ( 2 q 5 6 -(CH2) NR8c(=NR9)NRsR6 or ~(CH2)n~2l0 where in~ege- fr~m l to 6 and each of R5and R6 is independen~ly SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/00~38 - 5 - 2, ,' v7 hydrogen or alkyl, or R5 and R6 together with the nitroger.
atom to whlch they are bonded form a 5-, 6- or 7-memDere~
ring with an optional oxygen or sulphur atom or ar, optionally substituted second nltrogen atom in the -ing, R7 is alkyl or -(CH2)nNR5R6, R8 is hydrogen or alkyl, R~
is hydrogen or alkyl or Rg and R5 together wi,h .he nitrogen atoms to which they are bonded form an o?_ion211y substi.uted 5-, 6- or 7-membered rins, and Rlo is an optionally substituted piperidyl ring;
m is 1 or 2, and q is 2 to 4; and R~ i~ hydrogen, alkyl, and -CH2-(CH2)~ORl1 or -CH2-(CH2)nOCOR12 or o where n is an integer from 1 to 6; Rl1, R12 and R13 are hydrogen or C1_6alkyl; and R14 is hydroxy or -O-C1_6alkyl or -NR5R6 (where R~ and R6 may be linked to form a heterocyclic ring).
Unless otherwise specified, each aikyl group is prefer2b7y â Cl_8 group, more preferably Cl_6, and may be a strâig..t chain o~ branched.
?~ is p-cferably hydroge.., methyl c- e~hyl, espec~211y hydroge~.
Vaiues ,o_ R' include hydrogen, me_hi , ethyl, iso?ro?y_ and n-_u.yi. As an alkyl g-oup, R is p-efer2bly methyl cr ethyl.
P~2 is preferably a C4 alkyl grou?, such as n-but~l, iso-bu~yl or se~-butyl, especially iso-butyl.
SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/~3X
Z~.,7 - 6 -Values for R3 include -(CH2)nNR5R6 where R5 and R6 are hydrogen or methyl, -(CH2)nNHCOR7 where R7 is -(CH2~mNR5RC
in which m is l and R5 and R6 are hydrogen, ~(CH2)nCONH(CH2)qNR5R6 where q is 2 and R5 and R6 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring, -(CH2)nNR8C(=NRg)NR5R6 where RS, R6, R8 and Rg are all hydrogen, -(CH2)nNR8C(=NRg)NR5R6 where R5 and Rg together with the nitrogen atoms to whi-:^
they are bonded form an optionally substituted 2-imidazollnyl group, -(CH2)nRl0where Rlo is optionally substituted piperidyl,and n is an integer f-om l to 4.
Most preferably R3 is -(CH2)nNR5R6 where n is 3 or 4 and R5 and R6 are both hydrogen or methyl, -(CH2)4NHCOR7 whe~e 7 is CH2NH2, CH2CONH(CH2)2NR5R6 where R5 and R6 are joined together to form a pyrrolidine ring, -(CH2)nNR8C(=NRg)NR5R6 where n is 3 or 4 and R5~ R6, R8 and Rg are all hydrogen, -(CH2)4NR8C(=NRg)NR5R6 where R5 and Rg together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazollnyl sroup and R6 and R8 are both hydrogen, -(CH2)nNHC(=NH)NH2 where n is 3 or 4, and -CH2Rlo where Rlo is 4-piperidyl.
Drefe-re^ values for R4 are methyl, ethyl, -(CH2)2OCH3, 25 -cn (CH3)CO2CH3 and -(CH2)2OH, especially methyl and (CH2) 2 m~ he com?ounds of formula (I) may orm salts with bases e.g. sodium hydroxide. The com?ou-.ds cf fo-mula (I) h-J^
a basic r.itrogen a;om and may form aci~ av._i~ion salts e.c. wi.:. hydrochloric acid. Sucr. com?ounds form part c-r he present invention.
SUBSTITUTE SHEET
~O91/15507 PCT/CB91/0053X
2 ~i~,7 Where compounds of formula (I), or salts thereof, form solvates or hydrates, these also form an aspect of the invention.
.he compounds of formula (I) have at least two, and may have three or more asymmetric centres and therefore exis.
in more than one stereoisomeric form. The inventlor.
extends to all such forms and to mixtures thereof, including racemates, and diastereoisomeric mixtu-es.
Preferred isomers are those having the S-configuration a.
the chiral centre bearing R2 and the S-configuration at the chiral centre bearing R3, marked with an asterisk in formula (I).
The compounds of formula (I) or their salts, solvates or hydrates are preferably in pharmaceutically acceptable form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels.
mhe compounds of formula (I) or their salts, solvates G-hydrates are preferably in substantially pure form.
. substantially pure form will generally contain at leas~
50% by welght, preferably 75%, mo-e preferably 90% and s.ill more preferably 95% or 99% o- more of the compound cf formula I or its salt or solvate.
Compounds of formula (I) or their salts, solvates or hydrates may be isolated as crystalline solids or in tr.-form of foams or gums.
~. preferred pharmaceutlcally accep~able form is the crystalline _orm.
SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/0053g 2;. ~ ~;~ v ~
The present invention provides the compounds of formula (I) or pharmaceutically acceptable salts, solvates or hydrates thereof for use as active therapeutic agents, particularly as agents for treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs, such 2S
musculo-skeletal disorders resulting from collagenoly~i^
activity, pa-ticularly rheumatism and/or a-thritic conditions, and tissue remodelling.
Compounds of formula (I) also have potential utility i-.
the treatment of cancer; for preventing myelin degrada~ o~.
in the central and peripheral nervous system; and in c~ne-conditions in which members of the collagenase family G--neutral metalloproteases have pathological or other roieC.
The present invention also provides a process.for thepreparation of a compound of formula (I) which comprises converting a group R20 to hydrogen by cleaving a group R20 from a compound of formula (II):
OR2l L ~ ~ N
(II
whe-ein R20 is alkyl, optionally substituted phenyl o~
o?tionally substituted benzyl and R2l is hydrogen, alkyl, optiona ly substituted phenyl or optionally substituted benzyl and Rl, R2, R3 and R4 are as defined in formulz (I), and where necessary, converting R2l to hydrogen, an-cptionaliy thereafter converting the compound of formulc (I) to a further compound of formula (I).
WO91/15507 PCT/GB91/G0~3X
- 9 - 2 ~. , , Cleavage of R20, and where necessary R21, may be c2r_1ed out in aqueous acid or alkali or using a trimethylsilyl halide, preferably bromotrimethylsilane, in an ine-.
solvent, for example dichloromethane or acetonitrile.
Benzyl esters may alternatively be removed by hydrogenolysis or other standard debenzylation procedu~es.
Phenyl residues may be removed by hydrogenation ove-platinum oxide.
When both R20 and R21 are alkyl, cleavage o R20 only, _c sive to a compound of formula (II) in which R20 is hydrogen and R21 alkyl, which is a compound of fOr~.ULC (T) in which R is alkyl, may be carried out by treatment wi-h excess alkali under mild conditions, for example with aaueous sodium hydroxide in an alcoholic solvent a~ roor, temperature.
Similarly, where R20 is optionally substituted benzyl and R21 is alkyl, the benzyl group only may be cleaved by hydrogenation to give a compound of formula (II) in which R20 is hydrogen and R21 is alkyl.
Cleavage of an R21 alkyl group may thereafter be ca--iG^
out as described above to give a compound Oc formula (~) 2~ ir, which R is hydrogen.
~hen R ir, 2 compound of formula (I) is hydrogen ar,d R2- i a compound cf formula (II) is not hyàrogen, then cleavage cf both R21 and R20 is conveniently effected in a s_n~le reac~ion. Dreferably R20 and R21 are both alkyl, suc: as methyl c- e,hyl, or benzyl.
', will be appreciated that compounds Or formula (II) ir.
which R21 is hydrogen are themselves compounds of the 3~ invention of formula (I).
SUBSTITUTE SHEET
WO91/15507 PCT/CB91/ ~ 38 ~ ~ ~?'~ lO -A compound of formula (I) in which R3 is -(CH2)nNR8C(=NRg)NR5R6 in which R5, R6, R8 and Rg are as defined in formula (I) may be prepared by reacting ~
compound of formula (I) in which R3 is -(CH2)nNR5R6 in which R5 and R6 are either both hydrogen or one is hydrogen and the other is alkyl with a compound of formuia (IIA) NRg RI5-S ~ NR5R6 (IIA) or a salt thereof in which R5, R6 and Rg are as defined in formula (I) and Rls is Cl-6alkYl- The reaction may be carried out in the presence of a base such as sodium bicarbonate in a suitable solvent such as water.
Compounds of formula (II) may be prepared by treating a compound c' formula (III):
Rl 2 O = P ~ N ~
OR2l (III) SUBSTITUTE SHEET
WO91/15507 PCT/GB91/0053g Z,~ ,-j~'?',,7 n which Rl, R2, R20 and R2l are as defined in formula (II) (except that R2l is not H), with a compound o' formula (IV):
~2N
I~
¦ N~R4 (IV) in which R3 and R4 are as defined in formula (I), and any reactive amine group in R3 is in protected form.
The reac.ion is preferably carried out in the presence of a coupling agent, such as dicyclohexylcarbodiimide or l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride in the presence of l-hydroxybenzotriazole, or using l,l'-carbonyldiimidazole, in an inert solvent such as dichloromethane or acetonitrile.
Compouncs of the fo-mula (II) in which R3 is -(Cn2)n-Rl0 where n and Rlo are as defined in formula (I) can be prepared from the compound of formula (II) in which R3 ls 2~ -(C;2)r-Z where n is as defined i^, fo-mula (I) and Z is an o?.ionally subs'ituted pyridyl ring, by hydrogenation in the presence of a noble metal catalys..
It will be appreciated that when R3 is in protected form, the ?rc~ecting group may be chose~. to undergo concomitant cleavage with R20 and/or R2l.
SU~3STITUTE SHEET
WO91/15507 PCT1GB91tO0538 .J, .~7 --i2--Selective cleavage of the group R2l may then be carried out using the procedures described above fo; the preparation of compounds of formula (I) to give compounds of formula (II) in which R2l is hydrogen.
s The in~ermediate compounds cf formula (I-T) may be prepared by treating a compound of formu!a (V) o_ 2 s21-thereof:
Rl ~20 ~
O _ p NH2 (v) in which Rl, R20 and R2l are as defined in formula (III), with a compound of formula (VIA) or (VIB) or a salt thereof:
R~5 CO2Rl7 O C2Rl7 (VI~.) in whlch R2 is as de-ined ir. formul2 ( ), R,6 is a ie2vi.^
g-~? such 2S haloger., me.hanesulpho..yloxy c-t- luorome.hanesulphonyloxy and Rl7-s hydrogen o_ c c2-boxyl protecting g-oup, and there-~te- removing 2r. R~-, ca-boxyi pro~ec:ins g-oup. The pre erred method is the rea-:lo.; c_ (V) with (VI~.).
WO91/15507 PCT/GB91/00~38 - l3 _ 2~ ,7 When a compound of formula (VIB) is used, the reductlve amination may be carried out by hydrogenation over a noble metal catalyst such as palladium on carbon or by reac~io~.
with sodium cyanoborohydride at pH 6 to 7. Lower alkyl alcohol solvents such as methanol and ethanol are suitab~e for both reactions. These reactions may be carried o the presence of molecular sieves.
A hydrogenation reaction is preferred bu_ this ?rocess precludes the use of compounds of formulae (V) anc (V -) any of R20~ R2l or Rl7 is benzy'. ~refe-2bly c carboxyl protecting group is a methyl or e~hyl es,e-.
Ester protecting groups may be removed under standard basic hydrolysis conditions using dilute base such as I
Normal aqueous sodium hydroxide in methanol, or aaueous potassium hydroxide in l,4-dioxane.
When the compound of formula (V) is in the form of the free base, the compound of formula (VIB) is suitably an ~-keto ester (Rl7 = alkyl).
~hen the compound of formula (V) is a sal~, suc,- as ~he hydrochloride salt, the compound of formula (VIB) is ~ui.ably a salt of an ~-keto acid (Rl7 = Y.), ~or exam-le the sodium salt.
The p-eparation of compounds of formula (~) uslns 2 compound of formula (VIA) may be ca-r~ed ou_ unde-standa_d alkylation c,onditions. A halogen leaving g-ou~
is prefe-ably bromine and an oxycen-base~ leavln g-o_- s preferably ~-ifluoromethanesulphonyloxy.
SUBSTITUTE SHEET
~r ~
Compounds of formula (III) may alternatively be prepare~
by condensing a compound of formula (VII) or a salt thereof:
H2N CO2Rl7 (VII) in which R2is as defined in form_la (I) and Rl7 is a carboxyl protecting group with an aldehyde, Rl-CHO i., which Rl is as defined in formula (I~ and trea~in~ the condensation product with an appropriate dialkyl or trialkyl phosphite, for example dimethyl phosphite, and thereafter removing the carboxyl protecting group. The carboxyl group is conveniently protected as an alkyl or benzyl ester which may be removed using standard hydrolysis or hydrogenation conditions.
As described above in connection with reductive amination of compounds of formula (VIB), where a benzyl protecting group Rl7 is removed by hydrogenation then R20 and R21 2re restricted to alkyl.
~lternatively, compounds of formula (II) in which R20 and R2l are alkyl or optionally subst-~uted benzyl may be prepared by the reaction of a com?ound of formulz (VII, ):
U2N ~ H ~ ~ ~ R4 (VIII) SUE~STITUTE SHEET
WO91/15507 PCT/GB91/~K38 ~. S,~7 in which R2, R3 and R4 are as defined in formula (I), wit:r.
a compound o' formula (IX):
R
~ol (IX) in which Rl lS as defined in fcrmula (T), R20 and R21 are alkyl, optionally substituted phenyl, or op~ionally substituted benzyl and Rl6 is a leaving group as defined for formula (VIA), in the presence of a base such as 1~ triethylamine or Proton Sponge (l,8-bis(dimethylamino)-naphthalene), or using anhydrous potassium carbonate in an alcoholic solvent.
Where Rl6 is an oxygen-based leaving group, for example trifluoromethanesulphonyloxy, which is preferred, displacement of the leaving group is conveniently carried out in the presence of Proton Sponge in an inert solven~
such as acetonitrile or dichloromethane, over a period o_ several dAys in the absence of licht.
A further alternative preparation of com?ounds of formula (III) may be carried out by reacting a compound of formulc (IX) as hereinbefore defined with a compound of formula (~II) in which Rl7 is a carboxyl protecting group, usins conditions as described for the reaction of compounds o' formula (VIII) with compounds of _ormula (IX), and thereafte- removing the protectin_ group Rl7.
SUBSTITUTE~ SHEET
WO91/15507 PCT/GB91/~053 Suitable carboxyl protecting groups include alkyl, benzyl, trialkylsilyl, and trialkylsilylethyl groups. A
trialkylsilyl protecting group, for exam?le trimethylsilyl, is especially useful in that it may be readily incorporated, ln situ, for example by addition c hexamethyldisilazane to the reactants in acetonlt-ile r, the presence of triethylamine, and selectively removed ir.
aqueous methanol, without imposing any limitations Gr. the value of R20 and R2l. Other silylating agents include trimethylsilyl chloride and N,N-diethyltrimethylsilylamine.
An Rl7 alkyl carboxyl protecting group may be removed by base hydrolysis, for example using sodium hydroxide in aqueous methanol or potassium hydroxide in aqueous l,4-dioxane.
It will be appreciated that where the carboxyl protecting group Rl7 is alkyl, R20 and R2l may be alkyl, phenyl or benzyl derivatives, but where Rl7 is a benzyl group, R2C
and R2l are limited to alkyl and phenyl.
When compounds of formula (III) are prepared by this route, i~ is preferred that R20 and R2l are benzyl and R~, is t-ifluoromethanesulphonyloY.y in the compound of formu c (IX) and Rl7 is trimethylsllyl o- methyl in the com?oun~
Or formula (VII).
Com?ounds of formula (V II) may be p-e?ared by trez~ing 2 compound of formula (VII):
H2N CG2Rl7 ~VII) ~;UBSTITUTE SHEEl ~91/1~507 PCTtGB9l/~K38 25. ~ ~,.~.~,7 - i7 -in which R2 is as defined in fGrmula (I), R17 is hydrogen and wherein the amino group is optionally protected, wl~h a compound of formula (IV) as hereinbefore ds'ined, in the presence of a coupling agent as hereinbefore describec f~~
the preparation of compounds of formula (II) from compounds of formulae (III) and (IV).
Compounds of formula (IX) may be prepared f~O,.
hydroxyalkylphosphonate derivatives by conve-sion o-^ ths hydroxyl group to the leaving grou? R16 by conven_iona methods. For example, where R16 is tri rl uo-omethane-sulphonyloxy, trifiuoromethanesulphonic anhydride may De added to a solution of the hydroxyalkylphos?honate in an inert solvent such as dichloromethane, the reaction beir.-carried out at reduced tempe-ature under an nert atmosphere, according to the general method of E. Vedejs et al., Journal of Organic Chemistry 50, 2165, (1985).
Hydroxyalkylphosphonate compounds may in turn be prepared by reaction of the corresponding phosphite, for exa~ple dibenzylphosphite, with an aldehyde R1-CHG in which Rl is 2S defined ir. formula (I) accordins tO the general methc~
Gf F. Ie~.ie--Boulle~ and A. Foucaud, Syn~hesis, 915 (i982). ~enzyl and alkyl phos?hites are eithe-commercially available compounas or can be ?repared ^ror.commerc aliy available s~ar~ins ma~erials by stanaard me_hods.
Tntermediate compounds cr formula (~) are ei~he- known 3G compounds o- may be preparec fror. known aminoalkyl phosphonic a_id de-ivatives using standard ?rocedures tc in~roduce ~20 and R21 as required-~-otec~ion of the amine functisn du-ing these reac~ions m2y be necessary.
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~3 f ~ ~ ?'~
Z~.. ,.~,;~,7 Introduction of an R20 or R2l methyl group may be effected by reaction with diazomethane in a suitable inert solven~
Compounds of formula (V) of fixed configuration may be prepared by the general method of R. Jacquier et al., Phosphorus and Sulfur 36, 73, (1988).
Compounds of formula (IV) may be prepared from amino acid derivatives, many of which are commercially available, by conventional alkylation or coupling reactions.
Thus a compound of formula (IV) may be prepared from a compound of formula (X):
NH
Q ~ ~ ~ R4 (X) Q is (CH2)n-Z, -(CH2)nNH2, -(CH2)nNR8C(=NH)NH2, -(CH2)nNR8C(=NH)NO2 or -(CH2)nCO2H, n, Rq and R8 are as defined in formula (I), Z is optionally substituted 2~ pyridyl and Y is a nitrogen protection group, by conversion of Q to R3 and removal of the nitrogen protection group, Y. It will be appreciated tha~ the conversion of Q to R3 may be most readily effected at a later stage, for example conversion of (CH2)nNHC(=NH)NHNO2 to (CH2)nNHC(=NH)NH2 by hydrogenation can be concomitant with hydrogenolysis of R20 and R2l benzyl groups.
SUESTITUTE SHEET
~O91/15507 PCT/GB91/~38 A compound of formula ~IV) in which R3 7 S - (C~2) n~ 520 m~y ~e prepared by alkylation of a compound of formula ~X) ~.
which Q is -(CH2)nNH2 using standard alkylation procedures.
A compound of formula (IV) in which R3 is -(CH2)rNi'-:CG~
may be prepared by reaction of a compound of formula (Y.) in which Q is -(CH2)nNH2 with a carboxylic acid R7CO2;-, in the presence of a cou?ling agent.
A compound of formula (IV) in which R3 is ~(CH2)nCONH(CH2)qNR5R6 may be prepared by reac~ic-. o. c compound of formula (X) in which Q is -(CH2)nCO2~. wltn an amine derivative, NH2(CH2)qNR5R6 in the presence of a coupling agent, and thereafter if R5 or R6 is hydrogen optionally protecting the basic nitrogen atom.
A compound of formula (IV) in which R3 is -(CH2)nNR8C(=NRg)NR5R6 may be prepared from a compound of formula (X) in which Q is -(CH2)nNR8C(=NH)NH2 or -(CH2)nNH2 by N-alkylation and optionally therea-ter p-otecring the basic nitrogen atoms.
~ compound o the formula (IV) in which R3 is -(CH2)r~~10 2; where ~10 is a piperidyl grou? r.~ay be prepared by hyc-oger.ation of a compound o_ _^rmula (X) in whic:l ?~ s -(C:i2)r~Z and optionally thereafrer p-otecting the ? pe-icyl nitrogen atom.
Sui,able ni~rogen protectior. g.ou?s ro- y and fo- an;
p~imary amino function in R3 inciude t-butoxycarbonyl ~BOC) and benzyloxycarbonyi g.ou?s. When R3 is -~CH2)r-R1o where R1o is 9-pipe-_dyl, a suitable nitrogen protec_ing g.oup includes the benzyloxycarbonyl c-ou^.
SUE~STI~Ur~ S~EET
2r~.c~7 Nitrogen protection groups may be removed by standard methods. A t-butoxycarbonyl group may be removed by treatment with trifluoroacetic acid at reduced temperature. Benzyloxycarbonyl groups may be removed by catalytic hydrogenation.
A compound of formula (X) may be prepared from a com?ou?.d of formula (XI):
l~H
Q ~ OH
(XI) wherein Q' is Q in protected form or Q' is a precursor tO
Q and Y and Q are as defined for formula (X).
The reaction may be carried out by reaction with an amine, NH2R4, using standard procedures for forming an amide f-om a carboxylic acid and an amine, for example using a coupling agent such as l,l'-carbonyldiimidazole, l,3-dicyclohexylcarbodiimide or l-ethyl-3-[3-(dime~hy -amino)propyl~carbodiimide, or in the presence of ethyl chloroformate.
Compouncs o' formula (XI) are known compounds o- raJ b-?repared from known starting mate-ials by standa--methods.
-or exa.m~le the compound of form-~12 (XI) in whic- ~' is (CH2)4NHC(O)OCH2Ph and Y is t-butoxycarbonyl is de-ived 'rom lys~ne and is commercially available.
SUP,STITUTE S~EET
wo 91/15507 Pcr/GBgl/0053g - 21 - 2~ -?,7i'~,7 The compound of formula (XI) in which Q' is C;i2CO2C~2Ph and Y is t-butoxycarbonyl is derived from aspa-tic acid and is commercially available.
Compounds of formula (IIA) and (IIB) are commercially available or may be prepared from known star~ing mate-ia s using standard procedures.
The compound of formula (XI) in which Q' is 2 grou?
-(CH2)n~Z where Z is 4-pyridyl and v is t-bu.o:~yca-bonyl is prepared according to the method of 2.L. ~ xler and C.
Niemann, J. Or~. Chem., _, 575 (1958).
The compounds of formula (VII) are either known amino aci~
derivatives or can be made from these derivatives by known methods. Compounds of formula (VIA) and (VIB) are either known compounds or may be prepared from known compounds by known methods.
The intermediates of formula (II) disclosed herein are novel compounds and form an aspect of the present invention as do the described processes for their preparation.
Where obtainable, pharmaceu~ically acceptable salts o' the compounds of formula (I) may be formed conven.ionally by rea-tion with the approp-iate ac d c- base. Solvates may be formed by crystallization from tne appropriate solven~.
30 P.s men.ioned previously, the com?ounds of fo-mula (I) exist in more than one diastereoiso,leric form. Where .he processes of the invention produce m,ixtures thereof, the individual isomers may be separa~ed one from another by chromatography e.g. HPLC.
3~
SUBSTITUTE SHFET
WO91/15507 PCT/GB9l/ ~ 3~
2. . ~ - 22 -Alternatively, separate diastereoisomerlc compounds of formula (I) can be obtained by using stereoisomerically pure starting materials or by separatin~ desired isomers of intermediates at any stage in the overall synthetic process, and converting these intermedia.es to compounds of formula (I) It will be appreciated that where a sinsle ~iasteresisvme-of a compound of formula (I) is p-epa_ed by more than cnG
process variant as hereinbefore described, each of whic-allows a different chiral centre to be defined, it may be possible to deduce the configuration a_ a chi-al centre which is not pre-determined using a par~icular process variant Furthermore, it will be appreciated tha. although the absolute configuration at a particular chiral centre may not be known, it is possible to characterise a given diastereoisomer relative to its epimer by reference to the direction in which the plane of polarised light is rotated The present invention further provides a pharmaceu_lca' composition, which comprises a compoun~ of ~ormula (I),o-2 pharmaceutically acceptable salt o- sGlva-e thereof, and 2 pharmaceu~ically acceptable car-ie-A composition of this invention is uce~ the trea.men~
o. musculo-skeletal disorders, pa-t ~u_crl; arthritic diseases and for modulation of tissue -emode'llns A composition of the invention also has potential utili.y in the treatmen~ of cancer; for preven~ing myelin degradation in the cent-al and pe-i-he-al nervous system;
SU~STITUTE S: lE~T
WO91/15~07 PCT/GB91/00~38 - 23 - ~ ~j~. ~ 7 and in other conditions in which members of the collagenase family of neutral metallopro~eases have patholosical or other roles.
A composition of the invention, which may be prepared by admixture, may contain a diluer.t, binder, filler, disinteg-ant, flavouring agent, colourinc agent, lubrica~.~
o- prese-vative in conventional manner. These conventional excipients may be em?loyed i~ conventional manner, for example as in the prepara~ior. of compositions of related peptide enzyme inhibitors, su^h as the ACE
inhibito- enalapril.
A composition of the invention may be ada?ted for oral, topical, rectal or parenteral administra-ion but oral administration is preferred. Parenteral compositions may be administered intravenously, intramuscularly, intra-articularly, intradermally, subcutaneously or into the cerebro-spinal fluid.
Preferab1y, a pharmaceutical composition of the inventic-.
ls in u-. ~ dosage form and in a form adap~ed for use ir.
the medical or veterinarial fields. ~o- example, such ?re?ara~_ons may be in a pack form a-cor.?anied by writte-.
or printe inst-uctions fo- use as an acent in the ~-eatmer.~ or p-ophylaxis of any of Lhe disorders mentior.ed above .
~he sui.abie dosa e range for the com?ounds of the inven~ion may va-y from compound to com?ound and may depend on the condition to be t-eated. ~t will also depend, inter alia, upon the relation o potency to aDsorbability and the mode of administr2~ion choser..
SUBSTITUTE SHEET
WO91/15507 PCT/GBg1/~3g ~f ~ 3 .,7 2 4 The compound or composition of the invention may be formulated for administration by any route, the preferred route depending upon the disorder for which treatment is required, and is preferably in unit dosage form or in 2 form that a human patient may administer to himself in a single dosage.
Compositions may, îor example, be in the form of tablets, capsules, sachets, vials, powders, c-anules, iozenges, reconstitutable powders, or liquid preparations, fo-example solutions or suspensions, or su??osito ies.
The compositions, for example those suitable for oral administration, may contain conventional excipients such as binding agents, for example syrup, acacia, gela.in, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or phzrm2ceutic211y acceptable wetting agents such as sodium lauryl sulphate.
