New Potent Bisubstrate Inhibitors of Histone Acetyltransferase p300: Design, Synthesis and Biological Evaluation

Article abstract of DOI:10.1111/j.1747-0285.2010.01056.x

Bisubstrate-type compound Lys-CoA has been shown to inhibit the p300 histone acetyl transferase activity efficiently and may constitute a lead compound for a novel class of anticancer therapeutics. Based on this strategy, we synthesized a series of CoA derivatives and evaluated these molecules for their activity as p300 histone acetyltransferases inhibitor. The best activity was obtained with compound 3 bearing a C-5 spacing linker that connects the CoA moiety to a tert-butyloxycarbonyl (Boc) group. Based on docking simulations, this inhibitor exhibits favorable interactions with two binding areas, namely pockets P1 and P2, within the active site.

Full text of DOI:10.1111/j.1747-0285.2010.01056.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01056.x
Chem Biol Drug Des 2011; 77: 86–92
Research Letter
New Potent Bisubstrate Inhibitors of Histone
Acetyltransferase p300: Design, Synthesis and
Biological Evaluation
Franciane Ho A Kwie1,2, Martine Briet2,3
David Soupaya1,2, Pascal Hoffmann1,2
,
regulation of gene expression, cell growth, or differentiation (2,3).
Among these modifications, the functional implications of reversible
acetylation ⁄ deacetylation processes have been the most widely
studied. These chemical modifications are ensured by the coordi-
nated actions of two superfamilies of enzymes, i.e. histone acetyl-
transferases (HATs) and histone deacetylases (HDACs), which
catalyze the addition and the removal, respectively, of acetyl units
at the e-amino group of lysine residues in the lysine N-terminal
tails (4–6). Nuclear HATs can be grouped into four families based
on sequence homology: the GNAT (PCAF, GCN5), the MYST (Esa1,
Tip 60), the p300 ⁄ CBP, and the rtt109 families (7,8). Dysfunction of
p300 has been implicated in diseases such as inflammatory pro-
cesses, Huntington's disease, cardiovascular diseases, diabetes
mellitus, AIDS, and some cancers (8–10). These observations lead
to consideration of modulators (activators or inhibitors) of p300 as
potential next generation therapeutics. Moreover, such compounds
would also be useful for probing the functional significance of the
histone acetyltransferase activity in vivo. To date, a limited number
of HAT inhibitors have been described which can be divided into
three main groups: (i) naturally occurring compounds, such as curcu-
min, inhibitor of p300 (11,12), garcinol and anacardic acid for p300
and PCAF (13–15) (ii) small synthetic molecules such as isothiazol-
ones, which inhibit both p300 and PCAF (16,17) or more recently
compound C646 which is a selective inhibitor of p300 (18,19) (iii)
compounds structurally related to CoA ('bisubstrate inhibitors')
based on a mechanism approach. These include LysCoA for
p300 ⁄ CBP, histone H3 peptide-CoA analog for PCAF ⁄ GCN5 (20,21),
and H4K16-CoA for Tip60 and Esa1 (22). In this study, we report
the design and the preparation of a series of S-substituted coen-
zyme A derivatives based on the recently published crystal structure
of a semisynthetic heterodimeric p300 HAT domain in complex with
Lys-CoA (23). The authors reveal the presence of an additional elec-
tron rich pocket (P2) at about 10 ꢀ away from the binding pocket
required to accommodate the lysine moiety (P1), the two pockets
being connected by an electronegative groove (G) (Figure 1). Based
on these observations, we designed and synthesized a series of
bisubstrate-type compounds to explore the structural basis of P2
site and to take advantage of that pocket for the development of
inhibitors with improved activity. The new bisubstrate compounds
consist of (i) an S-substituted coenzyme A, (ii) a flexible linker and
(iii) a 'P2-exploring' group of lipophilic or hydrophilic nature
('capping group') (24). More conformationally constrained compounds
bearing either a piperazine or benzyl group were also prepared. The
inhibitory activities of all compounds (compounds 1–13) were
evaluated on p300 HAT.
