Novel quinolone-based potent and selective HDAC6 inhibitors: Synthesis, molecular modeling studies and biological investigation

Article abstract of DOI:10.1016/j.ejmech.2020.112998

In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6 (HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of potency and selectivity of these products was performed by employing computational studies which led to the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116) and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9 and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good solubility and stability profile.

Full text of DOI:10.1016/j.ejmech.2020.112998

Journal Pre-proof
Novel quinolone-based potent and selective HDAC6 inhibitors: synthesis, molecular
modelling studies and biological investigation
Nicola Relitti, A. Prasanth Saraswati, Giulia Chemi, Margherita Brindisi, Simone Brogi,
Daniel Herp, Karin Schmidtkunz, Fulvio Saccoccia, Giovina Ruberti, Cristina Ulivieri,
Francesca Vanni, Federica Sarno, Lucia Altucci, Stefania Lamponi, Manfred Jung,
Sandra Gemma, Stefania Butini, Giuseppe Campiani
PII:
S0223-5234(20)30970-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112998
EJMECH 112998
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 August 2020
Revised Date: 3 November 2020
Accepted Date: 3 November 2020
Please cite this article as: N. Relitti, A.P. Saraswati, G. Chemi, M. Brindisi, S. Brogi, D. Herp, K.
Schmidtkunz, F. Saccoccia, G. Ruberti, C. Ulivieri, F. Vanni, F. Sarno, L. Altucci, S. Lamponi, M. Jung,
S. Gemma, S. Butini, G. Campiani, Novel quinolone-based potent and selective HDAC6 inhibitors:
synthesis, molecular modelling studies and biological investigation, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112998.
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Novel quinolone-based potent and selective HDAC6 inhibitors: synthesis, molecular modelling
studies and biological investigation
b
Nicola Relitti a,1, A. Prasanth Saraswati a,1, Giulia Chemi a,2, Margherita Brindisi a,3, Simone Brogi , Daniel
c
c
d
Herp , Karin Schmidtkunz , Fulvio Saccoccia , Giovina Ruberti d, Cristina Ulivieri e, Francesca Vanni e,
a
c
a
a
Federica Sarnof, Lucia Altuccif, Stefania Lamponi , Manfred Jung , Sandra Gemma , Stefania Butini ,
Giuseppe Campiani a,*
a
Department of Biotechnology, Chemistry and Pharmacy, DoE Department of Excellence 2018-2022, University of
Siena, via Aldo Moro 2, 53100, Siena, Italy
b Department of Pharmacy, University of Pisa, via Bonanno 6, 56126, Pisa, Italy
c
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstraße 25, 79104, Freiburg,
Germany
d
Institute of Biochemistry and Cell Biology, CNR, Campus A. Buzzati-Traverso. Via E. Ramarini 32, 00015
Monterotondo (Rome) Italy
e Department of Life Sciences, University of Siena, via Aldo Moro 2, 53100, Siena, Italy
f Department of Precision Medicine, University of Campania Luigi Vanvitelli, Vico L. de Crecchio 7, 80138, Naples,
Italy
Corresponding author.
E-mail address: campiani@unisi.it (G. Campiani)
1 These authors equally contributed.
2
Present address: Wellcome Centre for Anti-Infectives Research, Drug Discovery Unit, Division of Biological
Chemistry and Drug Discovery, University of Dundee, DD1 5EH Dundee, United Kingdom
3
Present address: Department of Pharmacy, University of Napoli Federico II, DoE Department of Excellence 2018-
2022, Via D. Montesano 49, 80131, Napoli, Italy
Abstract
In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6
(HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for
HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of
potency and selectivity of these products was performed by employing computational studies which led to
the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These
compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116)
and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell
death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the
cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9
and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-
116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of

these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good
solubility and stability profile.
Keywords
HDAC6 inhibitors, HDAC, quinolone synthesis, cancer, structure-activity relationships, molecular modeling
Highlights
•
•
•
•
•
•
Rational design and synthesis of novel quinolone-based selective HDAC6 inhibitors
Compounds 7g and 7k were the most potent compounds against HDAC6
These compounds possess a good selectivity towards HDAC6 over the 1 and 8 isoforms
Compound 7g showed a strong reduction in cell viability against HCT-116 cells
Induction of apoptosis was observed after the treatment with 7g and 7k
No mutagenicity and cytotoxicity were observed for these compounds
Graphical abstract
Abbreviations
AcHsp90 (AcLys294), acetylated Hsp90 in lysine 294; AcTub, acetylated α-tubulin; DCM, dichloromethane;
DIBAL, diisobutylaluminum hydride; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; H3K9/14ac,
acetylated histone H3 in lysine 9 and 14; HCT-116, colon cancer cells; HDAC, histone deacetylase; HDACi,
HDAC inhibitors; Hsp90, heat-shock protein 90; PARP, Poly(ADP-ribose) polymerase; PTSA, p-
toluenesulfonic acid; PTSH, p-toluenesulfonyl hydrazide; SAR, structure-activity relationship; SP, standard
precision; TFA, trifluoroacetic acid; THF; tetrahydrofuran; U937, histiocytic lymphoma; ZBG, zinc binding
group.
1. Introduction
Histone deacetylases (HDACs) are enzymes involved in the removal of acetyl groups from histones, thus
modulating gene transcription. Their over-expression has been ascertained in solid cancers, hematological
malignancies, and parasitic disorders, making them a viable target for developing novel chemotherapeutic
agents [1,2], as well as in rare diseases [3]. Of the 18 identified HDAC isoforms (subdivided into 4 classes, I-
IV), HDAC6 is unique owing to the presence of a zinc finger ubiquitin binding domain and its localization.
Structurally, it contains two catalytic domains, DD1 and DD2, present at the N-terminal and the central
region, respectively. With regard to its localization, HDAC6 is a cytoplasmic enzyme (unlike HDAC1, 2 and
3, that are nuclear localized and HDAC8, that can show both nuclear and cytoplasmic spreading) which can

shuttle between cytoplasm and the nucleus in response to cellular signaling [4]. Apart from its action on
histones it also exerts deacetylase activity on non-histone substrates such as α-tubulin, heat-shock protein 90
(Hsp90), peroxiredoxin, cortactin, and tau [3]. This specific feature of HDAC6 is responsible for its unique
role in the pathogenesis of cancer and a number of rare diseases such as idiopathic pulmonary fibrosis,
inherited retinal disorders, Rett syndrome, and Charcot-Marie-Tooth disease [3,5]. Comparatively, HDAC10
that has a closer structural correlation with HDAC6 has a very weak activity as a lysine deacetylase and its
role has so far been described mostly for autophagy promotion [6].
Several HDAC inhibitors (HDACi) have successfully reached the clinic and are being used as therapeutic
tools to treat different malignances. Representative HDACi include: vorinostat (1, SAHA, 1) [7] and
romidepsin (2) [8] for the treatment of cutaneous T-cell lymphomas, panobinostat (3) [9] for multiple
myeloma, and belinostat (4) [10] for relapsed or refractory peripheral T-cell lymphoma. These compounds
behave as pan-HDACi since they inhibit several HDAC isoforms, thus causing many side effects. The
discovery of Tubastatin A (5) a tetrahydropyrido[4,3-b]indole hydroxamic acid, as a selective HDAC6i has
paved the way for the development of isoform-selective HDACi. However, a potent inhibition of HDAC10
isoform has been reported after exposure with 5 [11–13]. Identification of isoform-selective compounds is
vital for dissecting the biological roles of specific HDACs and for developing safer therapeutic tools. The
general pharmacophoric model of HDACi encompasses three key components namely: the surface
recognition group (cap), the zinc binding group (ZBG) and a linker bridging the two portions. Based on this
model several selective HDAC6 inhibitors have been developed mainly by modifying the cap and the linker
portions, while retaining hydroxamate as the ZBG [2,14,15].
Many natural products have been identified possessing potent anticancer properties, which have inspired
chemists to use them as building blocks in the synthesis of novel chemotherapeutics [16–19]. In this context,
functionalized 4-arylquinolin-2(1H)-ones represent an attractive scaffold for developing biologically active
molecules, including the orally active anticancer agent tipifarnib, that is currently undergoing clinical
investigation [20]. Based on an exhaustive literature search, we identified the quinolone moiety as a
promising cap group due to its lipophilicity in vitro. This system ideally fits in the cap region of the HDACi
pharmacophore [21]. Many quinolone containing compounds are well known as chemotherapeutics (e.g.
ciprofloxacin, norfloxacin) and anticancer agents (voreloxin) [22]. Quinoline-based anticancer agents exert
their activity by playing a role in the DNA intercalation process, inhibiting topoisomerase II activity and
tubulin polymerization [23].

