U.S. patent application number 15/882510 was filed with the patent office on 2018-06-07 for cyclic depsipeptide compounds and their uses.
The applicant listed for this patent is Cypralis Limited. Invention is credited to Hans Georg Fliri, Rhonan Lee Ford, Antonio Kuok Keong Vong.
Application Number | 20180153958 15/882510 |
Document ID | / |
Family ID | 47225510 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180153958 |
Kind Code |
A1 |
Fliri; Hans Georg ; et
al. |
June 7, 2018 |
CYCLIC DEPSIPEPTIDE COMPOUNDS AND THEIR USES
Abstract
The present invention relates to novel cycloundecadepsipeptide
compounds and their analogues which bind and inhibit cyclophilins,
have reduced immunosuppressive activity and improved
physicochemical properties including water solubility. The present
invention further relates to pharmaceutical compositions containing
said depsipeptide compounds and their analogues for use in the
treatment or prevention of diseases and pathologies which may be
ameliorated by the inhibition of cyclophilin activity.
Inventors: |
Fliri; Hans Georg;
(Cambridge, GB) ; Ford; Rhonan Lee; (Cambridge,
GB) ; Vong; Antonio Kuok Keong; (Cambridge,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cypralis Limited |
Cambridge |
|
GB |
|
|
Family ID: |
47225510 |
Appl. No.: |
15/882510 |
Filed: |
January 29, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15187039 |
Jun 20, 2016 |
9895412 |
|
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15882510 |
|
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14433327 |
Apr 2, 2015 |
9382295 |
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PCT/GB2013/052570 |
Oct 2, 2013 |
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15187039 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 31/12 20180101; A61P 43/00 20180101; A61P 9/00 20180101; A61P
29/00 20180101; C07K 11/02 20130101; A61P 3/10 20180101; A61P 11/00
20180101; A61P 9/10 20180101; A61P 31/10 20180101; A61P 3/04
20180101; A61P 25/00 20180101; C07K 7/56 20130101; A61K 38/15
20130101; A61P 33/00 20180101 |
International
Class: |
A61K 38/15 20060101
A61K038/15; C07K 11/02 20060101 C07K011/02; C07K 7/56 20060101
C07K007/56 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2012 |
GB |
1217560.0 |
Claims
1. A compound having the formula (1): ##STR00115## or a
pharmaceutically acceptable salt, tautomer or N-oxide thereof,
wherein L represents an optionally partially unsaturated chain of
1-6 carbon atoms which may be optionally branched, R1 and R2 may
independently represent H, alkyl, substituted alkyl, optionally
substituted cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl or optionally R1 and R2 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted, X represents H, OH, OC(.dbd.O)-alkyl,
OC(.dbd.O)-substituted alkyl, O-alkyl, O-substituted alkyl or
carbonyl (.dbd.O), R.sub.a represents hydrogen, alkyl, substituted
alkyl, alkoxy, substituted alkoxy, alkylthio, substituted alkylthio
or .dbd.CH.sub.2, and R.sub.b represents hydrogen or is absent
where R.sub.a is .dbd.CH.sub.2.
2. The compound according to claim 1 wherein R1 and R2 together
with the nitrogen atom to which they are attached form a 4-7
membered aryl, cycloalkyl or heterocyclic ring which may be further
fused or optionally substituted.
3. The compound according to claim 2 wherein R1 and R2 together
with the nitrogen atom to which they are attached form a 5-7
membered cycloalkyl or heterocyclic ring which may be further fused
or optionally substituted.
4. The compound according to claim 3 wherein NR1R2 is a ring
selected from: ##STR00116##
5. The compound according to claim 1 wherein L is
--(CH.sub.2).sub.n-- where n is 1-4.
6. The compound according to claim 1 wherein R1 and R2 are
independently H, alkyl or substituted alkyl.
7. The compound according to claim 6 wherein R1 and R2 are
independently H, alkyl or alkyl substituted with an aliphatic or
aromatic ring optionally containing one or more heteroatoms.
8. The compound according to claim 7 wherein R1 and/or R2 are
selected from H, methyl, ethyl, isopropyl,
(CH.sub.2).sub.n-pyridyl, (CH.sub.2).sub.n-pyrazoyl,
(CH.sub.2).sub.n-tetrazoyl, (CH.sub.2).sub.n-1,4-dioxanyl,
(CH.sub.2).sub.n--OMe, (CH.sub.2).sub.n-tetrahydropyranyl,
(CH.sub.2).sub.n--CN where n is 1, 2 or 3.
9. The compound according to claim 1 wherein X represents H or OH
or O-alkyl.
10. The compound according to claim 1 wherein R.sub.a and R.sub.b
represent hydrogen.
11. The compound according to claim 1 which is a compound selected
from the group consisting of:
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridin-4-ylmethyl-am-
ino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-2-(pyr-
idin-2-yl)-ethyl-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-(2-met-
hyl-2H-pyrazol-3-ylmethyl)-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-(1H-te-
trazol-5-ylmethyl)-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic
acid-(O-methyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-([1,4]dioxan-2-
-ylmethyl-methyl-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[3-trifluorome-
thyl-piperidinyl]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[(2-methoxy-et-
hyl)-methyl-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(3-methoxy-aze-
tidinyl)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-(tetra-
hydro-pyran-4-yl)-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal};
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(8-oxa-3-aza-bicyclo[3.2.1]oc-
tane)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(thiomorpholin-
e 1,1-dioxide)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(4,4-difluoro-piperidine)-hex-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-((4-fluoro-piperidin-4-yl)-me-
thanol)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--((S)-1-Pyrrolidin-2-yl-metha-
nol)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(3-methylamino-propionitrile)-
-hexanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridin-2-yl-amine)-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--((R)-3-Methyl-morpholine)-he-
xanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(bis-pyridin-2-ylmethyl-amine-
)-hexanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridin-2-ylmethyl-am-
ino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--(N'-methylpiperazine)-hexano-
ic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5
S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(4-phenyl-1-piperidyl)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5S)-1-benzylamino-3-methyl-5-methylamino-4-hydroxy-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal];
cyclo-[(3R,4R,5S)-4-hydroxy-1-isopropylamino-3-methyl-5-methylamino-hexan-
oic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]; and
cyclo-[(2R,3R,4S)-4-hydroxy-1-(2-hydroxymethyl-piperidin-1-yl)-3-methyl-5-
-methylamino-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] or a
pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition containing the compound according
to claim 1.
13. A pharmaceutical composition containing a compound according to
claim 11.
14. A method of treating a disorder, wherein the disorder is
selected from the group consisting of chronic and acute
inflammatory disorders, comprising administering an effective
amount of the compound of formula 1 as described in claim 1 to a
subject in need thereof.
15. A method of treating a disorder, wherein the disorder is
selected from the group consisting of chronic and acute
inflammatory disorders, comprising administering an effective
amount of a compound according to claim 11 to a subject in need
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/187,039, filed Jun. 20, 2016, which is a
continuation of U.S. patent application Ser. No. 14/433,327, filed
Apr. 2, 2015, now U.S. Pat. No. 9,382,295, which is a 35 U.S.C.
.sctn. 371 national stage filing of International Application No.
PCT/GB2013/052570, filed on Oct. 2, 2013, which claims the benefit
of United Kingdom Patent Application No. 1217560.0, filed on Oct.
2, 2012. Each of the foregoing applications are incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel
cycloundecadepsipeptide compounds and their analogues which bind
and inhibit cyclophilins, have reduced immunosuppressive activity
and improved physicochemical properties including water solubility,
and inhibit extracellularly secreted cyclophilins or their ability
to bind their cognate cell surface receptors, and inhibit
intracellular cyclophilins. The present invention further relates
to pharmaceutical compositions containing said depsipeptide
compounds and their analogues for use in the treatment or
prevention of diseases and pathologies.
BACKGROUND OF THE INVENTION
[0003] It is now well established that cyclophilins represent one
family of a large group of proteins which all possess
peptidyl-prolyl cis/trans isomerase (PPIase) activity, the other
families being FK-506-binding proteins and parvulins. Cyclophilins
are ubiquitous enzymes, being found in all living organisms with
high structural conservation across species. In humans there are
seven major cyclophilins: cyclophilin-A, cyclophilin-B,
cyclophilin-C, cyclophilin-D, cyclophilin-E, cyclophilin-40, and
NK-cyclophilin. The most abundant protein is cyclophilin-A, which
accounts for 0.6% of total cytosolic protein, whilst cyclophilin-D
is found predominantly in cell mitochondria. Cyclophilin-B and
cyclophilin-C are located largely in the endoplasmic reticulum
whilst cyclophilin-E is located in the cell nucleus. Cyclophilin-40
is found in the cytosol, as is NK-cyclophilin (named as such
because it was first discovered in human natural killer cells). The
cyclophilins have also been observed to translocate between
cellular compartments and, under certain circumstances, to be
secreted, properties which contribute to their physiological
functions. Cyclophilins have the specific enzymatic capability of
accelerating the rate of cis/trans isomerisation of peptidyl-prolyl
bonds and speed up the rate of folding of newly synthesised or
denatured proteins. PPIases also play a role in the repair of
proteins which have been damaged through exposure of cells to
oxidation, ultraviolet radiation, thermal stress and pH changes.
Cyclophilins A and B can be secreted from cells and the secreted
proteins act as pro-inflammatory cytokines. Additionally,
cyclophilins play a role in intracellular protein trafficking and
cyclophilin-D has a modulatory role in the opening of the
mitochondrial permeability transition pore.
[0004] The best known ligand and inhibitor for cyclophilins is
cyclosporin A (CsA), binding to cyclophilins A, B, and D with
nanomolar affinity. The well-known immunosuppressive activity of
CsA is manifested not through cyclophilin inhibitory activity, but
is a property of the complex formed with cyclophilin A: The whole
complex binds to the protein calcineurin, a phosphatase whose
activity is essential to initiate lymphokine gene transcription and
the immune response. Thus, immunosuppression is the result of the
formation of a ternary complex cyclosporin/cyclophilin
A/calcineurin and displayed only by cyclosporin A and a few
selected analogues or derivatives.
Cyclophilin-Mediated Diseases
[0005] Of particular importance for the present invention is that
cyclophilins have been found to be involved in many diseases,
including chronic and acute inflammatory disorders, malignancies,
viral, fungal and parasitic infections, central nervous system
disorders, tissue degenerative disorders, and ischemia/reperfusion
injury, cardiovascular disease, respiratory disease, metabolic
syndrome, obesity, diabetes and diseases associated with
mitochondrial dysfunction.
[0006] Cyclophilins have an important role in viral life-cycles and
the anti-viral activity of the non-immunosuppresive analogue
NIM-811, and by implication that of cyclosporin A, has been linked
to the inhibition of cyclophilin A. Cyclosporin A and analogues
have been shown to have activity against HIV, HCV and HBV and
several drugs are undergoing clinical trials for treatment of HCV
[Fischer G., Gallay P., Hopkins S. (2010) Cyclophilin inhibitors
for the treatment of HCV infection. Curr. Opin. Investig. Drugs
11:911-918]. Studies suggesting an involvement of cyclophilins in
the life-cycles of other viruses, for example severe acute
respiratory coronavirus, vaccinia virus, papilloma virus, and
herpes simplex virus, suggest that cyclophilin inhibitors may also
have utility for the treatment of infection by other viruses. The
activity of cyclophilin inhibitors against HCV suggests that they
might also be efficacious against other viruses [Zhou D., Mei Q.,
Li J., He H., (2012) Cyclophilin A and viral infections. Biochem.
Biophys. Res. Comm. 424:647-650], such as the Flaviviridae viruses,
for example Dengue virus, yellow fever virus and West Nile
virus.
[0007] The immune-suppressive functions of cyclosporin A have long
been recognised and important to its clinical use in
transplantation, however it and the nonimmune-suppressive analogues
mediate anti-inflammatory actions through their activity at
cyclophilins also. Furthermore, although cyclophilin A, B, C, D are
the most studied, to date seventeen cyclophilins have been
identified in the human genome and knowledge of their physiological
functions is incomplete. Furthermore, there is evidence that
cyclophilins may be secreted and act as mediators signalling via
cell surface receptors. CD147 has been identified as one such
receptor and is implicated in the functions of cyclophilins in
inflammation [Yurchenko V., Constant S., Eisenmesser E., Bukrinsky
M. (2010) Cyclophilin-CD147 interactions: a new target for
anti-inflammatory therapeutics. Clin. Exp. Immunol. 160:305-317]. A
significant body of research has implicated cyclophilins in the
control of inflammatory processes involved in the aetiology of
inflammatory disease [Kovarik J. (2013) From immunosuppression to
immunomodulation current principles and future strategies.
Pathobiology 80:275-281] including but not limited to: arthritis,
such as rheumatoid arthritis; respiratory diseases, such as
allergic asthma, allergic rhinitis, chronic asthma, COPD, pulmonary
hypertension; dermatological diseases, such as psoriasis, atopic
dermatitis, urticaria, mastocytosis; opthalmalogical diseases, such
as uveitis, conjunctivitis, dry eye, age-related macular
degeneration; inflammatory bowel disease; periodontitis; lupus.
Also an increasing body of evidence now suggests that chronic
inflammatory processes may underlie chronic diseases leading to
tissue degeneration or fibrosis or metabolic instability,
suggesting that ligands of cyclophilins may have utility in chronic
diseases including: degenerative disorders of the nervous system,
such as Alzheimer's disease, multiple sclerosis, amyotrophic
lateral sclerosis, Parkinson's disease, Huntington's chorea,
glaucoma; cardiovascular diseases, such as myocarditis,
cardiomyopathy, atherosclerosis, restenosis; bowel diseases such as
ulcerative colitis, Crohn's disease; liver disease, such as
cirrhosis or alcohol or non-alcohol fatty liver disease; metabolic
syndrome and diabetes.
[0008] Cyclophilins are regulated during disease and changes in
expression have been associated with cancer [Lee J. and Kim S S.
(2010) Current implications of cyclophilins in human cancers. J.
Exp. Clin. Cancer Res. 29:97]. The receptor for secreted
cyclophilin, CD147 has also been associated with cancer and linked
to proliferation, tumour invasiveness and chemo-resistance.
Conversely cyclophilin D activity has been associated with the
mitochondrial transition pore and the cell death induced by several
anti-cancer regimens. Cyclophilin inhibitors, optimised for
selectivity between cyclophilins or tissue or subcellular
distribution, may have utility in a wide range of cancer types
including but not limited to, melanoma, lymphoma, epithelial
ovarian, breast, prostate, oral, endometrial, hepatic, pancreatic,
or skin cancers.
[0009] It should be noted that the relative roles of secreted
cyclophilins, or of a particular cyclophilin, gives rise to the
opportunity to design pan-cyclophilin inhibitors or inhibitors with
selective inhibitory profiles, or with selective distribution
patterns across tissues or physiological compartments, in order to
optimise the therapeutic index for a given disease.
[0010] Of particular importance for the present invention is
cyclophilin D (PPIF), which is located in the mitochondrial matrix
where it serves as a modulatory component of the mitochondrial
permeability transition pore (MPTP). Mitochondrial permeability
transition is a phenomenon induced by high levels of matrix calcium
and is characterized by the opening of the MPTP. Activation of the
MPTP results in loss of the mitochondrial membrane potential,
expansion of the matrix, rupture of the mitochondrial outer
membrane and release of mitochondrial proteins into the cytosol
where they activate cellular death programs. The induction of the
MPTP also causes mitochondria to become depolarized, which
negatively affects the production of ATP, the cell's main source of
energy, creating an energy deficit in the cell. Much research has
found that the fate of the cell after an insult depends on the
extent of permeability transition; if it occurs to only a slight
extent and the energy deficit is transient, the cell may recover,
whereas occurrence to increasingly larger extents activates the
death programs leading first to apoptosis and in the extreme to
necrosis.
[0011] The central role of cyclophilin D in this chain of events
has been further substantiated by gene deletion or silencing
experiments.
[0012] The induction of mitochondrial permeability transition is
implicated in many diseases and conditions including: traumatic or
acute events such as ischaemia/reperfusion injury to organs,
traumatic brain injury, spinal cord injury, stroke, myocardial
infarction; and chronic diseases involving the progressive loss or
dysfunction of tissues leading to associated 5 symptoms, such as
congestive heart failure, cardiac diseases, CNS disorders [Martin L
J. (2012) Biology of mitochondria in neurodegenerative diseases.
Prog. Mol. Biol. Transl. Sci. 107:355-415] leading to dementia or
psychiatric impairments, Alzheimer's disease, Parkinson's disease,
Huntingdon's chorea; epilepsy; amyotrophic lateral sclerosis;
multiple sclerosis; muscular dystrophies; glaucoma; macular
degeneration; diabetic retinopathy; retinal dystrophies, such as
retinitis pigmentosa; liver disease; diseases caused by
mitochondrial dysfunction, such as Leber's hereditary optic
neuropathy (LHON), Friedreich's ataxia, MELAS syndrome, myoclonic
epilepsy with ragged red fibres (MERRF); cell death due to
infection, toxins or metabolic abnormality.
[0013] Accordingly, the MPTP is accepted as a therapeutic target
for pharmacological intervention to block tissue and nerve damage
originating from mitochondrial energy deficiencies and
abnormalities. Inhibiting the opening of the MPTP by inhibition of
cyclophilin D can treat and/or prevent disorders and diseases
associated with mitochondrial dysfunction.