,
1,2
Marie Maturano1,2, Frederic Rodriguez
,
´
´
Casimir Blonski1,2, Christian Lherbet1,2 and
1,2,
´
Cecile Baudoin-Dehoux
*
1CNRS, Laboratoire de Synthꢀse et Physico-Chimie de Molꢁcules
d'IntꢁrÞt Biologique, SPCMIB, UMR-5068, 118 Route de Narbonne,
F-31062 Toulouse Cedex 9, France
2Universitꢁ de Toulouse, UPS, Laboratoire de Synthꢀse et Physico-
Chimie de Molꢁcules d'IntꢁrÞt Biologique, SPCMIB, 118 route de
Narbonne, F-31062 Toulouse Cedex 9, France
3CNRS, Laboratoire de Biologie Cellulaire et Molꢁculaire du
Contrꢂle de la Prolifꢁration (LBCMCP), UMR 508, 118 Route de
Narbonne, F-31062 Toulouse Cedex 9, France
*Corresponding author: Cꢁcile Baudoin-Dehoux,
baudoin@chimie.ups-tlse.fr
Bisubstrate-type compound Lys-CoA has been
shown to inhibit the p300 histone acetyl transfer-
ase activity efficiently and may constitute a lead
compound for a novel class of anticancer thera-
peutics. Based on this strategy, we synthesized a
series of CoA derivatives and evaluated these
molecules for their activity as p300 histone acety-
ltransferases inhibitor. The best activity was
obtained with compound 3 bearing a C-5 spacing
linker that connects the CoA moiety to a tert-
butyloxycarbonyl (Boc) group. Based on docking
simulations, this inhibitor exhibits favorable inter-
actions with two binding areas, namely pockets
P1 and P2, within the active site.
Key words: bisubstrate inhibitor, chemical biology, drug design,
HAT, p300
Received 26 July 2010, revised 29 September 2010 and accepted for
publication 14 October 2010
The structural unit of chromatin, the nucleosome, encompasses 147
base pairs of DNA wrapped around an octamer of core histone
proteins in 1.7 turns (1). Histones are subjected to extensive post-
translational covalent modifications of their tail regions including
acetylation, methylation, phosphorylation or ubiquitinylation,
sumoylation, and ⁄ or ADP ribosylation, which play a key role in the
86

New Bisubstrate Inhibitors of p300
solution (3.7 mL). After an overnight stirring, the aqueous phase
was washed with CH2Cl2 (5 · 20 mL) and then lyophilized. The
desired product was purified by reserved-phase HPLC (TFA
0.05% ⁄ CH3CN 95 ⁄ 5).
A
N-[2-(S-coenzyme A)acetyl-N-tert-butyloxycarbonyl]
1
propanamide (1). Yield: 83%; H NMR (D2O, 300 MHz) d 0.75
(s, 3H); 0.88 (s, 3H); 1.41 (s, 9H); 1.68 (quint, J = 6.8 Hz, 2H); 2.46
(t, J = 6.5 Hz, 2H); 2.69 (t, J = 6.6 Hz, 2H); 3.08 (t, J = 6.7 Hz, 2H);
3.23 (t, J = 6.9 Hz, 2H); 3.27 (s, 2H); 3.36 (t, J = 6.6 Hz, 2H); 3.47
(t, J = 6.5 Hz, 2H); 3.55 (dd, J = 4.8 Hz, J = 9.8 Hz, 1H); 3.83 (dd,
J = 4.9 Hz, J = 9.7 Hz, 1H); 4.02 (s, 1H); 4.25 (m, 2H); 4.59 (m, 1H);
4.84 (m, 2H); 6.17 (d, J = 6.7 Hz, 1H); 8.24 (s, 1H); 8.55 (s, 1H); 13C
NMR (D2O, 100 MHz) d 175.2; 174.4; 173.0; 158.7; 156.1; 153.4;
149.9; 140.7; 119.1; 87.0; 84.4; 74.7; 74.6; 74.5; 72.4; 66.2; 38.9;
38.8; 38.1; 36.0; 35.9; 35.4; 31.8; 29.0; 28.3; 21.4; 18.7; LRMS: (ESI,
m ⁄ z) calc. for C31H54N9O19P3S: 981.2. Found: 489.9 (M-2H)2)
.
B
Linker
Capping
CoA
S
N-[2-(S-coenzyme A)acetyl-N-tert-butyloxycarbonyl]
1
O
butanamide (2). Yield: 89%; H NMR (D2O, 300 MHz) d 0.76 (s,
3H); 0.89 (s, 3H); 1.30 (m, 2H); 1.42 (s, 9H); 1.51 (m, 4H); 2.47 (t,
J = 6.5 Hz, 2H); 2.69 (t, J = 6.7 Hz, 2H); 3.04 (t, J = 6.7 Hz, 2H);
3.20 (t, J = 6.8 Hz, 2H); 3.26 (s, 2H); 3.37 (t, J = 6.6 Hz, 2H); 3.48
(t, J = 6.6 Hz, 2H); 3.56 (dd, J = 4.7 Hz, J = 9.9 Hz, 1H); 3.84 (dd,
J = 4.8 Hz, J = 9.6 Hz, 1H); 4.03 (s, 2H); 4.25 (m, 2H); 4.60 (m, 1H);
4.85 (m, 2H); 6.18 (d, J = 6.7 Hz, 1H); 8.26 (s, 1H); 8.56 (s, 1H); 13C
NMR (D2O, 100 MHz) d 175.2; 174.4; 172.8; 156.1; 153.4; 149.9;
119.1; 87.0; 84.3; 74.7; 74.6; 74.4; 72.4; 66.2; 40.4; 40.1; 38.9; 36.0;
35.9; 35.4.; 31.7; 29.1; 28.5; 28.3; 23.8; 21.4; 18.6; LRMS: (ESI, m ⁄ z)
calc. for C33H58N9O19P3S: 1009.3. Found: 1008.5 (M-H)).
