Largazole

Transformed Mammary Epithelial Cells (MDAMB-231)

GI50: 7.7nMLC50: 117nM

Nontransformed Mammary Epithelial Cells (NMuMG)

GI50: 122nMLC50: 272nM

Fibroblastic osteosarcoma cells (U2OS) GI50: 55nMLC50: 94nM

Nontransformed Fibroblast (NIH3T3) GI50: 480nMLC50: >8µM

Colon Cancer Line (HT29) GI50: 12nMLC50: 22nM

Neuroblastoma (IMR-32) GI50: 16nMLC50: 22nM

S NH

O O OSN

S

NNH

O

O

Taori, K.; Paul, V. J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 1806

Thiol AnaloguesHCT-116 growth inhibition (nM)

HCT-116 HDAC cellular assay (nM)

HeLa nuclear extracts HDAC’s (nM)

44±10 51±3 37±11

38±5 209±15 42±29

33±2 50±18 52±27

S NH

O O OSN

S

NNH

O

O

S NH

O O OSN

S

NNH

O

O

HS NH

O OSN

S

NNH

O

O

Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455

Analogues with no ThiolHCT-116 growth inhibition (nM)

HCT-116 HDAC cellular assay (nM)

HeLa nuclear extracts HDAC’s (nM)

> 10000 > 10000 > 10000

> 10000 > 10000 > 10000HO N

H

O OSN

S

NNH

O

O

NH

O OSN

S

NNH

O

O

Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455

HDAC Selectivity of LargazoleHDAC 1 (class I) (nM) HDAC 6 (class II) (nM)

Largazole 25 ± 11 5700 ± 3600

Largazole Thiol 2.5 ± 1.4 380 ± 76

Trichostatin A 4.9 ± 0.8 18 ± 12

Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455

Ki values HDAC 1 (nM) HDAC 2 (nM) HDAC 3 (nM) HDAC 6 (nM)

Largazole 20 21 48 >1000

Largazole Thiol 0.07 0.07 0.17 25

FK228 0.12 0.14 0.28 35

SAHA 10 10 15 9

Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M. Journal of the American Chemical Society 2008, 130, 11219.

• Antiproliferative effects of largazole (red) and largazole thiol (blue)

• human malignant melanoma cell lines• Largazole is more potent in antiproliferative effect

than largazole thiolBowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M. Journal of the American Chemical Society 2008, 130, 11219.

Linker Lengthn HCT-116 growth inhibition

(nM) (GI50)HeLa nuclear extracts HDAC’s (nM) (IC50)

1 >10000 >20000

2 6.8±0.6 32±13

3 620±50 7600±900

4 2500±600 4100±430

NH

O OSN

S

NNH

O

Sn-C7H15

On

O

Linker size of 4 is optimized for HDAC inhibition

Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021

Valine Residue and C17 VariationHCT-116 growth inhibition (nM)

HeLa nuclear extracts HDAC’s (nM)

Largazole 6.8±0.6 32±13

21±2 72±21

3900±450 >20000C7H15 S N

H

O O OSN

S

NNH

O

O

-Valine Residue can be replaced-Stereochemistry at C17 center is important

C7H15 S NH

O O OSN

S

NNH

O

O

Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021

HDAC SelectivityHDAC 1 (class I) (nM) HDAC 6 (class II) (nM)

Largazole 7.6 1800

690 >10000

1900 >10000

44 3300

Trichostatin A 5.2 1.8C7H15 S N

H

O O OSN

S

NNH

O

O

NH

O OSN

S

NNH

O

O

Sn-C7H15

O

NH

O OSN

S

NNH

O

O

Sn-C7H15

O

Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021

MDA-MB231 (nM) HME (nM)

Largazole 71 >600

Terminal olefin (no linker) >600 >600

>600 >600

>600 >600

>600 >600

S NH

O OH OSN

S

NNH

MeO2C

O

O NH

O O OSN

S

NNH

O

O

6

NH

O O OSN

S

NNH

O

O

6

-Breast cancer cell line vs. normal mammary epithelial cells-Crystal Violet Dye

Nasveschuk, C. G.; Ungermannova, D.; Liu, X.; Phillips, A. J. Organic Letters 2008, 10, 3595

Linker Analogues

• None of these analogues were active up to 5µM.– Human Epithelial Carcinoma Cell Line (A432)– Preadipocyte Cell Line (3T3L1)

• No free thiol can be formed.• Synthetic Largazole showed weak selectivity for the cancer cell line GI50 = 49 vs

127nM• Largazole Thiol showed weaker potency but better selectivity GI50 = 126 vs

1200nM

NH

O OSN

S

NNH

O

O

NH

O OSN

S

NNH

O

Br

O

O NH

O O OSN

S

NNH

O

O

Seiser, T.; Kamena, F.; Cramer, N. Angewandte Chemie International Edition 2008, 47, 6483

