U.S. patent application number 17/055937 was filed with the patent office on 2021-07-29 for inorganic salts of nicotinamide mononucloetide as anti-aging agents.
The applicant listed for this patent is Jumpstart Fertility Pty Ltd, Life Biosciences, Inc.. Invention is credited to Roland DOLLE, Rohan David JOYCE, Sebastian Mario MARCUCCIO, Simon TUCKER, Michel WATHIER.
Application Number | 20210230208 17/055937 |
Document ID | / |
Family ID | 1000005564409 |
Filed Date | 2021-07-29 |
United States Patent
Application |
20210230208 |
Kind Code |
A1 |
MARCUCCIO; Sebastian Mario ;
et al. |
July 29, 2021 |
INORGANIC SALTS OF NICOTINAMIDE MONONUCLOETIDE AS ANTI-AGING
AGENTS
Abstract
The present invention relates to inorganic salts of nicotinamide
mononucleotides and compositions of Formula I, useful in the
treatment of disorders and diseases associated with deficiencies in
NAD.sup.+: ##STR00001## wherein A, M, k, R.sup.1 and R.sup.2 are as
described herein.
Inventors: |
MARCUCCIO; Sebastian Mario;
(Scoresby, AU) ; JOYCE; Rohan David; (Noble Park,
AU) ; WATHIER; Michel; (Allston, MA) ; DOLLE;
Roland; (Boston, MA) ; TUCKER; Simon; (Coogee,
AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Biosciences, Inc.
Jumpstart Fertility Pty Ltd |
Boston
Coogee |
MA |
US
AU |
|
|
Family ID: |
1000005564409 |
Appl. No.: |
17/055937 |
Filed: |
May 15, 2019 |
PCT Filed: |
May 15, 2019 |
PCT NO: |
PCT/US2019/032432 |
371 Date: |
November 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62671807 |
May 15, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2500/33 20130101;
C12N 5/0603 20130101; C12N 2517/10 20130101; C12N 2501/999
20130101; C12N 2500/32 20130101; C12N 5/0609 20130101; C07H 19/048
20130101; A61P 15/08 20180101 |
International
Class: |
C07H 19/048 20060101
C07H019/048; C12N 5/075 20060101 C12N005/075; C12N 5/073 20060101
C12N005/073; A61P 15/08 20060101 A61P015/08 |
Claims
1. A salt of Formula (I): ##STR00028## or enantiomer, stereoisomer,
or tautomer thereof, wherein A is NR.sup.aR.sup.b; M.sup.1 is an
inorganic cation; R.sup.1 and R.sup.2 are independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8, provided M.sup.1 is not Na.
2. The salt of claim 1, wherein M.sup.1 is a cation selected from
the group consisting of Li.sup.+, K.sup.+, Rb.sup.+, Cs.sup.+,
Be.sup.2+, Mg.sup.2+, Ca.sup.2+, Sr.sup.2+, Zn.sup.2+, and
Ba.sup.2+.
3. The salt of any one of claims 1 to 2, wherein M.sup.1 is
K.sup.+.
4. The salt of any one of claims 1 to 2, wherein M.sup.1 is
Li.sup.+.
5. The salt of any one of claims 1 to 2, wherein M.sup.1 is
Rb.sup.+.
6. The salt of any one of claims 1 to 5, wherein R.sup.1 is H.
7. The salt of any one of claims 1 to 5, wherein R.sub.1 is
C.sub.1-C.sub.3alkyl.
8. The salt of any one of claims 1 to 5, wherein R.sup.2 is H.
9. The salt of any one of claims 1 to 5, wherein R.sup.2 is
C.sub.1-C.sub.3alkyl.
10. The salt of claim 1, selected from the group consisting of:
##STR00029##
11. A pharmaceutical composition comprising a salt of any one of
claims 1 to 10 and a pharmaceutically acceptable carrier.
12. A method of treating or preventing an age-related infertility
comprising administering to a subject in need thereof, an effective
amount of a salt of Formula I: ##STR00030## or enantiomer,
stereoisomer, or tautomer thereof, wherein A is NR.sup.aR.sup.b;
M.sup.1 is an inorganic cation; R.sup.1 and R.sup.2 are
independently H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8.
13. A method of treating or preventing infertility comprising
administering to a subject in need thereof, an effective amount of
a salt of Formula I: ##STR00031## or enantiomer, stereoisomer, or
tautomer thereof, wherein A is NR.sup.aR.sup.b; M.sup.1 is an
inorganic cation; R.sup.1 and R.sup.2 are independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.3cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8.
14. A method of improving oocyte or blastocyst quality and
maturation comprising contacting the oocyte or blastocyst with an
effective amount of a salt of Formula I: ##STR00032## or
enantiomer, stereoisomer, or tautomer thereof, wherein A is
NR.sup.aR.sup.b; M.sup.1 is an inorganic cation; R.sup.1 and
R.sup.2 are independently H, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl,
(C.sub.1-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.1-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8 prior to implantation into a subject in need of
treatment of age-related infertility.
15. A method of improving oocyte or blastocyst quality and
maturation comprising contacting the oocyte or blastocyst with an
effective amount of a salt of ##STR00033## or enantiomer,
stereoisomer, or tautomer thereof, wherein A is NR.sup.aR.sup.b;
M.sup.1 is an inorganic cation; R.sup.1 and R.sup.2 are
independently H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8 prior to implantation into a subject in need of
treatment of age-related infertility.
16. The method of claim 14 or 15 where the oocyte or blastocyst is
cultured in an IVF media containing the salt.
17. Use of a salt of Formula I: ##STR00034## or enantiomer,
stereoisomer, or tautomer thereof, wherein A is NR.sup.aR.sup.b;
M.sup.1 is an inorganic cation; R.sup.1 and R.sup.2 are
independently H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8 in the manufacture of a medicament for treating
an age-related disorder.
18. Use of a salt of Formula I: ##STR00035## or enantiomer,
stereoisomer, or tautomer thereof, wherein A is NR.sup.aR.sup.b;
M.sup.1 is an inorganic cation; R.sup.1 and R.sup.2 are
independently H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8 in the manufacture of a medicament for treating
infertility.
19. Use of a salt of Formula I: ##STR00036## or enantiomer,
stereoisomer, or tautomer thereof, wherein A is NR.sup.aR.sup.b;
M.sup.1 is an inorganic cation; R.sup.1 and R.sup.2 are
independently H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8 in the manufacture of a medicament for treating
age related infertility.
20. A process for the preparation of a salt of Formula I:
##STR00037## comprising contacting the compound of Formula II
##STR00038## with a metal-alkali hydroxide under conditions
effective to produce the product salt of Formula I.
21. The process of claim 20, wherein the metal-alkali hydroxide is
added dropwise into a solution of the compound of Formula II.
22. A cell culture medium for in vitro fertilization comprising: a
salt of Formula I: ##STR00039## or enantiomer, stereoisomer, or
tautomer thereof, wherein A is NR.sup.aR.sup.b; M.sup.1 is an
inorganic cation; R.sup.1 and R.sup.2 are independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8; and culturing agents.
23. The cell culture medium of claim 22, wherein the culturing
agent is an inorganic salt, an energy substrate, an amino acid, a
chelator, a pH indicator, an antibiotic, a serum, a vitamin, a
growth factor, or any combination thereof.
24. The cell culture medium of claim 23, wherein the inorganic salt
is calcium chloride, magnesium chloride, magnesium sulfate,
potassium chloride, sodium bicarbonate, sodium chloride, monosodium
phosphate, disodium phosphate, or any combination thereof.
25. The cell culture medium of claim 23, wherein the energy
substrate is glucose, pyruvate, lactate, pyruvate, or any
combination thereof.
26. The cell culture medium of claim 23, wherein the amino acid is
an essential amino acid.
27. The cell culture medium of claim 26, wherein the essential
amino acid is arginine, cysteine, glutamine, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
threonine, tryptophan, tyrosine, valine, or any combination
thereof.
28. The cell culture medium of claim 23, wherein the amino acid is
a non-essential amino acid.
29. The cell culture medium of claim 28, wherein the non-essential
amino acid is alanine, asparagine, aspartate, glutamate, proline,
serine, or any combination thereof.
30. The cell culture medium of claim 23, wherein the chelator is
clathro chelate, acetyl acetone, amino polycarboxylic acid, ATMP,
BAPTA, BDTH2, citric acid, cryptand, deferasirox,
2,3-dihydrobenzoic acid, 2,3-dimercapto-1-propane sulfonic acid,
dimercapto succinic acid, DOTA, DTPMP, EDDHA, EDDS, EDTMP,
etidronic acid, fura-2, gluconic acid, homocitric acid, imino
diacetic acid, Indo-1, nitrile triacetic acid, pentetic acid
(DTPA), phosphonate, phytochelati, poly aspartic acid, sodium poly
aspartate, trisodium citrate, transferrin, EDTA, EGTA, or any
combination thereof.
31. The cell culture medium of claim 23, wherein the pH indicator
is phenol red, bromothymol blue, alizarin red, 9-aminoacridine, or
any combination thereof.
32. The cell culture medium of claim 23, wherein the antibiotic is
actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin,
gentamicin, kanamycin, neomycin, penicillin, polymyxin B,
streptomycin, or any combination thereof.
33. The cell culture medium of claim 23, wherein the serum is human
serum albumin, bovine serum albumin, fetal bovine serum, synthetic
serum, or any combination thereof.
34. The cell culture medium of claim 23, wherein the vitamin is
ascorbic acid, biotin, menadione sodium bisulfite, mitomycin C,
pyridoxamine dihydrochloride, retinyl acetate, (-)-riboflavin,
(+)-sodium L-ascorbate, (+)-.alpha.-tocopherol, vitamin B.sub.12,
thiamine hydrochloride, i-inositol, pyridoxal hydrochloride,
nicotinamide, folic acid, D-calcium pantothenate, choline chloride,
or any combination thereof.
35. The cell culture medium of claim 23, wherein the growth factor
is adrenomedullin, angiopoietin, bone morphogenetic proteins,
macrophage colony-stimulating factor (M-CSF), granulocyte
colony-stimulating factor (G-CSF), granulocyte macrophage
colony-stimulating factor (GM-CSF), epidermal growth factor,
ephrins, erythropoietin, fibroblast growth factor, growth
differentiation factor-9, hepatocyte growth factor, insulin,
insulin-like growth factors, interleukins, keratinocyte growth
factor, migration-stimulating factor, macrophage-stimulating
protein, myostatin, neurotrophins, t-cell growth factor,
thrombopoietin, transforming growth factor, tumor necrosis
factor-alpha, vascular endothelial growth factor, or any
combination thereof.