[0014] Agents that are known to block cyclophilin functions include
the immune suppressant cyclosporin A (CsA) and some of its
non-immunosuppressive derivatives such as
N-methyl-Val-4-cyclosporin or NIM-811 (N-methyl-Ile-4-cyclosporin),
2-aminoethoxydiphenyl borate (2-APB), sanglifehrin, and bongkrekic
acid. Cyclosporin A has been in clinical use as immunosuppressant
to prevent rejection of organ transplants since 1983. While
life-saving for organ transplant recipients, the drug has a narrow
therapeutic index that limits its wider use. Toxicities and adverse
effects include renal and hepatotoxicity, hypertension, gingival
hyperplasia, hirsutism, nausea, headache, tremors and paresthesia.
Some of these effects are associated to the mechanism of
immunosuppression, which is generally considered an undesired
property except in transplantation. In fact, this activity
predisposes subjects to an increased risk of viral infections and
malignancies. Cyclosporin is very insoluble in aqueous systems and
as a consequence it is usually administered by mouth in form of
suspensions or emulsions. Non-aqueous formulations have been
developed for intravenous administration, but the additives used in
these preparations are often associated with adverse effects
themselves, notably anaphylactic reactions. There is thus a
pronounced therapeutic need to develop novel cyclophilin inhibitors
that have reduced or no immunosuppressive activity, and that are
permeable and soluble, such that they can easily be formulated as
therapeutic agents. The present invention provides compounds that
fulfil these requirements.
Cycloundecadepsipeptides--Cyclophilin Inhibitors with Reduced
Immunosuppression
[0015] The first cycloundecadepsipeptide to be identified to be a
potent inhibitor of cyclophilins has the structure shown in formula
A.
##STR00001##
[0016] In accordance with WO 2011/141891, this compound can also be
described as
Cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal).
Compounds of this family can generally be designated as
##STR00002##
[0017] In which AXX.sub.1 is
N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)-threonine, and D-Hiv is
(D)-2-hydroxyisovalerianic acid.
[0018] The main drawback to using the compound of formula A per se
for the treatment of the above mentioned indications is its high
level of immunosuppressive activity and its poor aqueous
solubility.
[0019] More recently, new cycloundecadepsipeptides which retain
their ability to bind cyclophilins but with significantly reduced
immunosuppressive properties have been disclosed (WO2010/052559
A1). This application claims the use of certain
cycloundecadepsipeptides as compounds for treating viral
infections, notably by Hepatitis C.
[0020] In a further disclosure, (WO2011/141891 A1) report the use
of certain cycloundecadepsipeptides as compounds for treating cell
death associated disorders or diseases.
[0021] The structural modifications described in these patents
serve to eliminate the immunosuppressive properties from the parent
molecule. However, they do not achieve improvement of the low
solubility of the molecules in aqueous systems.
[0022] It was now found that certain modifications of the residue 1
of the cycloundecadepsipeptide have not only good solubility in
aqueous solvents but at the same time also strongly reduced
immunosuppressive activity.
SUMMARY OF THE INVENTION
[0023] According to one aspect, compounds of the present invention
comprise novel cycloundecadepsipeptides which have reduced
immunosuppressive activity. According to another aspect, the
compounds have improved water solubility. According to another
aspect, the compounds have high affinity for cyclophilins,
including cyclophilin-A and cyclophilin-D. According to other
aspects, compounds of the present invention comprise
cycloundecadepsipeptide analogues that are useful in respect of
treating cyclophilin-mediated diseases or conditions and for
developing therapies with respect to such diseases and
conditions.
[0024] Disclosed herein are cycloundecadepsipeptides compounds
which can be designated as
##STR00003##
[0025] In which AXX.sub.1 contains a nitrogen atom in the side
chain, as further described below, and, AXX.sub.2 is Abu, Val, Thr,
Thr(OMe), Thr(OAc), Thr(OCOCH.sub.2CH.sub.2CH.sub.2OH), or an
alternative threonine ester or threonine-O-alkyl or substituted
O-alkyl moiety, Nva, 5-hydroxy-Nva (Hnv) or a moiety of type
C(.dbd.O)CH.sub.3 or C(.dbd.N--Y)CH.sub.3 where Y is OH, NH.sub.2
or O- or N-alkyl or substituted alkyl versions thereof;
[0026] AXX.sub.3 is optionally substituted alkylene, D-MeAla,
D-3-fluoro-MeAla, D-MeSer, D-MeSer(OAc),
D-MeSer(OCH.sub.2CH.sub.2OH), D-MeSer(OCH.sub.2CH.sub.2NEt.sub.2),
D-MeAsp(OMe) or a D-amino acid with a side chain selected from
hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,
alkylthio or substituted alkylthio;
[0027] AXX.sub.4 is MeLeu, MeIle, MeMet, MeVal, MeThr, MeThr(OAc),
MeAla, EtVal, EtIle, EtPhe, EtTyr, EtThr(OAc), MeThr(OAc), MeTyr,
MeTyr(OAc), MeTyr(OMe), MePhe, MeMet(Ox) wherein the sulphur atom
of methionine is sulphoxide or sulphone;
[0028] AXX.sub.5 is Leu, Val, Ile, Gly, Abu;
[0029] AXX.sub.6 is MeAla, Sar, MeLeu;
[0030] AXX.sub.7 is Gly, Ala;
[0031] D-Hiv is (D)-2-hydroxyisovalerianic acid;
[0032] AXX.sub.9 is MeLeu;
[0033] AXX.sub.10 is Leu; and
[0034] AXX.sub.11 is MeVal.
[0035] In accordance with the aspects of the invention there is
provided a compound having the formula (1):
##STR00004##
or a pharmaceutically acceptable salt, tautomer or N-oxide thereof,
wherein:
[0036] L represents a bond or an optionally substituted, optionally
partially unsaturated chain of 1-6 carbon atoms with optional
additional heteroatoms atoms in the chain, and may be optionally
branched and optionally linked to R.sub.1 to form a ring structure
containing one or more nitrogen atoms,
[0037] Q represents a primary, secondary or tertiary covalent bond,
a carbonyl group and optionally a linking group to R1,
[0038] R1 and R2 may be absent or independently represent H, alkyl,
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, optionally substituted heteroaryl, --COR.sub.3,
--CO.sub.2R.sub.3, --OR.sub.4, --NR.sub.4R.sub.5,
CONR.sub.4R.sub.5, --C(.dbd.NR.sub.6)NR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)OR.sub.3 and optionally R1 and R2 may together
with the nitrogen atom to which they are attached form a 4-7
membered aryl, cycloalkyl or heterocyclic ring which may be further
fused or optionally substituted,
[0039] R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0040] R4 and R5 independently represent H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl and optionally R4 and R5 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted,
[0041] R6 represents H, alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0042] X represents H, OH, OC(.dbd.O)-alkyl, OC(.dbd.O)-substituted
alkyl, O-alkyl, O-substituted alkyl, carbonyl (.dbd.O) or imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5,
[0043] R.sub.a represents hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or
optionally substituted alkylene, and
[0044] R.sub.b represents hydrogen or is absent.
[0045] The group L-Q-NR1R2 may comprise a primary, secondary or
tertiary amino group attached via an optionally substituted alkyl
linker. The group L-Q-NR1R2 may comprise --C(.dbd.O)--NR1R2.
[0046] The group L-Q-NR1R2 may comprise a primary or secondary
amide, urea, amidine, guanidine or carbamate group attached via an
optionally substituted alkyl linker. Q may be a carbonyl group such
that amide may be of orientation --C(.dbd.O)N as well as
--NC(.dbd.O). Where Q is a carbonyl group, L may be absent.
[0047] The group L-Q-NR1R2 may comprise a C.dbd.N double bond
moiety, for example C.dbd.N--OH, C.dbd.N--OR, C.dbd.N--NH2,
C.dbd.N--NHR or C.dbd.N--NRR. The group at AXX2 may also comprise a
carbonyl or a C.dbd.N double bond moiety, for example C.dbd.N--OH,
C.dbd.N--OR, C.dbd.N--NH2, C.dbd.N--NHR or C.dbd.N--NRR.
[0048] The group L-Q-NR1R2 may comprise a nitrogen containing
heterocyclic ring. The heterocyclic ring may be a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted.
[0049] Disclosed herein are pharmaceutical compositions containing
a compound disclosed herein.
[0050] Compounds disclosed herein may be used in the manufacture of
medicaments. The medicaments or pharmaceutical compositions may be
used in the treatment or prevention of diseases, pathologies or
symptomatology which may be ameliorated by inhibition of
cyclophilin activity.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Compounds of the present invention comprise novel
cycloundecadepsipeptides which have reduced immunosuppressive
activity. According to another aspect, the compounds have improved
water solubility. According to another aspect, the compounds have
high affinity for cyclophilins, including cyclophilin-A and
cyclophilin-D. According to other aspects, compounds of the present
invention comprise cycloundecadepsipeptide analogues that are
useful in respect of treating cyclophilin-mediated diseases or
conditions and for developing therapies with respect to such
diseases and conditions.
[0052] In accordance with the aspects of the invention there is
provided a compound having the formula (1):
##STR00005##
or a pharmaceutically acceptable salt, tautomer or N-oxide thereof,
wherein:
[0053] L represents an optionally substituted, optionally partially
unsaturated chain of 1-6 carbon atoms with optional additional
heteroatoms atoms in the chain, and may be optionally branched and
optionally linked to R.sub.1 to form a ring structure containing
one or more nitrogen atoms,
[0054] Q represents a primary, secondary or tertiary covalent bond,
a carbonyl group and optionally a linking group to R1,
[0055] R1 and R2 may be absent or independently represent H, alkyl,
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, optionally substituted heteroaryl, --COR.sub.3,
--CO.sub.2R.sub.3, --OR.sub.4, --NR.sub.4R.sub.5,
--CONR.sub.4R.sub.5, --C(.dbd.NR.sub.6)NR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)OR.sub.3 and optionally R1 and R2 may together
with the nitrogen atom to which they are attached form a 4-7
membered aryl, cycloalkyl or heterocyclic ring which may be further
fused or optionally substituted,
[0056] R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0057] R4 and R5 independently represent H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl and optionally R4 and R5 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted,
[0058] R6 represents H, alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0059] X represents H, OH, OC(.dbd.O)-alkyl, OC(.dbd.O)-substituted
alkyl, O-alkyl, O-substituted alkyl, carbonyl (.dbd.O) or imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5,
[0060] R.sub.a represents hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or
optionally substituted alkylene, and
[0061] R.sub.b represents hydrogen or is absent.
[0062] The group L-Q-NR1R2 may comprise a primary, secondary or
tertiary amino group attached via an optionally substituted alkyl
linker. The group L-Q-NR1R2 may comprise --C(.dbd.O)--NR1R2.
[0063] The group L-Q-NR1R2 may comprise a primary or secondary
amide, urea, amidine, guanidine or carbamate group attached via an
optionally substituted alkyl linker. Q may be a carbonyl group such
that amide may be of orientation --C(.dbd.O)N as well as
--NC(.dbd.O). Where Q is a carbonyl group, L may be absent.
[0064] The group L-Q-NR1R2 may comprise a C.dbd.N double bond
moiety, for example C.dbd.N--OH, C.dbd.N--OR, C.dbd.N--NH2,
C.dbd.N--NHR or C.dbd.N--NRR.
[0065] The group L-Q-NR1R2 may comprise a nitrogen containing
heterocyclic ring. The heterocyclic ring may be a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted.
[0066] Linking moiety L may be a bond, or 1-6 carbon atoms. L may
contain one or more heteroatoms in the chain. L may contain O, N or
S atoms interspersed between the carbon atoms. L may contain a
branch point. L may contain one or more double or triple bonds such
that L may be partially unsaturated. L may link with R1 or R2 to
form a ring containing one or more nitrogen atoms. L may be
(CH.sub.2)n where n is 1-4. L may contain a single double bond. L
may be CH.dbd.CH-- or CH.dbd.CH--CH.sub.2--. L may be absent where
Q is a carbonyl group.
[0067] Moiety Q may be a covalent bond. Q may be a primary (single)
covalent bond, where both R1 and R2 are present. Q may be a
secondary covalent (double) bond, where only a single R1 group is
present. Q may be a tertiary covalent (triple) bond to make a cyano
(CN) group where R1 and R2 are absent. Q may be a carbonyl group
such that Q-N is a C(.dbd.O)--N amide group. Q may link with R1 or
R2 to form a ring containing one or more nitrogen atoms.
[0068] Exemplary compounds may be where R1 and R2 are together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted or optionally partially unsaturated.
Exemplary rings include optionally substituted morpholinyl,
optionally substituted piperazinyl, optionally substituted
oxazepinyl, optionally substituted pyrrolidinyl, optionally
substituted piperidinyl, optionally substituted fused pyrrolidinyl,
optionally substituted thiomorpholinyl, or the S oxides thereof.
The ring may be fused to form a bicyclic system.
[0069] R1 and R2 may be absent or independently represent H, alkyl,
substituted alkyl, --COR.sub.3, --CO.sub.2R.sub.3, --OR.sub.4,
--NR.sub.4R.sub.5, CONR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)NR.sub.4R.sub.5, --C(.dbd.NR.sub.6)OR.sub.3 and
optionally R1 and R2 may together with the nitrogen atom to which
they are attached form a 4-7 membered aryl, cycloalkyl or
heterocyclic ring which may be further fused or optionally
substituted.
[0070] R1 and/or R2 may be H. R1 and/or R2 may be alkyl or
substituted alkyl. R1 and/or R2 may be methyl or ethyl. R1 and/or
R2 may be isopropyl. R1 and/or R2 may be aryl or substituted aryl.
R1 and/or R2 may be heteroaryl or substituted heteroaryl. R1 and/or
R2 may be an alkyl group substituted with a further ring. The ring
may be aliphatic or aromatic, and may contain one or more
heteroatoms. R1 and/or R2 may be of type CH.sub.2-ring or
CH.sub.2--CH.sub.2-ring. The ring may be a 2, 3 or 4-pyridyl. The
ring may be a 5 membered heteroaryl ring. The 5 membered ring may
contain 2, 3 or 4 nitrogen atoms. The ring may be a 6 membered
ring. The 6 membered ring may contain one or two oxygen or nitrogen
atoms. In each case the specified ring may be optionally further
substituted. R1 and/or R2 may be an alkyl group substituted with a
heteroatom in the alkyl chain. R1 and/or R2 may be a group of type
CH.sub.2--CH.sub.2--O-alkyl. R1 and/or R2 may be a cycloalkyl or
heterocycloalkyl group. The cycloalkyl or heterocycloalkyl group
may be further substituted.
[0071] Exemplary groups for R1 and/or R2 include methyl, ethyl,
isopropyl, (CH.sub.2)n-pyridyl, (CH.sub.2)n-pyrazoyl,
(CH.sub.2)n-tetrazoyl, --(CH.sub.2)n-1,4-dioxanyl, (CH.sub.2)n-OMe,
(CH.sub.2)n-tetrahydropyranyl, (CH.sub.2)n-CN where n is 1, 2 or
3.
[0072] R1 or R2 may represent an amide COR.sub.3 where R3
represents alkyl, substituted alkyl, cycloalkyl, optionally
substituted aryl, or optionally substituted heteroaryl. R1 or R2
may represent an amide COCH.sub.3. R1 or R2 may represent a
carbamate CO.sub.2R.sub.3 where R3 represents alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl. R1 or R2 may represent an oxime or
hydroxylamine OR.sub.4 where R4 represents H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl. R1 or R2 may represent an hydrazone
NR.sub.4R.sub.5 where R4 and R5 independently represent H, alkyl,
substituted alkyl, cycloalkyl, optionally substituted aryl, or
optionally substituted heteroaryl and optionally R4 and R5 may
together with the nitrogen atom to which they are attached form a
4-7 membered aryl, cycloalkyl or heterocyclic ring which may be
further fused or optionally substituted. R1 or R2 may represent
--C(.dbd.NR.sub.6)NR.sub.4R.sub.5 or --C(.dbd.NR.sub.6)OR.sub.3
where R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl
and R4 and R5 independently represent H, alkyl, substituted alkyl,
cycloalkyl, optionally substituted aryl, or optionally substituted
heteroaryl and optionally R4 and R5 may together with the nitrogen
atom to which they are attached form a 4-7 membered aryl,
cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted and R6 represents H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl.
[0073] Exemplary compounds include those based around formula
##STR00006##
or a pharmaceutically acceptable salt, tautomer or N-oxide thereof,
wherein:
[0074] R1 and R2 may independently represent H, alkyl, substituted
alkyl, optionally substituted cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, --COR.sub.3,
--CO.sub.2R.sub.3, --OR.sub.4, --NR.sub.4R.sub.5,
--CONR.sub.4R.sub.5, --C(.dbd.NR.sub.6)NR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)OR.sub.3 and optionally R1 and R2 may together
with the nitrogen atom to which they are attached form a 4-7
membered aryl, cycloalkyl or heterocyclic ring which may be further
fused or optionally substituted,
[0075] R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0076] R4 and R5 independently represent H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl and optionally R4 and R5 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted,
[0077] R6 represents H, alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl,
[0078] X represents H, OH, OC(.dbd.O)-alkyl, OC(.dbd.O)-substituted
alkyl, O-alkyl, O-substituted alkyl, carbonyl (.dbd.O) or imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5,
[0079] R.sub.a represents hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or
optionally substituted alkylene, and
[0080] R.sub.b represents hydrogen or is absent.