Figure 1: Substrate-binding surface and design of bisubstrate
inhibitors (A) View of the putative substrate-binding site of the
p300 histone acetyltransferase domain according to reference (23).
The putative binding site is shown as an electrostatic surface.
Pocket 1, pocket 2, and the connecting groove are annotated as P1,
P2, and G, respectively (B) Design of new bisubstrate inhibitors.
Bisubstrate inhibitors are composed of a CoASH moiety, a carb-
oxymethylene bridge, a linker with or without flexibility and a cap-
ping. These features are shown in relation to a schematic view of
p300 histone acetyltransferase binding surface to illustrate how
these different components may contribute to molecular recognition.
N-[2-(S-coenzyme A)acetyl-N-tert-butyloxycarbonyl]
1
butanamide (3). Yield: 77%; H NMR (D2O, 300 MHz) d 0.76 (s,
3H); 0.89 (s, 3H); 1.42 (s, 9H); 1.51 (m, 4H); 2.48 (t, J = 6.5 Hz, 2H);
2.70 (t, J = 6.6 Hz, 2H); 3.07 (t, J = 6.6 Hz, 2H); 3.22 (t, J = 6.2 Hz,
2H); 3.28 (s, 2H); 3.37 (t, J = 6.6 Hz, 2H); 3.49 (t, J = 6.6 Hz, 2H);
3.56 (dd, J = 4.9 Hz, J = 9.8 Hz, 1H); 3.84 (dd, J = 4.9 Hz,
J = 9.8 Hz, 1H); 4.03 (s, 1H); 4.26 (m, 2H); 4.60 (m, 1H); 4.83 (m,
2H); 6.19 (d, J = 6.9 Hz, 2H); 8.27 (s, 1H); 8.57 (s,1H); 13C NMR
(D2O, 100 MHz) d 175.2; 174.4; 172.8; 156.1; 153.4; 149.9; 140.4;
119.1; 87.0; 84.5; 74.8; 74.6; 74.2; 72.3; 66.3; 40.1; 39.9; 38.8; 36.0;
35.9; 35.3; 31.7; 28.2; 27.0; 26.2; 21.4; 18.7; LRMS: (ESI, m ⁄ z) calc.
Methods and Materials
General
All chemicals were purchased from Aldrich or Sigma. All the reac-
tions were carried out over argon atmosphere. All the solvents
were distilled prior to use. Analytical thin layer chromatography
was performed on MERCK F254 plates.1H NMR and 13C spectra
were recorded on a Brꢁker Avance (300 MHz and 100 MHz, respec-
tively). Mass spectra were obtained from the Paul Sabatier Univer-
sity Mass spectrometry service. The chemical ionization (DCI ⁄ NH3)
mass spectra were recorded on TSQ 7000 thermoelectron spectrom-
eters and the electrospray mass spectra on API 365 Perkin-Elmer
Sciex or Applied biosystems spectrometers. Analytical and prepara-
tive HPLC were carried out on reverse phase Hyperprep (Hypersyl)
C18 columns using linear gradients of H2O ⁄ 0.05% TFA versus ace-
tonitrile. LysCoA was synthesized as described in reference (25).
for C32H56N9O19P3S: 995.3. Found: 497.0 (M-2H)2)
.
N-[2-(S-coenzyme A)acetyl-N-benzoyl]propanamide (4).
