Neuroprotective chemicals and methods for identifying and using same
Provided herein are methods for identifying a compound having cell-protective (e.g., neuroprotective) activity. Compounds identified therefrom are also provided. These compounds can be used to treat various diseases, disorders, or conditions associated with, for example, unwanted cell death.
1. A method for identifying a compound having cell-protective activity, comprising:
incubating a test compound with a nicotinamide phosphoribosyltransferase (NAMPT);
measuring an activity of the NAMPT; and
identifying the test compound as having cell-protective activity when there is an increase in the activity of the NAMPT compared to a control that is not incubated with the test compound, wherein the test compound is a compound having formula (I):
wherein:
each of R 1 , R 2 , R 3 , and R 4 is independently selected from hydrogen, halo, hydroxyl, sulfhydryl, C 1 -C 6 alkoxy, C 1 -C 6 thioalkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 thiohaloalkoxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkynyl, cyclopropyl, —N 3 , cyano, —NH 2 , —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), nitro, —C(O)O(C 1 -C 6 alkyl), —C(O)NH 2 , —C(O)NH(C 1 -C 6 alkyl), —C(O)N(C 1 -C 6 alkyl) 2 , —S(O) 2 NH 2 , —S(O) 2 NH(C 1 -C 6 alkyl), and —S(O) 2 N(C 1 -C 6 alkyl) 2 ;
R and R′ are defined according to (1), (2), (3) or (4) below:
(1) R and R′ together with C 2 and C 3 , respectively, form a fused phenyl ring having formula (II):
wherein each of R 5 , R 6 , R 7 , and R 8 is independently selected from hydrogen, halo, hydroxyl, sulfhydryl, C 1 -C 6 alkoxy, C 1 -C 6 thioalkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 thiohaloalkoxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkynyl, cyclopropyl, —N 3 , cyano, —NH 2 , —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), nitro, —C(O)O(C 1 -C 6 alkyl), —C(O)NH 2 , —C(O)NH(C 1 -C 6 alkyl), —C(O)N(C 1 -C 6 alkyl) 2 , —S(O) 2 NH 2 , —S(O) 2 NH(C 1 -C 6 alkyl), and —S(O) 2 N(C 1 -C 6 alkyl) 2 ; or
(2) R and R′ together with C2 and C3, respectively, form a fused heteroaryl ring containing 6 ring atoms, wherein from 1-2 independently selected ring atoms is N; and wherein said heteroaryl ring is optionally substituted with from 1-2 independently selected R b ; or
(3) R and R′ together with C 2 and C 3 , respectively, form a fused heterocyclic ring containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected R a ; or
(4) each of R and R′ is, independently, hydrogen, C 1 -C 6 alkyl, or C 1 -C 6 haloalkyl; each of L 1 and L 2 is, independently, C 1 -C 3 alkylene, which is optionally substituted with from 1-2 independently selected R c ;
A is:
(i) CR A1 R A2 , wherein each of R A1 and R A2 is independently selected from hydrogen, halo, C 1 -C 3 alkyl, OR 9 , wherein R 9 is hydrogen or C 1 -C 3 alkyl that is optionally substituted with hydroxyl or C 1 -C 3 alkoxy, or a double bond formed between A and one of L 1 and L 2 ; or
(ii) C⊚O; or
(iii) C 3 -C 5 cycloalkylene that is (a) substituted with 1 oxo; and (b) optionally further substituted with from 1-4 independently selected R a ; or
(iv) heterocycloalkylene containing from 3-5 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heterocycloalkylene is (a) substituted with 1 oxo; and (b) is optionally further substituted with from 1-4 independently selected R a ;
Z is:
(i) —NR 10 R 11 ; or
(ii) —C(O)NR 10 R 11 ; or
(iii) —OR 12 ; or
(iv) —S(O) n R 13 , wherein n is 0, 1, or 2; or
(v) heterocycloalkenyl containing from 5-6 ring atoms, wherein from 1-3 of the ring atoms is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocycloalkenyl is optionally substituted with from 1-4 independently selected R a ; or
(vi) C 6 -C 10 aryl that is optionally substituted with from 1-4 independently selected R b ; or
(vii) heteroaryl containing from 5-14 ring atoms, wherein from 1-6 of the ring atoms is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-4 independently selected R b ; or
(viii) C 8 -C 14 arylcycloalkyl, wherein:
(1) the aryl portion is optionally substituted with from 1-4 independently selected R b , and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ; or
(ix) arylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) the aryl portion from is optionally substituted with from 1-4 independently selected R b , and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ; or