S-acetyl, disulfide analoguesLargazole Free

ThiolTSA

60nM 38nM 50nM 70nM 300nM

S NH

O O OSN

S

NNH

O

O

SS N

H

O OSN

S

NNH

O

N

O

-ED50 values from reporter gene assay in HEK293 cell promoted by cytomegalovirus (CMV) (nM)-Ability of being hydrolized to free thiol

Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 2008, 2483

• SEAP (Secreted Alkaline Phosphatase) reporter gene

• Cytomegalovirus promoter• Induction of promoter by largazole analogues• Measured Phosphatase units to calculate ED50

values

Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 2008, 2483

Isostere AnaloguesHDAC 1 HDAC 2 HDAC 3 HDAC 6

Largazole 40 42 96 >1000

Free Thiol 0.1 0.8 1 40

>3000 >3000 >3000 >3000

0.9 4 4 1500

S NH

O NH OSN

S

NNH

O

O

HS NH

NH OSN

S

NNH

O

O

-Purified Human HDAC’s IC50 values in nM-Probably due to changed conformational space on nitrogen vs. oxygen

Bowers, A. A.; Greshock, T. J.; West, N.; Estiu, G.; Schreiber, S. L.; Wiest, O.; Williams, R. M.; Bradner, J. E. Journal of the American Chemical Society 2009, 131, 2900

IC50 of Human HDACs of Other Analogues

HDAC1 (µM) HDAC2 (µM) HDAC3 (µM) HDAC6 (µM)

Largazole Thiol 0.0012 0.0035 0.0034 0.049

Enantiomer 1.2 3.1 1.9 2.2

C-2 Epimer 0.030 0.082 0.084 0.68

0.11 0.80 0.58 13

>30 >30 >30 >30C7H15 S N

H

O O OSN

S

NN

O

O

NH

O OSN

S

NNH

O

O

CO2H

Bowers, A. A.; West, N.; Newkirk, T. L.; Troutman-Youngman, A. E.; Schreiber, S. L.; Wiest, O.; Bradner, J. E.; Williams, R. M. Organic Letters 2009, 11, 1301

ContinuedHDAC1 (µM) HDAC2 (µM) HDAC3 (µM) HDAC6 (µM)

0.27 4.1 4.1 >30

23 29 14 >30

0.67 1.6 0.96 0.7

1 1.9 1.5 0.24

NH

NH

O OSN

S

NNH

O

O

NH2

NH

O OSN

S

NNH

O

O

HN

H2N

NH

NH

O OSN

S

NNH

O

O

HS

O

NH

O OSN

S

NNH

O

O

HN

HS

O Bow

ers,

A. A

.; W

est,

N.;

New

kirk

, T. L

.; Tr

outm

an-Y

oung

man

, A. E

.; Sc

hrei

ber,

S. L

.; W

iest

, O

.; Br

adne

r, J.

E.; W

illia

ms,

R. M

. Org

anic

Lett

ers

2009

, 11,

130

1

ContinuedHDAC1 (µM) HDAC2 (µM) HDAC3 (µM) HDAC6 (µM)

0.0019 0.0048 0.0038 0.13

0.077 0.12 0.085 >30

0.00032 0.00086 0.0011 0.029

Disulfide Homodimer 0.00069 0.0017 0.0015 0.045

HS NH

O OSN

S

NNH

O

HO

HS NH

O OSN

S

NNH

O

O

HS NH

O O

S

NNH

O

O

N

HS NH

O OON

O

NNH

O

O

Bow

ers,

A. A

.; W

est,

N.;

New

kirk

, T. L

.; Tr

outm

an-Y

oung

man

, A. E

.; Sc

hrei

ber,

S. L

.; W

iest

, O

.; Br

adne

r, J.

E.; W

illia

ms,

R. M

. Org

anic

Lett

ers

2009

, 11,

130

1

• Pyridine substitution of thiazole resulted in increased potency

• Methyl group in C-7 position is not important• Linker analogues did not result in increased

potency• Enantiomer potency decreased by 3 orders of

magnitude

• Also synthesized but not tested

NH

O OSN

S

NNH

O

O

HS

O

NH

O OSN

S

NNH

O

O

HS

O

Bowers, A. A.; West, N.; Newkirk, T. L.; Troutman-Youngman, A. E.; Schreiber, S. L.; Wiest, O.; Bradner, J. E.; Williams, R. M. Organic Letters 2009, 11, 1301

C-7 demethylationHDAC1 HDAC2 HDAC3 HDAC6 HCT-116

Largazole 0.0137 0.190 0.245 11.5 0.025±0.004

2.0 18.5 16.8 14.7 24.1±3.0

NA NA NA NA >100

Macrocycle dimer with one octanoyl linker

NA 51.8 22.6 NA 26.8±3.7

S NH

O O OSN

S

NNH

O

O

S NH

O O OSN

S

NNH

O

O

-Purified Human HDAC’s. IC50 Values in µM-C-7 demethylation decreases potency-C17 stereochemistry is crucial-Macrocycle dimer was surprisingly active in HDAC 2 and 3 but not 1

