Quinoline-based p300 histone acetyltransferase inhibitors with Pro-apoptotic activity in human leukemia U937 cells

Article abstract of DOI:10.1002/cmdc.201300536

Chemical manipulations performed on 2-methyl-3-carbethoxyquinoline (1), a histone acetyltransferase inhibitor previously identified by our research group and active at the sub-millimolar/millimolar level, led to compounds bearing higher alkyl groups at th

Full text of DOI:10.1002/cmdc.201300536

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DOI: 10.1002/cmdc.201300536
Quinoline-Based p300 Histone Acetyltransferase Inhibitors
with Pro-apoptotic Activity in Human Leukemia U937 Cells
Alessia Lenoci,[a] Stefano Tomassi,[b] Mariarosaria Conte,[c] Rosaria Benedetti,[c]
Veronica Rodriguez,[a] Simone Carradori,[a] Daniela Secci,[a] Sabrina Castellano,[d]
Gianluca Sbardella,[d] Patrizia Filetici,[e] Ettore Novellino,[b] Lucia Altucci,[c, f] Dante Rotili,*[a]
and Antonello Mai*[a, g]
Chemical manipulations performed on 2-methyl-3-carbethoxy-
quinoline (1), a histone acetyltransferase inhibitor previously
identified by our research group and active at the sub-milli-
molar/millimolar level, led to compounds bearing higher alkyl
groups at the C2-quinoline or additional side chains at the C6-
quinoline positions. Such compounds displayed at least three-
fold improved inhibitory potency toward p300 protein lysine
acetyltransferase activity; some of them decreased histone H3
and H4 acetylation levels in U937 cells and induced high de-
grees of apoptosis (three compounds >10-fold higher than
compound 1) after treatment of U937 cells.
ed with transcriptionally active euchromatin, whereas histone
hypoacetylation involves gene silencing and arrest of transcrip-
tion (heterochromatin).[3] Nuclear KATs can be grouped into at
least four different families, based on sequence similarity with
the acetyltransferase domain and biochemical mechanism of
acetyl transfer: the GNAT (Gcn5-related N-acetyltransferase)
family (KAT2), including GCN5 and PCAF; the p300/CBP (CREB
binding protein) family (KAT3); the MYST family (KAT6), named
for the founding members MOZ, Ybf2/Sas3, Sas2, and Tip60;
and the Rtt109 family, named for identification as regulator of
Ty1 transposition gene product 109 (KAT11).[4]
The KAT p300 and its paralogue CBP (KAT3B and -3A) have
more than 75 identified histone and non-histone substrates, in-
cluding p53, MyoD, STAT3, and NF-kB, and are able to interact
with and bind more than 400 cellular partners.[5] The broad
substrate specificity of p300 can be explained by its unique
catalytic mechanism proposed after elucidation of the X-ray
crystal structure of p300 in complex with the bi-substrate ana-
logue Lys-CoA.[6] It consists in an initial stable binding of
acetyl-CoA to p300, followed by a weak and transient interac-
tion with the histone substrate for acetyl transfer (Theorell–
Chance mechanism).[6] The intrinsic acetyltransferase activity of
p300 is weak, but it is amplified after autoacetylation.[5d] SIRT2,
a NAD+-dependent deacetylase, counterbalances this acetylat-
ing activity;[7] however, p300 is also able to inactivate SIRT2
through acetylation, thereby boosting robust p300 autoacety-
lation and catalytic activity.[8]
Reversible protein acetylation is controlled by the opposite
action of protein lysine acetyltransferases (KATs) and deacety-
lases (KDACs).[1] Respectively, such enzymes add (“writers”) or
remove (“erasers”) an acetyl group to or from the e-amino
group of lysine residues in histone tails as well as in non-his-
tone proteins.[2] In histones, these modifications affect the ac-
cessibility of transcription factors to DNA and regulate gene
expression. In particular, hyperacetylated histones are associat-
[a] Dr. A. Lenoci, Dr. V. Rodriguez, Dr. S. Carradori, Prof. D. Secci, Dr. D. Rotili,
Prof. A. Mai
Department of Drug Chemistry & Technologies
Sapienza University of Rome
P.le Aldo Moro 5, 00185 Rome (Italy)
E-mail: antonello.mai@uniroma1.it
[b] Dr. S. Tomassi, Prof. E. Novellino
Aberrant expression of p300 has been reported in advanced
human malignancies such as prostate, liver, and breast cancer,
seeming to be correlated with poor prognosis. CBP and p300
have also been found to be involved in multiple rare chromo-
somal translocations associated with acute myeloid leukemia
(AML).[9] At the molecular level, activation of STAT3, NF-kB, and
HIF-1a by p300 can lead to cytokine- or hypoxia-induced
cancer cell survival and proliferation,[10] and acetylation by
p300 of proteins involved in metabolism, autophagy, and mo-
tility may have a significant impact on cancer metastasis.[11]
Therefore, p300 is a reliable target to inhibit for obtaining an
anticancer effect.
