GLIKOGÉN FOSZFORILÁZ GÁTLÁS - KFKIcheminfo/osztaly/felolvas/fo2012/... · 2011-12-27 ·...

GLIKOGÉN FOSZFORILÁZ GÁTLÁS MONOSZACHARID SZÁRMAZÉKOKKAL

Somsák László

Debreceni Egyetem, Szerves Kémiai Tanszék

MTA Kémiai Tudományok Osztálya felolvasóülése

Budapest, 2011. dec. 13.

http://www.diabetesatlas.org/map

2030 Prevalence of diabetes mellitus in the population (age: 20-79 years)

http://www.idf.org

http://www.diabetesatlas.org/map

Type 1 diabetes Total insulin deficiency Type 2 diabetes Impaired insulin secretion or insulin resistance

Prevalence of diabetes mellitus in the population (age: 20-79 years)

Current therapeutic agents for type 2 diabetes

Source: D. E. Moller, Nature, 2001, 414, 821-827.

Oral hypoglycaemic agents are inadequate for 30-40 % of the patients

(A. S. Wagman, J. M. Nuss, Curr. Pharma. Design, 2001, 7, 417-450)

Drug class Site(s) of

action Molecular target Adverse effects

Sulphonylureas Pancreatic

b-cells

SU receptor

K+ ATP channel

Hypoglycaemia,

weight gain

Metformin -

Biguanides

(muscle)

Unknown Gastrointestinal

disturbances,

lactic acidosis

Acarbose Intestine a-glucosidase Gastrointestinal

disturbances

Thiazolidinediones Fat, muscle,

PPARg Weight gain,

oedema, anaemia

Insulin Liver,

muscle, fat

Insulin receptor Hypoglycaemia,

weight gain

Investigational targets/strategies for type 2 diabetes

Insulin Improving Combination SGLT-2

secretagogues insulin action therapies inhibitors

Strategies altering lipid metabolism Nutritional therapy

Inhibition of hepatic glucose production

Target Function

Glucagon Enhances hepatic glucose output

GCK Catalyzes the first step of glycolysis

6PF-2-K/F-2,6-P2ase Regulator of glycolytic and gluconeogenic rates

through production of F-2,6-P2

G-6-Pase Catalyzes the last step of gluconeogenesis

F-1,6-P2ase Regulates gluconeogenic rates

GSK3 Inhibits glycogen synthase

Glycogen phosphorylase Catalyzes the conversion of glycogen

to glucose-1-phosphate monomers

Source: Morral, N., Trends Endocrin. Metab., 2003, 14, 169-175.

Diseased states claimed to be affected by glycogen phosphorylase inhibitors

Type 2 diabetes mellitus

Early cardiac and cardiovascular disease

in non-diabetics (treatment and/or prevention)

Cardiac arrhythmias (stabilization)

Ischemic injury (protection)

Tumour growth (prevention)

Structure and binding sites of glycogen phosphorylase (rabbit muscle GP, RMGP) Figure by courtesy of E. D. Chrysina (Institute for Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Athens, Greece).

Reviews:

Oikonomakos,

Curr. Protein Pept. Sci.,

2002, 2, 561-586.

Somsák et al.,

Curr. Med. Chem.,

2008, 15, 2933-2983.

Thematic issue (Guest

editor: L. Somsák)

Mini-Rev. Med. Chem.,

2010, 10, 1091-1193.

Early glucose analogue inhibitors of RMGPb (Ki [mM])

Pioneered by Fleet, Johnson, and Oikonomakos

1700 7400

370 440

Review: Somsák et al., Curr. Pharma. Design, 2003, 9, 1177-1189.

OHHN CH3

OH HN OCH3

Attempted synthesis of spiro-hydantoin analogues

Remy et al.,

Can. J. Chem.,

1980, 58, 2660-2665.

Descotes, G.

Bull. Soc. Chim. Belges

1982, 91, 973-983.