Solid compositions may be obtained by conventional me~hod--2~ o- blending, 'illing, tableting o- the like. Repeated ~lending operations may be used to d stribute the 2c.ive agen. throughou those com?ositions employing large quantities of fillers. When the com?osition is in the fo~m of a tabiet, powder, o- lozenge, any carrie- suitabl-'o- formulating solid pharmaceutic21 com?ositions may b-used, examples being magnesium stearz-e, starch, glucose, lactose, sucrose, rice flour and cha k. Table_s may be coa~ed according to methods well ~nown in normal pharmaceu~ical practice, in particulz- with an ente-ic SUE~STl-rUTE SHEE~T
WO91/15507 PCT/GB9~/00~38 ~ ,7 coating. The composition may also be i-. the form of ar ingestible capsule, for example of gelat n containing the compound, if desired with a carrier or o_her excipients.
For example, in a hard gelatin capsule containing the required amount of a compound of the invention in the fo-m of a powder or granulate in intimate mi~ re with 2 lubricant, such as magnesium stearate, a filler, such 2s microcrystalline cellulose, and a disin-egrar , such as sodium starch glycollate.
Compositions for oral administration as :iqui~s may be -the form of, for example, emulsions, syr~ps, o- el-i: -s, or may be presented as a dry product fo- reconstitutior.
with water or other suitable vehicle be~ore use. Sucn liquid compositions may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acaciai aqueous or non-aqueous vehicles, which include edible oils, for example almond oil, fractionated coconu~ oil, oily es~e-s, for example esters of glycerine, or propylene glycol, c-ethyl alcohol, glycerine, water or norm21 s21ine;
preservatives, for example methyl or prcpyl p-hydroxybenzoate or sorbic acid; and desired conventional flavouring or colourin agen.ts.
The compounds cr this invention may als- be anminis eren by a non-oral route. In accordar.ce wi.-. ro-~~ir.e prarmaceutical procedure, the compositivns m2y be fo-mulated, for example for rectal administr2~ion 2s 2 su?pository or for parenteral admin-str2~ior in a-lnjectable form. For injection, fo~ ex2mple by intra-articular injection or by .njectio~ ir._o th^
cerebro-spinal fluid or via othe- routes whieh will gai-.
SUBSTITUTE SHEET
~r~
~ 26 -access to sites of demyelination, such as by intramuscular, intradermal or subcutaneous injection, as freely soluble solutions or as poorly dispersed depot stores, the compounds of the invention may be presented in an aqueous or non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable liquid, e.g. sterile pyrogen-free water or a parenterally acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, anti-oxidants or other preservatives, buffers o-solutes to render the solution isotonic with the blood,thickening agents, suspending agents or other pharmaceutically acceptable additives. Such forms will be presented in sterile unit dose form such as ampoules or disposable injectlon devices or in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn or a solid form or concentrate which can be used to prepare an injectable formulation.
For topical and percutaneous administration, the preparations may also be presented as an ointment, cream, lotion, gel, spray, aerosol, wash, skin paint or patch.
A unit dose for treating diseases in which enzymes of the collagenzse family are involved will generally contain from 10 to 1000 mg and preferably will contain from 10 to 500 ms, in particular 10, 50, 100, 150, 200, 250, 300, 35C, 400, 450 or 500 ms. The composition may be administered one or more times a day, for example 2, 3 o-4 times daily, so that the total daily dose for a 70 kg adult will normally be in ~he range 10 to 3000 mg. Such a dosage corresponds to approximately 0.15 to 50 mg/kg per day. Alternatively, in particular fc- injection, the unit dose will contain from 2 to 200 mg of a compound of the invention and be administered in multiples, if desired, to 3~ sive the desired daily dose.
SUBSTITUTE SHEET
The present invention relates to novel ?hosphorus derivatives, processes for their preparation and ~he - us-5 in medicine. In particular, the preser.~ invention -ela-es to thei- use as inhibitors of enzymes o- ths col_agen2^--family o- neutral metallo?roteases, fc- _re-~-n~ 2--"-' _:^
and othe- diseases.
10 The mammalian collagenase familv c~ er.zvmes com-~-ises c numbe- c- proteases, exem?lified Dy in_e-s-i~i2: (-Y?-- --collagenase itsel r, the stromelysins (a so know. 2s proteoglycanases or transins), fibroblas. and polymorphonuclear leucocyte gelat nases (also know-. zc 15 collagen-IV-ases), and 'pump-1' (putative me.allo?ro~e2s_ 1, uterine metalloprotease). Membership of the mammalian collagenase family of proteases is evident by possessior.
of a number of highly characteristic and experimentally verifiable properties. [Goldberc et a'., v. Bic'. Chem.
20 26iC, 6600, 1986; Whitham et al., Biocherr. ~. 2~^, 5'3, 1986, Breathnach e~ 2'., Nucleic Acids Res., ~ c, 198?~ ller et al., Biochem. v., 253, 1~ " ' 98~; C~~
e- al., J. Biol. Chem., 263, 657v, 198&; Mu-phy ~- c ., ~iochem. J., 258, 463, 198C; Quar.-~n e~ a ., ~iocne. .
(N.Y.), 2~, 5327, 198g; ~ir3ced21-.~.anser., ~. Or2' ?a_~
'7, 445, 1988~.
The range o- therapeuti- a??lica- ions o- the inver.~io-described hereinafte- reflects th- fundamer._21 -~ie ^-c^liage-. and othe- p~o~einaceous subst-2res c~ tr~2 collagenase family or enzymes ir. ~he conne-_ive _-ssu-ma~~ _hroughou. the body. A? :-cat ons exter.d _^
c'irica' interven.ions in many d_seases an~ ?henomer.-lnvolvi:sg the dest-uc.ior. o_ col_agen ar.~ o~he- conne^-: :-~issùe comporen~s, and also normr o- ciso-aere^ _:ssu-remodel ing.
SUBSTITUTE SH~ET
WO9ltlSS07 PCT/GB9l/00~38 ~7 ~ 2 --Inhibitors of the collagenase family of enzymes are considered to provide useful treatments for:
arthritic diseases, such as rheumatoid and osteo-arthritis, soft tissue rheumatism, polychond-itis and tendonitis; bone resorption diseases, such as osteoporosis, Paget's disease, hyperpara-hyroidism and cholesteatoma; the enhanced collagen des~ruc~ion tha-occurs in association with diabetes; the recessive classes of dystrophic epidermolysis bullosa; pe-iodontal dlsease and related consequences of sinsival ?rodu_~ion cS
collagenase, or of PMNL collagenase -elease f^l'owln~
cellular infiltration to inflamed gir.siva, _ncluding by combating the greater susceptibili.y of diabetes ?atients to periodontal disease; corneal ulce-ati3r., e.g. that induced by alkali or other burns, by radiation, by v tamin E or retinoid deficiency; ulceration of the skin and gastro-intestinal tract, and abnormal wound healing;
post-operative conditions, including colonic anastomosis, in which collagenase levels are raised; cancer, where members of the collagenase family of enzymes have been implicated in the neo~ascularizat on required tO support tumour growth and survival [-. 3asse~ e. a ~ . ~ Nature, 3a8, 699, 1990] in the tissue remodelling required to accommoda.e the growing primary a-.d secor.d2ry tumours, and in the penetration of tumour cells througr. the basemen_ membrane of the vascular walls du- n me~as~asis; and demyelinating diseases of the cen.-a` and pe-ipheral nervous systems, including syndromes ln whi-h myelin loss is the p-imary pathological even~ an^ .h3se ir. whic:r.
demyelination follows axonal a~-o-;.y. ~he degrad2tion of myelin in these diseases, exem?ii ed by ~ultiple sclerosis, is mediated by membe-s ~- .he c~llagenase family cc enzymes.
SUBSTITU~E S~E~T
~091/15507 PCT/GB91/0053 - 3 - ~ .,7 As a particular example of the therapeutic value of inhibitors of the collagenase family of enzymes such as are disclosed in the present invention, chronic arthritic diseases leading to extensive loss of the collagen, proteoglycan and elactin components of the cartilage, bone and tendons within the joints, should be amenable to treatment with inhibitors of the collagenases, proteoglycanases (stromelysins) and gelatinases cu--en;ly thought to be the major enzymes involved.
These enzymes have been detected in extracts of synovia and cartilage tissue, and have also been extensively studied in tissue cultures of a wide range of connective tissues. Apzrt from control of the biosynthesis, secretion and activation of the enzymes, the most important natural regulation of these enzymes in normal and diseased states, is considered to be the endogenous production of inhibitors such as the family of Tissue Inhibitor of Metalloproteases (TIMPS), and alpha-2 macroglobulin. An imbalance between the local levels of the proteolytic enzymes and natural inhibitors wlll allow destruction of connective tissue components to occur.
The compounds described in the present invention, being synthetic and low molecular weight inhibitors of this family of enzymes, offer a therapeutically useful way -:
which a more normal or non-pathological balance between inhib~tion and enzymic activity can be restored: they thus act to complement and supplement the endogenous enzyme inhibitors. Indeed, because these enzymes usually act only within restricted pericellular environments, before being inactivated by inhibitors c -culating in the blood and present in most inflammatory exudates, the low molecular weight inhibitors disclosed here may be more SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/~53g ~-. . ,~. .~7 effective than endogenous proteinaceous inhibitors tha~
are excluded by their size from the localized regions of connective tissue destruction.
European Patent Application 88310492.9 (Beecham Group) discloses a class of phosphorus derivatives having activity as inhibitors of collagenase and utility ln the treatment of rheumatoid arthritis and related diseases ir, which collagenolytic activity is a contributing facto~.
Novel structurally related compounds have now been discovered, which are collagenase inhibitors and thus ~~
potential utility in the treatment of diseases in which collagenolytic activity and tissue remodelling is implicated.
According to the present invention there is provided a compound of general formula (I), or a salt, solvate or hydrate thereof:
OR N ~ H ~ N
(I) in which, 2 is hydrogen, Cl_6 alkyl or optionally substituted benzyl;
Rl is hydrogen or Cl_6 alkyl;
R2 is C3-6 alkyl;
(CH ) NR R6 ~(CH2)nNHcoR7~ -(CH2)ncON ( 2 q 5 6 -(CH2) NR8c(=NR9)NRsR6 or ~(CH2)n~2l0 where in~ege- fr~m l to 6 and each of R5and R6 is independen~ly SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/00~38 - 5 - 2, ,' v7 hydrogen or alkyl, or R5 and R6 together with the nitroger.
atom to whlch they are bonded form a 5-, 6- or 7-memDere~
ring with an optional oxygen or sulphur atom or ar, optionally substituted second nltrogen atom in the -ing, R7 is alkyl or -(CH2)nNR5R6, R8 is hydrogen or alkyl, R~
is hydrogen or alkyl or Rg and R5 together wi,h .he nitrogen atoms to which they are bonded form an o?_ion211y substi.uted 5-, 6- or 7-membered rins, and Rlo is an optionally substituted piperidyl ring;
m is 1 or 2, and q is 2 to 4; and R~ i~ hydrogen, alkyl, and -CH2-(CH2)~ORl1 or -CH2-(CH2)nOCOR12 or o where n is an integer from 1 to 6; Rl1, R12 and R13 are hydrogen or C1_6alkyl; and R14 is hydroxy or -O-C1_6alkyl or -NR5R6 (where R~ and R6 may be linked to form a heterocyclic ring).
Unless otherwise specified, each aikyl group is prefer2b7y â Cl_8 group, more preferably Cl_6, and may be a strâig..t chain o~ branched.
?~ is p-cferably hydroge.., methyl c- e~hyl, espec~211y hydroge~.
Vaiues ,o_ R' include hydrogen, me_hi , ethyl, iso?ro?y_ and n-_u.yi. As an alkyl g-oup, R is p-efer2bly methyl cr ethyl.
P~2 is preferably a C4 alkyl grou?, such as n-but~l, iso-bu~yl or se~-butyl, especially iso-butyl.
SUBSTITUTE SH~ET
WO91/15507 PCT/GB91/~3X
Z~.,7 - 6 -Values for R3 include -(CH2)nNR5R6 where R5 and R6 are hydrogen or methyl, -(CH2)nNHCOR7 where R7 is -(CH2~mNR5RC
in which m is l and R5 and R6 are hydrogen, ~(CH2)nCONH(CH2)qNR5R6 where q is 2 and R5 and R6 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring, -(CH2)nNR8C(=NRg)NR5R6 where RS, R6, R8 and Rg are all hydrogen, -(CH2)nNR8C(=NRg)NR5R6 where R5 and Rg together with the nitrogen atoms to whi-:^
they are bonded form an optionally substituted 2-imidazollnyl group, -(CH2)nRl0where Rlo is optionally substituted piperidyl,and n is an integer f-om l to 4.
Most preferably R3 is -(CH2)nNR5R6 where n is 3 or 4 and R5 and R6 are both hydrogen or methyl, -(CH2)4NHCOR7 whe~e 7 is CH2NH2, CH2CONH(CH2)2NR5R6 where R5 and R6 are joined together to form a pyrrolidine ring, -(CH2)nNR8C(=NRg)NR5R6 where n is 3 or 4 and R5~ R6, R8 and Rg are all hydrogen, -(CH2)4NR8C(=NRg)NR5R6 where R5 and Rg together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazollnyl sroup and R6 and R8 are both hydrogen, -(CH2)nNHC(=NH)NH2 where n is 3 or 4, and -CH2Rlo where Rlo is 4-piperidyl.
Drefe-re^ values for R4 are methyl, ethyl, -(CH2)2OCH3, 25 -cn (CH3)CO2CH3 and -(CH2)2OH, especially methyl and (CH2) 2 m~ he com?ounds of formula (I) may orm salts with bases e.g. sodium hydroxide. The com?ou-.ds cf fo-mula (I) h-J^
a basic r.itrogen a;om and may form aci~ av._i~ion salts e.c. wi.:. hydrochloric acid. Sucr. com?ounds form part c-r he present invention.
SUBSTITUTE SHEET
~O91/15507 PCT/CB91/0053X
2 ~i~,7 Where compounds of formula (I), or salts thereof, form solvates or hydrates, these also form an aspect of the invention.
.he compounds of formula (I) have at least two, and may have three or more asymmetric centres and therefore exis.
in more than one stereoisomeric form. The inventlor.
extends to all such forms and to mixtures thereof, including racemates, and diastereoisomeric mixtu-es.
Preferred isomers are those having the S-configuration a.
the chiral centre bearing R2 and the S-configuration at the chiral centre bearing R3, marked with an asterisk in formula (I).
The compounds of formula (I) or their salts, solvates or hydrates are preferably in pharmaceutically acceptable form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels.
mhe compounds of formula (I) or their salts, solvates G-hydrates are preferably in substantially pure form.
. substantially pure form will generally contain at leas~
50% by welght, preferably 75%, mo-e preferably 90% and s.ill more preferably 95% or 99% o- more of the compound cf formula I or its salt or solvate.
Compounds of formula (I) or their salts, solvates or hydrates may be isolated as crystalline solids or in tr.-form of foams or gums.
~. preferred pharmaceutlcally accep~able form is the crystalline _orm.
SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/0053g 2;. ~ ~;~ v ~
The present invention provides the compounds of formula (I) or pharmaceutically acceptable salts, solvates or hydrates thereof for use as active therapeutic agents, particularly as agents for treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs, such 2S
musculo-skeletal disorders resulting from collagenoly~i^
activity, pa-ticularly rheumatism and/or a-thritic conditions, and tissue remodelling.
Compounds of formula (I) also have potential utility i-.
the treatment of cancer; for preventing myelin degrada~ o~.
in the central and peripheral nervous system; and in c~ne-conditions in which members of the collagenase family G--neutral metalloproteases have pathological or other roieC.
The present invention also provides a process.for thepreparation of a compound of formula (I) which comprises converting a group R20 to hydrogen by cleaving a group R20 from a compound of formula (II):
OR2l L ~ ~ N
(II
whe-ein R20 is alkyl, optionally substituted phenyl o~
o?tionally substituted benzyl and R2l is hydrogen, alkyl, optiona ly substituted phenyl or optionally substituted benzyl and Rl, R2, R3 and R4 are as defined in formulz (I), and where necessary, converting R2l to hydrogen, an-cptionaliy thereafter converting the compound of formulc (I) to a further compound of formula (I).
WO91/15507 PCT/GB91/G0~3X
- 9 - 2 ~. , , Cleavage of R20, and where necessary R21, may be c2r_1ed out in aqueous acid or alkali or using a trimethylsilyl halide, preferably bromotrimethylsilane, in an ine-.
solvent, for example dichloromethane or acetonitrile.
Benzyl esters may alternatively be removed by hydrogenolysis or other standard debenzylation procedu~es.
Phenyl residues may be removed by hydrogenation ove-platinum oxide.
When both R20 and R21 are alkyl, cleavage o R20 only, _c sive to a compound of formula (II) in which R20 is hydrogen and R21 alkyl, which is a compound of fOr~.ULC (T) in which R is alkyl, may be carried out by treatment wi-h excess alkali under mild conditions, for example with aaueous sodium hydroxide in an alcoholic solvent a~ roor, temperature.
Similarly, where R20 is optionally substituted benzyl and R21 is alkyl, the benzyl group only may be cleaved by hydrogenation to give a compound of formula (II) in which R20 is hydrogen and R21 is alkyl.
Cleavage of an R21 alkyl group may thereafter be ca--iG^
out as described above to give a compound Oc formula (~) 2~ ir, which R is hydrogen.
~hen R ir, 2 compound of formula (I) is hydrogen ar,d R2- i a compound cf formula (II) is not hyàrogen, then cleavage cf both R21 and R20 is conveniently effected in a s_n~le reac~ion. Dreferably R20 and R21 are both alkyl, suc: as methyl c- e,hyl, or benzyl.
', will be appreciated that compounds Or formula (II) ir.
which R21 is hydrogen are themselves compounds of the 3~ invention of formula (I).
SUBSTITUTE SHEET
WO91/15507 PCT/CB91/ ~ 38 ~ ~ ~?'~ lO -A compound of formula (I) in which R3 is -(CH2)nNR8C(=NRg)NR5R6 in which R5, R6, R8 and Rg are as defined in formula (I) may be prepared by reacting ~
compound of formula (I) in which R3 is -(CH2)nNR5R6 in which R5 and R6 are either both hydrogen or one is hydrogen and the other is alkyl with a compound of formuia (IIA) NRg RI5-S ~ NR5R6 (IIA) or a salt thereof in which R5, R6 and Rg are as defined in formula (I) and Rls is Cl-6alkYl- The reaction may be carried out in the presence of a base such as sodium bicarbonate in a suitable solvent such as water.
Compounds of formula (II) may be prepared by treating a compound c' formula (III):
Rl 2 O = P ~ N ~
OR2l (III) SUBSTITUTE SHEET
WO91/15507 PCT/GB91/0053g Z,~ ,-j~'?',,7 n which Rl, R2, R20 and R2l are as defined in formula (II) (except that R2l is not H), with a compound o' formula (IV):
~2N
I~
¦ N~R4 (IV) in which R3 and R4 are as defined in formula (I), and any reactive amine group in R3 is in protected form.
The reac.ion is preferably carried out in the presence of a coupling agent, such as dicyclohexylcarbodiimide or l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride in the presence of l-hydroxybenzotriazole, or using l,l'-carbonyldiimidazole, in an inert solvent such as dichloromethane or acetonitrile.
Compouncs of the fo-mula (II) in which R3 is -(Cn2)n-Rl0 where n and Rlo are as defined in formula (I) can be prepared from the compound of formula (II) in which R3 ls 2~ -(C;2)r-Z where n is as defined i^, fo-mula (I) and Z is an o?.ionally subs'ituted pyridyl ring, by hydrogenation in the presence of a noble metal catalys..
It will be appreciated that when R3 is in protected form, the ?rc~ecting group may be chose~. to undergo concomitant cleavage with R20 and/or R2l.
SU~3STITUTE SHEET
WO91/15507 PCT1GB91tO0538 .J, .~7 --i2--Selective cleavage of the group R2l may then be carried out using the procedures described above fo; the preparation of compounds of formula (I) to give compounds of formula (II) in which R2l is hydrogen.
s The in~ermediate compounds cf formula (I-T) may be prepared by treating a compound of formu!a (V) o_ 2 s21-thereof:
Rl ~20 ~
O _ p NH2 (v) in which Rl, R20 and R2l are as defined in formula (III), with a compound of formula (VIA) or (VIB) or a salt thereof:
R~5 CO2Rl7 O C2Rl7 (VI~.) in whlch R2 is as de-ined ir. formul2 ( ), R,6 is a ie2vi.^
g-~? such 2S haloger., me.hanesulpho..yloxy c-t- luorome.hanesulphonyloxy and Rl7-s hydrogen o_ c c2-boxyl protecting g-oup, and there-~te- removing 2r. R~-, ca-boxyi pro~ec:ins g-oup. The pre erred method is the rea-:lo.; c_ (V) with (VI~.).
WO91/15507 PCT/GB91/00~38 - l3 _ 2~ ,7 When a compound of formula (VIB) is used, the reductlve amination may be carried out by hydrogenation over a noble metal catalyst such as palladium on carbon or by reac~io~.
with sodium cyanoborohydride at pH 6 to 7. Lower alkyl alcohol solvents such as methanol and ethanol are suitab~e for both reactions. These reactions may be carried o the presence of molecular sieves.
A hydrogenation reaction is preferred bu_ this ?rocess precludes the use of compounds of formulae (V) anc (V -) any of R20~ R2l or Rl7 is benzy'. ~refe-2bly c carboxyl protecting group is a methyl or e~hyl es,e-.
Ester protecting groups may be removed under standard basic hydrolysis conditions using dilute base such as I
Normal aqueous sodium hydroxide in methanol, or aaueous potassium hydroxide in l,4-dioxane.
When the compound of formula (V) is in the form of the free base, the compound of formula (VIB) is suitably an ~-keto ester (Rl7 = alkyl).
~hen the compound of formula (V) is a sal~, suc,- as ~he hydrochloride salt, the compound of formula (VIB) is ~ui.ably a salt of an ~-keto acid (Rl7 = Y.), ~or exam-le the sodium salt.
The p-eparation of compounds of formula (~) uslns 2 compound of formula (VIA) may be ca-r~ed ou_ unde-standa_d alkylation c,onditions. A halogen leaving g-ou~
is prefe-ably bromine and an oxycen-base~ leavln g-o_- s preferably ~-ifluoromethanesulphonyloxy.
SUBSTITUTE SHEET
~r ~
Compounds of formula (III) may alternatively be prepare~
by condensing a compound of formula (VII) or a salt thereof:
H2N CO2Rl7 (VII) in which R2is as defined in form_la (I) and Rl7 is a carboxyl protecting group with an aldehyde, Rl-CHO i., which Rl is as defined in formula (I~ and trea~in~ the condensation product with an appropriate dialkyl or trialkyl phosphite, for example dimethyl phosphite, and thereafter removing the carboxyl protecting group. The carboxyl group is conveniently protected as an alkyl or benzyl ester which may be removed using standard hydrolysis or hydrogenation conditions.
As described above in connection with reductive amination of compounds of formula (VIB), where a benzyl protecting group Rl7 is removed by hydrogenation then R20 and R21 2re restricted to alkyl.
~lternatively, compounds of formula (II) in which R20 and R2l are alkyl or optionally subst-~uted benzyl may be prepared by the reaction of a com?ound of formulz (VII, ):
U2N ~ H ~ ~ ~ R4 (VIII) SUE~STITUTE SHEET
WO91/15507 PCT/GB91/~K38 ~. S,~7 in which R2, R3 and R4 are as defined in formula (I), wit:r.
a compound o' formula (IX):
R
~ol (IX) in which Rl lS as defined in fcrmula (T), R20 and R21 are alkyl, optionally substituted phenyl, or op~ionally substituted benzyl and Rl6 is a leaving group as defined for formula (VIA), in the presence of a base such as 1~ triethylamine or Proton Sponge (l,8-bis(dimethylamino)-naphthalene), or using anhydrous potassium carbonate in an alcoholic solvent.
Where Rl6 is an oxygen-based leaving group, for example trifluoromethanesulphonyloxy, which is preferred, displacement of the leaving group is conveniently carried out in the presence of Proton Sponge in an inert solven~
such as acetonitrile or dichloromethane, over a period o_ several dAys in the absence of licht.
A further alternative preparation of com?ounds of formula (III) may be carried out by reacting a compound of formulc (IX) as hereinbefore defined with a compound of formula (~II) in which Rl7 is a carboxyl protecting group, usins conditions as described for the reaction of compounds o' formula (VIII) with compounds of _ormula (IX), and thereafte- removing the protectin_ group Rl7.
SUBSTITUTE~ SHEET
WO91/15507 PCT/GB91/~053 Suitable carboxyl protecting groups include alkyl, benzyl, trialkylsilyl, and trialkylsilylethyl groups. A
trialkylsilyl protecting group, for exam?le trimethylsilyl, is especially useful in that it may be readily incorporated, ln situ, for example by addition c hexamethyldisilazane to the reactants in acetonlt-ile r, the presence of triethylamine, and selectively removed ir.
aqueous methanol, without imposing any limitations Gr. the value of R20 and R2l. Other silylating agents include trimethylsilyl chloride and N,N-diethyltrimethylsilylamine.
An Rl7 alkyl carboxyl protecting group may be removed by base hydrolysis, for example using sodium hydroxide in aqueous methanol or potassium hydroxide in aqueous l,4-dioxane.
It will be appreciated that where the carboxyl protecting group Rl7 is alkyl, R20 and R2l may be alkyl, phenyl or benzyl derivatives, but where Rl7 is a benzyl group, R2C
and R2l are limited to alkyl and phenyl.
When compounds of formula (III) are prepared by this route, i~ is preferred that R20 and R2l are benzyl and R~, is t-ifluoromethanesulphonyloY.y in the compound of formu c (IX) and Rl7 is trimethylsllyl o- methyl in the com?oun~
Or formula (VII).
Com?ounds of formula (V II) may be p-e?ared by trez~ing 2 compound of formula (VII):
H2N CG2Rl7 ~VII) ~;UBSTITUTE SHEEl ~91/1~507 PCTtGB9l/~K38 25. ~ ~,.~.~,7 - i7 -in which R2 is as defined in fGrmula (I), R17 is hydrogen and wherein the amino group is optionally protected, wl~h a compound of formula (IV) as hereinbefore ds'ined, in the presence of a coupling agent as hereinbefore describec f~~
the preparation of compounds of formula (II) from compounds of formulae (III) and (IV).
Compounds of formula (IX) may be prepared f~O,.
hydroxyalkylphosphonate derivatives by conve-sion o-^ ths hydroxyl group to the leaving grou? R16 by conven_iona methods. For example, where R16 is tri rl uo-omethane-sulphonyloxy, trifiuoromethanesulphonic anhydride may De added to a solution of the hydroxyalkylphos?honate in an inert solvent such as dichloromethane, the reaction beir.-carried out at reduced tempe-ature under an nert atmosphere, according to the general method of E. Vedejs et al., Journal of Organic Chemistry 50, 2165, (1985).