[0081] X represents H, OH, OC(.dbd.O)-alkyl, OC(.dbd.O)-substituted
alkyl, O-alkyl, O-substituted alkyl, carbonyl (.dbd.O) or imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5. Where X is
OH, the amino acid is threonine. The hydroxyl moiety of the
threonine can be in the form of an ester or O-alkyl group where the
ester or alkyl group is optionally substituted. For example, the
amino acid may be Thr(OMe), Thr(OAc),
Thr(OCOCH.sub.2CH.sub.2CH.sub.2OH), or an alternative threonine
ester or threonine-O-alkyl or substituted O-alkyl moiety. The ester
can be in the form OC(.dbd.O)-alkyl or OC(.dbd.O)-substituted
alkyl. X can represent a group of type --OCOR.sub.3 or
--OCO.sub.2R.sub.3, where R3 represents alkyl, substituted alkyl,
cycloalkyl, optionally substituted aryl, or optionally substituted
heteroaryl. X can represent a group of type --OR.sub.4 where R4
represents H, alkyl, substituted alkyl, cycloalkyl, optionally
substituted aryl, or optionally substituted heteroaryl. X can be
present as a carbonyl group (.dbd.O). X can be present as an imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5 where R4 and
R5 independently represent H, alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl
and optionally R4 and R5 may together with the nitrogen atom to
which they are attached form a 4-7 membered aryl, cycloalkyl or
heterocyclic ring which may be further fused or optionally
substituted.
[0082] R.sub.a represents hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or
optionally substituted alkylene. Ra includes substituted alkyl
groups of type --S--R7, --CH2-S--R7 and the sulfoxide and sulfone
analogues thereof where R7 represents H, alkyl or substituted
alkyl.
[0083] Exemplary groups for Ra include: .dbd.CH.sub.2;
--CH.sub.2SH; --CH.sub.2--S--(CH.sub.2).sub.nN--R.sub.4R.sub.5,
where R4 and R5 independently represent H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl and optionally R4 and R5 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted and n is 1-4;
--CH.sub.2--S--(CH.sub.2)n-aryl where n is 1-4;
--CH.sub.2--S--(CH.sub.2).sub.n-heteroaryl where n is 1-4;
--CH.sub.2--S--CH.sub.3; --CH.sub.2--S-cycloalkyl; CH.sub.2--S--
heterocycloalkyl; --CH.sub.2--S--(CH.sub.2).sub.nCOOR4 where R4
represents H, alkyl, substituted alkyl, cycloalkyl, optionally
substituted aryl, or optionally substituted heteroaryl and n is
1-4; --CH.sub.2--S--(CH.sub.2).sub.n--CH.dbd.CH.sub.2 where n is
1-4; --CH.sub.2--S--(CH.sub.2).sub.nN--C(.dbd.NH)--NH.sub.2 where n
is 1-4. In each example given above, the sulphur may be oxidised to
the sulfoxide or sulfone, and formulas can be represented as
--CH.sub.2--S(.dbd.O).sub.m--(CH.sub.2)-- where m is 0-2.
[0084] Further exemplary groups for Ra can be found in publication
US2012/0088734, the contents of which are incorporated herein.
[0085] R.sub.b represents hydrogen or is absent where Ra is
alkylene.
[0086] Exemplary compounds may include a compound of formula 1
wherein L is a chain of 1-6 carbon atoms, Q is a primary covalent
bond or a carbonyl group and R1 and R2 are together with the
nitrogen atom to which they are attached form a 4-7 membered aryl,
cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted.
[0087] Exemplary compounds may include a compound of formula 1
wherein L is a chain of 1-6 carbon atoms, Q is a primary covalent
bond or a carbonyl group and R1 and R2 are together with the
nitrogen atom to which they are attached form a 5-7 membered
cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted.
[0088] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from --(CH.sub.2)n-NR1R2
where n is 1-4 and R1 and R2 may independently represent H, alkyl,
substituted alkyl or may together with the nitrogen atom to which
they are attached form a 4-7 membered aryl, cycloalkyl or
heterocyclic ring which may be further fused or optionally
substituted.
[0089] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from
--(CH.sub.2).sub.n--S--(CH.sub.2).sub.m--NR1R2 where n is 1-4, m is
1-4 and R1 and R2 may independently represent H, alkyl, substituted
alkyl or may together with the nitrogen atom to which they are
attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring
which may be further fused or optionally substituted.
[0090] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from
--(CH.sub.2).sub.n--CO--NR1R2 where n is 1-4 and R1 and R2 may
independently represent H, alkyl, substituted alkyl or may together
with the nitrogen atom to which they are attached form a 4-7
membered aryl, cycloalkyl or heterocyclic ring which may be further
fused or optionally substituted.
[0091] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from
--(CH.sub.2).sub.n--S--(CH.sub.2).sub.m--CO--NR1R2 where n is 1-4,
m is 1-4 and R1 and R2 may independently represent H, alkyl,
substituted alkyl or may together with the nitrogen atom to which
they are attached form a 4-7 membered aryl, cycloalkyl or
heterocyclic ring which may be further fused or optionally
substituted.
[0092] Exemplary structures for --NR1R2 include
##STR00007##
[0093] Exemplary compounds include a compound of formula 1 wherein
L is a C1-6 alkyl group with 0-1 heteroatom substituents, and 0-1
double bonds, Q is a primary covalent bond and R1 and R2 are
independently H, alkyl or substituted alkyl groups.
[0094] Exemplary compounds include a compound of formula 1 wherein
L or Q is linked to R.sub.1 to form a ring structure containing one
or more nitrogen atoms.
[0095] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from
--(CH.sub.2).sub.n--NR1R2 where n is 1-4, R1 is H or alkyl, and R2
represents H, COR.sub.3, --CO.sub.2R.sub.3, --CONR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)NR.sub.4R.sub.5, or --C(.dbd.NR.sub.6)OR.sub.3
where R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl,
R4 and R5 independently represent H, alkyl, substituted alkyl,
cycloalkyl, optionally substituted aryl, or optionally substituted
heteroaryl and optionally R4 and R5 may together with the nitrogen
atom to which they are attached form a 4-7 membered aryl,
cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted, and R6 represents H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl,
[0096] Exemplary compounds may include a compound of formula 1
wherein the group L-Q-NR1-R2 is selected from
--(CH.sub.2).sub.n--S--(CH.sub.2).sub.m--NR1R2 where n is 1-4, m is
1-4, R1 is H or alkyl, and R2 represents --COR.sub.3,
--CO.sub.2R.sub.3, --CONR.sub.4R.sub.5,
--C(.dbd.NR.sub.6)NR.sub.4R.sub.5, or --C(.dbd.NR.sub.6)OR.sub.3
where R3 represents alkyl, substituted alkyl, cycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl,
R4 and R5 independently represent H, alkyl, substituted alkyl,
cycloalkyl, optionally substituted aryl, or optionally substituted
heteroaryl and optionally R4 and R5 may together with the nitrogen
atom to which they are attached form a 4-7 membered aryl,
cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted, and R6 represents H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl,
[0097] Exemplary structures for N--COR.sub.3, --CO.sub.2R.sub.3,
--CONR.sub.4R.sub.5, --C(.dbd.NR.sub.6)NR.sub.4R.sub.5, or
--C(.dbd.NR.sub.6)OR.sub.3 include:
##STR00008##
where the arrows indicated positions which can be further
substituted. Groups may include
##STR00009##
[0098] Exemplary compounds include a compound of formula 1 wherein
Q is a secondary covalent bond, R1 is absent and R2 is --OR.sub.4
or --NR.sub.4R.sub.5, where R4 and R5 independently represent H,
alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl,
or optionally substituted heteroaryl and optionally R4 and R5 may
together with the nitrogen atom to which they are attached form a
4-7 membered aryl, cycloalkyl or heterocyclic ring which may be
further fused or optionally substituted.
[0099] Exemplary compounds include those shown below. In the
diagram below, the L-Q-NR1-R2 group is depicted from the cyclic
peptide ring.
##STR00010## ##STR00011## ##STR00012##
[0100] Exemplary amide structures of type CONR1R2 include
##STR00013## ##STR00014## ##STR00015##
[0101] Examples of structures where L or Q are linked to R1
include:
##STR00016## ##STR00017## ##STR00018## ##STR00019##
where R is one or more optional substituents on the aromatic
ring.
[0102] Alternative structures are
##STR00020##
where R is independently H, alkyl or substituted alkyl.
[0103] Alternative structures contain a ring containing one or more
nitrogen atoms. The ring may be aromatic, as shown above. The ring
may be an optionally substituted 6 membered aromatic ring. The ring
may be a pyridyl or pyrazinyl ring. The ring may be of formula
##STR00021##
where R is independently H, alkyl or substituted alkyl.
[0104] In each of the C1-6 chains shown above, the chains can
contain one or more double bonds. The chains can be (CH.sub.2) n
where n is 1-6 or can contain a CH.dbd.CH group as well as further
CH.sub.2 groups up to a total of 6 carbon atoms.
[0105] The compound may be according to the formula
##STR00022##
or a pharmaceutically acceptable salt, tautomer or N-oxide thereof,
wherein
[0106] L represents a bond or an optionally substituted, optionally
partially unsaturated chain of 1-6 carbon atoms with optional
additional heteroatoms atoms in the chain, and may be optionally
branched,
[0107] Z represents N or CH,
[0108] R7 represents H, optionally substituted alkyl, NH2,
heterocycloalkyl, --NR.sub.4R.sub.5
[0109] R4 and R5 independently represent H, alkyl, substituted
alkyl, cycloalkyl, optionally substituted aryl, or optionally
substituted heteroaryl and optionally R4 and R5 may together with
the nitrogen atom to which they are attached form a 4-7 membered
aryl, cycloalkyl or heterocyclic ring which may be further fused or
optionally substituted
[0110] X represents H, OH, OC(.dbd.O)-alkyl, OC(.dbd.O)-substituted
alkyl, O-alkyl, O-substituted alkyl, carbonyl (.dbd.O) or imine
(.dbd.N--Y) where Y is --OR.sub.4 or --NR.sub.4R.sub.5,
[0111] R.sub.a represents hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or
optionally substituted alkylene, and
[0112] R.sub.b represents hydrogen or is absent,
[0113] R7 may be at any position of the pyridyl or pyrazyl group.
R7 may be H, methyl, or morpholinyl.
[0114] The disclosures herein include any pharmaceutically
acceptable salts. Where compounds are isomers, all chiral forms and
racemates are included. The disclosures include all solvates,
hydrates and crystal forms.
[0115] To the extent that any of the compounds described have
chiral centres, the present invention extends to all isomers of
such compounds, whether in the form of diastereomeric mixtures or
or separated diastereomers. The invention described herein relates
to all crystal forms, solvates and hydrates of any of the disclosed
compounds however so prepared. To the extent that any of the
compounds disclosed herein have acid or basic centres such as
carboxylates or amino groups, then all salt forms of said compounds
are included herein. In the case of pharmaceutical uses, the salt
should be seen as being a pharmaceutically acceptable salt.
[0116] Pharmaceutically acceptable salts that may be mentioned
include acid addition salts and base addition salts. Such salts may
be formed by conventional means, for example by reaction of a free
acid or a free base form of a compound with one or more equivalents
of an appropriate acid or base, optionally in a solvent, or in a
medium in which the salt is insoluble, followed by removal of said
solvent, or said medium, using standard techniques (e.g. in vacuo,
by freeze-drying or by filtration). Salts may also be prepared by
exchanging a counter-ion of a compound in the form of a salt with
another counter-ion, for example using a suitable ion exchange
resin.
[0117] Examples of pharmaceutically acceptable salts include acid
addition salts derived from mineral acids and organic acids, and
salts derived from metals such as sodium, magnesium, or preferably,
potassium and calcium or organic bases such as ethanolamine,
N,N-dialkylethanolamines, morpholine, etc.
[0118] Examples of acid addition salts include acid addition salts
formed with acetic, 2,2-dichloroacetic, citric, lactic, mandelic,
glycolic, adipic, alginic, aryl sulfonic acids (e.g.,
benzenesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic
and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L-aspartic,
benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric,
camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic,
caprylic, cinnamic, citric, cyclamic, dodecylsulfuric,
ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic,
formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic
(e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g.
L-glutamic), .alpha.-oxoglutaric, glycolic, hippuric, hydrobromic,
hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and
(.+-.)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic),
(.+-.)-DL-mandelic, metaphosphoric, methanesulfonic,
1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,
palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic,
4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic,
tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric
acids.
[0119] Particular examples of salts are salts derived from mineral
acids such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric and sulfuric acids; from organic acids, such
as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, arylsulfonic acids; and from metals
such as sodium, magnesium, or preferably, potassium and
calcium.
[0120] Also encompassed are any solvates of the compounds and their
salts. Preferred solvates are solvates formed by the incorporation
into the solid state structure (e.g. crystal structure) of the
compounds of the invention of molecules of a non-toxic
pharmaceutically acceptable solvent (referred to below as the
solvating solvent). Examples of such solvents include water,
alcohols (such as ethanol, isopropanol and butanol) and
dimethylsulfoxide. Solvates can be prepared by recrystallising the
compounds of the invention with a solvent or mixture of solvents
containing the solvating solvent. Whether or not a solvate has been
formed in any given instance can be determined by subjecting
crystals of the compound to analysis using well known and standard
techniques such as thermogravimetric analysis (TGE), differential
scanning calorimetry (DSC) and X-ray crystallography.
[0121] The solvates can be stoichiometric or non-stoichiometric
solvates. Particular solvates may be hydrates, and examples of
hydrates include hemihydrates, monohydrates and dihydrates.
[0122] For a more detailed discussion of solvates and the methods
used to make and characterise them, see Bryn et al., Solid-State
Chemistry of Drugs, Second Edition, published by SSCI, Inc of West
Lafayette, Ind., USA, 1999, ISBN 0-967-06710-3.
Definitions
Amino
[0123] Amino means NH.sub.2. Amino includes substituted amino.
Substituted amino means NHR or NR.sup.2R.sup.3 where R.sup.2 and
R.sup.3 are independent substituents or where NR.sup.2R.sup.3 forms
an optionally substituted 4 to 7 membered non-aromatic heterocyclic
ring optionally containing a second heteroatom ring member selected
from O, N and S and oxidised forms thereof.
[0124] Exemplary substituted amino groups include NMe.sub.2,
NEt.sub.2, piperidinyl, piperazinyl, morpholino, N-cyclohexyl,
where the rings may be further substituted.
Alkyl
[0125] Alkyl means an aliphatic hydrocarbon group. The alkyl group
may be straight or branched or cyclic. "Branched" means that at
least one carbon branch point is present in the group. Thus, for
example, tert-butyl and isopropyl are both branched groups. The
alkyl group may be a lower alkyl group. "Lower alkyl" means an
alkyl group, straight or branched, having 1 to about 6 carbon
atoms, e.g. 2, 3, 4, 5 or 6 carbon atoms.
[0126] Exemplary alkyl groups include methyl, ethyl, n-propyl,
i-propyl, n-butyl, t-butyl, s-butyl, n-pentyl, 2-pentyl, 3-pentyl,
n-hexyl, 2-hexyl, 3-hexyl, n-heptyl, 2-heptyl, 3-heptyl, 4-heptyl,
2-methyl-but-1-yl, 2-methyl-but-3-yl, 2-methyl-pent-1-yl,
2-methyl-pent-3-yl.
[0127] The alkyl group may be optionally substituted, e.g. as
exemplified below. The term alkyl also includes aliphatic
hydrocarbon groups such as alkenyl, and alkylidene and cycloalkyl,
cycloalkylidene, heterocycloalkyl and heterocycloalkylidene groups,
which may be further substituted.
Alkenyl
[0128] Alkenyl means an unsaturated aliphatic hydrocarbon group.
The unsaturation may include one or more double bond, one or more
triple bond or any combination thereof. The alkenyl group may be
straight or branched. "Branched" means that at least one carbon
branch point is present in the group. Any double bond may,
independently of any other double bond in the group, be in either
the (E) or the (Z) configuration.
[0129] The alkenyl group may be a lower alkenyl group. "Lower
alkenyl" means an alkenyl group, straight or branched, having 2 to
6 carbon atoms, e.g. 2, 3, 4, 5 or 6 carbon atoms. Exemplary
alkenyl groups include ethenyl, n-propenyl, i-propenyl,
but-1-en-1-yl, but-2-en-1-yl, but-3-en-1-yl, pent-1-en-1-yl,
pent-2-en-1-yl, pent-3-en-1-yl, pent-4-en-1-yl, pent-1-en-2-yl,
pent-2-en-2-yl, pent-3-en-2-yl, pent-4-en-2-yl, pent-1-en-3-yl,
pent-2-en-3-yl, pentadien-1-yl, pentadien-2-yl, pentadien-3-yl.
Where alternative (E) and (Z) forms are possible, each is to be
considered as individually identified. The alkenyl group may be
optionally substituted, e.g. as exemplified below. Alkenyl includes
cyano.
