Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon

Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.

Pharmacol. Exp. Ther. (JPET#172387)

SUPPLEMENTAL DATA

Anti-Colon Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase

Inhibitor

Yanxia Liu, Lilibeth A. Salvador, Seongrim Byeon, Yongcheng Ying, Jason C. Kwan,
Brian K. Law, Jiyong Hong, and Hendrik Luesch

Department of Medicinal Chemistry, University of Florida, 1600 SW Archer Road, Gainesville,

Florida 32610 (Y.L., L.A.S., J.C.K., H.L.), Department of Chemistry, Duke University, 124
Science Drive, Durham, North Carolina 27708 (S.B., Y.Y., J.H.), Department of Pharmacology
& Therapeutics, University of Florida, 1600 SW Archer Road, Gainesville, Florida 32610
(B.K.L.)

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)

Synthesis of N-methylated analogues of largazole. The N-methylated analogues of largazole

were synthesized as shown in Scheme S1.

Scheme S1. Synthesis of the N-methylated analogues of largazole. Reagents and conditions: (a)
NaH, MeI, DMF, 25 C, 48 h; (b) TFA, CH2Cl2, 25 C, 1 h; (c) 3, DMAP, CH2Cl2, 25 C, 1 h,
93% for three steps (4b); (d) 2,4,6-trichlorobenzoyl chloride, Et3N, THF, 0 C, 1 h; then N-BocN-methyl-L-valine (for 5a), N-Boc-L-valine (for 5b), DMAP, 25 C, 10 h, 95% (5a), 99% (5b);
(e) 0.5 N LiOH, THF, H2O, 0 C, 3 h; (f) TFA, CH2Cl2, 25 C, 2 h; (g) HATU, HOAt, i-Pr2NEt,
CH2Cl2, 25 C, 32 h, 35% for three steps (8a), 53% for three steps (8b); (h) 9, Grubbs' secondgeneration catalyst (50 mol%), toluene, reflux, 4 h, 35% (10a), 49% (10b).

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Compound 10a. 1H NMR (400 MHz, CDCl3) 7.63 (s, 1H), 6.70 (brs, 1H), 6.29 (d, J = 6.8
Hz, 1H), 5.82 (dt, J = 15.2, 6.8 Hz, 1H), 5.69 (t, J = 9.4 Hz, 1H), 5.47 (dd, J = 15.2, 8.4 Hz, 1H),
4.97 (dd, J = 16.8, 7.2 Hz, 1H), 4.37 (dd, J = 18.8, 4.8 Hz, 1H), 4.34 (d, J = 11.2 Hz, 1H), 3.38
(d, J = 11.6 Hz, 1H), 2.95 (s, 3H), 2.88 (t, J = 6.8 Hz, 2H), 2.71 (dd, J = 14.8, 10.4 Hz, 1H),
2.55-2.50 (m, 3H), 2.42-2.34 (m, 1H), 2.29 (q, J = 7.2 Hz, 2H), 1.70 (s, 3H), 1.66-1.61 (m, 2H),
1.29-1.25 (m, 8H), 1.25 (d, J = 6.8 Hz, 3H), 1.10 (d, J = 7.2 Hz, 3H), 0.88 (t, J = 7.2 Hz, 2H);
HRMS (FAB) found 636.2465 [calcd for C30H44N4O5S3 (M)+ 636.2474].
Compound 10b. 1H NMR (400 MHz, CDCl3) 7.74 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 6.01
(dd, J = 10.8, 6.8 Hz, 1H), 5.85 (dt, J = 15.6, 6.8 Hz, 1H), 5.60 (dd, J = 15.6, 7.2 Hz, 1H), 5.23
(d, J = 16.0 Hz, 1H), 4.46 (dd, J = 8.0, 3.6 Hz, 1H), 4.20 (d, J = 16.0 Hz, 1H), 4.05 (d, J = 11.2
Hz, 1H), 3.32 (d, J = 11.6 Hz, 1H), 2.94-2.88 (m, 2H), 2.86 (s, 3H), 2.56-2.45 (m, 2H), 2.52 (t, J
= 7.6 Hz, 2H), 2.31 (dt, J = 14.0, 7.2 Hz, 2H), 2.11-2.07 (m, 1H), 1.85 (s, 3H), 1.68-1.63 (m,
2H), 1.32-1.26 (m, 8H), 0.88 (t, J = 7.2 Hz, 3H), 0.70 (d, J = 6.8 Hz, 3H), 0.66 (d, J = 6.8 Hz,
3H); HRMS (FAB) found 636.2469 [calcd for C30H44N4O5S3 (M)+ 636.2474].