Figure 1. Representative structures of HDAC inhibitors (1-5) and general structure of the title compounds 6-8.
In our pursuit to identify novel cap groups for potent and selective HDAC6i [14,15,24], we exploited the
potential of a naturally occurring compound containing the quinolone skeleton as an effective versatile and
hindered cap-group suitable for a broad variety of scaffold decorations (as shown in the general structure of
Figure 1). By a virtual screening approach, we identified several cap-groups; a preliminary computational
investigation, using compound 6a, characterized by a viridicatin-based cap-group, showed that it perfectly
fits into the hydrophobic sub-pocket delimited by L712 and F642 (Danio rerio HDAC6 numbering,
corresponding to residues L749 and F679 in hHDAC6). Moreover, by our computational approach, we
observed that the cap-group of Tubastatin A and 6a into zfHDAC6 (PDB ID: 6THV) target the same region
of the enzyme as highlighted in Figure 2. Therefore, we selected this interesting quinolone-based cap-group
to rationally design and synthesize a new focused library of HDAC6 selective inhibitors as described below.
According to our preliminary in silico analysis and aiming at obtaining compounds with high selectivity
index towards HDAC6 (over HDAC1) to be screened against different cancer cell lines we synthesized a
library of quinolone-based hydroxamates. The synthesis of the 3-hydroxy-4-arylquinolin-2(1H)-ones moiety
was carried out by the application of a multicomponent ring-expansion recently reported by Tangella et al.
This protocol permits the conversion of isatin to viridicatin alkaloids after the in situ generation of α-
aryldiazomethanes in presence of an aldehyde. derivatives and inspired from a multicomponent protocol,
useful to access the 3-hydroxy-4-arylquinolin- 2(1H)-ones, we constructed a series of cap groups [25]. This
protocol enabled us to develop novel HDAC6i (compounds 6a-d, 7a-k and 8, Table 1) bearing N- or O-
appended linker moieties, and a variety of focused modifications at both their heterocyclic core and the
aromatic portions.

Figure 2. Docked pose of 6a into zfHDAC6 (PDB ID: 6THV), superposed to the crystallized inhibitor Tubastatin A
belonging to 6THV. Focus on residues in sticks that are the constituents of the hydrophobic sub-pocket explored by the
viridicatin cap-group (residues L712 and F642 in zfHDAC6 correspond to L749 and F679 in hHDAC6) is presented.
The picture was generated by means of PyMOL.
2. Chemistry
The structures of final compounds 6a-d, 7a-k and 8 are shown in Table 1. In order to achieve the synthesis
of the quinolone core of the compounds, the ring expansion procedure described by Tangella et al was
applied [25]. It is a multicomponent reaction involving: an aldehyde, activated with p-toluenesulfonyl
hydrazide (PTSH), and a N-substituted isatin in the presence of K2CO3 as base.
In Scheme 1 the synthesis of the final compounds 6a-d is described. Isatin (9) was alkylated with methyl 4-
(bromomethyl)benzoate and the resulting product 10 was subjected to the ring expansion procedure with
benzaldehyde or 3-pyridinecarboxaldehyde obtaining compounds 11a,b, respectively. As expected, a
transesterification with EtOH occurred in this step. Intermediates 11a,b were converted to their
corresponding hydroxamic acid derivatives 6a,d after treatment with a strong excess of aqueous NH2OH in
the presence of KOH. Intermediate 11a was also submitted to O-alkylation with MeI or benzyl bromide
providing derivatives 12a,b, respectively. These latter were converted into final compounds 6b,c upon
reaction with NH2OH and KOH.

Scheme 1. Synthesis of compounds 6a-d. Reagents and conditions: (a) methyl 4-(bromomethyl)benzoate, NaH,
DMF, 0 to 25 °C, 12 h, quantitative yield; (b) PTSH, benzaldehyde or 3-pyridinecarboxaldehyde, K2CO3, EtOH, 80 °C,
12 h, 32-64%; (c) NH2OH (50% water solution), KOH, DCM, MeOH, 25 °C, 3 h, 82-97%; (d) MeI, NaH, THF, 0 to 25
°C, 12 h; or BnBr, KI, K2CO3, DMF, 80 °C, 12 h, 29-64%.
In Scheme 2 is reported the synthesis of compounds 7a-c,h and 8. The ring expansion performed on isatin
(9) in the presence of benzaldehyde or 3-pyridinecarboxaldehyde provided quinolone derivatives 13a,b,
respectively. These latter were subjected to alkylation reaction with methyl 4-(bromomethyl)benzoate in the
presence of K2CO3 as the base to furnish the O-alkylated intermediates 14a,b. Treatment of 14a with
NH2OH in the presence of KOH led to compound 7a. Alternatively, alkylation of the lactam nitrogen of
14a,b with MeI or propargyl bromide followed by reaction with NH2OH, led to the N-alkyl derivatives
7b,c,h [26]. For the synthesis of compound 8, isatin was alkylated with MeI and successively subjected to
the ring expansion reaction with cyclohexanecarboxaldehyde obtaining compound 16. From this
intermediate, compound 8 was obtained as previously described.

Scheme 2. Synthesis of compounds 7a-c,h and 8. Reagents and conditions: (a) PTSH, benzaldehyde or 3-
pyridinecarboxaldehyde or cyclohexanecarboxaldehyde, K2CO3, EtOH, 80 °C, 12 h, 30-45%; (b) methyl 4-
(bromomethyl)benzoate, K2CO3, KI, DMF, 80 °C, 12 h, 20-74%; (c) NH2OH, KOH, DCM, MeOH, H2O, 25 °C, 3 h,
50-98%; (d) MeI/ propargyl bromide, NaH, THF, 0 to 25 °C, 12 h, 56% to quantitative yield.
In Scheme 3 is reported the synthesis of bromides 19 and 22 used as alkylating agents for the synthesis of
final compounds 7e, 7g and 7k. Following the procedure described by Liu et al. 4-pyridinecarboxaldehyde
(17) was reduced to alcohol 18 with NaBH4 and then converted to the corresponding bromide 19 after
treatment with PBr3 [27]. For the synthesis of the intermediate 22, 4-(bromomethyl)benzonitrile (20) was
reduced with DIBAL to the corresponding benzaldehyde 21, that was successively converted to dioxolane 22
with ethylene glycol in the presence of PTSA [28].
Scheme 3. Synthesis of bromo-derivatives 19 and 22. Reagents and conditions: (a) NaBH4, MeOH, 0 to 25 to 25 °C,
2 h, 80%; (b) 48% HBr, 100 °C, 4 h then PBr3, DCM, 45 °C, 4 h, 93%; (c) DIBAL, DCM, -78 to 0 °C, 1 h, quantitative
yield; (d) ethylene glycol, PTSA, toluene, 110 °C, 12 h, 69%.
In Scheme 4 the synthesis of final compounds 7d,e,i,j is described. Isatin was alkylated with benzyl bromide
or bromide 19 using NaH as the base to generate intermediates 23a,b [26]. These compounds were subjected
to the previously described ring expansion procedure with benzaldehyde or 3-pyridinecarboxaldehyde to
provide the desired 2-quinolones 24a-d that were converted to the final compounds 7d,e,i,j in the presence of
NH2OH.
Scheme 4. Synthesis of compounds 7d,e,i,j. Reagents and conditions: (a) BnBr or 19, NaH, DMF, 0 to 25 °C, 12 h,
38-74%; (b) PTSH, benzaldehyde or 3-pyridinecarboxaldehyde, K2CO3, EtOH, 80 °C, 12 h, 24-44%; (c) methyl 4-