Department of Pharmacy, University of Naples “Federico II”
Via D. Montesano 49, 80131 Naples (Italy)
[c] Dr. M. Conte, Dr. R. Benedetti, Prof. L. Altucci
Department of Biochemistry, Biophysics & General Pathology
Second University of Naples, Vico L. De Crecchio 7, 80138 Naples (Italy)
[d] Dr. S. Castellano, Prof. G. Sbardella
Department of Pharmacy, University of Salerno
Via Giovanni Paolo II 132, 84084 Fisciano (SA) (Italy)
[e] Dr. P. Filetici
Laboratory of Nucleic Acids
Institute of Molecular Biology & Pathology (CNR-IBPM)
Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome (Italy)
[f] Prof. L. Altucci
Institute of Genetics & Biophysics (CNR-IGB)
Via P. Castellino 111, 80131 Naples (Italy)
In the last decade, there was a boom in the design, synthe-
sis, and biological evaluation of KDAC inhibitors in cancer as
well as non-cancer diseases, and two of them [SAHA (vorino-
stat) and FK-228 (romidepsin)] have been approved by the US
Food and Drug Administration (FDA) for the treatment of
refractory cutaneous T-cell lymphoma.[12]
[g] Prof. A. Mai
Istituto Pasteur-Fondazione Cenci Bolognetti
Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300536.
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Only a few diverse KAT inhibitors have been described thus
far, belonging to the series of bi-substrate inhibitors,[13] natural
products,[14] or small molecules.[15] Focusing on p300, Lys-CoA
is an example of a bi-substrate inhibitor,[13a] curcumin and
plumbagin showed p300 specificity among the natural sub-
stances,[14c,d] isothiazolones have been reported as covalent
p300 inhibitors,[15d] and C646 was recently identified by virtual
screening.[15f]
chemical space around 1 by functionalizing its structure with
some additional groups, and hence introducing a hydroxy
group at the C6 position of the quinoline ring (compound 7),
which was in turn alkylated with a benzyl, 2-phenylethyl, 3-
phenypropyl, 4-phenylbutyl, 5-phenylpentyl, or 2-oxo-2-phe-
nylethyl chain (compounds 8–13). Furthermore, some lysine-
mimicking substituents were inserted at the C6-hydroxy group
of the quinoline scaffold, likely to improve the competition of
the compounds with the lysine substrate. Thus, C6-tert-butoxy-
carbonylaminopentyloxy-(14), -aminopentyloxy-(15), -(methyl-
amino)pentyloxy-(16), -(dimethylamino)pentyloxy-(17), -(n-pro-
In our experience, we have described some quinoline/hy-
droxyquinoline compounds as KAT inhibitors,[16] a number of
bis-3,5-dibromo-4-hydroxyphenyl derivatives defined as epige-
netic multiple ligands endowed with histone methyltransfer-
ase/sirtuin/p300 mixed inhibitory activities,[17] and a series of
long-chain alkylidenemalonates (LoCAMs) carrying the unique
profile of p300 inhibitors/PCAF activators.[18] Herein we report
the results of our studies performed on novel quinoline com-
pounds starting from the prototype MC1626 (1), identified
through a phenotypic screen in yeast and then evaluated in
human U937 leukemia cells.[16a,b] Once tested, 1 displayed both
inhibition of yeast cell growth and of human KAT activity in
U937 cell nuclear extracts at sub-millimolar concentrations, in-
ducing 27% apoptosis at 1 mm after 24 h incubation.[16b]
As the quinoline ring is a privileged scaffold in medicinal
chemistry, pursuing our interest in improving both KAT inhibi-
tory activity and pro-apoptotic effects on U937 cells, we start-
ed to explore the chemical space around the quinoline nucleus
by introducing some chemical modifications at fairly accessible
positions of the 2-methyl-3-carbethoxyquinoline scaffold.
