IP Library › Granted Patent US 10,106,833

Granted Patent B2

US 10,106,833 · App. 14/390,857 · Granted Oct 23, 2018

Methods and compounds for identifying glycosyltransferase inhibitors

Inventors: Daniel Evan Kahne (Brookline, MA); Suzanne Walker Kahne (Brookline, MA); Christian M. Gampe (Brighton, MA); Hirokazu Tsukamoto (Sendai, JP)

Assignee: President and Fellows of Harvard College

C12Q1/48 C07H11/04 C07H13/12 C07H15/26 G01N2333/91091 G01N2500/04 G01N2500/20

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Quick Facts

Patent No.: US 10,106,833
App. No.: 14/390,857
Granted: Oct 23, 2018
Kind: B2

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Abstract

The present invention provides moenomycin-based probe compounds of Formula (I) for use in screening inhibitors of bacterial glycosyltransferases. The present invention also provides bacterial glycosyltransferase screening assays using compounds of Formula (I).

Claims (163)

1. A compound of Formula (I):

or a salt thereof,

wherein

R 1 is -L-R P , —C(O)NH-L-R P , —CH 2 O-L-R P , or —C(O)O-L-R P ;

R 2 and R 3 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 4 is hydrogen or —WR 4a ;

W is —O— or —NH—;

R 4a is hydrogen, a hydroxyl protecting group, optionally substituted aliphatic, —C(O)R 10 , —C(O)NHR 8 , —C(═NR 8 )NHR 8 , or —C(O)OR 9 ;

R 5 is hydrogen or —NHR 8 ;

R 6 is hydrogen, —CH 3 , —CH 2 OR 9 , or —CH 2 OR CX ; wherein R CX is a carbohydrate moiety;

R 7 is hydrogen, —OR 9 , or —N(R 8 ) 2 ;

each R 8 is independently hydrogen, an amino protecting group, —C(O)R 10 , optionally substituted aliphatic, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl, or two R 8 groups on the same nitrogen may be taken together to form an optionally substituted heterocyclyl;

each R 9 is independently hydrogen, a hydroxyl protecting group, —C(O)R 10 , optionally substituted aliphatic, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl;

each R 10 is independently optionally substituted aliphatic, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl;

R a and R b are independently hydrogen or a hydroxyl protecting group;

G is an optionally substituted C 1-16 aliphatic group, wherein 0 to 10 methylene units are optionally replaced with —O—, —NR x —, —S—, —C(O)—, —C(═NR x ), —S(O)—, —SO 2 —, —N═N—, —C═N—, —N—O—, an optionally substituted arylene, an optionally substituted heterocyclylene, or an optionally substituted heteroarylene; wherein each instance of R x is independently hydrogen, optionally substituted aliphatic, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; or

G is a group of Formula (a), (b), or (c):

wherein a is 3, 4, or 5;

wherein

X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently hydrogen or halogen;

d is an integer between 1 and 25, inclusive; and

e is an integer of between 2 and 25, inclusive;

provided the sum of d and e is greater than 16; or

wherein

Y is —O—, —S—, —NR Y —, or an optionally substituted methylene group, wherein R Y is hydrogen, optionally substituted aliphatic, or an amino protecting group;

each instance of R c is independently —F, —Br, —I, —Cl, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, optionally substituted heteroaryl, —OR e , —SR e , —NHR e , or —N(R e ) 2 , wherein each instance of R e is independently hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R e groups are joined to form a 5- to 6-membered optionally substituted heterocycyl or optionally substituted heteroaryl ring;

each instance of R d is independently —F, —Br, —I, —Cl, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, optionally substituted heteroaryl, —OR f , —SR f , —NHR f , or —N(R f ) 2 , wherein each instance of R f is independently hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R f groups are joined to form a 5- to 6-membered optionally substituted heterocycyl or optionally substituted heteroaryl ring;

R z is hydrogen, —F, —Br, —I, —Cl, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, optionally substituted heteroaryl, —OR g , —SR g , —NHR g , or —N(R g ) 2 , wherein each instance of R g is independently hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted carbocycyl, optionally substituted heterocycyl, optionally substituted aryl, or optionally substituted heteroaryl or two R g groups are joined to form a 5- to 6-membered optionally substituted heterocycyl or optionally substituted heteroaryl ring;

each instance of n is, independently, 0, 1, 2, 3, or 4;

each instance of m is, independently, 0, 1, 2, 3, or 4; and

x is 1, 2, 3, 4, 5, or 6;

R YY is hydrogen or —OR XX ;

R XX is hydrogen, a hydroxyl protecting group, or a group of formula:

wherein

R 11 is hydrogen, optionally substituted aliphatic, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 12 and R 13 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 14 is hydrogen or —NHR 8 ;

R 15 is hydrogen, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 16 is hydrogen or —OR 9 ;

R 17 is hydrogen or —OR 9 ;

R 18 is hydrogen or —OR 9 ;

R 19a is hydrogen or —OR 9 ;

R 19b is hydrogen or —OR 9 ;

wherein a hydrogen radical on the compound of Formula (I) is replaced with -L-R P ;