(bromomethyl)benzoate, K2CO3, KI, DMF, 80 °C, 12 h, 62-30%; (d) NH2OH, KOH, DCM, MeOH, H2O, 25 °C, 3 h,
45-90%.
The synthesis of compound 7f is described in Scheme 5. Since alkylation of the isatin with 1-benzyl-4-bromo
or iodopiperidine failed in all the attempted conditions, N-alkylated isatin 28 was prepared starting from
piperidone (25) following a procedure described in a patent by Spear et al. [28]. 25 was first N-benzylated
and then subjected to a reductive amination protocol with aniline in presence of NaBH(OAc)3 providing
intermediate 27. This compound was treated with oxalyl chloride, obtaining an unstable intermediate that
was immediately treated with AlCl3 in DCM to afford isatin derivative 28 under Friedel-Craft conditions.
Ring expansion, alkylation, and final reaction with NH2OH were then performed as previously described,
leading to the formation of compound 7f.
Scheme 5. Synthesis of compound 7f. Reagents and Conditions: (a) Benzyl bromide, K2CO3, DMF, 80 °C, 12 h,
70%; (b) aniline, NaBH(OAc)3, DCM, AcOH, 0 to 25 °C, 12 h, 89%; (c) oxalyl chloride, DCM, 25 °C, 2 h; then AlCl3,
DCM, 40 °C, 2 h, 52%; (d) PTSH, benzaldehyde, K2CO3, EtOH, 80 °C, 12 h, 55%; (e) methyl 4-
(bromomethyl)benzoate, K2CO3, KI, DMF, 80 °C, 12 h, 15%; (f) NH2OH, KOH, DCM, MeOH, H2O, 25 °C, 3 h, 62%.
In Scheme 6 the synthesis of the final compounds 7g,k is described. Isatin was alkylated with bromide 22
affording intermediate 29. This compound was subjected to the previously described ring expansion
procedure to get the desired quinolones 30a,b. These intermediates were alkylated with methyl 4-
(bromomethyl)benzoate obtaining 31a,b which were subsequently deprotected in acidic medium, and the
corresponding free aldehydes were subjected to a reductive amination protocol with diethylamine in the
presence NaBH(OAc)3, affording the corresponding diethylamino derivatives, that were converted to the
final compounds 7g,k as previously described.

Scheme 6. Synthesis of compounds 7g,k. Reagents and conditions: (a) 22, NaH, DMF, 0 to 25 °C, 12 h, quantitative
yield; (b) PTSH, benzaldehyde or 3-pyridinecarboxaldehyde, K2CO3, EtOH, 80 °C, 12 h, 48%; (c) methyl 4-
(bromomethyl)benzoate, K2CO3, KI, DMF, 80 °C, 12 h, 55-80%; (d) 6 N HCl, THF, 25 °C, 1 h, quantitative yield; (e)
diethylamine, NaBH(OAC)3, DCM, AcOH, 0 to 25 °C, 12 h, 47-59%; (f) NH2OH, KOH, DCM, MeOH, H2O, 25 °C, 3
h, 74-80%.
3. Results and Discussion
The compounds herein developed were firstly tested in enzymatic assay to evaluate their HDAC6 inhibitory
profile compared with the HDAC1 and 8 isoforms. Successively, the ability of the best performing
compounds to induce the acetylation of histone, tubulin and HSP90 was evaluated, followed by assessment
of their anticancer properties in two cell lines (HCT-116 and U937). We have also determined key
pharmacokinetic parameters (solubility and chemical stability), toxicity and mutagenicity of selected
compounds.
Table 1. Novel HDAC6 inhibitors 6a-d, 7a-k and 8 against hHDAC1, as IC50 (µM), and hHDAC6, as IC50 (nM).a
Cpd
R
X
HDAC1 IC50
HDAC6 IC50
HDAC6/HDAC1

(µM)
11 ± 1
(nM)
H
CH
CH
308 ± 51
61.6 ± 7.5
3.6
73
6a
6b
Me
4.5 ± 0.31
CH
5.5 ± 0.3
84.5 ± 7.9
65
6c
50 96%
25 82%
10 36%
1 < 10%
6.6 ± 0.4
50 80%
25 46%
10 26%
1 < 10%
6.1 ± 0.9
50 97%
25 93%
10 76%
1 17%
H
H
N
295.6 ± 24.4
146.9 ± 14.2
n.d.
45
6d
7a
7b
7c
7d
CH
CH
CH
CH
Me
n.d.
74
518.9 ± 17.1
82.3 ± 11.4
176.0 ± 14.7
84.1 ± 9.9
n.d.
CH
CH
1.2 ± 0.2
14
7e
7f
50 79%
25 67%
10 46%
1 < 10%
870.4 ± 87.8
n.d.
CH
0.322 ± 0.073
6.9 ± 0.9
46
7g
Me
N
N
5.01 ± 0.44
2.5 ± 0.41
46 ± 5
109
96
7h
7i
25.6 ± 3.1
N
1.7 ± 0.31
45.3 ± 5.6
38
7j
N
0.319 ± 0.08
3.0 ± 0.18
11.5 ± 1.5
33.0 ± 2.6
29
91
7k
8
-
-
aEach value is the mean of at least three determinations; compounds were assayed at eight concentrations;
results are expressed with SD.
3.1. Structure-activity relationships (SAR)

From the enzymatic assay emerged a significant affinity of the molecules for the HDAC6 with selectivity
over HDAC1 and 8 isoforms (Tables 1 and 2). Starting from compound 6a (HDAC6 IC50 = 308 nM) we
decided to explore the effect of the introduction of alkyl groups such as methyl or benzyl substituent on the
oxygen at C3 (6b,c). These modifications led to a significant improvement in both the HDAC6 inhibition
potency (HDAC6 IC50 of 61 and 84 nM, for 6b and 6c respectively) and the selectivity over HDAC1
isoform. Successively we decided to explore the effect of replacing the 4-phenyl group with a more basic 3-
pyridyl group (compound 6d); this modification did not improve potency and led to a HDAC6 inhibition
(295 nM) similar to 6a. We then decided to interrogate the outcome of a switch of the acidic ZBG from the
N1 to the oxygen atom at C3; thus, we developed a series of analogues typified by 7a. This new prototypic
compound led to improved potency at HDAC6 over 6a (7a HDAC6 IC50 = 147 nM). Following exploration
of several substituents at N1 generated compounds 7b-g. This focused series of derivatives demonstrated that
the introduction of a basic lateral chain such as 4-pyridyl (7e) or N,N-diethylaminomethylbenzyl (7g)
determined a strong increase in HDAC6 inhibition (7e,g HDAC6 IC50 equal to 84 and 7 nM, respectively).
Also, it was shown that the introduction of large and/or alkyl substituents generated analogues with weaker
potency against HDAC6 (7b-d,f IC50 ranging from 82 to 870 nM). The substitution of the phenyl ring at C4
with a 3-pyridyl group (compounds 7h-k) resulted in a general increase of HDAC6 inhibition, when
compared to 7a, with IC50 values ranging from 12 to 46 nM. As observed for 7g, also compound 7k, bearing
a N,N-diethylaminomethylbenzyl at N1, showed the highest potency of this subgroup of analogues. From 7a,
the introduction of a methyl group at N1 and the replacement of the phenyl group at C4 with a cyclohexyl
group led to compound 8. This compound showed a good inhibitory profile for HDAC6 together with a
significant selectivity over HDAC1 (IC50 = 33 nM and selectivity index of 91).
In order to gain a full picture in terms of SAR analysis for the new series of compounds, we flanked in vitro
data with computational studies. Starting from the molecular docking calculations using Glide employing
Standard Precision (SP) as scoring function and visual inspection we were able to understand the general
trend of the preferred binding modes of quinolone derivatives on HDAC1 and HDAC6 isoforms (for more
details see the appropriate sections in the supplementary material file). Keeping on with the already
described inhibitors, for our newly developed derivatives we retained the para-substituted phenylhydroxamic
acid functionality, anchored to the benzyl linker, as the ZBG. Among the derivatives the polar contacts
established by the hydroxamic acid of the ZBG were similar and represented by three H-bonds established
between the carbonyl and Y782, the NH and G619, the OH and H610. Considering the specific contacts, we
primarily focused our attention to the best positioning of the ZBG on the quinolone scaffold (heterocyclic
nitrogen vs exocyclic hydroxyl group). Merging the results of the docking studies and the enzymatic tests we
observed that: when the ZBG is placed on the nitrogen atom as for compounds 6a-d the activity on HDAC6
slightly decreases when compared to the substitution on the oxygen atom (3-OH) (i.e. compounds 7a-k and
8). The binding mode of compounds is depicted in Figures 3 and Figures S1, S3. The whole orientation of
the scaffolds of compound 6a,b (Figures 3A,B), presented a similar binding mode, differing from that of the
other analogues as represented in Figures 3C-F and Figures S1-S3 (see details in the Supplementary Material

sections). Compound 7a (Figure 3C), with no substituents on N1, was able to establish two π-π stackings
with F620, F680 through the phenyl ring of the ZBG, and an additional H-bond between H651 and the
carbonyl of the quinolone core. The binding mode of compounds 7b-d,h,i,j is discussed in the
Supplementary Material section.