36. The cell culture medium of claim 22, wherein the cell culture
medium further comprises an oocyte, zygote, blastocyst, or any
combination thereof.
37. A salt of Formula I: ##STR00040## or enantiomer, stereoisomer,
or tautomer thereof, wherein A is NR.sup.aR.sup.b; M.sup.1 is an
inorganic cation; R.sup.1 and R.sup.2 are independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
or R.sup.1 and R.sup.2, together with the atom to which they are
attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; R.sup.a and R.sup.b are
independently, at each occurrence, H, or C.sub.1-C.sub.6alkyl,
wherein the alkyl is optionally substituted with one or more
substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; k is 1 or 2; and n is an
integer from 1 to 8. made by the process of claim 20.
38. The salt of claim 37, wherein the salt is ##STR00041##
Description
RELATED APPLICATIONS
[0001] The application claims priority to, and the benefit of, U.S.
Provisional Application No. 62/671,807, filed on May 15, 2018, the
contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE DISCLOSURE
[0002] The present invention relates to inorganic salts of
nicotinamide mononucleotides and compositions thereof useful in the
treatment of disorders and diseases associated with aging.
BACKGROUND OF THE DISCLOSURE
[0003] Aging is the result of complex interactions involving
biological, physical, and biochemical processes that cause
dysfunctions in cells and organs which manifests in a variety of
diseases and other outcomes. For example, female fecundity is
markedly sensitive to the effects of ageing. For example, the USA
Centers for Disease Control has reported that the percentage of
assisted reproductive technology (ART) associated pregnancies and
births percentages declined steadily among women in their mid-30s
onward from approximately 25% of ART cycles resulting in singleton
live births to 14% by the age of 40 (Centers for Disease Control
and Prevention, American Society for Reproductive Medicine, Society
for Assisted Reproductive Technology. 2011 Assisted Reproductive
Technology National Summary Report. Atlanta (Ga.): US Dept of
Health and Human Services; 2013). This trend is markedly increased
above the age of 40 with the CDC reporting that women older than
age 44 have a very low likelihood of success. The percentages of
live births and singleton live births declined to about 1% in this
group. It is generally considered that a woman's age is the most
important factor affecting the chance of a live birth when her own
eggs (oocytes) are used.
[0004] It is understood that the qualitative deterioration of
oocytes due to aging is a fundamental factor in the decline in
fertility. In older women, for example, the oocytes are reported to
be susceptible to abnormal chromosome division, exhibit decreased
mitochondrial quality, low ATP production, increased oxidative
stress, and decreased antioxidant levels (Nelson S M, Telfer E E,
Anderson R A. The ageing ovary and uterus: new biological insights.
Hum Reprod Update. 2013; 19:67-83; Wilding M. Potential long-term
risks associated with maternal aging (the role of the
mitochondria). Fertil Steril. 2015; 103:1397-401; 3. Meldrum D R,
Casper R F, Diez-Juan A, Simon C, Domar A D, Frydman R. Aging and
the environment affect gamete and embryo potential: can we
intervene? Fertil Steril. 2016; 105:548-59).
[0005] For all of the foregoing reasons, the oocyte represents an
excellent target tissue for the evaluation of therapeutic
modalities that are expected to have an impact upon the ageing
process and, furthermore, offer the prospect of addressing
age-related infertility.
[0006] One such possible therapeutic modality for treating ageing
comprises agents which boost therapeutic levels of NAD.sup.+.
NAD.sup.+ is an essential component of cellular processes necessary
to support various metabolic functions. The classic role of
NAD.sup.+ is a co-enzyme that catalyzes cellular redox reactions,
becoming reduced to NADH, in many fundamental metabolic processes,
such as glycolysis, fatty acid beta oxidation, or the tricarboxylic
acid cycle. In addition to playing these roles, NAD.sup.+ has a
critical role as the substrate of NAD.sup.+-consuming enzymes such
as poly-ADP-ribose polymerases (PARPs), sirtuins, and CD38/157
ectoenzymes. These NAD.sup.+-consuming enzymes have been known to
mediate many fundamental cellular processes.
[0007] There are five major precursors and intermediates to
synthesize NAD.sup.+: tryptophan, nicotinamide, nicotinic acid
(NA), nicotinamide riboside (NR), and nicotinamide mononucleotide
(NMN). NAD.sup.+ can be synthesized de novo by the conversion of
the amino acid tryptophan through multiple enzymatic steps to
nicotinic acid mononucleotide (NaMN). NaMN is converted to
nicotinic acid dinucleotide (NaAD.sup.+) by NMN/NaMN
adenylyltransferases (NMNATs) and then amidated to NAD.sup.+ by
NAD.sup.+ synthetase.
[0008] In mammals, a major pathway of NAD.sup.+ biosynthesis is the
salvage pathway from nicotinamide. Nicotinamide is converted to
NMN, a key NAD+intermediate, by nicotinamide
phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in this
pathway. NMNATs then convert NMN into NAD.sup.+. NAMPT plays a
critical role in regulating cellular NAD+levels. On the other hand,
nicotinic acid is converted to NaMN by nicotinic acid
phosphoribosyltransferase (NPT). NR needs to be converted to NMN by
nicotinamide ribose kinases, NMRK1 and NMRK2 (also known as NRK1
and NRK2), which phosphorylate NR 16.Maintenance of adequate
NAD.sup.+ biosynthesis is paramount for cell survival and function.
Derailment from normal NAD homeostasis substantially affects not
only the NAD.sup.+/NADH pool required for redox reactions but also
activities of NAD.sup.+-dependent enzymes for crucial cellular
functions.
[0009] It is now becoming a consensus that NAD.sup.+ levels decline
at cellular, tissue/organ, and organismal levels during the course
of aging. Activities of NAD-consuming enzymes are affected by this
NAD.sup.+ decline, contributing to a broad range of age-associated
pathophysiologies
[0010] Nicotinamide adenine dinucleotide is an enzyme co-factor
that is essential for the function of several enzymes related to
reduction-oxidation reactions and energy metabolism. (Katrina L.
Bogan & Charles Brenner, Nicotinic Acid, Nicotinamide and
Nicotinamide Riboside: A Molecular Evaluation of NAD.sup.+
Precursor Vitamins in Nutritions, 28, Annual Review of Nutrition
115 (2008)). NAD.sup.+ functions as an electron carrier in energy
metabolism of amino acids, fatty acids and carbohydrates (Bogan
& Brenner, Annu. Rev. Nutr. 2008, 28, 115-130). NAD.sup.+ is
critical for redox reactions and as a substrate for signaling by
the PARPs (poly adenoside diphophosphate-ribose polymerases) and
the sirtuins (SIRT1 to SIRT7), in the regulation of DNA repair,
energy metabolism, cell survival and circadian rhythms which have
all been shown to be critical in the ageing process (Bronkowski, M.
S. & Sinclair, D., Nat. Rev. Mole. Cell. Bio., 17, 679-690,
(2016)). Raising NAD.sup.+ concentrations delays aging in yeast,
files and mice (Mouchiroud et al. Cell 154, 464-471, (2014)). It
has recently also been demonstrated that NAD.sup.+ directly
regulates protein-protein interactions, the modulation of which may
protect against cancer and radiation exposure as well as having a
direct impact on aging (Li et al., Science 355, 1312-1317, 2017).
Thus increasing bodies of evidence support the idea that
interventions using NAD.sup.+ intermediates, such as NMN and NR,
can bolster the system by restoring the available NAD.sup.+ and
mitigate physiological decline associated with aging.
[0011] Although NAD can be synthesized de novo from the amino acid
tryptophan, this process does not occur in all tissues, requiring
most cells to rely on the salvage pathway (described above) for
regenerating NAD.sup.+ from other intracellular intermediates,
which are primarily made available through dietary sources
(Christopher R. Martens, et al., Nat. Commun. 9, 1286, (2018) and
Bogan, K. L. & Brenner, C., Annu. Rev. Nutr. 28, 115-130,
(2008)). Other NAD precursors like nicotinic acid and nicotinamide
can also be administered to boost NAD cellular bioavailability.
However, clinically relevant levels of nicotinic acid are
associated with undesirable flushing at therapeutic doses (MacKay,
D., Hathcock, J. & Guarneri, E., Nutr. Rev. 70, 357-366
(2012)), and nicotinamide does not reliably activate (and may even
inhibit) sirtuins despite raising concentrations of NAD (Bitterman,
K. J., et al., J. Biol. Chem. 277, 45099-45107 (2002); Guan, X., et
al., PLoS One. 9, e107729 (2014); and Trammell, S. A. et al. Nat.
Commun. 7, 12948 (2016)). Therefore, administration of nicotinic
acid or nicotinamide is unlikely to be widely adopted for
maintaining health and function with aging.
[0012] In contrast to nicotinic acid and nicotinamide,
administration of NAD.sup.+ metabolites such as nicotinamide
mononucleotide (NMN) or nicotinamide riboside (NR), appears to
increase levels of NAD.sup.+ and improves multiple physiological
functions in animal models (Yoshino, J. et al., Cell Metab. 14,
528-536 (2011); Mills, K. F. et al., Cell Metab. 24, 795-806
(2016); and Frederick, D. W. et al., Cell Metab. 24, 269-282
(2016)). At least one of these metabolites has been reported to be
well tolerated in humans leading to elevation of NAD levels and
improved physiological functions albeit that further studies are
required to confirm the findings of this exploratory study
(Christopher R. Martens, et al., Nat. Commun 9, 1286, (2018)).
Furthermore, a recent study showed that single doses of NR
stimulated blood cellular NAD.sup.+ metabolism in healthy humans in
a dose-dependent manner (Trammell, S. A. et al., Nat. Commun. 7,
12948 (2016)), showing the limitation of this metabolite. However,
many of the known NAD+ metabolites are unstable in a variety of
physiological environments and thus do not lend themselves to
viable pharmaceutical drugs for administration to patients in need
of such metabolites for boosting the NAD+ levels in said
patients.
[0013] Given the central role that NAD.sup.+ plays in critical
cellular and physiological pathways, developing novel stable agents
with improved properties that can elevate NAD.sup.+ levels in
disease states and/or during the aging process is necessary to
improve the human condition.
SUMMARY OF THE DISCLOSURE
[0014] Provided herein are inorganic salts of NMN which salts
surprisingly increase cellular NAD.sup.+ levels to a greater extent
than NMN.