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Synthesis of Phe, His, Asp, and Tyr analogues of largazole. The Phe, His, Asp, and Tyr
analogues of largazole were synthesized as shown in Scheme S2.

Scheme S2. Synthesis of the Phe, His, Asp, and Tyr analogues of largazole. Reagents and
conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et3N, THF, 0 C, 1 h; then Boc-L-Phe-OH (for
11a), N-Boc-L-His-OH (for 11b), Fmoc-Asp(OtBu)-OH (for 11c), Fmoc-Try(tBu)-OH (for
11d), DMAP, 25 C, 10 h, 100% (11a), 62% (11b), 100% (11c), 96% (11d); (b) 0.25 N LiOH,
THF, H2O, 0 C, 3 h; (c) For 13a and 13b: TFA, CH2Cl2, 25 C, 2 h; For 13c and 13d: Et2NH,
CH3CN, 25 C, 2 h; (d) HATU, HOAt, i-Pr2NEt, CH2Cl2, 25 C, 32 h, 30% for three steps (14a),
12% for three steps (14b), 18% for three steps (14c), 18% for three steps (14d); (e) (Boc)2O,
Et3N, DMAP, 50 C, 1 h, 41%; (f) 9, Grubbs second-generation catalyst (50 mol%), toluene,
reflux, 4 h, 59% (15a), 27% (16b), 34% (15c), 47% (15d); (g) TFA, CH2Cl2, 25 C, 14 h, 39%
(17b), 41% (16c), 100% (16d).
4

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Compound 15a. 1H NMR (400 MHz, CDCl3) 7.65 (s, 1H), 6.88 (d, J = 6.8 Hz, 2H), 6.786.62 (m, 3H), 6.01(brs, 1H), 5.81 (dt, J = 14.4, 6.8 Hz, 1H), 5.72 (m, 1H), 5.53 (dd, J = 15.6, 6.8
Hz, 1H), 4.90 (m, 1H), 4.80 (dd, J = 17.6, 6.4 Hz, 1H), 4.37 (d, J = 16.8 Hz, 1H), 4.13 (d, J =
11.6 Hz, 1H), 3.26 (d, J = 11.6 Hz, 1H), 3.20 (dd, J = 13.6, 2.8 Hz, 1H), 3.06, (dd, J = 13.6, 5.2
Hz, 1H), 2.91-2.81 (m, 2H), 2.66-2.52 (m, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.27 (q, J = 6.8 Hz, 2H),
1.83 (s, 3H), 1.63 (m, 2H), 1.28-1.23 (m, 8H), 0.87 (t, J = 6.8 Hz, 3H); HRMS (FAB) found
670.2316 [calcd for C33H42N4O5S3 (M)+ 670.2317].
Phe-dimer. 1H NMR (400 MHz, CDCl3) 7.81 (s, 2H), 7.11-6.89 (m, 10H), 5.88-5.68 (m,
1H), 5.59-5.55 (m, 3H), 5.19 (d, J = 10.4 Hz, 1H), 5.16 (d, J = 17.2 Hz, 2H), 4.93 (dd, J = 15.2,
6.4 Hz, 2H), 4.59 (dd, J = 15.6, 4.4 Hz, 2H), 4.19 (q, J = 5.6 Hz, 2H), 3.62 (d, J = 11.6 Hz, 2H),
3.14 (d, J = 11.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H), 2.87-2.82 (m, 4H), 2.69-2.56 (m, 4H), 2.52 (t,
J = 7.2 Hz, 2H), 2.32-2.24 (m, 2H), 1.62-1.56 (m, 8H), 1.32-1.22 (m, 8H), 0.88 (t, J = 6.8 Hz,
3H); MS (ESI) found 1177.4 [calcd for C56H66N8O9S5Na (M+Na)+ 1177.36].
Compound 17b. 1H NMR (400 MHz, CD3OD) 8.01 (s, 1H), 7.72 (s, 2H), 7.54 (brs, 1H),
7.17 (brs, 1H), 5.81 (dt, J = 15.2, 7.6 Hz, 1H), 5.76-5.71 (m, 1H), 5.62 (dd, J = 15.2, 6.8 Hz,
1H), 5.16 (d, J = 17.6 Hz, 1H), 4.96 (m, 1H), 4.39 (d, J = 17.6 Hz, 1H), 3.75 (d, J = 11.6 Hz,
1H), 3.48 (m, 1H), 3.14 (m, 1H), 3.04 (dd, J = 16.8, 10.8 Hz, 1H), 2.93 (m, 2H), 2.72 (dd, J =
16.8, 2.0 Hz, 1H), 2.55 (t, J = 7.6 Hz, 2H), 2.31 (dt, J = 12.0, 6.8, 2H), 1.76 (s, 3H), 1.68-1.59
(m, 2H), 1.32-1.29 (m, 8H), 0.90 (t, J = 7.2 Hz, 3H); HRMS (FAB) found 661.2305 [calcd for
C30H41N6O5S3 (M+H)+ 661.2300].