Hydroxyalkylphosphonate compounds may in turn be prepared by reaction of the corresponding phosphite, for exa~ple dibenzylphosphite, with an aldehyde R1-CHG in which Rl is 2S defined ir. formula (I) accordins tO the general methc~
Gf F. Ie~.ie--Boulle~ and A. Foucaud, Syn~hesis, 915 (i982). ~enzyl and alkyl phos?hites are eithe-commercially available compounas or can be ?repared ^ror.commerc aliy available s~ar~ins ma~erials by stanaard me_hods.
Tntermediate compounds cr formula (~) are ei~he- known 3G compounds o- may be preparec fror. known aminoalkyl phosphonic a_id de-ivatives using standard ?rocedures tc in~roduce ~20 and R21 as required-~-otec~ion of the amine functisn du-ing these reac~ions m2y be necessary.
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~3 f ~ ~ ?'~
Z~.. ,.~,;~,7 Introduction of an R20 or R2l methyl group may be effected by reaction with diazomethane in a suitable inert solven~
Compounds of formula (V) of fixed configuration may be prepared by the general method of R. Jacquier et al., Phosphorus and Sulfur 36, 73, (1988).
Compounds of formula (IV) may be prepared from amino acid derivatives, many of which are commercially available, by conventional alkylation or coupling reactions.
Thus a compound of formula (IV) may be prepared from a compound of formula (X):
NH
Q ~ ~ ~ R4 (X) Q is (CH2)n-Z, -(CH2)nNH2, -(CH2)nNR8C(=NH)NH2, -(CH2)nNR8C(=NH)NO2 or -(CH2)nCO2H, n, Rq and R8 are as defined in formula (I), Z is optionally substituted 2~ pyridyl and Y is a nitrogen protection group, by conversion of Q to R3 and removal of the nitrogen protection group, Y. It will be appreciated tha~ the conversion of Q to R3 may be most readily effected at a later stage, for example conversion of (CH2)nNHC(=NH)NHNO2 to (CH2)nNHC(=NH)NH2 by hydrogenation can be concomitant with hydrogenolysis of R20 and R2l benzyl groups.
SUESTITUTE SHEET
~O91/15507 PCT/GB91/~38 A compound of formula ~IV) in which R3 7 S - (C~2) n~ 520 m~y ~e prepared by alkylation of a compound of formula ~X) ~.
which Q is -(CH2)nNH2 using standard alkylation procedures.
A compound of formula (IV) in which R3 is -(CH2)rNi'-:CG~
may be prepared by reaction of a compound of formula (Y.) in which Q is -(CH2)nNH2 with a carboxylic acid R7CO2;-, in the presence of a cou?ling agent.
A compound of formula (IV) in which R3 is ~(CH2)nCONH(CH2)qNR5R6 may be prepared by reac~ic-. o. c compound of formula (X) in which Q is -(CH2)nCO2~. wltn an amine derivative, NH2(CH2)qNR5R6 in the presence of a coupling agent, and thereafter if R5 or R6 is hydrogen optionally protecting the basic nitrogen atom.
A compound of formula (IV) in which R3 is -(CH2)nNR8C(=NRg)NR5R6 may be prepared from a compound of formula (X) in which Q is -(CH2)nNR8C(=NH)NH2 or -(CH2)nNH2 by N-alkylation and optionally therea-ter p-otecring the basic nitrogen atoms.
~ compound o the formula (IV) in which R3 is -(CH2)r~~10 2; where ~10 is a piperidyl grou? r.~ay be prepared by hyc-oger.ation of a compound o_ _^rmula (X) in whic:l ?~ s -(C:i2)r~Z and optionally thereafrer p-otecting the ? pe-icyl nitrogen atom.
Sui,able ni~rogen protectior. g.ou?s ro- y and fo- an;
p~imary amino function in R3 inciude t-butoxycarbonyl ~BOC) and benzyloxycarbonyi g.ou?s. When R3 is -~CH2)r-R1o where R1o is 9-pipe-_dyl, a suitable nitrogen protec_ing g.oup includes the benzyloxycarbonyl c-ou^.
SUE~STI~Ur~ S~EET
2r~.c~7 Nitrogen protection groups may be removed by standard methods. A t-butoxycarbonyl group may be removed by treatment with trifluoroacetic acid at reduced temperature. Benzyloxycarbonyl groups may be removed by catalytic hydrogenation.
A compound of formula (X) may be prepared from a com?ou?.d of formula (XI):
l~H
Q ~ OH
(XI) wherein Q' is Q in protected form or Q' is a precursor tO
Q and Y and Q are as defined for formula (X).
The reaction may be carried out by reaction with an amine, NH2R4, using standard procedures for forming an amide f-om a carboxylic acid and an amine, for example using a coupling agent such as l,l'-carbonyldiimidazole, l,3-dicyclohexylcarbodiimide or l-ethyl-3-[3-(dime~hy -amino)propyl~carbodiimide, or in the presence of ethyl chloroformate.
Compouncs o' formula (XI) are known compounds o- raJ b-?repared from known starting mate-ials by standa--methods.
-or exa.m~le the compound of form-~12 (XI) in whic- ~' is (CH2)4NHC(O)OCH2Ph and Y is t-butoxycarbonyl is de-ived 'rom lys~ne and is commercially available.
SUP,STITUTE S~EET
wo 91/15507 Pcr/GBgl/0053g - 21 - 2~ -?,7i'~,7 The compound of formula (XI) in which Q' is C;i2CO2C~2Ph and Y is t-butoxycarbonyl is derived from aspa-tic acid and is commercially available.
Compounds of formula (IIA) and (IIB) are commercially available or may be prepared from known star~ing mate-ia s using standard procedures.
The compound of formula (XI) in which Q' is 2 grou?
-(CH2)n~Z where Z is 4-pyridyl and v is t-bu.o:~yca-bonyl is prepared according to the method of 2.L. ~ xler and C.
Niemann, J. Or~. Chem., _, 575 (1958).
The compounds of formula (VII) are either known amino aci~
derivatives or can be made from these derivatives by known methods. Compounds of formula (VIA) and (VIB) are either known compounds or may be prepared from known compounds by known methods.
The intermediates of formula (II) disclosed herein are novel compounds and form an aspect of the present invention as do the described processes for their preparation.
Where obtainable, pharmaceu~ically acceptable salts o' the compounds of formula (I) may be formed conven.ionally by rea-tion with the approp-iate ac d c- base. Solvates may be formed by crystallization from tne appropriate solven~.
30 P.s men.ioned previously, the com?ounds of fo-mula (I) exist in more than one diastereoiso,leric form. Where .he processes of the invention produce m,ixtures thereof, the individual isomers may be separa~ed one from another by chromatography e.g. HPLC.
3~
SUBSTITUTE SHFET
WO91/15507 PCT/GB9l/ ~ 3~
2. . ~ - 22 -Alternatively, separate diastereoisomerlc compounds of formula (I) can be obtained by using stereoisomerically pure starting materials or by separatin~ desired isomers of intermediates at any stage in the overall synthetic process, and converting these intermedia.es to compounds of formula (I) It will be appreciated that where a sinsle ~iasteresisvme-of a compound of formula (I) is p-epa_ed by more than cnG
process variant as hereinbefore described, each of whic-allows a different chiral centre to be defined, it may be possible to deduce the configuration a_ a chi-al centre which is not pre-determined using a par~icular process variant Furthermore, it will be appreciated tha. although the absolute configuration at a particular chiral centre may not be known, it is possible to characterise a given diastereoisomer relative to its epimer by reference to the direction in which the plane of polarised light is rotated The present invention further provides a pharmaceu_lca' composition, which comprises a compoun~ of ~ormula (I),o-2 pharmaceutically acceptable salt o- sGlva-e thereof, and 2 pharmaceu~ically acceptable car-ie-A composition of this invention is uce~ the trea.men~
o. musculo-skeletal disorders, pa-t ~u_crl; arthritic diseases and for modulation of tissue -emode'llns A composition of the invention also has potential utili.y in the treatmen~ of cancer; for preven~ing myelin degradation in the cent-al and pe-i-he-al nervous system;
SU~STITUTE S: lE~T
WO91/15~07 PCT/GB91/00~38 - 23 - ~ ~j~. ~ 7 and in other conditions in which members of the collagenase family of neutral metallopro~eases have patholosical or other roles.
A composition of the invention, which may be prepared by admixture, may contain a diluer.t, binder, filler, disinteg-ant, flavouring agent, colourinc agent, lubrica~.~
o- prese-vative in conventional manner. These conventional excipients may be em?loyed i~ conventional manner, for example as in the prepara~ior. of compositions of related peptide enzyme inhibitors, su^h as the ACE
inhibito- enalapril.
A composition of the invention may be ada?ted for oral, topical, rectal or parenteral administra-ion but oral administration is preferred. Parenteral compositions may be administered intravenously, intramuscularly, intra-articularly, intradermally, subcutaneously or into the cerebro-spinal fluid.
Preferab1y, a pharmaceutical composition of the inventic-.
ls in u-. ~ dosage form and in a form adap~ed for use ir.
the medical or veterinarial fields. ~o- example, such ?re?ara~_ons may be in a pack form a-cor.?anied by writte-.
or printe inst-uctions fo- use as an acent in the ~-eatmer.~ or p-ophylaxis of any of Lhe disorders mentior.ed above .
~he sui.abie dosa e range for the com?ounds of the inven~ion may va-y from compound to com?ound and may depend on the condition to be t-eated. ~t will also depend, inter alia, upon the relation o potency to aDsorbability and the mode of administr2~ion choser..
SUBSTITUTE SHEET
WO91/15507 PCT/GBg1/~3g ~f ~ 3 .,7 2 4 The compound or composition of the invention may be formulated for administration by any route, the preferred route depending upon the disorder for which treatment is required, and is preferably in unit dosage form or in 2 form that a human patient may administer to himself in a single dosage.
Compositions may, îor example, be in the form of tablets, capsules, sachets, vials, powders, c-anules, iozenges, reconstitutable powders, or liquid preparations, fo-example solutions or suspensions, or su??osito ies.
The compositions, for example those suitable for oral administration, may contain conventional excipients such as binding agents, for example syrup, acacia, gela.in, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or phzrm2ceutic211y acceptable wetting agents such as sodium lauryl sulphate.
Solid compositions may be obtained by conventional me~hod--2~ o- blending, 'illing, tableting o- the like. Repeated ~lending operations may be used to d stribute the 2c.ive agen. throughou those com?ositions employing large quantities of fillers. When the com?osition is in the fo~m of a tabiet, powder, o- lozenge, any carrie- suitabl-'o- formulating solid pharmaceutic21 com?ositions may b-used, examples being magnesium stearz-e, starch, glucose, lactose, sucrose, rice flour and cha k. Table_s may be coa~ed according to methods well ~nown in normal pharmaceu~ical practice, in particulz- with an ente-ic SUE~STl-rUTE SHEE~T
WO91/15507 PCT/GB9~/00~38 ~ ,7 coating. The composition may also be i-. the form of ar ingestible capsule, for example of gelat n containing the compound, if desired with a carrier or o_her excipients.
For example, in a hard gelatin capsule containing the required amount of a compound of the invention in the fo-m of a powder or granulate in intimate mi~ re with 2 lubricant, such as magnesium stearate, a filler, such 2s microcrystalline cellulose, and a disin-egrar , such as sodium starch glycollate.
Compositions for oral administration as :iqui~s may be -the form of, for example, emulsions, syr~ps, o- el-i: -s, or may be presented as a dry product fo- reconstitutior.
with water or other suitable vehicle be~ore use. Sucn liquid compositions may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acaciai aqueous or non-aqueous vehicles, which include edible oils, for example almond oil, fractionated coconu~ oil, oily es~e-s, for example esters of glycerine, or propylene glycol, c-ethyl alcohol, glycerine, water or norm21 s21ine;
preservatives, for example methyl or prcpyl p-hydroxybenzoate or sorbic acid; and desired conventional flavouring or colourin agen.ts.
The compounds cr this invention may als- be anminis eren by a non-oral route. In accordar.ce wi.-. ro-~~ir.e prarmaceutical procedure, the compositivns m2y be fo-mulated, for example for rectal administr2~ion 2s 2 su?pository or for parenteral admin-str2~ior in a-lnjectable form. For injection, fo~ ex2mple by intra-articular injection or by .njectio~ ir._o th^
cerebro-spinal fluid or via othe- routes whieh will gai-.
SUBSTITUTE SHEET
~r~
~ 26 -access to sites of demyelination, such as by intramuscular, intradermal or subcutaneous injection, as freely soluble solutions or as poorly dispersed depot stores, the compounds of the invention may be presented in an aqueous or non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable liquid, e.g. sterile pyrogen-free water or a parenterally acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, anti-oxidants or other preservatives, buffers o-solutes to render the solution isotonic with the blood,thickening agents, suspending agents or other pharmaceutically acceptable additives. Such forms will be presented in sterile unit dose form such as ampoules or disposable injectlon devices or in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn or a solid form or concentrate which can be used to prepare an injectable formulation.
For topical and percutaneous administration, the preparations may also be presented as an ointment, cream, lotion, gel, spray, aerosol, wash, skin paint or patch.
A unit dose for treating diseases in which enzymes of the collagenzse family are involved will generally contain from 10 to 1000 mg and preferably will contain from 10 to 500 ms, in particular 10, 50, 100, 150, 200, 250, 300, 35C, 400, 450 or 500 ms. The composition may be administered one or more times a day, for example 2, 3 o-4 times daily, so that the total daily dose for a 70 kg adult will normally be in ~he range 10 to 3000 mg. Such a dosage corresponds to approximately 0.15 to 50 mg/kg per day. Alternatively, in particular fc- injection, the unit dose will contain from 2 to 200 mg of a compound of the invention and be administered in multiples, if desired, to 3~ sive the desired daily dose.
SUBSTITUTE SHEET
5~07 PCT/GB91/~K38 - 27 - 2~ 7 The present invention additionally provides a method Gf treating conditions in which degradation of connective tissue and other proteinaceous components of the body occurs, such as rheumatism and/or arthritic conditions in mammals, such as humans, which comprises administering to the mammal in need of such treatment an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
The present invention also provides the use of a compound of formula (I) or a pharmaceutically ac-eptable salt thereof, for the manufacture of a medicament for use i?.
the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs such as rheumatism and/or arthritic conditions.
The following Descriptions and Examples illustrate the preparation of compounds of the invention. All temperatures are expressed in C.
SUBSTITUTE SHEET
W091/15507 PCT/GB91/ ~ 38 Z~ ~ 7 Descri~tion 1 Dibenzvl (l-hvdroxv~roPvl)~hos~honata (31) (PhCH20~P ~ OH
The general method of F. Texier-Boullet and A. Foucaud [Synthesis, 1982, 916] was employed. A mixture of dibenzyl phosphite (31.13 ml, 0.14 mole) and propionaldehyde (10.21 ml, 1 equiv.) was stirred at room temperature and basic alumina (70g) added in one portion.
After standing overnight at room temperature chloroform was added and the alumina collected and washed with chlo_oform. The filtrate was evaporated to dryness and the resulting clear oil chroma.ographed on silica gel (600g) with gradient elution (ether - 5% methanol/ether).
The title compound was obtained as a clear oil which solidified on standing (27.82g, 64%). A sample was recrystallized from ether/pentzne to give a white crystalline solid, m.p. 81-82C.
Founa: C,64.09; H,6.71. C'7n2109P1 requires C,63.74;
H,6.61%.
~ (~D~13): 1.04(3H,t,J=7Hz), 1.--1.95(2H,m), 2.27(1:;., D_ s), 3.8(1H, 2 overlapping t-iplets, J=5 and lOHz), 4.97-5.18(4H,m), 7.34(lOH,s).
WO91/15507 PCT/GB91/~K3g - 2g - '~
Description 2 DibenzYl ((l-trifluoromethanesul~honYloxY)Dro~y~hos-phonate (D2) (PhCH20)2P ~ - S"
The title compound was prepared by the general method of E. Vedejs et al. [J. Org. Chem. 1985, 50(12), 2165]. A
solution of dibenzyl (1-hydroxypropyl)phosphonate ~D1) (24.97g, 0.078 mole) in methylene chloride (180 ml) was cooled to -50C under N2. 2,6-Lutidine (11.12 ml, 0.095 mole) was added followed by trifluoromethane-sulphonic anhydride (15.1 ml, 0.0898 mole) keeping the temperature at -50C. The mixture was allowed to warm slowly to 0C and then taken into cold ether. The solution was subjected to a rapid aqueous work-u? by washing the organic layer with ice-cold water, dilute hydrochloric acid (x2) and finally brine. The organic layer was dried (anhydrous MgSO4) and evaporated to dryness to give the title compound as 2 pinkish orange oil (33.77g, 96%) which was used without further puri^ication.
~ (CDCl3): 1.08(3H,t,J=7Hz), 1.88(2H,m), 4.94(1H, 2 overlapping triplets,J=5.5 and 7Hz), 9.88-5.22(4:-,m) ar.d 7.35(lOH,m).
SUBSTITU~E SHEFI-WO91/15507 PCT/GB91/ ~ 38 ~,~s~,7 - 30 -Description 3 N-(1-(R)-Dibenzyloxyphos~hinylproDvl)-(S)-leucine (D3A) and N-(1-(S)-DibenzvloxvPhos~hinvlpropvl)-(S)-leucine (D3B) C2H5 ~ CH3 (phcH2o)2p N CO2H
O H
Method A
Following the general me.hod of US 4808741 for the preparation of leucine t_imethylsilyl es~er a mixture Oc 15 (S)-leucine (1.15g, 0.0088 mole), hexamethyldisilazane - (1.75 ml), and triethylamine (1.38 ml) in acetonitrile (13.5 ml) was heated at reflux for a total of 4h.
Dibenzyl ((1-trifluoromethanesulphonyloxy)propyl)-phosphonate (D2) (4.5g, 0.01 mole) was then added and the mixture maintained at 40-42C for 48h. The reaction can also be c2rried out at a~bient temperature. After coolinc the mixture was filtered, washed with methanol and the filtrate evaporated to dryness. The residue was taken up in chloroform and washed with dilute HCl (x2) and finally water. The chloroform layer was dried ~anhydrous Na2SO~), filte-ed and evaporated to dryness to give an orange gummy solid (3.67g). The crude product was tr-turated with the minimum volume of ether/pentane to give a white crystalline solid which after collection, washing with a little cold ether/pentane and drying gave the title compound, R,S isomer (D3A) (0.47g, 11%), m.p. 112-115C.
WO91/15507 PCT/GB91/~53g ~ . , ..~. ~,7 Observed Desorption CI (NH3) (M+H)+ 434. C23H32NO5P
requires M 433.
[a]D2o = -23.09 (c=0.97 MeOH).
Found: C,63.73; ~,7.42i N,3-23- C23H32NO5P requires C,63.73; H,7.44; N,3.23%.
(CDC13): 0.89 (6H,t), 1.03 (3H,t), 1.25-2.0 (5H,m), 2.7 (lH,m), 3.28 (2H, br s), 3.73 (lH, br t), 4.9-5.15 (4~., m), 7.35 (lOH, s).
The other isomer, N-(l-(S)-dibenzyloxyphos?hinylpropyl)-(S)-leucine (D3B), can be obtained by ?reparative HPLC
using a Hamilton PRP-1 column, 300 x 7.Omm, 264R with a 40:60 acetonitrile:water eluent mixture and a flow rate cf 4.0 ml/min. Under these conditions the R,S isomer (D3A) elutes first with a retention time of 34.6 min and the S,S
isomer (D3B) is well separated at 42.7 min.
For the isomer (D3B):
Observed FAB (M+H) 434. C23H32NO5p req (CDC13): 0.88 (6H,dd), 0.98 (3H,t), L.4 (lH,m), 1.52-1.
(4H,m), 2.72 (lH,m), 3.38 (lH,m), 9.9-5.15 (4H,m), 7.32 (10~, s) .
The S,S isomer (D3B) on coupling with (S)-amino acid derivatives leads to the S,S,S, se-ies.
Method 3 A mixture o~ (S)-leucine methyl es~er hydrochloride (0,543g; 0.003 mole), dibenzyl (1-~rifluoromethane-sulphonyloxy)propyl)-phosphonate (D2) (1.35g; 0.003 mole) and anhydrous potassium carbonate (1.0g) in methanol (2 ml) was heated at 50C, with sti-ring, for 4 hours and then lert at room temperature overnigh .
SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/00538 2~ 32 The reaction mixture was evaporated to dryness ln vacuo, and dissolved in chloroform (5 ml) and filtered. The filtrate, and washings, were combined and chromatographed on silica gel 60 (50g) using ethyl acetate-pentane ~
as the eluent, to affo-d a mixture of N-(l-(R)-dibenzyi-oxyphosphinylpropyl)-(S)-leucine methyl ester and N-(l-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine methyl ester as an oil (0.55g). The esters can be separated into the individual diastereoisomers by column chromatography on silica gel wlth initially 50% diethyl ether/pentane as eluent, rising to 100% diethyl ether.
The above mixture of esters (l.lg, 0.0025 mole) in methanol (9.0 ml) was treated with a solution of sodium hydroxide (O.llg; 0.00275 mole) in water (1.5 ml), and the solution was stirred at room temperature overnight. It was evaporated to one third volume, ln vacuo, taken in water and extracted with ether. The aqueous fraction was acidified with citric acid to pH 3-4 and then extracted (5x) with chloroform. The chloroform fraction was dried (Na2SO4) and evaporated to dryness in vacuo to give a mixture of the title compounds (D3A) and (D3B) as an oil that slowly solidified.
Trituration of the product with ether gave N-(l-(R)-dibenzyloxyphosphinylpropyl)-(s)-leucine (D3A) (0.34g) as a white crystalline solid, iden~ical to the product obtained by Method A.
Alternatively, the single isome- esters can be hydrolysed separately. For example N-(l-(S)-dibenzyloxyphosphinyl-propyl)-(S)-leucine methyl este- on hydrolysis by the above method gave N-(l-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3B), m.p. 71-73C.
3~
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~53g J~j,;,~"~7 Description 4 Na-tert-ButoxycarbonYl-NE-benzYloxvcarbonyl-(S)- lvsine methvlamide (D4) s H
H3C ~ ~ HCH3 H
H3C CH3 ~ ~ OCH2Ph A stirred solution of Na-tert-butoxycarbonyl-N-benzyloxycarbonyl-(S)-lysine (1.5s, 3.95 mmol) in anhydrous dichloromethane (30 ml) maintained at 0C was sequentially treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.9lg, 4.7 mmol) and 1-hydroxybenzotriazole (0.64g, 4.7 mmol). After 0.5 h, the mixture was warmed up to room temperature, methylamine bubbled through, and the resulting solution left stirring for 18 h. The solution was then filtered, washed with sat. aq. NaHCO3, dried over anhydrous magnesium sulphate and concentrated under reduced pressure to afford a solid.
Pu-ification by flash chromatography (5% methanol in chloroform) gave the title compound as a white solid 25 (1.18g).
(CDCl3): 1.3-1.85 (6H,m), i.~2 (9.~,s), 2.28 (3H,d), 3.~`, (2H,q), 4.05 (lH,m), ~.88 (lH,m), 5.08 (2A,s), 5.15 (lH,m), 6.2 (lH,m), 7.35 (5H,m).
SUBSTI~ SHEET
550~ PCTtGB91/0053 DescriPtion 5 Benzyloxv-carb~
trifluoroacetate Salt (D5~
0 N ~ OCH2ph (O C) Solution of the a i hloromethane (5 ml) ated under reducedAfter 0 5 h the sol n waS dried oVer ~ h Cl Purification. d as such Scri~tion 5 5D_~- S)-leucyl C2H5 ~ H H3 0 (PhcH O)p""~ ~ N ~ ~ CH3 ~ N ~ OcH2ph SlJBsTlTlJTE SHEET
"' 91/15507 PCI/GB91/00~38 s~ r ~ ~ ~7 ~ 35 ~
A solution of N~ (R)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (0.33g, 0.76 mmol) in anhydrous dichloromethane (10 ml) was cooled to 0C, and treated sequentially with 5 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.176g, 0.92 mmol) and 1-hydroxy-benzotriazole (0.124g, 0.92 mmol). After stirring for 0.5 h the reaction mixture was trea~ed with the salt (D~) (0.29g) followed by N,N-diisopropylethylamine (0.216g, 10 1.42 mmol). The mixture was stirred for 18 h at room temperature, then washed with sat. aq. NaHCO3 (2x20 ml), and sat. aq. NaCl (2x20 ml). The aqueous washes were back-extracted with dichloromethane and the combined organic fractions dried over anhydrous magnesium sulphate, 15 and evaporated in vacuo to yield an oil. On purification by flash chromatography l(MeOH:CH2Cl2) (1:20) v/v then (1:9) v/v], the title compound was isolated as a white solid (0.20g).
20 Observed FAB (M+H)+ 678. C38H5307N4 requires _ 677.
Descri~tion 7 Na-tert-Butoxvcarbonyl-NE- (N-benzvloxvcarbonvl-25 clvcvl)- (S)-lysine methvlamide (D7) H3C~ ~ NIIC~II H N/~OCH2Ph 3~
SUBSTITUTE SHEET
WO91/15507 PCT/GBgl/0053P--2 ~
A stirred solution of N-benzyloxycarbonyl-glycine (1.8g, 5.82 mmol) in anhydrous dichloromethane (60 ml) was treated with l-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.23g, 6.4 mmol) followed by l-hydroxybenzotriazole (0.98g, 7.2 mmol). Sti-ring was continued for 0.5 h then treated with ~ tert-butoxycarbonyl-(S)-lysine methylamide (1.51g, 5.82 mmol) diluted in dichloromethane (5 ml), and lef~ s.irring.
After 18 h the mixture was washed with sat. âC . NaHC03 (2x30 ml), dried over anhydrous masnesiur, sul?;.ate and evaporated in vacuo to yield a pale yellow o l.
Pu-ificatio~ by flash chromatography af-orded the title compound as a clear viscous oil which solidified on standing.
(CDCl3): 1.26-1.82 (6H,m), 1.42 (9H,s), 2.79 (3H,d), 3.26 (2H,m), 3.87 (2H,d), 4.05 (lH,m), 5.12 (2H,s), 5.28 (lH,m), 5.72 (lH,brs), 6.46 (lH,brs), 7.32-7.4 (5H,m).
Descri~tion 8 N-(N-Benzvloxvcarbonvlalvcvl)-(S)-lvsine methvlamide, trifluoroacetate salt (D8) TFA.H2N ~ NHCH~ H
H
O
SUE~STITUTE SHEET
~'~91/15~07 2~ ~ .`.'< ~, 7 PCT/GB91/0053X
A cool (0C) solution of the amide (D7) (O.lg, 0.22 mmol) in dichloromethane (5 ml) was treated wilh trifluoroacet c acid (2 ml). After stirring at 0C for 1 h, the solution was allowed to warm up to room temperatu-e. The solvent was evaporated under reduced pressure, to yield the crude product (D8) which was used without further pu-ification.