Figure 3. Docked poses into HDAC6 (PDB ID: 5EDU) of compounds 6a,b, 7a, 7g, 7k and 8 (panels A-F,
respectively). Compounds are represented as sticks. The residues of the active sites are represented as lines
and the whole protein is represented as cartoon. The Zn2+ ion is represented as a gray sphere. The polar
contacts are depicted as dotted lines while the coordination bond as plain lines. The pictures were generated
by means of PyMOL.
With the dual aim of increasing polar contacts within HDAC6 binding site while improving water solubility
of these molecules, we introduced some functionalities on the quinolone scaffold leading to compounds 6d,
7e-k bearing pyridine at C4, benzylpiperidine and diethylaminomethylbenzyl groups at N1 (Figures 3D-E for
compounds 7g and 7k). The introduction of a pyridin-3-yl instead of the phenyl ring at C4 (compound 6d,
Figure S1B), gave better results in terms of selectivity between the two investigated isoforms, compared to
the parent compound 6a. These findings prompted us to evaluate the introduction of the 4-methylenepyridine
system at N1 on the series of compound 7. Although establishing a polar contact through the nitrogen of the
pyridine with the backbone of F680, compound 7e (Figure S3A) did not show a satisfactory profile towards
the HDAC6 isoform, since a similar set of contacts were also established with the HDAC1 isoform. The
insertion of the different basic functionalities like the benzylpiperidine, as for compound 7f (Figure S3B),
resulted in an increase in the length of the molecule that precluded the formation of any relevant H-bond,
decreasing the HDAC6 inhibitory potency. On the contrary, the insertion of a diethylaminomethylbenzyl
portion at N1 as in compound 7g (Figure 3D) provided the most potent HDAC6 inhibitor, with a potency in
the low nanomolar range. As expected, the more flexible and protonatable diethylaminomethylbenzyl
substituent allowed to form not only a new and not previously observed H-bond with the D567 residue, but
also a strong salt bridge with the same residue. In summary, the introduction of a flexible and protonatable
moiety, able to establish a salt bridge and a polar contact with D567 in HDAC6, is a preferred choice to
improve the potency of enzyme inhibition. On the other hand, isoform selectivity was improved by
introducing at C4 the pyridin-3-yl, retaining a significant potency (7h-i, Figure S3C,D) and with over 100-
fold selectivity ratio towards HDAC6 over HDAC1. Compound 7k (Figure 2E) showed a different binding
mode compared to the other compounds of the series. With the introduction of the pyridine-4-yl, and
additional bond with H651 was established, maintaining the same inhibitory profile. Furthermore, taking into
consideration the hydrophobic residues present in the interaction site of HDAC6, we introduced a cyclohexyl
ring at C4 in place of the aromatic system, obtaining 8, the binding mode of which is shown in Figure 2F.
Owing to ring flexibility and lower steric hindrance of the methyl at N1 of 8, different residues were engaged
through hydrophobic interactions, namely F679, F680 and L749.
In addition, we performed molecular docking calculation for assessing the putative binding mode of the
developed compounds into HDAC1 enzyme. The output of this calculation is depicted in Figures S4-S6, and
the binding mode of all molecules is reported in the Supplementary Material.
3.2. hHDAC8 inhibition assays

Binding affinity studies towards hHDAC8 isoform were conducted on the best HDAC6 inhibitors (7g,k and
8) to have an indication of the potency and selectivity of these new quinolone-based derivatives also against
this isoform. Among the three tested derivatives, compound 7g demonstrated the highest selectivity with an
IC50 value of 0.55 µM (Table 2) and an index of 80 (HDAC8/HDAC6), while compound 8 was the least
potent with an IC50 value of 1.64 µM and a selectivity index of 50. These results highlighted a significant
selectivity profile of these compounds towards HDAC6.
Table 2. Inhibitory activity of compounds 7g,k and 8 as IC50 (µM), and apparent inhibitor constant (Kapp)
against the hHDAC8 enzyme
Cpd
IC50 (µM)a
Kapp (µM)a
7g
7k
8
TubA
0.695
n.d.b
23
0.55 ± 0.15
0.36 ± 0.1
80
0.49 ± 0.20
0.40 ± 0.1
42
1.64 ± 0.3
1.93 ± 0.2
50
HDAC8/HDAC6
aAll compounds were assayed at least two times, and the results are expressed with standard deviations.
dn.d. Not determined
3.3. Cell-based studies
The anticancer effect of compounds 7g and 7k, the best performing small-molecules emerged from the
HDAC6 in vitro experiments, and one of the starting hit 7a, were evaluated in cell-based MTT assays against
a colon cancer cell line (HCT-116) and a histiocytic lymphoma cell line (U937). All compounds showed a
good cytotoxic activity in HCT-116 when used at 50 µM for 24 h and 48 h of induction (Table 3 and Table
S1). However, 7g resulted to be the best molecule tested, with an IC50 of 3.6 µM after 48 h of induction,
causing a strong block of cell proliferation already at 25 µM (80%), and at 10 µM, after 24 and 48 h 50% and
90% respectively. Compound 7k, showed a potent cytotoxicity against HCT-116 cells after 48 h of induction
at all concentrations, showing 10 to 20% of cell survival. In accordance with HDAC enzymatic results,
compound 7a resulted the least potent molecule, showing the best results only when tested at 50 µM for 48 h
(10% cell viability). In U937 cells, these compounds were found to be less active, inducing the 60-70% of
cell proliferation reduction after 48 h at 50 µM (Table 3 and Table S2). Compound 7a was the most potent in
this cell line with respect to the other compounds.
Table 3. IC50 of 7a, 7k and 7g in HCT-116 and U937 cell lines.
IC50 (µM)
HCT-116
U937
NIH3T3
12 h
24 h
48 h
12 h
24 h
48 h
24 h
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
90
n.d.
90
6b
7a
7k
7g
33.4 ± 0.1
25.7 ± 0.25
11.6 ± 0.3
15.02 ± 0.12
8.4 ± 0.56
3.6 ± 0.29
27.9 ± 0.05
45.5 ± 0.37
40.6 ± 0.24
n.d.
13.5 ± 0.2
n.d.

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
18
8
n.d. Not determined
To assess the nature of cellular death occurring after 7a, 7g and 7k treatment, Poly(ADP-ribose)-Polymerase
(PARP) was evaluated in HCT-116 (Figure 4E,F) and U937 (Figure 5E,F) cell lines. A significant increase
of PARP cleavage levels was observed in the samples after 24 h of treatment with 7g and 7k, tested at 10 µM
, demonstrating the activation of apoptotic process [29].
Figure 4. Western blot analysis for cleaved PARP, AcTub, H3K9/14ac and Hsp90(AcLys294) on HCT-116
cancer cell line treated with the compounds 7a, 7g and 7k at 10 µM for 12 and 24 h. Tubastatin A (5 µM for
24 h) and SAHA (5 µM for 24 h) were the positive controls. GAPDH and H4 were used for the
normalization. Target levels were quantized using ImageJ software. **** p-value ≤ 0.0001, *** p-value ≤
0.001, ** p-value ≤ 0.01, * p-value ≤ 0.05, ns p-value > 0.05 vs control cells.