Yield: 74%; 1H NMR (D2O, 300 MHz) d 0.72 (s, 3H); 0.86 (s, 3H); 1.83
(qt, J = 6.8 Hz, 2H); 2.42 (t, J = 6.6 Hz, 2H); 2.66 (t, J = 6.7 Hz, 2H);
3.25 (s, 2H); 3.31 (m, 4H); 3.41 (m, 4H); 3.53 (dd, J = 4.8 Hz,
J = 9.8 Hz, 1H); 3.82 (dd, J = 4.8 Hz, J = 9.7 Hz, 1H); 4.00 (s, 1H);
4.24 (m, 2H); 4.58 (m, 1H); 4.83 (m, 2H); 6.13 (d, J = 6.5 Hz, 1H); 7.48
(m, 3H); 7.68 (m, 2H). 8.18 (s, 1H); 8.51 (s,1H); 13C NMR (D2O,
100 MHz) d 174.6; 173.8; 172.4; 170.5; 155.4; 152.7; 149.1; 139.5;
133.3; 132.0; 128.6; 126.8; 118.5; 86.4; 83.7; 74.1; 73.9; 73.8; 71.9;
65.5; 38.3; 37.4; 37.3; 35.4; 35.3; 34.9; 31.2; 28.0; 20.9; 18.1; LRMS
(ESI, m ⁄ z) calc. for C33H50N9O18P3S: 985.2. Found: 984.3 (M-H)).
General procedure for coupling Coenzyme A to
the carboxamide derivatives
To a solution of carboxamide compounds (compounds 15a–15i) in
THF (1 mL), was added a solution of CoASH in a pH = 8 aqueous
N-[2-(S-coenzyme A)acetyl-N-benzoyl]butanamide
1
(5). Yield: 83%; H NMR (D2O, 300 MHz) d 0.73 (s, 3H); 0.87 (s,
Chem Biol Drug Des 2011; 77: 86–92
87

Kwie et al.
3H); 1.62 (m, 2H); 2.40 (t, J = 6.6 Hz, 2H); 2.64 (t, J = 6.7 Hz, 2H);
3.23 (s, 2H); 3.32 (m, 4H); 3.41 (m, 4H); 3.54 (dd, J = 4.4 Hz,
J = 9.6 Hz, 1H); 3.82 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H); 4.01 (s, 1H);
4.25 (m, 2H); 4.58 (m, 1H); 4.83 (m, 2H); 6.15 (d, J = 6.8 Hz, 1H);
7.51 (m, 3H); 7.68 (m, 2H); 8.21 (s, 1H); 8.54 (s, 1H); 13C NMR (D2O,
100 MHz) d 174.6; 173.8; 172.4; 170.5; 155.5; 152.8; 149.2; 139.6;
133.5; 131.9; 128.6; 126.8; 118.5; 86.5; 84.0; 74.3; 74.0; 73.7; 71.8;
65.8; 39.5; 39.3; 38.3; 35.4; 35.3; 34.8; 31.2; 26.0; 25.9; 20.9; 18.0.
LRMS (ESI, m ⁄ z) calc. for C34H52N9O18P3S: 999.2. Found: 998.5
(M-H)).
Procedure for Boc removal
The protected compound was dissolved in a solution of TFA in CH2Cl2
(v ⁄ v 50 ⁄ 50). After stirring for 10 min, TFA was removed under
vacuum. Then, the residue was dissolved in H2O (10 mL) to be
lyophilized. Purification by reverse-phase HPLC (TFA 0.05% ⁄ CH3CN
95 ⁄ 5) and subsequent lyophilization afforded the desired product.
N-[2-(S-coenzyme A)acetyl]propane-1,3-diamine (7).
1
Yield: 38%; H NMR (D2O, 300 MHz) d 0.86 (s, 3H); 0.97 (s, 3H);
1.91 (m, 2H); 2.49 (t, J = 6.4 Hz, 2H); 2.71 (t, J = 6.8 Hz, 2H); 3.03
(t, J = 7.7 Hz, 2H); 3.31 (s, 2H); 3.37 (m, 4H); 3.51 (t, J = 6.3Hz,
2H); 3.65 (dd, J = 4.4Hz, J = 10.6 Hz, 1H); 3.89 (dd, J = 5.0 Hz,
J = 9.8 Hz, 1H); 4.05 (s, 1H); 4.30 (m, 2H); 4.63 (m, 1H); 4.90 (m,
2H); 6.24 (d, 1H, J = 6.7 Hz); 8.46 (s, 1H); 8.68 (s,1H); 13C NMR
(D2O, 100 MHz) d 174.8; 174.1; 173.1; 150.0; 145.4; 142.8; 118.6;
87.8; 83.4; 74.4; 74.1; 74.0; 72.3; 65.3; 38.5; 37.2; 36.7; 35.6; 35.5;
35.0; 31.4; 26.8; 21.0; 18.6; LRMS (ESI, m ⁄ z) calc. for
C26H46N9O17P3S: 881.2. Found: 880.4 (M-H)).