DescriPtion 9 ~-~N-((R)-1-Phos~hono~roDyl)-(S)-leucvl~-N-(N-benzvl-oxvcarbonvl~lvcvl)-(s)-lvsine methvlamid-, dibenzvl este-(D9) ~ CH3 O ~~N ~N J~ OCH2Ph A solution of N-(l-(R)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (0.43g, 1 mmol) in anhydrous dichloromethane 25 (20 ml) was treated with 1-(3-dimethylam_nopropyl)-3-e_hylcarbodiimide hydrochloride (0.21g, 1.1 mmol) and 1-hydroxybenzotriazole (0.162g, 1.2 mmo_). A_ter sti--in~
fo- 1 h, the salt (D8) (0.25g), diluted n di-hloromethzn^
(10 ml), W25 added followed by diisopropylethylamine (1 equiv.) stirring continued for a fu-the- 18 h. The mixture was then washed with sat. aq. Nz:;.CO3 (2x20 ml), dried ove- anhydrous magnesium sulphate and evaporated vacuo to afford a yellow oil. Pur 'icat on by flasA
SUBSTITUTE SHEE~
WO91/15507 PCT/GB91/0053~-2~ 7 chromatography ~(MeOH:CHC13)(1:15) v/v)] afforded the title compound as a white solid (0.4g).
~ (CDC13): 0.96 (6H,t), 1.05 (3H,t), 1.2-2.4 (13H,m), 2.-_ (3H,d), 3.18 (2H,m), 3.69 (lH,m), 3.82 (2H,d), 4.38 (l~,q), 4.88-5.03 (4H,m), 5.08 (2H,s), 6.35 (lH,t), 6.5_ (lH,t), 7.08 (lH,q), 7.22-7.4 (15H,m), 7.51 (lH,d).
Desc-i~tion 10 ~-.e-t-Butoxycarbonvl-(S)-aspartic acid methylamide (D10 H
H3C X ~ N ~ ~ CH3 H3C CH3O ~
To a solution of N-tert-butoxycarbonyl-(S)-aspartic acid ~-benzyl ester (66g) in dry tetrahydrofu-an (100 ml) at -10C was added diisopropylethylamine (38 ml) followed by ethyl chloroformate (23 ml) and a solution of methylamine (10g) in dry tetrahydrofuran (30 ml) was added. A'-er 0.5h the reaction mixture was evaporated to dryness ln vacuo and the residue, in ethyl acetate, was washeà with '0~ sodium carbonate, citric acid and water, and d--ed (~a2SO4). Evaporation to dryness in vacuo, followed by tri.uration with ethyl acetate-ether (1:1) affordea a so id (45g) that was hydrogenated in ethanol (600 ml) ove-i0~ palladium on carbon (8g) untll uptake of hydroaenceased. The reaction mixture was 'iltered, and the SVBSTITUTE SHEET
~91/15~07 ~ 7 PCT/CB91/00538 filtrate was evaporated to dryness to afford the title compound (D10) as a white solid (33.5g), m.p. 160-162C
(dec).
Found: C,48.69; H,7.35; N,11.12%. C1oH18N205 requ C,48.77; H,7.37; N,11.38%.
Description 11 N-tert-Butoxycarbonyl-~-~(2-~vrrolidinoe~hyl)amide -(S)-aspartic acid methvlamide (D11) H
H3C>~O ~N ~ ~ CH3 \I~ N N
O \J
To a solution of N-tert-butoxycarbonyl-(S)-aspartic acid methylamide (D10) (5g) in dichloromethane (50 ml) at 0C
was added 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (4.74g) and 1-hydroxybenzo.riazole (3.34g).
After 10 mins N-(2-aminoethyl)pyrrolidine (2.82g) w25 added dropwise, and the solution was stirred at 0C for 2h 2~ a~d then at room temperature overnigh~. The reactior.
mixture was washed with sodium bicarbona.e solution, wazer, and dried (Na2S04), and then evaporated to d-~nesc ir. vacuo to give the title compound (D11) (l.Og).
~ (CDCl3): 1.42(9H,s), 1.9(4H,m), 2.5-2.9(8H,m), 2.82(3H,d,J=5Hz), 3.38(2H,q,J=5Hz), 4.5(1H,m), 6.21(1H,m), 6.75(lH,m) and 7.02(lH,m).
SUBSTITUTE SHEET
The present invention also provides the use of a compound of formula (I) or a pharmaceutically ac-eptable salt thereof, for the manufacture of a medicament for use i?.
the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs such as rheumatism and/or arthritic conditions.
The following Descriptions and Examples illustrate the preparation of compounds of the invention. All temperatures are expressed in C.
SUBSTITUTE SHEET
W091/15507 PCT/GB91/ ~ 38 Z~ ~ 7 Descri~tion 1 Dibenzvl (l-hvdroxv~roPvl)~hos~honata (31) (PhCH20~P ~ OH
The general method of F. Texier-Boullet and A. Foucaud [Synthesis, 1982, 916] was employed. A mixture of dibenzyl phosphite (31.13 ml, 0.14 mole) and propionaldehyde (10.21 ml, 1 equiv.) was stirred at room temperature and basic alumina (70g) added in one portion.
After standing overnight at room temperature chloroform was added and the alumina collected and washed with chlo_oform. The filtrate was evaporated to dryness and the resulting clear oil chroma.ographed on silica gel (600g) with gradient elution (ether - 5% methanol/ether).
The title compound was obtained as a clear oil which solidified on standing (27.82g, 64%). A sample was recrystallized from ether/pentzne to give a white crystalline solid, m.p. 81-82C.
Founa: C,64.09; H,6.71. C'7n2109P1 requires C,63.74;
H,6.61%.
~ (~D~13): 1.04(3H,t,J=7Hz), 1.--1.95(2H,m), 2.27(1:;., D_ s), 3.8(1H, 2 overlapping t-iplets, J=5 and lOHz), 4.97-5.18(4H,m), 7.34(lOH,s).
WO91/15507 PCT/GB91/~K3g - 2g - '~
Description 2 DibenzYl ((l-trifluoromethanesul~honYloxY)Dro~y~hos-phonate (D2) (PhCH20)2P ~ - S"
The title compound was prepared by the general method of E. Vedejs et al. [J. Org. Chem. 1985, 50(12), 2165]. A
solution of dibenzyl (1-hydroxypropyl)phosphonate ~D1) (24.97g, 0.078 mole) in methylene chloride (180 ml) was cooled to -50C under N2. 2,6-Lutidine (11.12 ml, 0.095 mole) was added followed by trifluoromethane-sulphonic anhydride (15.1 ml, 0.0898 mole) keeping the temperature at -50C. The mixture was allowed to warm slowly to 0C and then taken into cold ether. The solution was subjected to a rapid aqueous work-u? by washing the organic layer with ice-cold water, dilute hydrochloric acid (x2) and finally brine. The organic layer was dried (anhydrous MgSO4) and evaporated to dryness to give the title compound as 2 pinkish orange oil (33.77g, 96%) which was used without further puri^ication.
~ (CDCl3): 1.08(3H,t,J=7Hz), 1.88(2H,m), 4.94(1H, 2 overlapping triplets,J=5.5 and 7Hz), 9.88-5.22(4:-,m) ar.d 7.35(lOH,m).
SUBSTITU~E SHEFI-WO91/15507 PCT/GB91/ ~ 38 ~,~s~,7 - 30 -Description 3 N-(1-(R)-Dibenzyloxyphos~hinylproDvl)-(S)-leucine (D3A) and N-(1-(S)-DibenzvloxvPhos~hinvlpropvl)-(S)-leucine (D3B) C2H5 ~ CH3 (phcH2o)2p N CO2H
O H
Method A
Following the general me.hod of US 4808741 for the preparation of leucine t_imethylsilyl es~er a mixture Oc 15 (S)-leucine (1.15g, 0.0088 mole), hexamethyldisilazane - (1.75 ml), and triethylamine (1.38 ml) in acetonitrile (13.5 ml) was heated at reflux for a total of 4h.
Dibenzyl ((1-trifluoromethanesulphonyloxy)propyl)-phosphonate (D2) (4.5g, 0.01 mole) was then added and the mixture maintained at 40-42C for 48h. The reaction can also be c2rried out at a~bient temperature. After coolinc the mixture was filtered, washed with methanol and the filtrate evaporated to dryness. The residue was taken up in chloroform and washed with dilute HCl (x2) and finally water. The chloroform layer was dried ~anhydrous Na2SO~), filte-ed and evaporated to dryness to give an orange gummy solid (3.67g). The crude product was tr-turated with the minimum volume of ether/pentane to give a white crystalline solid which after collection, washing with a little cold ether/pentane and drying gave the title compound, R,S isomer (D3A) (0.47g, 11%), m.p. 112-115C.
WO91/15507 PCT/GB91/~53g ~ . , ..~. ~,7 Observed Desorption CI (NH3) (M+H)+ 434. C23H32NO5P
requires M 433.
[a]D2o = -23.09 (c=0.97 MeOH).
Found: C,63.73; ~,7.42i N,3-23- C23H32NO5P requires C,63.73; H,7.44; N,3.23%.
(CDC13): 0.89 (6H,t), 1.03 (3H,t), 1.25-2.0 (5H,m), 2.7 (lH,m), 3.28 (2H, br s), 3.73 (lH, br t), 4.9-5.15 (4~., m), 7.35 (lOH, s).
The other isomer, N-(l-(S)-dibenzyloxyphos?hinylpropyl)-(S)-leucine (D3B), can be obtained by ?reparative HPLC
using a Hamilton PRP-1 column, 300 x 7.Omm, 264R with a 40:60 acetonitrile:water eluent mixture and a flow rate cf 4.0 ml/min. Under these conditions the R,S isomer (D3A) elutes first with a retention time of 34.6 min and the S,S
isomer (D3B) is well separated at 42.7 min.
For the isomer (D3B):
Observed FAB (M+H) 434. C23H32NO5p req (CDC13): 0.88 (6H,dd), 0.98 (3H,t), L.4 (lH,m), 1.52-1.
(4H,m), 2.72 (lH,m), 3.38 (lH,m), 9.9-5.15 (4H,m), 7.32 (10~, s) .
The S,S isomer (D3B) on coupling with (S)-amino acid derivatives leads to the S,S,S, se-ies.
Method 3 A mixture o~ (S)-leucine methyl es~er hydrochloride (0,543g; 0.003 mole), dibenzyl (1-~rifluoromethane-sulphonyloxy)propyl)-phosphonate (D2) (1.35g; 0.003 mole) and anhydrous potassium carbonate (1.0g) in methanol (2 ml) was heated at 50C, with sti-ring, for 4 hours and then lert at room temperature overnigh .
SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/00538 2~ 32 The reaction mixture was evaporated to dryness ln vacuo, and dissolved in chloroform (5 ml) and filtered. The filtrate, and washings, were combined and chromatographed on silica gel 60 (50g) using ethyl acetate-pentane ~
as the eluent, to affo-d a mixture of N-(l-(R)-dibenzyi-oxyphosphinylpropyl)-(S)-leucine methyl ester and N-(l-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine methyl ester as an oil (0.55g). The esters can be separated into the individual diastereoisomers by column chromatography on silica gel wlth initially 50% diethyl ether/pentane as eluent, rising to 100% diethyl ether.
The above mixture of esters (l.lg, 0.0025 mole) in methanol (9.0 ml) was treated with a solution of sodium hydroxide (O.llg; 0.00275 mole) in water (1.5 ml), and the solution was stirred at room temperature overnight. It was evaporated to one third volume, ln vacuo, taken in water and extracted with ether. The aqueous fraction was acidified with citric acid to pH 3-4 and then extracted (5x) with chloroform. The chloroform fraction was dried (Na2SO4) and evaporated to dryness in vacuo to give a mixture of the title compounds (D3A) and (D3B) as an oil that slowly solidified.
Trituration of the product with ether gave N-(l-(R)-dibenzyloxyphosphinylpropyl)-(s)-leucine (D3A) (0.34g) as a white crystalline solid, iden~ical to the product obtained by Method A.
Alternatively, the single isome- esters can be hydrolysed separately. For example N-(l-(S)-dibenzyloxyphosphinyl-propyl)-(S)-leucine methyl este- on hydrolysis by the above method gave N-(l-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3B), m.p. 71-73C.
3~
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~53g J~j,;,~"~7 Description 4 Na-tert-ButoxycarbonYl-NE-benzYloxvcarbonyl-(S)- lvsine methvlamide (D4) s H
H3C ~ ~ HCH3 H
H3C CH3 ~ ~ OCH2Ph A stirred solution of Na-tert-butoxycarbonyl-N-benzyloxycarbonyl-(S)-lysine (1.5s, 3.95 mmol) in anhydrous dichloromethane (30 ml) maintained at 0C was sequentially treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.9lg, 4.7 mmol) and 1-hydroxybenzotriazole (0.64g, 4.7 mmol). After 0.5 h, the mixture was warmed up to room temperature, methylamine bubbled through, and the resulting solution left stirring for 18 h. The solution was then filtered, washed with sat. aq. NaHCO3, dried over anhydrous magnesium sulphate and concentrated under reduced pressure to afford a solid.
Pu-ification by flash chromatography (5% methanol in chloroform) gave the title compound as a white solid 25 (1.18g).
(CDCl3): 1.3-1.85 (6H,m), i.~2 (9.~,s), 2.28 (3H,d), 3.~`, (2H,q), 4.05 (lH,m), ~.88 (lH,m), 5.08 (2A,s), 5.15 (lH,m), 6.2 (lH,m), 7.35 (5H,m).
SUBSTI~ SHEET
550~ PCTtGB91/0053 DescriPtion 5 Benzyloxv-carb~
trifluoroacetate Salt (D5~
0 N ~ OCH2ph (O C) Solution of the a i hloromethane (5 ml) ated under reducedAfter 0 5 h the sol n waS dried oVer ~ h Cl Purification. d as such Scri~tion 5 5D_~- S)-leucyl C2H5 ~ H H3 0 (PhcH O)p""~ ~ N ~ ~ CH3 ~ N ~ OcH2ph SlJBsTlTlJTE SHEET
"' 91/15507 PCI/GB91/00~38 s~ r ~ ~ ~7 ~ 35 ~
A solution of N~ (R)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (0.33g, 0.76 mmol) in anhydrous dichloromethane (10 ml) was cooled to 0C, and treated sequentially with 5 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.176g, 0.92 mmol) and 1-hydroxy-benzotriazole (0.124g, 0.92 mmol). After stirring for 0.5 h the reaction mixture was trea~ed with the salt (D~) (0.29g) followed by N,N-diisopropylethylamine (0.216g, 10 1.42 mmol). The mixture was stirred for 18 h at room temperature, then washed with sat. aq. NaHCO3 (2x20 ml), and sat. aq. NaCl (2x20 ml). The aqueous washes were back-extracted with dichloromethane and the combined organic fractions dried over anhydrous magnesium sulphate, 15 and evaporated in vacuo to yield an oil. On purification by flash chromatography l(MeOH:CH2Cl2) (1:20) v/v then (1:9) v/v], the title compound was isolated as a white solid (0.20g).
20 Observed FAB (M+H)+ 678. C38H5307N4 requires _ 677.
Descri~tion 7 Na-tert-Butoxvcarbonyl-NE- (N-benzvloxvcarbonvl-25 clvcvl)- (S)-lysine methvlamide (D7) H3C~ ~ NIIC~II H N/~OCH2Ph 3~
SUBSTITUTE SHEET
WO91/15507 PCT/GBgl/0053P--2 ~
A stirred solution of N-benzyloxycarbonyl-glycine (1.8g, 5.82 mmol) in anhydrous dichloromethane (60 ml) was treated with l-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.23g, 6.4 mmol) followed by l-hydroxybenzotriazole (0.98g, 7.2 mmol). Sti-ring was continued for 0.5 h then treated with ~ tert-butoxycarbonyl-(S)-lysine methylamide (1.51g, 5.82 mmol) diluted in dichloromethane (5 ml), and lef~ s.irring.
After 18 h the mixture was washed with sat. âC . NaHC03 (2x30 ml), dried over anhydrous masnesiur, sul?;.ate and evaporated in vacuo to yield a pale yellow o l.
Pu-ificatio~ by flash chromatography af-orded the title compound as a clear viscous oil which solidified on standing.
(CDCl3): 1.26-1.82 (6H,m), 1.42 (9H,s), 2.79 (3H,d), 3.26 (2H,m), 3.87 (2H,d), 4.05 (lH,m), 5.12 (2H,s), 5.28 (lH,m), 5.72 (lH,brs), 6.46 (lH,brs), 7.32-7.4 (5H,m).
Descri~tion 8 N-(N-Benzvloxvcarbonvlalvcvl)-(S)-lvsine methvlamide, trifluoroacetate salt (D8) TFA.H2N ~ NHCH~ H
H
O
SUE~STITUTE SHEET
~'~91/15~07 2~ ~ .`.'< ~, 7 PCT/GB91/0053X
A cool (0C) solution of the amide (D7) (O.lg, 0.22 mmol) in dichloromethane (5 ml) was treated wilh trifluoroacet c acid (2 ml). After stirring at 0C for 1 h, the solution was allowed to warm up to room temperatu-e. The solvent was evaporated under reduced pressure, to yield the crude product (D8) which was used without further pu-ification.
DescriPtion 9 ~-~N-((R)-1-Phos~hono~roDyl)-(S)-leucvl~-N-(N-benzvl-oxvcarbonvl~lvcvl)-(s)-lvsine methvlamid-, dibenzvl este-(D9) ~ CH3 O ~~N ~N J~ OCH2Ph A solution of N-(l-(R)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (0.43g, 1 mmol) in anhydrous dichloromethane 25 (20 ml) was treated with 1-(3-dimethylam_nopropyl)-3-e_hylcarbodiimide hydrochloride (0.21g, 1.1 mmol) and 1-hydroxybenzotriazole (0.162g, 1.2 mmo_). A_ter sti--in~
fo- 1 h, the salt (D8) (0.25g), diluted n di-hloromethzn^
(10 ml), W25 added followed by diisopropylethylamine (1 equiv.) stirring continued for a fu-the- 18 h. The mixture was then washed with sat. aq. Nz:;.CO3 (2x20 ml), dried ove- anhydrous magnesium sulphate and evaporated vacuo to afford a yellow oil. Pur 'icat on by flasA
SUBSTITUTE SHEE~
WO91/15507 PCT/GB91/0053~-2~ 7 chromatography ~(MeOH:CHC13)(1:15) v/v)] afforded the title compound as a white solid (0.4g).
~ (CDC13): 0.96 (6H,t), 1.05 (3H,t), 1.2-2.4 (13H,m), 2.-_ (3H,d), 3.18 (2H,m), 3.69 (lH,m), 3.82 (2H,d), 4.38 (l~,q), 4.88-5.03 (4H,m), 5.08 (2H,s), 6.35 (lH,t), 6.5_ (lH,t), 7.08 (lH,q), 7.22-7.4 (15H,m), 7.51 (lH,d).
Desc-i~tion 10 ~-.e-t-Butoxycarbonvl-(S)-aspartic acid methylamide (D10 H
H3C X ~ N ~ ~ CH3 H3C CH3O ~
To a solution of N-tert-butoxycarbonyl-(S)-aspartic acid ~-benzyl ester (66g) in dry tetrahydrofu-an (100 ml) at -10C was added diisopropylethylamine (38 ml) followed by ethyl chloroformate (23 ml) and a solution of methylamine (10g) in dry tetrahydrofuran (30 ml) was added. A'-er 0.5h the reaction mixture was evaporated to dryness ln vacuo and the residue, in ethyl acetate, was washeà with '0~ sodium carbonate, citric acid and water, and d--ed (~a2SO4). Evaporation to dryness in vacuo, followed by tri.uration with ethyl acetate-ether (1:1) affordea a so id (45g) that was hydrogenated in ethanol (600 ml) ove-i0~ palladium on carbon (8g) untll uptake of hydroaenceased. The reaction mixture was 'iltered, and the SVBSTITUTE SHEET
~91/15~07 ~ 7 PCT/CB91/00538 filtrate was evaporated to dryness to afford the title compound (D10) as a white solid (33.5g), m.p. 160-162C
(dec).
Found: C,48.69; H,7.35; N,11.12%. C1oH18N205 requ C,48.77; H,7.37; N,11.38%.
Description 11 N-tert-Butoxycarbonyl-~-~(2-~vrrolidinoe~hyl)amide -(S)-aspartic acid methvlamide (D11) H
H3C>~O ~N ~ ~ CH3 \I~ N N
O \J
To a solution of N-tert-butoxycarbonyl-(S)-aspartic acid methylamide (D10) (5g) in dichloromethane (50 ml) at 0C
was added 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (4.74g) and 1-hydroxybenzo.riazole (3.34g).
After 10 mins N-(2-aminoethyl)pyrrolidine (2.82g) w25 added dropwise, and the solution was stirred at 0C for 2h 2~ a~d then at room temperature overnigh~. The reactior.
mixture was washed with sodium bicarbona.e solution, wazer, and dried (Na2S04), and then evaporated to d-~nesc ir. vacuo to give the title compound (D11) (l.Og).
~ (CDCl3): 1.42(9H,s), 1.9(4H,m), 2.5-2.9(8H,m), 2.82(3H,d,J=5Hz), 3.38(2H,q,J=5Hz), 4.5(1H,m), 6.21(1H,m), 6.75(lH,m) and 7.02(lH,m).
SUBSTITUTE SHEET
7 PCT/GB91/ ~ 3~
2~ 7 Description 12 N-~N-((R)-1-DibenzyloxvPhosPhinvlPropvl)-(S) -leuc~ll -B-~(2-pyrrolidinoethvl)amidel-(S)-asPartic acld methvlamide (D12) C2H5 ~ H
(PhCH20)2P` ~ N ~ ~ ~ CH3 N ~
This compound was prepared from N-(1-(R)-dibenzyloxy-phosphinylpropyl)-(S)-leucine (D3A) and ~-[(2-pyrrolidinoethyl)amide]-(S)-aspartic 2^id methylamide (prepared from (Dll) by reaction with trifluoroacetic acid by the procedure described for (D5)) following the method of Description 11, m.p. 110-118C
(48% yield).
(CDCl3): 0.85(6H,t,J=5Hz), 1.05(3H,t,J=7Y.z), 1.4(3H,m), 1.6(3H,m), 1.85(approx. 6H,m), 2.6-2.85(approx. lOH,m), 3.3(1H,m), 3.45(1H,m), 3.65(1H,m), 4.85(1H,m), 5.0(4H,m), 7.~5(lOH,m), 7.5(lH,m) and 8.2(lH,d,J=7Hz).
Des-riDtion 13 N-~e-t-Butoxycarbonvl-N~-nitro-(S)-arainin.e methvlamide (D13) H
H3C ~ O ~N ~ NHCH3 NH
H3C CH3 N N~N02 H
3~
SU~3STITUTE SHEET
~ ~91/15507 2- ~ i. ;. 7 PCT/GB91/~K3g N-tert-Butoxycarbonyl-N~-nitro-~S)-arginine (3g, g 4 mmol), 1-hydroxybenzotriazole (2.88g, 18.8 mmol) and methylamine hydrochloride (1.26g, 18.7 mmol) were dissolved in dry dimethylformamide (50 ml) and cooled in an ice-salt bath to -10C. Diisopropylethylamine (3.3 rl, 18.78 mmol) was added followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.76g, 14.9 mmol). After lh, at 0C the reactio~. ~.ixrure was stirred for 18h at room temperature when t.l.c.
~(CHC13:MeOH:AcOH) (10:2:1) v/v] showe~ the reaction tc be complete. After evaporation to dryness the solid was dissolved in water and applied to a column of Dowex 50W-X8 (ammonium form). This was eluted with water and eluan~
containing W absorbing material was collected, concentrated and then shaken with Amberlite IRA-901 (acetate form) for lh. The resin was collected and washed with water. The filtrate and washings were combined and lyophilised to give the product (D13) as a chromatographically pure white solid (2.6g, 89%).
Observed FAB (M+H)+ 333- C18H2~65 reqUires M 332-Descri~tion 19 2~ - r~- ( (R)-l-Dibenzvloxy~hos~hinyl~ro~yl)-(S)-leucvl~
n ~ro-(S)-arqinine methvlamide (D1 G ) C2Hs ~ H
(PhCH2O)2~p~ NH ~ ~ 3 NH
SUBSTITUTE SHEET
~ i; ~7 ~ 42 -Na-tert-Butoxycarbonyl-N~-nitro-(S)-arginine methylamide ~D13) (0.142g, 0.43 mmol) was treated with 95~
trifluoroacetic acid in water (5 ml) for 0.5h at 0C.
~xcess acid was removed under reduced pressure and the residue twice evaporated with dry toluene. The residue was dissolved in dry dimethylformamide (2 ml) and the pH
adjusted to 8-9 by the addition of diisopropylethylamine.
This was added to a solution of N-(l-(R)-àibenzyloxyphosphinylpropyl)-(s)-leucine (D3A) (0.15g, 0.35 mmol) and l-hydroxybenzotriazole (0.095g, 0.62 mmol) in dimethylformamide (5 ml). The mixture was cooled to -10 to -15C and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.08g, 0.42 mmol) added.
The reaction mixture was allowed to warm to room ~emperature and stirred for 18h. The mixture was then evaporated to dryness and purified by silica gel chromatography [(MeOH:CHCl3) (1:9) v/v~. The product (D14) was obtained as a pale yellow foam. (0.16g, 71%).
Observed FAB (M+H) 648. C30H46N7O7P req DescriDtion 15 Na-tert-Butoxvcarbonyl-(R)-~-(4-Dvridvl)alanine (D15A) and 2~ a-tert-butoxvcarbonvl- (s) -B- (4-Dvridvl)alanine (DlSB) H
H3C ~ `t~ ~ OH
H~C CH3 N
SUBSTITUTE SHEET
" ~91/15507 ~ ~ i ~7 PCT/GB9lt00~38 Crude racemic ~-(4-pyridyl)alanine dihydrochloridel (0.88g, 3.66 mmol) was dissolved in water (30 ml) and solid NaHCO3 (1.51g) added. When dissolved, dioxan (30 ml) was added and the mixture cooled in an ice-salt bath to 0C. Di-tert-butyl dicarbonate (1.5g, 6.9 mmol) as a solution in dioxan (20 ml) was added and after lh at 0-4C, the mixture was stirred for 18h at room temperature.
The reaction mixture was evaporated to dryness and a mixture of ethyl acetate, water and acetic acid (S:l:l, v/v) added. The precipitated solid was filte~ed off and the filtrate evaporated in vacuo. The residue was triturated with several portions of hot ethanol. When cool, the extracts were combined and evaporated to dryness. This process was repeated until the solid residue redissolved readily in ethanol. Evaporation of this solution gave the title compound (Dl5) as a racemic mixture, contaminated with sodium acetate. This was used without further purification.
R.L. Bixler and C. Niemann, J. Org. Chem., 23, 575 (1958).