[0015] A first aspect of the present disclosure relates to salts of
Formula (I): (I),
##STR00002##
and enantiomers, stereoisomers, and tautomers thereof, wherein
[0016] A is NR.sup.aR.sup.b;
[0017] M.sup.1 is an inorganic cation;
[0018] R.sup.1 and R.sup.2 are independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or
--[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a,
[0019] or R.sup.1 and R.sup.2, together with the atom to which they
are attached, form a 5-membered heterocyclic ring optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.1-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl;
[0020] R.sup.a and R.sup.b are independently, at each occurrence,
H, or C.sub.1-C.sub.6alkyl, wherein the alkyl is optionally
substituted with one or more substituents selected from
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl; [0021] k is 1 or 2; and [0022]
n is an integer from 1 to 8, provided M.sup.1 is not Na
[0023] Another aspect of the present disclosure relates to a
pharmaceutical composition comprising a salt of Formula Ia, or a
pharmaceutically acceptable salt thereof, in association with a
pharmaceutically acceptable carrier.
[0024] Another aspect of the present disclosure relates to a method
of treating or preventing an age-related infertility comprising
administering to a subject in need thereof, an effective amount of
a salt of Formula (I), or enantiomer, stereoisomer, or tautomer
thereof.
[0025] Another aspect of the present disclosure relates to a method
of treating or preventing infertility comprising administering to a
subject in need thereof, an effective amount of a salt of Formula
(I), or enantiomer, stereoisomer, or tautomer thereof.
[0026] Another aspect of the present disclosure relates to a salt
of Formula (I), or enantiomer, stereoisomer, or tautomer thereof,
for use in the manufacture of a medicament for treating age related
infertility.
[0027] Another aspect of the present disclosure relates to a salt
of Formula (I), or enantiomer, stereoisomer, or tautomer thereof,
for use in the manufacture of a medicament for use in treating
infertility.
[0028] In another aspect, the present invention relates to a method
of treating or preventing an age-related disorder, comprising
administering to a subject in need thereof, a therapeutically
effective amount of a pharmaceutical composition of the salt of
Formula I.
[0029] Another aspect of the present disclosure relates to a method
of improving oocyte quality and maturation, comprising
administering to a subject in need thereof, a therapeutically
effective amount of a salt of Formula I.
[0030] Another aspect of the present disclosure relates to the use
of a salt of Formula (I), or enantiomer, stereoisomer, or tautomer
thereof, in the manufacture of a medicament for treating an
age-related disorder.
[0031] In another aspect, the invention comprises treatment of an
oocyte with a salt of Formula (I) ex vivo prior to implantation
into a subject, for the treatment of age-related infertility.
[0032] In another aspect, the invention comprises treatment of a
blastocyst with a salt of Formula (I) ex vivo prior to implantation
into a subject, for the treatment of age-related infertility.
[0033] In another aspect, the invention comprises treatment of an
oocyte with a salt of Formula (I) ex vivo prior to implantation
into a subject, for the treatment of infertility.
[0034] In another aspect, the invention comprises treatment of a
blastocyst with a salt of Formula (I) ex vivo prior to implantation
into a subject, for the treatment of infertility.
[0035] In another aspect, a salt of Formula (I) is provided as a
component in solution for use in treating a cell ex vivo for use in
the treatment of an age related disorder. In some embodiments, the
age related disorder is age-related infertility. In other aspects a
salt of Formula (I) is provided as a component in solution for use
in treating a cell ex vivo for use in the treatment of
infertility.
[0036] Another aspect of present disclosure relates to a process
for preparing salts of Formula (I), comprising contacting a
nicotinamide mononucleotide derivative of Formula II with a
metal-alkali hydroxide under suitable conditions effective to
produce the salt of Formula I.
[0037] The present disclosure also relates to methods of
accelerating recovery from a disease or disorder. The method
comprises administering to a subject in need thereof an effective
amount of a salt of Formula (I) in combination with the prescribed
treatment of said disease.
[0038] In another aspect, the present disclosure relates to a cell
culture medium for in vitro fertilization comprising: one or more
salts of Formula (I) and culturing agents.
[0039] Another aspect of the present disclosure relates to salts
Formula I prepared by a process comprising contacting a
nicotinamide mononucleotide derivative of Formula II with a
metal-alkali hydroxide under suitable conditions effective to
produce the salt of Formula I.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0040] The present disclosure relates to salts of Formula (I) and
compositions comprising the same, that are useful in treating or
preventing an age-related disorder such as age related infertility
amongst other such disorders. The methods of the present disclosure
can be used in the treatment of a variety of diseases and disorders
by preventing or ameliorating anti-aging processes and aiding
cellular restoration processes.
[0041] Salts of Formula (I) are potent and are efficacious at
clinically achievable doses; are stable in a variety of potential
dosage forms; possess acceptable solubility, acceptable pH, are
crystalline, have a reduced propensity to absorb water, display
ease of handling,--all of which are consistent with the
development, manufacture and use of a medicament. In addition, the
salts disclosed herein offer increased biological activity toward
increased cellular NAD.sup.+ levels.
[0042] In one aspect of disclosure relates to a salt of Formula
I
##STR00003##
wherein A, k, M.sup.1, R.sup.1 and R.sup.2 are as described
herein.
[0043] The articles "a" and "an" are used in this disclosure to
refer to one or more than one (i.e., to at least one) of the
grammatical object of the article. By way of example, "an element"
means one element or more than one element.
[0044] The term "and/or" is used in this disclosure to mean either
"and" or "or" unless indicated otherwise.
[0045] The term "optionally substituted" is understood to mean that
a given chemical moiety (e.g., an alkyl group) can (but is not
required to) be bonded other substituents (e.g., heteroatoms). For
instance, an alkyl group that is optionally substituted can be a
fully saturated alkyl chain (i.e., a pure hydrocarbon).
Alternatively, the same optionally substituted alkyl group can have
substituents different from hydrogen. For instance, it can, at any
point along the chain be bounded to a halogen atom, a hydroxyl
group, or any other substituent described herein. Thus the term
"optionally substituted" means that a given chemical moiety has the
potential to contain other functional groups, but does not
necessarily have any further functional groups. Suitable
substituents used in the optional substitution of the described
groups include, without limitation, halogen, oxo, --OH, --CN,
--COOH, --CH.sub.2CN, --O--(C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkyl, C.sub.1-C.sub.6 alkoxy,
(C.sub.1-C.sub.6)haloalkyl, C.sub.1-C.sub.6 haloalkoxy,
--O--(C.sub.2-C.sub.6) alkenyl, --O--(C.sub.2-C.sub.6) alkynyl,
(C.sub.2-C.sub.6) alkenyl, (C.sub.2-C.sub.6) alkynyl, --OH,
--OP(O)(OH).sub.2, --OC(O)(C.sub.1-C.sub.6) alkyl,
--C(O)(C.sub.1-C.sub.6)alkyl, --OC(O)O(C.sub.1-C.sub.6) alkyl,
--NH.sub.2, --NH((C.sub.1-C.sub.6) alkyl), --N((C.sub.1-C.sub.6)
alkyl).sub.2, --NHC(O)(C.sub.1-C.sub.6) alkyl,
--C(O)NH(C.sub.1-C.sub.6) alkyl, --S(O).sub.2(C.sub.1-C.sub.6)
alkyl, --S(O)NH(C.sub.1-C.sub.6) alkyl, and S(O)N((C.sub.1-C.sub.6)
alkyl).sub.2. The substituents can themselves be optionally
substituted. "Optionally substituted" as used herein also refers to
substituted or unsubstituted whose meaning is described below.
[0046] As used herein, the term "substituted" means that the
specified group or moiety bears one or more suitable substituents
wherein the substituents may connect to the specified group or
moiety at one or more positions. For example, an aryl substituted
with a cycloalkyl may indicate that the cycloalkyl connects to one
atom of the aryl with a bond or by fusing with the aryl and sharing
two or more common atoms.
[0047] As used herein, the term "unsubstituted" means that the
specified group bears no substituents.
[0048] Unless otherwise specifically defined, the term "aryl"
refers to cyclic, aromatic hydrocarbon groups that have 1 to 3
aromatic rings, including monocyclic or bicyclic groups such as
phenyl, biphenyl or naphthyl. Where containing two aromatic rings
(bicyclic, etc.), the aromatic rings of the aryl group may be
joined at a single point (e.g., biphenyl), or fused (e.g.,
naphthyl). The aryl group may be optionally substituted by one or
more substituents, e.g., 1 to 5 substituents, at any point of
attachment. Exemplary substituents include, but are not limited to,
--H, -halogen, --O--(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6)
alkyl, --O--(C.sub.2-C.sub.6) alkenyl, --O--(C.sub.2-C.sub.6)
alkynyl, (C.sub.2-C.sub.6) alkenyl, (C.sub.2-C.sub.6) alkynyl,
--OH, --OP(O)(OH).sub.2, --OC(O)(C.sub.1-C.sub.6) alkyl,
--C(O)(C.sub.1-C.sub.6) alkyl, --OC(O)O(C.sub.1-C.sub.6) alkyl,
NH.sub.2, NH((C.sub.1-C.sub.6) alkyl), N((C.sub.1-C.sub.6)
alkyl).sub.2, --S(O).sub.2--(C.sub.1-C.sub.6) alkyl,
--S(O)NH(C.sub.1-C.sub.6) alkyl, and S(O)N((C.sub.1-C.sub.6)
alkyl).sub.2. The substituents can themselves be optionally
substituted. Furthermore when containing two fused rings the aryl
groups herein defined may have a saturated or partially saturated
ring fused with a fully aromatic ring. Exemplary ring systems of
these aryl groups include, but are not limited to, phenyl,
biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl,
indanyl, indenyl, tetrahydronaphthalenyl, tetrahydrobenzoannulenyl,
and the like.