(PGO)n

(PGO)n (PGO)n

(PGO)n(PGO)n

Spiro-hydantoins and benzoyl-urea are equipotent inhibitors of RMGPb

R = CH3 Ki = 305 mM

R = C6H5 Ki = 4.6 mM

X = O Ki = 3 mM

X = S Ki = 5 mM

Oikonomakos et al.,

Eur. J. Biochem. 2002, 269, 1684-1696.

Syntheses of N-substituted-N’-b-D-glucopyranosylureas and related compounds

OAcAcO

ORHN NH2

OAcAcO

N C N R'O

R = Ac, Bz H

R' = (a) alkyl, aryl, aralkyl, (b) alkanoyl, aroyl, arylalkanoyl

(Cl3CO)2CO, CH2Cl2, H2O, NaHCO3, r. t.

H2, Ra-Ni

PPh3, CO2, NH3,

R'(b)Cl

1. PMe32. R'NCO H+/H2O

NH2.HO2CNH2

C10H7CONCO

NH4O2CONH2

PhNHCONH2

X = O, S, Se

OAcAcO

OR'NH2 O

(COCl)2

CNNO2, CH2Cl2O

CONH2O

HBr, AcOH

PhNCO (neat), reflux

PhCONHNH2, DCC, CH2Cl2, r. t.

Inhibition of RMGPb by N-acyl-N’-b-D-glucopyranosylureas

R Ki [mM] R Ki [mM]

–CH3 305 –H 4.6

15 –CH3 2.3

–C6H5 3.7

0.35 –CF3 1.8

–C(CH3)3 0.7

4.0 –NO2 3.3

–Cl 4.4

68 –NH2 6.0

–OH 6.3

No inhibition –OCH3 3.2

–COOH IC50 350

–COOCH3 4.0

linker Ki [mM]

NHCO 81 (144) 444 10

NHCONH 18 350 (IC50) 5.2

NHCONHCO 4.6 10 0.35

NHCONHCONH 21 - -

NHCONHCONHCO - - 45 %

(at 625 mM)

Influence of the linker length on the inhibition of RMGPb

linker Ar

linker Ki [mM]

NHCONHCH2 750 - -

NHCOCH2CH2 85 - -

NHCOCH=CH 18 - 3.5

NHCOC≡C 61 - -

NHCOCH2O 34 - -

NHCOOCH2 350 - -

NHCOCH2NH 70 - 142

NHCONHCO 2.3

NHCONHSO2 No inh. (Ar = 4-CH3-C6H4)

NHSO2NHCO No inh.

Influence of the linker composition on the inhibition of RMGPb I.

linker Ar

linker Ki [mM]

NHCOCONH 100 144 56

CONHCONH No inh. - -

CONHNHCO 22 %

(at 3.75 mM) - -

Influence of the linker composition on the inhibition of RMGPb II.

linker Ar

Binding of N-acetyl-b-D-glucopyranosylamine at the active site of muscle GPb

Anagnostou, E. et al.,

Bioorg. Med. Chem.,

2006, 14, 181-189.

OHHN CH3

Ki = 32 mM

Binding of TH at the active site of muscle GPb

Oikonomakos et al.,

Bioorg. Med. Chem.,

2002, 10, 261.

Binding of N-benzoyl-b-D-glucopyranosylamine and N-benzoyl-N’-b-D-glucopyranosyl urea at the active site of muscle GPb

O Ki = 81 mM

O O Ki = 4.6 mM

Interactions of N-benzoyl- and N-2-naphthoyl-N’-b-D-glucopyranosylureas in the b-channel of muscle GPb

Ki = 4.6 mM Ki = 0.35 mM

New N-b-D-glucopyranosyl derivatives tested as inhibitors of RMGPb

Gimisis, Mini-Rev. Med. Chem., 2010, 10, 1127-1138.

