U.S. patent application number 17/004434 was filed with the patent office on 2020-12-17 for novel rapamycin derivatives.
The applicant listed for this patent is NOVARTIS AG. Invention is credited to Simone BONAZZI, Michael CONNOLLY, David Jonathan GLASS, Manuel MIHALIC, Andrew William PATTERSON, Silvio ROGGO, Tea SHAVLAKADZE.
Application Number | 20200392159 17/004434 |
Document ID | / |
Family ID | 1000005051749 |
Filed Date | 2020-12-17 |
View All Diagrams
United States Patent
Application |
20200392159 |
Kind Code |
A1 |
BONAZZI; Simone ; et
al. |
December 17, 2020 |
NOVEL RAPAMYCIN DERIVATIVES
Abstract
The disclosure relates to compounds of formula (I) ##STR00001##
and pharmaceutically acceptable salts, and compositions thereof,
wherein the substituents are as defined herein. Also provided are
methods of making compounds of formula (I), and methods involving
the compounds or compositions for treating disorders and diseases
described herein.
Inventors: |
BONAZZI; Simone; (Cambridge,
MA) ; CONNOLLY; Michael; (Salem, MA) ; GLASS;
David Jonathan; (Cortland Manor, NY) ; MIHALIC;
Manuel; (Grenzach-Wyhlen, DE) ; PATTERSON; Andrew
William; (Somerville, MA) ; ROGGO; Silvio;
(Muttenz, CH) ; SHAVLAKADZE; Tea; (Hawthorne,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVARTIS AG |
Basel |
|
CH |
|
|
Family ID: |
1000005051749 |
Appl. No.: |
17/004434 |
Filed: |
August 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16141856 |
Sep 25, 2018 |
10800793 |
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17004434 |
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62563312 |
Sep 26, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 13/12 20180101; A61P 29/00 20180101; A61P 19/00 20180101; C07F
9/6561 20130101; A61P 25/02 20180101; A61P 19/10 20180101; A61P
9/10 20180101; A61P 1/16 20180101; A61P 11/00 20180101; A61P 9/12
20180101; A61P 37/00 20180101; A61P 3/10 20180101; A61P 31/10
20180101; A61P 25/16 20180101; A61P 35/00 20180101; C07D 498/18
20130101; A61P 3/04 20180101; A61P 25/28 20180101 |
International
Class: |
C07D 498/18 20060101
C07D498/18; A61P 1/16 20060101 A61P001/16; A61P 13/12 20060101
A61P013/12; A61P 35/00 20060101 A61P035/00; A61P 25/16 20060101
A61P025/16; A61P 37/00 20060101 A61P037/00; A61P 9/10 20060101
A61P009/10; A61P 19/10 20060101 A61P019/10; A61P 19/00 20060101
A61P019/00; A61P 29/00 20060101 A61P029/00; A61P 31/10 20060101
A61P031/10; A61P 19/02 20060101 A61P019/02; A61P 25/28 20060101
A61P025/28; A61P 3/10 20060101 A61P003/10; A61P 9/12 20060101
A61P009/12; A61P 11/00 20060101 A61P011/00; A61P 3/04 20060101
A61P003/04; C07F 9/6561 20060101 C07F009/6561; A61P 25/02 20060101
A61P025/02 |
Claims
1. A compound of formula (I) or a pharmaceutically acceptable salt
thereof, wherein ##STR00088## R.sub.1 is selected from the group
consisting of hydroxy, ##STR00089## and R.sub.2 is selected from
the group consisting of ##STR00090## wherein m is 0, 1, 2 or 3; n
is 1, 2 or 3; o is 1, 2, 3, 4, 5 or 6; p is 1, 2, 3, 4 or 5 q is 1,
2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4, 5 or 6; r is 2,
3 or 4; s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6; X
is O, S, NR.sub.6 or SO.sub.2; R.sub.3 is hydrogen, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkyl, C.sub.3-6cycloalkylC.sub.0-6alkyl or
phenylC.sub.0-6alkyl; R.sub.4 is hydrogen; R.sub.5 is hydrogen,
hydroxy or cyano; or R.sub.4 and R.sub.5 together form .dbd.O; and
R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-6cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
2. The compound of claim 1 or a pharmaceutically acceptable salt
thereof, wherein R.sub.1 is selected from the group consisting of
hydroxy, ##STR00091## and R.sub.2 is selected from the group
consisting of ##STR00092## wherein m is 0, 1, 2 or 3; n is 1, 2 or
3; o is 1, 2, 3, 4, 5 or 6; p is 1, 2, 3, 4 or 5 q is 1, 2, 3, 4 or
5 wherein the sum of p and q is 2, 3, 4, 5 or 6; r is 2, 3 or 4; s
is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6; X is O, S,
NR.sub.6 or SO.sub.2; R.sub.3 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl; R.sub.4
is hydrogen; R.sub.5 is hydrogen, hydroxy or cyano; or R.sub.4 and
R.sub.5 together form .dbd.O; and R.sub.6 is hydrogen,
C.sub.1-6alkyl, C.sub.3-8cycloalkylC.sub.0-6alkyl,
phenylC.sub.0-6alkyl, C.sub.1-6alkyl-CO--,
C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--, C.sub.1-6alkyl-SO.sub.2--
or C.sub.3-6cycloalkylC.sub.0-6alkyl-SO.sub.2--.
3. The compound according to claim 1 or 2 or a pharmaceutically
acceptable salt thereof, wherein R.sub.1 is hydroxy.
4. The compound according to any one of claims 1 to 3 or a
pharmaceutically acceptable salt thereof, wherein R.sub.2 is
##STR00093## and n is 1, 2 or 3.
5. The compound according to any one of claims 1 to 4 or a
pharmaceutically acceptable salt thereof, wherein R.sub.2 is
##STR00094##
6. The compound according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein said compound is selected from:
TABLE-US-00005 Structure ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103## ##STR00104## ##STR00105## ##STR00106##
7. The compound according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein said compound is ##STR00107##
8. The compound according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein said compound is ##STR00108##
9. The compound according to any one of claims 1 to 5 or a
pharmaceutically acceptable salt thereof, wherein said compound is
##STR00109##
10. A pharmaceutical composition comprising a therapeutically
effective amount of a compound according to any one of claims 1 to
9 or a pharmaceutically acceptable salt thereof and one or more
pharmaceutically acceptable carriers.
11. A pharmaceutical combination comprising a therapeutically
effective amount of a compound according to any one of claims 1 to
9 or a pharmaceutically acceptable salt thereof and one or more
therapeutically active agents.
12. A method of treating a disorder or a disease mediated by the
mTOR pathway in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a compound according to any one of claims 1 to 9 or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition of claim 10, or the pharmaceutical combination of claim
11.
13. A method of treating a disease or disorder in a subject,
wherein the target tissue, cell or organ associated with the
pathology of the disease or disorder has FKBP12 levels sufficient
to inhibit mTORC1, the method comprising administering to the
subject in need thereof a therapeutically effective amount of a
compound according to any one of claims 1 to 9 or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition of claim 10, or the pharmaceutical combination of claim
11.
14. A method of treating a disease or disorder in a subject having,
or previously determined as having, FKBP12 levels sufficient to
inhibit mTORC1, the method comprising administering to the subject
in need thereof a therapeutically effective amount of a compound
according to any one of claims 1 to 9 or a pharmaceutically
acceptable salt thereof, or the pharmaceutical composition of claim
10, or the pharmaceutical combination of claim 11.
15. The method of any one of claims 12 to 14, wherein the disease
or disorder is selected from sarcopenia, skin atrophy, cherry
angiomas, seborrheic keratoses, brain atrophy, atherosclerosis,
arteriosclerosis, pulmonary emphysema, osteoporosis,
osteoarthritis, high blood pressure, erectile dysfunction,
cataracts, macular degeneration, glaucoma, stroke, cerebrovascular
disease (strokes), chronic kidney disease, diabetes-associated
kidney disease, impaired hepatic function, liver fibrosis,
autoimmune hepatitis, endometrial hyperplasia, metabolic
dysfunction, renovascular disease, hearing loss, mobility
disability, cognitive decline, tendon stiffness, heart dysfunction
such as cardiac hypertrophy and/or systolic and/or diastolic
dysfunction and/or hypertension, heart dysfunction which results in
a decline in ejection fraction, immune senescence, Parkinson's
disease, Alzheimer's disease, cancer, immune-senescence leading to
cancer due to a decrease in immune-surveillance, infections due to
an decline in immune-function, chronic obstructive pulmonary
disease (COPD), obesity, loss of taste, loss of olfaction,
arthritis, and type II diabetes including complications stemming
from diabetes, such as kidney failure, blindness and
neuropathy.
16. The method of any one of claims 12 to 14, wherein the disorder
is liver fibrosis.
17. A method of treating a disease or disorder in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of a compound according to any one
of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or
the pharmaceutical composition of claim 10, or the pharmaceutical
combination of claim 11, wherein the disorder or disease is
selected from: Acute or chronic organ or tissue transplant
rejection; Transplant vasculopathies; Smooth muscle cell
proliferation and migration leading to vessel intimal thickening,
blood vessel obstruction, obstructive coronary atherosclerosis,
restenosis; Autoimmune diseases and inflammatory conditions;
Treatment and prevention of asthma; Multi-drug resistance (MDR);
Fungal infections; Inflammation; Infection; Age-related diseases;
Neurodegenerative diseases; Proliferative disorders, in particular
cancer; Seizures and seizure related disorders; and Mitochondrial
myopathy and mitochondrial stress.
18. The method of claim 17, wherein the disorder is a disorder that
includes the process of fibrosis and/or inflammation.
19. The method of claim 18, wherein the disorder is selected from
liver and kidney disorders.
20. The method of claim 19, wherein the liver disorder is selected
from: liver fibrosis, which occurs in end-stage liver disease;
liver cirrhosis; liver failure due to toxicity;
non-alcohol-associated hepatic steatosis or NASH; and
alcohol-associated steatosis.
21. The method of claim 19, wherein the kidney disorder is kidney
fibrosis.
22. The method of claim 21, wherein the kidney fibrosis occurs as a
result of acute kidney injury.
23. The method of claim 19, wherein the kidney disorder is chronic
kidney disorder.
24. The method of claim 19, wherein the kidney disorder is diabetic
nephropathy.
25. A method of treating an age-related disorder or disease in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of a compound according
to any one of claims 1 to 9 or a pharmaceutically acceptable salt
thereof, or the pharmaceutical composition of claim 10, or the
pharmaceutical combination of claim 11, wherein the disorder or
disease is selected from: sarcopenia, skin atrophy, cherry
angiomas, seborrheic keratoses, brain atrophy, atherosclerosis,
arteriosclerosis, pulmonary emphysema, osteoporosis,
osteoarthritis, high blood pressure, erectile dysfunction,
cataracts, macular degeneration, glaucoma, stroke, cerebrovascular
disease (strokes), chronic kidney disease, diabetes-associated
kidney disease, impaired hepatic function, liver fibrosis,
autoimmune hepatitis, endometrial hyperplasia, metabolic
dysfunction, renovascular disease, hearing loss, mobility
disability, cognitive decline, tendon stiffness, heart dysfunction
such as cardiac hypertrophy and/or systolic and/or diastolic
dysfunction and/or hypertension, heart dysfunction which results in
a decline in ejection fraction, immune senescence, Parkinson's
disease, Alzheimer's disease, cancer, immune-senescence leading to
cancer due to a decrease in immune-surveillance, infections due to
an decline in immune-function, chronic obstructive pulmonary
disease (COPD), obesity, loss of taste, loss of olfaction,
arthritis, and type II diabetes including complications stemming
from diabetes, such as kidney failure, blindness and
neuropathy.
26. A method of treating cancer in a subject, the method comprising
administering to the subject a therapeutically effective amount of
a compound according to any one of claims 1 to 9 or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition of claim 10, or the pharmaceutical combination of claim
11.
27. The method of claim 26, further comprising a PD-1/PDL-1
inhibitor.
28. The method of claim 26 or 27, wherein the cancer is selected
from renal cancer, renal cell carcinoma, colorectal cancer, uterine
sarcoma, endometrial uterine cancer, endometrial cancer, breast
cancer, ovarian cancer, cervical cancer, gastric cancer,
fibro-sarcoma, pancreatic cancer, liver cancer, melanoma, leukemia,
multiple myeloma, nasopharyngeal cancer, prostate cancer, lung
cancer, glioblastoma, bladder cancer, mesothelioma, head cancer,
rhabdomyosarcoma, sarcoma, lymphoma, and neck cancer.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/141,856 filed 25 Sep. 2018, which claims
priority to U.S. Patent Application No. 62/563,312 filed 26 Sep.
2017, each of which is incorporated herein by reference in its
entirety.
FIELD
[0002] The invention provides 32-deoxo-rapamycin derivatives, and
relates to their methods of use.
BACKGROUND
[0003] In mammalian cells, the target of rapamycin (mTOR) kinase
exists in two distinct multiprotein complexes, described as the
mTORC1 complex and the mTORC2 complex, both of which sense the
availability of nutrients and energy, and integrate inputs from
growth factors and stress signaling. The mTORC1 integrates signals
from growth factors and nutrients and controls cell growth and
metabolism. Laplante M. et al. Cell. (2012) 149(2):274-93. mTORC1
is a key regulator of protein translation and autophagy. The mTORC1
complex is sensitive to allosteric mTOR inhibitors such as
rapamycin and rapamycin analogs (so called `rapalogs`). Rapamycin
and previously-produced rapalogs' mode of action involves the
formation of an intracellular complex with FK506 binding proteins,
either FKBP12, FKBP51 or FKBP52 (these three FKBPs will be
referenced here as "FKBP" or "FKBPs"), followed by the binding of
the FKBP-rapalog complex to the FRB (FK506-rapamycin binding)
domain of mTOR. Marz A. M. et al. Mol Cell Biol. (2013)
33(7):1357-1367. Large FK506-Binding Proteins Shape the
Pharmacology of RapamycinSuch interaction of the FKBP-rapalog
complex with mTORC1 results in allosteric inhibition of the
complex. Rapamycin and rapalogs, such as RAD001 (everolimus;
Afinitor.RTM.), have gained clinical relevance by inhibiting the
activity of mTORC1, which is associated with both benign and
malignant proliferation disorders. Royce M. E. et al. Breast Cancer
(Auckl). (2015) 9:73-79; Pleniceanu O. et al. Kidney Int Rep.
(2018) 3(1):155-159.
[0004] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygoscopius, see e.g. McAlpine, J. B., et al., J.
Antibiotics (1991) 44:688; Schreiber, S. L.; et al., J. Am. Chem.
Soc. (1991) 113:7433; U.S. Pat. No. 3,929,992. The following
numbering convention for rapamycin and its derivatives used in this
document is shown below:
##STR00002##
[0005] Rapamycin is a potent immunosuppressant and has also been
shown to have antitumor and antifungal activity. It has been shown
to be useful in preventing or treating systemic lupus
erythematosus, pulmonary inflammation, insulin-dependent diabetes
mellitus, skin disorders such as psoriasis, smooth muscle cell
proliferation and intimal thickening following vascular injury,
adult T-cell leukemia/lymphoma, malignant carcinomas, cardiac
inflammatory disease, anemia and increased neurite outgrowth. Its
utility as a pharmaceutical, however, is restricted by its very low
and variable bioavailability. Moreover, Rapamycin is challenging to
formulate, making it difficult to obtain stable galenic
compositions.
[0006] To overcome these problems, numerous rapalogs have been
(semi)synthesized. Water soluble prodrugs have been prepared by
derivatizing rapamycin at C28 and 40 to form glycinate, propionate
and pyrrolidino butyrate prodrugs (U.S. Pat. No. 4,650,803). Other
analogs of rapamycin include monoacyl and diacyl analogs (U.S. Pat.
No. 4,316,885), acetal analogs (U.S. Pat. No. 5,151,413) silyl
ethers (U.S. Pat. No. 5,120,842), hydroxy esters (U.S. Pat. No.
5,362,718) as well as aryl, alkyl, alkenyl, and alkynyl analogs
(U.S. Pat. Nos. 5,665,772; 5,258,389; 6,384,046; WO97/35575).
Modifications to rapamycin include the demethylation, elimination
or replacement of one or more of the methoxy groups; elimination,
derivatization or replacement of one or more of the hydroxy
moieties; reduction, elimination or derivatization of one or more
of the ketone moieties; replacement of the 6-membered pipecolate
ring with a 5-membered prolyl ring; alternative substitution on the
cyclohexyl ring with a substituted cyclopentyl ring. Illustrative
examples of the patent literature in this field are U.S. Pat. No.
5,527,907, WO96/41865 and WO99/36553 describing a wide variety of
rapalogs with the aim to avoid the immunosuppressive side effects
of rapamycin. U.S. Pat. No. 5,985,890 discloses examples of 32
deoxo-rapamycin analogs including 32-deoxo-rapamycin itself. It is
described that these compounds have been reported to have an
improved pharmacologic profile over rapamycin and greater
stability.
[0007] The rapalogs described in the literature above have been
disclosed to be useful for the treatment of the same disorders as
rapamycin. U.S. Pat. Nos. 8,906,374 and 9,669,032 disclose the use
in cancer. U.S. Pat. No. 9,358,236 discloses the use in
neurodegenerative disorders.
[0008] In animal models, rapalogs extend lifespan and delay the
onset of age-related diseases. Aging, like other biological
processes, is regulated by signaling pathways such as the TOR
pathway (named "TOR" in this case, to include the yeast and C
elegans systems, where the mammalian equivalent [mTOR] is simply
called "TOR"]) and, in mammals, the mTORC1 pathway. Modulation of
TOR and mTORC1 signaling prolongs lifespan and delays the onset of
age-related diseases in a wide array of organisms, from flies to
mammals. For instance, inhibition of the TOR pathway by genetic
mutation extended lifespan in yeast, C elegans, and drosophila, and
inhibition of the mTORC1 pathway extended lifespan in mice
(Kaeberlein et al., Science (2005) 310:1193-1196; Kapahi et al.,
Curr Biol (2004) 14:885-890; Selman et al., Science (2009)
326:140-144; Vellai et al., Nature (2003) 426:620). In addition,
the mTORC1 inhibitor rapamycin extended the lifespan of mice even
when given late in life (Harrison et al., Nature (2009)
460(7253):392-395). These data raise the possibility that drugs
that target the mammalian TOR (mTOR) pathway will have therapeutic
effects in aging and age-related diseases in humans. For instance,
WO2008/022256 describes methods and topical formulations comprising
an mTOR inhibitor for treating or preventing an age-related
disease. A report of a clinical trial using rapamycin in elderly
men was described by M. Leslie in Science, 2013, 342. J. Mannick et
al. describe in Sci Transl Med. (2014) 6(268): 268ra179 that mTOR
inhibition improves the immune function in the elderly. However,
investigators have been wary of using currently available mTOR
inhibitors in human aging trials due to their side effects
(including immunosuppression, cytopenias, stomatitis, GI distress
and interstitial pneumonitis).
[0009] In animal and human studies of Focal Cortical Dysplasia
(FCD) and Tuberous Sclerosis Complex (TSC), the mTOR pathway is
implicated in mediating the cellular and molecular changes leading
to the formation of the cortical malformations and the expression
of epilepsy (Wong et al., Experimental Neurology (2013) 244:
22-26). Focal Cortical Dysplasia (FCD) is a malformation of
cortical development, which is the most common cause of refractory
epilepsy in the pediatric population and the second/third most
common etiology of medically intractable seizures in adults (Kabat
J, et al., Pol J. Radiology (2012) 77(2) 35-43). Mutations in the
tuberous sclerosis complex (TSC), including tuberous sclerosis
complex-1 (TSC1) and tuberous sclerosis complex-2 (TSC2), act
upstream of the mTOR pathway, leading to a wide spread development
of benign tumors, mental retardation, and a high incidence of
epilepsy (Manning et al., Identification of the tuberous sclerosis
complex-2 tumor suppressor gene product tuberin as a target of the
phosphoinositide 3-kinase/akt pathway, Mol. Cell, (2002) 10:
151-162; Inoki et al., Dysregulation of the TSC-mTOR pathway in
human Disease, Nat. Genet, (2005), 37:19-24; and Holmes and
Stafstrom, Tuberous sclerosis complex and epilepsy: recent
developments and future Challenges, Epilepsia, (2007)
48:617-630).
[0010] Aberrant mTOR activation interferes with normal brain
development and leads to epilepsy. Rapamycin treatment, which
inhibits the mTORC1 pathway, is shown to attenuate structural
abnormalities and reduce seizures in TSC and PTEN mouse models
(Ehninger et al., Reversal of learning deficits in a Tsc2+/- mouse
model of tuberous sclerosis; Nat. Med., (2008), 843-848; Meikle et
al., Response of a neuronal model of tuberous sclerosis to
mammalian target of rapamycin, mTOR inhibitors: effects on mTORC1
and Akt signaling lead to improved survival and function; J.
Neuroscience., (2008) 28:5422-5432; Zeng et al., Rapamycin prevents
epilepsy in a mouse model of tuberous sclerosis complex; Ann.
Neurol., (2008) 63:444-453; Ljungberg et al., Rapamycin suppresses
seizures and neuronal hypertrophy in a mouse model of cortical
dysplasia; (2009) pp. 389-398; and Zhou et al., Pharmacological
inhibition of mTORC1 suppresses anatomical, cellular, and
behavioral abnormalities in neural-specific Pten knock-out mice, J.
Neurosci., (2009), 29:1773-1783). Further, pharmacological
inhibition of the mTOR pathway, either before or immediately
following neurological insults, can prevent pathological changes in
animal brains and the development of spontaneous recurrent seizure
in an acquired epilepsy model (Zeng et al., The mammalian target of
rapamycin signaling pathway mediates epileptogenesis in a model of
temporal lobe epilepsy; J. Neurosci., (2009) pp. 6964-6972).
Rapamycin and rapalogs are therefore also considered to be of
potential value in such indications.
[0011] Mitochondrial myopathy (MM) is the most common manifestation
of adult-onset mitochondrial disease and shows a multifaceted
tissue-specific stress response: (1) transcriptional response,
including metabolic cytokines FGF21 and GDF15; (2) remodeling of
one-carbon metabolism; and (3) the mitochondrial unfolded protein
response. In Cell Metabolism 26, 419-428, Aug. 1, 2017, it is
described by Khan et al. that these processes are part of one
integrated mitochondrial stress response (ISRmt), which is
controlled by mTORC1 in skeletal muscle. A mtDNA replication defect
activates mTORC1, which drives an integrated mitochondrial stress
response through ATF4 activation, inducing de novo nucleotide and
serine synthesis, the 1 C-cycle, and FGF21 and GDF15 production.
mTORC1 inhibition by rapamycin downregulated all components of
ISRmt (the integrated mitochondrial stress response), improved all
MM hallmarks, and reversed the progression of even late-stage MM,
without inducing mitochondrial biogenesis. Rapamycin and rapalogs
are therefore also considered to be of potential value in such
indications.
[0012] There remains a need to provide new mTOR inhibitors that are
good drug candidates. In particular, preferred compounds should
have at least mTORC1 inhibitory capacity, and be well absorbed from
the gastrointestinal tract, be sufficiently metabolically stable
and possess favorable pharmacokinetic properties. Furthermore, the
ideal drug candidate will be able to exist in a physical form that
is stable, non-hygroscopic and appropriate for formulation.
SUMMARY
[0013] The compounds of structural formula (I) are mTORC1
inhibitors and are therefore potentially useful in the treatment of
a wide range of disorders, particularly age-related disorders, or
diseases and disorders currently approved for treatment using
rapalogs. Full reduction of the ketone at C32 and the replacement
of the C16 methoxy group by a cyclic N-containing aliphatic ring
system, such as a cyclic amine, amide or sultam, provides compounds
with the above-mentioned desired advantages, exhibiting a balance
of good potency, stability and bioavailability.