[0049] Unless otherwise specifically defined, "heteroaryl" means a
monovalent monocyclic or polycyclic aromatic radical of 5 to 24
ring atoms, containing one or more ring heteroatoms selected from
N, O, or S, the remaining ring atoms being C. Heteroaryl as herein
defined also means a bicyclic heteroaromatic group wherein the
heteroatom is selected from N, O, or S. The aromatic radical is
optionally substituted independently with one or more substituents
described herein. Examples include, but are not limited to, furyl,
thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl,
isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl,
thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl,
thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene,
triazolyl, triazinyl, imidazo[1,2-b]pyrazolyl,
furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl,
pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl,
pyrazolo[3,4-c]pyridinyl, thieno[3,2-c]pyridinyl,
thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl,
indolyl, indolinyl, indolinonyl, dihydrobenzothiophenyl,
dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl,
tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl,
quinolinyl, isoquinolinyl, 1,6-naphthyridinyl,
benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthyridinyl,
thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl,
[1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl,
pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl,
pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl,
pyrrolo[1,2-a]pyrimidinyl, tetrahydro pyrrolo[1,2-a]pyrimidinyl,
3,4-dihydro-2H-1.quadrature..sup.2-pyrrolo[2,1-b]pyrimidine,
dibenzo[b,d] thiophene, pyridin-2-one, furo[3,2-c]pyridinyl,
furo[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4] thiazinyl,
benzooxazolyl, benzoisoxazolyl, furo[2,3-b]pyridinyl,
benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine,
[1,2,4]triazolo[1,5-a]pyridinyl, benzo [1,2,3]triazolyl,
imidazo[1,2-a]pyrimidinyl, [1,2,4]triazolo[4,3-b]pyridazinyl,
benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole,
1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo
[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl,
thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl,
thieno[2,3-b]pyrrolyl, 3H-indolyl, and derivatives thereof.
Furthermore when containing two fused rings the heteroaryl groups
herein defined may have a saturated or partially saturated ring
fused with a fully aromaticring. Exemplary ring systems of these
heteroaryl groups include indolinyl, indolinonyl,
dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl,
thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine,
3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl,
indolyl, and dihydrobenzoxanyl.
[0050] Halogen or "halo" refers to fluorine, chlorine, bromine, or
iodine.
[0051] Alkyl refers to a straight or branched chain saturated
hydrocarbon containing 1-12 carbon atoms. Examples of a
(C.sub.1-C.sub.6) alkyl group include, but are not limited to,
methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl,
sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.
[0052] "Alkoxy" refers to a straight or branched chain saturated
hydrocarbon containing 1-12 carbon atoms containing a terminal "O"
in the chain, i.e., --O(alkyl). Examples of alkoxy groups include,
without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or
pentoxy groups.
[0053] "Alkenyl" refers to a straight or branched chain unsaturated
hydrocarbon containing 2-12 carbon atoms. The "alkenyl" group
contains at least one double bond in the chain. The double bond of
an alkenyl group can be unconjugated or conjugated to another
unsaturated group. Examples of alkenyl groups include ethenyl,
propenyl, n-butenyl, iso-butenyl, pentenyl, or hexenyl. An alkenyl
group can be unsubstituted or substituted. Alkenyl, as herein
defined, may be straight or branched.
[0054] "Alkynyl" refers to a straight or branched chain unsaturated
hydrocarbon containing 2-12 carbon atoms. The "alkynyl" group
contains at least one triple bond in the chain. Examples of alkenyl
groups include ethynyl, propargyl, n-butynyl, iso-butynyl,
pentynyl, or hexynyl. An alkynyl group can be unsubstituted or
substituted.
[0055] The term "alkylene" or "alkylenyl" refers to a divalent
alkyl radical. Any of the above mentioned monovalent alkyl groups
may be an alkylene by abstraction of a second hydrogen atom from
the alkyl. As herein defined, alkylene may also be a
C.sub.1-C.sub.6alkylene. An alkylene may further be a
C.sub.1-C.sub.4 alkylene. Typical alkylene groups include, but are
not limited to, --CH.sub.2--, --CH(CH.sub.3)--,
--C(CH.sub.3).sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--, --CH.sub.2C(CH.sub.3).sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
and the like.
[0056] "Cycloalkyl" means monocyclic or polycyclic saturated carbon
rings (e.g., fused, bridged, or spiro rings) containing 3-18 carbon
atoms (e.g., C.sub.3-C.sub.10). Examples of cycloalkyl groups
include, without limitations, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl,
bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl.
[0057] "Heterocyclyl" or "heterocycloalkyl" means monocyclic or
polycyclic rings (e.g., fused, bridged, or spiro rings) containing
carbon and heteroatoms taken from oxygen, nitrogen, or sulfur and
wherein there is not delocalized .quadrature. electrons
(aromaticity) shared among the ring carbon or heteroatoms. The
heterocycloalkyl can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-10-, 11-, or
12-membered ring. The heterocycloalkyl ring structure may be
substituted by one or more substituents. The substituents can
themselves be optionally substituted. Examples of heterocyclyl
rings include, but are not limited to, oxetanyl, azetadinyl,
tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, oxazolinyl,
oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl,
tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl,
thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl
S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl,
oxazolidinonyl, and homotropanyl. In accordance with the present
disclosure, 3- to 10-membered heterocyclyl refers to saturated or
partially saturated non aromatic rings structures containing
between 3 and 10 atoms in which there is at least one heteroatoms
selected from the group N, O, or S.
[0058] The term "hydroxyalkyl" means an alkyl group as defined
above, where the alkyl group is substituted with one or more --OH
groups. Examples of hydroxyalkyl groups include HO--CH.sub.2--,
HO--CH.sub.2--CH.sub.2-- and CH.sub.3--CH(OH)--.
[0059] The term "haloalkyl" as used herein refers to an alkyl
group, as defined herein, which is substituted one or more halogen.
Examples of haloalkyl groups include, but are not limited to,
trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl,
etc.
[0060] The term "haloalkoxy" as used herein refers to an alkoxy
group, as defined herein, which is substituted one or more halogen.
Examples of haloalkyl groups include, but are not limited to,
trifluoromethoxy, difluoromethoxy, pentafluoroethoxy,
trichloromethoxy, etc.
[0061] The term "cyano" as used herein means a substituent having a
carbon atom joined to a nitrogen atom by a triple bond, i.e.,
C.ident.N.
[0062] The term "amine" as used herein refers to primary
(R--NH.sub.2, R.noteq.H), secondary (R.sub.2--NH, R.sub.2.noteq.H)
and tertiary (R.sub.3--N, R.noteq.H) amines. A substituted amine is
intended to mean an amine where at least one of the hydrogen atoms
has been replaced by the substituent.
[0063] The term "amino" as used herein means a substituent
containing at least one nitrogen atom. Specifically, NH.sub.2,
--NH(alkyl) or alkylamino, --N(alkyl).sub.2 or dialkylamino,
amide-, carbamide-, urea, and sulfamide substituents are included
in the term "amino".
[0064] The term "oxo" as used herein refers to an ".dbd.O"
group.
[0065] The term "isomer" refers to salts and/or compounds that have
the same composition and molecular weight but differ in physical
and/or chemical properties. The structural difference may be in
constitution (geometric isomers) or in the ability to rotate the
plane of polarized light (stereoisomers). With regard to
stereoisomers, the salts of Formula (I) may have one or more
asymmetric carbon atom and may occur as racemates, racemic mixtures
and as individual enantiomers or diastereomers.
[0066] The term "cation" contemplates all organic and inorganic
positively charged ions. The cation in the compounds of formula (I)
may include, but are not limited to, those described in "Handbook
of Pharmaceutical Salts: Properties, Selections and Use", Stahl, H.
and Wermuth, C., ed., Verlag Helvetica Chemica Acta, Zurich,
Wiley-VCH, 2002.
[0067] The term "inorganic cation" refers to any positively charged
inorganic atom or group of atoms. Exemplary inorganic cations are
the alkali metals, (e.g., lithium, sodium, and potassium), the
alkaline earth metals (e.g., calcium and magnesium), manganic,
ferrous, cobalt, thallium, manganous, and ammonium
(NH.sub.4.sup.+).
[0068] The term "degrade," as used herein, means to chemically
decompose. Degradation is distinct from melting in that it involves
the breaking of chemical bonds or the formation of new chemical
bonds. Methods of degradation can include hydrolysis,
solventolysis, thermolysis, or oxidation.
[0069] The disclosure also includes pharmaceutical compositions
comprising an effective amount of a disclosed salt and a
pharmaceutically acceptable carrier. Representative
"pharmaceutically acceptable salts" include, e.g., water-soluble
and water-insoluble salts, such as the acetate, amsonate
(4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate,
bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate,
calcium, calcium edetate, camsylate, carbonate, chloride, citrate,
clavulariate, dihydrochloride, edetate, edisylate, estolate,
esylate, fumerate, fiunarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexafluorophosphate, hexylresorcinate,
hydrabamine, hydrobromide, hydrochloride, hydroxvnaphthoate,
iodide, isothionate, lactate, lactobionate, laurate, magnesium,
malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,
methylsulfate, mucate, napsylate, nitrate, N-methylglucamine
ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate,
pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate),
pantothenate, phosphate/diphosphate, picrate, polygalacturonate,
propionate, p-toluenesulfonate, salicylate, stearate, subacetate,
succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate,
teoclate, tosylate, triethiodide, and valerate salts.
[0070] A "patient" or "subject" is a mammal, e.g., a human, mouse,
rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate,
such as a monkey, chimpanzee, baboon or rhesus.
[0071] An "effective amount" when used in connection with a salt or
pharmaceutical composition is an amount effective for treating or
preventing a disease in a subject as described herein.
[0072] The term "carrier", as used in this disclosure, encompasses
carriers, excipients, and diluents and means a material,
composition or vehicle, such as a liquid or solid filler, diluent,
excipient, solvent or encapsulating material, involved in carrying
or transporting a pharmaceutical agent from one organ, or portion
of the body, to another organ, or portion of the body of a
subject.
[0073] As used herein, the terms "treat" or "treatment" are
synonymous with the term "prevent" and are meant to indicate a
postponement of development of diseases, preventing the development
of diseases, and/or reducing severity of such symptoms that will or
are expected to develop. Thus, these terms include ameliorating
existing disease symptoms, preventing additional symptoms,
ameliorating or preventing the underlying causes of symptoms,
inhibiting the disorder or disease, e.g., arresting the development
of the disorder or disease, relieving the disorder or disease,
causing regression of the disorder or disease, relieving a
condition caused by the disease or disorder, or stopping or
alleviating the symptoms of the disease or disorder.
[0074] The term "disorder" is used in this disclosure to mean, and
is used interchangeably with, the terms disease, condition, or
illness, unless otherwise indicated.
[0075] The term "administer", "administering", or "administration"
as used in this disclosure refers to either directly administering
a disclosed salt or a composition to a subject, or administering a
prodrug derivative or analog of the salt or composition to the
subject, which can form an equivalent amount of active salt within
the subject's body.