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Compound 16c. 1H NMR (500 MHz, CD3OD) 8.03 (s, 1H), 7.69 (d, J = 6.0 Hz, 1H), 5.80
(dt, J = 15.0, 6.5 Hz, 1H), 5.63-5.59 (m, 2H), 4.98 (dd, J = 17.5, 7.0 Hz, 1H), 4.59 (m, 1H), 4.48
(d, J = 17.5 Hz, 1H), 3.85 (d, J = 11.5 Hz, 1H), 3.41 (d, J = 11.0 Hz, 1H), 2.92 (ddd, J = 7.5, 7.5,
4.0 Hz, 2H), 2.85-2.67 (m, 2H), 2.55 (t, J = 7.5 Hz, 2H), 2.31 (q, J = 6.5 Hz, 2H), 1.77 (s, 3H),
1.64 (t, J = 7.0 Hz, 2H), 1.32-1.29 (m, 8H), 0.91 (t, J = 7.0 Hz, 3H); HRMS (ESI) found
639.2105 [calcd for C28H39N4O7S3 (M+H)+ 639.1981].
Compound 16d. 1H NMR (500 MHz, CDCl3) 7.71 (s, 1H), 7.30 (d, J = 6.0 Hz, 1H), 6.71
(d, J = 8.0 Hz, 2H), 6.26 (brs, 1H), 6.23 (d, J = 8.5 Hz, 2H), 5.84 (dt, J = 14.5, 7.0 Hz, 1H), 5.70
(m, 1H), 5.55 (dd, J = 15.5, 6.5 Hz, 1H), 4.94-4.89 (m, 2H), 4.37 (dd, J = 17.5, 3.5 Hz, 1H), 4.15
(d, J = 11.5 Hz, 1H), 3.28 (d, J = 11.5 Hz, 1H), 3.14 (dd, J = 13.5, 2.0 Hz, 1H), 2.98 (dd, J =
14.5, 5.5 Hz, 1H), 2.92-2.81 (m, 1H), 2.67 (m, 2H), 2.50 (t, J = 7.0 Hz, 1H), 2.29 (q, J = 7.0 Hz,
1H), 1.84 (s, 3H), 1.61 (m, 2H), 1.30-1.26 (m, 8H), 0.88 (t, J = 7.5 Hz, 3H); HRMS (FAB) found
687.2496 [calcd for C33H43N4O6S3 (M+H)+ 687.2344].

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)

Supplementary Tables
Supplementary Table S1. Relative mRNA expression of selected genes in HCT116 cells upon
largazole (20 nM) treatment (10 h)
Category
Cell cycle

Growth factor receptor signaling

Apoptosis
a

Symbol
CDKN1A (p21)
CDKN1C (p57)
CDKN2B (p19)
CDKN2D (p15)
CDK6
CCND1
EGFR
ERBB2 (HER-2)
ERBB3
HGFR (MET)
IRS-1
BCL2L11

Fold changea
+1.82 (+2.45b)
+3.19, +2.99, +2.78, +2.73, +2.54
+3.71
+3.26
2.05
2.01
2.54
2.28
2.02, 2.00
2.66, 2.53
7.90, 5.41, 4.36
+3.60, +3.10, +3.07, +2.38

For some transcripts there were several probes. b Value from RT-qPCR.