N-[2-(S-coenzyme A)acetyl-N-benzoyl]pentanamide
1
(6). Yield: 90%; H NMR (D2O, 300 MHz) d 0.75 (s, 3H); 0.89 (s,
3H); 1.35 (m, 2H); 1.56 (m, 4H); 2.43 (t, J = 6.5 Hz, 2H); 2.58 (t,
J = 6.6 Hz, 2H); 3.17-3.28 (m, 6H); 3.26 (t, J = 6.7 Hz, 2H); 3.26 (t,
J = 6.8 Hz, 2H); 3.57 (dd, J = 4.4 Hz, J = 9.4 Hz, 1H); 3.85 (dd,
J = 4.9 Hz, J = 9.7 Hz, 1H); 4.03 (s, 1H); 4.26 (m, 2H); 4.60 (m,
1H); 4.85 (m, 2H); 6.14 (d, J = 6.2 Hz, 1H); 7.49 (m, 3H); 7.68 (m,
2H); 8.18 (s, 1H); 8.52 (s, 1H); 13C NMR (D2O, 100 MHz) d 174.7;
173.7; 172.2; 170.4; 155.3; 152.6; 149.1; 139.6; 133.5; 131.9;
128.6; 126.8; 118.5; 86.4; 83.5; 74.1; 74.0; 73.9; 71.9; 65.5; 39.7;
39.5; 38.2; 35.4; 35.3; 34.8; 31.1; 28.0; 27.9; 23.4; 20.9; 18.1;
LRMS (ESI, m ⁄ z) calc. for C35H54N9O18P3S: 1013.3 Found: 1012.4
(M-H)).
N-[2-(S-coenzyme A)acetyl]propane-1,3-propanamine
1
(8). Yield: 41%; H NMR (D2O, 300 MHz) d 0.84 (s, 3H); 0.95 (s, 3H);
1.63 (m, 4H); 2.48 (t, J = 6.3 Hz, 2H); 2.68 (t, J = 6.6 Hz, 2H); 3.01 (t,
J = 7.2 Hz, 2H); 3.24 (t, J = 6.7 Hz, 2H); 3.27 (s, 2H); 3.36 (t,
J = 6.6 Hz, 2H); 3.49 (t, J = 6.3 Hz, 2H); 3.64 (m, 1H); 3.90 (m, 1H); 4.03
(s, 1H); 4.29 (m, 2H); 4.61 (m, 1H); 4.88 (m, 2H); 6.21 (d, J = 5.3 Hz, 1H);
8.44 (s, 1H); 8.67 (s,1H); 13C NMR (D2O, 100 MHz) d 174.9; 174.2;
172.8; 150.1; 144.8; 143.0; 87.7; 83.3; 74.52; 74.5; 74.4; 74.2; 72.2;
65.3; 39.3; 39.2; 38.6; 35.7; 35.6; 35.1; 31.4; 25.6; 24.4; 21.1; 18.6;
LRMS (ESI, m ⁄ z) calc. for C27H48N9O17P3S: 895.2. Found: 894.5 (M-H)).
N-[2-(S-Coenzyme A)acetyl]-N-(tert-butyl)piperazine-
1-carboxylate (11). Yield: 98%; 1H NMR (D2O, 300 MHz) d
0.76 (s, 3H); 0.90 (s, 3H); 1.46 (s, 9H); 2.48 (t, J = 6.4 Hz, 2H); 2.74
(t, J = 6.5 Hz, 2H); 3.39 (t, J = 6.5 Hz, 2H); 3.46-3.58 (m, 13H); 3.85
(dd, J = 4.6 Hz, J = 9.6 Hz, 1H); 4.03 (s, 1H); 4.26 (m, 2H); 4.60 (m,
2H); 4.85 (m, 2H); 6.18 (d, J = 6.7 Hz, 1H); 8.25 (s, 1H); 8.56 (s,1H);
13C NMR (D2O, 100 MHz) d 175.3; 174.4; 171.1; 156.8; 156.1;
153.4; 149.9; 140.1; 119.1; 87.1; 84.5; 74.8; 74.7; 74.3; 72.5; 66.2;
46.5; 42.6; 38.9; 36.1; 33.3; 31.7; 28.2; 21.5; 18.7; LRMS (ESI, m ⁄ z)
N-[2-(S-coenzyme A)acetyl]propane-1,3-butanamine
1
(9). Yield: 23%; H NMR (D2O, 300 MHz) d 0.85 (s, 3H); 0.96 (s,
3H); 1.39 (m, 2H); 1.55 (m, 2H); 1.67 (m, 2H); 2.49 (t, J = 6.3 Hz,
2H); 2.69 (t, J = 6.7 Hz, 2H); 2.99 (t, J = 7.6 Hz, 2H); 3.21 (t,
J = 6.9 Hz, 2H); 3.26 (s, 2H); 3.36 (t, J = 6.6 Hz, 2H); 3.49 (t, 2H,
J = 6.3 Hz); 3.65 (m, 1H); 3.90 (m, 1H); 4.04 (s, 1H); 4.30 (m, 2H);
4.62 (m, 1H); 4.90 (m, 2H); 6.21 (d, 1H, J = 5.4Hz); 8.44 (s, 1H); 8.67
(s, 1H); 13C NMR (D2O, 100 MHz) d 174.9; 174.2; 172.6; 150.0; 87.7;
83.5; 74.5; 74.4; 74.1; 72.2; 65.2; 39.6; 39.5; 38.6; 35.7; 35.6; 35.1;
31.4; 28.0; 26.6; 23.2; 21.1; 18.6; LRMS (ESI, m ⁄ z) calc. for
C28H50N9O17P3S: 909.2. Found: 908.5 (M-H)).