[0014] In one aspect, the disclosure provides compounds of the
structural formula (I):
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein
[0015] R.sub.1 is selected from the group consisting of
hydroxy,
##STR00004##
and
[0016] R.sub.2 is selected from the group consisting of
##STR00005##
wherein
[0017] m is 0, 1, 2 or 3;
[0018] n is 1, 2 or 3;
[0019] o is 1, 2, 3, 4, 5 or 6;
[0020] p is 1, 2, 3, 4 or 5
[0021] q is 1, 2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4,
5 or 6;
[0022] r is 2, 3 or 4;
[0023] s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6;
[0024] X is O, S, NR.sub.6 or SO.sub.2;
[0025] R.sub.3 is hydrogen, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl;
[0026] R.sub.4 is hydrogen;
[0027] R.sub.5 is hydrogen, hydroxy or cyano; or R.sub.4 and
R.sub.5 together form .dbd.O; and
[0028] R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
[0029] In an embodiment, the disclosure provides a pharmaceutical
composition comprising a therapeutically effective amount of a
compound of structural formula (I) or a pharmaceutically acceptable
salt thereof and one or more pharmaceutically acceptable
carriers.
[0030] In an embodiment, the disclosure provides a pharmaceutical
combination comprising a therapeutically effective amount of a
compound of structural formula (I) or a pharmaceutically acceptable
salt thereof and one or more therapeutically active agents.
[0031] In another aspect, the disclosure provides a method of
treating a disorder or a disease mediated by the mTOR pathway in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of a compound of
structural formula (I), or a pharmaceutical composition, or a
pharmaceutical combination thereof. In another aspect, the
disclosure provides a method of treating a disease or disorder in a
subject, wherein the target tissue or organ associated with the
pathology of the disease or disorder has FKBP12 levels sufficient
to inhibit mTORC1, the method comprising administering to the
subject in need thereof a therapeutically effective amount of a
compound of structural formula (I) or a pharmaceutically acceptable
salt thereof, or a pharmaceutical composition, or a pharmaceutical
combination thereof.
[0032] In an embodiment, the target tissue or organ associated with
the pathology of the disease or disorder to be treated with a
compound of structural formula (I) has FKBP12 levels sufficient to
inhibit mTORC1 are determined empirically, e.g., using an
FKBP12-specific inhibitor in comparison to rapamycin or RAD001.
[0033] In another aspect, the disclosure provides a method of
treating a disease or disorder in a subject having, or previously
determined as having, FKBP12 levels sufficient to inhibit mTORC1,
the method comprising administering to the subject in need thereof
a therapeutically effective amount of a compound of structural
formula (I) or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition thereof, or a pharmaceutical combination
thereof.
[0034] In an embodiment, the subject has, or is previously
determined to have, FKBP12 levels in the target tissue, organ or
cells sufficient to inhibit mTORC1.
[0035] In another aspect, the disclosure provides a method of
treating an age-related disease or disorder in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of a compound of structural
formula (I) or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition, or a combination thereof,
[0036] In an embodiment, the disease or disorder is selected from
sarcopenia, skin atrophy, cherry angiomas, seborrheic keratoses,
brain atrophy--also referred to as dementia, atherosclerosis,
arteriosclerosis, pulmonary emphysema, osteoporosis,
osteoarthritis, high blood pressure, erectile dysfunction,
cataracts, macular degeneration, glaucoma, stroke, cerebrovascular
disease (strokes), chronic kidney disease, diabetes-associated
kidney disease, impaired hepatic function, liver fibrosis,
autoimmune hepatitis, endometrial hyperplasia, metabolic
dysfunction, renovascular disease, hearing loss, mobility
disability (e.g., frailty), cognitive decline, tendon stiffness,
heart dysfunction such as cardiac hypertrophy and/or systolic
and/or diastolic dysfunction and/or hypertension, heart dysfunction
which results in a decline in ejection fraction, immune senescence,
Parkinson's disease, Alzheimer's disease, cancer, immune-senescence
leading to cancer due to a decrease in immune-surveillance,
infections due to an decline in immune-function, chronic
obstructive pulmonary disease (COPD), obesity, loss of taste, loss
of olfaction, arthritis, and type II diabetes including
complications stemming from diabetes, such as kidney failure,
blindness and neuropathy.
[0037] In another aspect, the disclosure provides a method of
treating a disease or disorder in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a compound of structural formula (I) or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition, or a pharmaceutical combination thereof, wherein the
disorder or disease is selected from: [0038] Acute or chronic organ
or tissue transplant rejection; [0039] Transplant vasculopathies;
[0040] Smooth muscle cell proliferation and migration leading to
vessel intimal thickening, blood vessel obstruction, obstructive
coronary atherosclerosis, restenosis; [0041] Autoimmune diseases
and inflammatory conditions; [0042] Treatment and prevention of
asthma; [0043] Multi-drug resistance (MDR); [0044] Fungal
infections; [0045] Inflammation; [0046] Infection; [0047]
Age-related diseases; [0048] Neurodegenerative diseases; [0049]
Proliferative disorders, in particular cancer; [0050] Seizures and
seizure related disorders; and [0051] Mitochondrial myopathy and
mitochondrial stress.
[0052] In another aspect, the disclosure provides a method of
treating cancer in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a compound of structural formula (I) or a pharmaceutically
acceptable salt thereof, or a pharmaceutical composition, or a
pharmaceutical combination thereof.
[0053] In an embodiment, the method further comprises a PD-1/PDL-1
inhibitor.
[0054] In an embodiment, the cancer is selected from renal cancer,
renal cell carcinoma, colorectal cancer, uterine sarcoma,
endometrial uterine cancer, endometrial cancer, breast cancer,
ovarian cancer, cervical cancer, gastric cancer, fibro-sarcoma,
pancreatic cancer, liver cancer, melanoma, leukemia, multiple
myeloma, nasopharyngeal cancer, prostate cancer, lung cancer,
glioblastoma, bladder cancer, mesothelioma, head cancer,
rhabdomyosarcoma, sarcoma, lymphoma, and neck cancer.
[0055] In an embodiment, the disorder is a liver disorder that
includes the process of fibrosis and/or inflammation, e.g., liver
fibrosis that occurs in end-stage liver disease; liver cirrhosis;
liver failure due to toxicity; non-alcohol-associated hepatic
steatosis or NASH; and alcohol-associated steatosis.
[0056] In an embodiment, the disorder is a kidney disorder that
includes the process of fibrosis or inflammation in the kidney,
e.g., kidney fibrosis, which occurs as a result of acute kidney
injury, leading to chronic kidney disease and diabetic
nephropathy.
[0057] In an embodiment, the disorder is a heart dysfunction, e.g.,
myocardial infarction or cardiac hypertrophy. In an embodiment, the
heart dysfunction is systolic and/or diastolic dysfunction. In an
embodiment, the heart dysfunction is hypertension. In an
embodiment, the heart dysfunction results in a decline in ejection
fraction.
[0058] In an embodiment, the disorder is an immune-senescence
leading to cancer due to a decrease in immune-surveillance.
[0059] In an embodiment, the disorder is cancer, including tumors
which are treated by immunotherapy, and those which have been
previously treated by either rapamycin, or Everolimus or another
rapalog. In an embodiment, the cancer includes tumors where the
mTOR pathway is shown to be activated, including settings where
there is a mutation in the Tsc1 gene, or where the tumor
microenvironment is appropriately treated by a rapalog.
[0060] The details of one or more embodiments of the disclosure are
set forth herein. Other features, objects, and advantages of the
disclosure will be apparent from the Figures, the Detailed
Description, the Examples, and the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1A is an image of a co-crystal structure of Example 1
with FKBP12. The absolute configuration at C16 is (S).
[0062] FIG. 1B shows the structure of Example 1 with the FKBP12
protein removed.
[0063] FIG. 2A is an image of a co-crystal structure of Example 2
with FKBP12. The absolute configuration at C16 is (R).
[0064] FIG. 2B shows the structure of Example 2 with the FKBP12
protein removed.
[0065] FIG. 3 is a line graph showing the pharmacokinetic profile
of Example 2 in rats following a single p.o. dose of 3 mg/kg. Y
axis--blood concentrations of Example 2 (nM). X axis--time (hours)
for blood collection following Example 2 administration. Data are
mean.+-.standard deviation from 3 rats.
[0066] FIG. 4A is a bar graph showing comparative blood
concentrations of RAD001 (open bars) and Example 2 (solid bars) in
rats orally given a single dose of either compound at 3, 10 and 30
mg/kg. Compound concentrations were measured 3 and 24 hours (h)
following dosing. Data are mean.+-.standard deviation from 5-6 rats
in each group. Rats where a compound was below quantification limit
were excluded from data analysis: this applies to RAD001 treated
groups. Asterisk (*) indicates significant difference between
respective RAD001 and Example 2 treated groups. ** P<0.01; ***
P<0.001; ****P<0.0001, t-tests. BQL--below quantification
limit. Y axis--compound concentrations in blood (nM). X axis--time
(3 and 24 hours) following oral dosing and orally given doses (3,
10 and 30 mg/kg).
[0067] FIG. 4B is a bar graph showing comparative brain
concentrations of RAD001 (open bars) and Example 2 (solid bars) in
rats orally given as a single dose of either compound at 3, 10 and
30 mg/kg. Compound concentrations were measured 3 and 24 hours (h)
following dosing. Data are mean.+-.standard deviation. 5-6 rats
were used in each group. Rats where a compound was below
quantification limit were excluded from data analysis: this applies
to RAD001 treated groups. Asterisk (*) indicates significant
difference between respective RAD001 and Example 2 treated groups.
** P<0.01; *** P<0.001; ****P<0.0001, t-tests. BQL--below
quantification limit. Y axis--compound concentrations in brain
(nM). X axis--time (3 and 24 hours) following dosing and orally
given doses (3, 10 and 30 mg/kg).
[0068] FIG. 4C shows blood concentration of Example 2 in rats
following intra venous (i.v.) and oral (p.o.) dosing. Y axis--blood
concentrations of Example 2 (nM). X axis--time (hours) for blood
collection following Example 2 administration. Data are
mean.+-.standard deviation from 3 rats.
[0069] FIG. 4D shows blood concentration of RAD001 in rats
following intra venous (i.v.) and oral (p.o) dosing. Y axis--blood
concentrations of RAD001 (nM). X axis--time (hours) for blood
collection following RAD001 administration. Data are
mean.+-.standard deviation from 3 rats.
[0070] FIGS. 5A-5D show that Example 2 inhibits the mTORC1 pathway
in the rat liver. Rats were given a single oral dose of Example 2
at 3 or 10 or 30 mg/kg, and liver samples were collected at 3 hours
(h) and 24 h following dosing. Rats treated with a vehicle (Veh)
were used as a control. (5A) and (5C) show immunoblot images of
phosphorylated (p-) and total (t-) S6 proteins in rat livers
treated with a vehicle or 3 or 10 or 30 mg/kg of Example 2 and
analyzed at 3 h (5A) and 24 h (5C) following treatment. Histograms
in (5B) and (5D) show densitometric quantification of p-S6 to t-S6
at 3 h (5B) and 24 h (5D) following treatment. In histograms (5B
and 5D), average arbitrary values that indicate p-S6/t-S6 ratios
are shown above each bar. X axes represent orally given doses (3,
10, 30 mg/kg). Y-axes represent arbitrary units. Six rats were used
in each experimental group. Data are mean.+-.standard deviation.
Data were analyzed with a one way ANOVA followed by Dunnett's
multiple comparison tests, where means from all groups were
compared to the vehicle treated group. ** P<0.01, ****
P<0.001, ns--not significant.
[0071] FIG. 6A-6C shows inhibition of S6K1 (Thr389) in wild-type
(6A), FKBP12 knock-down (6B) and FKBP12 knock-out (6C) 293T cells,
following treatment with RAD001 (dotted line) and Example 2 (solid
line). Cells were treated in triplicate. The Y axis represents
percent inhibition relative to the S6K1 (Thr389) level in cells
treated with media plus DMSO. The X axis represents concentrations
for RAD001 and Example 2.
DETAILED DESCRIPTION
[0072] In a first aspect, the disclosure provides a compound of the
formula (I)
##STR00006##
or a pharmaceutically acceptable salt thereof, wherein
[0073] R.sub.1 is selected from the group consisting of
hydroxy,
##STR00007##
and
[0074] R.sub.2 is selected from the group consisting of
##STR00008##
wherein
[0075] m is 0, 1, 2 or 3;
[0076] n is 1, 2 or 3;
[0077] o is 1, 2, 3, 4, 5 or 6;
[0078] p is 1, 2, 3, 4 or 5
[0079] q is 1, 2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4,
5 or 6;
[0080] r is 2, 3 or 4;
[0081] s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6;
[0082] X is O, S, NR.sub.6 or SO.sub.2;
[0083] R.sub.3 is hydrogen, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl;
[0084] R.sub.4 is hydrogen;
[0085] R.sub.5 is hydrogen, hydroxy or cyano; or R.sub.4 and
R.sub.5 together form .dbd.O; and
[0086] R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
[0087] In an embodiment, the disclosure provides a compound of the
formula (I)
##STR00009##
or a pharmaceutically acceptable salt thereof, wherein
[0088] R.sub.1 is selected from the group consisting of
hydroxy,
##STR00010##
and
[0089] R.sub.2 is selected from the group consisting of
##STR00011##
wherein
[0090] m is 0, 1, 2 or 3;
[0091] n is 1, 2 or 3;
[0092] o is 1, 2, 3, 4, 5 or 6;
[0093] p is 1, 2, 3, 4 or 5
[0094] q is 1, 2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4,
5 or 6;
[0095] r is 2, 3 or 4;
[0096] s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6;
[0097] X is O, S, NR.sub.6 or SO.sub.2;
[0098] R.sub.3 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl;
[0099] R.sub.4 is hydrogen;
[0100] R.sub.5 is hydrogen, hydroxy or cyano; or R.sub.4 and
R.sub.5 together form .dbd.O; and
[0101] R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-6cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
Definitions
[0102] Unless specified otherwise, the term "compounds of the
disclosure" or "compound of the disclosure" refers to compounds of
formula (I), and exemplified compounds, and salts thereof, as well
as all stereoisomers (including diastereoisomers and enantiomers),
rotamers, tautomers and isotopically labeled compounds (including
deuterium substitutions), as well as inherently formed
moieties.
[0103] As used herein, "" represents a portion of a variable bound
to the base molecule and includes both (R)- and
(S)-stereochemistry. For example, when R.sup.2 is
##STR00012##
"" represents the portion of R.sub.2 bound to C16 and includes both
(R)- and (S)-stereochemistry.
[0104] As used herein, the term "C.sub.1-6alkyl" refers to a
straight or branched hydrocarbon chain radical consisting solely of
carbon and hydrogen atoms, containing no unsaturation, having from
one to six carbon atoms, and which is attached to the rest of the
molecule by a single bond. The term "C.sub.1-4alkyl" is to be
construed accordingly. Examples of C.sub.1-6alkyl include, but are
not limited to, methyl, ethyl, n-propyl, 1-methylethyl
(iso-propyl), n-butyl, n-pentyl and 1,1-dimethylethyl
(t-butyl).
[0105] As used herein, the term "hydroxyC.sub.1-6alkyl" refers to
an alkyl group substituted with one or more --OH groups. Examples
of hydroxyC.sub.1-6alkyl groups include HO--CH.sub.2--,
HO--CH.sub.2CH.sub.2--, and --CH.sub.2--CH(OH)--.
[0106] As used herein, the term "C.sub.3-8cycloalkylC.sub.0-6alkyl"
refers to a stable monocyclic saturated hydrocarbon radical
consisting solely of carbon and hydrogen atoms, having from three
to eight carbon atoms, and which is attached to the rest of the
molecule by a single bond or by a C.sub.1-6alkyl radical as defined
above. Examples of C.sub.3-8cycloalkylC.sub.0-6alkyl include, but
are not limited to, cyclopropyl, cyclopropyl-methyl, cyclobutyl,
cyclobutyl-ethyl, cyclopentyl, cyclopentyl-propyl, cyclohexyl,
cyclohepty and cyclooctyl.
[0107] As used herein, the term "phenylC.sub.0-6alkyl" refers to a
phenyl ring attached to the rest of the molecule by a single bond
or by a C.sub.1-6alkyl radical as defined above. Examples of
phenylC.sub.0-6alkyl include, but are not limited to, phenyl and
benzyl.
[0108] As used herein, "hydroxy" or "hydroxyl" refers to --OH.
[0109] Various (enumerated) embodiments of the disclosure are
described herein. It will be recognized that features specified in
each embodiment may be combined with other specified features to
provide further embodiments of the disclosure.
[0110] Embodiment 1. A compound of formula (I) or a
pharmaceutically acceptable salt thereof, as described above.
[0111] Embodiment 2. A compound according to embodiment 1 or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is
hydroxy.
[0112] Embodiment 3. A compound according to embodiment 1 or 2 or a
pharmaceutically acceptable salt thereof, wherein R.sub.2 is
selected from
##STR00013##
in particular
##STR00014##
wherein m, n, X and R.sub.3 are as defined above.
[0113] Embodiment 4. A compound according to any one of embodiments
1 to 3 or a pharmaceutically acceptable salt thereof, wherein R
is
##STR00015##
and n is 1, 2 or 3. In an embodiment, R.sub.2 is
##STR00016##
[0114] Embodiment 5. A compound according to any one of embodiments
1 to 4, or a pharmaceutically acceptable salt thereof, wherein
R.sub.2 is
##STR00017##
[0115] Embodiment 6. A compound according to any one of embodiments
1 to 4 or a pharmaceutically acceptable salt thereof, wherein
R.sub.2 is
##STR00018##
[0116] Embodiment 7. A compound according to any one of embodiments
1 to 4 or a pharmaceutically acceptable salt thereof, wherein
R.sub.2 is
##STR00019##
In an embodiment, R.sub.3 is hydrogen, C.sub.1-6alkyl or
hydroxyC.sub.1-6alkyl.
[0117] Embodiment 8. A compound according any one of embodiments 1
to 5 of formula (I)-A:
##STR00020##
or a pharmaceutically acceptable salt thereof, wherein R.sub.1 is
selected from the group consisting of hydroxy,
##STR00021##
[0118] In an embodiment, R.sub.1 is hydroxyl. In an embodiment,
R.sub.2 is as defined in formula (I). In an embodiment, the C16
position has (R) stereochemistry. In an embodiment, the C16
position has (S) stereochemistry.
[0119] Embodiment 9. A compound according to any one of embodiments
1 to 8 or a pharmaceutically acceptable salt thereof, wherein said
compound is
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin (Compound
1):
##STR00022##
[0120] Embodiment 10. A compound according to any one of
embodiments 1 to 9 or a pharmaceutically acceptable salt thereof,
present as a single diastereoisomer at C16. In an embodiment, the
C16 position has (R) stereochemistry. In an embodiment, the C16
position has (S) stereochemistry.
[0121] Embodiment 11. A compound according to any one of
embodiments 1 to 9 or a pharmaceutically acceptable salt thereof,
present as a diastereoisomeric mixture at C16.
[0122] Embodiment 12. A compound according to any one of
embodiments 1 to 10 of formula (I)-B,
##STR00023##
or a pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.2 are as defined for formula (I). In an embodiment, R.sub.2
is
##STR00024##
[0123] Embodiment 13. A compound of formula (I) or a
pharmaceutically acceptable salt thereof, wherein said compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1):
##STR00025##
[0124] Embodiment 14. A compound of formula (I) or a
pharmaceutically acceptable salt thereof, wherein said compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2):
##STR00026##
[0125] Embodiment 15: A compound of formula (I) or a
pharmaceutically acceptable salt thereof, wherein said compound is
selected from:
TABLE-US-00001 Compound Structure Example 3 ##STR00027## Example 4
##STR00028## Example 5 ##STR00029## Example 6 Example 7
diastereomers at C16 ##STR00030## Example 8 Example 9 diastereomers
at C16 ##STR00031## Example 10 Example 11 diastereomers at C16
##STR00032## Example 12 Example 13 Single diastereomer at C16
##STR00033## Example 14 Example 15 diastereomers at C16
##STR00034## Example 16 ##STR00035## Example 17 ##STR00036##
Example 18 ##STR00037## Example 19 ##STR00038##
[0126] Definitions of specific functional groups and chemical terms
are described in more detail below. The chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.,
inside cover, and specific functional groups are generally defined
as described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in Thomas Sorrell, Organic Chemistry, University
Science Books, Sausalito, 1999; Smith and March, March's Advanced
Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc.,
New York, 2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern
Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge
University Press, Cambridge, 1987.
[0127] Depending on the choice of the starting materials and
procedures, the compounds of the disclosure can be present in the
form of one of the possible stereoisomers or as mixtures thereof
for stereocenters not fixed by formula (I), formula (I)-A and
formula (I)-B--for example as pure optical isomers, or as
stereoisomer mixtures, such as racemates and diastereoisomer
mixtures, depending on the number of asymmetric carbon atoms. The
disclosure is meant to include all such possible stereoisomers,
including racemic mixtures, diastereomeric mixtures and optically
pure forms. Optically active (R)- and (S)-stereoisomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. If the compound contains a double
bond, the substituent may be the E or Z configuration. If the
compound contains a disubstituted cycloalkyl, the cycloalkyl
substituent may have a cis- or trans-configuration. All tautomeric
forms are also intended to be included.
[0128] The term "tautomers" refer to compounds that are
interchangeable forms of a particular compound structure, and that
vary in the displacement of hydrogen atoms and electrons. Thus, two
structures may be in equilibrium through the movement of .pi.
electrons and an atom (usually H). For example, enols and ketones
are tautomers because they are rapidly interconverted by treatment
with either acid or base. Another example of tautomerism is the
aci- and nitro-forms of phenylnitromethane that are likewise formed
by treatment with acid or base. Tautomeric forms may be relevant to
the attainment of the optimal chemical reactivity and biological
activity of a compound of interest.
[0129] As used herein, the terms "salt" or "salts" refers to an
acid addition or base addition salt of a compound of the
disclosure. "Salts" include in particular "pharmaceutical
acceptable salts". The term "pharmaceutically acceptable salts"
refers to salts that retain the biological effectiveness and
properties of the compounds of the disclosure and, which typically
are not biologically or otherwise undesirable. In many cases, the
compounds of the disclosure are capable of forming acid and/or base
salts by virtue of the presence of amino and/or carboxyl groups or
groups similar thereto. Pharmaceutically acceptable salts are well
known in the art. For example, Berge et al., describe
pharmaceutically acceptable salts in detail in J. Pharmaceutical
Sciences, 1977, 66, 1-19, incorporated herein by reference.
[0130] Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids.
[0131] Inorganic acids from which salts can be derived include, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like.
[0132] Organic acids from which salts can be derived include, for
example, acetic acid, propionic acid, glycolic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic
acid, and the like.
[0133] Pharmaceutically acceptable base addition salts can be
formed with inorganic and organic bases.
[0134] Inorganic bases from which salts can be derived include, for
example, ammonium salts and metals from columns I to XII of the
periodic table. In certain embodiments, the salts are derived from
sodium, potassium, ammonium, calcium, magnesium, iron, silver,
zinc, and copper; particularly suitable salts include ammonium,
potassium, sodium, calcium and magnesium salts.
[0135] Organic bases from which salts can be derived include, for
example, primary, secondary, and tertiary amines, substituted
amines including naturally occurring substituted amines, cyclic
amines, basic ion exchange resins, and the like. Certain organic
amines include isopropylamine, benzathine, cholinate,
diethanolamine, diethylamine, lysine, meglumine, piperazine and
tromethamine.
[0136] In another aspect, the disclosure provides compounds in
acetate, ascorbate, adipate, aspartate, benzoate, besylate,
bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate,
camphorsulfonate, caprate, chloride/hydrochloride,
chlortheophyllonate, citrate, ethandisulfonate, fumarate,
gluceptate, gluconate, glucuronate, glutamate, glutarate,
glycolate, hippurate, hydroiodide/iodide, isethionate, lactate,
lactobionate, laurylsulfate, malate, maleate, malonate, mandelate,
mesylate, methylsulphate, mucate, naphthoate, napsylate,
nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate,
pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,
polygalacturonate, propionate, sebacate, stearate, succinate,
sulfosalicylate, sulfate, tartrate, tosylate trifenatate,
trifluoroacetate or xinafoate salt form.