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Supplementary Table S2. Genes regulated 2-fold by largazole but to a lesser extent by SAHA
and FK228 in HCT116 cells (see Supplementary Fig. S2B)

Symbol

Gene ID

CD24 molecule
collagen, type I, alpha 1
vesicle-associated membrane protein 1
(synaptobrevin 1)
histone cluster 2, H2be
HIST2H2BE
homolog of rat pragma of Rnd2
PRAGMIN
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide
DDX26B
26B
methionine sulfoxide reductase B3
MSRB3
protein phosphatase 4, regulatory subunit 4
PPP4R4
heat shock 70kDa protein 1A
HSPA1A
LOC100288387 similar to c-jun
transducin-like enhancer of split 3 (E(sp1) homolog,
TLE3
Drosophila)

CD24
COL1A1
VAMP1

Relative mRNA expression

(fold change)
Largazole SAHA FK228
2.54
1.42
1.47
2.42
1.65
1.52
2.26
1.55
1.68
2.15
2.14
2.12

1.73
1.66
1.77

1.73
1.34
1.66

2.06
2.05
2.03
2.03
2.03

1.67
1.80
0.63
1.69
1.76

1.62
1.62
1.58
1.64
1.49

ZNF18
EGLN3
SLC46A3
MPP7

zinc finger protein 18

egl nine homolog 3 (C. elegans)
solute carrier family 46, member 3
membrane protein, palmitoylated 7 (MAGUK p55
subfamily member 7)

2.03
2.01
2.01
2.02

1.78
1.63
1.73
1.60

1.77
1.44
1.58
1.62

IPP
C8orf58 ///
PDLIM2

intracisternal A particle-promoted polypeptide

chromosome 8 open reading frame 58 ///
PDZ and LIM domain 2 (mystique)

2.03
2.07

1.65
1.57

1.75
1.39

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)
Supplementary Table S3. Microsomal stability of largazole and largazole thiol adducta

Time (min)
0
5
15
30
Time (min)
0
5
15
30

Largazole
Microsomes only
Denatured microsomes +
NADPH
100
100
0.13 0.05
90 17
0.07 0.02
76 21
0.03 0.00
69 14
Largazole thiol adduct
Microsomes only
Denatured microsomes +
NADPH
0.00
Not detected
b
100
Not detected
124 23
Not detected
97 5
Not detected

Microsomes +
NADPH
100
0.03 0.01
0.04 0.01
0.04 0.01
Microsomes +
NADPH
100
19 3
2.8 1.2
0.96 0.27

Assays were done in triplicate. Values are expressed as % remaining. Mean values are shown S.D. b Defined as
maximum amount of thiol adduct formed from largazole.
a

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)

Supplementary Figures

Supplementary Fig. S1. Molecular Docking with selected largazole thiol analogues. The Phe
and Tyr analogue of largazole thiol are docked into a homology model of HDAC1 and an
overlay of both structures is shown. The Phe analogue of largazole thiol is able to bind in a
similar conformation as the Tyr analogue (see Fig. 3H), but is unable to form a hydrogen bond
with Glu 98.

10

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)

Supplementary Fig. S2. Transcriptional analysis of HCT116 cells treated with largazole, SAHA
or FK228. (A)

Time point optimization for transcriptomic analysis based on p21 marker

expression. Cells were treated for various time periods with drug concentrations that effectively
inhibit cellular HDACs responsible for histone H3 hyperacetylation (see Fig. 4A). RNA was
isolated and subjected to TaqMan-based RT-qPCR analysis for p21. Highest levels of p21
transcript were induced after 10 h of treatment by all three HDAC inhibitors. (B) The Venn
diagram depicts overlapping and non-overlapping genes that are modulated (directly or
indirectly) by these HDAC inhibitors (20 nM largazole, 20 nM FK228, 2 M SAHA) upon 10 h
of treatment ( 2-fold change, p < 0.01).

11

Liu Y, Salvador LA, Byeon S, Ying Y, Kwan JC, Law BK, Hong J, Luesch H (2010) Anti-Colon
Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor. J.
Pharmacol. Exp. Ther. (JPET#172387)

Supplementary Fig. S3. Stability of largazole in aqueous solution. Largazole was added to cell
growth medium or phosphate buffer at pH 4.0, 7.0, 8.0. Largazole was stable over a period of 24
h and no significant hydrolysis was observed. Largazole thiol was not detected by LC-MS. Error
bars indicate S.D. from duplicate experiments.

12