First, we prepared the methyl ester analogue of 1, com-
pound 2, and we replaced the methyl group at the quinoline
C2 position with higher alkyl chains such as ethyl (3), isopropyl
(4), n-propyl (5), and benzyl (6) groups. Then, considering 1 as
the active fragment against KAT, we thought to explore the
pylamino)pentyloxy-(18),
-(allylamino)pentyloxy-(20),
-(cyclopropylamino)pentyloxy-(19),
-(prop-2-yn-1-ylamino)pentyloxy-
(21), and -5-oxo-5-(prop-2-yn-1-ylamino)pentyloxy-(22) quino-
lines were prepared for testing as KAT inhibitors. Finally, we
also synthesized the C6-hexyloxy analogue 23 to dissect the
role of the lysine-mimicking amino terminal function in KAT
inhibition (Figure 1).
Compounds 1–7 were prepared by Friedlander condensa-
tion between the properly substituted ortho-nitrobenzalde-
hyde and the appropriate commercially available b-keto ester
at 708C in anhydrous ethanol in the presence of stannous
chloride, zinc chloride, and 3 ꢁ molecular sieves (Scheme 1).
Compounds 8–23 were synthesized by starting from 6-hy-
droxyquinoline 7 as depicted in Scheme 2. Alkylation of 7 in
dry acetonitrile at 958C with the appropriate (aryl)alkyl bro-
mide or iodide in the presence of anhydrous potassium car-
bonate and sodium iodide afforded compounds 8–13 and 23.
Similarly, compound 14 was obtained under the same condi-
tions and in the presence of tert-butyl (5-bromopentyl)carba-
mate 24, which was in turn synthesized from the correspond-
ing alcohol by reaction of tetrabromomethane and triphenyl-
phosphine in dry dichloromethane. The Boc-protected quino-
Figure 1. Novel quinoline-based KAT inhibitors.
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Table 1. p300 (KAT3B) inhibitory activities of 1–23 at fixed doses.[a]
Compd
Inhibition [%]
(100 mm)
(200 mm)
(400 mm)
1
2
3
4
5
6
7
8
22.6Æ2.1
20.5Æ0.6
33.4Æ0.3
27.0Æ0.8
42.8Æ0.1
60.3Æ1.5
16.5Æ1.0
62.0Æ1.3
5.6Æ2.8
10.6Æ0.2
6.7Æ0.2
0
ND
ND
35.6Æ1.9
ND
54.0Æ0.4
64.1Æ0.3
ND
65.3Æ0.5
ND
ND
ND
ND
67.3Æ5.8
ND
ND
ND
ND
ND
ND
ND
83.3Æ1.1
ND
ND
ND
ND
44.0Æ1.3
ND
67.6Æ3.4
66.3Æ0.4
ND
69.2Æ0.2
ND
ND
ND
ND
85.2Æ1.5
ND
ND
ND
ND
ND
ND
ND
96.4Æ0.8
ND
ND
Scheme 1. Reagents and conditions: a) ZnCl2, SnCl2, 3 ꢁ molecular sieves, dry
EtOH, 708C, 2–4 h.
9
line 14 was subsequently deprotected in acidic medium to
provide the desired amine hydrochloride 15 (Scheme 2). Reac-
tion between our common intermediate 7 and 1,5-dibromo-
pentane afforded bromide 25, which was then substituted
with the appropriate primary and secondary amines in dry
N,N-dimethylformamide (DMF) to obtain compounds 16–21
(Scheme 2). Finally, compound 22 was furnished by alkylation
of 7 with 5-bromo-N-(prop-2-yn-1-yl)pentanamide (26), which
was prepared by acylation of propargylamine with 5-bromo-
pentanoyl chloride in dry dichloromethane (Scheme 2).
Compounds 1–23 were tested against p300 (KAT3B)
(Table 1) and PCAF (KAT2B) (Table S1 in the Supporting Infor-
mation) at 100 mm. Compounds showing >30% inhibition
were also tested at 200 and 400 mm (Table 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
58.5Æ2.3
0
16.2Æ0.6
9.0Æ1.5
7.2Æ1.2
12.1Æ1.5
3.4Æ1.7
0
69.3Æ0.3
27.9Æ1.3
0
[a] Data are the mean ÆSD of n=2 independent experiments performed
in duplicate. ND: not determined.