[0137] Any formula given herein is also intended to represent
unlabeled forms as well as isotopically labeled forms of the
compounds. Isotopically labeled compounds have structures depicted
by the formulae given herein except that one or more atoms are
replaced by an atom having a selected atomic mass or mass number.
Isotopes that can be incorporated into compounds of the disclosure
include, for example, isotopes of hydrogen.
[0138] Further, incorporation of certain isotopes, particularly
deuterium (i.e., .sup.2H or D) may afford certain therapeutic
advantages resulting from greater metabolic stability, for example
increased in vivo half-life or reduced dosage requirements or an
improvement in therapeutic index or tolerability. It is understood
that deuterium in this context is regarded as a substituent of a
compound of the disclosure. The concentration of deuterium, may be
defined by the isotopic enrichment factor. The term "isotopic
enrichment factor" as used herein means the ratio between the
isotopic abundance and the natural abundance of a specified
isotope. If a substituent in a compound of this disclosure is
denoted as being deuterium, such compound has an isotopic
enrichment factor for each designated deuterium atom of at least
3500 (52.5% deuterium incorporation at each designated deuterium
atom), at least 4000 (60% deuterium incorporation), at least 4500
(67.5% deuterium incorporation), at least 5000 (75% deuterium
incorporation), at least 5500 (82.5% deuterium incorporation), at
least 6000 (90% deuterium incorporation), at least 6333.3 (95%
deuterium incorporation), at least 6466.7 (97% deuterium
incorporation), at least 6600 (99% deuterium incorporation), or at
least 6633.3 (99.5% deuterium incorporation). It should be
understood that the term "isotopic enrichment factor" can be
applied to any isotope in the same manner as described for
deuterium.
[0139] Other examples of isotopes that can be incorporated into
compounds of the disclosure include isotopes of hydrogen, carbon,
nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as
.sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18F .sup.31P,
.sup.32P, .sup.35S, .sup.36Cl, .sup.123I, .sup.124I, .sup.125I
respectively. Accordingly it should be understood that the
disclosure includes compounds that incorporate one or more of any
of the aforementioned isotopes, including for example, radioactive
isotopes, such as .sup.3H and .sup.14C, or those into which
non-radioactive isotopes, such as .sup.2H and .sup.13C are present.
Such isotopically labelled compounds are useful in metabolic
studies (with .sup.14C), reaction kinetic studies (with, for
example .sup.2H or .sup.3H), detection or imaging techniques, such
as positron emission tomography (PET) or single-photon emission
computed tomography (SPECT) including drug or substrate tissue
distribution assays, or in radioactive treatment of patients. In
particular, an .sup.18F or labeled compound may be particularly
desirable for PET or SPECT studies. Isotopically-labeled compounds
of the disclosure can generally be prepared by conventional
techniques known to those skilled in the art or by processes
analogous to those described in the accompanying Examples and
Preparations using an appropriate isotopically-labeled reagents in
place of the non-labeled reagent previously employed.
[0140] As used herein, the term "pharmaceutical composition" refers
to a compound of the disclosure, or a pharmaceutically acceptable
salt thereof, together with at least one pharmaceutically
acceptable carrier, in a form suitable for oral or parenteral
administration.
[0141] As used herein, the term "pharmaceutically acceptable
carrier" refers to a substance useful in the preparation or use of
a pharmaceutical composition and includes, for example, suitable
diluents, solvents, dispersion media, surfactants, antioxidants,
preservatives, isotonic agents, buffering agents, emulsifiers,
absorption delaying agents, salts, drug stabilizers, binders,
excipients, disintegration agents, lubricants, wetting agents,
sweetening agents, flavoring agents, dyes, and combinations
thereof, as would be known to those skilled in the art (see, for
example, Remington The Science and Practice of Pharmacy, 22.sup.nd
Ed. Pharmaceutical Press, 2013, pp. 1049-1070).
[0142] The term "a therapeutically effective amount" of a compound
of the disclosure refers to an amount of the compound of the
disclosure that will elicit the biological or medical response of a
subject, for example, reduction or inhibition of an enzyme or a
protein activity, or ameliorate symptoms, alleviate conditions,
slow or delay disease progression, or prevent a disease, etc. In an
embodiment, the term "a therapeutically effective amount" refers to
the amount of the compound of the disclosure that, when
administered to a subject, is effective to (1) at least partially
alleviate, prevent and/or ameliorate a condition, or a disorder or
a disease (i) mediated by the mTOR pathway, or (ii) associated with
mTOR activity, or (iii) characterized by activity (normal or
abnormal) of mTOR; or (2) reduce or inhibit the activity of mTOR;
or (3) reduce or inhibit the expression of mTOR. In an embodiment,
the term "a therapeutically effective amount" refers to the amount
of the compound of the disclosure that, when administered to a
cell, or a tissue, or a non-cellular biological material, or a
medium, is effective to at least partially reducing or inhibiting
the activity of mTOR; or at least partially reducing or inhibiting
the expression of mTOR.
[0143] As used herein, the term "subject" refers to primates (e.g.,
humans, male or female), dogs, cats, rabbits, guinea pigs, pigs,
rats and mice. In certain embodiments, the subject is a primate. In
yet other embodiments, the subject is a human.
[0144] The terms "administer," "administering," or
"administration," as used herein refers to implanting, absorbing,
ingesting, injecting, inhaling, or otherwise introducing an
inventive compound, or a pharmaceutical composition thereof.
[0145] As used herein, the term "inhibit", "inhibition" or
"inhibiting" refers to the reduction or suppression of a given
condition, symptom, or disorder, or disease, or a significant
decrease in the baseline activity of a biological activity or
process.
[0146] As used herein, the term "treat", "treating" or "treatment"
of any disease or disorder refers to alleviating, delaying the
onset of, ameliorating the disease or disorder (i.e., slowing or
arresting the development of the disease or at least one of the
clinical symptoms thereof); or alleviating or ameliorating at least
one physical parameter or biomarker associated with the disease or
disorder, including those which may not be discernible to the
patient. In some embodiments, "treatment," "treat," and "treating"
require that signs or symptoms of the disease, disorder or
condition have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease or condition. For example, treatment may
be administered to a susceptible individual prior to the onset of
symptoms (e.g., in light of a history of symptoms and/or in light
of genetic or other susceptibility factors). Treatment may also be
continued after symptoms have resolved, for example, to delay or
prevent recurrence.
[0147] As used herein, the term "prevent", "preventing" or
"prevention" of any disease or disorder refers to the prophylactic
treatment of the disease or disorder; or delaying the onset or
progression of the disease or disorder.
[0148] As used herein, "age-related disease or disorder" refers to
any disease or disorder whose incidence in a population or severity
in an individual correlates with the progression of age. More
specifically, an age-related disease or disorder is a disease or
disorder whose incidence is at least 1.5 fold higher among human
individuals greater than 65 years of age relative to human
individuals between the ages of 25-35. Examples of age-related
disorders include, but are not limited to: sarcopenia, skin
atrophy, cherry angiomas, seborrheic keratoses, brain atrophy--also
referred to as dementia, atherosclerosis, arteriosclerosis,
pulmonary emphysema, osteoporosis, osteoarthritis, high blood
pressure, erectile dysfunction, cataracts, macular degeneration,
glaucoma, stroke, cerebrovascular disease (strokes), chronic kidney
disease, diabetes-associated kidney disease, impaired hepatic
function, liver fibrosis, autoimmune hepatitis, endometrial
hyperplasia, metabolic dysfunction, renovascular disease, hearing
loss, mobility disability (e.g., frailty), cognitive decline,
tendon stiffness, heart dysfunction such as cardiac hypertrophy
and/or systolic and/or diastolic dysfunction and/or hypertension,
heart dysfunction which results in a decline in ejection fraction,
immune senescence, Parkinson's disease, Alzheimer's disease,
cancer, immune-senescence leading to cancer due to a decrease in
immune-surveillance, infections due to an decline in
immune-function, chronic obstructive pulmonary disease (COPD),
obesity, loss of taste, loss of olfaction, arthritis, and type II
diabetes including complications stemming from diabetes, such as
kidney failure, blindness and neuropathy.
[0149] As used herein, a subject is "in need of" a treatment if
such subject would benefit biologically, medically or in quality of
life from such treatment.
[0150] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided herein is intended
merely to better illuminate the disclosure and does not pose a
limitation on the scope of the disclosure otherwise claimed.
[0151] Any asymmetric atom (e.g., carbon or the like) of the
compound(s) of the disclosure can be present in racemic or
enantiomerically enriched, for example the (R)-, (S)- or
(R,S)-configuration. In certain embodiments, each asymmetric atom
has at least 50% enantiomeric excess, at least 60% enantiomeric
excess, at least 70% enantiomeric excess, at least 80% enantiomeric
excess, at least 90% enantiomeric excess, at least 95% enantiomeric
excess, or at least 99% enantiomeric excess in the (R)- or
(S)-configuration. Substituents at atoms with unsaturated double
bonds may, if possible, be present in cis- (Z)- or
trans-(E)-form.
[0152] Accordingly, as used herein a compound of the disclosure can
be in the form of one of the possible stereoisomers, rotamers,
atropisomers, tautomers or mixtures thereof, for example, as
substantially pure geometric (cis or trans) stereoisomers,
diastereomers, optical isomers (antipodes), racemates or mixtures
thereof.
[0153] Any resulting mixtures of stereoisomers can be separated on
the basis of the physicochemical differences of the constituents,
into the pure or substantially pure geometric or optical isomers,
diastereomers, racemates, for example, by chromatography and/or
fractional crystallization.
[0154] Any resulting racemates of compounds of the disclosure or of
intermediates can be resolved into the optical antipodes by known
methods, e.g., by separation of the diastereomeric salts thereof,
obtained with an optically active acid or base, and liberating the
optically active acidic or basic compound. In particular, a basic
moiety may thus be employed to resolve the compounds of the
disclosure into their optical antipodes, e.g., by fractional
crystallization of a salt formed with an optically active acid,
e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric
acid, di-O, O'p-toluoyl tartaric acid, mandelic acid, malic acid or
camphor-10-sulfonic acid. Racemic compounds of the disclosure or
racemic intermediates can also be resolved by chiral
chromatography, e.g., high pressure liquid chromatography (HPLC)
using a chiral adsorbent.
Methods of Making Compounds of Formula (I)
[0155] In another aspect, the disclosure provides a process for the
production of compounds of formula (I), formula (I)-A and formula
(I)-B. Compounds of formula (I), formula (I)-A and formula (I)-B
can be made according to the following process as described in
Schemes 1, 2 and 3:
##STR00039##
[0156] A compound of formula (I), in which R.sub.2 is as defined
under formula (I), may be obtained by reacting C32-deoxy rapamycin
(Intermediate 1) with R.sub.2--H, wherein R.sub.2 is as defined
under formula (I), in the presence of a suitable reagent for a
substitution reaction, e.g. p-toluenesulphonic acid, in the
presence of a suitable solvent, e.g. dichloromethane. Suitable
conditions are as follows:
[0157] 1) R.sub.2--H, p-toluenesulphonic acid-H.sub.2O,
dichloromethane, room temperature
[0158] 2) R.sub.2--H, trifluoroacetic acid, -40.degree. C.,
dichloromethane (see EP1212331B1)
[0159] 3) R.sub.2--H, 5M LiClO.sub.4, Et.sub.2O (0.1M), room
temperature (see TL, 1995, 43, 7823)
[0160] 4) R.sub.2--H, Cp.sub.2HfCl.sub.2--AgClO.sub.4 (Suzuki's
catalyst), 4A MS, dichloromethane, room temperature (see TL, 1995,
43, 7823)
[0161] 5) R.sub.2--H, BF.sub.3--OEt.sub.2 or Zn(OTf).sub.2, THF,
0.degree. C. (see TL, 1994, 37, 6835)
[0162] 6) R.sub.2--H, ZnCl.sub.2, dichloromethane, 0.degree. C.
(see JOC, 1994, 59, 6512).
[0163] C32-deoxy rapamycin used as the starting material can be
prepared by methods known in the art, e.g. as described in US
Patent Publication No. 005985890 or WO2007085400.
##STR00040##
[0164] A compound of formula (I)-A, wherein R.sub.1 is selected
from
##STR00041##
and R.sub.2 is as defined under formula (I), may be obtained by
reacting Intermediate 1 with R.sub.1--H or R.sub.1--X followed by
reaction with R.sub.2--H. In an embodiment, Intermediate 1 is
reacted with R.sub.1--H or R.sub.1--X under alkylation,
phosphination or esterification conditions to provide Intermediate
1-A. In an embodiment, Intermediate 1-A is reacted with R.sub.2--H
under substitution reaction conditions, e.g., as provided herein,
to afford a compound of formula (I)-A.
##STR00042##
[0165] A compound of formula (I)-C, wherein R.sub.1 is
##STR00043##
and R.sub.2 is as defined under formula (I), may be obtained by
reacting Intermediate 1 with R.sub.1--H followed by reaction with
R.sub.2--H. In an embodiment, Intermediate 1 is activated and
reacted under nucleophilic conditions to provide Intermediate 1-A.
In an embodiment, Intermediate 1-A is reacted with R.sub.2--H under
substitution reaction conditions, e.g., as provided herein, to
afford the compound of formula (I)-C.
[0166] The reactions can be effected according to conventional
methods, for example as described in the Examples.
[0167] The work-up of the reaction mixtures and the purification of
the compounds thus obtainable may be carried out in accordance with
known procedures.
[0168] Acid addition salts may be produced from the free bases in
known manner, and vice-versa.
[0169] Starting materials may be known or prepared according to
conventional procedures starting from known compounds, for example
as described in the Examples.
[0170] In another aspect, the disclosure provides a pharmaceutical
composition comprising a compound of the disclosure, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier. In a further embodiment, the composition
comprises at least two pharmaceutically acceptable carriers, such
as those described herein. The pharmaceutical composition can be
formulated for particular routes of administration such as oral
administration, parenteral administration (e.g. by injection,
infusion, transdermal or topical administration), and rectal
administration. Topical administration may also pertain to
inhalation or intranasal application. The pharmaceutical
compositions of the disclosure can be made up in a solid form
(including, without limitation, capsules, tablets, pills, granules,
powders or suppositories), or in a liquid form (including, without
limitation, solutions, suspensions or emulsions). Tablets may be
either film coated or enteric coated according to methods known in
the art. Typically, the pharmaceutical compositions are tablets or
gelatin capsules comprising the active ingredient together with one
or more of:
[0171] a) diluents, e.g., lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose and/or glycine;
[0172] b) lubricants, e.g., silica, talcum, stearic acid, its
magnesium or calcium salt and/or polyethyleneglycol; for tablets
also
[0173] c) binders, e.g., magnesium aluminum silicate, starch paste,
gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose
and/or polyvinylpyrrolidone; if desired
[0174] d) disintegrants, e.g., starches, agar, alginic acid or its
sodium salt, or effervescent mixtures; and
[0175] e) absorbents, colorants, flavors and sweeteners.
[0176] The compound of the disclosure, or a pharmaceutically
acceptable salt thereof, may also be in the form of a drug-eluting
stent, i.e. a stent coated with a compound of the disclosure, or a
pharmaceutically acceptable salt thereof.
[0177] The compounds of the disclosure in free form or in
pharmaceutically acceptable salt form, exhibit valuable
pharmacological properties, e.g. mTOR pathway modulating
properties, e.g. as indicated in vitro and in vivo tests as
provided in the next sections, and are therefore indicated for
therapy or for use as research chemicals, e.g. as tool
compounds.
[0178] Methods to measure potency of mTORC1 inhibitors are well
known in the art. Generally, potency is determined by IC50 values,
assessed by determining of inhibition of phosphorylation of S6,
which is in the mTORC1 signaling pathway. The IC50 values of the
mTORC1 inhibitors are compared to the IC50 value of rapamycin in
the same assay. An mTORC1 inhibitor having an IC50 value within
100-fold that of the IC50 of rapamycin in the same assay is
suitable for use in the disclosure--meaning a less potent rapalog
may still be desirable, so as to more easily achieve only partial
inhibition of mTORC1 activity in particular settings, and so as to
improve the ability to measure the molecule in the bloodstream
(since higher concentrations are necessary for a less potent
molecule)--which would be helpful in fine-tuning the blood
concentration/efficacy relationship.
[0179] Suitable assays to measure the potency of mTOR inhibitors
are described for instance in U.S. Pat. No. 5,665,772 as measured
by the IC50 value in an MLR (mixed lymphocyte reaction) assay
and/or in an IL-6 (interleukin-6)-dependent mediated proliferation
assay.
[0180] The MLR assay is typically carried out as follows: Spleen
cells (0.5.times.106) from Balb/c mice (female, 8-10 weeks) are
co-incubated for 5 days with 0.5.times.106 irradiated (2000 rads)
or mitomycin C treated spleen cells from CBA mice (female, 8-10
weeks). The irradiated allogeneic cells induce a proliferative
response in the Balb/c spleen cells, which can be measured by
labeled precursor incorporation into the DNA. Since the stimulator
cells are irradiated (or mitomycin C treated) they do not respond
to the Balb/c cells with proliferation but do retain their
antigenicity. The antiproliferative effect of the compounds tested
on the Balb/c cells is measured at various dilutions and the
concentration resulting in 50% inhibition of cell proliferation
(IC50) is calculated. The inhibitory capacity of the test sample
may be compared to rapamycin and expressed as a relative IC50 (i.e.
IC50 test sample/IC50 rapamycin).
[0181] The IL-6 mediated proliferation assay is typically carried
as follows: the assay uses an interleukin-6 (IL-6)-dependent mouse
hybridoma cell line and is performed in 96-well microtiter plates.
5000 cells/well are cultivated in serum-free medium (as described
by M. H. Schreier and R. Tees in Immunological Methods, I.
Lefkovits and B. Pernis, eds., Academic Press 1981. Vol. II, pp.
263-275), supplemented with 1 ng recombinant IL-6/ml. Following a
66 hour incubation in the absence or presence of a test sample,
cells are pulsed with 1 .mu.Ci (3-H)-thymidine/well for another 6
hours, harvested and counted by liquid scintillation.
(3-H)-thymidine incorporation into DNA correlates with the increase
in cell number and is thus a measure of cell proliferation. A
dilution series of the test sample allows the calculation of the
concentration resulting in 50% inhibition of cell proliferation
(IC50). The inhibitory capacity of the test sample may be compared
to rapamycin and expressed as a relative IC50 (i.e. IC50 test
sample/IC50 rapamycin).
[0182] Potency of mTOR inhibitors may also be determined using a
MEF TSC1-/- cell-based assay. MEF TSC1-/- cells are Mouse Embryonic
Fibroblasts deficient in the Tuberous sclerosis protein, TSC1,
which negatively regulates mTORC1 signaling. Thus, the deficiency
of TSC1 induces constitutive mTORC1 activation, resulting in
phosphorylation (activation) of the downstream proteins in the
mTORC1 signaling pathways. This cell-based assay is used to measure
inhibition (de-phosphorylation) of the mTORC1 signaling components
S6 and 4EBP1, by rapalogs or other mTOR inhibitors.
[0183] The assay is typically carried out as follows: MEF TSC1-/-
cells are plated on Poly-D-lysine coated 384 well Griener clear
bottom plates and incubated overnight at 37.degree. C., 5% CO2. On
the following day, cells are washed 8 times with "Hard starve"
solution (1 L DPBS+1 g D-(+) glucose+10 ml of 7.5% Sodium
Bicarbonate+20 ml of 1M HEPES) and incubated for further 2 hours in
the same solution. Cells are next treated with compounds with
decreasing concentrations (8 points at 3.16 fold dilutions) and
incubated for 2 hours at 37.degree. C., 5% CO2. Cells are fixed
with 4% paraformaldehyde for 30 min and washed 5 times with
TBS-EDTA followed by immuno-staining with florescent tag labeled
antibodies for pS6 and p4EBP1. Nuclei are visualized with Hoechst
staining. Cells are imaged using respective florescence channels
and the potency of mTOR inhibitors is defined by pS6 IC.sub.50
(nM).
Diseases and Disorders
[0184] Compounds of the disclosure may be useful in the prevention
or treatment of an indication or prodromal condition selected from:
[0185] Acute or chronic organ or tissue transplant rejection;
[0186] Transplant vasculopathies; [0187] Smooth muscle cell
proliferation and migration leading to vessel intimal thickening,
blood vessel obstruction, obstructive coronary atherosclerosis,
restenosis; [0188] Autoimmune diseases and inflammatory conditions;
[0189] Treatment and prevention of asthma; [0190] Multi-drug
resistance (MDR); [0191] Fungal infections; [0192] Inflammation;
[0193] Infection; [0194] Age-related diseases; [0195]
Neurodegenerative diseases; [0196] Proliferative disorders, in
particular cancer; [0197] Seizures and seizure related disorders;
[0198] Mitochondrial myopathy and mitochondrial stress; [0199]
Treatable conditions which have been shown to make age-related
diseases more likely, such as settings where there is an increase
in senescence inducing cytokines (e.g. IL6); [0200] Disorders that
include the process of fibrosis and/or inflammation, e.g., liver
and kidney disorders. Examples include, liver fibrosis, which
occurs in end-stage liver disease; liver cirrhosis; liver failure
due to toxicity; non-alcohol-associated hepatic steatosis or NASH;
and alcohol-associated steatosis. Another example is kidney
fibrosis, which occurs as a result of acute kidney injury, leading
to chronic kidney disease. Also, diabetic nephropathy can induce
kidney fibrosis and inflammation. Often kidney disease causes heart
failure, as a result of an increase in blood pressure; this can
also be associated with cardiac fibrosis. Rapalogs possess
preclinical efficacy in treating models of cardiac failure, and are
effective in decreasing liver fibrosis in patients who have
undergone liver transplants (Buss, S. J. et al. Beneficial effects
of Mammalian target of rapamycin inhibition on left ventricular
remodeling after myocardial infarction. J Am Coll Cardiol. (2009)
54(25): 2435-46; Buss, S. J. et al. Augmentation of autophagy by
mTOR-inhibition in myocardial infarction: When size matters.
Autophagy. (2010) 6(2):304-6; Villamil, F. G. et al. Fibrosis
progression in maintenance liver transplant patients with hepatitis
C recurrence: a randomized study of everolimus vs. calcineurin
inhibitors. Liver Int. (2014) 34(10):1513-21).
[0201] Treatment of acute or chronic organ or tissue transplant
rejection, include the treatment of recipients of e.g., heart,
lung, combined heart-lung, liver, kidney, pancreatic, skin or
corneal transplants. The compounds of the disclosure also indicated
for the prevention of graft-versus-host disease, such as following
bone marrow transplantation.
[0202] Transplant vasculopathies include atherosclerosis.
[0203] Autoimmune diseases and inflammatory conditions include in
particular inflammatory conditions with an etiology including an
autoimmune component such as arthritis (for example rheumatoid
arthritis, arthritis chronica progrediente and arthritis deformans)
and rheumatic diseases. Specific autoimmune diseases for which the
compounds of formula (I), formula (I)-A, formula (I)-B and formula
(I)-C may be employed include, autoimmune hematological disorders
(including e.g. hemolytic anemia, aplastic anemia, pure red cell
anaemia and idiopathic thrombocytopenia), systemic lupus
erythematosus, polychondritis, sclerodoma, Wegener granulamatosis,
dermatomyositis, chronic active hepatitis, myasthenia gravis,
psoriasis, Steven-Johnson syndrome, idiopathic sprue, autoimmune
inflammatory bowel disease (including e.g. ulcerative colitis and
Crohn's disease) endocrine ophthalmopathy, Graves disease,
sarcoidosis, multiple sclerosis, primary biliary cirrhosis,
juvenile diabetes (diabetes mellitus type I), uveitis (anterior and
posterior), keratoconjunctivitis sicca and vernal
keratoconjunctivitis, interstitial lung fibrosis, psoriatic
arthritis, glomerulonephritis (with and without nephrotic syndrome,
e.g. including idiopathic nephrotic syndrome or minimal change
nephropathy) and juvenile dermatomyositis.