Figure 5. Western blot analysis for cleaved PARP, AcTub, H3K9/14ac and Hsp90(AcLys294) on U937
cancer cell line treated with the compounds 7a, 7g and 7k at 10 µM for 12 and 24 h. Tubastatin A (5 µM for
24 h) and SAHA (5 µM for 24 h) were the positive controls. GAPDH and H4 were used for the
normalization. Target levels were quantized using ImageJ software. **** p-value ≤ 0.0001, *** p-value ≤
0.001, ** p-value ≤ 0.01, * p-value ≤ 0.05, ns p-value > 0.05 vs control cells.
To evaluate the capability of the compounds 7a, 7g and 7k to inhibit the HDACs, Western blot studies were
conducted in both cell lines analyzing the acetylation levels of alpha-tubulin and of the lysine 9 and 14 of
histone 3 (Figure 4 and 5). All molecules, tested at 10 µM, strongly enhanced the levels of AcTub in HCT-
116 (Figure 4C) and U937 (Figure 5C), with a fold ≥ 9, indicating the inhibition of cytoplasmic HDACs. The
analysis of histone acetylation levels showed that the amount of H3K9/14ac increased after 7a, 7g and 7k
treatment, respect to the control, suggesting a possible inhibition of nuclear HDACs may have also occurred.
However, in HCT-116 (Figure 4D) compounds 7a and 7g appeared to be more selective for cytoplasmic
HDACs, increasing the H3K9/14ac of 2/4-fold, compared to 7k (> 6-fold). In U937 cells (Figure 5D) only
compound 7a showed a low potency against nuclear HDACs.
The interplay between HDAC6 and Hsp90 has been widely demonstrated in the last decade. It was observed
that the over-acetylation of Hsp90, due to the inactivation or knockdown of HDAC6, determined the loss of
chaperone activity of this protein [30]. Therefore, we decided to evaluate Hsp90(AcLys294) after treatment
with 7a, 7g, and 7k. In HCT-116 (Figure 4E, F), but not in U937 (Figure 5E, F), 5, 7g and 7k increased the
levels of Hsp90(AcLys294) when tested at 10 µM for 12 or 24 h, suggesting the inhibition of HDAC6. In
U937, instead, only 7a raised the Hsp90ac levels respect to the control (Figure 5).
3.4. In silico prediction of selected drug-like properties and experimental evaluation of physico-
chemical properties

We predicted the ADME properties and the heart toxicity potential of the newly developed compounds by
means of QikProp, a software implemented in Maestro suite (QikProp, version 4.3, Schrödinger, LLC, New
York, NY, 2015). The output of the calculation is reported in Table S3. The predicted values for every
molecule can be considered acceptable since they were within the recommended range of the software.
Selected parameters were also assessed experimentally. The in vitro solubility and chemical stability of
selected compounds 6b, 7e, and 7k were measured by means of HPLC methods as reported in previous work
from us [31,32]. As expected, the presence of basic groups on compounds 7e,k improved the solubility
profile at pH = 3 with respect to compound 6b. Gratifyingly, our analysis also revealed that both 7e,k
exhibited a favorable chemical stability profile at acidic pH. In particular, the chemical stability was almost
quantitative for the selected compounds with more than 95% of the compound remaining unaltered at pH = 3
after 24 h (Table S4).
3.5. Mutagenicity profile evaluation for 6b, 7k, and 8.
Potential mutagenicity associated with the use of hydroxamic acid-based compounds poses a significant
challenge in terms of their drug-like profile [33]. To date, givinostat is the only compound under clinical
trials that exhibited no mutagenic effect, while the other FDA approved drugs 1-4 have shown mutagenicity
[34]. Therefore, we investigated for compounds 6b, 7k and 8 the mutagenic effect in the Salmonella
typhimurium strains TA98 and TA100. The Ames test is employed to detect potential risks of mutagenicity at
the early stages of drug development. The assay can be performed with or without the S9 fraction of rat liver.
This latter condition allows an in-depth investigation for evaluating the risk of mutagenicity derived from the
metabolites of the compounds under study. After applying both the experimental conditions, no mutagenic
effect was observed for compounds 6b, 7k and 8 in the Salmonella typhimurium strains TA98 and TA100 at
all concentrations (5-230 µM) (Figures S7-S9).
3.6. Lossen rearrangement study on compound 7k.
It has been observed that the leading cause of mutagenicity associated with the use of HDACi is related to a
Lossen’s rearrangement involving the hydroxamate group with the formation of the corresponding
isocyanate. This intermediate can trigger mutagenicity by damaging the DNA due to its susceptibility to
undergo nucleophilic attack [34]. In laboratory, this reaction can be performed on O-activated hydroxamic
acids under basic conditions. However, under physiological conditions, it has been reported that in case of 2-
naphthohydroxamic acid this reaction occurs by the involvement of acetyl-CoA, which activates the oxygen
by the addition of an acetyl group [35]. Because of the low acidity of hydroxamic acids, it has been
speculated that hydroxamate-based HDACi can reach the catalytic site as neutral molecules and get
deprotonated only after a strong metal chelation (e.g. zinc and potassium) [34,36]. In this way, a metal-
assisted Lossen’s rearrangement may occur leading to the formation of the isocyanate.
In order to check the susceptibility of our compounds to undergo Lossen’s rearrangement we investigated 7k
in terms of the formation of the corresponding isocyanate (by checking the conversion to its aniline

counterpart) by exposing it to one equivalent of K2CO3 in DMSO at 40 °C (Figures S10, S11). After 18 h we
observed by ESI-MS analysis that the predominant peaks were: the starting material (7k, [M+ H]+ = 563.2)
and the corresponding carboxylic acid ([M + H]+ = 548.2, Figure S11). From this study we can assume that
the absence of mutagenicity detected in the Ames assay can be explained by a low vulnerability of our
compounds to undergo Lossen’s rearrangement, thus confirming a safe profile for the HDACi described in
this paper.
3.7. Cytotoxic profile evaluation for 6b, 7g ,7k and 8.
Compounds 6b, 7k and 8 were further characterized in a cytotoxicity assay to establish their effect on mouse
fibroblasts NIH3T3 cells. Cell viability was measured by the Neutral Red Uptake test and data normalized as
% control. TC50 values of 90 µM were observed for 6b, 7k and 18 µM for 8, thus indicating a safe profile in
normal cells for compounds 6b and 7k, while a low cytotoxicity in NIH3T3 cells was observed with
compound 8 (Table 3 and Table S5). A second cytotoxicity study was performed on human peripheral blood
mononuclear cells (PBMC) after the treatment with compounds 7g and 7k for 24 and 48 h. Cell viability was
measured by using Guava® ViaCount™ Reagent. From cell viability study, was observed that compound
7k, after 24 h treatment, exhibited a low cytotoxic action only when tested at 90 µM, thus confirming a safe
profile for this compound. On the other hand, compound 7g, after 24 h treatment, demonstrated a strong
reduction in cell viability already when tested at 45 µM (Figure S12, panel A). No significant difference in
cytotoxicity were observed when the cells were treated for longer times (48 h, Figure S12, panel B).
4. Conclusion
In this work we describe the synthesis of quinolone-based HDAC inhibitors preferentially targeting the
isoform 6. The synthesis of the cap group was achieved by applying a multicomponent reaction involving
several substituted isatins, PTSH and a suitable aldehyde. This versatile synthetic strategy could be applied
for future scaffold modifications and for the development of optimized analogs. The design of the described
molecules was facilitated by applying a computational chemistry approach. The final compounds obtained
showed inhibition values in the nanomolar range for HDAC6 with a selectivity index up to 109-fold for
HDAC6 over HDAC1. Moreover, selected analogues showed a weak affinity for the hHDAC8 enzyme. The
most potent HDAC6 inhibitors were subjected to cell-based assays to check their anticancer potential. In
particular, compound 7g gave the best result in terms of cytotoxicity against colon cancer cells, inducing
90% cell death after 48 h at 10 µM, and after 12 h when tested at 50 and 25 µM, by triggering apoptosis. The
influence of the compounds on tubulin, histone and Hsp90 acetylation status was evaluated to establish their
selectivity towards HDAC6. Notably, all the tested compounds enhanced the levels of AcTub as a result of
the inhibition of cytoplasmic HDACs. In HCT-116, 7a and 7g showed a lower increase of H3K9/14ac, thus
indicating a lower selectivity for nuclear HDACs. Additionally, compounds 7g and 7k led to the increase of
Hsp90(AcLys294), a selective substrate of HDAC6. By applying HPLC protocols, compound 7k was found
soluble and stable in aqueous solution at pH = 3. Compounds 6b, 7k and 8 did not display any cytotoxic or