Alkylidene
[0130] Alkylidene means any alkyl or alkenyl group linked to the
remainder of the molecule via a double bond. The definitions and
illustrations provided herein for alkyl and alkenyl groups apply
with appropriate modification also to alkylidene groups.
Alkylthio
[0131] Alkylthio means any alkyl group containing a sulfur atom in
the carbon chain. The sulphur atom may be in the form of a
thioether (C--S--C), a sulfoxide (C--S(.dbd.O)--C) or sulfone
(C--S(.dbd.O).sub.2--C). Alkylthio groups may be further
substituted. Alkylthio groups include CH.sub.2--S--R where R is a
further alkyl, cycloalkyl or substituted alkyl group.
Cycloalkyl
[0132] Cycloalkyl means a cyclic non-aromatic hydrocarbon group.
The cycloalkyl group may include non-aromatic unsaturation. The
cycloalkyl group may have 3 to 6 carbon atoms, e.g. 3, 4, 5 or 6
carbon atoms. Exemplary cycloalkyl groups include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl,
cyclohexenyl.
[0133] The cycloalkyl group may be optionally substituted, as
defined below, e.g. as exemplified below. Exemplary substituted
cycloalkyl groups include mono- or poly-alkyl-substituted
cycloalkyl groups such as 1-methylcyclopropyl, 1-methylcyclobutyl,
1-methylcyclopentyl, 1-methylcyclohexyl, 2-methylcyclopropyl,
2-methylcyclobutyl, 2-methylcyclopentyl, 2-methylcyclohexyl,
1,2-dimethylcyclohexyl or 1,3-dimethylcyclohexyl.
Cycloalkylidene Group
[0134] Cycloalkylidene means any cycloalkyl group linked to the
remainder of the molecule via a double bond. The definitions and
illustrations provided herein for cycloalkyl groups apply with
appropriate modification also to cycloalkylidene groups.
Heterocycloalkyl
[0135] Heterocycloalkyl group means a non-aromatic cyclic group
which contains one or more heteroatoms in the ring. The
heterocycloalkyl group may contain O, N or S atoms. The
heterocycloalkyl group may be fully saturated or partially
unsaturated. The heterocycloalkyl group is typically monocyclic or
bicyclic, and more usually is monocyclic.
[0136] Exemplary heterocycloalkyl groups include azetidinyl,
pyrrolidinyl, piperidinyl, azepinyl, diazepinyl, dihydrofuranyl
(e.g. 2,3-dihydrofuranyl, 2,5-dihydrofuranyl),
4,5-dihydro-1H-maleimido, dioxolanyl, 2-imidazolinyl,
imidazolidinyl, isoxazolidinyl, morpholinyl, oxazolidinyl,
piperazinyl, pyrrolidinonyl, 2-pyrrolinyl, 3-pyrrolinyl,
sulfolanyl, 3-sulfolenyl, tetrahydrofuranyl, thiomorpholinyl,
dihydropyranyl (e.g. 3,4-dihydropyranyl, 3,6-dihydropyranyl),
dioxanyl, hexahydropyrimidinyl, 2-pyrazolinyl, pyrazolidinyl,
pyridazinyl, 4H-quinolizinyl, quinuclinyl, tetrahydropyranyl,
3,4,5,6-tetrahydropyridinyl, 1,2,3,4-tetrahydropyrimidinyl,
3,4,5,6-tetrahydropyrimidinyl, tetrahydrothiophenyl,
tetramethylenesulfoxide, thiazolidinyl, 1,3,5-triazinanyl,
1,2,4-triazinanyl, hydantoinyl, and the like. The point of
attachment may be via any atom of the ring system.
Heterocycloalkylidene Group
[0137] Heterocycloalkylidene means any heterocycloalkyl group
linked to the remainder of the molecule via a double bond. The
definitions and illustrations provided herein for heterocycloalkyl
groups apply with appropriate modification also to
heterocycloalkylidene groups.
Optionally Substituted
[0138] "Optionally substituted" as applied to any group means that
the said group may if desired be substituted with one or more
substituents, which may be the same or different. `Optionally
substituted alkyl` includes both `alkyl` and `substituted
alkyl`.
[0139] Examples of suitable substituents for "substituted" and
"optionally substituted" moieties include halo (fluoro, chloro,
bromo or iodo), C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, hydroxy,
C.sub.1-6 alkoxy, cyano, amino, nitro, C.sub.1-6 alkylamino,
C.sub.2-6 alkenylamino, di-C.sub.1-6 alkylamino, C.sub.1-6
acylamino, di-C.sub.1-6 acylamino, C.sub.1-6 aryl, C.sub.1-6
arylamino, C.sub.1-6 aroylamino, benzylamino, C.sub.1-6 arylamido,
carboxy, C.sub.1-6 alkoxycarbonyl or (C.sub.1-6 aryl)(C.sub.1-10
alkoxy)carbonyl, carbamoyl, mono-C.sub.1-6 carbamoyl, di-C.sub.1-6
carbamoyl or any of the above in which a hydrocarbyl moiety is
itself substituted by halo, cyano, hydroxy, C.sub.1-2 alkoxy,
amino, nitro, carbamoyl, carboxy or C.sub.1-2 alkoxycarbonyl. In
groups containing an oxygen atom such as hydroxy and alkoxy, the
oxygen atom can be replaced with sulphur to make groups such as
thio (SH) and thio-alkyl (S-alkyl). Optional substituents therefore
includes groups such as S-methyl. In thio-alkyl groups, the sulphur
atom may be further oxidised to make a sulfoxide or sulfone, and
thus optional substituents therefore includes groups such as
S(O)-alkyl and S(O).sub.2-alkyl.
[0140] Substitution may take the form of double bonds, and may
include heteroatoms. Thus an alkyl group with a carbonyl (C.dbd.O)
instead of a CH.sub.2 can be considered a substituted alkyl
group.
[0141] Substituted groups thus include for example CFH.sub.2,
CF.sub.2H, CF.sub.3, CH.sub.2NH.sub.2, CH.sub.2OH, CH.sub.2CN,
CH.sub.2SCH.sub.3, CH.sub.2OCH.sub.3, OMe, OEt, Me, Et,
--OCH.sub.2O--, CO.sub.2Me, C(O)Me, i-Pr, SCF.sub.3, SO.sub.2Me,
NMe.sub.2, CONH.sub.2, CONMe.sub.2 etc. In the case of aryl groups,
the substitutions may be in the form of rings from adjacent carbon
atoms in the aryl ring, for example cyclic acetals such as
O--CH.sub.2--O.
[0142] The term optionally substituted includes groups containing
both alkyl are aryl moieties such as benzyl, CH2-pyridyl or any
other group having a ring attached to an alkyl group. Thus
optionally substituted includes CH.sub.2-ring or
CH.sub.2--CH.sub.2-ring.
[0143] While it is possible for the active ingredients to be
administered alone it may be preferable to present them as
pharmaceutical formulations. The formulations of the invention,
both for veterinary and for human use, comprise at least one active
ingredient, together with one or more acceptable carriers and
optionally other therapeutic ingredients. The carrier(s) must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and physiologically innocuous to the
recipient thereof.
[0144] The term "pharmaceutical formulation" in the context of this
invention means a composition comprising an active agent and
comprising additionally one or more pharmaceutically acceptable
carriers. The composition may further contain ingredients selected
from, for example, diluents, adjuvants, excipients, vehicles,
preserving agents, fillers, disintegrating agents, wetting agents,
emulsifying agents, suspending agents including liposomes or
nanoparticulates, sweetening agents, flavouring agents, perfuming
agents, antibacterial agents, antifungal agents, lubricating agents
and dispersing agents, depending on the nature of the mode of
administration and dosage forms. The compositions may take the
form, for example, of tablets, dragees, powders, elixirs, syrups,
liquid preparations including suspensions, sprays, inhalants,
tablets, lozenges, emulsions, solutions, cachets, granules,
capsules and suppositories, as well as liquid preparations for
injections, including liposome preparations.
[0145] The formulations include but are not limited to those
suitable for the administration routes described herein. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. Techniques and formulations generally are found in
Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,
Pa.), herein incorporated by reference in its entirety. Such
methods include the step of bringing into association the active
ingredient with the carrier which constitutes one or more accessory
ingredients. In general the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then,
if necessary, shaping the product. Excipients include ascorbic acid
and other antioxidants, chelating agents such as EDTA,
carbohydrates such as dextrin, hydroxyalkylcellulose,
hydroxyalkylmethylcellulose, stearic acid and the like. The pH of
the formulations ranges from about 3 to about 11, but is ordinarily
about 7 to 10.
[0146] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous or non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The
active ingredient may also be administered as a bolus, electuary or
paste.
[0147] Tablets may optionally be coated or scored and optionally
are formulated so as to provide slow or controlled release of the
active ingredient. Tablets containing the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipient
which are suitable for manufacture of tablets are acceptable. These
excipients may be, for example, inert diluents, such as calcium or
sodium carbonate, lactose, lactose monohydrate, croscarmellose
sodium, povidone, calcium or sodium phosphate; granulating and
disintegrating agents, such as maize starch, or alginic acid;
binding agents, such as cellulose, microcrystalline cellulose,
starch, gelatin or acacia; and lubricating agents, such as
magnesium stearate, stearic acid or talc. Tablets may be uncoated
or may be coated by known techniques including microencapsulation
to delay disintegration and adsorption in the gastrointestinal
tract and thereby provide a sustained action over a longer period.
For example, a time delay material such as glyceryl monostearate or
glyceryl distearate alone or with a wax may be employed.
Formulations for oral use may be also presented as hard gelatin
capsules where the active ingredient is mixed with an inert solid
diluent, for example calcium phosphate or kaolin, or as soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, such as peanut oil, liquid paraffin or olive
oil.
[0148] For administration to the eye or other external tissues
e.g., mouth and skin, the formulations are preferably applied as a
topical ointment or cream containing the active ingredient(s) in an
amount of, for example, 0.075 to 20% w/w (including active
ingredient(s) in a range between 0.1% and 20% in increments of 0.1%
w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w
and most preferably 0.5 to 10% w/w. When formulated in an ointment,
the active ingredients may be employed with either a paraffinic or
a water-miscible ointment base. Alternatively, the active
ingredients may be formulated in a cream with an oil-in-water cream
base.
[0149] If desired, the aqueous phase of the cream base may include,
for example, at least 30% w/w of a polyhydric alcohol, i.e. an
alcohol having two or more hydroxyl groups such as propylene
glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol (including PEG 400) and mixtures thereof. The
topical formulations may desirably include a compound which
enhances absorption or penetration of the active ingredient through
the skin or other affected areas. Examples of such dermal
penetration enhancers include dimethyl sulphoxide and related
analogues.
[0150] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner. While the
phase may comprise merely an emulsifier (otherwise known as an
emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a
lipophilic emulsifier which acts as a stabilizer. It is also
preferred to include both an oil and a fat. Together, the
emulsifier(s) with or without stabilizer(s) make up the so-called
emulsifying wax, and the wax together with the oil and fat make up
the so-called emulsifying ointment base which forms the oily
dispersed phase of the cream formulations.
[0151] Emulgents and emulsion stabilizers suitable for use in the
formulation of the invention include Tween.RTM. 60, Span.RTM. 80,
cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl
mono-stearate and sodium lauryl sulfate.
[0152] The choice of suitable oils or fats for the formulation is
based on achieving the desired cosmetic properties. The cream
should preferably be a non-greasy, non-staining and washable
product with suitable consistency to avoid leakage from tubes or
other containers. Straight or branched chain, mono- or dibasic
alkyl esters such as di-isoadipate, isocetyl stearate, propylene
glycol diester of coconut fatty acids, isopropyl myristate, decyl
oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate
or a blend of branched chain esters known as Crodamol CAP may be
used, the last three being preferred esters. These may be used
alone or in combination depending on the properties required.
Alternatively, high melting point lipids such as white soft
paraffin and/or liquid paraffin or other mineral oils are used.
[0153] Aqueous suspensions of the invention contain the active
materials in admixture with excipients suitable for the manufacture
of aqueous suspensions. Such excipients include a suspending agent,
such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxy-benzoate, one or more colouring agents, one
or more flavouring agents and one or more sweetening agents, such
as sucrose or saccharin.
[0154] Oil suspensions may be formulated by suspending the active
ingredient in a vegetable oil, such as arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oral suspensions may contain a thickening agent, such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such
as those set forth herein, and flavouring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an antioxidant such as ascorbic
acid.
[0155] Dispersible powders and granules of the invention suitable
for preparation of an aqueous suspension by the addition of water
provide the active ingredient in admixture with a dispersing or
wetting agent, a suspending agent, and one or more preservatives.
Suitable dispersing or wetting agents and suspending agents are
exemplified by those disclosed above. Additional excipients, for
example sweetening, flavouring and colouring agents, may also be
present.
[0156] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, such as olive oil, castor oil or arachis oil, a long
or medium chain triglyceride, a mineral oil, such as liquid
paraffin, or a mixture of these. Suitable emulsifying agents
include naturally-occurring gums, such as gum acacia and gum
tragacanth, naturally occurring phosphatides, such as soybean,
phosphatidyl choline, glycerol, lecithin, esters or partial esters
or salts derived from fatty acids and hexitol anhydrides, such as
sorbitan monooleate, and condensation products of these partial
esters with ethylene oxide, polyoxyethylene sorbitan monooleate, or
a polyethoxylated castor oil such as Kolliphor EL, formerly known
as Cremophor EL.RTM.. The emulsion may also contain sweetening and
flavouring agents. Syrups and elixirs may be formulated with
sweetening agents, such as glycerol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative,
flavouring or a colouring agent.
[0157] The pharmaceutical compositions of the invention may be in
the form of a sterile injectable preparation, such as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned herein. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, such as a solution in
1,3-butane-diol or prepared as a lyophilized powder. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile fixed oils may conventionally be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0158] Formulations suitable for administration to the eye include
eye drops wherein the active ingredient is dissolved or suspended
in a suitable carrier, especially an aqueous solvent for the active
ingredient. The active ingredient is preferably present in such
formulations in a concentration of 0.5 to 20%, advantageously 0.5
to 10% particularly about 1.5% w/w.
[0159] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0160] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa
butter or a salicylate.
[0161] Formulations suitable for intrapulmonary or nasal
administration have a particle size for example in the range of 0.1
to 500 .mu.m (including particle sizes in a range between 0.1 and
500 .mu.m in increments such as 0.5 .mu.m, 1 .mu.m, 30 .mu.m, 35
.mu.m, etc.), which is administered by rapid inhalation through the
nasal passage or by inhalation through the mouth so as to reach the
alveolar sacs. Suitable formulations include aqueous or oily
solutions of the active ingredient. Formulations suitable for
aerosol or dry powder administration may be prepared according to
conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis of infections as described herein.
[0162] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0163] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0164] The formulations are presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injection, immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof of the active ingredient.
[0165] Compounds of the invention can also be formulated to provide
controlled release of the active ingredient to allow less frequent
dosing or to improve the pharmacokinetic or toxicity profile of the
active ingredient. Accordingly, the invention also provided
compositions comprising one or more compounds of the invention
formulated for sustained or controlled release.
[0166] The dosages may be varied depending upon the requirements of
the patient, the severity of the condition being treated, and the
compound being employed. Determination of the proper dosage for a
particular situation is within the skill of the art. Generally,
treatment is initiated with the smaller dosages which are less than
the optimum dose of the compound. Thereafter the dosage is
increased by small increments until the optimum effect under the
circumstances is reached. For convenience, the total daily dosage
may be divided and administered in portions during the day if
desired.
[0167] The magnitude of an effective dose of a compound will, of
course, vary with the nature of the severity of the condition to be
treated and with the particular compound and its route of
administration. The selection of appropriate dosages is within the
ability of one of ordinary skill in this art, without undue burden.
In general, the daily dose range may be from about 0.1 mg to about
100 mg per kg body weight of a human and non-human animal,
preferably from about 1 mg to about 50 mg per kg of body weight of
a human and non-human animal, and most preferably from about 3 mg
to about 30 mg per kg of body weight of a human and non-human
animal.
[0168] One or more compounds of the present invention may be
administered by any route appropriate to the condition to be
treated. Suitable routes include oral, rectal, nasal, topical
(including buccal and sublingual), vaginal and parenteral
(including subcutaneous, intramuscular, intravenous, intradermal,
intrathecal and epidural), and the like. It will be appreciated
that the preferred route may vary with for example the condition of
the recipient.
[0169] In another embodiment, the compounds of the present
invention may be combined with one or more active agents, e.g. each
at a therapeutic concentration as reported in the art. Non-limiting
examples of suitable combinations include combinations of one or
more compounds of the present invention with at least one or two or
more compounds or a pharmaceutically acceptable salt thereof
selected from one or more of the following groups. [0170] (i)
Cyclooxygenase inhibitors e.g. Aspirin; [0171] (ii) Calcium channel
antagonists e.g. Nifedipine (Adalat), Nicardipine (Cardene),
Diltiazem (Tildiem); [0172] (iii) Potassium channel activators (K+
ATP) e.g. Nicorandil; [0173] (iv) Compounds that acts by generating
nitric oxide (NO), NO donors or compounds that enhance NO release
e.g. Nitrates; [0174] (v) Angiotensin II receptor agonists e.g.