Desc-i~tion 16 Na-ter~-Butoxvcarbonvl-(R) -B- (4-pyridvl)alanine methvlamide (Dl6A) and N~-tert-butoxvcarbonYl-(S) -B- (4-~vridyl)alanine methylamide (D16B) H
H3C ~ O ~ N ~ NHCH3 H3C CH3 ~
'~
SU~3STITUTE SHEET
WO91/15507 PCT/GB91/00~38 Z, ~ 7 Crude racemic derivative (D15) (3.66 mmcl) was dissolve^
in dry dimethylformamide (30 ml) and 1-hydroxybenzo-triazole (1.13g, 7.38 mmol) and methylamine hydrochloria-(0.5g, 7.40 mmol) added. The mixture was cooled to -10~
in an ice-salt bath and diisopropyle_hylamine (' 3 ml, ~-mmol) added. After S minutes, 1-(3-dime~hylamino~ropyl)-3-ethylcarbodiimide hydrochloride (1.7g, 8.9 mmol) was added and after stirring for lh a, -5C, the mixture was stirred overnight at room temperature. Afte evapo_atio..
to dryness, dichloromethane (50 ml) was added and ther.
- extracted with several po_tions (3x15 ml) of sat. aq.
NaHCO3. The organic phase was dried ove- anhydrous magnesium sulphate, filtered and the solven_ removed ~ r vacuo. The residue was purified by silica gel chromatography [(MeOH:CHCl3) (1:9) v/v] to give the racemic title compound (D16) as a white solid (0.91g).
Observed FAB (M+H) 280- C14H21N33 reqUires M 279-DescriD.ion 17 N~-tert-Butoxvcarbonvl-(R)-~-(4-Di~e-idvl)alanine methYlamide (D17A) and ~'~-te_t-bu~oxvcarbonvl-(S) ~-(4-DiDeridvl)alanine methvlamide (Dl7B) H
H3C ~ O ~ N ~ NHCH3 H3C CH3 ~
~ NH
The mixtu;e o' isomers c' amid~ (D16) (0.5~, ;.79 mm_ ) was dissolved in glaci21 acetic a- d (4~ ml) and degasse-SUBSTITUTE SHEET
~'~91/15507 ~ r ~ ~ PCT/GB91/00538 under reduced pressure. A suspension of Adam's catalys~(0.2g) in glacial acetic acid (5 ml) was added and the mixture hydrogenated for 22h at atmospheric pressure. The reaction mixture was filtered through Kieselguhr and the solvent removed in vacuo to give the racemic product (Dl7) as a chromatographically pure oil (O.Slg).
Description l8 N~-tert-Butoxycarbonyl-(R) -B- (4-(N-benzvloxvcarbonvl)-Pi~eridvl)alanine methvlamide (Dl8A) and Na-te-t-butox~-carbonvl-(S)-~-(4-(N-benzyloxvcarbonvl)~iDeridvl)alanine methvlamide (Dl8B) H
H3C ~ ~fN ~ NHCH3 H3C CH3 ~
~ N ~ OCH2Ph O
Isomer mixture (Dl7) (0.51g, 1.79 mmol) was dissolved in a dioxan/water mixture (2:l, 30 ml) and the pH adjusted ~o 7 by the addition of solid NaHCO3. The mixture was cooled to 0C, a second portion of NaHCO3 (0.17g, 2 mmol) added followed by the portionwise addition of benzylchloroformate (0.3 ml, 2.1 mmol). Afte- lh at 0C, ~he mixture was stirred overnight a. room temperature.
After evaporation to dryness, the residue was taken up ir.
e-hyl acetate and extracted with 10% aq. citr.c acid (2Y.), sa~. aq. NaHCO3 (2x) and sat. aq. NaCl. The organic pnase W25 dried (anhydrous MgSO4), filtered and the solvent removed in vacuo. The resultant oil was purified by SUBSTITUTE SHEZ~T
WO91/15507 PCT/GB91/~3~-,7 silica gel chromatography [(CH2C12:MeOH)(l9:1)v/v] to gi~e the racemic title compound (D18) as an oil which crystallised on standing under 60-80 ligh~ petroleum (0.68g, 91%), m.p. 130-131C.
Observed FAB (M+H) 420. C22H33N3Os req~ires M 419-Description 19 Na-~N-((R)-1-Dibenzvloxvphos~hinvl~ropyl)-(S)-leucvll-(R)-~-(4-(N-benzvloxycarbonyl)~i~eridvl)alanine methylamide (D19A) and N-~N-((R)-1-dibenzyloxv~hos~r.invl~roDvl)-(S)-leucvll-(S)-~-(4-(N-benzvloxvcarbonvl)pi~eridvl)alanine methvlamide (D19B) CH3 C2H5 ~ H
(PhCH2O)2P ~ HCH~
O ~
~ ~ ~ OCH2Ph The protected amide isomer mixture (D18) (0.19g, 0.45 mmol) was treated with 95% trifluoroacetic acid in wate-(5 ml) for 0.Sh at 0C. Excess acid was -emoved under reduced pressure and the oily residue evaporated twice with dry toiuene. The residue W25 dissolved in dry dimethyl_ormamide (2 ml) and the pH adjusted to 8-9 by ~he addition o^ diisopropylethylamine. ~his w2S added to 2 solution of N-(l-(R)-dibenzyloxy?hos?ninylpropyl)-(S)-leucine (D3~.) (0.15g, 0.35 mmol) ana 1-hydroxybenzo-t~iazole (0.llg, 0.7 mmol) in dimetr.ylformamide (5 ml)~
The mixture was cooled to -10C and --(3-dimethylaminopropyl)-3-ethylca-bodiimide hydrochloriae SUBSTITUTE SHEET
91/15~07 ~,r~ 7 PCT/GB~1/00538 ~ 47 ~
(0.lg, 0.52 mmol) added. The mi~.ture was allowed to ~Jarm to room temperature and then stirred for 18h. The reaction mixture was evaporated to dryness in vacuo ana the residue redissolved in dichloromethane. This was extracted with 10% aq. citric acid, sat. aq. NaHCO3 (2x) sat. aq. NaCl and then dried over anhydrous magnesium sulphate. Evaporation in vacuo gave the title com?ound isomer mixture (D19) as an oil which was ?urified by silica gel chromatography [(CH2Cl2:MeOH)(g:1)].
Descri~tion 20 Na-tert-Butoxycarbonyl-N~-benzvloxvc2rbonvl-(S)-ornithine methvlamide (D20) H3C ~ O ~N ~ NHCH3 o H3C CH3 N ~ OCH2Ph A solution of N~-tert-butoxycarbony -N-benzyloxycarbonyl-(S)-ornithine (1.5g, 0.004 mol) in 2nhyd-ous dichlorome~hane (60 ml) maintained z- 0C was sequenliallv treated with 1-(3-dimethylaminopropy:)-3-ethylcarboàiimia^
hydrochloride (0.94g) and 1-hydroxyb-nzo.riazole (0.66g) and then left stirring fo_ 0.5h. Me-hylamine (excess) was bubbled th-ough, flushed with nitroc-n and the solutior.
extracted w-th dilute citric acid (40 ml) and brine (40 ml). The crganic phase was dried ar.d evaporated to yielc a viscous o-l. Purification by flas.. chromatography [2%
methanol ir, ethyl acetate~ afforded -he title compound (D20) as a white solid (1.2g).
SUBSTITUTE SHEET
WO91/15507 PCT/GB9l/0053~
2~
Observed FAB (M+H)+ 3~0. C1gH29N3Os requires M 379-DescriPtion 21 N~-Benzyloxvcarbonvl-(S)-ornithine methylamide, trifluoroacetate salt (D21) TFA.H2N~NHCH3 o N OCH2Ph A solution of the amide (D20) (0.14g) in dichloromethane (5 ml) maintained at 0C was treated with trifluoroacetic acid (2 ml). The solution was left stirring at room temperature for 2h, then solvent evaporated in vacuo to afford crude title compound (D21) as an oil. This was used as such without further purification.
Description 22 N~-[N-((R)-1-Dibenzyloxv~hos~hinvl~roDvl)-(S)-leucvll-N~-benzvloxycarbonvl-(S)-ornithine methvlamlde (D22) (PhCH20)2P~ ~ N ~ ~ NHCU3O
SUBSTITUTE SHEET
~'~91/15507 ~s ~,~ ,7 PCTIGB91/00~38 A stirred solution of N-(1-(R)-dibenzyloxyphosphinylpropyl~-(S)-leucine (D3A) (0.142g) ir anhydrous dichloromethane (15 ml) maintained at 0C w2S
treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.07g) and 1-hydroxybenzotriazole (0.04gg), After 0.Sh, the mixture was sequentially treated with crude salt (D21)and N,N-diisopropylethylamine (0.095g) and then left stirring at room temperature for 18h. The solution was then washed with lM aq. citric acid (10 ml), sat. aq. NaHCO3 (10 ml) and sat. aq. NaCl (10 ml). The organic fraction was dried and evaporated in vacuo to yield an oil. Purification by fiash chromatography (5%
methanol in chloroform) afforded the title compound (D22) as a white foam (0.095g).
1~
Observed FAB (M~H) 695- C37H51N47P reqUires _ Description 23 N~-tert-Butoxvcarbonvl-N-dimethyl-(S)-lvsine methYlamide (D23) H
H3C ~ O ~N ~ NHCH3 CH3 H3C CH3 ~ N~c~
A solution of the amide (D4) (0.~g) in methanol (100 ml) 30 w25 treated with 5% palladium on charcoal (lg). The suspension was diluted with 38% aqueous formaldehyde solution (6 ml) and hydrogenated at atmospheric pressure and ambient temperature for 48h. A further aliquot o~
catalyst (0.5g) and formaldehyde (3 ml) was added and SUBSTITUTE SHEET
WO91/15507 PCT/~B91/~3 ?~
hydrogenation continued for a further 24h. The solution was filtered through Kieselguhr, solvent evaporated in vacuo and the residue purified by flash chromatography [(CHCl3:MeOH:NH3)(12:2:1) v/v] to afford the title compound ~D23) (0.29g).
Observed M+ 287. C14H29N3O3 requires M 287.
Description 24 N~-Dimethvl-(S)-lvsine methvlamide, trifluoroacetate sal~
(D24) TFA.H2N ~ NHCH3,CH3.
A stirred solution of the amine (D23) (0.11g) maintained at 0C in dichloromethane (5 ml) was treated with tr,fluoroacetic acid (2 ml). The solution was stirred fc-lh, then solvent evaporated at reduced pressure to affor~
crude title compound (D24) as a clear oil. This W2S used as such without further purification.
~'~91/15507 ~ ,7 PCT/GB91/~538 Descri~tion 25 Na-[N-((R)-1-Dibenzvloxvphos~hinvlvropvl)-(S)-leucvll-N~-dimethvl-(S)-lvsine methvlamide (D25) ~ CH3 C2Hs ~ H
(PhCH2O)2P" ~ N ~ ~ N N_ CH
A stirred solution of N-(1-(R)-dibènzyloxy-phosphinylpropyl)-(S)-leucine (D3A) (0.132g) in anhydrous dichloromethane (20 ml) was treated sequentially with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.066g) and 1-hydroxybenzotriazole (0.042g). The mixture was left stirring for 0.5h, treated with amine salt (D24) and N,N-diisopropylethylamine (0.148g) and stirring continued for 4h at room temperature. The mixture was washed with water (2xlO ml), sat. aq. NaHCO3 (20 ml), dried over anhydrous magnesium sulphate and evaporated _ _zcuo to give a pale yellow oil. Purification by flasr.
chromatography [(CHCl3:MeOH:NH3)(15:2:0.5)v/v] afforded tne title compound (D25) as a white foa~, (O.llg).
Observed FA~ (M+H) 603. C32H51N4OsP req SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~K3~--. ., ... ,. ~, ~
- 52 ~
Description 26 o Na-tert-Butoxvcarbonvl-N-dimethvl-(S)-ornithine methvlamide (D26) H
H3C ~ `r~ N ~Jl~ ~HCH
H~C CH3 ~ - CH~
CH~
The title compound (D26) was prepared following the procedure described for the synthesis of Na-tert-butoxycarbonyl-N-dimethyl-(S)-lysine (D23) (yield: 69%).
Observed FAB (M+H) 274. C13H27N303 requ Description 27 N-Dimethvl-(S)-ornithine methvlamide, trifluoroacetate salt (D27) TFA H l~ HCH~
" ~'~`~-CH~
CH~
The titie compound (D27) was prepared following the ?rocedure described for the synthesis of NE-dimethyl-(S)-lysine methylamide, trifluoroacetate sal. (D24). The title compound (D27) was used without any formal purification.
~'~91/15507 ~$ ;~ ~ Y 7 PCT/GB91/00538 Description 28 N~-~N-((R)-1-Dibenzvloxyphosphinylpropyl)-(S)-leucvl!-.N-dimethYl-(S)-ornithine methylamide (D28) CH~
/~ CH3 C2H5 ~ H
(PhCH20)2P ` N ~f ~ NHCH3 --N~ 3 The title compound (D28) was prepared following the procedure described for the synthesis of the dibenzyl ester (D25). (Yield: 56%).
Observed FAB (M+H) 589. C31H49N4O5P requires _ 588.
Description 29 (R)-2-Hydroxv-4-methYlPentanoic acid (D29) /~ CH3 HO'^~CO2H
The title compound was prepared by modification of thC
method of G. Iwasaki et al, Chem. Pharm. Bull. 1989, 37(2), 280. A solution of sodium nitrlte (33.8g) i.i ~a i--(100 ml) was added dropwise over 1.75h to a sti.red SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/00~3 solution of D-leucine (20g, 0.15 mol) in 3N sulphuric acid (700 ml) and 50% aqueous acetic acid (500 ml) at 92-98C.
The mixture was heated for a further 3h at 98-99C, then cooled and extracted with diethyl ether (4x250 ml). The combined organic layers were dried with anhydrous magnesium sulphate, filtered and evaporated to àryness ~o give the title compound (D29) as a pale yellow solid (18.91g, 94%). A sample was recrystallized from CHCl3/pentane as white needles, m.p. 77.5-79C.
[a]22D = 26.41 (c=0.98 lN NaOH).
Description 30 Methvl (R)-2-hydroxy-4-methylpentanoate (D30) , HO'-~C02CH3 A mixture of acid (D29) (3.97g, 0.03 mol) in methanol (100 ml) and lN ethereal HCl (10 ml) was maintained at ambient temperature for 5 days. After evaporation to dryness ana purlfication by chromatography on silica gel with diethyl etner as eluant the title compound (D30) was obtained as 2 pale yellow oil (3.51g, 80%) and was carried forward without further purification.
SVBSTITUTE S~EET
~'^91/15~07 ~ ? ;,~ PCT/GB9l/00~3g Description 31 N-(1-(S)-DiethoxvPhosPhinYlpropvl)-(S)-leu-ine methv ester (D31) C2H5 ~--(C~H50~P N CO2CH3 The alcohol (D31) (0.696g, 0.0048 moi) ir. dichlorome~.lar.-(10 ml) was cooled to -60C and ther. 2,6-lu.idine (0.68 ml) followed by trifluoromethanesulphonic anhydride (0.92 ml) were added. After allowing to warm to 0C a sol~~~c~.
of (S)-1-aminopropylphosphonic acid, diethyl ester1 (0.93g, 0.0048 mol) in dichloromethane (10 ml) was addec, followed by proton sponge (1.02g). The mixture was stirred for 4 days in the dark under N2 and then filtered, the solid washed with chloroform and the filtrate washed with 10% citric acid (x2) followed by wate~. The organi_ layer was d-ied with anhydrous magnesium sulphate, filtered and evaporated to give the crude produc, as c -G-oil. Purification by column chroma,ogra~hy (first s_ --_ gel with g-adient elution 0-6~ MeO~./CHCl3, ~hen sil--2 ge with grad~ent elution 0-2% MeOH/EtO.C) gave the .it e compound (D31) as a pale yellow oil (o.48g~ 33%).
ODse~ved ~i 323.1862. C14H30NO5P requires 323.1858.
Other physical properties in accorc with .ne litera~_r--1 ~-A-0 401 963 SUBSTITUTE SHEET
WO91/15507 PCT/G891/0053~-2~ t;
Description 32 Na-~N-((S)-Diethoxvphos~hinylpropYl)-(S)-leucyll _~E_ benzyloxvcarbonvl-(S)-lvsine methvl amide (D32) ~ CH3 C2H5 ~ H
(C~H50)2P 1 N ~ ~ NHCH3 O H ~ N ~ OCH2Ph N-(1-(S)-Diethoxyphosphinylpropyl)-(S)-leucine1 was prepared from the corresponding methyl es.er (D31) by standard base hydrolysis. A solution of this acid (0.25g, 0.00081 mol) in dichloromethane (9 ml) w2S cooled to 0C
under N2 and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.17g) then 1-hydroxybenzotriazole (0.12g) were added and the mixture stirred for lh at this temperature. N-Benzyloxycarbonyl-(S)-lysine methylamide trifluoroacetate (D5) (from 0.0097 mol of the corresponding N-tert-butoxycarbonyl derivative (D4)) ~.
dichloromethane (5 ml) was added followed by diisopropylethylamine (0.35 ml) and the mixture stirrec z~
room temperature overnigh~. After dilu.ion with chloroform (20 ml) the mixture was ~ashed with 10% ci~_-ic acid (x2) and water. The organic laye_ was dried (znhydrous magnesium sulphate) and vola~ile material evaporated in vacuo to give a colourless gum.
Pu-ification by chromatography on sil iC2 gel (gradien_ elu_ion 0-5% MeOH/EtOAc) gave the produ~_ (D32) as a colou-less gum (0.36g, 76%).
Observed M+ 584.3346. C28H49N~O7P -equ -es 584.3339.
1 EP-~.-0 401 963 SUBSTITUTE SHEET
'~91/15507 ~1 ~ ~i' ~7 PCT/GB91/~38 Descri~tion 33 N~-~N-((S)-l-Diethoxyphos~hinyl~ropyl)-(S)-leucvll-(S)-lvsine methvlamide (D33) C2H5 ~ H
(C2H5O)2P l N ~ -~ ~ CH3 O ~ NH2 N-[N-((S)-l-Diethoxyphosphinylpropyl)-(S)-leucyl]-N-benzyloxycarbonyl-(S)-lysine methylamide (D32) (0.18g, 0.0003 mol) in methanol (25 ml) with 10% palladium on charcoal (0.2g) was hydrogenated at atmos?heric pressure overnight. After filtration through Kieselguhr and evaporation to dryness the title compound (D33) was obtained as a clear gum.
Observed M+ 450.2982. C20H43N4O5P requires 450.2972.
Descri~tion 34 N-Benzvloxycarbonyl-N~-tert-butoxvc2rbonvl-(S)-lysinP 2-hvdroxYethvlamide (D34) 3 CH3 \ / ~ ~ 2 SUE~STITUTE SHEET
WO91/15507 PCT/GB91/0~53~
~ ?'~ `;7 - 58 -A solution of NE-benzyloxycarbonyl-N~-ter~-butoxycarbonyi-(S)-lysine (5g, 0.0132 mol) in dichloromethane (50 ml~ was cooled to 0C and 1-hydroxybenzotriazole (1.78g) followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.52g) were added. The mixture was st~--e~
at 0C for lh and then ethanolamine (0.88g, 0.0144 mol) added in one portion. The solution was s.irred over,.lgh~
at room temperature, washed with water (2x30 ml), saturated sodium bicarbonate (30 ml) and finally wate- (30 ml). The organic laye- was dried with anhydrous magnes~
sulphate, filtered and evaporated to dryness to give _;~e product (D34) as a clear gum (S.44g, 97%) which was used without further purification.
Observed M+ 423.2365. C21H33N3O6 requires 423-2369-Description 35 N-Benzvloxvcarbonvl-(S)-lvsine 2-hvdroxvethvlamide, trifluoroacetate salt (D35) TFA H2~ ~ ~ ~ OH
H ~ ~ OCH2Ph ~-ifluoroacetic acid (30 ml) was added tG a stir-ed solu~ion of the lysine de-ivative (D34) (2.64g, 0.0062 mol) n d-chloromethane (S0 ml) at 0C. ~fter 3h stir-in a~ ice-bath temperature volatile material was removed SUBSTITUTE SHEET
~'~91/15507 PCT/GB91/00538 2 . i~;~ ~ 7 under reduced pressure to give the crude product (D35) ir.
quantitative yield. This was used in subsequent steps without further purification.
Observed FAB (M+H) 324- Cl6H25N34 requireS M
Descri~tion 36 ~a-~N-((R~-l-Dibenzvloxvphos~hinvl~-o~vl)-(S)-leucvll-(S)-~'E-benzvloxvcarbonvl-lvsine 2-hvdroxvethvlamide (D36) CH~
2 5 ~ H
(PhCH2O)2P` N ~ ~ N ~ OH
O ~ N ~ OCH2Ph A solution of N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (1.61g, 0.0037 mol) in dichloromethane (60 ml) was cooled to 0C and 1-hydroxybenzotriazole (0.78g, 0.0058 mol) was added followed by 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimiae hydrochloride (1.09g, 0.0057 mol). The mixture was sti-red fcr lh at ice-bath ~emperature and then a solution of the trifluoroacetate sal. (D35) (1.2 equivalents) i-, d chlo~ome.hane (15 ml) h'2S adde~ fcllowed by dlisopropyle_hylamine (3.36g) tG
ensu-e neutralization of excess .-i'luoroacetic acid.
Afte- stirring overnight at room ~emperature .he reac_ion mixture was washed with 10% acuec_s citric acid (2x20 ml), wate- (20 ml), sa~urated soaium ~ _arbonate (2~.20 ml) znd 'inally wate-. The organic laye- was dried (anhyarous magnesium sulphate) filterea a-,à evaporated tc dryness.
The residue was chromatographec o-, silica gel with 1:1 SUBSTITUTE SHEET
WO91/1~507 PCT/GB91/~3~-2 ~ 7 increasing percentage of methanol (to 10%). Further purification on silica gel with a gra ien. of 0-4~
MeOH/CHCl3 gave the title compound (D36) as a clea- gum (0.83g, 30%).
Observed M+ 738. C39H55N4O8P requires M 738.
Descri~tion 37 N~-~N-((S)-1-Dibenzvloxy~hosphinyl~ro~vl)-(S)-leucvl~-(S)-N~-benzvloxycarbonvl-lvsine 2-hvdroxy~~hvl2mide (D3 C2H5 ~ CH3 O
(PhCH20)2P 1N ~ ~ N ~ OH
~, N ~ OCH2Ph The title compound ~D37) (1.035g) W25 prepared from N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3B) (0.67g, 0.0015 mol) and the trifluoroacetate salt (D35) by the method given in Description 36.
Observed FAB (M+H)+ 739- C39~55N~-82 reqUires M 738-~91/15507 ~ PCT/GB91/0053g Description 38 N~-~(S)-tert-Butoxvcarbonvl-leucvll-N-benzvloxvcarb~nyl-(S)-lvsine methylamide (D38) ~ CH3 H3C ~ O N O ~ N ~ OCH2Ph N-tert-Butyloxycarbonyl-(S)-leucine (25g, 0.11 mol) was dissolved in dichloromethane (250 ml) and cooled to 0C.
l,1'-Carbonyldiimidazole (18g, 0.113 mol) was added and the mixture left stirring at 0C for 0.5h. The solu~ion was allowed to warm to room temperature for 15 mins and then recooled to 0C.
N-Benzyloxycarbonyl-(S)-lysine methylamide trifluoroacetate salt (D5) (47g, 0.118 mol) in dichloromethane (150 ml) was added followed by the immediate addition of diisopropylethylamine (30.5g, 0.236 mol). The reaction mixture was left sti-ring overnight 2-room temperature and then treated with water and ex~racted with chloroform. The combined organic layers were washed with dilute hydrochloric acid (2x100 ml), wate- (2x100 .1), aqueous sodium carbonate (3x100 ml) and brine. The organic layer was dried with anhydrous magnesium sulpha~e and evaporated to dryness to give the ti~le compound (D3 (50g, 93%) which was used withou, furthe- purification.
Observed (M+H)+ 507- C26H42N46 requires M 506.
WO 91/15507 PCI/GB91/005~"
~r ~. ~?'i~ ~
Description 39 Na-~(S)-Leucvll-Nf-benz~loxvcarbonvl-(5)-lvsine methvlamide (D39) TFA.H2N ~ ~ NHCH3 N~,~OCH2Ph o A cooled solution of the amide (D38) (50g, 0.099 mol) in dichloromethane (150 ml) was treated w th trifluoroacetic acid (150 ml). After 3h the solvent W2S evaporated under reduced pressure. The resulting oil was dissolved in methanol and acidified with dilute hyd-ochloric acid to give the hydrochloride salt. Water was added and non-basic organic material extracted out with ethyl acetate (2x200 ml). The aqueous layer was basi~ied to pH 8-9 with 10% sodium hydroxide and then extracted with ethyl acetate (3x150 ml). These combined organic extracts were dried ~Na2SO4) and evaporated to d-yness to cive the ~itle compound (D39) (20g, 50%) as 2 white solid.
Observed (M+H)~ 407. C21H3qN~O~ requ res ~ 406.
SUBSTITUTE SHE~T
'91/15507 ~,~ ~ ~; 7 PCT/CB91/00~3g Description 40 Na-~N-((R)-1-DibenzvloxvphosphinvlPropyl)-(S)-leucYll-N-benzvloxvcarbonvl-(S)-lvsine methvlamide and Na-[N-~(S)-1-Dibenzvloxv~hosphinvlpropyl)-(S)-leucyll-(S)-NE-benzvloxYcarbonYl-lysine methYlamide (D40) 2 2, H - H
~ N ~ OCH2Ph Dibenzyl ((1-trifluoromethanesulphonyloxy)propyl)-phosphonate (D2) (4.4g, 0.0098 mol) was dissolved in dry dichloromethane (20 ml). N~-[(S)-Leucyl]-N-benzyloxycarbonyl-(S)-lysine methylamide (D39) (4.0g, 0.0098 mol) and Proton Sponge (2.0g, 0.0098 mol) were added to the solution and the reaction mixture was stirred in the dark at room temperature for 10 days.
The solution was diluted further with chloroform, washed wl.h 10% citric acid (2xS0 ml) and water (3x50 ml), dried (anhydrous MgSO4) and evaporated to dryness to give an orange oil. This was purified by column chromatography 2~ (silica ge~, 2% MeOH/EtOAc) to give the mixture of title com?ounds (D40) (2.55g, 35%) as a clear oll.
Observed (M+H)+ 709. C38H53N47? requires M 708.
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/0053~
2 . ~.~ 64 -Descri~tion 41 N-~N-((S)-1-DibenzvloxyPhosphinylProPvl)-(S)-leucY
nitro-(S)-arqinine methYlamide (D41) 2 5 ~ H
(phcH2o)2p NH ~ 3 NH
The title compound (D41) (0.72g, 74%) was pre~ared fro~
N-tert-butoxycarbonyl-N~-nitro-(S)-arginine methylamide 15 (D13) (0.6g, 0.0018 mol) and N-((S)-1-diber.zyloxy-phosphinylpropyl)-(S)-leucine (D3B) (0.633g, 0.0014 mol) by the method described in Description 14 with the exception that dichloromethane was used as reaction solvent with sufficient dimethylformamide to effect solution.
Descri~tion 42 N-((R)-1-Dibenzvloxy~hos~hinylethyl)-(S)-leucine (D42A) and N-((S)-1-dibenzvloxY~hos~hinYle~hvl)-(S)-leucine (D42B) ~ CH~
(phcH2o)2p N ~
SUBSTITUTE SHE~T
~91/1~507 PCT/GB91/00538 ~ 7 The title mixture of diastereoisomers (D42) was prepared analogously to the method in Description 3, Method B, a5 c white solid.