mutagenic profile as assessed by cytotoxicity studies on mouse fibroblasts and Ames test on Salmonella
typhimurium strains. The absence of mutagenicity was further confirmed by the low susceptibility of
compound 7k to undergo Lossen’s rearrangement to generate its isocyanate derivative. Taken together, these
data highlight a promising antitumor potential for these quinolone-based HDAC6 selective inhibitors. These
evidences may pave the way to the development of more potent and selective optimized hits useful for a H2L
transition and for further biological investigation.
5. Experimental data
5.1. General information
Unless otherwise specified, materials were purchased from commercial suppliers and used without further
purification. Reaction progress was monitored by TLC using silica gel 60 F254 (0.040–0.063ꢀmm) with
1
13
detection by UV. Silica gel 60 (0.040–0.063ꢀmm) was used for column chromatography. H NMR and C
NMR spectra were recorded on a Varian 300ꢀMHz spectrometer or a Bruker 400ꢀMHz spectrometer by using
the residual signal of the deuterated solvent as internal standard. Splitting patterns are described as singlet
(s), doublet (d), triplet (t), quartet (q) and broad (br); the values of chemical shifts (δ) are given in ppm and
coupling constants (J) in Hertz (Hz). HPLC were performed with a Shimadzu Prominence apparatus
equipped with a scanning absorbance UV-VIS detector (Diode Array SPD-M20A) also equipped with a
thermostatic chamber or with Agilent 1100 Series equipped with UV-VIS detector. ESI-MS spectra were
performed by an Agilent 1100 Series LC/MSD spectrometer. HRESIMS were carried out by a Thermo
Finningan LCQ Deca XP Max ion-trap mass spectrometer equipped with Xcalibur software, operated in
positive ion mode. Infrared (IR) spectra were recorded in the neat on an Agilent Cary 630 Spectrometer FT-
IR instrument and are reported in reciprocal centimeters (cm−1). Melting points were detected by a BÜCHI
melting point B-450 and reported and °C. The yields are referred to purified products and are not optimized.
All moisture-sensitive reactions were performed under argon atmosphere using oven-dried glassware and
anhydrous solvents. Final compounds were analyzed by combustion analysis (C, H, N) to confirm purity
>95%.
5.2. Chemistry
5.2.1 Methyl 4-((2,3-dioxoindolin-1-yl)methyl)benzoate (10).
To a solution of isatin (300 mg, 2.03 mmol) in dry DMF, cooled at 0 °C, a 60% dispersion of NaH, in
mineral oil (95 mg, 2.38 mmol) was added and stirred for 5 min at 0 °C. Then, methyl 4-
(bromomethyl)benzoate (2.34 g, 10.20 mmol) was added and the reaction was allowed to reach 25 °C and
stirred for 12 h. Thereafter, a saturated solution of NH4Cl (5 mL) was added and the mixture was extracted
with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4 and
concentrated in vacuo. The residue was used in the next step without further purifications and affording 10
(2.00 g, quantitative yield) as a red solid. 1H NMR (300 MHz, CDCl3) δ 8.02 (d, J = 8.3 Hz, 2H), 7.63 (dd, J

= 7.5, 0.8 Hz, 1H), 7.48 (td, J = 7.8, 1.3 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.11 (td, J = 7.6, 0.8 Hz, 1H), 6.70
(d, J = 7.9 Hz, 1H), 4.98 (s, 2H), 3.90 (s, 3H). ESI-MS m/z: [M + H]+ 296.0.
5.2.2. Ethyl 4-((3-hydroxy-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)benzoate (11a).
A solution of benzaldehyde (379 mg, 3.57 mmol) and PTSH (665 mg, 3.57 mmol) in EtOH (17 mL) was
stirred at 25 °C for 2 h. After this, 10 (575 mg, 3.57 mmol) and K2CO3 (987 mg, 7.14 mmol) were added and
the reaction was stirred at 80 °C for 12 h. The solvent was evaporated, and the crude was purified by
1
chromatography on silica gel (20% EtOAc in petroleum ether) giving 11a (430 mg, 64%) as a red solid. H
NMR (300 MHz, CDCl3) δ 8.02 (d, J = 8.3 Hz, 2H), 7.65 – 6.99 (m, 11H), 5.74 (s, 2H), 4.49 – 4.25 (m, 2H),
1.37 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 166.2, 159.3, 140.8, 140.7, 133.6, 132.8, 130.8, 130.2,
129.9, 129.7, 128.6, 128.4, 127.5, 126.5, 124.2, 123.3, 122.3, 114.9, 61.0, 46.9, 14.3; ESI-MS m/z: [M + H]+
400.1; mp 187.3-190.0 °C.
5.2.3. Ethyl 4-((3-hydroxy-2-oxo-4-(pyridin-3-yl)quinolin-1(2H)-yl)methyl)benzoate (11b).
Starting from 10 (100 mg, 0.33 mmol) the title compound was obtained following the procedure described to
get 11a. The residue was purified by chromatography on silica gel (2% MeOH in DCM) affording 11b (43
1
mg, 32%) as a red solid. H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.57 (s,
1H), 7.92 (d, J = 8.6 Hz, 2H), 7.83 (dd, J = 7.8, 1.9 Hz, 1H), 7.56 (dd, J = 7.8, 4.8 Hz, 1H), 7.44 – 7.23 (m,
4H), 7.21 – 7.00 (m, 2H), 5.74 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); ESI-MS m/z: [M +
H]+ 401.1.
5.2.4. N-Hydroxy-4-((3-hydroxy-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)benzamide (6a).
To a solution of 11a (55 mg, 0.14 mmol) in a 2:1 mixture of DCM (4 mL): MeOH (2 mL), a 50% solution of
NH2OH in water (660 µL, 10.00 mmol) and a 4 M solution of KOH in methanol (1.25 mL, 5.00 mmol) were
added dropwise. The reaction was stirred at 25 °C for 3 h, then it was neutralized with 6 N HCl and
concentrated in vacuo. The residue was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH
in DCM) affording 6a (44 mg, 82%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.36
13
(s, 1H), 8.98 (s, 1H), 7.75 – 7.24 (m, 11H), 7.11 (d, J = 4.1 Hz, 2H), 5.69 (s, 2H); C NMR (75 MHz,
DMSO-d6) δ 164.4, 158.9, 142.0, 140.2, 134.0, 133.9, 132.2, 130.4, 128.9, 128.2, 127.8, 127.3, 127.0, 125.7,
124.3, 123.1, 122.2, 115.6, 45.9; ESI-MS m/z: [M - H]- 385.0. FT-IR (neat) νmax 3375, 3291, 3036, 1616,
1566, 1496, 1257, 1015 cm-1; mp 192.7-195.8 °C, decomposition; Anal. (C23H18N2O4) C, H, N.
5.2.5. N-Hydroxy-4-((3-hydroxy-2-oxo-4-(pyridin-3-yl)quinolin-1(2H)-yl)methyl)benzamide (6d).
Starting from 11b (20 mg, 0.05 mmol) the title compound was obtained following the procedure described to
get 6a. The residue was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH in DCM)
affording 15 (16 mg, 84%). 1H NMR (300 MHz, DMSO-d6) δ 11.15 (s, 1H), 9.74 (s, 1H), 8.99 (s, 1H), 8.65
(d, J = 3.5 Hz, 1H), 8.57 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.62 – 7.50 (m, 1H), 7.46