Losartan (Cozaar); [0175] (vi) Angiotensin converting enzyme (ACE)
inhibitors e.g. Enalapril (Vasotec/Renitec); [0176] (vii) Dual
angiotensin converting enzyme/neutral peptidase inhibitors
(ACE/NEP); [0177] (viii) Neutral peptidase inhibitors (NEP); [0178]
(ix) Endothelin antagonists of the ET-A and/or ET-B receptors e.g.
Bostentan (Tracleer); [0179] (x) Renin inhibitors e.g. Aliskiren
(Tekturna); [0180] (xi) Adenosine diphosphate (ADP) inhibitors
and/or P2Y12 receptor inhibitors e.g. Clopidogrel (Plavix); [0181]
(xii) Tissue plasminogen activators (e.g. Reteplase); [0182] (xiii)
Phosphodiesterase inhibitors e.g. Cilostazol (Pletal); [0183] (xiv)
Glycoprotein IIB/IIIA (integrin .alpha.IIbj33) inhibitora e.g.
murine-human chimeric antibodies e.g. Abciximab (ReoPro),
Eptifibatide (Integrilin) and/or synthetic peptides (e.g.,
Eptifibatide) and/or synthetic non-peptides e.g., Tirofiban
(Aggrastat); [0184] (xv) Adenosine reuptake inhibitor e.g.
Dipyridamole (Persantine); [0185] (xvi) Thromboxane inhibitors
and/or thromboxane synthase inhibitors and/or thromboxane receptor
antagonists e.g. Terutroban; [0186] (xvii) Prostacyclins e.g.
Epoprostenol (Flolan); [0187] (xviii) Aldosterone receptor
antagonists e.g Eplerinone (Inspra); [0188] (xix) Plasminogen
activators (PA), e.g. Alteplase and Tenecteplase; [0189] (xx)
Cholesterylester transfer protein inhibitors; [0190] (xxi)
Mevalonate decarboxylase antagonists (e.g., Hymeglusin), and/or a
squalene synthesis inhibitors (e.g., Zaragozic acid) and/or an
HMG-CoA reductase inhibitor/statin e.g. Atorvastatin (Lipitor);
[0191] (xxii) Calcium Channel blockers (CCB) e.g. Nifedipine
(Procardia); [0192] (xxiii) Beta adrenergic blocker e.g.
Propranolol (Inderal LA); [0193] (xxiv) An alpha adrenergic
blockers e.g. Doxazosin (Cardura); [0194] (xxv) ApoA-I mimics;
[0195] (xxvi) Na+/K+-ATPase membrane pump inhibitors; [0196]
(xxvii) Inotropic agents; [0197] (xxviii) Anti-fibrotic agents,
e.g., Amiloride; [0198] (xxix) Anticoagulant agent agents e.g.
Warfarin (Coumadin); [0199] (xxx) Compounds that protect against
cellular and/or mitochondrial oxidative damage by e.g. reactive
oxygen species (ROS) e.g. Mito-Q, Vitamin E; [0200] (xxxi) A
compound that enhances or facilitates or up-regulates mitochondrial
respiration e.g. Coenzyme Q10 mimics, Resveratrol; [0201] (xxxii)
An anti-diabetic agent e.g. Metformin and/or a PPAR modulator e.g.
Rosiglitazone; [0202] (xxxiii) Interferons, e.g., pegylated
rIFN-alpha 2b (PEG-Intron); [0203] (xxxiv) Ribavirin and its
analogs, e.g. Ribavirin (Rebetol, Copegus), and Taribavirin
(Viramidine); [0204] (xxxv) HCV NS3 protease inhibitors, e.g.
Boceprevir, Telaprevir; [0205] (xxxvi) Alpha-glucosidase 1
inhibitors, e.g. Celgosivir, Miglitol; [0206] (xxxvii)
Hepatoprotectants, e.g. Emericasan, [0207] (xxxviii) Nucleoside or
nucleotide inhibitors of HCV NS5B polymerase e.g. Valopicitabine;
[0208] (xxxix) Non-nucleoside inhibitors of HCV NS5B polymerase,
e.g., Filibuvir; [0209] (xl) HCV NS5A inhibitors, e.g. BMS-790052;
[0210] (xli) TLR-7 agonists, e.g., Imiquimod; [0211] (xlii)
Cyclophillin inhibitors, e.g, DEBIO-025, SCY-635, and NIM811;
[0212] (xliii) HCV IRES inhibitors, e.g, MCI-067; [0213] (xliv)
Entry or fusion inhibitors e.g. CCR5 receptor antagonists e.g.
Marovirac; e.g. HIV fusion inhibitors e.g. Enfuvirtide (Fusion);
[0214] (xlv) Nucleoside analogue reverse-transcriptase inhibitors
(NARTIs or NRTIs) e.g. Zidovudine (AZT), Emtricitabine; [0215]
(xlvi) Nucleotide analogue reverse-transcriptase inhibitors
(NtARTIs or NtRTIs) e.g. Tenofovir (Viriad); [0216] (xlvii)
Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) e.g.
Efavirenz (Sustiva); [0217] (xlviii) Integrase strand transfer
inhibitors (Integrase inhibitors) e.g. Elvitegravir; [0218] (xlix)
Protease inhibitors e.g. Saquinavir, Ritonavir; [0219] (l) Other
drugs for treating HIV and HCV viral infections infections, e.g.
Thymosin alpha 1 (Zadaxin), Nitazoxanide; [0220] (li)
Pharmacokinetic enhancers; [0221] (lii) Beta-adrenergic agonists
e.g. Salmeterol, Salbutamol; [0222] (liii) Anti-cholinergic agents
(muscarinic) e.g. Tiotropium; [0223] (liv) Methyxanthines e.g.
Theophyllin); [0224] (lv) Corticosteroids e.g. Dexamethasone,
Budesonide, Prednsione; [0225] (lvi) Vitamin D analogues e.g.
Calcipotriol, Calcitriol; [0226] (lvii) Retinoids e.g. Acitretin;
[0227] (lviii) Immunosuppressants e.g. Adalimumab, Etanercept,
Methotrexate; [0228] (lix) Reversible Acetylcholinesterase
inhibitors e.g. Donepezile (Aracept), Rivastigmine (Exelon) and
Galantamine (Reminyl); [0229] (lx) Other anticholinergics e.g.
Orphenadrine (Biorphen), Procyclidine (Arpicolin) Trihexyphenidyl
(Broflex); [0230] (lxi) N-methyl-D-aspartate receptor (NMDA
receptor) antagonists (glutamate site) e.g. Memantine (Abixa);
[0231] (lxii) NMDA receptor antagonists (glycine site) e.g.
Gavestinel; [0232] (lxiii) Indirect NMDA receptor antagonists e.g.
Lubeluzole (Prosynap); [0233] (lxiv) L-DOPA (Levodopa); [0234]
(lxv) Dopamine Agonists e.g. bromocriptine (Parlodel), Cabergoline
(Cabaser) pergolide, Pramipexole (Mirapexin), Apomorphine (Apo-GO);
[0235] (lxvi) Glutamate antagonists e.g. Amantadine (Symmetrel);
Selfotel [0236] (lxvii) Inhibitors of Catachol-O-Methyl-Transferase
(COMT) e.g. Entacapone (Comtess) Tolcapone (Tasmar); [0237]
(lxviii) Monoamine oxidase inhibitors e.g Rasagiline (Azilect),
Selegiline (Eldepryl, Zelapar); [0238] (lxix) Sodium channel
blockers e.g. Riluzole (Rilutek); [0239] (lxx) Gamma-aminobutyric
acid (GABA) agonists e.g. Piracetam (Nootropil), Clomethiazole;
[0240] (lxxi) Lipid peroxidation inhibitors e.g. Tirilazad; [0241]
(lxxii) Nootropic agents e.g. Citicholine [0242] (lxxiii) Estrogen
receptor antagonists e.g. Tamoxifen; [0243] (lxxiv) Aromatase
inhibitors e.g Anastrozole; [0244] (lxxv) DNA alkylating agents
e.g. Cyclophosphamide; [0245] (lxxvi) DNA intercalating agents e.g.
Anthracyclines e.g Doxorubicin, Epirubicin; [0246] (lxxvii)
Anti-mitotic agents e.g. Docetaxel, Paclitaxel; [0247] (lxxviii)
Thymidylate Synthase inhibitors e.g. 5-Flurouracil (5FU); [0248]
(lxxix) Antifolates e.g Methotrexate; [0249] (lxxx) Topoisomerase
inhibitors e.g. Topotecan (Hycamtin), Irinotecan; [0250] (lxxxi)
Platinum containing anti-neoplastic agents e.g. Cisplatin (Platin),
Carboplatin (Paraplatin), Oxaliplatin; [0251] (lxxxii) Inhibitors
of microtubule formation e.g. Vincristine (Oncovin), Vinblastine;
[0252] (lxxxiii) BCR-Abl Inhibitors e.g. Imatibib (Gleevec); [0253]
(lxxxiv) Inhibitors of HER2 expression e.g. Trastuzumab
(Herceptin);
[0254] A combination therapy described herein may be administered
as a simultaneous or sequential regimen. When administered
sequentially, the combination may be administered in two or more
administrations.
[0255] Co-administration of a compound of the invention with one or
more other active agents generally refers to simultaneous or
sequential administration of a compound of the invention and one or
more other active agents, such that therapeutically effective
amounts of the compound of the invention and one or more other
active agents are both present in the body of the patient.
[0256] Specific (non-limiting) examples of compounds include
TABLE-US-00001 Patent Example Structure No. Name ##STR00023## 2
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
morpholino-hexanoic acid-Thr- Sar-MeLeu-Leu-MeLeu-Ala-D-
Hiv-MeLeu-Leu-MeVal] ##STR00024## 3 cyclo-[(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1-N- (methyl-pyridin-4-ylmethyl-
amino)-hexanoic acid-Thr- Sar-MeLeu-Leu-MeLeu-Ala-D-
Hiv-MeLeu-Leu-MeVal] ##STR00025## 4 cyclo-{(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1-N- [methyl-2-(pyridin-2-yl)-ethyl-
amino]-hexanoic acid-Thr- Sar-MeLeu-Leu-MeLeu-Ala-D-
Hiv-MeLeu-Leu-MeVal} ##STR00026## 5 cyclo-{(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1-N- [methyl-(2-methyl-2H-pyrazol-
3-ylmethyl)-amino]-hexanoic acid-Thr-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal} ##STR00027## 6
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
[methyl-(1H-tetrazol-5- ylmethyl)-amino]-hexanoic acid-
Thr-Sar-MeLeu-Leu-MeLeu- Ala-D-Hiv-MeLeu-Leu-MeVal} ##STR00028## 7
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
morpholino-hexanoic acid-(O- methyl-Thr)-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00029## 8
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
([1,4]dioxan-2-ylmethyl-methyl- amino)-hexanoic acid-Thr-Sar-
MeLeu-Leu-MeLeu-Ala-D-Hiv- MeLeu-Leu-MeVal} ##STR00030## 9
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
[3-trifluoromethyl-piperidinyl]- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal} ##STR00031## 10
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
[(2-methoxy-ethyl)-methyl- amino]-hexanoic acid-Thr-Sar-
MeLeu-Leu-MeLeu-Ala-D-Hiv- MeLeu-Leu-MeVal} ##STR00032## 11
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(3-methoxy-azetidinyl)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal} ##STR00033## 12
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(methyl-(tetrahydro-pyran-4-yl)- amino)-hexanoic acid-Thr-Sar-
MeLeu-Leu-MeLeu-Ala-D-Hiv- MeLeu-Leu-MeVal} ##STR00034## 13
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
morpholino-hexanoic acid-Abu- Sar-MeLeu-Leu-MeLeu-Ala-D-
Hiv-MeLeu-Leu-MeVal] ##STR00035## 14 cyclo-[(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1-(6- methyl-pyridin-2-yl)-hept-1- enoic
acid-Thr-Sar-MeLeu-Leu- MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal]
##STR00036## 15 cyclo-{(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1- (pyrimidin-2-yl)-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu- MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal}
##STR00037## 16 cyclo-[(3R,4R,5S)-4-(hydroxy)-
3-methyl-5-(methylamino)-1- (pyrimidin-2-yl)-heptanoic acid-
Thr-Sar-MeLeu-Leu-MeLeu- Ala-D-Hiv-MeLeu-Leu-MeVal] ##STR00038## 17
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(6-
methyl-pyridin-2-yl)-heptanoic acid-Thr-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00039## 18
cyclo-{(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(6-
morpholin-4-yl-pyridin-2-yl)- hept-1-enoic acid-Thr-Sar-
MeLeu-Leu-MeLeu-Ala-D-Hiv- MeLeu-Leu-MeVal} ##STR00040## 19
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(6-
methyl-pyridin-2-yl)-hept-1- enoic acid-Abu-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00041## 20
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(6-
morpholin-4-yl-pyridin-2-yl)- heptanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00042## 21
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(6-
methyl-pyridin-2-yl)-heptanoic acid-Abu-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00043## 22
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(8-oxa-3-aza-bicyclo[3.2.1] octane)-hexanoic acid-Thr-
Sar-MeLeu-Leu-MeLeu-Ala-D- Hiv-MeLeu-Leu-MeVal] ##STR00044## 23
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(thiomorpholine 1,1-dioxide)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00045## 24
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(4,4-difluoro-piperidine)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00046## 25
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
((4-fluoro-piperidin-4-yl)- methanol)-hexanoic acid-Thr-
Sar-MeLeu-Leu-MeLeu-Ala-D- Hiv-MeLeu-Leu-MeVal] ##STR00047## 26
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
((S)-1-Pyrrolidin-2-yl- methanol)-hexanoic acid-Thr-
Sar-MeLeu-Leu-MeLeu-Ala-D- Hiv-MeLeu-Leu-MeVal] ##STR00048## 27
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(3-methylamino-propionitrile)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00049## 28
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(methyl-pyridin-2-yl-amine)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00050## 29
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
((R)-3-Methyl-morpholine)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00051## 30
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(bis-pyridin-2-ylmethyl-amine)- hexanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00052## 31
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(methyl-pyridin-2-ylmethyl- amino)-hexanoic acid-Thr-Sar-
MeLeu-Leu-MeLeu-Ala-D-Hiv- MeLeu-Leu-MeVal] ##STR00053## 32
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-N-
(N'-methylpiperazine)-hexanoic acid-Thr-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00054## 33
cyclo-[(2R,3R,4S)-1-(1H- benzoimidazol-2-yl)-2-methyl-4-
methylamino-3-hydroxy- pentanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00055## 34
cyclo-[(3R,4R,5S)-4-(hydroxy)- 3-methyl-5-(methylamino)-1-(4-
phenyl-1-piperidyl)-hexanoic acid-Thr-Sar-MeLeu-Leu-
MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal] ##STR00056## 35
cyclo-[(3R,4R,5S)-1- benzylamino-3-methyl-5- methylamino-4-hydroxy-
hexanoic acid-Thr-Sar-MeLeu- Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal]
##STR00057## 36 cyclo-[(3R,4R,5S)-1- benzylcarbamoyl-3-methyl-5-
methylamino-4-hydroxy- pentanoic acid-Thr-Sar-MeLeu-
Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal] ##STR00058## 37
cyclo-[(3R,4R,5S)-4-hydroxy-1- isopropylamino-3-methyl-5-
methylamino-hexanoic acid-Thr- Sar-MeLeu-Leu-MeLeu-Ala-D-
Hiv-MeLeu-Leu-MeVal] ##STR00059## 38 cyclo-[(3R,4R,5S)-1-(acetyl-
isopropyl-amino)-4-hydroxy-3- methyl-5-methylamino-hexanoic
acid-Thr-Sar-MeLeu-Leu- MeLeu-Ala-D-Hiv-MeLeu-Leu- MeVal]
##STR00060## 39 cyclo-[(2R,3R,4S)-4-hydroxy-1-
(2-hydroxymethyl-piperidin-1- yl)-3-methyl-5-methylamino- hexanoic
acid-Thr-Sar-MeLeu- Leu-MeLeu-Ala-D-Hiv-MeLeu- Leu-MeVal]
Methods for Preparing Compounds of the Invention
[0257] The skilled person will recognise that compounds of the
invention may be prepared, in known manner, in a variety of ways.
The routes below are merely illustrative of some methods that can
be employed for the synthesis of compounds of formula (1).
[0258] The present invention further provides a process for the
preparation of a compound of formula (1) in which L is
--CH.sub.2--, Q is a primary covalent bond and NR.sub.1R.sub.2 are
as defined in formula (1), by conversion of the alkenyl group
(AXX.sub.1) of Example 1, or a protected derivative thereof, to an
aldehyde by treatment with ozone in a suitable solvent such as
dichloromethane, in the temperature range of -80.degree. C. to
0.degree. C., followed by treatment of the resulting species with
an agent such as dimethylsulfide or triphenylphosphine at a
suitable temperature such as between -80.degree. C. to 40.degree.
C. The aldehyde or protected aldehyde may also be prepared by known
methods such as by treatment of sodium periodate/osmium tetraoxide
in combination with an oxidant such as N-methylmorpholine N-oxide
or hydrogen peroxide in a solvent combinations such as 1,4-dioxane,
tert-butanol and water. The resulting aldehyde (or lactol mixture)
can be converted to the compound of formula (1) by treatment with
an amine of formula HNR.sub.1R.sub.2, wherein R.sub.1 and R.sub.2
are as defined in formula (1), with a reducing agent such as sodium
triacetoxyborohydride or sodium cyanoborohydride, with or without
the presence of an acid such as acetic acid, in a solvent such as
dichloromethane. If necessary protecting groups such as
triethylsilyl groups can be removed with a reagent such as
triethylamine trihydrofluoride.