When tested on PCAF, the presented quinolines did not
exceed 25% inhibition at 100 mm (Table S3, Supporting Infor-
mation), and therefore this activity was not further investigat-
ed.
tivity.[16b] The methyl ester analogue 2 showed the same poten-
cy as 1, whereas derivatives in which the C2-methyl was re-
placed with a higher alkyl chain (3–6) were more efficient, with
2-benzyl-3-carbethoxyquinoline 6 being the most potent. The
Against p300, the prototype 1 displayed 22.6% inhibition at
100 mm, according to its sub-millimolar/millimolar range of ac-
Scheme 2. Reagents and conditions: a) appropriate (aryl)alkyl bromide/iodide, anhyd K2CO3, NaI, dry CH3CN, 958C, 4–8 h; b) 1,5-dibromopentane, anhydrous
K2CO3, dry CH3CN, 958C, 3 h; c) appropriate amine, anhydrous K2CO3, NaI, dry DMF, 608C, 2–5 h; d) CBr4, PPh3, dry CH2Cl2, RT, 2 h; e) 4n HCl in dioxane, dry
THF, RT, 25 h; f) Et3N, dry CH2Cl2, RT, 20 h.
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insertion of a hydroxy group at the C6 position of the quino-
line scaffold (compound 7) did not improve the potency of the
prototype, but it furnished the key intermediate for the synthe-
sis of C6-alkyloxy and -(amino)alkyloxy derivatives carrying
a further substituent in addition to the 2-methyl-3-carbethoxy-
quinoline fragment. The insertion of a benzyloxy group at C6
(compound 8) clearly increased the potency of 1, while the
use of longer chains such as 2-phenylethyloxy (9), 3-phenylpro-
pyloxy (10), 4-phenylbutyloxy (11), and 5-phenylpentyloxy (12)
did not. Interestingly, such inhibitory activity was fully restored
if a 2-oxo-2-phenethyloxy chain was introduced at C6, yielding
one of the most potent derivatives (13, IC50: 74.0Æ10.8 mm)
among those described herein.
When an (amino)pentyloxy chain, designed as a lysine-mim-
icking portion, was inserted at C6 of the quinoline ring, in gen-
eral, scarcely potent p300 inhibitors (if any) were obtained
(compounds 14–20), regardless of the length and complexity
of the alkyl substituent(s) introduced at the amino group, or of
the change of such amino group into the corresponding tert-
butyl carbamate. The sole important exception to this behavior
was compound 21, which carries a C6-(prop-2-yn-1-ylamino)-
pentyloxy chain linked to the 2-methyl-3-carbethoxyquinoline
fragment, and is the most potent p300 inhibitor described
herein (IC50: 57.5Æ8.2 mm). Replacing the propargylamino func-
tion with the corresponding amide (22) or deleting the amino
function altogether (23) furnished less potent or totally in-
active compounds, respectively.
Figure 2. Effects of compounds 5, 6, 13, and 21 (at 100 mm) on acetylation
levels of histone H3, histone H4, and a-tubulin in human leukemia U937
cells; ctrl=control; A, SAHA 24 h; B, SAHA 1 h; C, SAHA 1 h washout +
SAHA 24 h; D, compound 5; E, SAHA 1 h washout + 5; F, compound 6; G,
SAHA 1 h washout + 6; H, compound 13; I, SAHA 1 h washout + 13; J,
compound 21; K, SAHA 1 h washout + 21.
Selected compounds 5, 6, 13, and 21 were tested in human
U937 leukemia cells at 100 mm to study their effects on acetyla-
tion levels of histones H3, H4, and a-tubulin, as functional
assays for KAT inhibition in cells. The assays were performed
either by treating the cells directly with the compounds, or
after pre-treatment (1 h) with 5 mm SAHA and subsequent
washout, to increase the starting signals of acetylation for H3,
H4, and a-tubulin (Figure 2).
Western blot analyses showed that 5, 6, and 21 are highly
efficient in decreasing the acetyl-H3 and acetyl-H4 signals with
respect to control, with or without SAHA pre-treatment,
whereas 13 was observed to be ineffective under both condi-
tions. In addition, among the tested compounds, only 21 de-
creased a-tubulin acetylation levels (Figure 2).