Chen, F.; Gao, A.-H.; Li, J.; Nan, F.-J. ChemMedChem 2009, 4, 1269

colorectal carcinoma cell lineHCT-116

human lung cancer cell lineA549

human embryonic kidney cell lineHEK293

human embryonic lung fibroblast cell lineHLF

Largazole 0.08 0.32 1.36 0.98

R = i-Pr, 18Z >10 >10 >10 >10

R = i-Bu, 18E 0.56 3.28 8.95 6.12

R = i-Bu, 18Z >10 >10 >10 >10

R = Bn, 18E 0.26 0.77 2.57 1.43

R = Bn, 18Z >10 >10 >10 >10

R = p-OH-Bn, 18E 0.39 1.46 >100 >100

R = p-OH-Bn, 18Z >10 >10 >10 >10

S NH

O O OSN

S

NNH

O

R

O

GI50 values in µM for antiproliferative studies of several cancer cell linesZeng, X.; Yin, B.; Hu, Z.; Liao, C.; Liu, J.; Li, S.; Li, Z.; Nicklaus, M. C.; Zhou, G.; Jiang, S. Organic Letters 2010, 12, 1368

Double Bond Analogues

• Z-isomers cannot chelate Zn2+ ions, making them inactive

• Replacing valine with tyrosine resulted in less potency but better selectivity over normal cells.

Zeng, X.; Yin, B.; Hu, Z.; Liao, C.; Liu, J.; Li, S.; Li, Z.; Nicklaus, M. C.; Zhou, G.; Jiang, S. Organic Letters 2010, 12, 1368

C-7 Variations

IC50 HDAC1 (nM) IC50 HDAC4 (µM)

Largazole 11.4 3.0

X = H 13.7 7.5

X = Et 161.3 4.9

X = Bn 4.5 3.0

4.7 3.0

S NH

O O OSN

S

NNH

O

XO

S NH

O O OSN

S

NNH

O

O

-Purified Recombinant HDAC’s-Size of substituent on C-7 position does not affect activity

Souto, J. A.; Vaz, E.; Lepore, I.; Poppler, A.-C.; Franci, G.; Alvarez, R.; Altucci, L.; de Lera, A. n. R. Journal of Medicinal Chemistry 2010, 53, 4654

HDAC inhibition (nM) MCF-7 inhibition (nM)

Largazole 572±29 5±1

Free Thiol 0.043±0.026 277±130

17.2±2.3 377±62

0.17±0.05 2458±1135

3.15±0.35 >10000

0.99±0.07 5902±1698

HS NH

O OSN

S

NNH

O

O

HS NH

O OSN

S

NNH

O

O

HS NH

O OSN

NH

O

O NH O

HS NH

O OSN

S

NNH

O

O

Ph

Bene

lkeb

ir, H

.; M

arie

, S.;

Hay

den,

A. L

.; Ly

le, J

.; Lo

adm

an, P

. M.;

Crab

b, S

. J.;

Pack

ham

, G

.; G

anes

an, A

. Bio

orga

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&am

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edic

inal

Che

mist

ry 2

011,

19,

365

0.

• Valine, glycine, and thiazoline groups could be replaced with slight decrease in activity

• Cell growth inhibition does not correlate necessarily with HDAC inhibition

• Half life of compounds is a concern• No specification on what HDAC was used

Benelkebir, H.; Marie, S.; Hayden, A. L.; Lyle, J.; Loadman, P. M.; Crabb, S. J.; Packham, G.; Ganesan, A. Bioorganic & Medicinal Chemistry, In Press, Corrected Proof

Amino Acid ReplacementHCT116 (GI50 nM) HDAC1 (IC50 nM)

Valine (Largazole) 10 7.6

Phenylalanine 81 54

Histidine 77 44

Tyrosine 34 23

Aspartic Acid 74 61

N-Me-Gly 2700 Nd

N-Me-Val 10000 Nd

Phe-Dimer 4000 Nd

Liu, Y.; Salvador, L. A.; Byeon, S.; Ying, Y.; Kwan, J. C.; Law, B. K.; Hong, J.; Luesch, H. Journal of Pharmacology and Experimental Therapeutics 2010, 335, 351.

S NH

O OSN

S

NNH

O

O

N

S NH

O OSN

S

NNH

O

O

S

S NH

O OSN

S

NNH

O

O

OH

27 28 29

Bhansali, P.; Hanigan, C. L.; Casero, R. A.; Tillekeratne, L. M. V. Journal of Medicinal Chemistry 2011, 54, 7453.