[0259] Compounds of formula (1) wherein L-Q-NR.sub.1R.sub.2 make up
a group of formula (2)
##STR00061##
may be conveniently prepared by treatment of the aldehyde derived
from Example 1 above, or a protected derivative thereof, with an
alkyl-phosphosnium salt or alkyl organophosphinate ester in
combination with a base such as lithium diisopropylamide, sodium
hexamethyldisilazide, potassium hexamethyldisilazide, butyl
lithium, potassium tert-butoxide, sodium hydride in a solvent such
as THF or toluene at a temperature in the range of -80.degree. C.
to 50.degree. C., preferably -80.degree. C. to room temperature. If
necessary protecting groups such as triethylsilyl groups can be
removed with a reagent such as triethylamine trihydrofluoride.
[0260] Compounds of formula (1) wherein L-Q-NR.sub.1R.sub.2 make up
a group of formula (3)
##STR00062##
may be conveniently prepared by treatment of compounds of formula
(2) as defined above with hydrogen in a solvent such as ethyl
acetate or isopropanol with a catalyst such as 5-10% palladium on
carbon.
[0261] Compounds of formula (1) wherein L-Q-NR.sub.1R.sub.2 make up
a group of formula (4)
##STR00063##
may be conveniently prepared by treatment of the aldehyde derived
from Example 1 above, or a protected derivative thereof, with the
corresponding unsubstituted or substituted diamine in a solvent
such as acetonitrile, with an oxidant such as air or oxygen at a
temperature in the range of 0.degree. C. to 50.degree. C.,
preferably room temperature. If necessary protecting groups such as
triethylsilyl groups can be removed with a reagent such as
triethylamine trihydrofluoride.
[0262] Compounds of formula (1) in which L is a bond, Q is a
carbonyl and NR.sub.1R.sub.2 are as defined in formula (1), by
conversion of the aldehyde derived from Example 1 above, or a
protected derivative thereof, to a carboxylic acid with an oxidant
such as sodium chlorite, in the presence of an alkene such as
2,3-dimethylbut-2-ene, in a solvent such as tetrahydrofuran,
tert-butanol and buffered aqueous at a temperature in the range of
-20.degree. C. to 50.degree. C., preferably room temperature.
Subsequent amide formation using standard conditions such as
(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)met-
haniminium hexafluorophosphate and triethylamine with an amine of
formula HNR.sub.1R.sub.2 in a solvent such as dichloromethane
followed by removal of protecting groups, if required, such as
triethylsilyl groups with a reagent such as triethylamine
trihydrofluoride.
[0263] Compounds of formula (1) wherein L-Q-NR.sub.1R2 make up a
group of formula (5)
##STR00064##
may be conveniently prepared by reaction of a group of formula (1)
wherein L is --CH.sub.2--, Q is a primary covalent bond and
NR.sub.1 are as defined in formula (1) with a group of formula
R.sub.3CO.sub.2H under standard coupling conditions (as above) or
with a group of formula R.sub.3COCl, (R.sub.3CO).sub.2O in a
solvent such as dichloromethane and a base such as triethylamine,
pyridine or diisopropylamine.
[0264] Compounds of the invention include those where X is H, i.e.
the amino acid at position 2 is Abu. Such compounds can be prepared
via deoxygenation of the amino acid threonine at said position to
turn the CH(OH)CH.sub.3 into CH.sub.2CH.sub.3. Thus included within
the scope of the invention is the use of a thionochloroformate to
produce a compound of type C--OC(.dbd.S)OR, which can then be
turned into a C--H analogue. The compounds of the invention can be
produced by reacting phenyl thionochloroformate with the
2-threonine hydroxyl group. The invention as disclosed herein
therefore includes the use of a thionoformate, for example as shown
in example intermediate 13(i):
##STR00065##
in the preparation of compounds as disclosed herein.
Methods for the Preparation of Compounds of the Invention
Example 1: Production of
Cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal) by
Fermentation of Strain NRRL-18230
##STR00066##
[0266] Cylindrotrichum sp. NRRL-18230 was sourced from the culture
collection at the Agricultural Research Service, US Dept of
Agriculture, US, and cultured on malt yeast agar (MYA: 2% malt
extract, 0.4% yeast extract, 2% agar in deionised water) at
22.degree. C. Starting material was generated by suspending ten 0.5
cm.sup.2 plugs taken from the growing edges of a mature agar plate
culture in sterile distilled water (10 ml) containing glass beads
(2.5-3.5 mm diameter, 5 ml) and shaking vigorously to cause
homogenisation. A seed culture was generated by aseptically
inoculating each of three 250 ml conical flasks containing 10 0 ml
malt yeast broth (MYB: 2% malt extract and 0.4% yeast extract in
deionised water at native pH) with 2 ml of the starting material
and culturing at 22.degree. C. and 150 rpm on a rotating shaker.
After 11 days the mycelial pellets from the first seed stage were
macerated with glass beads in distilled water and a second seed
stage was generated by inoculating each of fifteen 250 ml conical
flasks containing 100 ml MYB medium with 10% v/v of the macerated
material and culturing at 22.degree. C. and 150 rpm. After a
further 14 days, a production stage was initiated by inoculating
each of six 5 L Erlenmeyer flasks containing 2.5 L MYB medium with
250 ml of macerated material from mycelia pellets produced from the
second seed stage. The cultures were grown at 100 rpm and
22.degree. C. and harvested after 14 days when titres of
cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal) had
reached a plateaux, as determined by sampling and analysing by
reverse phase HPLC. The harvest biomass was collected by
centrifugation at 3000 rpm for 15 minutes using a Beckman J6B
Centrifuge. The resulting pellet was extracted by homogenising the
biomass with portions of ethyl acetate (3.times.2.5 L) followed by
intermittent stirring over several hours to allow extraction to
occur. This process was similarly repeated with methanol
(2.times.1.5 L). The ethyl acetate and methanol extracts were
separately concentrated by rotary evaporation. The ethyl acetate
extract was defatted by dissolving in acetonitrile (300 ml) and
extracting with n-hexane (2.times.300 ml). The combined hexane
layers were back-extracted with acetonitrile (300 ml) and then the
acetonitrile layers were combined and dried to yield 1.2 g of
acetonitrile-soluble material. The methanol extract was similarly
defatted to yield 2.7 g of acetonitrile-soluble material. The
acetonitrile-soluble samples from the ethyl acetate and methanol
extracts were dissolved and combined in 1:1 n-hexane:ethyl acetate
(10 ml) and purified by column chromatography on silica gel (35-70
.mu.m, column: o 8 cm.times.16 cm) eluting initially with
n-hexane:ethyl acetate (1:1) followed by ethyl acetate and then
ethyl acetate-methanol (98:2 followed by 96:4), with all mobile
phases containing 0.1% formic acid. Fractions found to contain only
the compound of interest, as determined by analysis using reversed
phase HPLC with evaporative light scattering detection, were
combined and concentrated in vacuo to yield pure
Cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(1.248 g).
Example 2: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00067##
[0267] i) Preparation of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]}
##STR00068##
[0269]
Cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(Example 1) (0.124 g, 0.1 mmol) was dissolved in dry
dichloromethane (16 ml) and added to a 3-neck flask equipped with a
glass inlet tube (for nitrogen/ozone addition) with an outlet
connected to a Dreschler bottle containing 2 M potassium iodide
solution. The reaction mixture was cooled to -78.degree. C. using a
solid CO.sub.2/acetone bath under a nitrogen atmosphere. When the
temperature of the reaction vessel had stabilised, ozone was
bubbled through the reaction mixture until it became a pale blue
colour (approx. 3-5 minutes). The ozone supply was removed and dry
nitrogen gas was then bubbled through the reaction mixture until
the blue colour disappeared. Dimethylsulphide (0.038 mL) was then
added, and the reaction mixture was allowed to warm to room
temperature over 3 hours. After this time, the reaction mixture was
washed with brine then dried (Na.sub.2SO.sub.4), filtered and
evaporated under reduced pressure to yield the crude title compound
as a mixture of aldehyde-lactol which was used in the next step
without isolation. ESMS MNa+1257.1, MK+1273.3
Example 2: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
[0270] To a stirred solution of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.123 g, 0.1 mmol) in dry dichloromethane (10 ml) was
added morpholine (0.044 ml, 0.5 mmol), sodium triacetoxyborohydride
(0.089 g, 0.5 mmol) and the reaction mixture was stirred at room
temperature for 18 h. After this time, additional amounts of
morpholine (0.044 ml, 0.5 mmol) and sodium triacetoxyborohydride
(0.089 g, 0.5 mmol) were added and the reaction mixture was stirred
at 40.degree. C. for 4.5 h. After this time, additional amounts of
morpholine (0.025 ml, 0.28 mmol) and sodium triacetoxyborohydride
(0.089 g, 0.5 mmol) were added and the reaction mixture was stirred
at room temperature for a further 23 h. The reaction mixture was
diluted with dichloromethane, washed with a saturated aqueous
solution of sodium bicarbonate, brine, then the organic phase was
dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was
purified by SCX chromatography using a solvent gradient of 100%
ethanol to 0.21 M trimethylamine in ethanol followed by MPLC
chromatography using a solvent gradient of 100% dichloromethane to
92% dichloromethane/8% ethanol to give title compound as a white
solid. ESMS MH+ 1306.6
Example 3: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridi-
n-4-ylmethyl-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00069##
[0272] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and methyl-pyridin-4-ylmethyl-amine to afford
the product (12 mg). ESMS MH+ 1341.6
Example 4: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-2-(pyr-
idin-2-yl)-ethyl-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-Me Val}
##STR00070##
[0274] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and methyl-2-(pyridin-2-yl)-ethyl-amine to
afford the product (22 mg). ESMS MH+ 1355.5
Example 5: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-(2-met-
hyl-2H-pyrazol-3-ylmethyl)-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00071##
[0276] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and
methyl-(2-methyl-2H-pyrazol-3-ylmethyl)-amine to afford the product
(37 mg). ESMS MH+ 1344.6
Example 6: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[methyl-(1H-te-
trazol-5-ylmethyl)-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00072##
[0278] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and methyl-(1H-tetrazol-5-ylmethyl)-amine to
afford the product (10 mg). ESMS MNa+1353.2
Example 7: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic
acid-(O-methyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00073##
[0279] i) Preparation of
Cyclo-(MeBmt-(O-methyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal-
)
##STR00074##
[0281]
Cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(Example 1) (120 mg, 0.1 mmol) was dissolved in dry dichloromethane
(2 mL) at room temperature and under nitrogen. To the reaction
mixture was added powdered 3 .ANG. molecular sieves (120 mg),
trimethyloxonium tetrafluoroborate (142 mg, 0.96 mmol) and Proton
sponge (247 mg, 1.15 mmol). The mixture was stirred for 1.5 hours,
diluted with dichloromethane and washed with 1 M HCl (lx). The
organic layer was dried over a hydrophobic frit and concentrated in
vacuo. The product was purified by silica gel chromatography using
iso-hexanes/acetone (1/1) as eluent to afford the target compound
as viscous clear oil (110 mg, 87%). ESMS MH+ 1261.69
Example 7: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic
acid-(O-methyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
[0282]
Cyclo-(MeBmt-(O-methyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-
-MeVal) (Example 7, step i) (100 mg, 0.08 mmol) was dissolved in
dry dichloromethane (40 mL) and added to a 3-neck flask equipped
with a glass inlet tube (for nitrogen/ozone addition) with an
outlet connected to a Dreschler bottle containing 2 M potassium
iodide solution. The reaction mixture was cooled to -78.degree. C.
using a solid CO.sub.2/acetone bath under a nitrogen atmosphere.
When the temperature of the reaction vessel had stabilised, ozone
was bubbled through the reaction mixture until it became a pale
blue colour (approx. 3-5 minutes). The ozone supply was removed and
dry nitrogen gas was then bubbled through the reaction mixture
until the blue colour disappeared. Dimethylsulfide (0.058 mL, 0.8
mmol) was then added, and the reaction mixture was allowed to warm
to room temperature over 3 hours. After this time the solvent was
removed in vacuo to yield a foam. This was dissolved in dry
dichloromethane (0.5 mL) and was added morpholine (0.069 mL, 0.8
mmol) followed by sodium triacetoxyborohydride (0.168 g, 0.8 mmol)
at room temperature and under nitrogen. The reaction mixture was
stirred at 40.degree. C. for 18 h. After this time, additional
amounts of morpholine (0.035 mL, 0.04 mmol) and sodium
triacetoxyborohydride (0.085 g, 0.4 mmol) were added and the
reaction mixture was stirred at 40.degree. C. for 2 h. After this
time the reaction mixture was diluted with dichloromethane and
quenched with 2 M HCl). The organic layer was separated and washed
with a saturated aqueous solution of sodium bicarbonate. The
organic phase was dried through a hydrophobic frit and evaporated.
The product was purified by silica gel chromatography using
dichloromethane/ethanol (15/1 then 9/1) as eluent to afford the
target compound as a white solid (45 mg, 43%). ESMS MH+ 1320.49
Example 8: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-([1,4]dioxan-2-
-ylmethyl-methyl-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00075##
[0284] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and [1,4]dioxan-2-ylmethyl-methyl-amine to
afford the product (21 mg). ESMS MH+ 1351
Example 9: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[3-trifluorome-
thyl-piperidinyl]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00076##
[0286] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and 3-trifluoromethyl-piperidine to afford the
product (27 mg). ESMS MH+ 1373.1
Example 10: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-[(2-methoxy-et-
hyl)-methyl-amino]-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00077##
[0288] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and (2-methoxy-ethyl)-methyl-amine to afford
the product (19 mg). ESMS MH+ 1308
Example 11: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(3-methoxy-aze-
tidinyl)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00078##
[0290] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and 3-methoxy-azetidine to afford the product
(23 mg). ESMS MH+ 1307.1
Example 12: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-(tetra-
hydro-pyran-4-yl)-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00079##
[0292] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) and methyl-(tetrahydro-pyran-4-yl)-amine to
afford the product (26 mg). ESMS MH+ 1334.7
Example 13: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00080##
[0293] i) Preparation of
Cyclo-[MeBmt-(O-phenoxythiocarbonyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-Me-
Leu-Leu-MeVal]
##STR00081##
[0295] To a stirred solution of
cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(Example 1) (187 mg, 0.15 mmol) in dichloromethane (1 mL) was added
pyridine (1 mL) and DMAP (0.2 g, 1.6 mmol). Phenyl
thionochloroformate (0.44 mL, 3.2 mmol) was added dropwise. The
mixture was left to stir at room temperature for 18 h. The reaction
mixture was treated with cold 0.1 M hydrochloric acid and extracted
with dichloromethane. Combined organic layers were dried over
sodium sulphate, filtered and the filtrate was evaporated to give
an oily residue. It was purified by silica gel chromatography
eluting with isopropyl alcohol in dichloromethane (0-10%) to afford
product (130 mg). ESMS MH+ 1383
ii) Preparation of
Cyclo-(MeBmt-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
##STR00082##
[0297] To a solution of
cyclo-[MeBmt-(O-phenoxythiocarbonyl-Thr)-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-Me-
Leu-Leu-MeVal] (Example 13, step i) (130 mg, 0.09 mmol) in
anhydrous toluene (1 mL) was added tributyltin hydride (82 mg, 0.28
mmol) and AIBN (5 mg). The reaction vial was capped and the
contents were purged with nitrogen. It was heated at 100.degree. C.
for 6 h. The reaction mixture was purified on a silica gel column
by eluting it with isopropyl alcohol in dichloromethane (0-10%) to
afford the product (81 mg). ESMS MH+ 1231
iii) Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00083##
[0299] The titled compound was prepared by the method of Example 2
(Step i) using
cyclo-(MeBmt-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(Example 13, step ii) to afford the product (93 mg) as an
aldehyde-lactol mixture. ESMS MH+ 1201 (M.sup.+-H.sub.2O)
Example 13: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-morpholino-hex-
anoic acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
[0300] The titled compound was prepared by the method of Example 2
(Step ii) using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-h-
exanoic acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 13, step iii) and morpholine to afford the product (28
mg). ESMS MH+ 1290.5
Example 14: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00084##
[0302] To a solution of
(6-methyl-pyridin-2-ylmethyl)-triphenyl-phosphonium bromide (131
mg, 0.29 mmol) in anhydrous THF (1 mL) at -78.degree. C. under
nitrogen, was added a 0.6 M solution of NaHMDS in toluene (0.4 mL).