13
– 7.24 (m, 4H), 7.22 – 6.96 (m, 2H), 5.70 (s, 2H); C NMR (75 MHz, DMSO-d6) δ 164.3, 158.7, 150.8,
149.4, 142.9, 140.1, 138.2, 133.9, 132.3, 130.0, 127.8, 127.5, 127.0, 125.3, 124.0, 123.4, 121.9, 121.0, 115.7,
46.0; ESI-MS m/z: [M - H]+ 386.0. Anal. (C22H17N3O4) C, H, N.
5.2.6. Ethyl 4-((3-methoxy-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)benzoate (12a).
To a solution of 11a (100 mg, 0.25 mmol) in dry THF (3 mL) cooled at 0 °C, a 60% dispersion of NaH in
mineral oil (15 mg, 0.38 mmol) was added. The mixture was stirred at 0 °C for 30 min, then MeI (78 µL,
1.25 mmol) was added. The reaction was allowed to reach 25 °C and stirred for 12 h. After this time a
saturated solution of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL). The
combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by
chromatography on silica gel (25% EtOAc in Petroleum ether) affording 12a (30 mg, 29%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.11 – 7.90 (m, 2H), 7.58 – 7.44 (m, 3H), 7.44 – 7.14 (m, 7H), 7.15 – 6.99 (m,
1H), 5.69 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H); ESI-MS m/z: [M + H]+
414.0.
5.2.7. Ethyl 4-((3-(benzyloxy)-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)benzoate (12b).
To a solution of 11a (50 mg, 0.125 mmol) and benzyl bromide (16 µL, 0.137 mmol) in anhydrous DMF (2
mL) was added K2CO3 (34 mg, 0.25 mmol) followed by KI (2 mg, 0.01 mmol). Reaction was refluxed for 8
h at 80 °C. Reaction mixture was then partitioned between aqueous saturated NH4Cl and EtOAc, and the
organic layer was washed with a saturated solution of NaHCO3 (5 mL) and brine (5 mL), dried over Na2SO4
and concentrated in vacuo. The residue was purified by chromatography on silica gel (25% EtOAc in
petroleum ether) affording 12b (39 mg, 64%) as a pale-yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.03 (d, J =
8.3 Hz, 2H), 7.56 – 7.40 (m, 3H), 7.40 – 7.13 (m, 10H), 7.13 – 6.94 (m, 3H), 5.73 (s, 2H), 5.15 (s, 2H), 4.37
(q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H); ESI-MS m/z: [M + H]+ 490.0.
5.2.8. N-Hydroxy-4-((3-methoxy-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)benzamide (6b).
Starting from 12a (30 mg, 0.07 mmol) the title compound was obtained following the procedure described to
get 6a. The residue was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH in DCM)
1
affording 6b (28 mg, 97%) as a white solid. H NMR (300 MHz, CDCl3) δ 11.15 (s, 1H), 9.00 (s, 1H), 7.69
(d, J = 7.9 Hz, 2H), 7.61 – 7.21 (m, 9H), 7.21 – 6.95 (m, 2H), 5.65 (s, 2H), 3.71 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) δ 164.4, 158.8, 144.8, 140.3, 137.7, 136.4, 133.7, 132.2, 129.7, 129.5, 128.9, 128.6, 127.7, 127.2,
+
127.0, 123.0, 121.1, 115.6, 60.1, 45.6; ESI-MS m/z: [M + H]+ 401.0; HRMS-ESI m/z: calcd for C24H21N2O4
[M+H]+: 401,1496; found: 401.1488; FT-IR (neat) νmax 3217, 2920, 2854, 1719, 1630, 1595, 1456, 1259,
1091, 1016 cm-1; mp 161.0-164.0 °C, decomposition; Anal. (C24H20N2O4) C, H, N.
5.2.9. 4-((3-(Benzyloxy)-2-oxo-4-phenylquinolin-1(2H)-yl)methyl)-N-hydroxybenzamide (6c).

Starting from 12b (27 mg, 489.56 mmol) the title compound was obtained following the procedure described
to get 6a. The residue was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH in DCM)
1
affording 6c (22 mg, 85%) as a light brown solid. H NMR (300 MHz, DMSO-d6) δ 11.16 (s, 1H), 9.01 (s,
1H), 7.70 (d, J = 7.9 Hz, 2H), 7.59 – 6.81 (m, 16H), 5.70 (s, 2H), 5.07 (s, 2H). 13C NMR (75 MHz, DMSO-
d6) δ 164.4, 159.1, 143.4, 140.3, 138.3, 137.3, 136.5, 133.6, 132.2, 129.8. 129.6, 128.8, 128.6 (2C), 128.5,
128.3, 127.7, 127.2, 127.1, 123.0, 121.2, 115.6, 73.5, 45.6; ESI-MS m/z: [M + H]+ 476.9; Anal. (C30H24N2O4
)
C, H, N.
5.2.10. 3-Hydroxy-4-phenylquinolin-2(1H)-one (13a).
A solution of benzaldehyde (100 mg, 0.94 mmol) and PTSH (175 mg, 0.94 mmol) in EtOH (5 mL) was
stirred at 25 °C for 2 h. After this, isatin (138 mg, 0.94 mmol) and K2CO3 (260 mg, 1.88 mmol) were added
and the reaction was stirred at 80 °C for 12 h. The solvent was evaporated, and the crude was purified by
chromatography on silica gel (20% EtOAc in petroleum ether) giving 13a (95 mg, 42%) as a brown solid.
1HNMR (300 MHz, DMSO-d6) δ 12.19 (s, 1H), 9.16 (s, 1H), 7.57 – 7.35 (m, 3H), 7.34 – 7.18 (m, 4H), 7.09
– 6.94 (m, 2H); ESI-MS m/z: [M + Na]+ 260.1.
5.2.11. 3-Hydroxy-4-(pyridin-3-yl)quinolin-2(1H)-one (13b).
Starting from 3-pyridinecarboxaldehyde (181 mg, 1.69 mmol) and isatin (500 mg, 1.69), the title compound
was obtained following the procedure previously described for compound 13a. The crude was purified by
1
chromatography on silica gel (5% MeOH in DCM) giving 13b (180 mg, 45%) as a red solid. H NMR (300
MHz, DMSO-d6) δ 12.27 (s, 1H), 9.49 (s, 1H), 8.62 (dd, J = 4.8, 1.7 Hz, 1H), 8.53 (dd, J = 2.2, 0.8 Hz, 1H),
7.85 – 7.71 (m, 1H), 7.62 – 7.46 (m, 1H), 7.42 – 7.25 (m, 2H), 7.17 – 7.05 (m, 1H), 7.05 – 6.94 (m, 1H);
ESI-MS m/z: [M + H]+ 239.0.
5.2.12. Methyl 4-(((2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (14a).
To a solution of 13a (100 mg, 0.42 mmol) in dry DMF (4 mL) methyl 4-(bromomethyl)benzoate (145 mg,
0.63 mmol), KI (7 mg, 0.04 mmol) and K2CO3 (116 mg, 0.84 mmol) were added. The reaction mixture was
stirred at 80 °C for 12 h. Thereafter the mixture was cooled down to 25 °C and a saturated solution of NH4Cl
(10 mL) was added. The mixture was extracted with EtOAc (10 mL) and the organic layer was washed with
a saturated solution of NaHCO3 (5 mL) and brine (5 mL). Then it was dried over Na2SO4 and concentrated in
vacuo. The crude was purified by chromatography on silica gel (33% EtOAc in petroleum ether) giving 14a
(61 mg, 38%) as a pale brown solid. 1H NMR (300 MHz, CDCl3) δ 12.95 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H),
13
7.57 – 7.35 (m, 6H), 7.33 – 7.01 (m, 5H), 5.24 (s, 2H), 3.89 (s, 3H); C NMR (75 MHz, CDCl3) δ 167.0,
161.0, 143.7, 142.2, 140.3, 140. 3, 135.5, 133.2, 129.7, 129.4, 129.1, 128.7, 128.3, 127.9, 126.7, 122.8,
120.8, 115.9, 73.4, 57.0; ESI-MS m/z: [M + H]+ 386.1; m.p,: 195.0-197.9 °C.
5.2.13. Methyl 4-(((2-oxo-4-(pyridin-3-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (14b).