L is a covalent bond, —NR y —, —N(R y )C(O)—, —N(R y )C(O)N(R y )—, —N(R y )C(S)N(R y )—, —C(O)N(R y )—, —N(R y )SO 2 —, —SO 2 N(R y )—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO 2 —, optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, or an optionally substituted aliphatic linker, wherein one or more methylene units of the aliphatic linker are optionally replaced by —NR y —, —N(R y )C(O)—, —N(R y )C(O)N(R y )—, —N(R y )C(S)N(R y )—, —C(O)N(R y )—, —N(R y )SO 2 —, —SO 2 N(R y )—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO 2 —, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein R y is hydrogen, C 1-6 alkyl, or —C(O)C 1-6 alkyl; and

R P is selected from the group consisting of ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, enzymes, calorimetric labels, magnetic labels, and haptens.

2. A compound of Formula (I):

or a salt thereof,

wherein

R 1 is hydrogen, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 2 and R 3 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 4 is hydrogen or —WR 4a ;

W is —O— or —NH—;

R 4a is hydrogen, a hydroxyl protecting group, optionally substituted aliphatic, —C(O)R 10 , —C(O)NHR 8 , —C(═NR 8 )NHR 8 , or —C(O)OR 9 ;

R 5 is hydrogen or —NHR 8 ;

R 6 is hydrogen, —CH 3 , —CH 2 OR 9 , or —CH 2 OR CX ; wherein R CX is a carbohydrate moiety;

R 7 is hydrogen, —OR 9 , or —N(R 8 ) 2 ;

each R 10 is independently optionally substituted aliphatic, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl;

R a and R b are independently hydrogen or a hydroxyl protecting group;

G is a group of Formula (a), (b), or (c):

wherein a is 3, 4, or 5;

wherein

X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently hydrogen or halogen;

d is an integer between 1 and 25, inclusive; and

e is an integer of between 2 and 25, inclusive;

provided the sum of d and e is greater than 16; or

wherein

Y is —O—, —S—, —NR Y —, or an optionally substituted methylene group, wherein R Y is hydrogen, optionally substituted aliphatic, or an amino protecting group;

each instance of n is, independently, 0, 1, 2, 3, or 4;

each instance of m is, independently, 0, 1, 2, 3, or 4; and

x is 1, 2, 3, 4, 5, or 6;

R YY is hydrogen or —OR XX ;

R XX is hydrogen, a hydroxyl protecting group, or a group of formula:

wherein

R 11 is hydrogen, optionally substituted aliphatic, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 12 and R 13 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 14 is hydrogen or —NHR 8 ;

R 15 is hydrogen, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 16 is hydrogen or —OR 9 ;

R 17 is hydrogen or —OR 9 ;

R 18 is hydrogen or —OR 9 ;

R 19a is hydrogen or —OR 9 ;

R 19b is hydrogen or —OR 9 ;

wherein a hydrogen radical on the compound of Formula (I) is replaced with -L-R P :

L is —NHC(S)NH—, —C(O)CH 2 —NHC(S)NH—,

and

R P is selected from the group consisting of ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, enzymes, calorimetric labels, magnetic labels, and haptens.

3. The compound of claim 2 , wherein R YY is —OH or —OR XX , and R XX is a group of formula:

4. The compound of claim 2 , wherein R XX is a group of formula:

5. The compound of claim 2 , wherein R 6 is —CH 2 OH or —CH 2 OR CX ; wherein R CX is a carbohydrate moiety.

6. The compound of claim 2 , wherein G is

7. The compound of claim 2 , wherein R P is selected from the group consisting of Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680, AMCA, AMCA-S, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, aminomethylcoumarin, carbocyanine, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, coumarin, coumarin 343, cyanine dyes, dansyl, dapoxyl, dialkylaminocoumarin, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, DM-NERF, eosin, erythrosin, fluorescein, FAM, hydroxycoumarin, IRD40, IRD 700, IRD 800, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOE), lissamine rhodamine B, Marina Blue, merocyanine, methoxycoumarin, naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, oxonol dyes, Pacific Blue, phycoerythrin, PyMPO, pyrene, rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, rhodol green, styryl dyes, 2′,4′,5′,7′-tetrabromosulfone-fluorescein, tetramethyl-rhodamine (TMR), carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X, 5(6)-carboxyfluorescein, 2,7-dichlorofluorescein, N,N-bis(2,4,6-trimethylphenyl)-3,4,9,10-perylenebis(dicarboximide), HPTS, ethyl eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green 520, ATTO 465, ATTO 488, ATTO 495, Y0Y0-1,5-FAM, BCECF, dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-I, SYTOX Green, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3, Fluo-4, fluoro-emerald, YoYo-I ssDNA, YoYo-I dsDNA, YoYo-I, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX, Spectrum Green, Spectrum Red, NeuroTrace 500525, NBD-X, MitoTracker Green FM, LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP (post-activation), FLASH-CCXXCC, Azami Green monomeric, Azami Green, green fluorescent protein (GFP), EGFP, Kaede Green, 7-benzylamino-4-nitrobenz-2-oxa-1,3-diazole, Bexl, doxorubicin, Lumio Green, and SuperGlo GFP.