The mixture was left to stir from -78.degree. C. to -30.degree. C.
over 40 min. It was re-cooled to -78.degree. C. A solution of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 2,
step i) (60 mg, 49 .mu.mol) in anhydrous THF (1 mL) was added
dropwise. The resulting mixture was left to stir at room
temperature for 72 h. It was treated with saturated ammonium
chloride and extracted with dichloromethane. The organic layer was
collected and evaporated. The crude oily residue was eluted on a
silica gel column (10 g SiO2) with isopropyl alcohol in
dichloromethane (0-15%) to give the titled compound (19 mg). ESMS
MH+ 1324
Example 15: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(pyrimidin-2-yl)-
-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00085##
[0304] The titled compound was prepared by the method of Example 14
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) and triphenyl-pyrimidin-2-ylmethyl-phosphonium bromide
to afford the product (56 mg). ESMS MH+ 1311
Example 16: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(pyrimidin-2-yl)-
-heptanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00086##
[0306] A solution of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(pyrimidin-2-yl)-
-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example
15) (49 mg, 37 .mu.mol) in ethyl acetate (20 mL) was passed through
a 10% palladium on activated carbon cartridge on an H-Cube.RTM. at
1 mL/min at room temperature. The solution was evaporated and dried
under reduced pressure to afford the product as colourless solid
(27 mg). ESMS MH+ 1313.6
Example 17: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-heptanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00087##
[0308] The titled compound was prepared by the method of Example 16
using
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example
14) to afford the product (30 mg). ESMS MH+ 1326.7
Example 18: Preparation of
cyclo-{(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-morpholin-4-y-
l-pyridin-2-yl)-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal}
##STR00088##
[0310] The titled compound was prepared by the method of Example 14
using
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 2,
step i) and
(6-morpholin-4-yl-pyridin-2-ylmethyl)-triphenyl-phosphonium bromide
to afford the product (47 mg). ESMS MH+ 1395
Example 19: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-hept-1-enoic
acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00089##
[0312] The titled compound was prepared by the method of Example 14
using
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example
13, Step iii) and
(6-methyl-pyridin-2-ylmethyl)-triphenyl-phosphonium bromide to
afford the product (7 mg). ESMS MH+ 1308.8
Example 20: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-morpholin-4-y-
l-pyridin-2-yl)-heptanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00090##
[0314] The titled compound was prepared by the method of Example 16
using
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-morpholin-4-y-
l-pyridin-2-yl)-hept-1-enoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example
18).
to afford the product (33 mg). ESMS MH+ 1397
Example 21: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-heptanoic
acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00091##
[0316] The titled compound was prepared by the method of Example 16
using
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(6-methyl-pyridi-
n-2-yl)-hept-1-enoic
acid-Abu-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example
19) to afford the product (5.3 mg). ESMS MH+ 1310.7
Example 22: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(8-oxa-3-aza-b-
icyclo[3.2.1]octane)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00092##
[0318] Dry dichloromethane (10 mL) was added to a mixture of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.080 g, 0.065 mmol) and
8-oxa-3-aza-bicyclo[3.2.1]octane (0.097 g, 0.65 mmol). The mixture
was stirred at room temperature for 10 min, and then the solvent
was evaporated (repeated twice). Dry dichloromethane (10 mL) was
added and followed by triethylamine (0.090 mL, 0.65 mmol), and
sodium triacetoxyborohydride (0.137 g, 0.65 mmol). The reaction
mixture was stirred at 40.degree. C. for 18 h. The reaction mixture
was diluted with dichloromethane, washed with a saturated aqueous
solution of sodium bicarbonate, and then the organic phase was
separated and evaporated. The residue was purified by SCX
chromatography using a solvent gradient of 100% ethanol to 2 M
trimethylamine in ethanol followed by MPLC chromatography using a
solvent gradient of 100% dichloromethane to 85% dichloromethane/15%
ethanol to give 37 mg of title compound as a white solid. ESMS MH+
1332.5
Example 23: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(thiomorpholin-
e 1,1-dioxide)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00093##
[0320] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.070 g, 0.057 mmol) and thiomorpholine 1,1-dioxide
(0.077 g, 0.57 mmol) to afford the product (11 mg). ESMS MH+
1355.4
Example 24: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(4,4-difluoro--
piperidine)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00094##
[0322] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.070 g, 0.057 mmol) and 4,4-difluoro-piperidine (0.089
g, 0.57 mmol) to afford the product (40 mg). ESMS MH+ 1340.6
Example 25: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-((4-fluoro-pip-
eridin-4-yl)-methanol)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00095##
[0324] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.070 g, 0.057 mmol) and
(4-fluoro-piperidin-4-yl)-methanol (0.096 g, 0.57 mmol) to afford
the product (37 mg). ESMS MH+ 1353.8
Example 26: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--((S)-1-Pyrrol-
idin-2-yl-methanol)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00096##
[0326] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.060 g, 0.049 mmol) and (S)-1-Pyrrolidin-2-yl-methanol
(0.049 g, 0.49 mmol) to afford the product (44 mg). ESMS MH+
1320.5
Example 27: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(3-methylamino-
-propionitrile)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00097##
[0328] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.060 g, 0.049 mmol) and 3-methylamino-propionitrile
(0.041 g, 0.49 mmol) to afford the product (41 mg). ESMS MH+
1303.6
Example 28: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridi-
n-2-yl-amine)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00098##
[0330] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.050 g, 0.040 mmol) and methyl-pyridin-2-yl-amine
(0.044 g, 0.40 mmol) to afford the product (17 mg). ESMS MH+
1327.6
Example 29: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--((R)-3-Methyl-
-morpholine)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00099##
[0332] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.050 g, 0.040 mmol) and (R)-3-methyl-morpholine (0.041
g, 0.40 mmol) to afford the product (31 mg). ESMS MH+ 1320.7
Example 30: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(bis-pyridin-2-
-ylmethyl-amine)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00100##
[0334] The titled compound was prepared by the method of Example 22
using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (0.058 g, 0.047 mmol) and bis-pyridin-2-ylmethyl-amine
(0.094 g, 0.47 mmol) to afford the product (16 mg). ESMS MH+
1418.5
Example 31: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N-(methyl-pyridi-
n-2-ylmethyl-amino)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00101##
[0336] The titled compound was prepared by the method of Example 2,
step ii using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-he-
xanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) (0.132 g, 0.108 mmol) and
methyl-pyridin-2-ylmethyl-amine (0.092 g, 0.753 mmol) to afford the
product (20 mg). ESMS MH+ 1341.6
Example 32: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-N--(N'-methylpip-
erazine)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00102##
[0338] The titled compound was prepared by the method of Example 2,
step ii using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-he-
xanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) (0.070 g, 0.057 mmol) and N-methyl piperazine
(0.040 g, 0.397 mmol) to afford the product (27 mg). ESMS MH+
1317.3
Example 33: Preparation of
cyclo-[(2R,3R,4S)-1-(1H-benzoimidazol-2-yl)-2-methyl-4-methylamino-3-hydr-
oxy-pentanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00103##
[0339] i) Preparation of
cyclo-[(5R,6R,7S)-5-methyl-7-methylamino-6-triethylsilanyloxy-oct-2-enoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00104##
[0341] To a stirred solution of
cyclo-(MeBmt-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal)
(Example 1) (1.17 g, 940 .mu.mol) in dry dichloromethane (12 mL),
was added triethylamine (261 .mu.L, 1.9 mmol) and triethylsilyl
trifluoromethanesulfonate (424 .mu.L, 1.9 mmol), and the reaction
was stirred for 16 hours. After this time, additional triethylamine
(523 .mu.L, 3.8 mmol) and triethylsilyl trifluoromethanesulfonate
(848 .mu.L, 3.8 mmol) and the reaction was stirred for 4 hours.
After this time, water (10 mL) was added and the layers were
separated using a phase-sep cartridge. The organics were
concentrated under reduced pressure, and purified by MPLC using a
solvent gradient of 100% isohexane to 40% acetone/60% isohexane, to
give the title compound. ESMS MH+ 1476.3 .sup.1H NMR (300 MHz,
CDCl.sub.3) gave 4 characteristic NH amide peaks at 8.10 (d, 1H),
7.96 (d, 1H), 6.97 (d, 1H), 6.85 (d, 1H)
ii) Preparation of
cyclo[(3R,4R,5S)-4-hydroxy-3-methyl-5-methylamino-1-oxo-hexanoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00105##
[0343] The product obtained in
cyclo-[(5R,6R,7S)-5-methyl-7-methylamino-6-triethylsilanyloxy-oct-2-enoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 33,
step i) (500 mg, 339 .mu.mol) was dissolved in dry dichloromethane
(50 ml) and added to a 3-neck flask equipped with a glass inlet
tube (for nitrogen/ozone addition) with an outlet connected to a
Dreschler bottle containing 2 M potassium iodide solution. The
reaction mixture was cooled to -78.degree. C. using a solid
CO.sub.2/acetone bath under a nitrogen atmosphere. When the
temperature of the reaction vessel had stabilised, ozone was
bubbled through the reaction mixture until it became a pale blue
colour (approx. 3-5 minutes). The ozone supply was removed and dry
nitrogen gas was then bubbled through the reaction mixture until
the blue colour disappeared. Dimethylsulphide (99 .mu.L, 1.4 mmol)
was then added, and the reaction mixture was allowed to warm to
room temperature over 3 hours. After this time, the reaction
mixture was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and evaporated under reduced pressure to yield the title
compound. ESMS (M+-TES) 1350.0
[0344] .sup.1H NMR (300 MHz, CDCl.sub.3) gave 4 characteristic NH
amide peaks at 8.10 (d, 1H), 7.91 (d, 1H), 7.03 (d, 1H), 6.84 (d,
1H) along with a characteristic aldehyde peak at 9.76 (s, 1H)
iii) Preparation of
cyclo-[(2R,3R,4S)-1-(1H-benzoimidazol-2-yl)-2-methyl-4-methylamino-3-trie-
thylsilanyloxy-pentanoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00106##
[0346] To a stirred solution of the product obtained in
cyclo[(3R,4R,5S)-4-hydroxy-3-methyl-5-methylamino-1-oxo-hexanoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 33,
step ii) (150 mg, 102 mol) in acetonitrile (3 mL) was added
1,2-benzenediamine (11 mg, 102 .mu.mol), and the reaction was
stirred for 168 hours at room temperature while a stream of
compressed air was bubbled through. The reaction was concentrated
under reduced pressure, and the residue was purified by MPLC using
a solvent gradient of 100% dichloromethane to 10% isopropanol/90%
dichloromethane, to give the title compound. ESMS (M+H) 1552.8
Example 33: Preparation of
cyclo-[(2R,3R,4S)-1-(1H-benzoimidazol-2-yl)-2-methyl-4-methylamino-3-hydr-
oxy-pentanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
[0347] To a stirred solution of the product obtained in
cyclo-[(2R,3R,4S)-1-(1H-benzoimidazol-2-yl)-2-methyl-4-methylamino-3-trie-
thylsilanyloxy-pentanoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal](Example 33,
step iii) (49 mg, 32 .mu.mol) in acetonitrile (1 mL) was added
triethylamine trihydrofluoride (51 .mu.L, 320 .mu.mol), and the
reaction was stirred at room temperature for 16 hours. The reaction
was concentrated under reduced pressure, and the residue was
purified by MPLC using a solvent gradient of 100% dichloromethane
to 20% isopropanol/80% dichloromethane, to give the title compound.
ESMS (M+H) 1323.8
Example 34: Preparation of
cyclo-[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-(4-phenyl-1-pipe-
ridyl)-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00107##
[0349] To a stirred solution of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (50 mg, 41 .mu.mol) in dry dichloromethane (1 mL) was
added 4-phenylpiperidine (26 mg, 162 .mu.mol), sodium
triacetoxyborohydride (51 mg, 243 .mu.mol) and the reaction mixture
was stirred at room temperature for 16 h. The reaction mixture was
diluted with dichloromethane, washed with a saturated aqueous
solution of sodium bicarbonate, brine and the organic phase was
evaporated. The residue was purified by MPLC using a solvent
gradient of 100% dichloromethane to 90% dichloromethane/10%
isopropanol to give the title compound. ESMS MH+ 1380.5
Example 35: Preparation of
cyclo-[(3R,4R,5S)-1-benzylamino-3-methyl-5-methylamino-4-hydroxy-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00108##
[0351] To a stirred solution of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (120 mg, 82 .mu.mol) in dry dichloromethane (1 ml) was
added benzylamine (53 .mu.L, 492 .mu.mol), sodium
triacetoxyborohydride (104 mg, 492 .mu.mol) and the reaction
mixture was stirred at room temperature for 16 h. The reaction
mixture was diluted with dichloromethane, washed with a saturated
aqueous solution of sodium bicarbonate, brine, then the organic
phase was evaporated. The residue was purified by MPLC using a
solvent gradient of 100% dichloromethane to 90% dichloromethane/10%
isopropanol to give a crude intermediate that was dissolved in
acetonitrile (1 mL) and treated with triethylamine trihydrofluoride
(77 .mu.L, 472 .mu.mol), and stirred for 16 hours at room
temperature. The reaction was concentrated under reduced pressure
and purified by MPLC using a solvent gradient of 100%
dichloromethane to 80% dichloromethane/20% isopropanol to give the
title compound. ESMS (M+H) 1326.8
Example 36: Preparation of
cyclo-[(3R,4R,5S)-1-benzylcarbamoyl-3-methyl-5-methylamino-4-hydroxy-pent-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00109##
[0352] i) Preparation of
cyclo-[(3R,4R,5S)-3-methyl-5-methylamino-4-triethylsilanyloxy-hexanedioic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00110##
[0353] To a stirred solution of
cyclo[(3R,4R,5S)-4-hydroxy-3-methyl-5-methylamino-1-oxo-hexanoic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 33,
step ii) (200 mg, 140 .mu.mol) in tetrahydrofuran (1 mL) and
tert-butanol (1 mL), was added 2,3-dimethylbut-2-ene (81 .mu.L, 680
.mu.mol), followed by a solution of dibasic sodium hydrogen
phosphate (58 mg, 410 .mu.mol) in water (0.5 mL). A solution of
sodium chlorite (37 mg, 410 .mu.mol) in water (0.5 mL) was then
added and the reaction was stirred for 4 hours at room temperature.
The reaction was diluted with water (10 mL) and the organics were
extracted into tert-butylmethylether (3.times.5 mL). The combined
organics were dried using a phase-sep cartridge, and concentrated
under reduced pressure to give the title compound. ESMS (M+-TES)
1382.8 .sup.1H NMR (300 MHz, CDCl.sub.3) gave 4 characteristic NH
amide peaks at 8.08 (d, 1H), 7.91 (d, 1H), 7.02 (d, 1H), 6.83 (d,
1H)
ii) Preparation of
cyclo-[(3R,4R,5S)-1-benzylcarbamoyl-3-methyl-5-methylamino-4-triethylsila-
nyloxy-pentanoic acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00111##
[0355] To a stirred solution of
cyclo-[(3R,4R,5S)-3-methyl-5-methylamino-4-triethylsilanyloxy-hexanedioic
acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 36,
step i) (100 mg, 68 .mu.mol) in dichloromethane (1 mL), was added
triethylamine (18 .mu.L, 135 .mu.mol), benzylamine (15 .mu.L, 135
.mu.mol) and
(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)met-
haniminium hexafluorophosphate (26 mg, 68 .mu.mol), the reaction
was then stirred for 16 hours. The reaction was diluted with water
(5 mL) and the organics separated using a phase-sep cartridge. The
aqueous was re-extracted with dichloromethane (4 mL) and passed
through a phase-sep cartridge. The combined organics were
concentrated under reduced pressure to give the title compound as a
crude intermediate which was carried forward. ESMS (M-TES+NH.sub.4)
1473.0
Example 36: Preparation of
cyclo-[(3R,4R,5S)-1-benzylcarbamoyl-3-methyl-5-methylamino-4-hydroxy-pent-
anoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
[0356] To a stirred solution of the crude
cyclo-[(3R,4R,5S)-1-benzylcarbamoyl-3-methyl-5-methylamino-4-triethylsila-
nyloxy-pentanoic acid-(2S,3S)-2-amino-3-triethylsilanyloxy-butyric
acid-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] (Example 36,
step ii) (100 mg, 64 .mu.mol) in acetonitrile (1 mL) was added
triethylamine trihydrofluoride (52 .mu.L, 320 .mu.mol) and the
reaction was stirred for 16 hours. Additional triethylamine
trihydrofluoride (52 .mu.L, 320 .mu.mol) was added and the reaction
stirred for an additional 24 hours. Additional triethylamine
trihydrofluoride (105 .mu.L, 640 .mu.mol) was added and the
reaction was stirred for an additional 72 hours. The reaction was
concentrated under reduced pressure, and purified by MPLC eluting
with 100% dichloromethane to 12% isopropanol/88% dichloromethane to
give the title compound. ESMS (M+H) 1340.6
Example 37: Preparation of
cyclo-[(3R,4R,5S)-4-hydroxy-1-isopropylamino-3-methyl-5-methylamino-hexan-
oic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00112##
[0358] To a stirred solution of
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] } (Example
2, step i) (240 mg, 194 .mu.mol) in dry dichloromethane (2 ml) was
added isopropylamine (69 mg, 1.2 mmol), sodium
triacetoxyborohydride (250 mg, 1.2 mmol) and the reaction mixture
was stirred at room temperature for 72 h. The reaction mixture was
diluted with dichloromethane, washed with a saturated aqueous
solution of sodium bicarbonate, brine and then the organic phase
was evaporated. The residue was purified by SCX chromatography
using a solvent gradient of 100% ethanol to 0.21M trimethylamine in
ethanol to give the title compound. ESMS (M+H) 1278.6
Example 38: Preparation of
cyclo-[(3R,4R,5S)-1-(acetyl-isopropyl-amino)-4-hydroxy-3-methyl-5-methyla-
mino-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00113##
[0360] To a stirred solution of
cyclo-[(3R,4R,5S)-4-hydroxy-1-isopropylamino-3-methyl-5-methylamino-hexan-
oic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal](Example
37) (81 mg, 63 .mu.mol) in dichloromethane (2 mL) was added
triethylamine (53 .mu.L, 39 mg, 380 .mu.mol) followed by acetyl
chloride (27 .mu.L, 30 mg, 380 .mu.mol), and the reaction mixture
was stirred at room temperature for 1 h. The reaction mixture was
concentrated under reduced pressure and then partitioned between
dichloromethane (2 mL) and water (2 mL). The organics were
separated using a hydrophobic frit, and purified by SCX
chromatography using 100% ethanol to give the title compound. ESMS
(M+H) 1320.7
Example 39: Preparation of
cyclo-[(2R,3R,4S)-4-hydroxy-1-(2-hydroxymethyl-piperidin-1-yl)-3-methyl-5-
-methylamino-hexanoic
acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal]
##STR00114##
[0362] The titled compound was prepared by the method of Example 2,
step ii using
cyclo-{[(3R,4R,5S)-4-(hydroxy)-3-methyl-5-(methylamino)-1-oxo-he-
xanoic acid-Thr-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal] }
(Example 2, step i) (0.070 g, 0.057 mmol) and 2-piperidinemethanol
(0.039 g, 0.340 mmol) to afford the product (27 mg). ESMS MH+
1334.8
Biological and Physical Properties
[0363] Example Compounds are listed and described in Table 1,
below.