Observed FAB (M+H) 420- C22H30NO5P -equires M 419.
DescriPtion 43 Na-~N-((R)-l-Dibenzvloxv~hos~hinvlethvl)-(S)-leucv'l-(S)-N-benzvloxycarbonvl-lvsine methvlamiàe (D43A) and N~-~N-((S)-l-Dlbenzyloxvphos~hinvlethvl)-(S)-leucvll-(Ci-N-benzvloxvcarbonvl-l~sine methvlamide ~D43B) C~
CH ~ CH~ O
OCH2Ph The title compound (D43) was obtained as a clear oil (1.0g, 63~) from the acid (D42) (l.0g, 0.002 mol) following the general method of Description 6.
Observed FAB (M+H) 695- C37n5lN4o72 requires M 69 SUBSTlTUTE SHEET
W091/15507 PCr/GB91/00~?~-2 . , i~; 7 Example 1 N-[N-((R)-1-PhosPhonopro~yl)-(S)-leucvll-(S)-lvsine methvlamide (El) C H ~ CH3 (HO)P"`~ N ~ N ~ NHCH3 ~ NH2 The dibenzyl ester (D6) (O.lOSg, 0.16 mmol) was dissolved in ethanol (90 ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated 1n vacuo to give the title compound (El) (O.Olg).
Observed FAB (M+H) 395. C16H3505N4P req (CDC13/CD30D): 0.95 (6H,dd), 1.08 (3H,t), 1.4-2.0 (9H,m), 2.65 (lH,m), 2.75 (3H,s), 2.92 (2H,m), 3.45 (2H,m), 4.18 (lH,m), 4.4 (lH,m).
SUBSTITUTE SHEET
~'~91/15507 Z' PCT/GB91/00538 Examole 2 Na-~N-((R)-1-Phosphonopro~vl)-(S)-leucvll-N~-qlycyl-(S)-lvsine methylamide (E2) ~ CH3 (HO)20 H N ~ NH~
The dibenzyl ester (D9) (0.43g) was dissolved in ethanc (60 ml) and hydrogenated over 10% palladium on charcoal 2.
atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated ln vacuo to give the title compound (E2) (0.059g).
Observed FAB (M+H)+ 452- ClgH38o6N5p requires M 45i-(CD30D): 0.78-0.95 (9H,m), 1.19-1.9 (~lH,m), 2.31 (lH,m), 2.62 (3H,s), 3.C;-3.25 (2H,m), 3.3G-3.65 (2~.,m), 3.83-3.92 (lH,bt), 4.15-4.3 (lH,t).
SUBSTITUTE SHEET
Z, ~ ,.";.` 7 - 6 8 Exam~le 3 N-[N-((R)-l-phosphonopro~yl)-(s)-leucv~ -r(2-Pvrrolidinoethyl)amidel-(s)-asD2rtic acid methvla~.ido (_3, CHl - ~ ~ H
(H0)2P N ~ ~ NHCH
~ N
o This compound was prepared from N-[N-[(R)-dibenzyloY.v-phosphinylpropyl]-(S)-leucine-~-[(2-pyr-olidinoethy')-amide]]-(S)-aspartic acid methylamide (D12) by hydrogenation over 10~ palladium on cn2rcoal at atmospheric pressure. m.p. 130-135C (95% yield).
(CD30D): 1.0(6H,t,J=6Hz), 1.1(3H,t,J=7kz), 1.55-1.8(4H,m), 2.0(lH,m), 2.15(4~.,m), 2.6-2.85( 6H, m), 3.3-3.75(approx. 8H,m), 4.1(lH,t,J=6Hz) and 4.75(1H,t,J=5Hz).
_xam~le 4 ~ '-((R)-l-~hos~hono~-o-~vl)-(S)-:eu~ -(^)-a-~ ',r-methvlamide (E4) C H ~ CH3 (HO)P""~ N ~ N ~ ~lHCH3 NH
- N NH
H
SUBSTITUTE SHEET
~"~91/15507 2 . ~- ~,.; ;,7 PCT/GBg1/0053g The fully protected derivative (D14) was dissolved in a mixture of glacial acetic acid (40 ml) and water (iO ml) and hydrogenated over 10% palladium on charcoal cata'ys.
for 18h at atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated in vacuo to give the title compound (E4) (0.085g).
Observed FAB (M+H) 423. C16H35N605P req ~ (CD30D): 0.95(6H,dd), 1.03(3H,t), 1.5-i.94(9r.,m), 2.56(lH,m), 2.72(3H,s), 3.17(2H,m), 4.02(i~,t), 4.37(lH,t).
Exam~le 5 N~-~N-((R)-l-PhosphonoPropvl)-(S)-leucyll-(R)-~-(4-PiPeridvl)alanine methvlamide (E5A) and N~-~N-((R)-l-PhosPhonoproPvl)-(S)-leucvll-(S) -B- (4-Pi~eridvl)alanine methYlamide (E5B) C2H5 ~ H
(HO)P~ N ~ N ~ CH3 ~
Tne fully protected derivative (Dl9) (0.24g) was disso~ve-in ethanoi (50 ml) and hydrogena~e~ over 10% palladium c-cha-coal at atmospheric pressure. ml he solution was ~lltered through Kieselguhr and solven. evaporated ~
vacuo. The residue was tritura~ed with ether to remove slisht impu~ities leaving the prod ~~ (E5) as an o f-whi-_ solid (0.13g).
SVBSTITUTE SHEET
WO91/15507 PCT/GB91/~39--~?~.:7 Observed FAB (M+H)+ 421- cl8H37N4o5p requireS M 420-(CD30D): 0.95(6H,dd), 1.02,1.05(3H, overlapplng triplets), 1.4-2.0(12H,m), 2.4-2.55(1H, 2 overlappins m), 2.71(3H,d), 2.81-3.03(2H,m), 3.34(4H,br m), 3.68-3.81(1H, dt), 4.40(lH,m).
Exam~le 6 N-~N-((R)-1-Phos~honopro~vl)-(S)-leucvll-(S)-orn-t:rine methvlamide (E6) HO~P" ~ N ~ nHCH3 O H O
A solution of phosphonic diester (D22) (0.07g) in methanol (20 ml) was treated with 5% palladium on charcoal. The suspension was hydrogenated at atmospheric pressure for 24h, filtered through Kieselguhr and solvent evapora_ed _~
vacuo. The residue was triturated with diethyl ethe- (5 ml) to give the title compound (E6) as a white solid 25 (0.03g), m.p. 165-169C.
Observed FAB (M+H) 381- cl5H33N4o5? requi-es M 38C-~ (CDCl3/CD30D): 1.0(6H,dd), 1.12 (3r., t), 1.6-1.9(8H,m), 2.05(1H,m), 2.7(lH,m), 2.75(3H,s), 2.9(2H,t), 4.42(1Y,m), ~.55(lH,t).
The S,S,S diastereoisomer is prepared by a similar metho~.
SUBSTITUTE SI~EET
~ '91/15507 2~ ~ ~~ PCTtGBgl/00538 Exam~le 7 N~-~N-((R)-1-Phosphonopropyl)-(S)-leucYll-N~-dimethvl-(S)-lysine methvlamide (E7) C~ 5 ~ H
(HO)p~`~ N ~ N = ,CH3 A solution of phosphonic diester (D25) (0.75g) in methanol (20 ml) was treated with 5% palladium on charcoal (O.OSg) and hydrogenated at atmospheric pressure for 24h. The solution was filtered through Kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether (10 ml) to give the title compound (E7) as white solid (0.045g).
Observed FAB (M+H)~ 423. C18H39N405P requ (CD30D): 1.0(6H,dd), 1.1(3H,t), 1.3-1.85(lOH,m), 2.0(lH,m), 2.6(lH,m), 2.75(3H,s), 2.8(6H,s), 3.05(2H,t), 4.3_(2H,m).
SU~3STITUTE SHEET
WO91/15507 PCT/GBg1/~?-~, ,, ,, ~, J - 72 -Exam~le 8 N~-~N-((R)-1-Phosphonopro~vl)-(S)-leucvll-N-dimethvl-(5)-ornithine methylamide (E8) 2 5 ~ ~ O
(HO)~P` ~ HCH
O H O
\ ~",CHt CH~
The title compound (E8) was prepared from the dibenzyl ester (D28) following the procedure described for the synthesis of N~-[N-((R)-l-phosphonopropyl)-(S)-leucyl] -N-dimethyl-(S)-lysine methylamide (~7). (Yield: 74%). m.p.
88.5-90C.
Observed FAB (M+H) 409- Cl7H37N4o5? requires M 408-Exam~le 9 N~-~N-((S)-1-Phosphono~ropvl)-(S)-leucvll-(S)-lysine methvlamide, hvdrobromide salt (_9) ~ CH
2 ~ O
- ~ NH2.HBr SUBSTITUTE SHEET
.,7 ' ~91/15507 PCT/GB91/00538 Bromotrimethylsilane (0.48 ml, 12 equiv.) was added ~o a solution of the diethyl ester (D33) (0.14g, 0.0024 mol) -dry acetonitrile (10 ml) and stirred at room temperature for 3 days.
The resulting yellow solution W25 evaporated to dryness and treated with methanol/wate-. After evaporation and repeated treatment with methanol/water the product (~9) was obtained in quantitative yield as a pale orange foam.
Observed FAB (M+H)+ 395- cl6H35N4o5p reauireS M 39~-(CD30D): 1.01(6H,dd), 1.18(3H,t), 1.3-2.15(11H,m), 2.74(3H,s), 2.98(2H,t), 3.19(1H,m), 4.3(1H,m), 4.42(1H,t).
Example 10 Na-~N-((R)-1-PhosPhonopropyl)-(S)-leucYll-(S)~lysine 2-hvdroxyethvlamide (E10) ~ CH3 C2H5 ~ H
(HO)2p~ ~ N ~ ~ NH2 A solution of the dibenzyl ester (D3~) (0.8g, 0.001~ mol in methanol (100 ml) with 10% palladium on charcoal W2S
hydrogenated at atmospheric pressure ove-night. After filtration through Kieselguhr and evaporation to dryness the residue was taken up in doubly Gist lled water, refiltered and freeze-dried to give the title compound ~F10) as a white foam (0.39g, 85%).
/
SUBSTITUTE SHEET
WO91/15507 P~T/GB91/ ~ Y
~;~ . ' J '~ V~
Observed FAB (M+H)+ 425. C17H37N406P requ (CD30D): 0.98(6H,dd), 1.05(3H,t), 1.3-2.05(1lH, overlapping m), 2.54(1H,br m), 2.92(2H,m), 3.3(2H,m, overlaps CHD20D solvent signal), 3.6(2H,t), 4.10(1H,b- m), 4.38(lH,dd).
Exam~le 11 ~-~N-((S)-1-Phos~hono~ro~vl)-(S)-leucvll-(S)-lvsine 2-hvdroxyethvlamide (E11) C ~ CH~
(HO)2P ~ ~ ~ ~ OH
O H I ~ NH2 The title compound (E11) was obtained by atmospheric pressure hydrogenation of the dibenzyl ester (D37) by the method given in Example 10.
0bserved FAB (M+H)+ 425- C17H3~N406P requires M 424-~ (CD30D): 0.97 (6H,dd), 1.12 (3H,t) 1.35-2.10 (llH, ove-lapping m), 2.46 (l..,m), 2.g5 (2H,m), 3.32 (2H,m, ove-laps CHD20D solven. signal), 3.53 (lH,m), 3.65 (2H,m), 4.49 (l:.,m).
S~ ST)TUTE S~1EET
2,.i. _;.'.~,7 ~'~91/15507 PCT/CB91/~38 Example 12 Na-[N-((R)-1-PhosPhono~ropyl)-(S)-leucvll-(S)-lvsine methylamide and Na-rN-((S)-1-phosphonopro~vl)-(5)-leucvll-(S)-lvsine methylamide (E12) (H)2P N ~ ~ NHCH3 o H --NH2 The mixture of dibenzyl esters (D40) (2.55 g, 0.004 mol) in ethanol (100 ml) was hydrogenated over 10% palladium on charcoal at atmospheric pressure. The solution was filtered through Kieselguhr and the solvent evaporated in vacuo to give the title compound (E12) as a white solid (l.Og, 71%).
Observed FAB (M+H)+ 395. C16H35N405P requi Exam~le 13 N-rN-(~S)-1-Phos~hono~roPvl)-(S)-leucvll-(S)-ar~inine methvlamide (E13) 2 5 ~H
)2P NH~ NHCH3 NH
S~ ~TITUTF SHEET
WO91/15507 PCT/GB91/~K3 The title compound (E13) (0.5g, 90%) was ~repared from the dibenzyl ester (D41) (0.633g, 0.014 mol) by the method o' Example 4.
Observed FAB (M+H)+ 423- C16H35N6O5P requires M 422-(CD30D): 0.97(6H,t), 1.10(3H,t), 1.49-1.95(9H,m), 2.50(lH,m), 3.21(2H,m), 3.60(lH,m), 4.94(lH,m).
Example 14 Na-~N-((R)-1-Phosphono~ro~vl)-(S)-leucvll-(S)-homoarainine methvlamide, disodium salt (E14A) and N~-~N-((S)-1-phos~hono~ropvl-(S)-leucvll-(S)-homoarsinine methvlamide, disodium salt (E14B) ~ HCH3 C2H5 ~ H O N ~ NH2 NH
The mixture of R,S,S and S,S,S isomers (~12) (34.5mg, 0.087 mmol) in water (0.5 ml) was treatec with sodium bicarbonate (44 mg, 6 equiv.) followed by 2-methyl-2-thiopseudourea sulphate (24.3 mg, 1 equiv.) and stirre~ z-room temperature for 3h. Additional por~ions of sodium bicarbonate (8.5 mg) and 2-methyl-2-~hio?seudourea (15 mg) were added and the mixture then heated a~ 70C for i.5h.
Purification by reverse phase chromatogra?hy gave the title compound mixture of isomers (c14) 2S a white solic.
Observed FAB (M+H)+ 481. C17H35N6O-~Na2 requires M 480.
SU~3STITUTE SHEET
~91/lS507 ~ ~ ~_7 PCT/GB91/~K38 Example 15 N-~N-((R)-1-Phosphonopropvl)-(S)-leucvll-(S)-N-~2-imidazolinvl)-lvsine methvlamide disodium salt (E15A) and N~-~N-((S)-1-Phosphonopropvl)-(S)-leucvll-(S)-NE-(2-imidazolinvl)-lysine methvlamide disodium salt (E15B) l 5 ~ H
H O -N ~
The title mixture of diastereoisomers (E15) was prepared from the phosphonic acid mixture of isomers (E12) (36 9 mg, 0.0935 mmol), 2-methylthio-2-imidazoline hydroiodide (45.6 mg and, after 3h, 13.9 mg) and sodium bicarbonate (47 mg and, after 3h, 7.8 mg) by the general method of Example 14.
Observed FAB (M+H)+ 507- Cl9H37N605P~a2 requireS
SUBSTlTUTE SHEET
WO91/15507 PCT/CB91/ ~ 3'~
Z~-~, ,"~,7 - 78 -Example 16 Na-[N-(~R)-1-Phosphonoethyl)-(S)-leucvll-(S)-lvsine methvlamide (E16A) and Na-~N-((S)-1-phosphonoethvl)-(S)-leucvll-(S)-lvsine methvlamide (E16B) CH~
~ H
(HO),P ~ ~ ~ NHCH~
~ NH2 The dibenzyl ester (D43) (l.O.g, 0.0014 mol) was hydrogenated at atmospheric pressure by the method of Example 1 to give the title compound mixture (E16) in quantitative yield as a white crystalline solid.
Observed FAB (M+H) 381. C15H33N4O5P requi SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~053 r~
COLLAGENASE INHIBITOR ASSAY
The test is performed essentially as in Cawston and Barrett, Anal. Biochem. 99, 340-345 (1979). Compounds for testing are dissolved in methanol by sonication and added to collagenase (purified from culture supernatants from the human lung fibroblast cell line, WI-38) in buffer. After a 5 min pre-incubation at 37C, the assay tubes are cooled to 4C and 3H-acetylated rat skin type T
collagen is added. The assay tubes a-e incubated at 37C
overnight. The 3H-collagen forms insoluble fibrils, which are the substrate for the enzyme.
To terminate the assay, the assay tubes are spun at 12000 t5 rpm for 15 minutes. ~ndigested 3H-collagen is pelleted, while digested 3H-collagen is found as soluble peptides in the supernatant. A sample of the supernatant is taken for liquid scintillation counting.
The activity of collagenase inhibitors (IC50: 50%
inhibitory concentration) is expressed as that concentration of compound that inhibits a known ~standzrd) concentration of enzyme by 50%.
The compounds of Examples El-E9 had IC50 values between l.8 x l0 7 and 2.2 x l0 5M.
SUBSTITUTE SHEET
2~ 7 Description 12 N-~N-((R)-1-DibenzyloxvPhosPhinvlPropvl)-(S) -leuc~ll -B-~(2-pyrrolidinoethvl)amidel-(S)-asPartic acld methvlamide (D12) C2H5 ~ H
(PhCH20)2P` ~ N ~ ~ ~ CH3 N ~
This compound was prepared from N-(1-(R)-dibenzyloxy-phosphinylpropyl)-(S)-leucine (D3A) and ~-[(2-pyrrolidinoethyl)amide]-(S)-aspartic 2^id methylamide (prepared from (Dll) by reaction with trifluoroacetic acid by the procedure described for (D5)) following the method of Description 11, m.p. 110-118C
(48% yield).
(CDCl3): 0.85(6H,t,J=5Hz), 1.05(3H,t,J=7Y.z), 1.4(3H,m), 1.6(3H,m), 1.85(approx. 6H,m), 2.6-2.85(approx. lOH,m), 3.3(1H,m), 3.45(1H,m), 3.65(1H,m), 4.85(1H,m), 5.0(4H,m), 7.~5(lOH,m), 7.5(lH,m) and 8.2(lH,d,J=7Hz).
Des-riDtion 13 N-~e-t-Butoxycarbonvl-N~-nitro-(S)-arainin.e methvlamide (D13) H
H3C ~ O ~N ~ NHCH3 NH
H3C CH3 N N~N02 H
3~
SU~3STITUTE SHEET
~ ~91/15507 2- ~ i. ;. 7 PCT/GB91/~K3g N-tert-Butoxycarbonyl-N~-nitro-~S)-arginine (3g, g 4 mmol), 1-hydroxybenzotriazole (2.88g, 18.8 mmol) and methylamine hydrochloride (1.26g, 18.7 mmol) were dissolved in dry dimethylformamide (50 ml) and cooled in an ice-salt bath to -10C. Diisopropylethylamine (3.3 rl, 18.78 mmol) was added followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.76g, 14.9 mmol). After lh, at 0C the reactio~. ~.ixrure was stirred for 18h at room temperature when t.l.c.
~(CHC13:MeOH:AcOH) (10:2:1) v/v] showe~ the reaction tc be complete. After evaporation to dryness the solid was dissolved in water and applied to a column of Dowex 50W-X8 (ammonium form). This was eluted with water and eluan~
containing W absorbing material was collected, concentrated and then shaken with Amberlite IRA-901 (acetate form) for lh. The resin was collected and washed with water. The filtrate and washings were combined and lyophilised to give the product (D13) as a chromatographically pure white solid (2.6g, 89%).
Observed FAB (M+H)+ 333- C18H2~65 reqUires M 332-Descri~tion 19 2~ - r~- ( (R)-l-Dibenzvloxy~hos~hinyl~ro~yl)-(S)-leucvl~
n ~ro-(S)-arqinine methvlamide (D1 G ) C2Hs ~ H
(PhCH2O)2~p~ NH ~ ~ 3 NH
SUBSTITUTE SHEET
~ i; ~7 ~ 42 -Na-tert-Butoxycarbonyl-N~-nitro-(S)-arginine methylamide ~D13) (0.142g, 0.43 mmol) was treated with 95~
trifluoroacetic acid in water (5 ml) for 0.5h at 0C.
~xcess acid was removed under reduced pressure and the residue twice evaporated with dry toluene. The residue was dissolved in dry dimethylformamide (2 ml) and the pH
adjusted to 8-9 by the addition of diisopropylethylamine.
This was added to a solution of N-(l-(R)-àibenzyloxyphosphinylpropyl)-(s)-leucine (D3A) (0.15g, 0.35 mmol) and l-hydroxybenzotriazole (0.095g, 0.62 mmol) in dimethylformamide (5 ml). The mixture was cooled to -10 to -15C and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.08g, 0.42 mmol) added.
The reaction mixture was allowed to warm to room ~emperature and stirred for 18h. The mixture was then evaporated to dryness and purified by silica gel chromatography [(MeOH:CHCl3) (1:9) v/v~. The product (D14) was obtained as a pale yellow foam. (0.16g, 71%).
Observed FAB (M+H) 648. C30H46N7O7P req DescriDtion 15 Na-tert-Butoxvcarbonyl-(R)-~-(4-Dvridvl)alanine (D15A) and 2~ a-tert-butoxvcarbonvl- (s) -B- (4-Dvridvl)alanine (DlSB) H
H3C ~ `t~ ~ OH
H~C CH3 N
SUBSTITUTE SHEET
" ~91/15507 ~ ~ i ~7 PCT/GB9lt00~38 Crude racemic ~-(4-pyridyl)alanine dihydrochloridel (0.88g, 3.66 mmol) was dissolved in water (30 ml) and solid NaHCO3 (1.51g) added. When dissolved, dioxan (30 ml) was added and the mixture cooled in an ice-salt bath to 0C. Di-tert-butyl dicarbonate (1.5g, 6.9 mmol) as a solution in dioxan (20 ml) was added and after lh at 0-4C, the mixture was stirred for 18h at room temperature.
The reaction mixture was evaporated to dryness and a mixture of ethyl acetate, water and acetic acid (S:l:l, v/v) added. The precipitated solid was filte~ed off and the filtrate evaporated in vacuo. The residue was triturated with several portions of hot ethanol. When cool, the extracts were combined and evaporated to dryness. This process was repeated until the solid residue redissolved readily in ethanol. Evaporation of this solution gave the title compound (Dl5) as a racemic mixture, contaminated with sodium acetate. This was used without further purification.
R.L. Bixler and C. Niemann, J. Org. Chem., 23, 575 (1958).
Desc-i~tion 16 Na-ter~-Butoxvcarbonvl-(R) -B- (4-pyridvl)alanine methvlamide (Dl6A) and N~-tert-butoxvcarbonYl-(S) -B- (4-~vridyl)alanine methylamide (D16B) H
H3C ~ O ~ N ~ NHCH3 H3C CH3 ~
'~
SU~3STITUTE SHEET
WO91/15507 PCT/GB91/00~38 Z, ~ 7 Crude racemic derivative (D15) (3.66 mmcl) was dissolve^
in dry dimethylformamide (30 ml) and 1-hydroxybenzo-triazole (1.13g, 7.38 mmol) and methylamine hydrochloria-(0.5g, 7.40 mmol) added. The mixture was cooled to -10~
in an ice-salt bath and diisopropyle_hylamine (' 3 ml, ~-mmol) added. After S minutes, 1-(3-dime~hylamino~ropyl)-3-ethylcarbodiimide hydrochloride (1.7g, 8.9 mmol) was added and after stirring for lh a, -5C, the mixture was stirred overnight at room temperature. Afte evapo_atio..
to dryness, dichloromethane (50 ml) was added and ther.
- extracted with several po_tions (3x15 ml) of sat. aq.
NaHCO3. The organic phase was dried ove- anhydrous magnesium sulphate, filtered and the solven_ removed ~ r vacuo. The residue was purified by silica gel chromatography [(MeOH:CHCl3) (1:9) v/v] to give the racemic title compound (D16) as a white solid (0.91g).
Observed FAB (M+H) 280- C14H21N33 reqUires M 279-DescriD.ion 17 N~-tert-Butoxvcarbonvl-(R)-~-(4-Di~e-idvl)alanine methYlamide (D17A) and ~'~-te_t-bu~oxvcarbonvl-(S) ~-(4-DiDeridvl)alanine methvlamide (Dl7B) H
H3C ~ O ~ N ~ NHCH3 H3C CH3 ~
~ NH
The mixtu;e o' isomers c' amid~ (D16) (0.5~, ;.79 mm_ ) was dissolved in glaci21 acetic a- d (4~ ml) and degasse-SUBSTITUTE SHEET
~'~91/15507 ~ r ~ ~ PCT/GB91/00538 under reduced pressure. A suspension of Adam's catalys~(0.2g) in glacial acetic acid (5 ml) was added and the mixture hydrogenated for 22h at atmospheric pressure. The reaction mixture was filtered through Kieselguhr and the solvent removed in vacuo to give the racemic product (Dl7) as a chromatographically pure oil (O.Slg).
Description l8 N~-tert-Butoxycarbonyl-(R) -B- (4-(N-benzvloxvcarbonvl)-Pi~eridvl)alanine methvlamide (Dl8A) and Na-te-t-butox~-carbonvl-(S)-~-(4-(N-benzyloxvcarbonvl)~iDeridvl)alanine methvlamide (Dl8B) H
H3C ~ ~fN ~ NHCH3 H3C CH3 ~
~ N ~ OCH2Ph O
Isomer mixture (Dl7) (0.51g, 1.79 mmol) was dissolved in a dioxan/water mixture (2:l, 30 ml) and the pH adjusted ~o 7 by the addition of solid NaHCO3. The mixture was cooled to 0C, a second portion of NaHCO3 (0.17g, 2 mmol) added followed by the portionwise addition of benzylchloroformate (0.3 ml, 2.1 mmol). Afte- lh at 0C, ~he mixture was stirred overnight a. room temperature.
After evaporation to dryness, the residue was taken up ir.
e-hyl acetate and extracted with 10% aq. citr.c acid (2Y.), sa~. aq. NaHCO3 (2x) and sat. aq. NaCl. The organic pnase W25 dried (anhydrous MgSO4), filtered and the solvent removed in vacuo. The resultant oil was purified by SUBSTITUTE SHEZ~T
WO91/15507 PCT/GB91/~3~-,7 silica gel chromatography [(CH2C12:MeOH)(l9:1)v/v] to gi~e the racemic title compound (D18) as an oil which crystallised on standing under 60-80 ligh~ petroleum (0.68g, 91%), m.p. 130-131C.