-CH3 -(CH2)2SCH3

Ki [mM] 510 1200 350

Alexacou et al., Bioorg. Med. Chem., 2010, 18, 7911-7922.

IC50 [mM] 33 5.7 28

No H-bond between His377 main chain CO and N1-H.

Novel design principle for inhibitors of GP

Ki = 0.35 mM

- rigid, spirobicyclic

stucture

- H-bridge

- interactions in the

b-channel

- lack of H-bridge

X = O Ki = 3.1 mM

X = S Ki = 5.1 mM

Leads:

X = O,S, NH Target

compounds:

His377

b-channel

New spirocyclisation of (a-D-gluco-hept-2-ulopyranosyl-bromide)onamide

Somsák & Nagy, Tetrahedron-Asymmetry, 2000, 11, 1719-1724.

BzOBzO

NaOMeMeOH

CH3NO2, 80 oC

S8, N2 atm.

H2NCNH2

acetone, refluxor EtOH, MW

H2NCNHPh

DMF, 80 oC

conv. 72 %, yield 82 %

79 % 92 %

BzOBzO

By-product in each reaction:

NH4SCN,

Acylation of glucopyranosylidene-spiro-iminothiazolones

BzOBzO

NaOMeMeOH

BzOBzO

dry Py, r. t.R

R = Yield (%) Yield (%)

Me-CO 76 N-deacylation to give

iPr-CO 81 unprot. thiazolone

tBu-CO 82 26

Ph-CO 84 58

1-Naphth-CO 73 56

2-Naphth-CO 89 50

4-Me-Ph-SO2 96 (conv. 95 %) 60

1-Naphth-SO2 91 (conv. 75 %)

2-Naphth-SO2 97 (conv. 76 %)

Alkylation of glucopyranosylidene-spiro-iminothiazolones

BzOBzO

NaOMeMeOH

BzOBzO

BrCH2CH2Br

BzOBzO

K2CO3, DMF, r. t.

NaOMeMeOH

K2CO3, DMF, r. t.

BrCH2CH2CH2Br

67 % 48 %

70 % 48 %

Inhibition of RMGPb by glucopyranosylidene-spiro-iminothiazolone derivatives

RR = H

Ki = 14 mM Ki = 137 mM Ki = 9 mM

Ki = 8 mM Ki = 4 mM Ki = 16 mM

47 % inhibition at 1 mM

No inhibition up to 1 mM

Ki = 5.1 mM

Binding of the spiro-iminothiazolone at the active site of RMGPb and comparison to spiro-thiohydantoin

Ki = 14 mM Ki = 5.1 mM

OAcAcO

AcOAcO

ArC(Cl)NOH NBS

Synthesis of glucopyranosylidene-spiro-oxathiazolines

94 36 78

79 69 71

79 30 65

90 46 90

Yields Ar

R = H R = Ac

Somsák, Praly, et al., Bioorg. Med. Chem. Lett., 2008, 18, 5680-5683,

Bioorg. Med. Chem. 2009, 17, 5696-5707.

Attempted synthesis of a glucopyranosylidene-spiro-oxadiazoline

NBS, CHCl3

AcOAcO

1. PMe3, PhCH3, CH2Cl2

AcOAcO

Somsák, Praly, et al., Bioorg. Med. Chem. 2009, 17, 5696-5707.

Attempted synthesis of a glucopyranosylidene-spiro-oxadiazoline

AcOAcO

1. PMe3, PhCH3, CH2Cl2

AcOAcO

Somsák, Praly, et al., Bioorg. Med. Chem. 2009, 17, 5696-5707.

Synthesis of glycopyranosylidene-spiro-isoxazolines

R = Ac, Bz, Bn, Et3Si

Ra-Ni, TsNHNH2,

AcOH-H2O-Py, rt

2-ethanesulfonyl-benzothiazole, LiHMDS, -78 °C, DBU, THF

1,4-dioxane reflux

R'C(Cl)=NOH, Et3N,CH2Cl2, rt

Praly et al.,

Tetrahedron

2006, 47,

6143-6147.