[0364] Data showing cyclophilin A (Cyp A) inhibitory activity,
cyclophilin D (Cyp D) inhibitory activity, immunosuppressive
potential, and aqueous solubility for selected compounds are
described in Table 1. General procedures and protocols of assays
used to obtain the data are given below.
[0365] The data shows that cycloundecadepsipeptide compounds as
described herein, are potent inhibitors of cyclophilin A and
cyclophilin D (Ki<300 nM), as measured by the protease-free
PPIase assay; have significantly less immunosuppressive activity
than the compound of Example 1, as measured by the CaN assay in the
presence of Cyp-A; and have water solubility at least equal to or
greater than that of Example 1, as measured by the water solubility
assay.
TABLE-US-00002 TABLE 1 Solubility Compound CypA Ki nM CypD Ki nM
CaN + CypA nM ( M) Compound 1.7 3.2 70 10-25 of formula A (Example
1) Example 2 9.2 6.5 >1,000 >100 Example 3 +++ +++ -- 50-75
Example 4 +++ +++ -- 25-50 Example 5 +++ +++ -- 50-75 Example 6 +++
+++ -- 25-50 Example 7 ++ +++ -- 50-75 Example 8 ++ +++ -- >100
Example 9 +++ +++ -- 10-25 Example 10 ++ +++ -- >100 Example 11
+++ +++ -- >100 Example 12 ++ +++ -- >100 Example 13 +++ +++
-- 25-50 Example 14 +++ +++ -- 10-25 Example 15 +++ +++ >1,000
10-25 Example 16 +++ +++ -- 25-50 Example 17 +++ +++ >1,000
10-25 Example 18 +++ +++ -- 10-25 Example 19 +++ +++ -- 10-25
Example 20 +++ +++ >5,000 10-25 Example 21 +++ +++ -- 10-25
Example 22 +++ +++ >10,000 25-50 Example 23 +++ +++ >10,000
75-100 Example 24 +++ +++ -- 25-50 Example 25 + + -- >100
Example 26 + ++ -- >100 Example 27 ++ +++ -- <100 Example 28
+++ +++ -- 25-50 Example 29 ++ ++ -- >100 Example 30 ++ +++ --
10-25 Example 31 ++ +++ -- 50-75 Example 32 + ++ -- -- Example 33
+++ +++ -- 25-50 Example 34 +++ +++ -- 10-25 Example 35 ++ +++ --
25-50 Example 36 +++ +++ -- 25-50 Example 37 ++ +++ -- >100
Example 38 ++ +++ -- 50-75 Example 39 + ++ >100 Results of CypA
and CypD screening assay of Example compounds. + CypA/D inhibition
Ki between 100 nM to 300 nM, ++ CypA/D inhibition Ki between 25 nM
to 100 nM +++ CypA/D inhibition Ki between 1 nM to 25 nM
General Procedures and Assays
*Protease-Free PPIase Assay
[0366] The protease-free PPIase assay measures the rate of cis to
trans conversion of a peptide substrate catalyzed by the enzymes
cyclophilin A and cyclophilin D. Addition of a cyclophilin
inhibitor (e.g., a test compound) slows the catalyzed rate and a
K.sub.i value is obtained. A K.sub.i value of less than 30 nM
demonstrates that the test compound is a potent inhibitor of
cyclophilin A or cyclophilin D.
Materials
[0367] Assay Buffer:
[0368] 35 mM HEPES pH 7.8, filtered through a 0.2 m filter. 50
.mu.M DTT was added prior to use each day and then the buffer was
stored on ice. [0369] Enzyme: Human recombinant cyclophilin A (Cyp
A) (Sigma C3805) enzyme was diluted to 1 .mu.M with enzyme dilution
buffer (20 mM HEPES pH 7.8, 40% glycerol, 50 .mu.M DTT and 1 .mu.M
BSA) and stored at -20 OC. [0370] Substrate:
Succinimide-Ala-Ala-Pro-Phe-p-nitroanailide (SUC-AAPF-pNA) (from
Bachem AG, L-1400), 20 mg/ml prepared in 0.5 M LiCl in
trifluoroethanol.
Method
[0371] All readings were taken with an Agilent 8453
Spectrophotometer which consists of a cuvette holder, stirrer and
chiller to maintain a stirred cuvette temperature of
10.0.+-.0.1.degree. C. The temperature is monitored by the use of
temperature probe. To prevent UV degradation of test compounds, the
light below 290 nm was blocked using a glass slide in the light
path. 1.5 ml of assay buffer was put into a 3 ml quartz cuvette and
cooled to 10.0.+-.0.1.degree. C. while stirring (vigorous but not
so fast as to produce cavitation). The inhibitor was diluted in
100% DMSO, and then added to the assay to a maximum final
concentration of 0.5% DMSO in the assay. A blank spectrum was
obtained, then 3 .mu.L of enzyme was added (2 nM final
concentration) and then 3 .mu.L substrate (60 NM final
concentration) added. The absorbance was measured at 330 nm for 300
s or 500 s for blank runs (NOTE: the substrate must be added in one
quick injection and the measurements started immediately to
minimize mixing errors).
[0372] A first order rate equation was fitted to the absorbance
data, for each concentration of inhibitor, to obtain the rate
constant (the first 10 to 15 seconds were excluded as mixing causes
errors in this portion of curve). The catalytic rate was calculated
from the enzymatic rate constant minus the background rate
constant. An exponential curve was generated using the catalytic
rate constants versus the inhibitor concentration to obtain the
K.sub.i value for the inhibitor. The K.sub.i value is indicative of
the binding affinity between the test compound and cyclophilin A or
cyclophilin D.
**Calcineurin Phosphatase (CaN) Assay
[0373] The calcineurin phosphatase assay is a means for estimating
the immunosuppressive potential of a test compound. Calcineurin is
a serine-threonine protein phosphatase that on activation
dephosphorylates members of the nuclear factor of activated T cells
(NFAT), which are important in T lymphocyte activation. Some
cyclophilin A inhibitors, such as cyclosporin A (CsA) or compound
1, when bound to cyclophilin A (Cyp A) markedly inhibit calcineurin
activity, resulting in very significant immunosuppressive effects.
Alternatively, in the presence of cyclophilin A, some cyclophilin A
inhibitors show reduced calcineurin inhibition and thus less
immunosuppression or do not inhibit calcineurin activity at all and
show no immunosuppressive effects.
[0374] To investigate the immunosuppressive potential of exemplary
compounds of Formula 1, which are novel cycloundecadepsipeptide
compounds, their ability to inhibit calcineurin activity was
measured in the presence of Cyp A.
[0375] The CaN assay kit used is based on a colorimetric assay for
measuring calcineurin phosphatase activity, and it is commercially
available (Enzo Life Sciences and Calbiochem). Calmodulin is also
required for calcineurin activity and RII phosphopeptide is used as
an efficient peptide substrate for calcineurin. We have modified
the method to enable measurement of Cyp A-dependent and Cyp
A-independent inhibition of calcineurin through the addition of Cyp
A in a 1:1 complex with the inhibitor. The detection of free
phosphate released is based on the classic Malachite green
assay.
Materials:
[0376] Enzo Life Sciences CaN Assay Kit: BML-AK804 [0377] 2.times.
assay buffer: 100 mM Tris, pH7.5, 200 mM NaCl, 12 mM MgCl.sub.2, 1
mM DTT, 0.05% NP-40, 1 mM CaCl.sub.2 [0378] Malachite Green: BIOMOL
Green.TM. reagent [0379] Calmodulin (Human, recombinant): was
thawed on ice, diluted 1:50 with 2.times. assay buffer, and then
stored on ice. [0380] Calcineurin: was thawed quickly, stored on
ice immediately, diluted 1:12.5 with 1.times. assay buffer, and
then stored on ice. [0381] R-II Substrate: 915 .mu.L ultrapure
water (UPW) was added to the 1.5 mg vial substrate to give a final
concentration of 0.75 mM. [0382] Inhibitors: 2.5 mM inhibitor in
100% DMSO. [0383] Cyp A: recombinant human CypA (Sigma C3805), 1
mg/ml; Recombinant 6-his tagged CypA prepared by the Univ. of
Edinburgh was also used. Comparison of the results showed that both
enzymes gave identical results.
Method
[0384] Inhibitor dilutions: inhibitor compounds were diluted in UPW
in polypropylene low-binding 96 well plates at 5.times. the final
assay concentration. For samples `without Cyp A`, a 4-point
dilution series of the inhibitor was prepared in duplicate to
obtain a final assay concentration of 10, 1, 0.1 and 0.01 .mu.M.
For samples `with Cyp A`, a 7-point dilution series was prepared to
obtain a 1:1 complex of the inhibitor with CypA; the inhibitor and
Cyp A final assay concentrations of 10, 3.33, 1.11, 0.37, 0.12,
0.04, 0.014 .mu.M were prepared. Cs A inhibitor controls were also
prepared to obtain a final concentration of 10 .mu.M Cs A with and
without M Cyp A.
[0385] Assay Setup: using the half area 96 well plates supplied
with the kit, 10 .mu.l UPW was added to duplicate wells to provide
the non-inhibited control. 10 .mu.l of the inhibitor or the
inhibitor/Cyp A complex was added to the appropriate sample wells.
25 .mu.l of the 2.times. assay buffer with CaM was added to all
wells, then 5 .mu.l of CaN was added to all wells (40 U per well
final concentration) except duplicate `no calcineurin blank` wells
to which 5 .mu.L 1.times. assay buffer was added. The assay plate
was placed in an oven at 30.degree. C. for 15 minutes to
equilibrate to the reaction temperature. The reaction was started
by the addition of 10 .mu.l RII-peptide (0.15 mM final
concentration). The reaction was allowed to proceed at 30.degree.
C. for a time period in which the reaction is linear for about 60
minutes. The reaction was then terminated by adding 100 .mu.l of
the Malachite Green reagent. The color was allowed to develop for
15-30 minutes at room temperature before the absorbance at 620 nm
was measured using a plate reader (Molecular Devices--SpectraMax
M5). The data were analyzed by subtracting `no Calcineurin blank`
from all the absorbance readings and plotting the background
corrected absorbances against Log.sub.10 inhibitor concentration. A
sigmoidal-dose response curve was fitted to the data using GraphPad
Prism Software.
[0386] The compound 1 is a potent inhibitor of calcineurin activity
and therefore a potent immunosuppressive. It exerts its
immunosuppressive activity by binding to cyclophilin A to form a
complex, which then binds to calcineurin and thereby inhibits
calcineurin activity. As shown in table 1, compound 1 has an
IC.sub.50 value of 70 nM in the calcineurin/cyclophilin A assay.
Thus, compounds with values higher than 70 nM in this assay will be
predictably less immunosuppressive than the compound of example 1.
As can be seen from table 1, cycloundecadepsipeptide compound 2
produces significantly higher values than 70 nM in this assay and
therefore are significantly less immunosuppressive than compound
1.
***Water Solubility Assay (Measured in pH 7.8 Buffer)
[0387] The aqueous solubility of compounds 1 and 2 in buffer (pH
7.8) was measured by recording the onset of precipitation of the
compounds as a function of increasing concentration. The onset of
precipitation, if it occurred, was detected by an increase in
absorbance at 650 nm.
Materials
[0388] Assay Buffer: 35 mM HEPES pH 7.8
[0389] Stock solutions of Control and Test Compounds: 10 mM in 100%
DMSO
Method
[0390] 10 mM stock solutions of control and test compounds were
prepared in 100% DMSO. A series of dilutions were prepared from the
stock in DMSO so that the final concentrations in the assay were 0,
3.33, 10, 25, 50, 75 and 100 .mu.M and DMSO was limited to 1%.
[0391] Assay buffer (247.5 .mu.l) was placed into flat bottomed
transparent 96-well plate. For blank samples DMSO (2.5 .mu.l) was
added. For test and control samples 2.5 .mu.l of the appropriate
DMSO dilution stocks were added to the appropriate well. All test
and control compounds were performed in triplicate.
[0392] The plates were sealed with adhesive plate seal and shaken
at 250 rpm at 25.degree. C. for 18 h on a plate shaker.
[0393] After incubation the plate seals were taken off and any
bubbles observed in wells removed. The plates were read on a
SpectraMaxM5 with a 5 s pre-shake at 650 nm.
[0394] Data files were transferred to the appropriate worksheet and
the solubility range of the compounds was calculated from the
data.
[0395] The values shown in the tables indicate the concentration in
M (micromolar) at which the compound remains in solution.
[0396] As shown in table 1, the compound of example 1 has an onset
of precipitation between 10-25 M concentration of water. Compounds
measured to precipitate at higher concentrations of water than
10-25 .mu.M thus have better aqueous solubility (e.g. the compound
of example 2).
Rat Pharmacokinetic Data
[0397] Rat pharmacokinetic data was obtained for example 2,
following iv dosing at 1 mg/kg and po dosing at 10 mg/kg and
individual analysis of plasma and whole blood samples at various
time points was carried out.
Whole Blood
[0398] Following intravenous administration of the compound of
example 2 at a nominal dose of 1 mg/kg, the mean value of systemic
clearance was 1.16 L/hr/kg, which corresponded to 35.0% of hepatic
blood flow in rats (3.31 L/hr/kg). The mean half-life (T.sub.1/2)
was 6.14 hr.
[0399] The mean value of C.sub.max (at 5 minutes after dosing)
following IV administration at a nominal dose of 1 mg/kg was 643.18
.mu.g/L. The mean values of AUC.sub.(0-4) and AUC.sub.(0-.infin.)
were 826.92 and 858.75 hr*g/L.
[0400] The mean volume of distribution at the terminal phase was
10.32 L/kg, which corresponded to 15.40-fold of the total body
water (0.67 L/kg) in rats.
[0401] Following oral administration of the compound of example 2
at a dose of 10 mg/kg, the mean values of C.sub.max and T.sub.max
were 678.45 ng/mL and 2.00 hr; the mean values of AUC.sub.(0-t) and
AUC.sub.(0-.infin.) were 5934.43 and 6021.60 ng/mL*hr; the mean
value of half-life (T.sub.1/2) was 4.03 hr. The mean value of
bioavailability was 70.12%.
Plasma
[0402] Following intravenous administration of example 2 at a
nominal dose of 1 mg/kg, the mean value of systemic clearance was
4.18 L/hr/kg, which corresponded to 1.26-fold of hepatic blood flow
in rats (3.31 L/hr/kg). The mean half-life (T.sub.1/2) was 4.96
hr.
[0403] The mean value of C.sub.max (at 5 minutes after dosing)
following IV administration at a nominal dose of 1 mg/kg was 274.94
.mu.g/L. The mean values of AUC.sub.(0-t) and AUC.sub.(0-.infin.)
were 197.06 and 238.98 hr*g/L.
[0404] The mean volume of distribution at the terminal phase was
29.94 L/kg, which corresponded to 44.69-fold of the total body
water (0.67 L/kg) in rats.
[0405] Following oral administration of example 2 at a dose of 10
mg/kg, the mean values of C.sub.max and T.sub.max were 328.35 ng/mL
and 2.00 hr; the mean values of AUC.sub.(0-t) and
AUC.sub.(0-.infin.) were 2245.93 and 2291.04 ng/mL*hr; the mean
value of half-life (T.sub.1/2) was 4.40 hr. The mean value of
bioavailability was 95.87%.
[0406] In whole blood, the mean volume of distribution at the
terminal phase was 10.32 L/kg whereas in plasma the mean volume of
distribution at the terminal phase was 29.94 L/kg, indicating only
a .about.3-fold difference in blood to plasma ratio. This is
significantly less than the 10-40 fold blood to plasma ratio
typically observed with cyclophilin inhibitors such as BC556
(Gregory M A et al, Poster presented at EASL Meeting, Barcelona,
April 2012). This data suggests that compound 2 and other compounds
of Formula 1 may have improved whole animal tissue distribution
over many other cyclophilin inhibitors.
* * * * *