U937 cells were also treated with 5, 6, 13, and 21 at 100 mm
for 30 h to determine effects on cell-cycle progression, apopto-
sis, and granulocytic differentiation. SAHA (5 mm) was used as
a positive control. Under the tested conditions, the com-
pounds did not show clear alteration of cell cycle, with the ex-
ception of 21, which showed a slight increase in the G2/M
phase percentage, and 5, the effect on cell cycle of which
could not be clearly evaluated due to the intense extent of
apoptosis (see below; Figure 3A). The percentage of cells in
the pre-G1 peak was taken as an index of apoptosis: in this
assay, 5 displayed extensive apoptosis, and 6 and 21 induced
apoptosis to 39 and 15%, respectively, relative to control (Fig-
ure 3B). Granulocytic differentiation was evaluated as the per-
centage of cells positive to the superficial antigen CD11c and
negative to propidium iodide (CD11c+/PIÀ cells): in general,
low differentiation activity was detected with the tested com-
Figure 3. Effects on a) cell cycle, b) apoptosis induction, and c) granulocytic
differentiation in U937 cells treated with 5, 6, 13, and 21 for 30 h at 100 mm.
SAHA (5 mm) was used as a positive control.
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pounds, with 5 and 21 reaching ~6% of CD11c+/PIÀ cells (Fig-
ure 3C).
mixtures or after extraction involved the use of a rotary evaporator
operating at a reduced pressure of ~20 Torr. Organic solutions
were dried over anhydrous Na2SO4. Elemental analysis was used to
determine purity of the described compounds, which were deter-
mined in all cases to be >95 %, with analytical results within
0.40 % of theoretical values. All chemicals were purchased from
Sigma–Aldrich s.r.l. (Milan, Italy) or from TCI Europe N.V. (Zwijn-
drecht, Belgium) and were of the highest purity available. As
a rule, samples prepared for physical and biological studies were
dried under high vacuum over P2O5 for 20 h at temperatures rang-
ing from 25 to 408C, depending on the sample melting point.
In conclusion, we performed chemical manipulations on the
scaffold of 1, a KAT inhibitor prototype previously disclosed by
us and active at sub-millimolar/millimolar levels, to improve its
potency both in enzyme and cellular assays. Through replace-
ment of the C2-methyl substituent of 1 with n-propyl (5) or
benzyl (6) groups, as well as by inserting additional 2-oxo-2-
phenethyloxy (13) or (prop-2-yn-1-ylamino)pentyloxy (21) side
chains at the C6 position of the quinoline ring of compound 1,
we obtained two- to threefold increased potency of such de-
rivatives against p300 (Table 1), with 21 being the most potent
(IC50: 57.5 mm). Compounds 5, 6, 13, and 21 (100 mm) were
tested in human leukemia U937 cells to determine their effects
on histones H3, H4, and a-tubulin acetylation levels, as func-
tional assays for KAT inhibition in cells. Western blot results
showed a pronounced decrease in H3 and H4 acetylation in
cells treated with 5 and 21, whereas 6 was less efficient and
13 totally inactive, likely due to cell permeability problems. Ac-
cordingly, when the selected compounds were tested in U937
cells to study cell cycle, apoptosis induction, and cell differen-
tiation (at 100 mm for 30 h), 5 effected extensive apoptosis, fol-
lowed by 6 (39%) and 21 (15%). In a similar assay, the proto-
type 1 induced 27% apoptosis when tested at 1 mm (10ꢂ).[16]
The intense degree of apoptosis displayed by 5 correlates well
with its ability to highly decrease H3 and H4 acetylation levels
(Figure 2), but is not in full agreement with enzyme inhibition
data (21, for instance, was the most potent KAT inhibitor in
enzyme assays, strongly hypoacetylated H3, H4, and a-tubulin,
and induced 15% apoptosis in U937 cells); therefore, for com-
pound 5, off-target effects in producing apoptosis cannot be
ruled out. Compound 13 did not show any effect on cell cycle
or in apoptosis induction. Despite low efficiency, 5, 6, and 21
also displayed slight granulocytic differentiation under the con-
ditions tested. Taking into account both enzyme (p300 KAT)
and cellular data, compounds 5 and 21 can be retained as val-
uable starting points for further exploration of the chemical
space around the quinoline nucleus, in order to acquire addi-
tional data and to improve the potency of the compounds.
Example procedures for the syntheses of final compounds 1–23
are reported below. Chemical, physical, and analytical data for the
final compounds (1–23) are reported in the Supporting Informa-
tion, along with the syntheses and chemical and physical data of
intermediate compounds 24–26.
Example procedure for the preparation of 1–7: Ethyl 6-hydroxy-
2-methylquinoline-3-carboxylate (7):
A 250 mL round-bottom
flask, equipped with a stir bar and reflux condenser, was flame
dried under an atmosphere of N2. 5-Hydroxy-2-nitrobenzaldehyde
(1 equiv, 2.2 g, 13.2 mmol) and ethyl acetoacetate (1.2 equiv,
2.0 mL, 15.8 mmol) were added, followed by 80 mL of anhyd EtOH.