Observed FAB (M+H) 420. C22H33N3Os req~ires M 419-Description 19 Na-~N-((R)-1-Dibenzvloxvphos~hinvl~ropyl)-(S)-leucvll-(R)-~-(4-(N-benzvloxycarbonyl)~i~eridvl)alanine methylamide (D19A) and N-~N-((R)-1-dibenzyloxv~hos~r.invl~roDvl)-(S)-leucvll-(S)-~-(4-(N-benzvloxvcarbonvl)pi~eridvl)alanine methvlamide (D19B) CH3 C2H5 ~ H
(PhCH2O)2P ~ HCH~
O ~
~ ~ ~ OCH2Ph The protected amide isomer mixture (D18) (0.19g, 0.45 mmol) was treated with 95% trifluoroacetic acid in wate-(5 ml) for 0.Sh at 0C. Excess acid was -emoved under reduced pressure and the oily residue evaporated twice with dry toiuene. The residue W25 dissolved in dry dimethyl_ormamide (2 ml) and the pH adjusted to 8-9 by ~he addition o^ diisopropylethylamine. ~his w2S added to 2 solution of N-(l-(R)-dibenzyloxy?hos?ninylpropyl)-(S)-leucine (D3~.) (0.15g, 0.35 mmol) ana 1-hydroxybenzo-t~iazole (0.llg, 0.7 mmol) in dimetr.ylformamide (5 ml)~
The mixture was cooled to -10C and --(3-dimethylaminopropyl)-3-ethylca-bodiimide hydrochloriae SUBSTITUTE SHEET
91/15~07 ~,r~ 7 PCT/GB~1/00538 ~ 47 ~
(0.lg, 0.52 mmol) added. The mi~.ture was allowed to ~Jarm to room temperature and then stirred for 18h. The reaction mixture was evaporated to dryness in vacuo ana the residue redissolved in dichloromethane. This was extracted with 10% aq. citric acid, sat. aq. NaHCO3 (2x) sat. aq. NaCl and then dried over anhydrous magnesium sulphate. Evaporation in vacuo gave the title com?ound isomer mixture (D19) as an oil which was ?urified by silica gel chromatography [(CH2Cl2:MeOH)(g:1)].
Descri~tion 20 Na-tert-Butoxycarbonyl-N~-benzvloxvc2rbonvl-(S)-ornithine methvlamide (D20) H3C ~ O ~N ~ NHCH3 o H3C CH3 N ~ OCH2Ph A solution of N~-tert-butoxycarbony -N-benzyloxycarbonyl-(S)-ornithine (1.5g, 0.004 mol) in 2nhyd-ous dichlorome~hane (60 ml) maintained z- 0C was sequenliallv treated with 1-(3-dimethylaminopropy:)-3-ethylcarboàiimia^
hydrochloride (0.94g) and 1-hydroxyb-nzo.riazole (0.66g) and then left stirring fo_ 0.5h. Me-hylamine (excess) was bubbled th-ough, flushed with nitroc-n and the solutior.
extracted w-th dilute citric acid (40 ml) and brine (40 ml). The crganic phase was dried ar.d evaporated to yielc a viscous o-l. Purification by flas.. chromatography [2%
methanol ir, ethyl acetate~ afforded -he title compound (D20) as a white solid (1.2g).
SUBSTITUTE SHEET
WO91/15507 PCT/GB9l/0053~
2~
Observed FAB (M+H)+ 3~0. C1gH29N3Os requires M 379-DescriPtion 21 N~-Benzyloxvcarbonvl-(S)-ornithine methylamide, trifluoroacetate salt (D21) TFA.H2N~NHCH3 o N OCH2Ph A solution of the amide (D20) (0.14g) in dichloromethane (5 ml) maintained at 0C was treated with trifluoroacetic acid (2 ml). The solution was left stirring at room temperature for 2h, then solvent evaporated in vacuo to afford crude title compound (D21) as an oil. This was used as such without further purification.
Description 22 N~-[N-((R)-1-Dibenzyloxv~hos~hinvl~roDvl)-(S)-leucvll-N~-benzvloxycarbonvl-(S)-ornithine methvlamlde (D22) (PhCH20)2P~ ~ N ~ ~ NHCU3O
SUBSTITUTE SHEET
~'~91/15507 ~s ~,~ ,7 PCTIGB91/00~38 A stirred solution of N-(1-(R)-dibenzyloxyphosphinylpropyl~-(S)-leucine (D3A) (0.142g) ir anhydrous dichloromethane (15 ml) maintained at 0C w2S
treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.07g) and 1-hydroxybenzotriazole (0.04gg), After 0.Sh, the mixture was sequentially treated with crude salt (D21)and N,N-diisopropylethylamine (0.095g) and then left stirring at room temperature for 18h. The solution was then washed with lM aq. citric acid (10 ml), sat. aq. NaHCO3 (10 ml) and sat. aq. NaCl (10 ml). The organic fraction was dried and evaporated in vacuo to yield an oil. Purification by fiash chromatography (5%
methanol in chloroform) afforded the title compound (D22) as a white foam (0.095g).
1~
Observed FAB (M~H) 695- C37H51N47P reqUires _ Description 23 N~-tert-Butoxvcarbonvl-N-dimethyl-(S)-lvsine methYlamide (D23) H
H3C ~ O ~N ~ NHCH3 CH3 H3C CH3 ~ N~c~
A solution of the amide (D4) (0.~g) in methanol (100 ml) 30 w25 treated with 5% palladium on charcoal (lg). The suspension was diluted with 38% aqueous formaldehyde solution (6 ml) and hydrogenated at atmospheric pressure and ambient temperature for 48h. A further aliquot o~
catalyst (0.5g) and formaldehyde (3 ml) was added and SUBSTITUTE SHEET
WO91/15507 PCT/~B91/~3 ?~
hydrogenation continued for a further 24h. The solution was filtered through Kieselguhr, solvent evaporated in vacuo and the residue purified by flash chromatography [(CHCl3:MeOH:NH3)(12:2:1) v/v] to afford the title compound ~D23) (0.29g).
Observed M+ 287. C14H29N3O3 requires M 287.
Description 24 N~-Dimethvl-(S)-lvsine methvlamide, trifluoroacetate sal~
(D24) TFA.H2N ~ NHCH3,CH3.
A stirred solution of the amine (D23) (0.11g) maintained at 0C in dichloromethane (5 ml) was treated with tr,fluoroacetic acid (2 ml). The solution was stirred fc-lh, then solvent evaporated at reduced pressure to affor~
crude title compound (D24) as a clear oil. This W2S used as such without further purification.
~'~91/15507 ~ ,7 PCT/GB91/~538 Descri~tion 25 Na-[N-((R)-1-Dibenzvloxvphos~hinvlvropvl)-(S)-leucvll-N~-dimethvl-(S)-lvsine methvlamide (D25) ~ CH3 C2Hs ~ H
(PhCH2O)2P" ~ N ~ ~ N N_ CH
A stirred solution of N-(1-(R)-dibènzyloxy-phosphinylpropyl)-(S)-leucine (D3A) (0.132g) in anhydrous dichloromethane (20 ml) was treated sequentially with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.066g) and 1-hydroxybenzotriazole (0.042g). The mixture was left stirring for 0.5h, treated with amine salt (D24) and N,N-diisopropylethylamine (0.148g) and stirring continued for 4h at room temperature. The mixture was washed with water (2xlO ml), sat. aq. NaHCO3 (20 ml), dried over anhydrous magnesium sulphate and evaporated _ _zcuo to give a pale yellow oil. Purification by flasr.
chromatography [(CHCl3:MeOH:NH3)(15:2:0.5)v/v] afforded tne title compound (D25) as a white foa~, (O.llg).
Observed FA~ (M+H) 603. C32H51N4OsP req SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~K3~--. ., ... ,. ~, ~
- 52 ~
Description 26 o Na-tert-Butoxvcarbonvl-N-dimethvl-(S)-ornithine methvlamide (D26) H
H3C ~ `r~ N ~Jl~ ~HCH
H~C CH3 ~ - CH~
CH~
The title compound (D26) was prepared following the procedure described for the synthesis of Na-tert-butoxycarbonyl-N-dimethyl-(S)-lysine (D23) (yield: 69%).
Observed FAB (M+H) 274. C13H27N303 requ Description 27 N-Dimethvl-(S)-ornithine methvlamide, trifluoroacetate salt (D27) TFA H l~ HCH~
" ~'~`~-CH~
CH~
The titie compound (D27) was prepared following the ?rocedure described for the synthesis of NE-dimethyl-(S)-lysine methylamide, trifluoroacetate sal. (D24). The title compound (D27) was used without any formal purification.
~'~91/15507 ~$ ;~ ~ Y 7 PCT/GB91/00538 Description 28 N~-~N-((R)-1-Dibenzvloxyphosphinylpropyl)-(S)-leucvl!-.N-dimethYl-(S)-ornithine methylamide (D28) CH~
/~ CH3 C2H5 ~ H
(PhCH20)2P ` N ~f ~ NHCH3 --N~ 3 The title compound (D28) was prepared following the procedure described for the synthesis of the dibenzyl ester (D25). (Yield: 56%).
Observed FAB (M+H) 589. C31H49N4O5P requires _ 588.
Description 29 (R)-2-Hydroxv-4-methYlPentanoic acid (D29) /~ CH3 HO'^~CO2H
The title compound was prepared by modification of thC
method of G. Iwasaki et al, Chem. Pharm. Bull. 1989, 37(2), 280. A solution of sodium nitrlte (33.8g) i.i ~a i--(100 ml) was added dropwise over 1.75h to a sti.red SUBSTITUTE SHEET
WO91/15~07 PCT/GB91/00~3 solution of D-leucine (20g, 0.15 mol) in 3N sulphuric acid (700 ml) and 50% aqueous acetic acid (500 ml) at 92-98C.
The mixture was heated for a further 3h at 98-99C, then cooled and extracted with diethyl ether (4x250 ml). The combined organic layers were dried with anhydrous magnesium sulphate, filtered and evaporated to àryness ~o give the title compound (D29) as a pale yellow solid (18.91g, 94%). A sample was recrystallized from CHCl3/pentane as white needles, m.p. 77.5-79C.
[a]22D = 26.41 (c=0.98 lN NaOH).
Description 30 Methvl (R)-2-hydroxy-4-methylpentanoate (D30) , HO'-~C02CH3 A mixture of acid (D29) (3.97g, 0.03 mol) in methanol (100 ml) and lN ethereal HCl (10 ml) was maintained at ambient temperature for 5 days. After evaporation to dryness ana purlfication by chromatography on silica gel with diethyl etner as eluant the title compound (D30) was obtained as 2 pale yellow oil (3.51g, 80%) and was carried forward without further purification.
SVBSTITUTE S~EET
~'^91/15~07 ~ ? ;,~ PCT/GB9l/00~3g Description 31 N-(1-(S)-DiethoxvPhosPhinYlpropvl)-(S)-leu-ine methv ester (D31) C2H5 ~--(C~H50~P N CO2CH3 The alcohol (D31) (0.696g, 0.0048 moi) ir. dichlorome~.lar.-(10 ml) was cooled to -60C and ther. 2,6-lu.idine (0.68 ml) followed by trifluoromethanesulphonic anhydride (0.92 ml) were added. After allowing to warm to 0C a sol~~~c~.
of (S)-1-aminopropylphosphonic acid, diethyl ester1 (0.93g, 0.0048 mol) in dichloromethane (10 ml) was addec, followed by proton sponge (1.02g). The mixture was stirred for 4 days in the dark under N2 and then filtered, the solid washed with chloroform and the filtrate washed with 10% citric acid (x2) followed by wate~. The organi_ layer was d-ied with anhydrous magnesium sulphate, filtered and evaporated to give the crude produc, as c -G-oil. Purification by column chroma,ogra~hy (first s_ --_ gel with g-adient elution 0-6~ MeO~./CHCl3, ~hen sil--2 ge with grad~ent elution 0-2% MeOH/EtO.C) gave the .it e compound (D31) as a pale yellow oil (o.48g~ 33%).
ODse~ved ~i 323.1862. C14H30NO5P requires 323.1858.
Other physical properties in accorc with .ne litera~_r--1 ~-A-0 401 963 SUBSTITUTE SHEET
WO91/15507 PCT/G891/0053~-2~ t;
Description 32 Na-~N-((S)-Diethoxvphos~hinylpropYl)-(S)-leucyll _~E_ benzyloxvcarbonvl-(S)-lvsine methvl amide (D32) ~ CH3 C2H5 ~ H
(C~H50)2P 1 N ~ ~ NHCH3 O H ~ N ~ OCH2Ph N-(1-(S)-Diethoxyphosphinylpropyl)-(S)-leucine1 was prepared from the corresponding methyl es.er (D31) by standard base hydrolysis. A solution of this acid (0.25g, 0.00081 mol) in dichloromethane (9 ml) w2S cooled to 0C
under N2 and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.17g) then 1-hydroxybenzotriazole (0.12g) were added and the mixture stirred for lh at this temperature. N-Benzyloxycarbonyl-(S)-lysine methylamide trifluoroacetate (D5) (from 0.0097 mol of the corresponding N-tert-butoxycarbonyl derivative (D4)) ~.
dichloromethane (5 ml) was added followed by diisopropylethylamine (0.35 ml) and the mixture stirrec z~
room temperature overnigh~. After dilu.ion with chloroform (20 ml) the mixture was ~ashed with 10% ci~_-ic acid (x2) and water. The organic laye_ was dried (znhydrous magnesium sulphate) and vola~ile material evaporated in vacuo to give a colourless gum.
Pu-ification by chromatography on sil iC2 gel (gradien_ elu_ion 0-5% MeOH/EtOAc) gave the produ~_ (D32) as a colou-less gum (0.36g, 76%).
Observed M+ 584.3346. C28H49N~O7P -equ -es 584.3339.
1 EP-~.-0 401 963 SUBSTITUTE SHEET
'~91/15507 ~1 ~ ~i' ~7 PCT/GB91/~38 Descri~tion 33 N~-~N-((S)-l-Diethoxyphos~hinyl~ropyl)-(S)-leucvll-(S)-lvsine methvlamide (D33) C2H5 ~ H
(C2H5O)2P l N ~ -~ ~ CH3 O ~ NH2 N-[N-((S)-l-Diethoxyphosphinylpropyl)-(S)-leucyl]-N-benzyloxycarbonyl-(S)-lysine methylamide (D32) (0.18g, 0.0003 mol) in methanol (25 ml) with 10% palladium on charcoal (0.2g) was hydrogenated at atmos?heric pressure overnight. After filtration through Kieselguhr and evaporation to dryness the title compound (D33) was obtained as a clear gum.
Observed M+ 450.2982. C20H43N4O5P requires 450.2972.
Descri~tion 34 N-Benzvloxycarbonyl-N~-tert-butoxvc2rbonvl-(S)-lysinP 2-hvdroxYethvlamide (D34) 3 CH3 \ / ~ ~ 2 SUE~STITUTE SHEET
WO91/15507 PCT/GB91/0~53~
~ ?'~ `;7 - 58 -A solution of NE-benzyloxycarbonyl-N~-ter~-butoxycarbonyi-(S)-lysine (5g, 0.0132 mol) in dichloromethane (50 ml~ was cooled to 0C and 1-hydroxybenzotriazole (1.78g) followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.52g) were added. The mixture was st~--e~
at 0C for lh and then ethanolamine (0.88g, 0.0144 mol) added in one portion. The solution was s.irred over,.lgh~
at room temperature, washed with water (2x30 ml), saturated sodium bicarbonate (30 ml) and finally wate- (30 ml). The organic laye- was dried with anhydrous magnes~
sulphate, filtered and evaporated to dryness to give _;~e product (D34) as a clear gum (S.44g, 97%) which was used without further purification.
Observed M+ 423.2365. C21H33N3O6 requires 423-2369-Description 35 N-Benzvloxvcarbonvl-(S)-lvsine 2-hvdroxvethvlamide, trifluoroacetate salt (D35) TFA H2~ ~ ~ ~ OH
H ~ ~ OCH2Ph ~-ifluoroacetic acid (30 ml) was added tG a stir-ed solu~ion of the lysine de-ivative (D34) (2.64g, 0.0062 mol) n d-chloromethane (S0 ml) at 0C. ~fter 3h stir-in a~ ice-bath temperature volatile material was removed SUBSTITUTE SHEET
~'~91/15507 PCT/GB91/00538 2 . i~;~ ~ 7 under reduced pressure to give the crude product (D35) ir.
quantitative yield. This was used in subsequent steps without further purification.
Observed FAB (M+H) 324- Cl6H25N34 requireS M
Descri~tion 36 ~a-~N-((R~-l-Dibenzvloxvphos~hinvl~-o~vl)-(S)-leucvll-(S)-~'E-benzvloxvcarbonvl-lvsine 2-hvdroxvethvlamide (D36) CH~
2 5 ~ H
(PhCH2O)2P` N ~ ~ N ~ OH
O ~ N ~ OCH2Ph A solution of N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) (1.61g, 0.0037 mol) in dichloromethane (60 ml) was cooled to 0C and 1-hydroxybenzotriazole (0.78g, 0.0058 mol) was added followed by 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimiae hydrochloride (1.09g, 0.0057 mol). The mixture was sti-red fcr lh at ice-bath ~emperature and then a solution of the trifluoroacetate sal. (D35) (1.2 equivalents) i-, d chlo~ome.hane (15 ml) h'2S adde~ fcllowed by dlisopropyle_hylamine (3.36g) tG
ensu-e neutralization of excess .-i'luoroacetic acid.
Afte- stirring overnight at room ~emperature .he reac_ion mixture was washed with 10% acuec_s citric acid (2x20 ml), wate- (20 ml), sa~urated soaium ~ _arbonate (2~.20 ml) znd 'inally wate-. The organic laye- was dried (anhyarous magnesium sulphate) filterea a-,à evaporated tc dryness.
The residue was chromatographec o-, silica gel with 1:1 SUBSTITUTE SHEET
WO91/1~507 PCT/GB91/~3~-2 ~ 7 increasing percentage of methanol (to 10%). Further purification on silica gel with a gra ien. of 0-4~
MeOH/CHCl3 gave the title compound (D36) as a clea- gum (0.83g, 30%).
Observed M+ 738. C39H55N4O8P requires M 738.
Descri~tion 37 N~-~N-((S)-1-Dibenzvloxy~hosphinyl~ro~vl)-(S)-leucvl~-(S)-N~-benzvloxycarbonvl-lvsine 2-hvdroxy~~hvl2mide (D3 C2H5 ~ CH3 O
(PhCH20)2P 1N ~ ~ N ~ OH
~, N ~ OCH2Ph The title compound ~D37) (1.035g) W25 prepared from N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3B) (0.67g, 0.0015 mol) and the trifluoroacetate salt (D35) by the method given in Description 36.
Observed FAB (M+H)+ 739- C39~55N~-82 reqUires M 738-~91/15507 ~ PCT/GB91/0053g Description 38 N~-~(S)-tert-Butoxvcarbonvl-leucvll-N-benzvloxvcarb~nyl-(S)-lvsine methylamide (D38) ~ CH3 H3C ~ O N O ~ N ~ OCH2Ph N-tert-Butyloxycarbonyl-(S)-leucine (25g, 0.11 mol) was dissolved in dichloromethane (250 ml) and cooled to 0C.
l,1'-Carbonyldiimidazole (18g, 0.113 mol) was added and the mixture left stirring at 0C for 0.5h. The solu~ion was allowed to warm to room temperature for 15 mins and then recooled to 0C.
N-Benzyloxycarbonyl-(S)-lysine methylamide trifluoroacetate salt (D5) (47g, 0.118 mol) in dichloromethane (150 ml) was added followed by the immediate addition of diisopropylethylamine (30.5g, 0.236 mol). The reaction mixture was left sti-ring overnight 2-room temperature and then treated with water and ex~racted with chloroform. The combined organic layers were washed with dilute hydrochloric acid (2x100 ml), wate- (2x100 .1), aqueous sodium carbonate (3x100 ml) and brine. The organic layer was dried with anhydrous magnesium sulpha~e and evaporated to dryness to give the ti~le compound (D3 (50g, 93%) which was used withou, furthe- purification.
Observed (M+H)+ 507- C26H42N46 requires M 506.
WO 91/15507 PCI/GB91/005~"
~r ~. ~?'i~ ~
Description 39 Na-~(S)-Leucvll-Nf-benz~loxvcarbonvl-(5)-lvsine methvlamide (D39) TFA.H2N ~ ~ NHCH3 N~,~OCH2Ph o A cooled solution of the amide (D38) (50g, 0.099 mol) in dichloromethane (150 ml) was treated w th trifluoroacetic acid (150 ml). After 3h the solvent W2S evaporated under reduced pressure. The resulting oil was dissolved in methanol and acidified with dilute hyd-ochloric acid to give the hydrochloride salt. Water was added and non-basic organic material extracted out with ethyl acetate (2x200 ml). The aqueous layer was basi~ied to pH 8-9 with 10% sodium hydroxide and then extracted with ethyl acetate (3x150 ml). These combined organic extracts were dried ~Na2SO4) and evaporated to d-yness to cive the ~itle compound (D39) (20g, 50%) as 2 white solid.
Observed (M+H)~ 407. C21H3qN~O~ requ res ~ 406.
SUBSTITUTE SHE~T
'91/15507 ~,~ ~ ~; 7 PCT/CB91/00~3g Description 40 Na-~N-((R)-1-DibenzvloxvphosphinvlPropyl)-(S)-leucYll-N-benzvloxvcarbonvl-(S)-lvsine methvlamide and Na-[N-~(S)-1-Dibenzvloxv~hosphinvlpropyl)-(S)-leucyll-(S)-NE-benzvloxYcarbonYl-lysine methYlamide (D40) 2 2, H - H
~ N ~ OCH2Ph Dibenzyl ((1-trifluoromethanesulphonyloxy)propyl)-phosphonate (D2) (4.4g, 0.0098 mol) was dissolved in dry dichloromethane (20 ml). N~-[(S)-Leucyl]-N-benzyloxycarbonyl-(S)-lysine methylamide (D39) (4.0g, 0.0098 mol) and Proton Sponge (2.0g, 0.0098 mol) were added to the solution and the reaction mixture was stirred in the dark at room temperature for 10 days.
The solution was diluted further with chloroform, washed wl.h 10% citric acid (2xS0 ml) and water (3x50 ml), dried (anhydrous MgSO4) and evaporated to dryness to give an orange oil. This was purified by column chromatography 2~ (silica ge~, 2% MeOH/EtOAc) to give the mixture of title com?ounds (D40) (2.55g, 35%) as a clear oll.
Observed (M+H)+ 709. C38H53N47? requires M 708.
SUBSTITUTE SHEET
WO91/15507 PCT/GB91/0053~
2 . ~.~ 64 -Descri~tion 41 N-~N-((S)-1-DibenzvloxyPhosphinylProPvl)-(S)-leucY
nitro-(S)-arqinine methYlamide (D41) 2 5 ~ H
(phcH2o)2p NH ~ 3 NH
The title compound (D41) (0.72g, 74%) was pre~ared fro~
N-tert-butoxycarbonyl-N~-nitro-(S)-arginine methylamide 15 (D13) (0.6g, 0.0018 mol) and N-((S)-1-diber.zyloxy-phosphinylpropyl)-(S)-leucine (D3B) (0.633g, 0.0014 mol) by the method described in Description 14 with the exception that dichloromethane was used as reaction solvent with sufficient dimethylformamide to effect solution.
Descri~tion 42 N-((R)-1-Dibenzvloxy~hos~hinylethyl)-(S)-leucine (D42A) and N-((S)-1-dibenzvloxY~hos~hinYle~hvl)-(S)-leucine (D42B) ~ CH~
(phcH2o)2p N ~
SUBSTITUTE SHE~T
~91/1~507 PCT/GB91/00538 ~ 7 The title mixture of diastereoisomers (D42) was prepared analogously to the method in Description 3, Method B, a5 c white solid.
Observed FAB (M+H) 420- C22H30NO5P -equires M 419.
DescriPtion 43 Na-~N-((R)-l-Dibenzvloxv~hos~hinvlethvl)-(S)-leucv'l-(S)-N-benzvloxycarbonvl-lvsine methvlamiàe (D43A) and N~-~N-((S)-l-Dlbenzyloxvphos~hinvlethvl)-(S)-leucvll-(Ci-N-benzvloxvcarbonvl-l~sine methvlamide ~D43B) C~
CH ~ CH~ O
OCH2Ph The title compound (D43) was obtained as a clear oil (1.0g, 63~) from the acid (D42) (l.0g, 0.002 mol) following the general method of Description 6.
Observed FAB (M+H) 695- C37n5lN4o72 requires M 69 SUBSTlTUTE SHEET
W091/15507 PCr/GB91/00~?~-2 . , i~; 7 Example 1 N-[N-((R)-1-PhosPhonopro~yl)-(S)-leucvll-(S)-lvsine methvlamide (El) C H ~ CH3 (HO)P"`~ N ~ N ~ NHCH3 ~ NH2 The dibenzyl ester (D6) (O.lOSg, 0.16 mmol) was dissolved in ethanol (90 ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated 1n vacuo to give the title compound (El) (O.Olg).
Observed FAB (M+H) 395. C16H3505N4P req (CDC13/CD30D): 0.95 (6H,dd), 1.08 (3H,t), 1.4-2.0 (9H,m), 2.65 (lH,m), 2.75 (3H,s), 2.92 (2H,m), 3.45 (2H,m), 4.18 (lH,m), 4.4 (lH,m).
SUBSTITUTE SHEET
~'~91/15507 Z' PCT/GB91/00538 Examole 2 Na-~N-((R)-1-Phosphonopro~vl)-(S)-leucvll-N~-qlycyl-(S)-lvsine methylamide (E2) ~ CH3 (HO)20 H N ~ NH~
The dibenzyl ester (D9) (0.43g) was dissolved in ethanc (60 ml) and hydrogenated over 10% palladium on charcoal 2.
atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated ln vacuo to give the title compound (E2) (0.059g).
Observed FAB (M+H)+ 452- ClgH38o6N5p requires M 45i-(CD30D): 0.78-0.95 (9H,m), 1.19-1.9 (~lH,m), 2.31 (lH,m), 2.62 (3H,s), 3.C;-3.25 (2H,m), 3.3G-3.65 (2~.,m), 3.83-3.92 (lH,bt), 4.15-4.3 (lH,t).
SUBSTITUTE SHEET
Z, ~ ,.";.` 7 - 6 8 Exam~le 3 N-[N-((R)-l-phosphonopro~yl)-(s)-leucv~ -r(2-Pvrrolidinoethyl)amidel-(s)-asD2rtic acid methvla~.ido (_3, CHl - ~ ~ H
(H0)2P N ~ ~ NHCH
~ N
o This compound was prepared from N-[N-[(R)-dibenzyloY.v-phosphinylpropyl]-(S)-leucine-~-[(2-pyr-olidinoethy')-amide]]-(S)-aspartic acid methylamide (D12) by hydrogenation over 10~ palladium on cn2rcoal at atmospheric pressure. m.p. 130-135C (95% yield).
(CD30D): 1.0(6H,t,J=6Hz), 1.1(3H,t,J=7kz), 1.55-1.8(4H,m), 2.0(lH,m), 2.15(4~.,m), 2.6-2.85( 6H, m), 3.3-3.75(approx. 8H,m), 4.1(lH,t,J=6Hz) and 4.75(1H,t,J=5Hz).
_xam~le 4 ~ '-((R)-l-~hos~hono~-o-~vl)-(S)-:eu~ -(^)-a-~ ',r-methvlamide (E4) C H ~ CH3 (HO)P""~ N ~ N ~ ~lHCH3 NH
- N NH
H
SUBSTITUTE SHEET
~"~91/15507 2 . ~- ~,.; ;,7 PCT/GBg1/0053g The fully protected derivative (D14) was dissolved in a mixture of glacial acetic acid (40 ml) and water (iO ml) and hydrogenated over 10% palladium on charcoal cata'ys.
for 18h at atmospheric pressure. The solution was filtered through Kieselguhr and solvent evaporated in vacuo to give the title compound (E4) (0.085g).