[0204] Treatment of multi-drug resistance (MDR) includes enhancing
the efficacy of other chemotherapeutic agents in the treatment and
control of multidrug resistant conditions such as multidrug
resistant cancer or multidrug resistant AIDS. MDR is particularly
problematic in cancer patients and ADS patients who will not
respond to conventional chemotherapy because the medication is
pumped out of the cells by Pgp.
[0205] Infection includes infection by pathogens having Mip or
Mip-like factors.
[0206] Age-related diseases include: sarcopenia, skin atrophy,
cherry angiomas, seborrheic keratoses, brain atrophy--also referred
to as dementia, atherosclerosis, arteriosclerosis, pulmonary
emphysema, osteoporosis, osteoarthritis, high blood pressure,
erectile dysfunction, cataracts, macular degeneration, glaucoma,
stroke, cerebrovascular disease (strokes), chronic kidney disease,
diabetes-associated kidney disease, impaired hepatic function,
liver fibrosis, autoimmune hepatitis, endometrial hyperplasia,
metabolic dysfunction, renovascular disease, hearing loss, mobility
disability (e.g., frailty), cognitive decline, tendon stiffness,
heart dysfunction such as cardiac hypertrophy and/or systolic
and/or diastolic dysfunction and/or hypertension, heart dysfunction
which results in a decline in ejection fraction, immune senescence,
Parkinson's disease, Alzheimer's disease, cancer, immune-senescence
leading to cancer due to a decrease in immune-surveillance,
infections due to an decline in immune-function, chronic
obstructive pulmonary disease (COPD), obesity, loss of taste, loss
of olfaction, arthritis, and type II diabetes including
complications stemming from diabetes, such as kidney failure,
blindness and neuropathy.
[0207] Neurodegenerative diseases include Huntington's Disease,
Parkinson's disease, spinocerebellar ataxia type 3, Alzheimer's
disease, motor neuron disease and peripheral neuropathy.
[0208] Proliferative disorders include cancer. Such conditions
include those listed in U.S. Pat. No. 9,669,032, in particular
renal cancer, renal cell carcinoma, colorectal cancer, uterine
sarcoma, endometrial uterine cancer, endometrial cancer, breast
cancer, ovarian cancer, cervical cancer, gastric cancer,
fibro-sarcoma, pancreatic cancer, liver cancer, melanoma, leukemia,
multiple myeloma, nasopharyngeal cancer, prostate cancer, lung
cancer, glioblastoma, bladder cancer, mesothelioma, head cancer,
rhabdomyosarcoma, sarcoma, lymphoma, or neck cancer.
[0209] Seizures and seizure related disorders include West
syndrome, Focal Cortical Dysplasia (FCD), tuberous sclerosis
complex (TSC), childhood absence epilepsy, benign focal epilepsies
of childhood, juvenile myoclonic epilepsy (JME), temporal lobe
epilepsy, frontal lobe epilepsy, refractory epilepsy,
Lennox-Gastaut syndrome, occipital lobe epilepsy, Proteus syndrome,
hemi-megalencephaly syndrome (HMEG), megalencephaly syndrome (MEG),
megalencephaly-capillary malformation (MCAP) and
megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome
(MPPH).
[0210] Mitochondrial myopathy and mitochondrial stress are
mitochondrial disorders as described in Chinnery, P. F. (2015);
EMBO Mol. Med. 7, 1503-1512; Koopman, W. J. et al., (2016); EMBO
Mol. Med. 8, 311-327 and Young, M. J., and Yound and Copeland, W.
C. (2016); Curr. Opin. Genet. Dev. 38, 52-62.
[0211] Treatable conditions which have been shown to make
age-related diseases more likely include senescence, e.g., immune
senescence. This is diagnosed by (i) an increase in circulating
cytokines, such as IL-6, but also by (ii) senescent cells found in
muscle, kidney, liver, brain, neurons, liver, pancreas, or the
heart; or also (iii) a decline in the efficiency of DNA-repair,
which can be shown by an increase in transcription of repetitive
elements, including transposon-encoded genes. For background, see
Baker, D. J. et al, Nature, 2016; 530(7589):184-9. doi:
10.1038/nature16932. Epub 2016 Feb. 3.
Methods of Treatment and Uses
[0212] The disclosure provides the use of a compound of the
disclosure for use in therapy. In a further embodiment, the therapy
is selected from a disease or disorder or co-morbidity, which may
be treated by modulation of the mTOR pathway. In an embodiment, the
disease is selected from the afore-mentioned list, in an embodiment
an age-related disease, such as respiratory tract infection-related
morbidity in the elderly.
[0213] The disclosure provides a compound of the disclosure for use
in therapy. In a further embodiment, the therapy is selected from a
disease that may be treated by modulation of the mTOR pathway. In
an embodiment, the disease is selected from the afore-mentioned
list, in an embodiment an age-related disease, such as respiratory
tract infection-related morbidity in the elderly.
[0214] The disclosure provides the use of a compound of the
disclosure for the manufacture of a medicament. In a further
embodiment, the medicament is for prevention or treatment of a
disease which may be treated by modulation of the mTOR pathway. In
an embodiment, the disease is selected from the afore-mentioned
list, in an embodiment an age-related disease, such as respiratory
tract infection-related morbidity in the elderly.
[0215] In one aspect, the disclosure provides a method of treating
a disorder or a disease mediated by the mTOR pathway in a subject
in need thereof, the method comprising administering to the subject
a therapeutically effective amount of a compound of formula (I),
formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof.
[0216] In one aspect, the disclosure provides a method of treating
a disease or disorder in a subject, wherein the target tissue or
organ associated with the pathology of the disease or disorder has
FKBP12 levels sufficient to inhibit mTORC1, the method comprising
administering to the subject in need thereof a therapeutically
effective amount of a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, a pharmaceutical composition comprising a compound of
formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
combination comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof.
[0217] In one aspect, the disclosure provides a method of treating
a disease or disorder in a subject having, or previously determined
as having, FKBP12 levels sufficient to inhibit mTORC1, the method
comprising administering to the subject in need thereof a
therapeutically effective amount of a compound of formula (I),
formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof.
[0218] In an embodiment, the disease or disorder is selected from
sarcopenia, skin atrophy, cherry angiomas, seborrheic keratoses,
brain atrophy, atherosclerosis, arteriosclerosis, pulmonary
emphysema, osteoporosis, osteoarthritis, high blood pressure,
erectile dysfunction, cataracts, macular degeneration, glaucoma,
stroke, cerebrovascular disease (strokes), chronic kidney disease,
diabetes-associated kidney disease, impaired hepatic function,
liver fibrosis, autoimmune hepatitis, endometrial hyperplasia,
metabolic dysfunction, renovascular disease, hearing loss, mobility
disability, cognitive decline, tendon stiffness, heart dysfunction
such as cardiac hypertrophy and/or systolic and/or diastolic
dysfunction and/or hypertension, heart dysfunction which results in
a decline in ejection fraction, immune senescence, Parkinson's
disease, Alzheimer's disease, cancer, immune-senescence leading to
cancer due to a decrease in immune-surveillance, infections due to
an decline in immune-function, chronic obstructive pulmonary
disease (COPD), obesity, loss of taste, loss of olfaction,
arthritis, and type II diabetes including complications stemming
from diabetes, such as kidney failure, blindness and
neuropathy.
[0219] In an embodiment, the disorder is liver fibrosis.
[0220] In one aspect, the disclosure provides a method of treating
a disease or disorder in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of a compound of formula (I), formula (I)-A, formula (I)-B
and formula (I)-C or a pharmaceutically acceptable salt thereof, a
pharmaceutical composition comprising a compound of formula (I),
formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
combination comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, wherein the disorder or disease is selected from:
[0221] Acute or chronic organ or tissue transplant rejection;
[0222] Transplant vasculopathies; [0223] Smooth muscle cell
proliferation and migration leading to vessel intimal thickening,
blood vessel obstruction, obstructive coronary atherosclerosis,
restenosis; [0224] Autoimmune diseases and inflammatory conditions;
[0225] Treatment and prevention of asthma; [0226] Multi-drug
resistance (MDR); [0227] Fungal infections; [0228] Inflammation;
[0229] Infection; [0230] Age-related diseases; [0231]
Neurodegenerative diseases; [0232] Proliferative disorders, in
particular cancer; [0233] Seizures and seizure related disorders;
and [0234] Mitochondrial myopathy and mitochondrial stress.
[0235] In an embodiment, the disorder is a disorder that includes
the process of fibrosis and/or inflammation.
[0236] In an embodiment, the disorder is selected from liver and
kidney disorders.
[0237] In an embodiment, the liver disorder is selected from: liver
fibrosis, which occurs in end-stage liver disease; liver cirrhosis;
liver failure due to toxicity; non-alcohol-associated hepatic
steatosis or NASH; and alcohol-associated steatosis.
[0238] In an embodiment, the kidney disorder is kidney
fibrosis.
[0239] In an embodiment, the kidney fibrosis occurs as a result of
acute kidney injury.
[0240] In an embodiment, the kidney disorder is chronic kidney
disorder.
[0241] In an embodiment, the kidney disorder is diabetic
nephropathy.
[0242] In one aspect, the disclosure provides a method of treating
an age-related disorder or disease in a subject in need thereof,
the method comprising administering to the subject a
therapeutically effective amount of a compound of formula (I),
formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, wherein the disorder or
disease is selected from: sarcopenia, skin atrophy, cherry
angiomas, seborrheic keratoses, brain atrophy, atherosclerosis,
arteriosclerosis, pulmonary emphysema, osteoporosis,
osteoarthritis, high blood pressure, erectile dysfunction,
cataracts, macular degeneration, glaucoma, stroke, cerebrovascular
disease (strokes), chronic kidney disease, diabetes-associated
kidney disease, impaired hepatic function, liver fibrosis,
autoimmune hepatitis, endometrial hyperplasia, metabolic
dysfunction, renovascular disease, hearing loss, mobility
disability, cognitive decline, tendon stiffness, heart dysfunction
such as cardiac hypertrophy and/or systolic and/or diastolic
dysfunction and/or hypertension, heart dysfunction which results in
a decline in ejection fraction, immune senescence, Parkinson's
disease, Alzheimer's disease, cancer, immune-senescence leading to
cancer due to a decrease in immune-surveillance, infections due to
an decline in immune-function, chronic obstructive pulmonary
disease (COPD), obesity, loss of taste, loss of olfaction,
arthritis, and type II diabetes including complications stemming
from diabetes, such as kidney failure, blindness and
neuropathy.
[0243] In one aspect, the disclosure provides a method of treating
cancer in a subject, the method comprising administering to the
subject a therapeutically effective amount of a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof.
[0244] In an embodiment, the method further comprises a PD-1/PDL-1
inhibitor.
[0245] In an embodiment, the cancer is selected from renal cancer,
renal cell carcinoma, colorectal cancer, uterine sarcoma,
endometrial uterine cancer, endometrial cancer, breast cancer,
ovarian cancer, cervical cancer, gastric cancer, fibro-sarcoma,
pancreatic cancer, liver cancer, melanoma, leukemia, multiple
myeloma, nasopharyngeal cancer, prostate cancer, lung cancer,
glioblastoma, bladder cancer, mesothelioma, head cancer,
rhabdomyosarcoma, sarcoma, lymphoma, and neck cancer.
[0246] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use as a
medicament.
[0247] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the prevention
or treatment of a disorder or disease mediated by the mTOR
pathway.
[0248] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the prevention
or treatment of a disorder or disease selected from: [0249] Acute
or chronic organ or tissue transplant rejection; [0250] Transplant
vasculopathies; [0251] Smooth muscle cell proliferation and
migration leading to vessel intimal thickening, blood vessel
obstruction, obstructive coronary atherosclerosis, restenosis;
[0252] Autoimmune diseases and inflammatory conditions; [0253]
Treatment and prevention of asthma; [0254] Multi-drug resistance
(MDR); [0255] Fungal infections; [0256] Inflammation; [0257]
Infection; [0258] Age-related diseases; [0259] Neurodegenerative
diseases; [0260] Proliferative disorders, in particular cancer;
[0261] Seizures and seizure related disorders; [0262] Mitochondrial
myopathy and mitochondrial stress; and [0263] Treatable conditions
which have been shown to make age-related diseases more likely,
such as settings where there is an increase in senescence inducing
cytokines.
[0264] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the prevention
or treatment of a disorder or disease that includes the process of
fibrosis and/or inflammation.
[0265] In an embodiment, the disorder is selected from liver and
kidney disorders.
[0266] In an embodiment, the liver disorder is selected from: liver
fibrosis, which occurs in end-stage liver disease; liver cirrhosis;
liver failure due to toxicity; non-alcohol-associated hepatic
steatosis or NASH; and alcohol-associated steatosis.
[0267] In an embodiment, the kidney disorder is kidney fibrosis,
which occurs as a result of acute kidney injury.
[0268] In an embodiment, the kidney disorder is chronic kidney
disorder.
[0269] In an embodiment, the kidney disorder is diabetic
nephropathy.
[0270] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the prevention
or treatment of an age-related disorder or disease selected from
sarcopenia, skin atrophy, cherry angiomas, seborrheic keratoses,
brain atrophy--also referred to as dementia, atherosclerosis,
arteriosclerosis, pulmonary emphysema, osteoporosis,
osteoarthritis, high blood pressure, erectile dysfunction,
cataracts, macular degeneration, glaucoma, stroke, cerebrovascular
disease (strokes), chronic kidney disease, diabetes-associated
kidney disease, impaired hepatic function, liver fibrosis,
autoimmune hepatitis, endometrial hyperplasia, metabolic
dysfunction, renovascular disease, hearing loss, mobility
disability (e.g., frailty), cognitive decline, tendon stiffness,
heart dysfunction such as cardiac hypertrophy and/or systolic
and/or diastolic dysfunction and/or hypertension, heart dysfunction
which results in a decline in ejection fraction, immune senescence,
Parkinson's disease, Alzheimer's disease, cancer, immune-senescence
leading to cancer due to a decrease in immune-surveillance,
infections due to an decline in immune-function, chronic
obstructive pulmonary disease (COPD), obesity, loss of taste, loss
of olfaction, arthritis, and type II diabetes including
complications stemming from diabetes, such as kidney failure,
blindness and neuropathy.
[0271] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the prevention
or treatment of cancer.
[0272] In one aspect, the disclosure provides a compound of formula
(I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, a pharmaceutical
composition comprising a compound of formula (I), formula (I)-A,
formula (I)-B and formula (I)-C or a pharmaceutically acceptable
salt thereof, or a pharmaceutical combination comprising a compound
of formula (I), formula (I)-A, formula (I)-B and formula (I)-C or a
pharmaceutically acceptable salt thereof, for use in the treatment
of renal cancer, renal cell carcinoma, colorectal cancer, uterine
sarcoma, endometrial uterine cancer, endometrial cancer, breast
cancer, ovarian cancer, cervical cancer, gastric cancer,
fibro-sarcoma, pancreatic cancer, liver cancer, melanoma, leukemia,
multiple myeloma, nasopharyngeal cancer, prostate cancer, lung
cancer, glioblastoma, bladder cancer, mesothelioma, head cancer,
rhabdomyosarcoma, sarcoma, lymphoma, or neck cancer.
[0273] In an embodiment, the disclosure provides a compound
##STR00044##
or a pharmaceutically acceptable salt thereof for use in the
treatment of an age-related disease, such as respiratory tract
infection-related morbidity in the elderly. In an embodiment, the
compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0274] In an embodiment, the disorder or disease is selected from:
[0275] Acute or chronic organ or tissue transplant rejection;
[0276] Transplant vasculopathies; [0277] Smooth muscle cell
proliferation and migration leading to vessel intimal thickening,
blood vessel obstruction, obstructive coronary atherosclerosis,
restenosis; [0278] Autoimmune diseases and inflammatory conditions;
[0279] Treatment and prevention of asthma; [0280] Multi-drug
resistance (MDR); [0281] Fungal infections; [0282] Inflammation;
[0283] Infection; [0284] Age-related diseases; [0285]
Neurodegenerative diseases; [0286] Proliferative disorders, in
particular cancer; [0287] Seizures and seizure related disorders;
[0288] Mitochondrial myopathy and mitochondrial stress; and [0289]
Treatable conditions which are shown to make age-related diseases
more likely, such as settings where there is an increase in
senescence inducing cytokines (e.g. IL6).
[0290] In an embodiment, the disorder or disease is an age-related
disorder or disease selected from sarcopenia, skin atrophy, cherry
angiomas, seborrheic keratoses, brain atrophy--also referred to as
dementia, atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, high blood pressure, erectile
dysfunction, cataracts, macular degeneration, glaucoma, stroke,
cerebrovascular disease (strokes), chronic kidney disease,
diabetes-associated kidney disease, impaired hepatic function,
liver fibrosis, autoimmune hepatitis, endometrial hyperplasia,
metabolic dysfunction, renovascular disease, hearing loss, mobility
disability (e.g., frailty), cognitive decline, tendon stiffness,
heart dysfunction such as cardiac hypertrophy and/or systolic
and/or diastolic dysfunction and/or hypertension, heart dysfunction
which results in a decline in ejection fraction, immune senescence,
Parkinson's disease, Alzheimer's disease, cancer, immune-senescence
leading to cancer due to a decrease in immune-surveillance,
infections due to an decline in immune-function, chronic
obstructive pulmonary disease (COPD), obesity, loss of taste, loss
of olfaction, arthritis, and type II diabetes including
complications stemming from diabetes, such as kidney failure,
blindness and neuropathy.
[0291] In an embodiment, the disorder or disease includes the
process of fibrosis and/or inflammation, e.g., liver and kidney
disorders. In an embodiment, the disorder is a kidney disorder. In
an embodiment, the disorder is a liver disorder. Examples include,
liver fibrosis, which occurs in end-stage liver disease; liver
cirrhosis; liver failure due to toxicity; non-alcohol-associated
hepatic steatosis or NASH; and alcohol-associated steatosis.
Another example is kidney fibrosis, which occurs as a result of
acute kidney injury, leading to chronic kidney disease. Also,
diabetic nephropathy can induce kidney fibrosis and inflammation.
Often kidney disease causes heart failure, as a result of an
increase in blood pressure; this can also be associated with
cardiac fibrosis. Rapalogs possess preclinical efficacy in treating
models of cardiac failure, and are effective in decreasing liver
fibrosis in patients who have undergone liver transplants.
[0292] The therapeutically effective dosage of a compound, the
pharmaceutical composition, or the combinations thereof, is
dependent on the species of the subject, the body weight, age and
individual condition, the disorder or disease or the severity
thereof being treated. A physician, clinician or veterinarian of
ordinary skill can readily determine the effective amount of each
of the active ingredients necessary to prevent, treat or inhibit
the progress of the disorder or disease.
[0293] The above-cited dosage properties are demonstrable in vitro
and in vivo tests using advantageously mammals, e.g., mice, rats,
dogs, monkeys or isolated organs, tissues and preparations thereof.
The compounds of the disclosure may be applied in vitro in the form
of solutions, e.g., aqueous solutions, and in vivo either
enterally, parenterally, advantageously intravenously, e.g., as a
suspension or in aqueous solution. The dosage in vitro may range
between about 10-3 molar and 10-9 molar concentrations. A
therapeutically effective amount in vivo may range depending on the
route of administration, between about 0.1-500 mg/kg, or between
about 0.1-500 mg per subject.
[0294] The compound of the disclosure may be administered either
simultaneously with, or before or after, one or more other
therapeutic agent. The compound of the disclosure may be
administered separately, by the same or different route of
administration, or together in the same pharmaceutical composition
as the other agents. A therapeutic agent is, for example, a
chemical compound, peptide, antibody, antibody fragment or nucleic
acid, which is therapeutically active or enhances the therapeutic
activity when administered to a patient in combination with a
compound of the disclosure.
[0295] In an embodiment, the disclosure provides a product
comprising a compound of the disclosure and at least one other
therapeutic agent as a combined preparation for simultaneous,
separate or sequential use in therapy. In an embodiment, the
therapy is the treatment of a disease or condition via partial or
full inhibition of mTOR. Products provided as a combined
preparation include a composition comprising the compound of the
disclosure and the other therapeutic agent(s) together in the same
pharmaceutical composition, or the compound of the disclosure and
the other therapeutic agent(s) in separate form, e.g. in the form
of a kit.
[0296] In an embodiment, the disclosure provides a pharmaceutical
composition comprising a compound of the disclosure and another
therapeutic agent(s). Optionally, the pharmaceutical composition
may comprise a pharmaceutically acceptable carrier, as described
above.
[0297] In an embodiment, the disclosure provides a kit comprising
two or more separate pharmaceutical compositions, at least one of
which contains a compound of the disclosure. In an embodiment, the
kit comprises means for separately retaining said compositions,
such as a container, divided bottle, or divided foil packet. An
example of such a kit is a blister pack, as typically used for the
packaging of tablets, capsules and the like.
[0298] The kit of the disclosure may be used for administering
different dosage forms, for example, oral and parenteral, for
administering the separate compositions at different dosage
intervals, or for titrating the separate compositions against one
another. To assist compliance, the kit of the disclosure typically
comprises directions for administration.
[0299] In the combination therapies of the disclosure, the compound
of the disclosure and the other therapeutic agent may be
manufactured and/or formulated by the same or different
manufacturers. Moreover, the compound of the disclosure and the
other therapeutic may be brought together into a combination
therapy: (i) prior to release of the combination product to
physicians (e.g. in the case of a kit comprising the compound of
the disclosure and the other therapeutic agent); (ii) by the
physician themselves (or under the guidance of the physician)
shortly before administration; (iii) in the patient themselves,
e.g. during sequential administration of the compound of the
disclosure and the other therapeutic agent.
[0300] Accordingly, the disclosure provides the use of a compound
of the disclosure for preventing or treating a disease or condition
via partial or full inhibition of mTOR, wherein the medicament is
prepared for administration with another therapeutic agent. The
disclosure also provides the use of another therapeutic agent for
preventing or treating a disease or condition mediated by mTOR
inhibition, wherein the medicament is administered with a compound
of the disclosure.
[0301] The disclosure also provides a compound of formula (I),
formula (I)-A and formula (I)-B or a pharmaceutically acceptable
salt thereof for use in a method of preventing or treating a
disease or condition mediated by mTOR inhibition, wherein the
compound of the disclosure is prepared for administration with
another therapeutic agent. The disclosure also provides another
therapeutic agent for use in a method of preventing or treating a
disease or condition mediated by mTOR inhibition, wherein the other
therapeutic agent is prepared for administration with a compound of
formula (I), formula (I)-A and formula (I)-B or a pharmaceutically
acceptable salt thereof. The disclosure also provides a compound of
formula (I), formula (I)-A and formula (I)-B or a pharmaceutically
acceptable salt thereof for use in a method of preventing or
treating a disease or condition mediated by mTOR inhibition,
wherein the compound is administered with another therapeutic
agent. The disclosure also provides another therapeutic agent for
use in a method of preventing or treating a disease or condition
mediated by mTOR inhibition, wherein the other therapeutic agent is
administered with a compound of formula (I), formula (I)-A and
formula (I)-B or a pharmaceutically acceptable salt thereof.
[0302] The disclosure also provides the use of a compound of
formula (I), formula (I)-A and formula (I)-B or a pharmaceutically
acceptable salt thereof for preventing or treating a disease or
condition mediated by mTOR, wherein the patient has previously
(e.g. within 24 hours) been treated with another therapeutic agent.