Anhyd SnCl2 (5 equiv, 12.5 g, 65.8 mmol), ZnCl2 (5 equiv, 9.0 g,
65.8 mmol) and 3 ꢁ molecular sieves (~1 g) were added to the so-
lution. This mixture was then heated at 708C under N2 atmos-
phere; the reaction was finished after 2 h. The mixture was cooled
to RT and filtered. The EtOH was removed in vacuo. The residue
was then made basic with 10% aq NaHCO3 (100 mL), transferred to
a 500 mL separator funnel, and extracted with EtOAc (4ꢂ100 mL).
The organic extracts were combined, washed thoroughly with sa-
turated aq NaCl, dried over anhyd Na2SO4, filtered and concentrat-
ed in vacuo. Purification by recrystallization from EtOAc gave 7 as
a white solid (2.1 g, 68%):[19] mp: 211–2138C (EtOAc); 1H NMR
(400 MHz, [D6]DMSO): d=1.37 (t, 3H, COOCH2CH3), 2.78 (s, 3H,
CH3), 4.37 (q, 2H, COOCH2CH3), 7.26 (d, 1H, quinoline proton), 7.40
(dd, 1H, quinoline proton), 7.83 (d, 1H, quinoline proton), 8.62 (s,
1H, quinoline proton), 10.11 ppm (s, 1H, OH); MS (ESI): m/z=232
[M+H]+.
Example procedure for the preparation of 8–14,22–23: Ethyl 2-
methyl-6-phenethoxyquinoline-3-carboxylate (9):
A
round-
bottom flask containing a solution of 7 (1 equiv, 0.43 mmol,
100 mg) in dry CH3CN (2 mL) was treated with, in sequence, NaI
(1.3 equiv, 0.56 mmol, 84 mg), anhyd K2CO3 (1.75 equiv, 0.76 mmol,
104.6 mg) and 2-bromoethylbenzene (1.5 equiv, 0.56 mmol,
76.7 mL). The mixture was then stirred at 958C for 5 h. The reaction
mixture was stopped by the addition of saturated aq NaCl (40 mL)
and extracted with EtOAc (4ꢂ50 mL). The organic layers were com-
bined, washed with saturated aq NaCl, dried over anhyd Na2SO4,
filtered and concentrated in vacuo. Purification by chromatography
on a silica gel column (EtOAc/n-hexane, 1:9) gave compound 9 as
a white powder (85.1 mg, 59%): mp: 68–718C (cyclohexane);
1H NMR (400 MHz, CDCl3): d=1.47 (t, 3H, COOCH2CH3), 2.97 (s, 3H,
CH3), 3.20 (t, 2H, CH2CH2O), 4.32 (t, 2H, CH2CH2O), 4.45 (q, 2H,
COOCH2CH3), 7.13 (d, 1H, quinoline proton), 7.35–7.39 (m, 5H,
phenyl proton), 7.45 (dd, 1H, quinoline proton), 7.94 (d, 1H, quino-
line proton), 8.61 ppm (s, 1H, quinoline proton); MS (ESI): m/z=
336 [M+H]+.
Experimental Section
Chemistry
General: Melting points (m.p.) were determined on a Bꢃchi 530
melting point apparatus. 1H NMR spectra were recorded at
400 MHz using a Bruker AC 400 spectrometer; chemical shifts (d)
are reported in parts per million (ppm) relative to the internal ref-
erence (Me4Si). Mass spectra (MS) were recorded on an API-TOF
Mariner instrument (Perspective Biosystems, Stratford, TX, USA);
samples were injected by a Harvard pump at a flow rate of 5–
10 mL minÀ1, infused using an electrospray (ESI) system.