[0076] The term "oocyte" is synonymous with the term "egg" and is
used in this disclosure to mean a female mammalian gametocyte or
germ cell.
[0077] The term "zygote" is used in this disclosure to mean a
fertilized egg or a diploid cell formed from the fusion of an
oocyte and sperm during fertilization.
[0078] The term "blastocyst" is used in this disclosure to mean a
mammalian embryo in which cellular differentiation has occurred
consisting of an inner cell mass or embryoblast, a cavity, and an
outer layer or trophoblast.
Salts of the Present Disclosure
[0079] The present disclosure relates to salts of Formula (I), and
enantiomers, stereoisomers, and tautomers thereof, which are useful
for the treatment of diseases and disorders associated with aging
and cellular restoration such as age related infertility.
[0080] In some embodiments, the salt of Formula I has the structure
of Formula (Ia)
##STR00004##
[0081] In some embodiments, the salt of Formula I has the structure
of Formula Ib:
##STR00005##
[0082] In some embodiments, the salt of Formula I has the structure
of Formula Ic:
##STR00006##
[0083] In some embodiments, the salt of Formula I has the structure
of Formula Id:
##STR00007##
[0084] In some embodiments, the salt of Formula I has the structure
of Formula Ie:
##STR00008##
[0085] In some embodiments, the salt of Formula I has the structure
of Formula If:
##STR00009##
[0086] In some embodiments, the salt of Formula I has the structure
of Formula Ig:
##STR00010##
[0087] In some embodiments, the salt of Formula I has the structure
of Formula Ik:
##STR00011##
[0088] In some embodiments, the salt of Formula I has the structure
of Formula I:
##STR00012##
[0089] In some embodiments, the salt of Formula I has the structure
of Formula Im:
##STR00013##
[0090] In some embodiments, the salt of Formula I has the structure
of Formula In:
##STR00014##
[0091] In some embodiments, the salt of Formula I has the structure
of Formula Io:
##STR00015##
[0092] In some embodiments, the salt of Formula I has the structure
of Formula Ip:
##STR00016##
[0093] In some embodiments, the salt of Formula I has the structure
of Formula Iq:
##STR00017##
[0094] In some embodiments, the salt of Formula I has the structure
of Formula Ir:
##STR00018##
[0095] In some embodiments, the salt of Formula I has the structure
of Formula Is:
##STR00019##
[0096] In one embodiment of the salt of Formula I, A is
NR.sup.aR.sup.b.
[0097] In another embodiment of the salt of Formula I, M.sup.1 is a
cation. In another embodiment, M.sup.1 is cationic atoms. In
another embodiment, M.sup.1 is, but not limited to, Li.sup.+,
Na.sup.+, K.sup.+, Rb.sup.+, Cs.sup.+, Be.sup.2+, Mg.sup.2+,
Ca.sup.2+, Sr.sup.2+, Zn.sup.2+, and Ba.sup.2+. In another
embodiment, M.sup.1 is Li.sup.+. In another embodiment, M.sup.1 is
Na.sup.+. In another embodiment, M.sup.1 is K.sup.+. In another
embodiment, M.sup.1 is Rb.sup.+. In another embodiment, M.sup.1 is
Cs.sup.+. M.sup.1 can also be divalent cations such as Ca.sup.2+,
Be.sup.2+, Mg.sup.2+, Ca.sup.2+, Sr.sup.2+, Zn.sup.2+, and
Ba.sup.2+. In one embodiment, M.sup.1 is Ca.sup.2+.
[0098] In some embodiments of the invention, R.sup.a is
independently, at each occurrence H, or C.sub.1-C.sub.6 alkyl. In
other embodiments, R.sup.a is H. In other embodiments, R.sup.a is
C.sub.1-C.sub.6alkyl. In other embodiments, R.sup.a is
C.sub.1-C.sub.6alkyl substituted with one or more substituents
selected from C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, or
(C.sub.0-C.sub.3alkylene)heteroaryl. In other embodiments, R.sup.a
is C.sub.1-C.sub.6alkyl substituted with one or more substituents
selected from C.sub.1-C.sub.6alkyl. In other embodiments, R.sup.a
is C.sub.1-C.sub.6alkyl substituted with one or
C.sub.2-C.sub.6alkenyl. In other embodiments, R.sup.a is
C.sub.1-C.sub.6alkyl substituted with one or more
C.sub.2-C.sub.6alkynyl. In other embodiments, R.sup.a is
C.sub.1-C.sub.6alkyl substituted with one or more
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl. In other
embodiments, R.sup.a is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)heterocycloakyl. In other
embodiments, R.sup.a is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl. In other
embodiments, R.sup.a is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)heteroaryl. In other embodiment
R.sup.a is methyl. In other embodiment R.sup.a is methyl
substituted with one or more substituents selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, or
(C.sub.0-C.sub.3alkylene)heteroaryl.
[0099] In some embodiments of the invention, R.sup.b is
independently, at each occurrence H, or C.sub.1-C.sub.6 alkyl. In
other embodiments, R.sup.b is H. In other embodiments, R.sup.b is
C.sub.1-C.sub.6alkyl. In other embodiments, R.sup.b is
C.sub.1-C.sub.6alkyl substituted with one or more substituents
selected from C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, or
(C.sub.0-C.sub.3alkylene)heteroaryl. In other embodiments, R.sup.b
is C.sub.1-C.sub.6alkyl substituted with one or more substituents
selected from C.sub.1-C.sub.6alkyl. In other embodiments, R.sup.b
is C.sub.1-C.sub.6alkyl substituted with one or
C.sub.2-C.sub.6alkenyl. In other embodiments, R.sup.b is
C.sub.1-C.sub.6alkyl substituted with one or more
C.sub.2-C.sub.6alkynyl. In other embodiments, R.sup.b is
C.sub.1-C.sub.6alkyl substituted with one or more
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl. In other
embodiments, R.sup.b is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)heterocycloakyl. In other
embodiments, R.sup.b is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl. In other
embodiments, R.sup.b is C.sub.1-C.sub.6alkyl substituted with one
or more (C.sub.0-C.sub.3alkylene)heteroaryl. In other embodiment R
is methyl. In other embodiment R.sup.b is methyl substituted with
one or more substituents selected from C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, or
(C.sub.0-C.sub.3alkylene)heteroaryl.
[0100] In a further embodiment, R is independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.3R.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a.
In another embodiment, R.sup.1 is H. In another embodiment, R.sup.1
is C.sub.1-C.sub.6alkyl. In another embodiment, R.sup.1 is
C.sub.1-C.sub.6haloalkyl. In another embodiment, R.sup.1 is
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl. In another
embodiment, R.sup.2 is --C(O)OR. In another embodiment, R.sup.2 is
--[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a. In another embodiment,
R.sup.1 is C(O)C.sub.1-C.sub.6alkyl.
[0101] In one embodiment, R.sup.2 is independently H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl, --C(O)OR.sup.a,
--C(O)NR.sup.aR.sup.b, or --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a.
In another embodiment, R.sup.2 is H. In another embodiment, R.sup.2
is C.sub.1-C.sub.6alkyl. In another embodiment, R.sup.2 is
C.sub.1-C.sub.6haloalkyl. In another embodiment, R.sup.2 is
(C.sub.0-C.sub.3alkylene)C(O)C.sub.1-C.sub.6alkyl. In another
embodiment, R.sup.2 is --C(O)OR.sup.a. In another embodiment,
R.sup.2 is --[CH.sub.2--CH.sub.2--O].sub.n--R.sup.a. In another
embodiment, R.sup.1 is C(O)C.sub.1-C.sub.6alkyl.
[0102] In a further embodiment of the salts of the Formula I,
R.sup.1 and R.sup.2, together with the atom to which they are
attached, may form a 5-membered heterocyclic ring. In yet a further
embodiment of the salts of the Formula I, R.sup.1 and R.sup.2,
together with the atom to which they are attached, may form a
5-membered heterocyclic ring substituted with one or more
substituents selected from C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
(C.sub.0-C.sub.3alkylene)C.sub.3-C.sub.8cycloakyl,
(C.sub.0-C.sub.3alkylene)heterocycloakyl,
(C.sub.0-C.sub.3alkylene)C.sub.6-C.sub.14aryl, and
(C.sub.0-C.sub.3alkylene)heteroaryl.
[0103] In another embodiment, n at each occurrence is 1, 2, 3, 4,
5, 6, 7, or 8. In another embodiment, n is 1. In another
embodiment, n is 2. In another embodiment, n is 3. In another
embodiment, n is 4. In another embodiment, n is 5. In another
embodiment, n is 6. In another embodiment, n is 7. In another
embodiment, n is 8.
[0104] In one embodiment, k is 1. In another embodiment, k is
2.
[0105] In another embodiment, the salt of Formula (I) is selected
from the group:
##STR00020##
Method for Preparation of the Salts
[0106] The salts of the present disclosure may be made by a variety
of methods, including standard chemistry. Suitable synthetic routes
are depicted in the Schemes given below.
[0107] Another aspect of present disclosure relates to a process
for preparing the salts of Formula (I). The process comprises
contacting a nicotinamide mononucleotide derivative of Formula (II)
with a metal-alkali hydroxide under suitable conditions effective
to produce the product salt of Formula (I)
[0108] In one embodiment, the process of preparing the instant
salts involves nitrogenating the atmosphere under which the
reaction occurs. In another embodiment, the first step in preparing
the instant salts involves charging the nucletotide with nitrogen
then adding deionized water to create a solution of NMN. In one
embodiment the second step involves adding the metal-alkali
hydroxide dropwise to the resulting solution of NMN to elevate the
pH to about 6.5.
[0109] In one embodiment of the invention, the process of preparing
the salt of Formula (I) involves contacting a compound of Formula
(II)
##STR00021##
with a metal-alkali hydroxide under conditions effective to produce
the salt of Formula (I).
[0110] In one embodiment, the metal-alkali hydroxide is selected
from LiOH, NaOH, KOH, Mg(OH).sub.2, Ca(OH).sub.2, Zn(OH).sub.2, and
Ba(OH).sub.2.
[0111] The process described herein provides an order of addition
of the materials, reagents and reactants to not only achieve a high
degree of purity of the salt of Formula (I), but also a stable form
of the salt of Formula (I). The reversal of the order of addition
described above, i.e. addition of solid mononucleotide (NMN) to
aqueous metal-alkali hydroxide leads to a substantially impure
product which is susceptible to premature degradation. Thus the
formation of the salts is not an inevitable consequence of simply
mixing the materials, reagents and reactants in any order, rather
the described step-wise addition appears to produce a more stable
and soluble salt of Formula (I) as compared to products produced by
mixing the reagents in the reverse order.