The disclosure also provides the use of another therapeutic agent
for preventing or treating a disease or condition mediated by mTOR,
wherein the patient has previously (e.g. within 24 hours) been
treated with a compound of formula (I), formula (I)-A and formula
(I)-B or a pharmaceutically acceptable salt thereof.
[0303] Specific individual combinations which may provide
particular treatment benefits include a combination of a
compound
##STR00045##
or a pharmaceutically acceptable salt thereof and a catalytic mTOR
inhibitor, in particular one as mentioned above. In an embodiment,
the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0304] In an embodiment, the disclosure provides a product
comprising a compound
##STR00046##
or a pharmaceutically acceptable salt thereof and a catalytic mTOR
inhibitor, in particular one as mentioned above as a combined
preparation for simultaneous, separate or sequential use in
therapy. In an embodiment, the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0305] In an embodiment, the disclosure provides a pharmaceutical
composition comprising a compound
##STR00047##
or a pharmaceutically acceptable salt thereof and a catalytic mTOR
inhibitor, in particular one as mentioned above, and a
pharmaceutically acceptable carrier. In an embodiment, the compound
is (S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0306] In an embodiment, the other therapeutic agent is selected
from a CD4 lymphocyte depleting agent, such as an anti-CD4 antibody
or antigen-binding fragment thereof, such as a humanized anti-CD4
antibody, e.g. zanolimumab. Such a therapeutic agent may in
particular be used for the treatment of a proliferative disorder,
in particular cancer. See also U.S. Pat. Nos. 8,906,374 and
9,427,463 for such a combination therapy.
[0307] Specific individual combinations which may provide
particular treatment benefits include a combination of a
compound
##STR00048##
or a pharmaceutically acceptable salt thereof and a CD4 lymphocyte
depleting agent, in particular one as mentioned above. In an
embodiment, the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0308] In an embodiment, the disclosure provides a product
comprising a compound
##STR00049##
or a pharmaceutically acceptable salt thereof and a CD4 lymphocyte
depleting agent, in particular one as mentioned above as a combined
preparation for simultaneous, separate or sequential use in
therapy. In an embodiment, the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0309] In an embodiment, the disclosure provides a pharmaceutical
composition comprising a compound
##STR00050##
or a pharmaceutically acceptable salt thereof, a CD4 lymphocyte
depleting agent, in particular one as mentioned above, and a
pharmaceutically acceptable carrier. In an embodiment, the compound
is (S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0310] In an embodiment, the other therapeutic agent is selected
from an agent that modulates the activity of immunoinhibitory
proteins, such as PD-1/PDL-1, such as an anti-PD-1 antibody or
PDL-1 antibody. Anti-PD-1 antibodies which are useful as such a
therapeutic agent are as disclosed in U.S. Pat. No. 9,683,048. Such
a therapeutic agent may be used in the treatment of cancer, in
particular for cancer immunotherapy.
[0311] Specific individual combinations which may provide
particular treatment benefits include a combination of a
compound
##STR00051##
or a pharmaceutically acceptable salt thereof and an agent that
modulates the activity of immunoinhibitory proteins, such as PD-1,
in particular one as mentioned above. In an embodiment, the
compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0312] In an embodiment, the disclosure provides a product
comprising a compound
##STR00052##
or a pharmaceutically acceptable salt thereof and an agent that
modulates the activity of immunoinhibitory proteins, such as PD-1,
in particular one as mentioned above as a combined preparation for
simultaneous, separate or sequential use in therapy. In an
embodiment, the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
[0313] In an embodiment, the disclosure provides a pharmaceutical
composition comprising
##STR00053##
or a pharmaceutically acceptable salt thereof, an agent that
modulates the activity of immunoinhibitory proteins, such as PD-1,
in particular one as mentioned above, and a pharmaceutically
acceptable carrier. In an embodiment, the compound is
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 1) or a pharmaceutically acceptable salt thereof. In an
embodiment, the compound is
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
(Example 2) or a pharmaceutically acceptable salt thereof.
Examples
[0314] The disclosure sets for the following examples. The
synthetic and biological examples described in this application are
offered to illustrate the compounds, pharmaceutical compositions,
and methods provided herein and are not to be construed in any way
as limiting their scope.
[0315] The compounds provided herein can be prepared from readily
available starting materials using modifications to the specific
synthesis protocols set forth below that would be well known to
those of skill in the art. It will be appreciated that where
typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the
particular reactants or solvents used, but such conditions can be
determined by those skilled in the art by routine optimization
procedures.
[0316] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions. The
choice of a suitable protecting group for a particular functional
group as well as suitable conditions for protection and
deprotection are well known in the art. For example, numerous
protecting groups, and their introduction and removal, are
described in Greene et al., Protecting Groups in Organic Synthesis,
Second Edition, Wiley, New York, 1991, and references cited
therein.
[0317] Rapamycin and its derivatives, for example, compounds of
formula (I), formula (I)-A and formula (I)-B exist as a solvent and
pH dependent equilibrium of six-membered and seven-membered
hemi-ketal forms shown below as E and F (Schemes 4 and 5). See The
Journal of Antibiotics (Tokyo) (1991) 44(6):688-90; and Tetrahedron
Letters (1992) 33(33):4139-4142. Rapamycin and its derivatives also
exist as a mixture of cis- and trans-amides shown below as E, H, J
and K (Schemes 4 and 5). [See Mierke, D. F. Schmieder, P., Karuso,
P. and Kessler, H. (1991), Conformational Analysis of the cis- and
trans-isomers of FK506 by NMR and Molecular Dynamics. Helvetica
Chimica Acta, 74: 1027-1047.] The NMR characterization data shown
in the examples corresponds only to the major equilibrium form
observed under the reported deutero solvent conditions.
##STR00054##
wherein:
[0318] R.sub.1 is selected from the group consisting of
hydroxy,
##STR00055##
and, and
[0319] R.sub.2 is selected from the group consisting of
##STR00056##
wherein
[0320] m is 0, 1, 2 or 3;
[0321] n is 1, 2 or 3;
[0322] o is 1, 2, 3, 4, 5 or 6;
[0323] p is 1, 2, 3, 4 or 5
[0324] q is 1, 2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4,
5 or 6;
[0325] r is 2, 3 or 4;
[0326] s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6;
[0327] X is O, S, NR.sub.6 or SO.sub.2;
[0328] R.sub.3 is hydrogen, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl;
[0329] R.sub.4 is hydrogen and R.sub.5 is hydrogen, hydroxy or
cyano or R.sub.4 and R.sub.5 together form .dbd.O; and
[0330] R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
[0331] In an embodiment, R.sub.1 is hydroxy.
[0332] In an embodiment, R.sub.2 is
##STR00057##
and n is 1, 2 or 3. In an embodiment, R.sub.2 is
##STR00058##
##STR00059##
wherein:
[0333] R.sub.1 is
##STR00060##
and
[0334] R.sub.2 is selected from the group consisting of
##STR00061##
wherein
[0335] m is 0, 1, 2 or 3;
[0336] n is 1, 2 or 3;
[0337] o is 1, 2, 3, 4, 5 or 6;
[0338] p is 1, 2, 3, 4 or 5
[0339] q is 1, 2, 3, 4 or 5 wherein the sum of p and q is 2, 3, 4,
5 or 6;
[0340] r is 2, 3 or 4;
[0341] s is 2, 3 or 4 wherein the sum of r and s is 4, 5 or 6;
[0342] X is O, S, NR.sub.6 or SO.sub.2;
[0343] R.sub.3 is hydrogen, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl or phenylC.sub.0-6alkyl;
[0344] R.sub.4 is hydrogen and R.sub.5 is hydrogen, hydroxy or
cyano or R.sub.4 and R.sub.5 together form .dbd.O; and
[0345] R.sub.6 is hydrogen, C.sub.1-6alkyl,
C.sub.3-8cycloalkylC.sub.0-6alkyl, phenylC.sub.0-6alkyl,
C.sub.1-6alkyl-CO--, C.sub.3-8cycloalkylC.sub.0-6alkyl-CO--,
C.sub.1-6alkyl-SO.sub.2-- or
C.sub.3-8cycloalkylC.sub.0-6alkyl-SO.sub.2--.
[0346] In an embodiment, R.sub.2 is
##STR00062##
and n is 1, 2 or 3. In an embodiment, R.sub.2 is
##STR00063##
[0347] In an embodiment, compounds of formula (I), formula (I)-A
and formula (I)-B exist as a solvent and pH dependent equilibrium
of six-membered and seven-membered hemi-ketal forms shown below as
E-1 and F-1 (Scheme 6). In an embodiment, compounds of formula (I),
formula (I)-A and formula (I)-B exist as a mixture of cis- and
trans-amides E-1 and H-1.
##STR00064##
Preparation of Compounds
[0348] Compounds of the disclosure can be prepared as described in
the following Examples.
Abbreviations
[0349] 4-EP 4-ethyl pyridine AcOH acetic acid ACN acetonitrile Aq
aqueous
C Celsius
[0350] d doublet dd doublet of doublets DCM dichloromethane
DIPEA N,N-diisopropylethylamine
[0351] DMSO dimethylsulfoxide EDTA ethylenediaminetetraacetic acid
ESIMS Electrospray ionization mass spectrometry EtOAc ethyl acetate
EtOH ethanol FA formic acid g gram h hour(s) HPLC high performance
liquid chromatography HRMS High resolution Mass Spectrometry iPrOH
2-propanol or isopropanol L liter LC liquid chromatography LCMS
liquid chromatography and mass spectrometry MeOH methanol MS mass
spectrometry M molar m multiplet min minutes mL milliliter(s) .mu.M
micromolar m/z mass to charge ratio N.sub.2 nitrogen gas nM
nanomolar NMR nuclear magnetic resonance .sup.1HNMR: Proton Nuclear
Magnetic Resonance spectroscopy
PEI Polyethylenimine
[0352] PPU Propyl-pyridyl-urea pTsOH p-toluenesulfonic acid prep
preparative rac racemic rpm revolutions per minute r.t. room
temperature s singlet sat. saturated
SFC Supercritical Fluid Chromatography
[0353] t triplet TCEP tris(2-carboxyethyl)phosphine TFA
trifluoroacetic acid THF tetrahydrofuran TLC thin layer
chromatography vol. volume
Methods Employed in the Purification of the Examples
[0354] Purification of intermediates and final products was carried
out via either normal or reverse phase chromatography.
[0355] Normal phase chromatography was carried out using prepacked
SiO.sub.2 cartridges (e.g., RediSep@ Rf columns from Teledyne Isco,
Inc.) eluting with gradients of appropriate solvent systems (e.g.,
hexanes and ethyl acetate; DCM and MeOH; or unless otherwise
indicated).
SFC was carried out using the methods described below: Method 1:
Princeton PPU 5 .mu.m (100A) column (30.times.250 mm);
CO.sub.2/MeOH Method 2: Princeton 4-EP 5 .mu.m (60A) column
(30.times.250 mm); CO.sub.2/MeOH Method 3: Reprospher PEI 5 .mu.m
(100A) column (30.times.250 mm); CO.sub.2/MeOH Gradients were
selected based on analytical separation. Reverse phase preparative
HPLC was carried out using the methods described below: Method
1:(Agilent) Phenomenex Luna C18; 5 .mu.m column (30.times.250 mm);
0.1% formic acid and 5% water in acetonitrile; 0.1% formic acid and
5% acetonitrile in water. Gradients were selected based on
analytical separation. Method 2: (EZprep) YMC Actus Triart C18; 5
.mu.m column (20.times.150 mm); acetonitrile/water. Gradients were
selected based on analytical separation. Chiral preparative HPLC
was carried out using the methods described below: Method 1:
Chiralpak IC; 5 .mu.m column (20.times.250 mm); n-Heptane/DCM/EtOH
Method 2: Chiralpak ID; 5 .mu.m column (20.times.250 mm);
n-Heptane/DCM/iPrOH Gradients were selected based on analytical
separation. LC/MS method: Column: Acquity UPLC BEH C18, 130
Angstrom, 1.7 uM, 2.1 mm.times.50 mm
Temperature: 50.degree. C.
Injection: 1 uL
[0356] Solvent A: water+5 mM ammonium hydroxide Solvent B:
acetonitrile+5 mM ammonium hydroxide
Gradient:
TABLE-US-00002 [0357] Time (min) Flow Rate A % B % Curve Initial
1.000 98.0 2.0 Initial 4.40 1.0 2.0 98.0 6 5.15 1.0 2.0 98.0 6 5.19
1.0 98.0 2.0 6
.sup.1H NMR Instruments:
[0358] Bruker UltraShield.TM. Advance III HD 400 MHz with a
cryo-DCI probe. Data were processed with MestReNova 11.0
software.
Preparation of Intermediates 1 to 7
Intermediate 1: C32-Deoxo-Rapamycin
##STR00065##
[0360] Intermediate 1 was prepared according to procedures known in
the literature including those disclosed in US Patent Publication
No. 005985890 and WO2007/085400 A1, each of which is incorporated
by reference herein in its entirety.
Intermediate 2: RAD001 (Everolimus; Afinitor@)
##STR00066##
[0362] Intermediate 2 was prepared according to procedures known in
the literature including those disclosed in WO2012103959, which is
incorporated herein by reference herein in its entirety.
Intermediate 3:
##STR00067##
[0363] Intermediate 3 was prepared in two steps through
Intermediate A as shown below:
##STR00068##
Step 1. Synthesis of Intermediate A
[0364] 2-((tert-butyldimethylsilyl)oxy)ethanol (0.471 g, 2.67 mmol)
was dissolved in anhydrous toluene (0.95 mL) in a reaction vial.
The vial was capped and then was vacuum purged with nitrogen.
NN-diisopropylethylamine (DIPEA) (0.490 ml, 2.81 mmol) was added
via syringe. The mixture was chilled to 0.degree. C. in an
ice-water bath. Triflic anhydride (Tf.sub.2O) (0.438 ml, 2.59 mmol)
was added dropwise at 0.degree. C. over a period of about two
minutes. The reaction mixture was stirred at 0.degree. C. for 30
minutes.
[0365] The vial was lifted out of the cold bath. DIPEA (0.490 ml,
2.81 mmol) was added via syringe. The vial was opened and solid
Intermediate 1 (0.600 g, 0.667 mmol) was quickly added in one
portion. The vial was quickly recapped and the mixture was vacuum
purged with nitrogen. Toluene (0.5 mL) was added.
[0366] The reaction was stirred at 40.degree. C. under nitrogen
overnight. The reaction was diluted with saturated aqueous
NaHCO.sub.3. The quenched mixture was extracted with EtOAc five
times. The organic extracts were combined, dried over
Na.sub.2SO.sub.4, vacuum filtered through celite and concentrated
to afford a waxy white solid crude product.
[0367] The crude product was purified by silica gel flash column
chromatography (0-35% acetone-heptane, gradient elution, 40 g
silica column, TLC in 35% acetone-heptane, visualize under UV) to
give the desired intermediate A (0.245 g, 0.231 mmol, 34.7% yield)
as a glass which was immediately used "as is" in the following
step.
[0368] Intermediate A: ESIMS [M+NH4] 1076.1, ESIMS [M-H]
1056.0.
Step 2. Synthesis of Intermediate 3
[0369] Intermediate A (0.135 g, 0.128 mmol) was dissolved in
anhydrous THF (1.2 mL) in a glass reaction vial. The vial was
capped and the mixture was vacuum purged twice with nitrogen. The
mixture was chilled to 0.degree. C. in an ice-water bath.
HF-Pyridine (0.12 mL, 1.332 mmol) was added dropwise via syringe
over a period of 30 seconds. The reaction was stirred at 0.degree.
C. for 60 minutes.
[0370] The reaction mixture was added dropwise to a saturated
aqueous NaHCO.sub.3 solution. The quenched mixture was extracted
five times with EtOAc. The organic extracts were combined, dried
over Na.sub.2SO.sub.4, vacuum filtered through celite and
concentrated to afford a white solid crude product.
[0371] The crude product was purified by silica gel flash column
chromatography (0-50% Acetone-heptane gradient elution, 40 g silica
column, TLC in 50% Acetone-heptane, visualize under UV) to give
Intermediate 3 (0.087 g, 0.092 mmol, 72.2% yield) as a white
solid.
Intermediate 3: ESIMS [M+NH4] 962.0, ESIMS [M-H] 943.0
[0372] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.46-6.25 (m,
2H), 6.19-6.09 (m, 1H), 5.91 (m, 1H), 5.55 (m, 1H), 5.34-5.26 (m,
1H), 5.26-5.15 (m, 1H), 4.88-4.64 (m, 2H), 4.11 (m, 1H), 3.94-3.82
(m, 1H), 3.79 (m, 1H), 3.73-3.63 (m, 3H), 3.63-3.47 (m, 4H), 3.45
(m, 4H), 3.33 (m, 3H), 3.28-3.15 (m, 2H), 3.13 (s, 3H), 3.08 (m,
1H), 2.80 (m, 1H), 2.32 (m, 3H), 2.14 (m, 2H), 2.07-1.94 (m, 2H),
1.94-1.72 (m, 5H), 1.72-1.56 (m, 8H), 1.56-1.40 (m, 3H), 1.39-1.21
(m, 7H), 1.21-1.09 (m, 1H), 1.08-0.99 (m, 8H), 0.98-0.83 (m, 9H),
0.73 (q, J=12.0 Hz, 1H).
[0373] Intermediate 4
##STR00069##
[0374] Intermediate 1 (0.233 g, 0.259 mmol) was combined with
2,6-di-tert-butyl-4-methylpyridine (0.425 g, 2.071 mmol) in
anhydrous dichloromethane (2.6 mL). The reaction mixture was vacuum
purged once with nitrogen. The reaction mixture was chilled to
0.degree. C. in an ice-water bath. Solid dimethylphosphinic
chloride (0.146 g, 1.294 mmol) was added in one portion. The
reaction mixture was stirred at 0.degree. C. for 80 minutes.
[0375] The reaction was diluted with saturated aqueous NaHCO.sub.3
and was extracted several times with EtOAc. The organic extracts
were combined, dried over Na.sub.2SO.sub.4, decanted and
concentrated to give a colorless tar crude product (0.768 g).
[0376] The crude product was purified by silica gel flash column
chromatography (0-80% Acetone-heptane gradient elution, 24 g silica
column, TLC in 80% EtOAc-heptane, visualize under UV). Product
containing fractions were pooled and concentrated to give
Intermediate 4 (0.087 g, 0.089 mmol, 34.4% yield) as a white
solid.
Intermediate 4: ESIMS [M+NH4] 993.7, ESIMS [M-H] 974.7.
[0377] HRMS: Calculated: 999.5812 (as sodium adduct). Found:
999.5807.
[0378] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 6.47-6.26 (m,
2H), 6.22-6.08 (m, 1H), 6.02-5.83 (m, 1H), 5.54 (m, 1H), 5.35-5.26
(m, 1H), 5.21 (m, 1H), 4.85-4.76 (m, 1H), 4.12 (m, 2H), 3.93-3.81
(m, 1H), 3.67 (t, J=7.7 Hz, 1H), 3.62 (d, J=6.7 Hz, 1H), 3.60-3.53
(m, 1H), 3.53-3.44 (m, 1H), 3.42-3.36 (m, 3H), 3.32 (m, 3H),
3.28-3.18 (m, 1H), 3.13 (m, 3H), 3.05 (m, 1H), 2.82 (m, 1H),
2.42-2.21 (m, 3H), 2.16-2.08 (m, 3H), 1.99 (m, 1H), 1.95-1.83 (m,
1H), 1.83-1.72 (m, 4H), 1.71-1.57 (m, 9H), 1.57-1.43 (m, 12H), 1.39
(m, 1H), 1.34-1.20 (m, 4H), 1.20-1.10 (m, 1H), 1.05 (m, 4H), 1.00
(d, J=6.5 Hz, 3H), 0.95 (dd, J=6.6, 2.1 Hz, 3H), 0.92 (d, J=6.6 Hz,
3H), 0.91-0.84 (m, 4H), 0.77 (q, J=12.1 Hz, 1H).
Intermediate 5
##STR00070##
[0380] Intermediate 1 (4.372 g, 4.86 mmol) was dissolved in
anhydrous dichloromethane (20 mL). Anhydrous toluene was added (20
mL). The reaction mixture was evaporated to dryness on a rotary
evaporator. This azeotropic drying process was repeated twice
more.
[0381] The dried starting material was combined with 2,6-lutidine
(1.388 ml, 11.91 mmol) in anhydrous dichloromethane (58 mL). The
flask was capped and the mixture was twice vacuum purged with
nitrogen. The mixture was chilled to -30 C in an
acetonitrile/dry-ice bath.
[0382] Triflic anhydride (1.209 ml, 7.16 mmol) was added dropwise
via syringe over a period of four minutes. The reaction mixture was
stirred at -30.degree. C. for 30 minutes. The reaction mixture was
transferred to a 0.degree. C. ice-water bath and was stirred for 20
minutes at 0.degree. C.
[0383] The reaction mixture was placed on the rotary evaporator and
was concentrated without heat. Isopropyl acetate (22 mL) was added.
Tetrazole (1.170 g, 16.71 mmol) was added in one portion. The flask
was quickly capped and was twice vacuum purged with nitrogen.
N,N-diisopropylethylamine (4.18 ml, 23.94 mmol) was added via
syringe over a period of one minute. The reaction mixture was
stirred overnight at room temperature.
[0384] The reaction mixture was concentrated on a rotary
evaporator. The concentrate was purified by normal-phase silica gel
flash column chromatography (0 to 40% acetone-heptane gradient
elution, 80 g silica column, TLC 40% acetone-heptane, visualize
under UV).
[0385] The second eluting peak fractions (as determined by UV
absorbance at 279 nm) were pooled and concentrated to give
Intermediate 5 (2.194 g, 2.304 mmol, 47.4% yield) as a white
solid.
Intermediate 5: ESIMS [M+NH4] 969.8, ESIMS [M-H] 950.8.
[0386] .sup.1H NMR (400 MHz, DMSO-de) .delta. 9.33 (d, J=6.4 Hz,
1H), 6.58-6.41 (m, 2H), 6.35-6.14 (m, 2H), 6.09-5.98 (m, 1H),
5.55-5.43 (m, 1H), 5.19 (m, 1H), 5.05 (m, 1H), 5.00-4.93 (m, 1H),
4.87-4.79 (m, 1H), 4.67-4.56 (m, 1H), 3.98-3.87 (m, 1H), 3.87 (d,
J=6.9 Hz, 1H), 3.61 (m, 2H), 3.55 (dd, J=11.8, 1.9 Hz, 1H),
3.49-3.38 (m, 1H), 3.31-3.17 (m, 4H), 3.10 (m, 4H), 3.04 (s, 3H),
2.88-2.75 (m, 1H), 2.29-2.09 (m, 3H), 2.07-1.86 (m, 3H), 1.88-1.60
(m, 9H), 1.59-1.44 (m, 7H), 1.43-1.01 (m, 11H), 0.96 (t, J=7.1 Hz,
5H), 0.95-0.77 (m, 7H), 0.72 (m, 4H).
Intermediate 6:
##STR00071##
[0388] 2-ethoxyethanol (0.538 ml, 5.55 mmol) was combined in
anhydrous toluene (2.0 mL) and anhydrous dioxane (0.22 mL).
N,N-diisopropylethylamine (1.067 ml, 6.11 mmol) was added via
syringe. The reaction mixture was twice vacuum purged with
nitrogen. The mixture was chilled to 0.degree. C. in an ice-water
bath.
[0389] Triflic anhydride (0.901 ml, 5.33 mmol) was added dropwise
via syringe over a period of two minutes. The reaction was stirred
at 0.degree. C. for 15 minutes. The cold bath was removed and the
reaction was allowed to equilibrate to room temperature over 15
minutes.
[0390] N,N-diisopropylethylamine (1.067 ml, 6.11 mmol) was added
dropwise via syringe over a period of 30 seconds. Intermediate 1
(1.00 g, 1.111 mmol) was added in one portion. The reaction mixture
was quickly capped and vacuum purged with nitrogen. Anhydrous
toluene (2.0 mL) and anhydrous dioxane (0.22 mL) were added via
syringe. The reaction mixture was stirred at 55.degree. C. for 24
hours.