All reactions were routinely checked by thin-layer chromatography
1
(TLC) and H NMR. TLC was performed on aluminum-backed silica
Ethyl
6-((5-aminopentyl)oxy)-2-methylquinoline-3-carboxylate
gel plates (Alufolien Kieselgel 60 F254, Merck) with spots visualized
by UV light or an alkaline KMnO4 solution. All solvents were re-
agent grade and, when necessary, were purified and dried by stan-
dard methods before use. The removal of solvent from reaction
hydrochloride (15): A solution of 14 (1 equiv, 0.4 mmol, 170 mg) in
dry THF (10 mL) was treated with 4n HCl in dioxane (60 equiv,
24.5 mmol, 6.1 mL) at 08C, and the resulting reaction mixture was
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left under stirring at RT for 25 h. After the end of the reaction, the
resulting precipitate was isolated by filtration, and washed first
with dry THF and then dry Et2O to give 15 as a white solid
(103.3 mg, 87%): mp: 213–2158C (EtOAc); 1H NMR (400 MHz,
[D6]DMSO): d=1.39 (t, 3H, COOCH2CH3), 1.52 (t, 2H,
H2NCH2CH2CH2), 1.65 (t, 2H, H2NCH2CH2), 1.83 (t, 2H, H2N(CH2)3CH2),
2.82 (m, 2H, H2NCH2), 2.98 (s, 3H, CH3), 4.14 (t, 2H, H2N(CH2)4CH2O),
4.41 (q, 2H, COOCH2CH3), 7.67 (dd, 1H, quinoline proton), 7.75 (d,
1H, quinoline proton), 7.92 (br, 3H, NH2·HCl), 8.18 (d, 1H, quinoline
proton), 9.10 ppm (s, 1H, quinoline proton); MS (ESI): m/z=317
[M+H]+ (relative to free amine).
and lysed using a lysis buffer supplemented with protease and
phosphatase inhibitors (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1%
NP-40, 10 mm NaF, 0.1 mm Na3VO4, 40 mgmLÀ1 PMSF, 20 gmLÀ1
aprotinin, 20 mgmLÀ1 leupeptin, 2 mgmLÀ1 antipain, 10 mm para-
nitrophenyl phosphate (pNPP), 10 mgmLÀ1 pepstatin A and 20 nm
okadaic acid). Then the cells were centrifuged for 15 min at 48C
(13000 rpm), and the protein concentration of the supernatant
was determined by colorimetric assay (Bio-Rad, Italy). The cell ex-
tracts were diluted 1:1 in 2X Laemli sample buffer (0.217m Tris-HCl,
pH 8.0, 52.17% sodium dodecyl sulfate (SDS), 17.4% glycerol,
0.026% bromophenol blue, 8.7% b-mercaptoethanol), and then
were boiled for 3 min. Equal amounts of protein (50 micrograms)
were run and separated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). Primary antibodies used were: anti-alpha tubulin ace-
tylated (Sigma) and anti-ERK1 (Santa Cruz Biotechnology).
Example procedure for the preparation of 16–21: Ethyl 6-((5-(al-
lylamino)pentyl)oxy)-2-methylquinoline-3-carboxylate (20):
A
round-bottom flask was charged, in sequence, with 25 (1 equiv,
0.26 mmol, 100 mg), dry DMF (3.6 mL), NaI (1.1 equiv, 0.29 mmol,
43.4 mg), anhyd K2CO3 (1.5 equiv, 0.39 mmol, 54.5 mg) and allyla-
mine (15 equiv, 3.9 mmol, 0.3 mL). The reaction mixture was left
under stirring at 608C for 4 h. Upon completion, the reaction was
cooled and then quenched with saturated aq NaCl (20 mL). The so-
lution was then extracted with EtOAc (4ꢂ40 mL). The combined
organic phases were washed with saturated aq NaCl, dried over
anhyd Na2SO4, filtered and concentrated in vacuo. Purification by
chromatography on a silica gel column (CHCl3/MeOH, 25:1) gave
20 as a white solid (78.8 mg, 85%): mp: 104–106 (petroleum
ether); 1H NMR (400 MHz, CDCl3): d=1.47 (t, 3H, COOCH2CH3),
1.57–1.62 (m, 2H, NHCH2CH2CH2), 1.66–1.73 (m, 2H, NHCH2CH2),
1.86–1.93 (m, 2H, NH(CH2)3CH2), 2.31 (br, 1H, NH), 2.73 (t, 2H,
NHCH2CH2), 2.96 (s, 3H, CH3), 3.35 (d, 2H, CH2 =CHCH2NH), 4.08 (t,
2H, NH(CH2)4CH2O), 4.45 (q, 2H, COOCH2CH3), 5.16–5.19 (d, 1H,
CHH=CHCH2NH), 5.23–5.27 (d, 1H, CHH=CHCH2NH), 5.91–6.01 (m,
1H, CHH=CHCH2NH), 7.11 (d, 1H, quinoline proton), 7.43 (dd, 1H,
quinoline proton), 7.94 (d, 1H, quinoline proton), 8.63 ppm (s, 1H,
quinoline proton); MS (ESI): m/z=357 [M+H]+.