(x) heteroarylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ; or
(xi) heteroarylcycloalkyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
each of R 10 and R 11 is independently selected from the substituents delineated collectively in (a) through (1) below:
(a) hydrogen;
(b) C 6 -C 10 aryl that is optionally substituted with from 1-4 R b ;
(c) heteroaryl containing from 5-14 ring atoms, wherein from 1-6 of the ring atoms is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-4 R b ;
(d) C 1 -C 6 alkyl or C 1 -C 6 haloalkyl, each of which is optionally substituted with from 1-3 R d ;
(e) —C(O) (C 1 -C 6 alkyl), —C(O)(C 1 -C 6 haloalkyl), —C(O)O(C 1 -C 6 alkyl), —S(O) 2 (C 1 -C 6 alkyl), —S(O) 2 (C 6 -C 10 aryl), or —S(O) 2 (C 1 -C 13 heteroaryl), wherein the C 6 -C 10 aryl and C 1 -C 13 heteroaryl are each independently optionally substituted with 1-4 R b ;
(f) C 2 -C 6 alkenyl or C 2 -C 6 alkynyl;
(g) C 8 -C 14 arylcycloalkyl, wherein:
(1) the aryl portion is optionally substituted with from 1-4 independently selected R b , and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
(h) arylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) the aryl portion from is optionally substituted with from 1-4 independently selected R b , and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ;
(i) heteroarylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ;
(j) heteroarylcycloalkyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
(k) C 3 -C 8 cycloalkyl or C 3 -C 8 cycloalkenyl, each of which is optionally substituted with from 1-4 independently selected R a ; and
(l) C 7 -C 12 aralkyl, wherein the aryl portion is optionally substituted with from 1-4 independently selected R b ,
provided that one of R 10 and R 11 must be selected from (b), (c), (g), (h), (i), (j), and (k);
R 12 is:
(i) C6-C10 aryl that is optionally substituted with from 1-4 R b ; or
(ii) heteroaryl containing from 5-14 ring atoms, wherein from 1-6 of the ring atoms is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-4 R b ; or
(iii) C 1 -C 6 alkyl or C 1 -C 6 haloalkyl, each of which is substituted with from 1-3 R d ; or
(iv) C 8 -C 14 arylcycloalkyl, wherein:
(1) the aryl portion is optionally substituted with from 1-4 independently selected R b , and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ; or
(v) arylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) the aryl portion from is optionally substituted with from 1-4 independently selected R b , and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ; or
(vi) heteroarylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ;
or
(vii) heteroarylcycloalkyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
R 13 is:
(i) C 6 -C 10 aryl that is optionally substituted with from 1-4 R b ; or
(ii) heteroaryl containing from 5-14 ring atoms, wherein from 1-6 of the ring atoms is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-4 R b ; or
(iii) C 8 -C 14 arylcycloalkyl, wherein:
(1) the aryl portion is optionally substituted with from 1-4 independently selected R b , and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
or
(iv) arylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) the aryl portion from is optionally substituted with from 1-4 independently selected R b , and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ;
or
(v) heteroarylheterocyclyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) from 1-2 of the ring atoms of the heterocyclyl portion is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclyl portion is optionally substituted with from 1-3 independently selected R a ;
or
(vi) heteroarylcycloalkyl containing from 8-14 ring atoms, wherein:
(1) from 1-2 of the ring atoms of the heteroaryl portion is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; and wherein said heteroaryl portion is optionally substituted with from 1-3 independently selected R b ; and
(2) the cycloalkyl portion is optionally substituted with from 1-4 independently selected R a ;
R a at each occurrence is, independently selected from halo, hydroxyl, C 1 -C 6 alkoxy, C 1 -C 6 thioalkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 thiohaloalkoxy, oxo, thioxo, ═NH, ═N(C 1 -C 6 alkyl), C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, —NH 2 , —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), and cyano;