Observed FAB (M+H) 423. C16H35N605P req ~ (CD30D): 0.95(6H,dd), 1.03(3H,t), 1.5-i.94(9r.,m), 2.56(lH,m), 2.72(3H,s), 3.17(2H,m), 4.02(i~,t), 4.37(lH,t).
Exam~le 5 N~-~N-((R)-l-PhosphonoPropvl)-(S)-leucyll-(R)-~-(4-PiPeridvl)alanine methvlamide (E5A) and N~-~N-((R)-l-PhosPhonoproPvl)-(S)-leucvll-(S) -B- (4-Pi~eridvl)alanine methYlamide (E5B) C2H5 ~ H
(HO)P~ N ~ N ~ CH3 ~
Tne fully protected derivative (Dl9) (0.24g) was disso~ve-in ethanoi (50 ml) and hydrogena~e~ over 10% palladium c-cha-coal at atmospheric pressure. ml he solution was ~lltered through Kieselguhr and solven. evaporated ~
vacuo. The residue was tritura~ed with ether to remove slisht impu~ities leaving the prod ~~ (E5) as an o f-whi-_ solid (0.13g).
SVBSTITUTE SHEET
WO91/15507 PCT/GB91/~39--~?~.:7 Observed FAB (M+H)+ 421- cl8H37N4o5p requireS M 420-(CD30D): 0.95(6H,dd), 1.02,1.05(3H, overlapplng triplets), 1.4-2.0(12H,m), 2.4-2.55(1H, 2 overlappins m), 2.71(3H,d), 2.81-3.03(2H,m), 3.34(4H,br m), 3.68-3.81(1H, dt), 4.40(lH,m).
Exam~le 6 N-~N-((R)-1-Phos~honopro~vl)-(S)-leucvll-(S)-orn-t:rine methvlamide (E6) HO~P" ~ N ~ nHCH3 O H O
A solution of phosphonic diester (D22) (0.07g) in methanol (20 ml) was treated with 5% palladium on charcoal. The suspension was hydrogenated at atmospheric pressure for 24h, filtered through Kieselguhr and solvent evapora_ed _~
vacuo. The residue was triturated with diethyl ethe- (5 ml) to give the title compound (E6) as a white solid 25 (0.03g), m.p. 165-169C.
Observed FAB (M+H) 381- cl5H33N4o5? requi-es M 38C-~ (CDCl3/CD30D): 1.0(6H,dd), 1.12 (3r., t), 1.6-1.9(8H,m), 2.05(1H,m), 2.7(lH,m), 2.75(3H,s), 2.9(2H,t), 4.42(1Y,m), ~.55(lH,t).
The S,S,S diastereoisomer is prepared by a similar metho~.
SUBSTITUTE SI~EET
~ '91/15507 2~ ~ ~~ PCTtGBgl/00538 Exam~le 7 N~-~N-((R)-1-Phosphonopropyl)-(S)-leucYll-N~-dimethvl-(S)-lysine methvlamide (E7) C~ 5 ~ H
(HO)p~`~ N ~ N = ,CH3 A solution of phosphonic diester (D25) (0.75g) in methanol (20 ml) was treated with 5% palladium on charcoal (O.OSg) and hydrogenated at atmospheric pressure for 24h. The solution was filtered through Kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether (10 ml) to give the title compound (E7) as white solid (0.045g).
Observed FAB (M+H)~ 423. C18H39N405P requ (CD30D): 1.0(6H,dd), 1.1(3H,t), 1.3-1.85(lOH,m), 2.0(lH,m), 2.6(lH,m), 2.75(3H,s), 2.8(6H,s), 3.05(2H,t), 4.3_(2H,m).
SU~3STITUTE SHEET
WO91/15507 PCT/GBg1/~?-~, ,, ,, ~, J - 72 -Exam~le 8 N~-~N-((R)-1-Phosphonopro~vl)-(S)-leucvll-N-dimethvl-(5)-ornithine methylamide (E8) 2 5 ~ ~ O
(HO)~P` ~ HCH
O H O
\ ~",CHt CH~
The title compound (E8) was prepared from the dibenzyl ester (D28) following the procedure described for the synthesis of N~-[N-((R)-l-phosphonopropyl)-(S)-leucyl] -N-dimethyl-(S)-lysine methylamide (~7). (Yield: 74%). m.p.
88.5-90C.
Observed FAB (M+H) 409- Cl7H37N4o5? requires M 408-Exam~le 9 N~-~N-((S)-1-Phosphono~ropvl)-(S)-leucvll-(S)-lysine methvlamide, hvdrobromide salt (_9) ~ CH
2 ~ O
- ~ NH2.HBr SUBSTITUTE SHEET
.,7 ' ~91/15507 PCT/GB91/00538 Bromotrimethylsilane (0.48 ml, 12 equiv.) was added ~o a solution of the diethyl ester (D33) (0.14g, 0.0024 mol) -dry acetonitrile (10 ml) and stirred at room temperature for 3 days.
The resulting yellow solution W25 evaporated to dryness and treated with methanol/wate-. After evaporation and repeated treatment with methanol/water the product (~9) was obtained in quantitative yield as a pale orange foam.
Observed FAB (M+H)+ 395- cl6H35N4o5p reauireS M 39~-(CD30D): 1.01(6H,dd), 1.18(3H,t), 1.3-2.15(11H,m), 2.74(3H,s), 2.98(2H,t), 3.19(1H,m), 4.3(1H,m), 4.42(1H,t).
Example 10 Na-~N-((R)-1-PhosPhonopropyl)-(S)-leucYll-(S)~lysine 2-hvdroxyethvlamide (E10) ~ CH3 C2H5 ~ H
(HO)2p~ ~ N ~ ~ NH2 A solution of the dibenzyl ester (D3~) (0.8g, 0.001~ mol in methanol (100 ml) with 10% palladium on charcoal W2S
hydrogenated at atmospheric pressure ove-night. After filtration through Kieselguhr and evaporation to dryness the residue was taken up in doubly Gist lled water, refiltered and freeze-dried to give the title compound ~F10) as a white foam (0.39g, 85%).
/
SUBSTITUTE SHEET
WO91/15507 P~T/GB91/ ~ Y
~;~ . ' J '~ V~
Observed FAB (M+H)+ 425. C17H37N406P requ (CD30D): 0.98(6H,dd), 1.05(3H,t), 1.3-2.05(1lH, overlapping m), 2.54(1H,br m), 2.92(2H,m), 3.3(2H,m, overlaps CHD20D solvent signal), 3.6(2H,t), 4.10(1H,b- m), 4.38(lH,dd).
Exam~le 11 ~-~N-((S)-1-Phos~hono~ro~vl)-(S)-leucvll-(S)-lvsine 2-hvdroxyethvlamide (E11) C ~ CH~
(HO)2P ~ ~ ~ ~ OH
O H I ~ NH2 The title compound (E11) was obtained by atmospheric pressure hydrogenation of the dibenzyl ester (D37) by the method given in Example 10.
0bserved FAB (M+H)+ 425- C17H3~N406P requires M 424-~ (CD30D): 0.97 (6H,dd), 1.12 (3H,t) 1.35-2.10 (llH, ove-lapping m), 2.46 (l..,m), 2.g5 (2H,m), 3.32 (2H,m, ove-laps CHD20D solven. signal), 3.53 (lH,m), 3.65 (2H,m), 4.49 (l:.,m).
S~ ST)TUTE S~1EET
2,.i. _;.'.~,7 ~'~91/15507 PCT/CB91/~38 Example 12 Na-[N-((R)-1-PhosPhono~ropyl)-(S)-leucvll-(S)-lvsine methylamide and Na-rN-((S)-1-phosphonopro~vl)-(5)-leucvll-(S)-lvsine methylamide (E12) (H)2P N ~ ~ NHCH3 o H --NH2 The mixture of dibenzyl esters (D40) (2.55 g, 0.004 mol) in ethanol (100 ml) was hydrogenated over 10% palladium on charcoal at atmospheric pressure. The solution was filtered through Kieselguhr and the solvent evaporated in vacuo to give the title compound (E12) as a white solid (l.Og, 71%).
Observed FAB (M+H)+ 395. C16H35N405P requi Exam~le 13 N-rN-(~S)-1-Phos~hono~roPvl)-(S)-leucvll-(S)-ar~inine methvlamide (E13) 2 5 ~H
)2P NH~ NHCH3 NH
S~ ~TITUTF SHEET
WO91/15507 PCT/GB91/~K3 The title compound (E13) (0.5g, 90%) was ~repared from the dibenzyl ester (D41) (0.633g, 0.014 mol) by the method o' Example 4.
Observed FAB (M+H)+ 423- C16H35N6O5P requires M 422-(CD30D): 0.97(6H,t), 1.10(3H,t), 1.49-1.95(9H,m), 2.50(lH,m), 3.21(2H,m), 3.60(lH,m), 4.94(lH,m).
Example 14 Na-~N-((R)-1-Phosphono~ro~vl)-(S)-leucvll-(S)-homoarainine methvlamide, disodium salt (E14A) and N~-~N-((S)-1-phos~hono~ropvl-(S)-leucvll-(S)-homoarsinine methvlamide, disodium salt (E14B) ~ HCH3 C2H5 ~ H O N ~ NH2 NH
The mixture of R,S,S and S,S,S isomers (~12) (34.5mg, 0.087 mmol) in water (0.5 ml) was treatec with sodium bicarbonate (44 mg, 6 equiv.) followed by 2-methyl-2-thiopseudourea sulphate (24.3 mg, 1 equiv.) and stirre~ z-room temperature for 3h. Additional por~ions of sodium bicarbonate (8.5 mg) and 2-methyl-2-~hio?seudourea (15 mg) were added and the mixture then heated a~ 70C for i.5h.
Purification by reverse phase chromatogra?hy gave the title compound mixture of isomers (c14) 2S a white solic.
Observed FAB (M+H)+ 481. C17H35N6O-~Na2 requires M 480.
SU~3STITUTE SHEET
~91/lS507 ~ ~ ~_7 PCT/GB91/~K38 Example 15 N-~N-((R)-1-Phosphonopropvl)-(S)-leucvll-(S)-N-~2-imidazolinvl)-lvsine methvlamide disodium salt (E15A) and N~-~N-((S)-1-Phosphonopropvl)-(S)-leucvll-(S)-NE-(2-imidazolinvl)-lysine methvlamide disodium salt (E15B) l 5 ~ H
H O -N ~
The title mixture of diastereoisomers (E15) was prepared from the phosphonic acid mixture of isomers (E12) (36 9 mg, 0.0935 mmol), 2-methylthio-2-imidazoline hydroiodide (45.6 mg and, after 3h, 13.9 mg) and sodium bicarbonate (47 mg and, after 3h, 7.8 mg) by the general method of Example 14.
Observed FAB (M+H)+ 507- Cl9H37N605P~a2 requireS
SUBSTlTUTE SHEET
WO91/15507 PCT/CB91/ ~ 3'~
Z~-~, ,"~,7 - 78 -Example 16 Na-[N-(~R)-1-Phosphonoethyl)-(S)-leucvll-(S)-lvsine methvlamide (E16A) and Na-~N-((S)-1-phosphonoethvl)-(S)-leucvll-(S)-lvsine methvlamide (E16B) CH~
~ H
(HO),P ~ ~ ~ NHCH~
~ NH2 The dibenzyl ester (D43) (l.O.g, 0.0014 mol) was hydrogenated at atmospheric pressure by the method of Example 1 to give the title compound mixture (E16) in quantitative yield as a white crystalline solid.
Observed FAB (M+H) 381. C15H33N4O5P requi SUBSTITUTE SHEET
WO91/15507 PCT/GB91/~053 r~
COLLAGENASE INHIBITOR ASSAY
The test is performed essentially as in Cawston and Barrett, Anal. Biochem. 99, 340-345 (1979). Compounds for testing are dissolved in methanol by sonication and added to collagenase (purified from culture supernatants from the human lung fibroblast cell line, WI-38) in buffer. After a 5 min pre-incubation at 37C, the assay tubes are cooled to 4C and 3H-acetylated rat skin type T
collagen is added. The assay tubes a-e incubated at 37C
overnight. The 3H-collagen forms insoluble fibrils, which are the substrate for the enzyme.
To terminate the assay, the assay tubes are spun at 12000 t5 rpm for 15 minutes. ~ndigested 3H-collagen is pelleted, while digested 3H-collagen is found as soluble peptides in the supernatant. A sample of the supernatant is taken for liquid scintillation counting.
The activity of collagenase inhibitors (IC50: 50%
inhibitory concentration) is expressed as that concentration of compound that inhibits a known ~standzrd) concentration of enzyme by 50%.
The compounds of Examples El-E9 had IC50 values between l.8 x l0 7 and 2.2 x l0 5M.
SUBSTITUTE SHEET
Claims (16)
1. A compound of the formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:
(I) in which, R is hydrogen, C1-6 alkyl or optionally substituted benzyl;
R1 is hydrogen or C1-6 alkyl;
R2 is C3-6 alkyl;
R3 is -(CH2)nNR5R6, -(CH2)nNHCOR7, -(CH2)nCONH(CH2)qNR5R6, -(CH2)nNR8C(=NR9)NR5R6 or -(CH2)n-R10 where n is an integer from 1 to 6 and each of R5 and R6 is independently hydrogen or alkyl, or R5 and R6 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring, R7 is alkyl or -(CH2)nNR5R6, R8 is hydrogen or alkyl, R9 is hydrogen or alkyl or R9 and R5 together with the nitrogen atoms to which they are bonded form an optionally substituted 5-, 6- or 7-membered ring, and R10 is an optionally substituted piperidyl ring m is 1 or 2, and q is 2 to 4; and R4 is hydrogen, alkyl, and -CH2-(CH2)nOR11 or -CH2-(CH2)nOCOR12 or where n is an integer from 1 to 6; R11, R12 and R13 are hydrogen or C1-6alkyl; and R14 is hydroxy or -O-C1-6alkyl or -NR5R6 (where R5 and R6 may be linked to form 2 heterocyclic ring).
(I) in which, R is hydrogen, C1-6 alkyl or optionally substituted benzyl;
R1 is hydrogen or C1-6 alkyl;
R2 is C3-6 alkyl;
R3 is -(CH2)nNR5R6, -(CH2)nNHCOR7, -(CH2)nCONH(CH2)qNR5R6, -(CH2)nNR8C(=NR9)NR5R6 or -(CH2)n-R10 where n is an integer from 1 to 6 and each of R5 and R6 is independently hydrogen or alkyl, or R5 and R6 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring, R7 is alkyl or -(CH2)nNR5R6, R8 is hydrogen or alkyl, R9 is hydrogen or alkyl or R9 and R5 together with the nitrogen atoms to which they are bonded form an optionally substituted 5-, 6- or 7-membered ring, and R10 is an optionally substituted piperidyl ring m is 1 or 2, and q is 2 to 4; and R4 is hydrogen, alkyl, and -CH2-(CH2)nOR11 or -CH2-(CH2)nOCOR12 or where n is an integer from 1 to 6; R11, R12 and R13 are hydrogen or C1-6alkyl; and R14 is hydroxy or -O-C1-6alkyl or -NR5R6 (where R5 and R6 may be linked to form 2 heterocyclic ring).
2. A compound according to claim 1 in which R is hydrogen, methyl or ethyl.
3. A compound according to either of claims 1 or 2 in which R1 is hydrogen, methyl, ethyl, isopropyl or n-butyl.
4. A compound according to any one of claims 1 to 3 in which R2 is n-butyl, iso-butyl or sec-butyl.
5. A compound according to any one of claims 1 to 4 in which R3 is -(CH2)nNR5R6 where R5 and R6 are hydrogen or methyl, -(CH2)nNHCOR7 where R7 is -(CH2)mNR5R6 in which m is 1 and R5 and R6 are hydrogen, -(CH2)nCONH(CH2)qNR5R6 where q is 2 and R5 and R6 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring, -(CH2n NR8C(=NR9)NR5R6 where R5, R6, R8 and R9 are all hydrogen, -(CH2)nNR8C(=NR9)NR5R6 where R5 and R9 together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazolinyl group, -(CH2)nR10 where R10 is optionally substituted piperidyl, and n is an integer from 1 to 4.
6. A compound according to any one of claims 1 to 5 in which R4 is methyl, ethyl, -(CH2)2OCH3, -CH(CH3)CO2CH3 and -(CH2)2OH.
7. A compound according to any one of claims 1 to 6 in which R is hydrogen, R1 is methyl or ethyl, R2 is iso-butyl, R3 is -(CH2)nNR5R6 where n is 3 or 4 and R5 and R6 are both hydrogen or methyl, -(CH2)4NHCOR7 where R7 is -CH2NH2, -CH2CONH(CH2)2NR5R6 where R5 and R6 are joined together to form a pyrrolidine ring, -(CH2)nNR8C(=NR9)NR5R6 where n is 3 or 4 and R5, R6, R8 and R9 are all hydrogen, -(CH2)4NR8C(=NR9)NR5R6 where R5 and R9 together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazolinyl group and R6 and R8 are both hydrogen, -(CH2)nNHC(=NH)NH2 where n is 3 or 4, and -CH2R10 where R10 is 4-piperidyl; and R4 is methyl or -(CH2)2OH.
8. A compound according to any one of claims 1 to 7 in which the chiral centre marked with an asterisk in formula (I) has the S-configuration.
9. A compound according to claim 1 which is N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-glycyl-(S)-lysine methylamide;
N-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-.beta.-[(2-pyrrolidinoethyl)amide]-(S)-aspartic acid methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(R)-.beta.-(4-piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-.beta.-(4-piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-ornithine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-ornithine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide, hydrobromide salt;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine 2-hydroxyethylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine 2-hydroxyethylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-homoarginine methylamide, disodium salt;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-homoarginine methylamide, disodium salt;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-N.epsilon.-(2-imidazolinyl)-lysine methylamide, disodium salt;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-N.epsilon.-(2-imidazolinyl)-lysine methylamide, disodium salt;
N.alpha.-[N-((R)-1-phosphonoethyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonoethyl)-(S)-leucyl]-(S)-lysine methylamide.
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-glycyl-(S)-lysine methylamide;
N-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-.beta.-[(2-pyrrolidinoethyl)amide]-(S)-aspartic acid methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(R)-.beta.-(4-piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-.beta.-(4-piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-ornithine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-ornithine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide, hydrobromide salt;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine 2-hydroxyethylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine 2-hydroxyethylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-homoarginine methylamide, disodium salt;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-homoarginine methylamide, disodium salt;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-N.epsilon.-(2-imidazolinyl)-lysine methylamide, disodium salt;
N.alpha.-[N-((S)-1-phosphonopropyl)-(S)-leucyl]-(S)-N.epsilon.-(2-imidazolinyl)-lysine methylamide, disodium salt;
N.alpha.-[N-((R)-1-phosphonoethyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((S)-1-phosphonoethyl)-(S)-leucyl]-(S)-lysine methylamide.
10. A process for the preparation of a compound as claimed in claim 1 which process comprises converting a group R20 to hydrogen by cleaving a group R20 from a compound of formula (II):
(II) wherein R20 is alkyl, optionally substituted phenyl, or optionally substituted benzyl and R21 is hydrogen, alkyl, optionally substituted phenyl, or optionally substituted benzyl and R1, R2, R3 and R4 are as defined in formula (I), and where necessary, converting R21 to hydrogen, and optionally thereafter converting the compound of formula (I) to a further compound of formula (I).
(II) wherein R20 is alkyl, optionally substituted phenyl, or optionally substituted benzyl and R21 is hydrogen, alkyl, optionally substituted phenyl, or optionally substituted benzyl and R1, R2, R3 and R4 are as defined in formula (I), and where necessary, converting R21 to hydrogen, and optionally thereafter converting the compound of formula (I) to a further compound of formula (I).
11. A compound of the formula (II) as defined in claim 10 subject to the proviso that R21 is not hydrogen.
12. A compound according to claim 11 which is N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-benzyloxycarbonyl-(s)-lysine methylamide;
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-(N-benzyl-oxycarbonylglycyl)-(S)-lysine methylamide, dibenzyl ester;
N-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-.beta.-[(2-pyrrolidinoethyl)amide]-(S)-aspartic acid methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.omega.-nitro-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(R)-.beta.-(4-(N-benzyloxycarbonyl)piperidyl)alanine methylamide;
Na-[N-((R)-l-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-.beta.-(4-(N-benzyloxycarbonyl)piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-benzyloxycarbonyl-(S)-ornithine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-ornithine methylamide;
N.alpha.-[N-((S)-1-diethoxyphosphinylpropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine 2-hydroxye_hylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine 2-hydroxye-hylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.epsilon.-benzyloxycarbonyl-lysine methylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.omega.-nitro-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylethyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine methylamide; and N.alpha.-[N-((S)-1-dibenzyloxyphosphinylethyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine methylamide.
N.alpha.-[N-((R)-1-phosphonopropyl)-(S)-leucyl]-N.epsilon.-(N-benzyl-oxycarbonylglycyl)-(S)-lysine methylamide, dibenzyl ester;
N-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-.beta.-[(2-pyrrolidinoethyl)amide]-(S)-aspartic acid methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.omega.-nitro-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(R)-.beta.-(4-(N-benzyloxycarbonyl)piperidyl)alanine methylamide;
Na-[N-((R)-l-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-.beta.-(4-(N-benzyloxycarbonyl)piperidyl)alanine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-benzyloxycarbonyl-(S)-ornithine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.epsilon.-dimethyl-(S)-ornithine methylamide;
N.alpha.-[N-((S)-1-diethoxyphosphinylpropyl)-(S)-leucyl]-(S)-lysine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine 2-hydroxye_hylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine 2-hydroxye-hylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-(S)-N.epsilon.-benzyloxycarbonyl-lysine methylamide;
N.alpha.-[N-((S)-1-dibenzyloxyphosphinylpropyl)-(S)-leucyl]-N.omega.-nitro-(S)-arginine methylamide;
N.alpha.-[N-((R)-1-dibenzyloxyphosphinylethyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine methylamide; and N.alpha.-[N-((S)-1-dibenzyloxyphosphinylethyl)-(S)-leucyl]-(S)-N.alpha.-benzyloxycarbonyl-lysine methylamide.
13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, solvate or hydrate thereo, and a pharmaceutically acceptable carrier.
14. A compound according to any one of claims 1 to 9 or a pnarmaceutically acceptable salt, solvate or hydrate thereo, for use as an active therapeutic substance.
15. A compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, solvate or hydrate thereof, for use in the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs.
16. The use of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, solvate or hydrate thereof, in the manufacture of a medicament for the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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GB9008078.9 | 1990-04-10 | ||
GB909008078A GB9008078D0 (en) | 1990-04-10 | 1990-04-10 | Novel compounds |
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CA2080227A1 true CA2080227A1 (en) | 1991-10-11 |
Family
ID=10674183
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA002080227A Abandoned CA2080227A1 (en) | 1990-04-10 | 1991-04-05 | Phosphonopeptides with collagenase inhibiting activity |
Country Status (10)
Country | Link |
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EP (1) | EP0527764A1 (en) |
JP (1) | JPH05505820A (en) |
AU (1) | AU634383B2 (en) |
CA (1) | CA2080227A1 (en) |
GB (1) | GB9008078D0 (en) |
IE (1) | IE911187A1 (en) |
NZ (1) | NZ237733A (en) |
PT (1) | PT97276A (en) |
WO (1) | WO1991015507A1 (en) |
ZA (1) | ZA912572B (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0520573A1 (en) * | 1991-06-27 | 1992-12-30 | Glaxo Inc. | Cyclic imide derivatives |
GB9122859D0 (en) * | 1991-10-28 | 1991-12-11 | Smithkline Beecham Plc | Novel compounds |
GB9200826D0 (en) * | 1992-01-15 | 1992-03-11 | Celltech Ltd | Peptidyl derivatives |
GB9211706D0 (en) * | 1992-06-03 | 1992-07-15 | Celltech Ltd | Peptidyl derivatives |
GB9211707D0 (en) * | 1992-06-03 | 1992-07-15 | Celltech Ltd | Peptidyl derivatives |
US5326760A (en) * | 1992-06-29 | 1994-07-05 | Glaxo, Inc. | Aminobutanoic acid compounds having metalloprotease inhibiting properties |
US5470834A (en) * | 1993-10-06 | 1995-11-28 | Florida State University | Sulfoximine and suldodiimine matrix metalloproteinase inhibitors |
US5455262A (en) * | 1993-10-06 | 1995-10-03 | Florida State University | Mercaptosulfide metalloproteinase inhibitors |
US6037472A (en) * | 1993-11-04 | 2000-03-14 | Syntex (U.S.A.) Inc. | Matrix metalloprotease inhibitors |
US5840698A (en) * | 1994-10-27 | 1998-11-24 | Affymax Technologies N.V. | Inhibitors of collagenase-1 and stormelysin-I metalloproteases, pharmaceutical compositions comprising same and methods of their use |
US5831004A (en) | 1994-10-27 | 1998-11-03 | Affymax Technologies N.V. | Inhibitors of metalloproteases, pharmaceutical compositions comprising same and methods of their use |
AU6951296A (en) * | 1995-08-08 | 1997-03-05 | Fibrogen, Inc. | C-proteinase inhibitors for the treatment of disorders related to the overproduction of collagen |
JP3973748B2 (en) * | 1998-01-14 | 2007-09-12 | 花王株式会社 | Hair growth inhibitor |
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GB8726714D0 (en) * | 1987-11-14 | 1987-12-16 | Beecham Group Plc | Compounds |
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1990
- 1990-04-10 GB GB909008078A patent/GB9008078D0/en active Pending
-
1991
- 1991-04-05 WO PCT/GB1991/000538 patent/WO1991015507A1/en not_active Application Discontinuation
- 1991-04-05 JP JP91506980A patent/JPH05505820A/en active Pending
- 1991-04-05 CA CA002080227A patent/CA2080227A1/en not_active Abandoned
- 1991-04-05 EP EP91907465A patent/EP0527764A1/en not_active Withdrawn
- 1991-04-05 AU AU76711/91A patent/AU634383B2/en not_active Ceased
- 1991-04-08 PT PT97276A patent/PT97276A/en not_active Application Discontinuation
- 1991-04-08 ZA ZA912572A patent/ZA912572B/en unknown
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ZA912572B (en) | 1992-02-26 |
GB9008078D0 (en) | 1990-06-06 |
PT97276A (en) | 1992-01-31 |
AU7671191A (en) | 1991-10-30 |
IE911187A1 (en) | 1991-10-23 |
JPH05505820A (en) | 1993-08-26 |
AU634383B2 (en) | 1993-02-18 |
EP0527764A1 (en) | 1993-02-24 |
WO1991015507A1 (en) | 1991-10-17 |
NZ237733A (en) | 1993-09-27 |
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