Tóth & Somsák,

J. Chem. Soc.

Perkin 1,

2001, 942-943.

Org. Biomol.

Chem.,

2003, 1, 4039-4046.

Carbohydrate

Chemistry: Proven

Synthetic Methods,

2011, in press. Praly et al.,

Bioorg. Med.

Chem.,

2009, 17,

7368-7380.

Inhibition of RMGPb (Ki [µM]) by glycopyranosylidene-spiro-heterocycles

26 19.6 no inh.

at 625 mM

8.2 6.6

0.16 0.63 no inh.

at 625 mM

Somsák, Praly et al.,

Bioorg. Med. Chem. Lett.,

2008, 18, 5680-5683.

Bioorg. Med. Chem.,

2009, 17, 5696-5707.

Praly et al.,

Bioorg. Med. Chem.,

2009, 17, 7368-7380.

Modification of N-acyl-b-D-glucopyranosylamines by non-classical bioisosteres*

OHHN R

linker

linkers

*Patani, G. A.; LaVoie, E. J., Chem. Rev., 1996, 96, 3147-3176.

Lima, L. M. A.; Barreiro, E. J., Curr. Med. Chem., 2005, 12, 23-49.

Synthesis of 1-D-glucopyranosyl-4-substituted-1,2,3-triazoles

OAcAcO

AcOAcO

BzOBzO

R = CH2OH, COOEt(Me), Ph, 1-naphthyl, 2-naphthyl

1. R-C CH [1 eq in a) and b); 2 eq in c]

a) 0.015 eq CuSO4, 0.2 eq L-ascorbic acid, water, 70 °C

b) 0.075 eq CuSO4, 0.2 eq L-ascorbic acid, water, 70 °C

c) 0.11 eq CuSO4, 0.3 eq L-ascorbic acid, DMSO, 70 °C

2. NaOMe (cat), MeOH, rt

Bokor et al., Bioorg. Med. Chem. 2010, 18, 1171-1180.

OHHN R

Similarity of the amide moiety and the 1,2,3-triazole ring:

size, dipolar character, and H-bond acceptor capacity

(Angell & Burgess, Chem. Soc. Rev. 2007, 36, 1674-1689).

Binding of glucopyranosylamides and -1,2,3-triazoles at the catalytic site of RMGPb I.

Ki = 151 mM O

Ki = 81 mM

Chrysina et al., Tetrahedron:

Asymm. 2009, 20, 733-740.

Ki = 13 mM

Ki = 16 mM

Chrysina et al., Tetrahedron:

Asymm. 2009, 20, 733-740.

Binding of glucopyranosylamides and -1,2,3-triazoles at the catalytic site of RMGPb II.

R = OH R = F

6.1 3640

7.7 4010

New N-b-D-glucopyranosyl heterocycles tested as inhibitors of RMGPb (Ki [mM])

Gimisis,

Mini-Rev. Med. Chem.,

2010, 10, 1127-1138.

Tsirkone et al.,

Bioorg. Med. Chem.,

2010, 18, 3413–3425.

Syntheses of C-glucopyranosyl-oxadiazoles

R'COClR = Ac, Bz, Bn H

ORHC NNHCOR'

R'COOH, DCC

Ra-Ni, NaH2PO2

R'CONHNH2

PhI(OAc)2

R'C(=NOH)Cl, Et3N

1. O-acylation2. cyclo- dehydration

R'CH(=NOH), NaOCl

Bu3SnN3

Me3SiN3, Bu2SnO

Tóth & Somsák, Carbohydr. Res., 2003, 338, 1319-1325.

Kun et al., Carbohydr. Res, 2011, 346, 1427-1438.

Tóth et al., Bioorg. Med. Chem., 2009, 17, 4773–4785.

Benltifa et al., Eur. J. Org. Chem., 2006, 4242.