R b at each occurrence is independently selected from the substituents delineated in (aa) through (dd) below:
(aa) C 1 -C 6 alkoxy; C 1 -C 6 haloalkoxy; C 1 -C 6 thioalkoxy; C 1 -C 6 thiohaloalkoxy; —O—(CH 2 ) 1-3 —[O(CH 2 ) 1-3 ] 1-3 —H; —C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), wherein the alkyl portion of each is optionally substituted with from 1-3 independently selected R e ;
(bb) halo; hydroxyl; cyano; nitro; —NH 2 ; azido; sulfhydryl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; —C(O)H; —C(O)(C 1 -C 6 alkyl); —C(O) (C 1 -C 6 haloalkyl); —C(O)OH; —C(O)O(C 1 -C 6 alkyl); —C(O)NH 2 ; —C(O)NH(C 1 -C 6 alkyl); —C(O)N(C 1 -C 6 alkyl) 2 ; —SO 2 (C 1 -C 6 alkyl); —SO 2 NH 2 ; —SO 2 NH(C 1 -C 6 alkyl); —SO 2 N(C 1 -C 6 alkyl) 2 ;
(cc) C 3 -C 6 cycloalkyl or heterocyclyl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein each of said phenyl and heterocyclyl is optionally substituted with from 1-3 independently selected R a ; and
(dd) phenyl or heteroaryl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heteroaryl is independently selected from N, NH, N(C 1 -C 3 alkyl), O, and S; wherein each of said phenyl and heteroaryl is optionally substituted with from 1-3 substituents independently selected from halo; hydroxyl; cyano; nitro; —NH 2 ; —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), C 1 -C 6 alkoxy; C 1 -C 6 haloalkoxy; C 1 -C 6 thioalkoxy; C 1 -C 6 thiohaloalkoxy; C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl;
R c at each occurrence is, independently selected from halo, C 1 -C 6 alkoxy, C 1 -C 6 thioalkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 thiohaloalkoxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, —NH 2 , —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), and cyano;
R d at each occurrence is, independently selected from hydroxyl, C 1 -C 6 alkoxy, C 1 -C 6 thioalkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 thiohaloalkoxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, —NH 2 , —NH(C 1 -C 6 alkyl), —N(C 1 -C 6 alkyl) 2 , —NHC(O)(C 1 -C 6 alkyl), and cyano; and
R e at each occurrence is, independently selected from hydroxyl, C 1 -C 6 alkoxy; C 1 -C 6 thioalkoxy; C 1 -C 6 haloalkoxy; C 1 -C 6 thiohaloalkoxy; —NH 2 ; —NH(C 1 -C 6 alkyl); —N(C 1 -C 6 alkyl) 2 ; —NHC(O)(C 1 -C 6 alkyl); cyano; —C(O)H; —C(O)(C 1 -C 6 alkyl); —C(O)(C 1 -C 6 haloalkyl); —C(O)OH; —C(O)O(C 1 -C 6 alkyl); —C(O)NH 2 ; —C(O)NH(C 1 -C 6 alkyl); —C(O)N(C 1 -C 6 alkyl) 2 ; —SO 2 (C 1 -C 6 alkyl); —SO 2 NH 2 ; —SO 2 NH(C 1 -C 6 alkyl); —SO 2 N(C 1 -C 6 alkyl) 2 ; and L 3 -(C 1 -C 6 alkylene)-biotin, where in L 3 is a —O—, —NH—, —NCH 3 —, —C(O)—, —C(O)NH—, —C(O)NCH 3 —, —NHC(O)—, or —NCH 3 C(O)—.
2. The method of claim 1 , wherein in the incubating step, the NAMPT is recombinantly produced.
3. The method of claim 1 , wherein in the incubating step, the NAMPT is present at about 0.1 to about 10 μM, about 0.5 to about 5 μM, or about 1 μM.
4. The method of claim 1 , further comprising incubating the test compound with nicotinamide, phosphoribosylpyrophosphate, alcohol dehydrogenase, nicotinamide mononucleotide adenylyl-transferase, and an alcohol.
5. The method of claim 4 , wherein the alcohol is ethanol.
6. The method of claim 1 , wherein the measuring step comprises determining an amount of nicotinamide adenine dinucleotide reduced form (NADH) and/or nicotinamide mononucleotide (NMN).
7. The method of claim 6 , wherein the amount of NADH and/or NMN is measured by ultra violet light, mass spectrometry or fluorescence.
8. The method of claim 7 , wherein the amount of NADH is measured by optical density 340 nm.
9. The method of claim 6 , wherein an increase in the amount of NADH and/or NMN is indicative of increased NAMPT activity.
10. The method of claim 1 , wherein the test compound having said cell-protective activity is determined to bind and/or activate the NAMPT.
11. The method of claim 1 , wherein the cell-protective activity is neuroprotective activity which includes one or more of: promoting survival, health, integrity, growth, development and/or function of neurons, protecting neurons from cell death, apoptosis and/or degeneration, and/or stimulating neurogenesis.