[0391] The reaction mixture was diluted with brine and was
extracted several times with EtOAc. The organic extracts were
combined, dried over Na.sub.2SO.sub.4, decanted and concentrated to
give a colorless tar crude product (4.25 g).
[0392] The crude product was purified by silica gel flash column
chromatography (0-50% Acetone-heptane gradient elution, 80 g silica
column, TLC in 40% Acetone-heptane, visualize under UV). Product
containing fractions were pooled and concentrated to give
Intermediate 6 (0.443 g, 0.456 mmol, 41.0% yield) as a colorless
glass.
Intermediate 6: ESIMS [M-H] 970.9.
[0393] HRMS: calculated--989.6678 (as ammonium adduct);
found--989.6655.
[0394] calculated--994.6232 (as sodium adduct);
found--994.6215.
Intermediate 7:
##STR00072##
[0395] Intermediate 7 was prepared in two steps through
Intermediate B as shown below:
##STR00073##
Step 1. Synthesis of Intermediate B
[0396] 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (0.400 g,
2.296 mmol) and triethylamine (0.320 ml, 2.296 mmol) were combined
dissolved in anhydrous THF (7.6 mL). The reaction mixture was
chilled to 0.degree. C. 2,4,6-Trichlorobenzoyl chloride (0.359 ml,
2.296 mmol) was added via syringe. The reaction was stirred at
0.degree. C. for ten minutes. The cold bath was removed and the
reaction was stirred at room temperature for 2.5 hours. A white
solid precipitated.
[0397] The reaction mixture was filtered through a syringe filter.
The filter was rinsed with THF (2 mL). The filtrates were combined
and concentrated on the rotary evaporator leaving a colorless
tar.
[0398] To the concentrate, toluene (7.6 mL) was added. Intermediate
1 (1.447 g, 1.607 mmol) was added in one portion followed by
4-dimethylaminopyridine (0.281 g, 2.296 mmol). The reaction mixture
was stirred at room temperature overnight.
[0399] The mixture was diluted with water and was extracted four
times with EtOAc. The organic extracts were combined, dried over
Na.sub.2SO.sub.4, decanted and concentrated to give a yellow tar
crude product (2.15 g).
[0400] The crude product was purified by silica gel flash column
chromatography (0-40% Acetone-heptane gradient elution, 40 g silica
column, TLC in 40% Acetone-heptane, visualize under UV). Product
containing fractions were pooled and concentrated to give
Intermediate B (0.438 g, 0.415 mmol, 18.1% yield) as a white
foam.
[0401] Intermediate B: ESIMS [M+NH4] 1074.0, ESIMS [M-H]
1055.1.
[0402] Step 2. Synthesis of Intermediate 7 Intermediate B (0.431 g,
0.408 mmol) was dissolved in THF (4 mL). HCl (1M, aq.) (2.0 ml,
2.00 mmol) was added via syringe. The reaction was stirred at room
temperature for 36 hours.
[0403] The reaction mixture was diluted with water and was
extracted four times with EtOAc. The organic extracts were
combined, dried over Na.sub.2SO.sub.4, decanted and concentrated to
give a faint yellow tar. The tar was dissolved in dichloromethane
and was diluted with heptane. The mixture was concentrated and
dried on high vacuum to give Intermediate 7 (0.407 g, 0.400 mmol,
98% yield) as a white solid.
Intermediate 7: ESIMS [M+NH4] 1034.0, ESIMS [M-H] 1015.0.
[0404] HRMS calculated--1033.6576 (as ammonium adduct);
found--1033.6588.
[0405] calculated--1038.6130 (as sodium adduct);
found--1038.6138.
Example 1:
(S)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
Example 2:
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-rapamycin
##STR00074##
[0407] To a stirring solution of Intermediate 1 (2.0 g, 2.222 mmol)
and isothiazolidine 1,1-dioxide (4.04 g, 33.3 mmol, 15 equivalents)
in anhydrous dichloromethane (5.6 mL) was added p-toluenesulphonic
acid.cndot.H.sub.2O (0.042 g, 0.222 mmol, 0.1 equivalent) in one
portion. The reaction was stirred at ambient temperature under a
nitrogen atmosphere for 34 minutes. The entire reaction mixture was
directly chromatographed by over silica (gradient elution from 100%
heptane to 40% acetone-heptane) to give a product mixture of both
diastereomers in a ratio of about 3:1 by UV absorbance at 279 nm on
LC/MS analysis.
[0408] The diastereomeric mixture was separated by normal phase
chromatography on silica (gradient elution from 100%
dichloromethane to 40% acetonitrile-dichloromethane).
[0409] The first eluting diastereomer (Rf .about.0.23 on silica TLC
developed in 30% acetonitrile-dichloromethane) affords Example 1
(S)-diastereomer as a white solid.
Example 1: ESIMS [M+NH4] 1006.7, ESIMS [M-H] 987.8
[0410] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 6.43 (dd,
J=14.9, 10.4 Hz, 1H), 6.35 (dd, J=14.9, 10.7 Hz, 1H), 6.16 (dd,
J=15.1, 10.2 Hz, 1H), 6.06-6.01 (m, 1H), 5.67 (dd, J=15.2, 8.6 Hz,
1H), 5.35 (dd, J=6.4, 1.8 Hz, 1H), 5.26 (d, J=9.6 Hz, 1H), 4.83
(td, J=6.6, 4.7 Hz, 1H), 4.12 (d, J=7.4 Hz, 1H), 3.88 (dd, J=11.1,
5.1 Hz, 1H), 3.84-3.77 (m, 1H), 3.63-3.60 (m, 2H), 3.51 (d, J=7.5
Hz, 1H), 3.46 (s, 3H), 3.45-3.42 (m, 3H), 3.35 (s, 3H), 3.29-3.14
(m, 3H), 3.06-2.96 (m, 2H), 2.93 (ddd, J=10.4, 6.4, 1.5 Hz, 1H),
2.44 (tt, J=8.6, 6.2 Hz, 1H), 2.37-2.23 (m, 4H), 2.22-2.14 (m, 2H),
2.03 (dt, J=12.3, 3.8 Hz, 1H), 1.96 (pd, J=6.4, 5.7, 3.5 Hz, 2H),
1.92-1.82 (m, 3H), 1.80-1.76 (m, 2H), 1.75 (d, J=1.2 Hz, 4H), 1.72
(d, J=3.1 Hz, 1H), 1.68 (d, J=1.3 Hz, 3H), 1.65-1.53 (m, 4H),
1.48-1.17 (m, 9H), 1.07 (d, J=6.5 Hz, 1H), 1.06 (s, 3H), 1.04 (d,
J=7.3 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H), 0.99 (dd, J=6.7, 2.3 Hz,
3H), 0.95 (d, J=6.8 Hz, 3H), 0.74 (q, J=11.9 Hz, 1H).
[0411] The second eluting diastereomer (Rf .about.0.16 on silica
TLC developed in 30% acetonitrile-dichloromethane) affords Example
2 (R)-diastereomer as a white solid
Example 2: ESIMS [M+NH4] 1006.9, ESIMS [M-H] 988.1
[0412] .sup.1H NMR (Chloroform-d) .delta. 6.48 (dd, J=14.7, 10.9
Hz, 1H), 6.24 (dd, J=14.6, 10.6 Hz, 1H), 6.16 (dd, J=14.9, 10.6 Hz,
1H), 6.01 (d, J=11.0 Hz, 1H), 5.38 (dd, J=14.9, 9.8 Hz, 1H), 5.23
(dd, J=6.2, 2.0 Hz, 1H), 5.12 (d, J=9.9 Hz, 1H), 4.73 (dd, J=12.1,
2.9 Hz, 1H), 4.65 (dt, J=8.3, 3.9 Hz, 1H), 4.14 (d, J=6.7 Hz, 1H),
3.97 (m, 1H), 3.74 (d, J=6.7 Hz, 1H), 3.62 (qd, J=13.9, 12.7, 5.5
Hz, 2H), 3.42 (s, 3H), 3.39 (m, 1H), 3.31 (s, 3H), 3.24 (ddd,
J=12.2, 7.5, 4.6 Hz, 1H), 3.10 (td, J=8.2, 3.8 Hz, 1H), 3.08 (s,
1H), 3.02-2.97 (m, 1H), 2.97-2.92 (m, 1H), 2.83-2.71 (m, 1H), 2.42
(ddt, J=13.1, 9.5, 6.4 Hz, 1H), 2.34 (d, J=4.3 Hz, 1H), 2.31 (s,
1H), 2.28-2.23 (m, 1H), 2.22-2.19 (m, 1H), 2.19 (s, 2H), 2.12-2.08
(m, 2H), 2.03-1.99 (m, 1H), 1.90 (s, 3H), 1.88 (s, 1H), 1.79 (s,
1H), 1.77 (s, 1H), 1.76 (s, 1H), 1.72-1.68 (m, 1H), 1.48 (s, 1H),
1.46 (s, 1H), 1.40 (d, J=3.0 Hz, 1H), 1.38 (s, 1H), 1.66 (d, J=3.0
Hz, 2H), 1.64 (d, J=2.9 Hz, 1H), 1.62 (s, 2H), 1.62 (s, 2H),
1.58-1.53 (m, 1H), 1.37 (s, 1H), 1.36 (d, J=2.3 Hz, 1H), 1.33 (d,
J=2.9 Hz, 1H), 1.30 (dd, J=6.7, 1.8 Hz, 1H), 1.28 (s, 2H), 1.28 (s,
2H), 1.24 (s, 1H), 1.09 (s, 1H), 1.07 (d, J=6.6 Hz, 3H), 1.05 (d,
J=6.6 Hz, 3H), 1.01 (d, J=3.2 Hz, 1H), 0.95 (d, J=6.8 Hz, 3H), 0.92
(s, 1H), 0.92-0.90 (m, 3H), 0.88 (d, J=6.8 Hz, 2H), 0.66 (q, J=12.0
Hz, 1H)
[0413] The absolute configurations of the C16 substituents in
Example 1 and Example 2 were determined by X-ray crystallographic
co-crystallization with FKBP12. [See Stuart L. Schrieber and Jon
Clardy, et al., Atomic Structure of the Rapamycin Humano
Immunophilin FKBP-12 Complex, J. Am. Chem. Soc., 1991, 113,
7433-7434.] Crystal structures are depicted in FIGS. 1A and 1B and
FIGS. 2A and 2B.
[0414] Pure FKBP12 (1-108) protein was concentrated to 9 mg/mL in
50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP. The complex
for co-crystallization was prepared by mixing the protein with 3 mM
of compound (from a 50 mM stock prepared in 90% dDMSO, 10% D2O).
The complex was incubated for two hours at 4.degree. C. then was
centrifuged at 10000 rpm for 2 minutes to remove any potential
pellet before crystallization. Co-crystals were obtained at
20.degree. C. and by sitting drop vapor diffusion using microseed
matrix screening [Allan D'Arcy et al., An automated microseed
matrix-screening method for protein crystallization, Acta Cryst.,
(2007) D63, 550-554.] The drops were made up of 200 nL of protein
solution, 160 nL of well solution and 40 nL seed-stock. Crystals
appeared within a few days in the A1 condition of the commercially
available "Ammonium sulfate" screen from Qiagen. The reservoir
solution consisted of 2.2M Ammonium sulfate. Crystals were
cryo-protected in reservoir solution supplemented with 20% Ethylene
Glycol and flash-frozen into liquid nitrogen. Data was collected at
the Swiss Light Source Facility (SLS, Villigen, Switzerland) on
beamline X10SA.
[0415] The data were processed with XDS (Kabsch, W. (2010), XDS.
Acta Cryst. D, 66: 125-132). The structures were determined by
molecular replacement (Collaborative Computational Project, Number
4 (1994). Acta Cryst. D50, 760-763) using previous FKBP12 X-ray
structures as search model. Programs REFMAC (Murshudov G N, Skubak
P, Lebedev A A, et al., REFMAC5 for the refinement of
macromolecular crystal structures. Acta Crystallographica Section
D: Biological Crystallography. 2011; 67 (Pt 4):355-367) and COOT
(Emsley P, Lohkamp B, Scott W G, Cowtan K. Features and development
of Coot, Acta Crystallographica Section D: Biological
Crystallography. 2010; 66 (Pt 4):486-501) were used for refinement
and model (re)building.
[0416] In the following examples, the absolute stereochemistry of
the C16 substituent was not determined by X-ray co-crystallization
and is not known. In some examples, only the major diastereomer
product from the reaction was isolated and characterized. In other
examples, each diastereomer was isolated and characterized without
absolute stereochemical assignment.
Example 3: C16-(4-oxoazetidin-2-yl)-C32-deoxo-rapamycin
##STR00075##
[0417] Example 3
[0418] *Absolute stereochemistry at C16 not assigned
[0419] To a solution of Intermediate 1 (130 mg, 0.144 mmol, 1.0 eq)
and azetidin-2-one (205 mg, 2.89 mmol, 20 eq) in acetonitrile (3
mL) was added para-toluenesulfonic acid monohydrate (82 mg, 0.433
mmol, 3 eq). The reaction mixture was stirred at room temperature
for 15 min and then was diluted with H.sub.2O and was extracted
with ethyl acetate. The organic extract was evaporated under
reduced pressure. The crude product was purified by
preparative-HPLC chromatography (method 1) followed by a SFC
purification (method 1) to give of Example 3 (2.5 mg, 1.7% yield)
as a white solid.
Example 3: ESIMS [M-H] 938.0
Exact Mass: 938.59
[0420] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.45 (dd,
J=14.0, 11.0 Hz, 1H), 6.28-6.11 (m, 2H), 5.98 (d, J=10.9 Hz, 1H),
5.95-5.85 (m, 1H), 5.50 (dd, J=14.2, 9.5 Hz, 1H), 5.06 (d, J=4.7
Hz, 1H), 5.02-4.97 (m, 1H), 4.94 (d, J=9.8 Hz, 1H), 4.63-4.53 (m,
2H), 4.34-4.22 (m, 1H), 3.99-3.92 (m, 1H), 3.84-3.74 (m, 1H),
3.61-3.53 (m, 1H), 3.49 (d, J=7.3 Hz, 1H), 3.46-3.39 (m, 1H),
3.37-3.25 (m, 3H), 3.22-3.12 (m, 4H), 3.10-3.04 (m, 1H), 3.04-2.95
(m, 1H), 2.92-2.65 (m, 4H), 2.27-2.11 (m, 2H), 2.10-1.99 (m, 2H),
1.96-1.87 (m, 1H), 1.84-1.70 (m, 5H), 1.69-1.37 (m, 15H), 1.37-1.05
(m, 8H), 1.03-0.93 (m, 4H), 0.90 (d, J=6.4 Hz, 3H), 0.88-0.83 (m,
5H), 0.79 (d, J=6.7 Hz, 3H), 0.75 (d, J=6.7 Hz, 3H), 0.59 (q,
J=11.8 Hz, 1H).
Example 4:
C16-(3-methylimidazolidin-2-one-1-yl)-C32-deoxo-rapamycin
##STR00076##
[0421] Example 4
[0422] *Absolute stereochemistry at C16 not assigned
[0423] To a solution of Intermediate 1 (100 mg, 0.111 mmol, 1.0 eq)
and 1-methylimidazolidin-2-one (167 mg, 1.66 mmol, 15 eq) in DCM (2
mL) was added para-toluenesulfonic acid monohydrate (63 mg, 0.333
mmol, 3 eq). The reaction mixture was stirred at room temperature
for 24 h. The mixture was diluted with H.sub.2O and was extracted
with DCM. The organic extract was evaporated under reduced
pressure. The crude product was purified by preparative-HPLC
chromatography (method 1) to afford Example 4 (9.0 mg, 8% yield) as
a white solid.
Example 4: ESIMS [M-H] 966.5
Exact Mass: 967.61
[0424] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. 0.59 (q, J=11.9
Hz, 1H), 0.74 (d, J=6.7 Hz, 3H), 0.79 (d, J=6.7 Hz, 3H), 0.80-0.90
(m, 8H), 0.92-1.00 (m, 4H), 1.04-1.10 (m, 1H), 1.12-1.34 (m, 7H),
1.34-1.67 (m, 14H), 1.69 (s, 3H), 1.71-1.78 (m, 2H), 1.88-1.94 (m,
1H), 1.96-2.08 (m, 3H), 2.10-2.25 (m, 2H), 2.60-2.69 (m, 4H),
2.80-2.94 (m, 2H), 3.07-3.20 (m, 5H), 3.21-3.26 (m, 2H), 3.33 (s,
3H), 3.41-3.48 (m, 1H), 3.56-3.66 (m, 3H), 3.99 (dd, J=6.5, 3.5 Hz,
1H), 4.53-4.63 (m, 3H), 4.95-5.00 (m, 2H), 5.07 (d, J=4.8 Hz, 1H),
5.35 (s, 1H), 5.45 (dd, J=14.9, 9.8 Hz, 1H), 5.98 (d, J=11.0 Hz,
1H), 6.15 (dd, J=14.8, 10.7 Hz, 1H), 6.23 (dd, J=14.6, 10.7 Hz,
1H), 6.44 (dd, J=14.6, 11.0 Hz, 1H).
Example 5:
C16-(2-hydroxyethyl)-2-oxoimidazolidin-1-yl)-C32-deoxo-rapamyci-
n
##STR00077##
[0425] Example 5
[0426] *Absolute stereochemistry at C16 not assigned
[0427] To a solution of Intermediate 1 (100 mg, 0.111 mmol, 1.0 eq)
and 1-(2-hydroxyethyl)limidazolidin-2-one (289 mg, 1.66 mmol, 15
eq) in DCM (2 mL) was added 4-methylbenzenesulfonic acid
monohydrate (119 mg, 0.626 mmol, 3 eq). The reaction was stirred at
room temperature overnight. The reaction was diluted with H.sub.2O
and was extracted with DCM. The organic extract was evaporated
under reduced pressure. The crude product was purified by
preparative HPLC chromatography (method 1) followed chiral
preparative HPLC (method 2) to yield Example 5 (13.7 mg, 12% yield)
as a white solid.
Example 5: ESIMS [M+H] 998.5
Exact Mass: 997.62
[0428] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.45 (dd,
J=14.3, 11.0 Hz, 1H), 6.23 (dd, J=14.3, 10.6 Hz, 1H), 6.14 (dd,
J=14.6, 10.6 Hz, 1H), 5.98 (d, J=10.9 Hz, 1H), 5.44 (dd, J=14.5,
9.8 Hz, 1H), 5.28 (s, 1H), 5.07 (d, J=4.7 Hz, 1H), 5.02-4.96 (m,
2H), 4.67 (t, J=5.3 Hz, 1H), 4.64-4.53 (m, 3H), 4.03-3.96 (m, 1H),
3.71-3.57 (m, 3H), 3.52-3.42 (m, 2H), 3.42-3.29 (m, 6H), 3.22-3.07
(m, 7H), 2.95-2.79 (m, 2H), 2.71-2.56 (m, 1H), 2.27-1.87 (m, 6H),
1.80-1.36 (m, 20H), 1.36-1.12 (m, 6H), 1.11-1.02 (m, 1H), 1.00-0.91
(m, 4H), 0.91-0.76 (m, 11H), 0.74 (d, J=6.7 Hz, 3H), 0.59 (q,
J=11.9 Hz, 1H).
Example 6:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-rapamycin
(Diastereomer 1)
Example 7:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-rapamycin
(Diastereomer 2)
##STR00078##
[0429] Example 6 and Example 7
[0430] *Absolute stereochemistry at C16 not assigned
[0431] Diastereomer 1: To a solution of Intermediate 1 (150 mg,
0.167 mmol, 1.0 eq) and 1,2-thiazetidine 1,1-dioxide (89 mg, 0.833
mmol, 5 eq) in DCM (3 mL) was added zinc(II) chloride (1M solution
in diethyl ether, 0.5 mL, 0.500 mmol, 3 eq) at 0.degree. C. The
reaction mixture was stirred at room temperature for 2 h. The
mixture was diluted with H.sub.2O and was extracted with ethyl
acetate. The organic extract was evaporated under reduced pressure.
The crude product of the diastereomeric mixture was separated by
flash chromatography (silica; MeCN/DCM 0:100 to 100:0).
[0432] Final purification of the first eluting diastereomer by
preparative HPLC (method 2) yielded Example 6 (18 mg, 10.7% yield)
as a white solid.
Example 6: ESIMS [M+NH.sub.4] 992.6, [M+FA-H] 1019.6
Exact Mass: 974.55
[0433] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.39 (dd,
J=14.8, 9.8 Hz, 1H), 6.34 (dd, J=14.8, 10.3 Hz, 1H), 6.18-6.10 (m,
1H), 6.02 (d, J=10.3, 1.5 Hz, 1H), 5.66 (dd, J=15.1, 8.6 Hz, 1H),
5.38 (s, 1H), 5.32 (dd, J=6.4, 1.8 Hz, 1H), 5.23 (d, J=9.6 Hz, 1H),
4.87-4.76 (m, 1H), 4.12-4.06 (m, 1H), 3.99 (ddd, J=12.0, 8.2, 6.2
Hz, 1H), 3.92-3.86 (m, 1H), 3.84 (dd, J=10.7, 4.4 Hz, 1H),
3.81-3.74 (m, 1H), 3.63-3.59 (m, 2H), 3.59 (d, J=3.6 Hz, 1H), 3.46
(d, J=7.6 Hz, 1H), 3.43 (s, 3H), 3.40-3.37 (m, 1H), 3.32 (s, 3H),
3.04 (ddd, J=8.2, 5.8, 3.8 Hz, 1H), 3.00-2.94 (m, 1H), 2.94-2.89
(m, 1H), 2.88-2.84 (m, 1H), 2.64 (d, J=6.7 Hz, 1H), 2.47-2.38 (m,
1H), 2.36-2.27 (m, 2H), 2.21-2.12 (m, 1H), 2.03-1.97 (m, 1H),
1.95-1.77 (m, 7H), 1.76-1.67 (m, 5H), 1.65 (s, 3H), 1.60-1.50 (m,
6H), 1.47-1.39 (m, 2H), 1.38-1.27 (m, 4H), 1.25-1.10 (m, 3H),
1.08-1.04 (m, 1H), 1.03-1.01 (m, 3H), 1.00 (d, J=1.4 Hz, 3H),
0.99-0.97 (m, 3H), 0.97 (d, J=6.6 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H),
0.75-0.66 (m, 1H).
[0434] Final purification of the second eluting diastereomer using
SFC chromatography (method 2) yielded Example 7 (15.8 mg, 9.2%
yield) as a white solid.
Example 7: ESIMS [M+NH.sub.4] 992.7, [M+FA-H] 1019.6
Exact Mass: 974.55
[0435] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.46 (dd,
J=14.2, 10.9 Hz, 1H), 6.22 (dd, J=14.2, 10.6 Hz, 1H), 6.14 (dd,
J=14.5, 10.6 Hz, 1H), 6.00 (d, J=10.9 Hz, 1H), 5.38 (dd, J=14.5,
9.6 Hz, 1H), 5.27-5.19 (m, 1H), 5.12-5.05 (m, 1H), 4.66-4.59 (m,
1H), 4.59-4.53 (m, 1H), 4.25 (d, J=1.8 Hz, 1H), 4.17-4.06 (m, 3H),
4.02 (ddd, J=11.8, 8.0, 3.5 Hz, 1H), 3.66 (d, J=7.0 Hz, 1H),
3.60-3.52 (m, 2H), 3.43-3.34 (m, 4H), 3.28 (s, 3H), 3.17-3.07 (m,
1H), 3.03-2.95 (m, 1H), 2.95-2.89 (m, 1H), 2.84-2.73 (m, 1H),
2.65-2.62 (m, 1H), 2.35-2.26 (m, 1H), 2.27-2.21 (m, 1H), 2.21-2.12
(m, 3H), 2.12-2.05 (m, 1H), 2.02-1.98 (m, 1H), 1.97-1.95 (m, 3H),
1.91-1.83 (m, 1H), 1.82-1.70 (m, 3H), 1.70-1.51 (m, 10H), 1.50-1.17
(m, 10H), 1.11-1.06 (m, 1H), 1.06-1.02 (m, 6H), 1.01-0.96 (m, 1H),
0.93 (d, J=6.8 Hz, 4H), 0.89 (d, J=6.6 Hz, 3H), 0.86 (d, J=6.8 Hz,
3H), 0.64 (q, J=11.9 Hz, 1H).