calc. for C32H54N9O19P3S: 993.8 Found: 496.0 (M-2H)2)
.
N-[2-(S-coenzyme A)acetyl]-N-phenyl(piperazin-1-yl)-
1-methanone (12). Yield: 35%; 1H NMR (D2O, 300 MHz): d
0.76 (s, 3H, Me); 0.90 (s, 3H); 2.46 (t, J = 6.6Hz, 2H); 2.73 (t,
J = 6.1 Hz, 2H); 3.23 (t, J = 6.6 Hz, 2H); 3.36-3.60 (m, 13H); 3.88 (s,
1H); 4.03 (dd, J = 4.2Hz, J = 9.4 Hz, 1H); 4.26 (m, 2H); 4.61 (m, 1H);
4.86 (m, 2H); 6.16 (d, J = 5.6 Hz, 1H); 7.47 (m, 5H); 8.23 (s, 1H);
8.55 (s,1H); 13C NMR (D2O, 100 MHz) d 174.6; 173.7; 172.6; 170.4;
155.1; 152.4; 149.0; 139.8;133.8; 130.5; 128.7; 126.6; 118.3; 86.3;
83.4; 74.0; 73.8; 73.7; 71.7; 65.3; 46.9; 45.4; 42.2; 41.9; 38.2; 35.4;
35.3; 32.5; 31.0; 20.7; 18.1; LRMS (ESI, m ⁄ z) calc. for
C34H50N9O18P3S: 997.2. Found: 996.8 (M-H)).
N-[2-(S-coenzyme A)acetyl]piperazine (10). Yield: 34%;
1H NMR (D2O, 300 MHz) d 0.83 (s, 3H); 0.94 (s, 3H); 2.50 (m, 2H);
2.74 (m, 2H); 3.29-3.44 (m, 8H); 3.48-3.59 (m, 4H); 3.65 (m, 1H) 3.87
(m, 3H); 4.04 (s, 1H); 4.30 (m, 2H); 4.62 (m, 2H); 4.89 (m, 2H); 6.23
(d, 1H, J = 5.2 Hz); 8.46 (s, 1H); 8.68 (s, 1H); 13C NMR (D2O,
100 MHz) d 174.7; 174.0; 170.5; 149.8; 144.4; 142.3; 87.5; 82.8;
74.3; 74.2; 74.1; 72.0; 64.9; 43.1; 42.9; 38.9; 38.2; 35.5; 35.4; 32.5;
31.1; 20.9; 18.3; LRMS (ESI, m ⁄ z) calc. for C27H46N9O17P3S: 892.2.
Found: 892.4 (M-H)).
N-[2-(S-coenzyme A)acetyl]-benzylamine (13). Yield:
1
81%; H NMR (D2O, 300 MHz): d 0.72 (s, 3H); 0.86 (s, 3H); 2.39 (t,
J = 6.3 Hz, 2H); 2.62 (t, J = 6.6 Hz, 2H); 3.28 (m, 4H); 3.41 (t,
J = 6.9 Hz, 2H); 3.53 (m, 1H); 3.81 (m, 1H); 3.99 (s, 1H); 4.22 (s
broad, 2H); 4.33 (s, 2H); 4.57 (s, 1H); 4.78 (m, 2H); 6.12 (d,
J = 6.3 Hz, 1H); 7.25 (m, 5H); 8.17 (s, 1H); 8.50 (s, 1H); 13C NMR
(D2O, 100 MHz) d 18.1; 20.8; 31.2; 34.8; 35.3; 35.4; 38.3; 43.3; 65.5;
71.8; 73.8; 73.9; 74.1; 83.7; 86.4; 127.3; 127.4; 128.7; 137.6; 152.8;
155.5; 172.5; 173.8; 174.7. LRMS (ESI, m ⁄ z): calc. for
C30H45N8O17P3S: 914.2. Found: 913.3 (M-H)).