Inhibition of RMGPb (Ki [µM]) by C-glucopyranosyl-oxadiazoles

-CH3 145 - no inh.

at 625 mM

at 625 mM 64

at 625 mM

at 625 mM 19

no inh.

at 625 mM

at 625 mM 2.4 38

Tóth et al.,

Bioorg. Med. Chem.,

2009, 17, 4773–4785.

Benltifa et al.,

Eur. J. Org. Chem.,

2006, 4242.

Hadady et al., Arkivoc.,

2004, vii, 140-149.

Chrysina et al., Prot. Sci.,

2005, 14, 873-878.

Inhibition of RMGPb by homotrivalent glucose derivatives

Cecioni et al., New J. Chem. 2009, 33, 148-156.

at 625 mM No inh.

at 625 mM 35 %

at 625 mM

Inhibition of RMGPa by heterobivalent glucose-pentacyclic triterpene derivatives

Oleanolic acid (OA) R1 = H, R2 = H, R3 = CH3

Ursolic acid (UA) R1 = H, R2 = CH3, R3 = H

Maslinic acid (MA) R1 = OH, R2 = H, R3 = CH3

Acid R n IC50 [mM]

OA Ac - 337

OA H - 26

UA Ac - 51

UA H - 45

MA Ac - no inh.

MA H - 779

OA Ac 1 68

UA Ac 10 65

MA Ac 1 78

O OROR

Cheng et al., New J. Chem. 2010, 34, 1450-1464.

Physiological effects of TH treatment in streptozotocin-induced diabetic rats

The results are the mean ± SD of four independent experiments.

Docsa et al., Mol. Med. Rep., 2011, 4, 477-481.

Intravenous

administration of

TH to Zucker

diabetic fatty rats

significantly

decreased

hepatic glycogen

phosphorylase a

levels, and the

activation of

synthase was

initiated without

any delay. Blood glucose Liver glycogen

Summary

Glucose-derived compounds » N-acyl-N’-b-D-glucopyranosylureas » glucopyranosylidene-spiro-heterocycles (isoxazolines, oxathiazolines) » C-b-D-glucopyranosyl heterocycles having large aromatic substituents inhibit glycogen phosphorylase in the nanomolar range.

Further inhibitor design may focus on the interactions in the b-channel of the enzyme.

Physiological investigations show glucopyranosylidene-spiro-thiohydantoin to act towards normalizing serum glucose and liver glycogen levels in diabetic rats.

Organic synthesis

Dr. CZIFRÁK, Katalin

Dr. KOVÁCS, László

Dr. NAGY, Veronika

Dr. HADADY, Zsuzsa

Dr. TÓTH, Marietta

Dr. FELFÖLDI, Nóra

Dr. BOKOR, Éva

KRAKOMPERGER, Attila

TELEPÓ, Katalin

KÓNYA, Bálint

HÜSE, Csaba

KUN, Sándor

KÓDER, Lászlóné

Dr. PRALY, Jean-Pierre

Dr. VIDAL, Sébastien

Prof. Dr.

GYÖRGYDEÁK, Zoltán

Participants

Financial support: Hungarian Scientific Research Fund (OTKA CK77712), TÁMOP 4.2.1/B-09/1/KONV-2010-0007 project co-financed by the European Union and the European Social Fund.

Structure elucidation

Prof. Dr.

SZILÁGYI, László

Prof. Dr.

E. KÖVÉR, Katalin

Enzyme kinetics

Department of Medical

Chemistry, University

of Debrecen

Prof. Dr. GERGELY, Pál

DOCSA, Tibor

Protein crystallography Institute of Organic and Pharmaceutical Chemistry,

The National Hellenic Research Foundation,

Athens, Greece

Dr. CHRYSINA, Evangelia D.

Dr. LEONIDAS, Demetres D.

Dr. ZOGRAPHOS, Spyros E. and others

Dr. OIKONOMAKOS, Nikos G.

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