8. The compound of claim 2 of formula:

9. The compound of claim 2 , wherein:

R 1 is —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

one of R 2 and R 3 is hydrogen, and the other is —OR 9 ;

R 4 is —W—R 4a ;

R 5 is —NHR 8 ;

R 6 is —CH 3 , —CH 2 OR 9 , or —CH 2 OR CX ;

R 7 is —OR 9 or —N(R 8 ) 2 ;

R 11 is —CH 3 , —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

one of R 12 and R 13 is hydrogen, and the other is —OR 9 ;

R 14 is —NHR 8 ; and

one of R 19a and R 19b is hydrogen, and the other is —OR 9 .

10. The compound of claim 3 , wherein R 11 is —CH 2 OH.

11. The compound of claim 2 , wherein R P is selected from the group consisting of fluorescein, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 2′,4′,5′,7′-tetrabromosulfone-fluorescein, 2,7-dichlorofluorescein, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, and 5(6)-carboxyfluorescein.

12. The compound of claim 11 , wherein R P is fluorescein.

13. The compound of claim 3 , wherein R 11 is —CH 3 .

14. The compound of claim 2 , wherein R 12 is hydrogen; and R 13 is —OH.

15. The compound of claim 2 , wherein G is of Formula (a).

16. The compound of claim 2 , wherein G is of Formula (b).

17. A compound of Formula (I):

or a salt thereof,

wherein

R 1 is hydrogen, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 2 and R 3 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 4 is hydrogen or —WR 4a ;

W is —O— or —NH—;

R 4a is hydrogen, a hydroxyl protecting group, optionally substituted aliphatic, —C(O)R 10 , —C(O)NHR 8 , —C(═NR 8 )NHR 8 , or —C(O)OR 9 ;

R 5 is hydrogen or —NHR 8 ;

R 6 is hydrogen, —CH 3 , —CH 2 OR 9 , or —CH 2 OR CX ; wherein R CX is a carbohydrate moiety;

R 7 is hydrogen, —OR 9 , or —N(R 8 ) 2 ;

each R 10 is independently optionally substituted aliphatic, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl;

R a and R b are independently hydrogen or a hydroxyl protecting group;

G is a group of Formula (a), (b), or (c):

wherein a is 3, 4, or 5;

wherein

X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently hydrogen or halogen;

d is an integer between 1 and 25, inclusive; and

e is an integer of between 2 and 25, inclusive;

provided the sum of d and e is greater than 16; or

wherein

Y is —O—, —S—, —NR Y —, or an optionally substituted methylene group, wherein R Y is hydrogen, optionally substituted aliphatic, or an amino protecting group;

each instance of n is, independently, 0, 1, 2, 3, or 4;

each instance of m is, independently, 0, 1, 2, 3, or 4; and

x is 1, 2, 3, 4, 5, or 6;

R YY is hydrogen or —OR XX ;

R XX is hydrogen, a hydroxyl protecting group, or a group of formula:

wherein

R 11 is hydrogen, optionally substituted aliphatic, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 12 and R 13 are independently hydrogen, optionally substituted aliphatic, —OR 9 , —N(R 8 ) 2 , or —C(O)NHR 8 ;

R 14 is hydrogen or —NHR 8 ;

R 15 is hydrogen, —C(O)NHR 8 , —CH 2 OR 9 , or —C(O)OR 9 ;

R 16 is hydrogen or —OR 9 ;

R 17 is hydrogen or —OR 9 ;

R 18 is hydrogen or —OR 9 ;

R 19a is hydrogen or —OR 9 ;

R 19b is hydrogen or —OR 9 ;

wherein a hydrogen radical on the compound of Formula (I) is replaced with -L-R P ;

L is an optionally substituted aliphatic linker wherein one methylene unit is replaced by tetrazolyl or NHC(S)NH—; and one or more additional methylene units are optionally replaced by —NR y —, —N(R y )C(O)—, —N(R y )C(O)N(R y )—, —N(R y )C(S)N(R y )—, —C(O)N(R y )—, —N(R y )SO 2 —, —SO 2 N(R y )—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, or —SO 2 —; wherein R y is hydrogen, C 1-6 alkyl, or —C(O)C 1-6 alkyl; and

R P is selected from the group consisting of ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, enzymes, calorimetric labels, magnetic labels, and haptens.

Assignments (2)

CONFIRMATORY LICENSE Recorded Nov 18, 2014

From: HARVARD UNIVERSITY

To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT

Reel/Frame 034282/0876 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 16, 2014

From: KAHNE, DANIEL; WALKER KAHNE, SUZANNE; GAMPE, CHRISTIAN; TSUKAMOTO, HIROKAZU

To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Reel/Frame 033965/0864 →

Continuity (2)

Provisional Application 61621229 · Apr 6, 2012

Related Publication 20150079618A1 · Mar 19, 2015