Largazole

Transformed Mammary Epithelial Cells (MDAMB-231) Nontransformed Mammary Epithelial Cells (NMuMG) Fibroblastic osteosarcoma cells (U2OS) Nontransformed Fibroblast (NIH3T3) Colon Cancer Line (HT29)*

GI50: 7.7nM LC50: 117nM GI50: 122nM LC50: 272nM GI50: 55nM LC50: 94nM GI50: 480nM LC50: >8M GI50: 12nM LC50: 22nM

Neuroblastoma (IMR-32)*

GI50: 16nM LC50: 22M

Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455 *Taori, K.; Paul, V. J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 1806

Thiol Analogues
HCT-116 growth HCT-116 HDAC inhibition (nM) cellular assay (nM) 4410 513 HeLa nuclear extracts HDACs (nM) 3711

385

20915

4229

332

5018

5227

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

Analogues with no Thiol

HCT-116 growth inhibition (nM) > 10000 HCT-116 HDAC cellular assay (nM) > 10000 HeLa nuclear extracts HDACs (nM) > 10000

> 10000

> 10000

> 10000

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

 HCT-116 cell line growth inhibition experiment HDAC assays were performed with cell permeable fluorescent substrate Cytotoxicity for HT-116 cells not reported

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

Importance of Thiol
Need thiol to chelate Zn2+ Analogue is active if hydrolysis to thiol is possible Structural similarity to FK228

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

Importance of Octanoyl Residue

Free thiol is better at inhibiting HDAC Octanoyl moeity increases cell permeability Antiproliferation studies
Largazole (Red)
IC50: 45-315 nM

Free Thiol (Blue)

IC50: 360-2600 nM

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

 Proliferation studies of various malignant human melanoma cell lines Cell viability assay based on ATP content

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

Features of Efficient HDAC inhibitors

Hydrophobic region that binds the rim of the active site a coordinating group (warhead) to chelate Zn2+ at the bottom of the channel a five- to seven-atom linker from the hydrophobic region to the coordinating group

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

Linker Length
n 1 2 3 4 HCT-116 growth inhibition (nM) >10000 6.80.6 62050 2500600 HeLa nuclear extracts HDACs (nM) >20000 3213 7600900 4100430

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 Variation

HCT-116 growth inhibition (nM) Largazole 6.80.6 212 HeLa nuclear extracts HDACs (nM) 3213 7221

3900450

>20000

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

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

MDA-MB231 (nM) Largazole Terminal olefin (no linker) 71 >600 >600

HME (nM) >600 >600 >600

>600

>600

>600

>600

-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 5M.

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

S-acetyl, disulfide analogues

Largazole Free Thiol

60nM

38nM

50nM

70nM

-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 Analogues
HDAC 1 Largazole Free Thiol 40 0.1 >3000 HDAC 2 HDAC 3 42 0.8 >3000 96 1 >3000 HDAC 6 >1000 40 >3000

0.9

1500

-Purified Human HDACs 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 (M) HDAC2 (M) HDAC3 (M) HDAC6 (M) Largazole Thiol Enantiomer C-2 Epimer 0.0012 1.2 0.030 0.11 0.0035 3.1 0.082 0.80 0.0034 1.9 0.084 0.58 0.049 2.2 0.68 13

>30

>30

>30

>30

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

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

1 1.9 1.5 0.24 1.6 0.96 0.7

23

0.67

0.27

HDAC1 (M)

Continued

29 14 >30

4.1 4.1 >30

HDAC2 (M) HDAC3 (M) HDAC6 (M)

HDAC1 (M) 0.0019

HDAC2 (M) HDAC3 (M) HDAC6 (M) 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

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

Continued

 Pyridine substitution of thiazol 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

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 demethylation
HDAC1 HDAC2 HDAC3 HDAC6 Largazole 0.0137 0.190 2.0 18.5 0.245 16.8 11.5 14.7 HCT-116 0.0250.0004 24.13.0

NA

NA

NA

NA

>100

Macrocycle dimer

NA

51.8

22.6

NA

26.83.7

-Purified Human HDACs. IC50 Values in M -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 line HCT-116 Largazole R = i-Pr, 18Z R = i-Bu, 18E R = i-Bu, 18Z R = Bn, 18E R = Bn, 18Z R = p-OH-Bn, 18E R = p-OH-Bn, 18Z 0.08 >10 0.56 >10 0.26 >10 0.39 >10

human lung cancer cell line A549 0.32 >10 3.28 >10 0.77 >10 1.46 >10

human embryonic kidney cell line HEK293 1.36 >10 8.95 >10 2.57 >10 >100 >10

human embryonic lung fibroblast cell line HLF 0.98 >10 6.12 >10 1.43 >10 >100 >10

GI50 values in M for antiproliferative studies of several cancer cell lines

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

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) Largazole X=H X = Et X = Bn 11.4 13.7 161.3 4.5 4.7

IC50 HDAC4 (M) 3.0 7.5 4.9 3.0 3.0

-Purified Recombinant HDACs -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

Largazole 57229

Free Thiol 17.22.3

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

0.990.07 >10000 59021698 24581135

3.150.35

0.170.05

0.0430.026 37762 277130

HDAC inhibition (nM) 51 MCF-7 inhibition (nM)

 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

Recent Analogues