Example 8:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-(2-hydroxy-
ethoxy)-rapamycin (diastereomer 1)
Example 9:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-(2-hydroxy-
ethoxy)-rapamycin (diastereomer 2)
##STR00079##
[0436] Example 8 and Example 9
[0437] *Absolute stereochemistry at C16 not assigned
[0438] To a solution of Intermediate 3 (50 mg, 0.053 mmol, 1.0 eq)
and 1,2-thiazetidine 1,1-dioxide (56.7 mg, 0.530 mmol, 10 eq) in
DCM (1 mL) was added zinc(II) chloride (1M solution in diethyl
ether, 0.265 mL, 0.265 mmol, 5 eq). The reaction mixture was
stirred at room temperature for 1.5 hours. The reaction was diluted
with H.sub.2O and was extracted with ethyl acetate. The organic
extract was evaporated under reduced pressure. The crude product of
the diastereomeric mixture was separated by flash chromatography
(silica; MeCN/DCM 0:100 to 100:0).
[0439] Final purification of the first eluting diastereomer using
preparative HPLC (method 2) yielded Example 8 (3.1 mg, 5.5% yield)
as a white solid.
Example 8: ESIMS [M+NH.sub.4] 1037.0, [M+FA-H] 1064.1
Exact Mass: 1018.58
[0440] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.: 6.53-6.44 (m,
1H), 6.41 (dd, J=11.0, 5.6 Hz, 1H), 6.29-6.14 (m, 2H), 6.07 (dd,
J=11.0, 1.5 Hz, 1H), 5.54 (dd, J=14.6, 9.1 Hz, 1H), 5.06 (d, J=8.9
Hz, 1H), 4.99-4.93 (m, 1H), 4.92-4.83 (m, 1H), 4.70-4.60 (m, 1H),
4.49-4.42 (m, 1H), 4.13-4.02 (m, 3H), 3.92-3.86 (m, 1H), 3.69-3.64
(m, 1H), 3.59-3.54 (m, 1H), 3.52-3.42 (m, 5H), 3.33 (s, 3H),
3.30-3.27 (m, 1H), 3.13 (s, 3H), 3.10-3.02 (m, 2H), 3.02-2.94 (m,
2H), 2.90-2.80 (m, 1H), 2.30-2.21 (m, 1H), 2.21-2.10 (m, 1H),
2.10-2.00 (m, 2H), 2.00-1.87 (m, 3H), 1.82 (s, 3H), 1.81-1.62 (m,
6H), 1.58-1.54 (m, 1H), 1.53-1.47 (m, 5H), 1.47-1.35 (m, 5H),
1.36-1.23 (m, 2H), 1.23-1.09 (m, 4H), 1.06-1.01 (m, 1H), 1.01-0.95
(m, 6H), 0.90-0.87 (m, 1H), 0.87-0.83 (m, 3H), 0.81 (d, J=6.7 Hz,
3H), 0.76-0.70 (m, 4H), 0.68-0.60 (m, 1H).
[0441] Final purification of the second eluting diastereomer using
preparative HPLC (method 2) yielded Example 9 (2.8 mg, 4.9% yield)
as a white solid.
Example 9: ESIMS [M+NH4] 1037.2, [M+FA-H] 1064.3
Exact Mass: 1018.58
[0442] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.:6.48-6.40 (m,
1H), 6.27-6.19 (m, 1H), 6.19-6.13 (m, 1H), 6.10-6.05 (m, 1H), 5.52
(dd, J=14.2, 9.3 Hz, 1H), 5.13-5.00 (m, 2H), 4.96 (d, J=9.4 Hz,
1H), 4.67-4.57 (m, 1H), 4.48-4.40 (m, 1H), 4.17-4.10 (m, 2H),
4.10-4.02 (m, 2H), 3.98-3.90 (m, 1H), 3.56-3.49 (m, 2H), 3.50-3.43
(m, 4H), 3.37 (d, J=11.2 Hz, 1H), 3.33 (s, 3H), 3.23-3.15 (m, 1H),
3.15-3.10 (m, 4H), 3.07-2.95 (m, 2H), 2.75-2.64 (m, 1H), 2.27-2.14
(m, 2H), 2.11-2.00 (m, 2H), 2.00-1.89 (m, 3H), 1.82 (s, 3H),
1.76-1.65 (m, 2H), 1.65-1.56 (m, 3H), 1.56-1.49 (m, 7H), 1.48-1.36
(m, 3H), 1.34-1.23 (m, 3H), 1.23-1.04 (m, 5H), 1.01-0.94 (m, 4H),
0.90 (d, J=6.4 Hz, 3H), 0.88-0.83 (m, 5H), 0.80 (d, J=6.7 Hz, 3H),
0.75 (d, J=6.7 Hz, 3H), 0.62 (q, J=11.8 Hz, 1H).
Example 10:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-dimethylphosphinyl-r-
apamycin (Diastereomer 1)
Example 11:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-dimethylphosphinyl-r-
apamycin (Diastereomer 2)
##STR00080##
[0443] Example 10 and Example 11
[0444] *Absolute stereochemistry at C16 not assigned
[0445] To a solution of Intermediate 4 (100 mg, 0.102 mmol, 1.0 eq)
and 1,2-thiazetidine 1,1-dioxide (54.9 mg, 0.512 mmol, 5 eq) in DCM
(5 mL) was added zinc(II) chloride (1M solution in diethyl ether,
0.512 mL, 0.512 mmol, 5 eq). The reaction mixture was stirred at
room temperature for 15 minutes. The reaction was diluted with
H.sub.2O and was extracted with ethyl acetate. The organic extract
was evaporated under reduced pressure. The crude product the
diastereomeric mixture was separated by SFC chromatography (method
3).
[0446] Final purification of the first eluting diastereomer using
preparative HPLC (method 2) yielded Example 10 (20.7 mg, 18.8%
yield) as a white solid.
Example 10: ESIMS [M+H] 1051.9, [M+FA-H] 1096.0
Exact Mass: 1050.56
[0447] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.49-6.39 (m,
2H), 6.30-6.14 (m, 2H), 6.11-6.01 (m, 1H), 5.60-5.50 (m, 1H),
5.06-5.00 (m, 1H), 4.98-4.93 (m, 1H), 4.87 (d, J=9.7 Hz, 1H),
4.67-4.58 (m, 1H), 4.17-4.01 (m, 3H), 4.01-3.92 (m, 1H), 3.92-3.85
(m, 1H), 3.73-3.64 (m, 1H), 3.60-3.52 (m, 1H), 3.50-3.42 (m, 1H),
3.35-3.26 (m, 4H), 3.15-3.12 (m, 3H), 3.11-3.01 (m, 2H), 3.01-2.95
(m, 1H), 2.88-2.79 (m, 1H), 2.29-2.20 (m, 1H), 2.20-2.12 (m, 1H),
2.10-1.99 (m, 2H), 1.99-1.87 (m, 3H), 1.82 (s, 3H), 1.80-1.71 (m,
1H), 1.71-1.62 (m, 4H), 1.58-1.54 (m, 1H), 1.52-1.48 (m, 5H),
1.47-1.31 (m, 13H), 1.28-1.11 (m, 4H), 1.07-1.02 (m, 1H), 1.01-0.95
(m, 8H), 0.87-0.84 (m, 3H), 0.82 (d, J=6.7 Hz, 3H), 0.79-0.76 (m,
1H), 0.74 (d, J=6.7 Hz, 3H), 0.72-0.66 (m, 1H).
[0448] Final purification of the second eluting diastereomer using
preparative HPLC (method 2) yielded Example 11 (13.4 mg, 12.2%
yield) as a white solid.
Example 11: ESIMS [M+H] 1051.8, [M+FA-H] 1095.8
Exact Mass: 1050.56
[0449] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.51-6.39 (m,
1H), 6.28-6.13 (m, 2H), 6.08 (d, J=11.1 Hz, 1H), 6.04-5.88 (m, 1H),
5.57-5.46 (m, 1H), 5.05 (s, 1H), 5.03 (d, J=5.7 Hz, 1H), 4.96 (d,
J=9.9 Hz, 1H), 4.67-4.56 (m, 1H), 4.18-4.11 (m, 2H), 4.09-4.02 (m,
2H), 3.99-3.89 (m, 2H), 3.60-3.49 (m, 2H), 3.37 (d, J=13.3 Hz, 1H),
3.30 (s, 3H), 3.18 (dt, J=7.7, 5.1 Hz, 1H), 3.15-3.11 (m, 4H),
3.07-3.00 (m, 1H), 2.73-2.63 (m, 1H), 2.28-2.13 (m, 2H), 2.13-2.05
(m, 2H), 2.05-1.97 (m, 2H), 1.97-1.88 (m, 1H), 1.83 (s, 3H),
1.80-1.69 (m, 1H), 1.68-1.58 (m, 4H), 1.58-1.48 (m, 7H), 1.48-1.43
(m, 1H), 1.42-1.34 (m, 10H), 1.34-1.28 (m, 1H), 1.28-1.22 (m, 2H),
1.22-1.13 (m, 2H), 1.13-1.03 (m, 1H), 1.03-0.94 (m, 5H), 0.94-0.88
(m, 4H), 0.86 (d, J=6.5 Hz, 3H), 0.81 (d, J=6.7 Hz, 3H), 0.75 (d,
J=6.6 Hz, 3H), 0.66 (q, J=12.0 Hz, 1H).
Example 12:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-(S)-(1H-tetrazol-1-y-
l)-rapamycin (Diastereomer 1)
Example 13:
C16-(1,1-dioxido-1,2-thiazetidin-2-yl)-C32-deoxo-C40-(S)-(1H-tetrazol-1-y-
l)-rapamycin (Diastereomer 2)
##STR00081##
[0450] Example 12 and Example 13
[0451] *Absolute stereochemistry at C16 not assigned
[0452] To a solution of Intermediate 5 (118 mg, 0.124 mmol, 1.0 eq)
and 1,2-thiazetidine 1,1-dioxide (66.4 mg, 0.620 mmol, 5 eq) in DCM
(6 mL) was added zinc(II) chloride (1M solution in diethyl ether,
0.620 mL, 0.620 mmol, 5 eq). The reaction mixture was stirred at
room temperature for three hours. The reaction was diluted with
H.sub.2O and was extracted with ethyl acetate. The organic extract
was evaporated under reduced pressure. The crude product of the
diastereomeric mixture was separated by SFC chromatography (method
3).
[0453] Final purification of the first eluting diastereomer using
preparative HPLC (method 2) yielded Example 12 (14.8 mg, 11.3%
yield) as a white solid.
Example 12: ESIMS [M+NH4] 1050.0, [M+FA-H] 1072.1
Exact Mass: 1026.57
[0454] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.32 (s, 1H),
6.52 (s, 1H), 6.45-6.37 (m, 1H), 6.31-6.14 (m, 2H), 6.10-6.04 (m,
1H), 5.60-5.49 (m, 1H), 5.23-5.12 (m, 1H), 5.07-5.01 (m, 1H),
4.99-4.93 (m, 1H), 4.87 (d, J=9.7 Hz, 1H), 4.69-4.59 (m, 1H),
4.15-4.02 (m, 3H), 3.94-3.84 (m, 1H), 3.72-3.54 (m, 3H), 3.51-3.42
(m, 1H), 3.27 (s, 4H), 3.11 (s, 3H), 3.07 (dd, J=7.3, 5.1 Hz, 1H),
3.01-2.91 (m, 1H), 2.85 (td, J=10.6, 9.5, 4.7 Hz, 1H), 2.28-2.13
(m, 3H), 2.10-2.00 (m, 2H), 1.98-1.88 (m, 3H), 1.82 (s, 3H),
1.80-1.75 (m, 1H), 1.73-1.67 (m, 4H), 1.59-1.53 (m, 4H), 1.53-1.45
(m, 6H), 1.45-1.34 (m, 3H), 1.33-1.20 (m, 3H), 1.20-1.07 (m, 3H),
1.05-1.01 (m, 1H), 1.00-0.94 (m, 6H), 0.88-0.82 (m, 6H), 0.80-0.75
(m, 1H), 0.73 (d, J=6.7 Hz, 3H).
[0455] Final purification of the second eluting diastereomer using
preparative HPLC (method 2) yielded Example 13 (12.3 mg, 9.4%
yield) as a white solid.
Example 13: ESIMS [M+H] 1027.7, [M-H] 1025.6, [M+FA-H] 1071.6
Exact Mass: 1026.57
[0456] .sup.1H NMR (400 MHz, DMSO-d.sub.6 .delta. 9.30 (s, 1H),
6.52-6.38 (m, 1H), 6.28-6.14 (m, 2H), 6.08 (d, J=11.1 Hz, 1H),
5.58-5.42 (m, 1H), 5.21-5.14 (m, 1H), 5.10-5.00 (m, 2H), 5.00-4.92
(m, 1H), 4.69-4.57 (m, 1H), 4.16-4.10 (m, 3H), 4.08-4.01 (m, 1H),
3.97-3.91 (m, 1H), 3.61 (dt, J=10.6, 4.1 Hz, 1H), 3.57-3.51 (m,
1H), 3.51-3.46 (m, 1H), 3.46-3.36 (m, 1H), 3.27 (s, 3H), 3.19-3.15
(m, 1H), 3.12 (s, 3H), 3.11-3.08 (m, 1H), 2.80-2.68 (m, 1H),
2.25-2.15 (m, 3H), 2.10-2.01 (m, 2H), 2.01-1.89 (m, 2H), 1.83 (s,
3H), 1.79-1.65 (m, 4H), 1.64-1.58 (m, 2H), 1.57-1.54 (m, 2H),
1.54-1.47 (m, 7H), 1.46-1.35 (m, 3H), 1.32-1.18 (m, 5H), 1.14-1.04
(m, 3H), 0.98 (d, J=6.6 Hz, 3H), 0.91-0.85 (m, 7H), 0.83 (d, J=6.7
Hz, 3H), 0.72 (d, J=6.6 Hz, 3H).
Example 14:
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-dimethylphosphinyl-rap-
amycin (Diastereomer 1)
Example 15:
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-dimethylphosphinyl-rap-
amycin (Diastereomer 2)
##STR00082##
[0457] Example 14 and Example 15
[0458] *Absolute stereochemistry at C16 not assigned
[0459] Intermediate 4 (0.146 g, 0.150 mmol) and isothiazolidine
1,1-dioxide (0.181 g, 1.496 mmol) were dissolved into anhydrous
acetonitrile (1.5 mL). para-Toluenesulfonic acid monohydrate (2.84
mg, 0.015 mmol) was added in one portion. The reaction mixture was
stirred at room temperature for two hours.
[0460] Aqueous saturated NaHCO.sub.3 was added. The mixture was
extracted several times with ethyl acetate. The organic extracts
were combined, dried over Na.sub.2SO, decanted and concentrated to
give a colorless tar crude product.
[0461] The crude product was dissolved in MeOH (2.5 mL) and was
purified in one injection via preparative-scale reverse phase
chromatography (40-90% acetonitrile-water plus 0.1% TFA modifier on
100 g C18 ISCO column).
[0462] The first eluting peak fractions were pooled and reduced to
about 1/3 volume on a rotary evaporator. The remaining solution was
made basic with saturated aqueous NaHCO.sub.4 and was extracted
several times with EtOAc. The organic extracts were combined, dried
over Na.sub.2SO.sub.4, decanted and concentrated to give Example 14
(0.055 g, 0.044 mmol, 29.3% yield) as a white solid.
Example 14: ESIMS [M+NH4] 1082.8, [M-H] 1063.7
[0463] HRMS: calculated for C55H89N2O14PSNa as sodium
adduct--1087.5670. Found--1087.5725.
[0464] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.45 (dd,
J=14.4, 10.9 Hz, 1H), 6.22 (dd, J=14.5, 10.6 Hz, 1H), 6.13 (dd,
J=14.6, 10.5 Hz, 1H), 5.98 (d, J=10.8 Hz, 1H), 5.35 (dd, J=14.7,
9.8 Hz, 1H), 5.23-5.17 (m, 1H), 5.10 (d, J=9.8 Hz, 1H), 4.67 (m,
2H), 4.47 (d, J=1.8 Hz, 1H), 4.18-4.04 (m, 2H), 4.03-3.91 (m, 1H),
3.72 (d, J=6.5 Hz, 1H), 3.68-3.48 (m, 2H), 3.38 (m, 4H), 3.28 (s,
3H), 3.26-3.12 (m, 2H), 3.12-2.91 (m, 4H), 2.74 (m, 1H), 2.48-2.26
(m, 3H), 2.26-2.12 (m, 3H), 2.12-2.04 (m, 2H), 1.87 (s, 3H),
1.85-1.72 (m, 4H), 1.72-1.54 (m, 12H), 1.54-1.43 (m, 6H), 1.43-1.33
(m, 3H), 1.33-1.21 (m, 2H), 1.21-1.09 (m, 2H), 1.03 (m, 7H),
1.00-0.78 (m, 9H), 0.72 (q, J=11.9 Hz, 1H).
[0465] The second eluting peak fractions were pooled and reduced to
about 1/3 volume on the rotary evaporator. The remaining solution
was made basic with saturated aqueous NaHCO.sub.3. The mixture was
extracted several times with ethyl acetate. The organic extracts
were combined, dried over Na2SO4, decanted and concentrated to give
Example 15 (0.010 g, 7.51 .mu.mol, 5.02% yield) as a white
solid.
Example 15: ESIMS [M+NH4] 1082.8, [M-H] 1063.8
[0466] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.36 (dd,
J=19.2, 10.3 Hz, 1H), 6.13 (m, 1H), 6.04-5.84 (m, 1H), 5.65 (m,
1H), 5.32 (m, 1H), 6.21 (m, 1H), 5.11 (m, 1H), 4.81 (dd, J=13.7,
7.5 Hz, 1H), 4.21-4.03 (m, 1H), 3.83 (dd, J=15.2, 5.1 Hz, 2H), 3.74
(d, J=13.2 Hz, 1H), 3.59 (dq, J=10.9, 6.8, 5.6 Hz, 2H), 3.52 (d,
J=7.1 Hz, 1H), 3.46-3.33 (m, 7H), 3.30 (m, 3H), 3.18 (m, 3H),
3.10-2.82 (m, 3H), 2.39 (t, J=4.1 Hz, 1H), 2.28 (m, 3H), 2.14 (m,
3H), 1.90-1.77 (m, 4H), 1.74 (m, 4H), 1.66 (d, J=10.4 Hz, 3H),
1.61-1.41 (m, 12H), 1.34-1.19 (m, 5H), 1.18-1.11 (m, 1H), 1.11-0.79
(m, 19H), 0.78-0.68 (m, 1H).
Example 16:
(R)-C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-(2-hydroxyethoxy)--
rapamycin
##STR00083##
[0467] Example 16
[0468] Absolute stereochemistry at C16 known to be (R) based on
using Example 2 as starting material.
[0469] Example 16 was prepared in a two-step procedure from
2-((tert-butyldimethylsilyl)oxy)ethanol and Example 2.
##STR00084##
Step 1. Preparation of Intermediate C
[0470] 2-((tert-butyldimethylsilyl)oxy)ethanol (0.142 g, 0.803
mmol) was dissolved in anhydrous toluene (0.4 mL).
N,N-diisopropylethylamine (0.147 ml, 0.843 mmol) was added via
syringe. The mixture was vacuum purged with nitrogen then was
chilled to 0 C in an ice-water bath. Triflic anhydride (0.132 ml,
0.779 mmol) was added via syringe over a period of 60 seconds. The
reaction was stirred at 0 C for 30 minutes.
[0471] N,N-diisopropylethylamine (0.147 ml, 0.843 mmol), toluene
(0.5 mL) and Example 2 (0.198 g, 0.200 mmol) were added. The ice
bath was removed and the reaction was stirred overnight at 40 C and
then for one hour at 55 C.
[0472] The reaction was diluted with saturated aqueous NaHCO3 and
was extracted four times with EtOAc. The organic extracts were
combined, dried over Na2SO4, vacuum filtered through celite, and
concentrated to give a crude product colorless oil.
[0473] The crude product was purified by silica gel flash column
chromatography (0-40% acetone-heptane, gradient elution, 12 g
silica column, TLC in 30% Acetone-heptane, visualize under UV) to
give Intermediate C (0.077 g, 0.067 mmol, 33.5% yield) as a white
solid.
Intermediate C: ESIMS [M+NH4] 1164.7, [M-H] 1147.0.
Step 2. Preparation of Example 16
[0474] Intermediate C (0.077 g, 0.067 mmol) was combined with
pyridine (5.43 .mu.l, 0.067 mmol) in anhydrous THF (0.7 mL). The
mixture was vacuum purged twice with nitrogen and then was chilled
to 0 C in an ice-water bath. HF-Pyridine (0.086 ml, 0.671 mmol) was
added dropwise via syringe over a period of 15 seconds. The
reaction was stirred at 0 C for 70 minutes.
[0475] The reaction was quenched with saturated aqueous NaHCO.sub.3
and was extracted several times with EtOAc. The organic extracts
were combined, dried over Na2SO4, vacuum filtered through celite
and concentrated to give a white solid crude product.
[0476] The crude product was purified by silica gel flash column
chromatography (0-40% acetone-heptane, gradient elution, 12 g
silica column, TLC in 40% EtOAc-heptane, visualize under UV) to
give Example 16 (0.049 g, 0.046 mmol, 69.0% yield) as a white
solid.
Example 16: ESIMS [M+NH4] 1050.9, [M-H] 1031.9
[0477] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 6.45 (dd,
J=14.6, 10.9 Hz, 1H), 6.22 (dd, J=14.7, 10.6 Hz, 1H), 6.13 (dd,
J=14.9, 10.6 Hz, 1H), 5.98 (d, J=10.9 Hz, 1H), 5.35 (dd, J=14.9,
9.8 Hz, 1H), 5.20 (d, J=5.9 Hz, 1H), 5.09 (d, J=9.9 Hz, 1H), 4.71
(d, J=12.3 Hz, 1H), 4.62 (m, 1H), 4.46 (s, 1H), 4.11 (d, J=6.8 Hz,
1H), 3.96 (t, J=11.3 Hz, 1H), 3.78 (m, 1H), 3.70 (m, 3H), 3.63-3.55
(m, 3H), 3.43 (s, 3H), 3.28 (s, 3H), 3.24-3.15 (m, 2H), 3.12-3.04
(m, 2H), 3.07-2.98 (m, 1H), 2.97 (q, J=7.9 Hz, 1H), 2.75 (m, 1H),
2.44-2.33 (m, 1H), 2.30 (M, 2H), 2.23 (m, 1H), 2.16 (m, 2H), 2.09
(d, J=13.1 Hz, 1H), 2.01 (m, 1H), 1.88 (s, 3H), 1.84 (d, J=13.1 Hz,
1H), 1.78-1.70 (m, 3H), 1.70-1.62 (m, 4H), 1.52 (dd, J=11.8, 7.7
Hz, 1H), 1.50-1.41 (m, 2H), 1.43-1.32 (m, 1H), 1.34-1.27 (m, 1H),
1.30-1.17 (m, 8H), 1.04 (dd, J=9.5, 6.6 Hz, 7H), 1.02-0.85 (m,
10H), 0.85 (d, J=6.8 Hz, 3H), 0.69 (q, J=12.0 Hz, 1H).