[0112] Although not wishing to be limited by theory it is
hypothesized that the glycosidic bond of the salt of Formula I is
susceptible to an increased risk of degradation under the
conditions resulting from the addition of solid NMN to aqueous base
yielding a substantially impure product comprising of the
degradation products nicotinamide and ribose rather than the
desired product of acceptable purity.
[0113] The instant disclosure also envisions analogous methods to
prepare the salts of Formula (I), known in the art of organic
synthesis as set forth in part by the following synthetic scheme
1.
[0114] Those skilled in the art will recognize if a stereocenter
exists in the salts of Formula (I). Accordingly, the present
includes both possible stereoisomers (unless specified in the
synthesis) and includes not only racemic salts but the individual
enantiomers and/or diastereomers as well. When a compound or salt
is desired as a single enantiomer or diastereomer, it may be
obtained by stereospecific synthesis or by resolution of the final
product or any convenient intermediate. Resolution of the final
product, an intermediate, or a starting material may be affected by
any suitable method known in the art. See, for example,
"Stereochemistry of Organic Compounds" by E. L. Eliel, S. H. Wilen,
and L. N. Mander (Wiley-lnterscience, 1994).
[0115] The salts and compounds described herein may be made from
commercially available starting materials or synthesized using
known organic, inorganic, and/or enzymatic processes.
[0116] The salts of Formula (I) can be prepared in a number of ways
well known to those skilled in the art of organic synthesis. By way
of example, salts of Formula (I) can be synthesized using the
methods described below, together with synthetic methods known in
the art of synthetic organic chemistry, or variations thereon as
appreciated by those skilled in the art. These methods include but
are not limited to those methods described below. Salts of Formula
(I) can be synthesized by following the steps outlined in General
Scheme 1 which comprises a sequence of assembling various
intermediates. Starting materials are either commercially available
or made by known procedures in the reported literature or as
illustrated.
##STR00022##
[0117] The salts of Formula (I) described herein can be prepared
according to the general procedures outlined in Scheme 1. A
condensation of the precursor and commercially available
nicotinamide mononucleotide is generally done with a metal-alkali
hydroxide in presence of suitable solvent (e.g., a mixture of
methanol and water). The mixture is heated to slowly evaporate the
solvents. The resulting mixture may also be freeze-dried to obtain
the desired product salt under suitable conditions of temperature
and pressure.
[0118] A mixture of enantiomers, diastereomers, cis/trans isomers
resulting from the process described above can be separated into
their single components by chiral salt technique, chromatography
using normal phase, reverse phase or chiral column, depending on
the nature of the separation.
[0119] It should be understood that in the description and formula
shown above, the groups M.sup.+ represent M.sup.1 as defined above,
except where otherwise indicated. Furthermore, for synthetic
purposes, the salts of General Scheme 1 are mere representative
with elected radicals to illustrate the general synthetic
methodology of the salts of Formula (I) as defined herein.
[0120] It is also understood that the salts disclosed herein
possess a neutral electrical charge and that the structure of
Formula I is only a representative of genus, which, if necessary,
may be balanced with a counterion to allow the salt to present a
neutral electrical charge. Such counterions may include, without
limitation, bromine, chlorine, and trifilates. In one embodiment,
the salt of this disclosure can be generated in situ without the
need to isolate from solution. In some embodiments, the salts
contemplated herein can be discrete 1:1 or 1:2 salts. The salts
described herein can also exist in other ratios, e.g., 1:1.5, 1:5,
and 1:10.
Methods of Using the Salts
[0121] Another aspect of the present disclosure relates to a method
of treating or preventing a disease or disorder associated with
aging, cellular degradation, and/or cellular restoration. Non
limiting examples of such diseases and disorders include
infertility, age related infertility, age-related loss of eye
function, reduction in bone density, obesity and insulin
insensitivity. In one embodiment, the salts of Formula (I) are
useful in the treatment of age related infertility. In another
embodiment the salts of Formula (I) are useful in the treatment of
fertility.
[0122] Another aspect of the present disclosure relates to a method
of treating or preventing an age-related disease or disorder. The
method comprises administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition of
the salt of Formula I.
[0123] Yet another aspect of the present disclosure relates to the
method of improving oocyte or blastocyst quality and maturation.
The method comprises contacting the oocyte or blastocyst for an
effective period of time with IVF media comprising a salt of
Formula (I).
[0124] In another aspect, the present disclosure provides media
containing a salt of Formula (I). The salts of Formula (I) are
useful in media for exposing eggs, oocytes and/or blastocytes for
periods of time necessary for enhancing NAD+ production prior to
implantation into a subject suffering from infertility or
age-related infertility. In some embodiments, media comprising a
salt of Formula (I) is provided. In some embodiments the media
comprises the various reagents and factors necessary for the egg,
oocyte or blastocyst depending on which stage of maturation and
development the egg, oocyte or blastocyst is in. For example, the
media can contain any of the agents or factors useful in IVF media
listed in Table 1 below:
TABLE-US-00001 TABLE 1 CULTURE MEDIA COMPONENTS Inorganic salts
Energy substrates (glucose, pyruvate and lactate) Essential amino
acids (arginine, cysteine, glutamine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
tyrosine and valine) Nonessential amino acids (alanine, asparagine,
asparatate, glycine, glutamate, proline and serine) Chelators pH
indicators Antibiotic agents (such as combination of penicillin and
streptomycin) Serum albumin Vitamins Growth factors (insulin or
GM-CSF, among others)
[0125] Also provided is a cell culture medium for in vitro
fertilization comprising: one or more salts of Formula (I) and
culturing agents.
[0126] In one embodiment, the culturing agent is an inorganic salt,
an energy substrate, an amino acid, a chelator, a pH indicator, an
antibiotic, a serum, a vitamin, a growth factor, or any combination
thereof. In one embodiment, the inorganic salt is calcium chloride,
magnesium chloride, magnesium sulfate, potassium chloride, sodium
bicarbonate, sodium chloride, monosodium phosphate, disodium
phosphate, or any combination thereof.
[0127] In one embodiment, the energy substrate is glucose,
pyruvate, lactate, pyruvate, or any combination thereof.
[0128] In one embodiment, the amino acid is an essential amino
acid. In one embodiment, the essential amino acid is arginine,
cysteine, glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, threonine, tryptophan,
tyrosine, valine, or any combination thereof.
[0129] In one embodiment, the amino acid is a non-essential amino
acid.
[0130] In one embodiment, the non-essential amino acid is alanine,
asparagine, aspartate, glutamate, proline, serine, or any
combination thereof.
[0131] In one embodiment, the chelator is clathro chelate, acetyl
acetone, amino polycarboxylic acid, ATMP, BAPTA, BDTH2, citric
acid, cryptand, deferasirox, 2,3-dihydrobenzoic acid,
2,3-dimercapto-1-propane sulfonic acid, dimercapto succinic acid,
DOTA, DTPMP, EDDHA, EDDS, EDTMP, etidronic acid, fura-2, gluconic
acid, homocitric acid, imino diacetic acid, Indo-1, nitrile
triacetic acid, pentetic acid (DTPA), phosphonate, phytochelati,
poly aspartic acid, sodium poly aspartate, trisodium citrate,
transferrin, EDTA, EGTA, or any combination thereof.
[0132] In one embodiment, the pH indicator is phenol red,
bromothymol blue, alizarin red, 9-aminoacridine, or any combination
thereof.
[0133] In one embodiment, the antibiotic is actinomycin D,
ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin,
kanamycin, neomycin, penicillin, polymyxin B, streptomycin, or any
combination thereof.
[0134] In one embodiment, the serum is human serum albumin, bovine
serum albumin, fetal bovine serum, synthetic serum, or any
combination thereof.
[0135] In one embodiment, the vitamin is ascorbic acid, biotin,
menadione sodium bisulfite, mitomycin C, pyridoxamine
dihydrochloride, retinyl acetate, (-)-riboflavin, (+)-sodium
L-ascorbate, (+)-.alpha.-tocopherol, vitamin B.sub.12, thiamine
hydrochloride, i-inositol, pyridoxal hydrochloride, nicotinamide,
folic acid, D-calcium pantothenate, choline chloride, or any
combination thereof.
[0136] In one embodiment, the growth factor is adrenomedullin,
angiopoietin, bone morphogenetic proteins, macrophage
colony-stimulating factor (M-CSF), granulocyte colony-stimulating
factor (G-CSF), granulocyte macrophage colony-stimulating factor
(GM-CSF), epidermal growth factor, ephrins, erythropoietin,
fibroblast growth factor, growth differentiation factor-9,
hepatocyte growth factor, insulin, insulin-like growth factors,
interleukins, keratinocyte growth factor, migration-stimulating
factor, macrophage-stimulating protein, myostatin, neurotrophins,
t-cell growth factor, thrombopoietin, transforming growth factor,
tumor necrosis factor-alpha, vascular endothelial growth factor, or
any combination thereof.
[0137] In one embodiment, the cell culture medium further comprises
an oocyte, zygote, blastocyst, or any combination thereof
[0138] Also, provided are kits for IVF media comprising various
agents, and factors necessary for oocyte or blastocyst maturation
including one or more salts of Formula (I). These agents and
cofactors can be dissolved in solution to create the IVF media
shortly before use in exposing an oocyte or blastocyst prior to
implanting into a patient in need of treatment for infertility or
age-related infertility.
[0139] The present invention also relates to the use of the salts
of Formula I and enantiomers, stereoisomers, and tautomers thereof
for the manufacture of medicaments for treating aging, cellular
restoration, cellular degradation, or infertility. In certain
embodiments the infertility treated is age-related infertility.
[0140] Another aspect of the present invention is a pharmaceutical
composition comprising the salt of Formula I and a pharmaceutically
acceptable carrier.
[0141] Another aspect of the present invention is a pharmaceutical
composition comprising the salt of Formula I and a pharmaceutically
acceptable carrier comprising therapeutically effective amounts of
one or more additional therapeutic agents.
[0142] In some embodiments, administration of a salt of Formula (I)
or a pharmaceutical composition comprising a salt of the present
invention and a pharmaceutically acceptable carrier induces a
change in the cell cycle or cell viability.
[0143] In some embodiments, administration of a salt of Formula (I)
or a pharmaceutical composition comprising a salt of the present
invention and a pharmaceutically acceptable carrier induces a
prophylactic change in the disorder or disease associated with
aging.