Biological assays
The assays were carried out in the presence of radioactive acetyl-
CoA as previously reported in reference (26). p300 enzyme was
obtained from Biomol. International. Peptide H4-8 was obtained
from Millegen. IC50 values were measured in the presence of 2 lM
88
Chem Biol Drug Des 2011; 77: 86–92

New Bisubstrate Inhibitors of p300
prepared from a standard procedure (27,28). The monobenzoylated
amine 14d was synthesized in a modest yield with benzoyl chloride
at )78 ꢀC (Scheme 1). To increase the yield compounds, 14e–f
were prepared by transamidation (29). The coupling of the monopro-
tected compounds 14a–g and commercially available benzoylpiper-
azine with bromoacetyl bromide was performed by slow addition at
)10 ꢀC and led to bromocarboxamide compounds 15a–h. The
reaction between benzylamine and bromoacetylbromide in the pres-
ence of triethylamine and catalytic 4-dimethylaminopyridine gives
the expected compound 15i in a similar yield.
a
H2N
NHBoc
n
O
n
H2N
NH2
Br
14a 3
14b 4
14c 5
c
n
RHN
N
H
n
b
n
R
Yield (%)
n = 3,4,5
15a 3 Boc 96
15b 4 Boc 31
15c 5 Boc 32
15d 3 Bz 11
15e 4 Bz 66
H2N
NHBz
n
n
Yield (%)
14d 3
14e 4
17
35
44
15f
5
Bz 15
14f
5
O
N
c
d
Then, bisubstrate inhibitors were prepared by coupling bromoacetyl
compounds with CoASH in aqueous solution at pH 8 as described
in Scheme 2. Thus, the capped CoA derivatives (compounds 1–6
and 11–13) were obtained in 80–90% yields. To explore the influ-
ence of the free-amine in the p300 HAT binding site, we decided to
cleave the Boc protecting group by TFA. After purification by HPLC,
1H NMR data of isolated compounds 7–10 revealed a mixture of
desired compounds with 2¢-regioisomers (iso-CoA derivatives).
Indeed, it is known that strongly acidic conditions lead to the
migration of the 3¢-phosphate group to the 2¢-position (30,31). The
NH
RN
HN
NH
RN
57%
Br
c
15g R = Boc
15h R = Bz
14g R = Boc
63%
R = Bz
O
e
Br
NH2
N
H
65%
15i
Scheme 1: Reagents and conditions: (a) see reference (28) (b)
PhCOCl 0.2 eq, DCM, )78 ꢀC then rt, 15 h for compound 14d;
PhCONH2 0.33 eq, reflux, 15 h for compounds 14e and 14f; (c)
BrCOCH2Br 0.5 eq, DCM, )10 ꢀC; (d) see reference (29) (e)
BrCOCH2Br 1 eq, Et3N 1.1 eq, DMAP 0.1 eq, CHCl3, )10 ꢀC.
1
percentage of 2¢-regioisomers was evaluated by H NMR on proton
H1¢ and ranges from 20 to 36%. HPLC separation of this mixture
was found to be awkward, and to date we do not succeed to sepa-
rate both regioisomers. Although Cole and coll. have shown than
deletion of the 3¢-phosphate led to an approximately 40-fold
decreased inhibition (25), we decided to include these four com-
pounds in enzymatic tests to get insight into the free-amine
influence on p300 HAT.
[3H]-acetyl coenzyme A, 267 lM of H4-8 peptide and 296 nM of
p300 (1284–1673) in 30 lL IPH buffer.
The overall thirteen CoA derivatives were tested for their ability to
act as inhibitors of recombinant p300 enzyme (1284–1660) and
were compared with the well-known Lys-CoA (25). As shown in
Table 1, the effect of the chain length in the N-Boc series is
Results and Discussion
The synthesis of bisubstrate derivatives begins with the synthesis
of bromocarboxamide compounds. Boc-monoprotected amines were
O
O
O
a
b
SCoA
Br
SCoA
• TFA
N
H
H2N
N
H
15a-f
RHN
N
H
n
RHN
n
n
′
+ 2 -regioisomers
n
R
Yield (%)
1
2
3
4
5
6
3
4
5
3
4
5
Boc 83
Boc 89
Boc 77
Bz 74
Bz 83
Bz 90
′
n
3
4
5
Yield (%) %2 -isomer
7
8
9
38
41
23
20
20
22
O
O
a
b
H
N
N
N
N
R
N
SCoA
R
N
• TFA
SCoA
SCoA
Br
O
′
R
Yield (%)
+ 2 -regioisomers
R
Yield (%) %2
′
-isomer
11 Boc 98
15g Boc
15h Bz
10 34
36
12 Bz
35
O
O
NH2
Br
a
81%
N
N
H
H
N
N
15i
13
N
N
O
P
OH
O
P
OH
OH
O
H
N
H
N
O
1′
CoASH
O
O
HS
O
2
′
O
H
H
OH
OH
OH
O
P
O
Scheme 2: Reagents and conditions: (a) CoASH lithium salt 0.5 eq, NaOH 1 M, pH = 8, THF, overnight; (b) TFA 50% in DCM starting from
compounds 1, 2, 3 or 11.