Example 17:
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-(S)-(1H-tetrazol-1-yl)-
-rapamycin
##STR00085##
[0478] Example 17
[0479] *Absolute stereochemistry at C16 not assigned
[0480] Intermediate 5 (0.109 g, 0.114 mmol) was combined with
isothiazolidine 1,1-dioxide (0.139 g, 1.145 mmol) in anhydrous
acetonitrile (1.1 mL). para-Toluenesulfonic acid monohydrate
(0.0022 g, 0.011 mmol) was added. The reaction was stirred at room
temperature for two hours.
[0481] The reaction was diluted with saturated aqueous NaHCO.sub.3.
The mixture was extracted several times with EtOAc. The organic
extracts were combined, dried over Na2SO, decanted and concentrated
to give a yellow tar crude product.
[0482] The crude product was purified by silica gel flash column
chromatography (0-40% acetone-heptane, gradient elution, 24 g
silica column, TLC in 40% acetone-heptane, visualize under UV) to
give Example 17 (0.053 g, 0.046 mmol, 40.0% yield) as a white
solid.
Example 17: ESIMS [M+H] 1041.8, [M-H] 1039.8
[0483] HRMS: calculated for C54H84N6012SNa--1063.5765.
Found--1063.5759.
[0484] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 8.86 (s, 1H),
6.39 (dd, J=14.7, 11.0 Hz, 1H), 6.22 (dd, J=14.7, 10.7 Hz, 1H),
6.11 (dd, J=15.0, 10.5 Hz, 1H), 5.99 (d, J=10.9 Hz, 1H), 5.34 (dd,
J=14.9, 9.8 Hz, 1H), 5.18 (d, J=5.7 Hz, 1H), 5.10 (d, J=9.8 Hz,
1H), 4.87 (m, 1H), 4.67 (m, 1H), 4.63 (d, J=12.2 Hz, 1H), 4.50 (s,
1H), 4.13 (d, J=6.1 Hz, 1H), 3.97 (t, J=11.4 Hz, 1H), 3.78 (d,
J=6.2 Hz, 1H), 3.69-3.62 (m, 1H), 3.55 (dt, J=11.2, 4.0 Hz, 1H),
3.50-3.39 (m, 1H), 3.39 (s, 3H), 3.38-3.30 (m, 1H), 3.27 (s, 3H),
3.25-3.13 (m, 1H), 3.07 (td, J=8.4, 3.8 Hz, 1H), 3.01 (m, 1H), 2.96
(q, J=7.9 Hz, 1H), 2.70-2.63 (m, 2H), 2.39 (m, 1H), 2.35-2.21 (m,
3H), 2.18 (d, J=13.7 Hz, 1H), 2.16-2.09 (m, 1H), 1.95-1.81 (m, 3H),
1.82 (s, 3H), 1.75 (m, 3H), 1.65 (s, 3H), 1.60 (m, 6H), 1.58-1.50
(m, 1H), 1.53-1.34 (m, 2H), 1.32 (m, 1H), 1.29 (m, 1H), 1.25 (m,
6H), 1.12 (m, 1H), 1.05 (d, J=6.6 Hz, 3H), 1.05-0.78 (m, 12H).
Example 18:
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-(2-ethoxyethoxy)-rapam-
ycin
##STR00086##
[0485] Example 18
[0486] *Absolute stereochemistry at C16 not assigned
[0487] Intermediate 6 (0.090 g, 0.093 mmol) was combined with
isothiazolidine 1,1-dioxide (0.112 g, 0.926 mmol) in anhydrous
dichloromethane (0.93 mL). para-Toluenesulfonic acid monohydrate
(1.761 mg, 9.26 .mu.mol) was added in one portion. The reaction was
stirred at room temperature for 100 minutes.
[0488] The entire reaction mixture was purified by silica gel flash
column chromatography (0-40% acetone-heptane, gradient elution, 24
g silica column, TLC in 40% acetone-heptane, visualize under UV) to
give a mixture of Example 18 and residual unreacted isothiazolidine
1,1-dioxide.
[0489] The mixed material was again purified by silica gel flash
column chromatography (0-50% acetonitrile-dichloromethane, gradient
elution, 24 g column, TLC in 30% acetonitrile-dichloromethane,
visualize under UV) to give Example 18 (0.021 g, 0.019 mmol, 20.4%
yield) as a white solid.
Example 18: ESIMS [M+NH4] 1078.9, [M-H] 1059.9
[0490] HRMS: calculated for C57H92N2O14SNa sodium
adduct--1083.6167. Found 1083.6151.
[0491] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 6.45 (dd,
J=14.5, 11.0 Hz, 1H), 6.21 (dd, J=14.6, 10.7 Hz, 1H), 6.14 (dd,
J=14.8, 10.8 Hz, 1H), 5.98 (d, J=10.9 Hz, 1H), 5.35 (dd, J=14.8,
9.8 Hz, 1H), 5.20 (d, J=5.8 Hz, 1H), 5.09 (d, J=9.8 Hz, 1H), 4.72
(d, J=12.1 Hz, 1H), 4.61 (m, 1H), 4.46 (s, 1H), 4.10 (d, J=6.9 Hz,
1H), 3.96 (t, J=11.4 Hz, 1H), **3.73 (t, J=5.5 Hz, 2H), 3.68 (d,
J=6.9 Hz, 1H), 3.59 (m, 4H), 3.53 (m, 2H), 3.46 (s, 3H), 3.28 (s,
3H), 3.21 (m, 1H), 3.13 (m, 1H), 3.07 (m, 2H), 3.05-2.99 (m, 1H),
2.97 (q, J=8.0 Hz, 1H), 2.78 (dq, J=14.4, 7.1 Hz, 1H), 2.44-2.33
(m, 1H), 2.31 (m, 2H), 2.27-2.19 (m, 1H), 2.19-2.12 (m, 2H),
2.07-1.94 (m, 2H), 1.88 (s, 3H), 1.83 (d, J=12.9 Hz, 1H), 1.74 (m,
3H), 1.66 (m, 7H), 1.56-1.44 (m, 1H), 1.43 (m, 2H), 1.41-1.15 (m,
12H), 1.04 (m, 7H), 0.91 (m, 8H), 0.84 (d, J=6.7 Hz, 3H), 0.70 (q,
J=12.0 Hz, 1H).
Example 19:
C16-(1,1-dioxidoisothiazolidin-2-yl)-C32-deoxo-C40-((3-hydroxy-2-(hydroxy-
methyl)-2-methylpropanoyl)oxy)-rapamycin
##STR00087##
[0492] Example 19
[0493] *Absolute stereochemistry at C16 not assigned
[0494] Intermediate 7 (0.088 g, 0.087 mmol) was combined with
isothiazolidine 1,1-dioxide (0.105 g, 0.866 mmol) in anhydrous
dichloromethane (0.87 mL). para-Toluenesulfonic acid monohydrate
(1.647 mg, 8.66 .mu.mol) was added. The reaction was stirred at
room temperature for 30 minutes.
[0495] The entire reaction mixture was purified by normal phase
flash column chromatography (0-60% acetonitrile-dichloromethane,
gradient elution, 12 g silica column, TLC in 30%
acetonitrile-dichloromethane) to give Example 19 (0.020 g, 0.017
mmol, 19.9% yield) as a white solid.
Example 19: ESIMS [M+NH4] 1123.0, [M-H] 1104.0
[0496] HRMS: Calculated for ammonium adduct
C58H92N2O16SNH4-1122.6511; found 1122.652.
[0497] .sup.1H NMR (600 MHz, Chloroform-d) .delta. 6.45 (dd,
J=14.7, 10.9 Hz, 1H), 6.22 (dd, J=14.7, 10.7 Hz, 1H), 6.13 (dd,
J=14.9, 10.6 Hz, 1H), 5.99 (d, J=10.9 Hz, 1H), 5.35 (dd, J=14.9,
9.8 Hz, 1H), 5.21 (d, J=5.8 Hz, 1H), 5.10 (d, J=9.9 Hz, 1H), 4.73
(ddd, J=11.0, 9.2, 4.7 Hz, 1H), 4.69 (d, J=12.4 Hz, 1H), 4.64 (m,
1H), 4.49 (s, 1H), 4.13 (d, J=6.5 Hz, 1H), 3.97 (t, J=11.3 Hz, 1H),
3.84 (dd, J=18.5, 11.4 Hz, 2H), 3.78-3.69 (m, 2H), 3.64 (m, 2H),
3.56 (td, J=13.5, 3.0 Hz, 1H), 3.43-3.31 (m, 4H), 3.28 (m, 3H),
3.26-3.17 (m, 2H), 3.12-2.92 (m, 3H), 2.76-2.68 (m, 1H), 2.45-2.34
(m, 1H), 2.31-2.21 (m, 3H), 2.20-2.10 (m, 3H), 2.06 (m, 1H), 1.87
(m, 4H), 1.75 (m, 3H), 1.71-1.59 (m, 3H), 1.60 (s, 3H), 1.60-1.50
(m, 1H), 1.48-1.39 (m, 1H), 1.42-1.31 (m, 2H), 1.33-1.20 (m, 8H),
1.11 (s, 3H), 1.11-1.05 (m, 2H), 1.03 (dd, J=18.2, 6.6 Hz, 6H),
0.99-0.82 (m, 10H), 0.79 (q, J=12.0 Hz, 1H).
Example 20: Biological Assays and Data
[0498] The activity of a compound according to the disclosure was
assessed by the following in vitro & in vivo methods.
Pharmacological Characterization
Materials and Methods
[0499] Cell-based assay for rapalog potency determination. Rapalog
potency was determined using MEF TSC1-/- cell based assay. MEF
TSC1-/- cells are Mouse Embryonic Fibroblasts deficient in Tuberous
sclerosis protein--TSC1, which negatively regulates mTORC1
signaling and thus display constitutive mTORC1 activation,
resulting in phosphorylation (activation) of downstream molecules.
This cell-based assay is used to measure inhibition
(de-phosphorylation) of S6 and 4EBP1 by rapalogs or other mTOR
inhibitors.
[0500] MEF TSC1-/- cells were plated on Poly-D-lysine coated 384
well Griener clear bottom plates and incubated overnight at
37.degree. C., 5% CO.sub.2. On the following day, cells were washed
8 times with "Hard starve" solution (1 L DPBS+1 g D-(+) glucose+10
ml of 7.5% Sodium Bicarbonate+20 ml of 1M HEPES) and incubated for
further 2 hours in the same solution. Cells were next treated with
compounds with decreasing concentrations (8 points at 3.16 fold
dilutions) and incubated for 2 hours at 37.degree. C., 5% CO.sub.2.
Cells were fixed with 4% paraformaldehyde for 30 min and washed 5
times with TBS-EDTA followed by immuno-staining with fluorescent
tag labeled antibodies for pS6 (Ser240/244) (Cell Signaling #9468)
and p4EBP1 (Thr 37/46) (Cell Signaling #5123). Nuclei were
visualized with Hoechst (ThermoFisher Scientific #H3570) staining.
Cells were imaged (InCell 600) using respective fluorescence
channels and the potency of mTOR inhibitors was defined by pS6
IC.sub.50 (nM).
[0501] Animal maintenance, treatment with compounds and tissue
collection. All procedures involving animals were approved by the
Institutional Animal Care and Use Committee of the Novartis
Institutes for Biomedical Research, Cambridge, Mass., USA. Adult
Sprague Dawley (SD) male rats were purchased from Envigo
(Indianapolis, USA) or Charles River (USA). Once imported, rats
were maintained at the specific pathogen free facilities with
controlled temperature and light (22.degree. C., 12-h light/12-h
dark cycle: lights on at 0600 h/lights off at 1800 h) and with ad
libitum access to food and water. Rats were acclimated for at least
3 days before experiments commenced.
[0502] Example 2 and RAD001 were formulated for oral (per os, p.o.)
dosing. Blank formulations (without Example 2 or RAD001) served as
vehicle controls. Rats received a single dosed of Example 2, RAD001
or a respective vehicle p.o. At pre-determined times following
treatment, rats were anesthetized with 3.5% isoflurane and
euthanized. Various organs were collected and frozen in liquid
nitrogen. Blood was collected via a tail vein or a cardiac puncture
(terminal) and frozen for further pharmacokinetics analyses. All
tissues were stored at -80.degree. C. until analyzed.
[0503] Determination of Example 2 and RAD001 concentrations in
blood and tissues. Concentrations were determined using HPLC/Mass
spectrometry.
[0504] Protein extraction and immunoblotting. For protein
extraction, snap frozen tissues were lysed in MSD Lysis Buffer
(MSD, Rockville, Md.), supplemented with complete EDTA free
protease inhibitor and PhosSTOP phosphatase inhibitor tablets
(Roche, Manheim, Germany), and centrifuged at 13,000 g for 20 min
at 4.degree. C. The resultant supernatant was used for
immunoblotting. Protein was quantified with BCA protein assay
(Thermo Scientific, MA). Samples were resolved on 4-20%
Criterion.TM. TGX.TM. Precast Midi Protein gels (Bio-Rad, CA) and
transferred onto nitrocellulose membranes (Bio-Rad, CA) using a
Trans Turbo Blot system (Bio-Rad, CA). Immunoblotting was performed
with antibodies to p-S6 and t-S6 from Cell Signaling Technologies
(all 1:1000 in TBS-T with 5% BSA). The `p` and `t` prefixes signify
`phosphorylated` and `total` forms respectively. HRP-conjugated
secondary antibodies against rabbit (#7074) were from Cell
Signaling Technologies, MA. The chemiluminescence signal was
generated using SuperSignal.TM. West Femto Enhanced
Chemiluminescent Substrate (#34095, Thermo Scientific, MA) or
Western Lightning@ Plus-ECL Enhanced Chemiluminescence Substrate
(NEL103001EA, Perkin Elmer, MA) and was captured using the ChemiDoc
MP Imaging System (Bio-Rad). Resultant digital images were
converted into a TIFF format and quantified using ImageJ
software.
[0505] Generating FKPB12 knock-down cells. A CRISPR/CAS9 vector
containing gRNA sequence that targets the FKBP12 C-terminus
(GCTTCTAAAACTGGAATGAC) was transfected into 293T cells. Selection
was performed with puromycin for 48 hours and cells were plated for
colony formation. FKBP12 knock-down clones were screened by
immunoblotting with an anti-FKBP12 antibody (Thermo Scientific,
Pierce #PA1-026A). Clones with significantly reduced FKBP12
(approximately 80% reduction relative to levels in unperturbed 293T
cells) were selected.
[0506] Generation of FKPB12 knock-out cells. A CRISPR/Cas9 system
was used to deliver ribonucleoprotein complexes containing guide
RNA (gRNA) sequence that targets FKBP12 (GCCACTACTCACCGTCTCCT) into
293T cells, using the Amaxa.RTM. 4D-Nucleofector.TM. X Kit (Lonza,
V4XC-2032). Cell clones were screened by immunoblotting with an
anti-FKBP12 antibody (Novus, NB300-508) and single clones
demonstrating a complete FKBP12 knock-out (no measurable FKBP12)
were selected.
[0507] Treatment of wild-type (WT), FKBP12 knock-down and FKBP12
knock-out 293T cells with RAD001 and Example 2. WT, FKBP12
knock-down and FKBP12 knock-out 293T cells were plated at a density
of 30,000 cells per well in poly-D-Lysine coated 96-well plates
(Corning, #354461) in Dulbecco's modified Eagle's medium
(ThermoFisher, #11995-065) supplemented with 10% fetal bovine serum
(ThermoFisher, #16140-071). Cells were incubated at 37.degree. C.,
5% CO.sub.2 for 48 hours until they reached .about.80% confluence.
Cells were treated with RAD001 and Example 2 using a 12-point dose
range from 1000 nM to 0.0033 nM for 2 hours at 37.degree. C. in
duplicates. Media supplemented with blank Dimethyl sulfoxide (DMSO)
was used as a control for both compounds. Phosphorylated amounts of
S6K1 (Thr389) were detected by a sandwich ELISA kit (Cell
signaling, #7063C) following the manufacture's protocol.
[0508] SPR assay to determine binding affinity to FK506-binding
proteins (FKBP).
[0509] N-terminal avi-his6 tagged FKBP fusions to FKBP12, FKBP51
and FKBP52 were expressed in E. coli and purified using standard
chromatography. Each protein was subsequently immobilized on a
streptavidin chip in a Biacore 8K SPR instrument (GE Healthcare).
Using single-cycle kinetics, compound titrations were flowed at 45
uL/min over each surface using 2-minute association and 30-minute
dissociation phases in a buffer containing 50 mM Tris pH 7.5/150 mM
NaCl/0.01% Tween 20/1 mM DTT/2% DMSO. The data was fit using low
molecular weight (LMVV) single-cycle kinetics. The equilibrium
dissociation constants (K.sub.D) are reported.
[0510] Differential pharmacology of rapalogs may be achieved in
different cell or tissue types depending on 1) the relative
abundance of FKBP homologs in these cells/tissues and 2) the
specificity of binding to these different FKBP homologs (Mol. Cell
Biol. (2013) 33:1357-1367).
Results
[0511] In vitro potency of mTOR inhibitors was defined by pS6
IC.sub.50 (nM) in MEF TSC1-/- cells.
TABLE-US-00003 Compound IC50 (nM) Rapamycin 0.050 RAD001 0.050
Intermediate 1 0.092 Intermediate 3 0.0591 Intermediate 4 0.066
Intermediate 5 0.106 Example 1 2.4 Example 2 0.274 Example 3 1.45
Example 4 >500 Example 5 153 Example 6 0.170 Example 7 0.300
Example 8 0.328 Example 9 1.45 Example 10 0.557 Example 11 3.33
Example 12 0.374 Example 13 0.656 Example 14 7.77 Example 15 3.2
Example 16 0.645 Example 17 1.1 Example 18 1.55
1050 values are calculated as the average from multiple assays. The
equilibrium dissociation constants (K.sub.D) to FKBP12, FKBP51, and
FKBP52.
TABLE-US-00004 FKBP Average Compound binding protein K.sub.D (nM)
RAD001 FKBP12: 500 FKBP51: 811 FKBP52: 1765 Example 1 FKBP12: 0.35
FKBP51: 153 FKBP52: 174 Example 2 FKBP12: 16 FKBP51: >10000
FKBP52: >10000 Example 6 FKBP12: 0.32 FKBP51: 39 FKBP52: 53
Example 7 FKBP12: 1.2 FKBP51: 287 FKBP52: 236 Example 10 FKBP12:
1.6 FKBP51: 95 FKBP52: 176 Example 14 FKBP12: 16 FKBP51: -- FKBP52:
542 Example 16 FKBP12: 35 FKBP51: 4064 FKBP52: 4073
[0512] Pharmacokinetic profile of Example 2 in rats. Rats aged 4
months were treated with a single p.o. dose of Example 2 at 3 mg/kg
and blood was collected at 0.25, 0.5, 1, 2, 4 and 7 h following
dosing. In this experiment, Example 2 was formulated in 15%
Polyethylene Glycol (PEG) 300, 7.5% Solutol HS15, 7.5% Cremophore
EL in MilliQ water. Concentrations of Example 2 were measured by
HPLC-MS (FIG. 3).
[0513] Comparative blood and brain concentrations of Example 2 and
RAD001 in rats.
[0514] Rats aged 4 months were treated p.o. with Example 2
(formulated in 15% PEG300, 7.5% Solutol HS15, 7.5% Cremophore EL in
MilliQ water) at 3, 10 and 30 mg/kg. In another experiment, rats
aged 8 weeks were treated p.o. with RAD001 (formulated as a
microemulsion at 2% (w/w) and diluted to a final concentration in
MilliQ water) at 3, 10 and 30 mg/kg. Blood and brain tissues were
collected at 3 hours (h) and 24 h following treatment to determine
concentrations of the compounds. When administered at the same
doses (3, 10 and 30 mg/kg), blood and brain concentrations for
Example 2 were higher compared with those for RAD001 (FIGS. 4A and
4B).
[0515] To compare bioavailability of Example 2 versus RAD001 in the
rat, compounds were formulated as solution formulations: Example 1
was formulated in 15% PEG300, 7.5% Solutol HS15, 7.5% Cremophore EL
in PBS and RAD001 was formulated in 10% PEG300, 10% Solutol HS15,
10% Cremophore EL in PBS. Compounds were administered to rats aged
7-9 weeks (N=3 per group) p.o. at 3 mg/kg and intravenously (i.v).
at 1 mg/kg (FIGS. 4C and 4D). For Example 2 and RAD001
respectively, bioavailability was 18% and 19%, i.v. terminal
half-life 9.9 h and 9.5 h, clearance 9 mL/min/kg and 32 mL/min/kg
and Vdss (volume of distribution at the steady state) 4.4 L/kg and
18.8 L/kg.
[0516] Example 2 inhibits mTORC1 pathway in the rat liver. The
ability of Example 2 to inhibit mTORC1 pathway in vivo was
determined in rats aged 4 months (FIG. 5A-5D). A single dose of
Example 2 at 3, 10 and 30 mg/kg resulted in a significant
de-phosphorylation (inactivation) of S6 in rat livers (compared
with the vehicle control), three hours following dosing (FIGS. 5A
and 5B). S6 remained inactivated 24 h following dosing with 10
(trend p=0.06) and 30 mg/kg (FIGS. 5C and 5D).
[0517] FKBP12 is required for the inhibitory effect of Example 2,
but not RAD001. Phosphorylated amounts of S6K1 (Thr389) were
measured in WT, FKBP12 knock-down and FKBP12 knock-out 293T cells,
treated with RAD001 or Example 2. S6K1 is a downstream target of
mTORC1 and phosphorylation of its rapalog-sensitive Thr389 site is
used as a functional readout of mTORC1 activity. Lee, C. H., Inoki,
K. and Guan, K. L. (2007). mTOR pathway as a target in tissue
hypertrophy. Annu. Rev. Pharmacol. Toxicol. 47, 443-467. In WT 293T
cells, both RAD001 and Example 2 were efficacious at inhibiting
S6K1 (Thr389) by .about.80% (FIG. 6A). In FKBP12 knock-down cells,
Example 2 failed to inhibit S6K1 (Thr389) phosphorylation to the
same level as RAD001: the maximum inhibition of S6K1 (Thr389) by
RAD001 was .about.70%, while inhibition achieved by Example 2 was
.about.40% (FIG. 6B). In FKBP12 knock-out cells, Example 2 failed
to inhibit S6K1 (Thr389) phosphorylation, while >60% inhibition
was still achieved by RAD001 (FIG. 6C). These results indicate that
pharmacological effects of Example 2 are restricted to FKBP12. Such
specificity of Example 2 to FKBP12 may facilitate targeting cells
and tissues with relatively high levels of FKBP12 expression, while
avoiding (or minimizing) side effects in the tissues where FKBP12
is weakly expressed.
EQUIVALENTS AND SCOPE
[0518] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The disclosure includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The disclosure includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0519] Furthermore, the disclosure encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims are introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the disclosure,
or aspects of the disclosure, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
disclosure or aspects of the disclosure consist, or consist
essentially of, such elements and/or features. It is also noted
that the terms "comprising" and "containing" are intended to be
open and permits the inclusion of additional elements or steps.
Where ranges are given, endpoints are included. Furthermore, unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are
expressed as ranges can assume any specific value or sub-range
within the stated ranges in different embodiments of the
disclosure, to the tenth of the unit of the lower limit of the
range, unless the context clearly dictates otherwise.
[0520] This application refers to various issued patents, published
patent applications, journal articles, and other publications, all
of which are incorporated herein by reference. If there is a
conflict between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the disclosure that falls within the prior
art may be explicitly excluded from any one or more of the claims.
Because such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
disclosure can be excluded from any claim, for any reason, whether
or not related to the existence of prior art.
[0521] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments described herein. The scope
of the present embodiments described herein is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. Those of ordinary skill in the art will appreciate
that various changes and modifications to this description may be
made without departing from the spirit or scope of the disclosure,
as defined in the following claims.
* * * * *