[0144] Administration of the disclosed salts can be accomplished
via any mode of administration for therapeutic agents. These modes
include systemic or local administration such as oral, nasal,
parenteral, transdermal, subcutaneous, vaginal, buccal, rectal or
topical administration modes.
[0145] Depending on the intended mode of administration, the
disclosed compositions can be in solid, semi-solid or liquid dosage
form, such as, for example, injectables, tablets, suppositories,
pills, time-release capsules, elixirs, tinctures, emulsions,
syrups, powders, liquids, suspensions, or the like, sometimes in
unit dosages and consistent with conventional pharmaceutical
practices. Likewise, they can also be administered in intravenous
(both bolus and infusion), intraperitoneal, subcutaneous or
intramuscular form, and all using forms well known to those skilled
in the pharmaceutical arts.
[0146] Pharmaceutical compositions can be prepared according to
conventional mixing, granulating or coating methods, respectively,
and the present pharmaceutical compositions can contain from about
0.1% to about 99%, from about 5% to about 90%, or from about 1% to
about 20% of the disclosed salt by weight or volume.
[0147] In one embodiment, the present invention relates to a method
of preparing a pharmaceutical composition of the present invention
by mixing at least one pharmaceutically acceptable salt of the
present invention, and, optionally, one or more pharmaceutically
acceptable carriers, additives, or excipients.
[0148] In another embodiment, the present invention relates to a
method of preparing a pharmaceutical composition of the present
invention by mixing at least one pharmaceutically acceptable salt
of the present invention and one or more additional therapeutic
agents.
[0149] Effective dosage amounts of the salts of Formula (I), when
used in the described methods, range from about 0.5 mg to about
5000 mg of the disclosed salt as needed to treat the condition.
Compositions for in vivo or in vitro use can contain about 0.5, 5,
20, 50, 75, 100, 150, 250, 500, 750, 1000, 1250, 2500, 3500, or
5000 mg of the disclosed salt or, in a range of from one amount to
another amount in the list of doses. In one embodiment, the
compositions are in the form of a tablet that can be scored.
[0150] The dosage regimen utilizing the disclosed salt is selected
in accordance with a variety of factors including type, species,
age, weight, sex and medical condition of the patient; the severity
of the condition to be treated; the route of administration; the
renal or hepatic function of the patient; and the particular
disclosed salt employed. A physician or veterinarian of ordinary
skill in the art can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progress of the condition.
[0151] Illustrative pharmaceutical compositions are tablets and
gelatin capsules comprising a salt of the Invention and a
pharmaceutically acceptable carrier, such as a) a diluent, e.g.,
purified water, triglyceride oils, such as hydrogenated or
partially hydrogenated vegetable oil, or mixtures thereof, corn
oil, olive oil, sunflower oil, safflower oil, fish oils, such as
EPA or DHA, or their esters or triglycerides or mixtures thereof,
omega-3 fatty acids or derivatives thereof, lactose, dextrose,
sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose
and/or glycine; b) a lubricant, e.g., silica, talcum, stearic acid,
its magnesium or calcium salt, sodium oleate, sodium stearate,
magnesium stearate, sodium benzoate, sodium acetate, sodium
chloride and/or polyethylene glycol; for tablets also; c) a binder,
e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose,
magnesium carbonate, natural sugars such as glucose or
beta-lactose, corn sweeteners, natural and synthetic gums such as
acacia, tragacanth or sodium alginate, waxes and/or
polyvinylpyrrolidone, if desired; d) a disintegrant, e.g.,
starches, agar, methyl cellulose, bentonite, xanthan gum, algic
acid or its sodium salt, or effervescent mixtures; e) absorbent,
colorant, flavorant and sweetener; f) an emulsifier or dispersing
agent, such as Tween 80, Labrasol, HPMC, DOSS, caproyl 909,
labrafac, labrafil, peceol, transcutol, capmul MCM, capmul PG-12,
captex 355, gelucire, vitamin E TGPS or other acceptable
emulsifier; and/or g) an agent that enhances absorption of the salt
such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400,
PEG200.
[0152] For preparing pharmaceutical compositions from the salts of
Formula (I), inert, pharmaceutically acceptable carriers can be
either solid or liquid. Solid form preparations include powders,
tablets, dispersible granules, capsules, cachets and suppositories.
The powders and tablets may be comprised of from about 5 to about
95 percent active ingredient. Suitable solid carriers are known in
the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar
or lactose. Tablets, powders, cachets and capsules can be used as
solid dosage forms suitable for oral administration. Examples of
pharmaceutically acceptable carriers and methods of manufacture for
various compositions may be found in A. Gennaro (ed.), Remington's
Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co.,
Easton, Pa.
[0153] Liquid form preparations include solutions, suspensions and
emulsions. For example, water or water-propylene glycol solutions
for parenteral injection or addition of sweeteners and opacifiers
for oral solutions, suspensions and emulsions. Liquid form
preparations may also include solutions for intranasal
administration.
[0154] Liquid, particularly injectable, compositions can, for
example, be prepared by dissolution, dispersion, etc. For example,
the disclosed salt is dissolved in or mixed with a pharmaceutically
acceptable solvent such as, for example, water, saline, aqueous
dextrose, glycerol, ethanol, and the like, to thereby form an
injectable isotonic solution or suspension. Proteins such as
albumin, chylomicron particles, or serum proteins can be used to
solubilize the disclosed compounds.
[0155] Parental injectable administration is generally used for
subcutaneous, intramuscular or intravenous injections and
infusions. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions or solid forms suitable
for dissolving in liquid prior to injection.
[0156] Aerosol preparations suitable for inhalation may include
solutions and solids in powder form, which may be in combination
with a pharmaceutically acceptable carrier, such as an inert
compressed gas, e.g., nitrogen.
[0157] Also included are solid form preparations that are intended
to be converted, shortly before use, to liquid form preparations
for either oral or parenteral administration. Such liquid forms
include solutions, suspensions and emulsions
EXAMPLES
[0158] The disclosure is further illustrated by the following
examples and synthesis schemes, which are not to be construed as
limiting this disclosure in scope or spirit to the specific
procedures herein described. It is to be understood that the
examples are provided to illustrate certain embodiments and that no
limitation to the scope of the disclosure is intended thereby. It
is to be further understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which may
suggest themselves to those skilled in the art without departing
from the spirit of the present disclosure and/or scope of the
appended claims.
[0159] The following salts disclosed herein were prepared using the
general synthetic methodology including without limitation reagents
such as KCl, NaCl, LiCl, NaOH, LiOH, or KOH. Suitable solvents such
as methanol, ethanol, water, acetic acid, ethylene glycol,
isopropanol were also used.
Abbreviations used in the following examples and elsewhere herein
are: [0160] AcOH acetic acid [0161] anh. anhydrous [0162] atm
atmosphere [0163] aq. aqueous [0164] br broad [0165] Boc
tert-butyloxycarbonyl [0166] brine saturated aqueous sodium
chloride [0167] n-BuLi n-butyllithium [0168] n-BuOH n-butanol
[0169] Calc'd calculated [0170] CDCl.sub.3 deuterated chloroform
[0171] CDI carbonyldiimidazole [0172] Chloroform-d deuterated
chloroform [0173] d doublet [0174] dd doublet of doublets [0175] dt
doublet of triplets [0176] D.sub.2O deuterated water (deuterium
oxide) [0177] DCE dichloroethane [0178] DCM dichloromethane [0179]
DIAD diisopropyl azodicarboxylate [0180] DIPEA
N,N-diisopropylethylamine [0181] DMAc N,N-dimethyl acetamide [0182]
DMAP N,N-dimethylpyridin-4-amine [0183] DME 1,2-dimethoxyethane
[0184] DMEDA N,N'-dimethylethylenediamine [0185] DMF
N,N-dimethylformamide [0186] DMSO dimethyl sulfoxide [0187]
DMSO-d.sub.6 deuterated dimethyl sulfoxide [0188] EDA
ethylenediamine [0189] EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [0190] Et.sub.2O
diethyl ether [0191] EtOAc ethyl acetate [0192] EtOH ethanol [0193]
ESI electrospray ionization [0194] g gram [0195] h hour(s) [0196] H
hydrogen [0197] .sup.1H NMR nuclear magnetic resonance (proton
nucleus) [0198] HATU
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate [0199] HBTU
3-[bis(dimethylamino)methylene]-3H-benzotriazol-1-oxide
hexafluorophosphate [0200] HOBt hydroxybenzotriazole [0201] HPLC
high pressure (or performance) liquid chromatography [0202] Hz
hertz [0203] J coupling constant [0204] KHCO.sub.3 potassium
bicarbonate [0205] KHMDS potassium hexamethyldisilazide [0206] KOAc
potassium acetate [0207] LCMS liquid chromatography mass
spectrometry [0208] LHMDS lihtium hexamethyldisilazide [0209] [#] M
molar concentration [0210] m multiplet [0211] [M+H].sup.+ molecular
ion plus hydrogen [0212] [M-tBu+H].sup.+ molecular ion minus
tert-butyl plus hydrogen [0213] mCPBA meta-chloroperoxybenzoic acid
[0214] Me.sub.2NH dimethylamine [0215] Me.sub.4NBr
tetramethylammonium bromide [0216] MeCN acetonitrile [0217]
MeNH.sub.2 methylamine [0218] MeOH methanol [0219] Methanol-d.sub.4
deuterated methanol [0220] 2-MeTHF 2-methyl tetrahydrofuran [0221]
mg milligram [0222] MHz megahertz [0223] min min [0224] mmol
millimole [0225] mL milliliter [0226] MS mass spectrometry [0227]
MS ES mass spectrometry electrospray [0228] Ms.sub.2O
methanesulfonic anhydride [0229] MTBE methyl tert-butyl ether
[0230] MW microwave [0231] m/z mass-to-charge ratio [0232] .mu.L
microliter [0233] N.sub.2 nitrogen [0234] NaHCO.sub.3 sodium
bicarbonate [0235] NMN nicotinamide mononucleotide [0236] NIS
N-iodosuccinimide [0237] NMP N-methyl-2-pyrrolidone [0238] NMR
nuclear magnetic resonance [0239] PEPPSI-iPr
[1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)pallad-
ium(II) dichloride [0240] PdCl.sub.2(Amphos)
bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)
dichloropalladium(II) [0241] Pd.sub.2(dba).sub.3
tris(dibenzylideneacetone)dipalladium(0) [0242] Pd(OAc).sub.2
palladium(II) acetate [0243] PdCl.sub.2(dppf)
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) [0244]
PdCl.sub.2(MeCN).sub.2 bis(acetonitrile)dichloropalladium(II)
[0245] PdCl.sub.2(PPh.sub.3).sub.2
bis(triphenylphosphine)palladium(II) dichloride [0246]
Pd(P(Cy).sub.3).sub.2Cl.sub.2
dichlorobis(tricyclohexylphosphine)palladium(II) [0247]
Pd(PPh.sub.3).sub.4 tetrakis(triphenylphosphine)palladium(0) [0248]
Pd(t-Bu.sub.3P).sub.2 bis(tri-tert-butylphosphine)palladium(0)
[0249] pH potential of hydrogen [0250] PMB 4-methoxybenzyl [0251]
PMBCl 4-methoxybenzyl chloride [0252] ppm parts per million [0253]
prep preparative [0254] py pyridine [0255] q quartet [0256] qd
quartet of doublets [0257] quant. quantitative [0258] quin.