Histone extraction: Cells were harvested, washed twice with cold
PBS, and lysed in triton extraction buffer (TEB): PBS containing
0.5% Triton X 100 (v/v), 2 mm PMSF, 0.02% (w/v) NaN3, at a cellular
density of 107 cells/mL for 10 min on ice, with gentle stirring. After
brief centrifugation (2000 rpm at 48C for 10 min), the supernatant
was removed, and the pellet was washed in half the volume of
TEB and centrifuged as before. The pellet was suspended in 0.2m
HCl at a cell density of 4ꢂ107 cells/mL, and acid extraction was left
to proceed overnight at 48C on a rolling table. Next, the samples
were centrifuged at 2000 rpm for 10 min at 48C, the supernatant
was removed, and the protein content was determined using
a Bradford assay. Antibodies against acetylated histones H3 and H4
(Upstate Biotechnologies) at concentrations of 2 mgmLÀ1 were
used. Ponceau red was used for normalization.
Flow cytometry: Cells were plated (2ꢂ105 cellsmLÀ1) and collected
after stimulation. They were then centrifuged (1200 rpm for 5 min
at 48C) and suspended in a solution of 1X PBS containing 0.1%
sodium citrate, 0.1% NP-40 and 50 mgmLÀ1 propidium iodide.
After 30 min incubation at room temperature in the dark, cell cycle
was evaluated by fluorescence-activated cell sorting (FACS) flow
cytometry (FACSCalibur, Becton Dickinson) and analyzed with the
program ModFit V3 (Verity Software House, Inc.).
Enzyme-based assays
KAT assays: The effects of test compounds on the catalytic activity
of p300 and PCAF were determined in a HotSpot HAT activity assay
(Reaction Biology Corp., Malvern, PA, USA), performed according to
the company’s instructions. In brief, the recombinant catalytic do-
mains of PCAF (aa 492–658) or p300 (aa 1284–1673) were incubat-
ed with histone H3 as a substrate (5 mm) and [acetyl-3H]-acetyl
coenzyme A (3.08 mm) as an acetyl donor in reaction buffer (50 mm
Tris-HCl (pH 8.0), 50 mm NaCl, 0.1 mm ethylenediaminetetraacetic
acid, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride
(PMSF), 1% DMSO) for 1 h at 308C in the presence or absence of
a dose titration of the test compound. Histone H3 acetylation was
assessed by liquid scintillation. Anacardic acid and curcumin served
as positive controls against PCAF and p300 activity, respectively.
Data at fixed doses were performed twice, each in duplicate. IC50
values were analyzed using Excel and GraphPad Prism 6.0 software
(GraphPad Software Inc., San Diego, CA, USA).
FACS analysis of apoptosis: Apoptosis was measured as the pre-G1
cell peak, analyzed by FACS with Cell Quest software (Becton Dick-
inson) as previously reported.[20]
FACS analysis of differentiation: After centrifugation, cells were re-
suspended in 10 mL of phycoerythrine-conjugated CD11c (CD11c-
PE, PharMingen). Control samples were incubated with 10 mL of
phycoerythrin-conjugated mouse IgG1; after incubation for 1 h at
48C in the dark, cells were washed with PBS and resuspended in
500 mL PBS containing propidium iodide (0.25 mgmLÀ1). The sam-
ples were then analyzed by flow cytometry.
Supporting Information
Synthetic procedures and characterization data for intermediate
and all remaining final compounds are given in the Supporting In-
formation, along with H NMR and mass spectra for compounds 1–
Cell-based assays
1
Cell culture: Cells were grown at 378C in a humidified atmosphere
containing 5% CO2 in RPMI-1640 medium (Sigma) supplemented
with 10% heat-inactivated fetal bovine serum (HyClone), penicil-
lin G (100 UmLÀ1), streptomycin (100 mgmLÀ1), l-glutamine (2 mm),
amphotericin B (250 mgmLÀ1). At the time indicated, inductions
were performed with ligands.
23. The results of the PCAF inhibition assay for compounds 1–23
are also given.
Acknowledgements
This work was supported by the Italian Ministry of Education,
Universities & Research–Investment Fund for Basic Research
Western blot analysis: After removal of the culture medium, the
cells were washed with cold 1X phosphate-buffered saline (PBS)
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Keywords: apoptosis · epigenetics · lysine acetyltransferases
(KATs) · leukemia · p300 · structure–activity relationships
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Received: December 17, 2013
Published online on February 6, 2014
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ChemMedChem 2014, 9, 542 – 548 548