Chem Biol Drug Des 2011; 77: 86–92
89

Kwie et al.
Table 1: In vitro inhibitory activity against p300 (1284–1673) of
bisubstrate-type inhibitors
p300
Structure
Compound n or R
IC50 (lM)
1
2
3
n = 3
n = 4
n = 5
3.6 € 1.5
0.4 € 0.1
0.07 € 0.02
H
N
BocHN
SCoA
SCoA
n
O
4
5
6
n = 3
n = 4
n = 5
0.7 € 0.1
0.80 € 0.07
0.30 € 0.07
H
H
Ph
N
N
n
O
O
7
8
9
n = 3
n = 4
n = 5
1.6 € 0.4
0.20 € 0.08
0.3 € 0.1
H
H2N
N
SCoA
n
Figure 2: Binding mode of best compound. Model clipping of
PDB structure 3BIY with continuum electrostatics coloring of molec-
ular surface, and compound 3 after docking (35,36). The figure
shows occupancy of cavities P1 and P2 by long chain (n = 5) and
Boc moieties, respectively, at right of the (yellow) overlapping loop
(upper clipping plane).
O
10
11
12
R = H
R = Boc 152 € 28
R = Bz
20 € 4
O
R N
N
28 € 8
SCoA
SCoA
13
None
209 € 51
H
N
O
Conclusion and Future Directions
Lys-CoA
0.5 € 0.2
In this paper, we report the synthesis of new series of bisubstrate
inhibitors. In the N-Boc series, we showed that the compound 3
bearing a 5-C chain length and a Boc moiety is well recognized in
the p300 binding site. Its efficiency can be explained by its capacity
to reach the pocket 2 discovered by Cole and coll. These results
will serve as starting point for further modifications in p300 inhibi-
tors, and studies are currently in progress in the laboratory to
design and prepare new analog inhibitors of compound 3. In future
experiments, rational drug design strategies using fragment
approaches, directed toward cavities P1 or P2, with or without com-
bination of CoA moiety, will also be investigated.
obvious. In fact, the compound 3 bearing the longest chain (n = 5)
is the most efficient, even better than Lys-CoA. Based on previous
studies of Cole and coll., the affinity displayed for the compound 3
could be attributed to the Boc group positioned in the pocket 2
(23). In the cases of N-benzoyl and free-amine series, no strong
dependencies were displayed between chain length and inhibitory
ability. To evaluate the impact of the chain on p300 binding site, a
more constrained compound, piperazine-CoA 10, was tested. How-
ever, in the light of the high IC50, compound 10 is not well recog-
nized by p300 HAT. This result could be explained by the steric
hindrance of the ring into the groove. Moreover, a decrease in the
inhibitory potency was also observed by adding more hindrance
with Boc- or benzoyl-protected piperazine (compound 11 and 12).
In the same way, poor inhibitory ability was found with aromatic
compound 13.
Acknowledgments
The authors are grateful to the 'Ministꢂre de l'Enseignement et de
la Recherche Scientifique' for financial support. We thank Dr. Cyril
Inard for advices and helpful discussions about this work and Mme
Chantal Zedde for help in HPLC purification.
To better understand the recognition of inhibitors at the molecular
level, preliminary docking studies (see Appendix S1) were carried
on these inhibitors using the PDB structure 3BIY (23,32). All candi-
dates bind like bisubstrate-type compounds but show poor correla-
tion between inhibitory activities and the different docking scores
used (Moldock, Plants, X-Score) (32–34). Nevertheless, analysis of
binding modes reveals first results related to the second cavity P2.
As it was hypothesized earlier, in the case of best compound 3
(R = Boc, n = 5), the chain length is sufficient to locate the Boc
moiety in P2 (Figure 2). Shorter chain lengths (n = 3 or 4) could not
permit the occupation of this cavity. The analysis of score compo-
nents such as hydrophobic and electrostatic contributions did not
give any clear affinity scale related to the electronegative groove or
P2 pocket occupation. However, these new results show that chain
length can have direct influences on the inhibitory activity.
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Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Chem Biol Drug Des 2011; 77: 86–92
91

Kwie et al.
Appendix S1. Experimental procedures.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
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