quintuplet [0259] quind quintuplet of doublets [0260] RBF
round-bottom flask [0261] Rt retention time [0262] rt room
temperature [0263] s singlet [0264] sat. saturated [0265] sat. aq.
saturated aqueous [0266] SEMCl 2-(trimethylsilyl)ethoxymethyl
chloride [0267] t triplet [0268] t-BuLi tert-butyllithium [0269] td
triplet of doublets [0270] TMS trimethylsilyl [0271] TMSCl
trimethylsilyl chloride [0272] tt triplet of triplets [0273] T3P
polyphosphonic anhydride [0274] TBAB tetrabutylammonium bromide
[0275] TEA triethylamine [0276] TFA trifluoroacetic acid [0277]
TFAA trifluoroacetic anhydride [0278] THF tetrahydrofuran [0279]
TLC thin layer chromatography [0280] TPPO triphenylphosphine oxide
[0281] XantPhos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
[0282] XPhos
2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl
Example 1. Synthesis of Sodium
((2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrof-
uran-2-yl)methyl phosphate (I-001)
##STR00023##
[0284] A 50 mL 3 N RBF fitted with a water condenser, rubber septum
and internal thermometer was charged with NMN (0.200 g, 0.598 mmol,
1 eq) and 4 ml of distilled deionised water and mixed to form a
solution. This solution was cooled using an ice/water bath to bring
the internal temperature below 10.degree. C. (pH of the solution
was 3.34). To this solution was then added dropwise 5.70 ml of 0.1M
NaOH solution slowly via syringe so as to prevent the temperature
from increasing. The pH after this addition was 7.01. The flask was
then removed and placed on the small kugel, attached to an oil pump
pulling less than 1 mBar at 30.degree. C. external (temperatures
above this lead to degradation of the product). This will slowly
remove water. Make sure the receiving flasks are cooled. Once dried
the product is rendered as a colourless solid. Yield: 87.8 mg
(83%); Melting point: 79-83.degree. C. (degradation, corrected)
outgassing at 130.degree. C.); Analytical data. .sup.1H-NMR (400
MHz, D.sub.2O) .delta.=9.62 (s, 1H), 9.35 (d, 1H), 9.01 (d, 1H),
8.32 (t, 1H), 6.21 (d, 1H), 4.69 (t, 1H), 4.65 (p, 1H), 4.50 (m,
1H), 4.23 (dq, 1H), 4.05 (dq, 1H) ppm
Example 2. Synthesis of Potassium ((2R,3S, 4R,
5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)m-
ethylphosphate (I-002)
##STR00024##
[0286] A 50 mL 3 N RBF fitted with a water condenser and internal
thermometer was charged with NMN (0.300 g, 0.897 mmol, 1 eq) and 5
ml of distilled deionised water and mixed to form a solution. This
solution was cooled using an ice/water bath to bring the internal
temperature below 10.degree. C. To this solution was then added
dropwise 8.79 ml of 0.1M KOH solution slowly so as to prevent the
temperature from increasing, the pH was 6.0-6.2. The flask was then
removed and the colourless solution frozen using liquid nitrogen.
While the flask was frozen it was connected to the freeze dryer.
This will slowly remove water. Once dried the product is rendered
as a colourless solid. Yield: 334.1 mg. Melting point:
105.3-111.8.degree. C. (degradation, corrected) outgassing at
130.degree. C.). Analytical data. .sup.1H-NMR (400 MHz, D.sub.2O)
.delta.=9.61 (s, 1H), 9.35 (d, 1H), 9.02 (d, 1H), 8.33 (dd, 1H),
6.21 (d, 1H), 4.68 (t, 1H), 4.65 (p, 1H), 4.50 (m, 1H), 4.25 (dq,
1H), 4.08 (dq, 1H) ppm
Example 3. Synthesis of Calcium
((2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrof-
uran-2-yl)methyl phosphate (I-003)
##STR00025##
[0288] A 50 mL 2 N RBF fitted with a water condenser, rubber septum
and internal thermometer, under nitrogen, was charged with NMN
(0.100 g, 0.299 mmol, 1 eq) and 15 ml of distilled deionised water
and mixed to form a solution. This solution was cooled using an
ice/water bath to bring the internal temperature below 5.degree. C.
To this solution was then added CaOH (11.1 mg, 0.149 mmol, 0.50
eq.). Over the next 10 minutes the stirred suspension turned to
solution and left only a very faint residue of solid. The resulting
solution was then freeze dried over night to give a colourless
solid. Yield: 0.103 g. Analytical data: .sup.1H-NMR (400 MHz,
D.sub.2O) .delta.=9.55 (s, 2H), 9.30 (d, 2H), 8.98 (d, 2H), 8.29
(app t, 2H), 6.18 (d, 2H), 4.62 (m, 4H), 4.46 (m, 2H), 4.22 (m,
2H), 4.03 (m, 2H) ppm.
Example 4. Synthesis of Magnesium
((2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrof-
uran-2-yl)methyl phosphate (I-004)
##STR00026##
[0290] A 50 mL 2 N RBF fitted with a water condenser, rubber septum
and internal thermometer, under nitrogen, was charged with NMN
(0.100 g, 0.299 mmol, 1 eq) and 15 ml of distilled deionised water
and mixed to form a solution. This solution was cooled using an
ice/water bath to bring the internal temperature below 5.degree. C.
To this solution was then added Magnesium Carbonate (Basic) (12.6
mg, 0.149 mmol, 0.50 eq.). Over the next 10 minutes the stirred
suspension turned to solution and left only a very faint residue of
solid. The pH was measured to be 5.8-6.1. The resulting solution
was then freeze dried over night to give a colourless solid. Yield:
0.105 g. Analytical data: .sup.1H-NMR (400 MHz, D.sub.2O)
.delta.=9.35 (s, 1H), 9.26 (d, 1H), 8.98 (d, 1H), 8.25 (t, 1H),
6.23 (d, 1H), 4.65 (p, 1H), 4.57 (t, 1H), 4.48 (m, 1H), 4.33 (dq,
1H), 4.19 (dq, 1H) ppm.
Example 5. Lithium
((2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrof-
uran-2-yl)methyl phosphate (I-005)
##STR00027##
[0292] A 50 mL 2 N RBF fitted with a water condenser and internal
thermometer was charged with NMN (0.100 g, 0.299 mmol, 1 eq) and 15
ml of distilled deionised water and mixed to form a solution or
faint suspension (Note 1) under nitrogen. This solution was cooled
using an ice/water bath (pH of solution=1.8). To this solution was
then added drop wise 2.84 ml of 0.1M LiOH solution slowly via
syringe so as to prevent the temperature from increasing. After
this addition the pH was 6.4-6.7. The solution was then transferred
into a 100 ml 1 N RBF and freeze dried over night. This yielded a
faint yellow to faint beige solid. Yield: 95.0 mg, 93.4%. Melting
point: .about.130.degree. C. (degradation-outgassing, corrected).
Analytical data: .sup.1H-NMR (400 MHz, D.sub.2O) .delta.=9.58 (s,
1H), 9.31 (d, 1H), 8.98 (d, 1H), 8.28 (t, 1H), 6.18 (d, 1H), 4.65
(t, 1H), 4.59 (p, 1H), 4.45 (m, 1H), 4.19 (dq, 1H), 4.02 (dq,
1H)ppm.
Example 6. NAD Cell Assays
[0293] NAD levels were assayed based on the NAD cycling method of
Zhu and Rand, PLoS One (2012), herein incorporated by reference.
COV434 cells were maintained in 6 well plates and treated with the
indicated compounds at a concentration of 200 uM for 4 hr. Media
was removed, plates were washed in cold PBS and cells were scraped
down in NAD extraction buffer containing 10 mM nicotinamide, 50 mM
Tris HCl, 0.1% Triton X-100. Cells were homogenised by sonication
for 5 seconds, and samples were centrifuged at 7,000 g for 5 min at
4 degrees. Aliquots were taken for later protein assay, and samples
were then passed through 10 kDa amicon filters at 14,000 g, 30 min
at 4 degrees to remove proteins from the sample. Each sample was
measured in technical triplicate, with 25 .mu.L sample added to 100
.mu.L ADH cycling mix (0.2 mg/ml alcohol dehydrogenase enzyme, 2%
ethanol, 100 mM Tris pH 8.5). Samples were allowed to cycle for 10
min at room temperature, followed by 50 .mu.L addition of an
MTT/PMS solution (0.1 mM phenazine methosulfate, 0.8 mM
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 100
mM Tris-HCl pH 8.5). Plates were then incubated for 15 min and
absorbance was measured at 570 nM. NAD concentrations were
extrapolated from a standard curve, and normalised to protein
concentrations determined by BCA protein assay.
[0294] The results of the assay described above are shown in Table
2 (below). The fold increase is obtained through a direct
comparison on a mole per mole between the compared salt and its
parent counterpart.
TABLE-US-00002 TABLE 2 Compound Fold increase Fold Increase
compared to ID compared to NMN NMN on a molar basis NMN 1.0 1.0
I-001 1.7 1.78
From the cellular NAD+ tests it was surprisingly and unexpectedly
found that inorganic salts of NMN increased the levels of NAD+ in
COV434 cells, which are derived from human ovarian cells, relative
to NMN. Such results demonstrate that inorganic salts of NMN can be
useful molecules for treating diseases and disorders related to
NAD+ deficiencies.
EQUIVALENTS
[0295] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
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