Starting from 13b (180 mg, 0.76 mmol) the title compound was obtained following the procedure previously
described for compound 14a. The crude was purified by chromatography on silica gel (50% EtOAc in
petroleum ether) giving 14b (60 mg, 20%) as a dark yellow oil. 1H NMR (300 MHz, CDCl3) δ 12.82 (s, 1H),
8.71 (dd, J = 5.0, 1.7 Hz, 1H), 8.61 – 8.47 (m, 1H), 7.98 – 7.81 (m, 2H), 7.64 – 7.32 (m, 4H), 7.24 – 7.01 (m,
4H), 5.45 – 5.15 (m, 2H), 3.89 (s, 3H); ESI-MS m/z: [M + H]+ 387.0.
5.2.14. N-Hydroxy-4-(((2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)oxy)methyl)benzamide (7a).
To a solution of 14a (19 mg, 0.049 mmol) in a 2:1 mixture of DCM (2 mL): MeOH (1 mL), a 50% solution
of NH2OH in water (329 µL, 4.90 mmol) and a 4 M solution of KOH in methanol (616 µL, 2.45 mmol) were
added dropwise. The reaction was stirred at 25 °C for 3 h, then it was neutralized with 6 N HCl and
concentrated in vacuo. The crude was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH
1
in DCM) giving 7a (11 mg, 55%) as a white solid. H NMR (300 MHz, DMSO-d6) δ 12.13 (s, 1H), 11.11 (s,
1H), 8.96 (s, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.50 – 7.30 (m, 5H), 7.28 – 7.14 (m, 2H), 7.12 – 7.00 (m, 3H),
7.00 – 6.88 (m, 1H), 5.07 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.3, 159.1, 144.2, 140.6, 138.4, 136.3,
133.7, 132.5, 129.8, 129.1, 128.7, 128.5, 128.0, 127.1, 126.2, 122.5, 120.3, 115.6, 72.8; ESI-MS m/z: [M +
H]+ 387.1; FT-IR (neat) νmax 3204, 3048, 2858, 1653, 1549, 1279, 1202, 1160, 989 cm-1; mp 204.8-205.4 °C,
decomposition; Anal. (C23H18N2O4) C, H, N.
5.2.15. N-Hydroxy-4-(((1-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)oxy)methyl)benzamide (7b).
Step 1: To a solution of 14a (16 mg, 0.04 mmol) in dry THF (1 mL) cooled at 0 °C, a 60% dispersion of
NaH in mineral oil (15 mg, 0.06 mmol) was added. The mixture was stirred at 0 °C for 30 min then MeI (8
µL, 0.12 mmol) was added. The reaction was allowed to reach 25 °C and stirred at this temperature for 12 h.
Then a saturated solution of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL).
The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The
crude was purified by chromatography on silica gel (50% EtOAc in petroleum ether) affording methyl 4-(((1-
methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (16 mg, quantitative yield) as a white
solid. 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 6.8 Hz, 2H), 7.57 – 7.32 (m, 5H), 7.32 – 7.17 (m, 3H), 7.17
– 6.95 (m, 3H), 5.13 (s, 2H), 3.95 – 3.79 (m, 6H); ESI-MS m/z: [M + H]+ 400.1.
Step 2: From methyl 4-(((1-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (22 mg,
0.05 mmol) the title compound was obtained following the procedure previously described for compound 7a.
The crude was purified by chromatography on silica gel (0.1% NH4OH, 10 % MeOH in DCM) giving 7b (14
mg, 64%). 1H NMR (300 MHz, DMSO-d6) δ 11.14 (s, 1H), 8.97 (s, 1H), 7.58-7.47 (m, 7H), 7.35 – 7.13 (m,
13
3H), 7.06 (dd, J = 8.2, 2.4 Hz, 3H), 5.04 (s, 2H), 3.76 (s, 3H) C NMR (75 MHz, MeOD) δ 166.4, 159.7,
143.0, 140.7, 139.2, 137.0, 133.2, 131.5, 129.4, 129.3, 128.1, 128.0, 127.9, 126.9, 126.5, 122.5, 121.0, 114.5,
73.0, 29.4; ESI-MS m/z: [M + H]+ 401.0; Anal. (C24H20N2O4) C, H, N.

5.2.16. N-Hydroxy-4-(((2-oxo-4-phenyl-1-(prop-2-yn-1-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzamide
(7c).
Step 1: To a solution of 14a (50 mg, 0.13 mmol) in dry DMF cooled at 0 °C, NaH (60% in mineral oil, 7 mg,
0.194) was added. The reaction was allowed to stir for 30 mins at the same temperature, then propargyl
bromide (19 µL, 0.129 mmol) was added. The reaction was allowed to reach 25 °C and stirred for 12 h. After
this time, a saturated solution of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5
mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo.
The crude was purified by chromatography on silica gel (25% EtOAc in petroleum ether) affording methyl 4-
(((2-oxo-4-phenyl-1-(prop-2-yn-1-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (31 mg, 56% yield) as a
1
brown solid. H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 7.5 Hz, 2H), 7.63 – 7.37 (m, 5H), 7.34 – 7.19 (m,
3H), 7.19 – 7.11 (m, 1H), 7.08 (d, J = 8.0 Hz, 2H), 5.23 (d, J = 2.5 Hz, 2H), 5.13 (s, 2H), 3.88 (s, 3H), 2.32
(s, 1H); ESI-MS m/z: [M + H]+ 424.1
Step 2: From methyl 4-(((2-oxo-4-phenyl-1-(prop-2-yn-1-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate
(30 mg, 0.07 mmol) the title compound was obtained following the procedure previously described for
compound 7a. The crude was purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH in DCM)
giving 7c (17 mg, 57%) as a brown solid. 1H NMR (300 MHz, MeOD) δ 11.13 (s, 1H), 8.97 (s, 1H), 7.73 –
7.53 (m, 4H), 7.53 – 7.39 (m, 3H), 7.35 – 7.13 (m, 3H), 7.15 – 6.96 (m, 3H), 5.23 (s, 2H), 5.05 (s, 2H), 3.31
(s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 164.3, 158.0, 143.4, 140.5, 138.4, 135.9, 133.4, 132.5, 129.7,
128.8 (2C), 128.7, 128.0, 127.3, 127.1, 123.3, 121.0, 115.6, 79.3, 75.3, 73.1, 32.2; ESI-MS m/z: [M + H]+
425.0. mp decomposition; Anal. (C26H20N2O4) C, H, N.
5.2.17.
N-Hydroxy-4-(((1-methyl-2-oxo-4-(pyridin-3-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzamide
(7h).
Starting from 14b (15 mg, 0.76 mmol) the title compound was obtained following the procedure previously
described for compound 7b, Step 1. The crude was purified by chromatography on silica gel (33% EtOAc in
petroleum
ether)
giving
methyl
4-(((1-methyl-2-oxo-4-(pyridin-3-yl)-1,2-dihydroquinolin-3-
yl)oxy)methyl)benzoate (10 mg, 66%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 8.68 (dd, J = 4.9, 1.7
Hz, 1H), 8.48 (dd, J = 2.2, 0.9 Hz, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.62 – 7.32 (m, 4H), 7.23 – 7.03 (m, 4H),
5.31 – 5.06 (m, 2H), 3.89 (s, 3H), 3.86 (s, 3H); ESI-MS m/z: [M + H]+ 401.1. Starting from methyl 4-(((1-
methyl-2-oxo-4-(pyridin-3-yl)-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (15 mg, 0.076 mmol) the title
compound was obtained following the procedure previously described for compound 7a. The crude was
purified by chromatography on silica gel (0.1% NH4OH, 10% MeOH in DCM) giving 7h (5 mg, 50%) as a
white solid. 1H NMR (300 MHz, MeOD) δ 8.61 (d, J = 4.8 Hz, 1H), 8.32 (s, 1H), 7.74 – 7.45 (m, 6H), 7.30 –
13
7.01 (m, 4H), 5.12 (d, J = 4.9 Hz, 2H), 3.88 (s, 3H); C NMR (75 MHz, MeOD) δ 166.3, 159.2, 149.3,
148.3, 143.4, 140.2, 138.4, 137.1, 134.8, 131.8, 130.1, 129.5, 128.2, 126.6, 126.2, 123.6, 122.8, 120.2, 114.7,
72.8, 29.2; ESI-MS m/z: [M + Na]+ 424.0; Anal. (C23H19N3O4) C, H, N.

5.2.18. 1-Methylindoline-2,3-dione (15) [26].
To a solution of isatin (1.0 g, 6.80 mmol) in dry DMF (4 mL), cooled at 0 °C, a 60% dispersion of NaH, in
mineral oil (275 mg, 6.80 mmol) was added and stirred for 5 min at 0 °C. Then, methyl iodide (635 µL,
10.20 mmol) was added and the reaction was allowed to reach 25 °C and stirred for 12 h. Thereafter, a
saturated solution of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL). The
combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude
was purified by chromatography on silica gel (25% EtOAc in petroleum ether) affording 15 (1.15 g,
1
quantitative yield) as a red solid. H NMR (300 MHz, CDCl3) δ 7.69 – 7.52 (m, 2H), 7.12 (td, J = 7.6, 0.9
Hz, 1H), 6.89 (dd, J = 8.6, 0.9 Hz, 1H), 3.25 (s, 3H). The experimental data are consistent with those
reported in literature [26].
5.2.19. 4-Cyclohexyl-3-hydroxy-1-methylquinolin-2(1H)-one (16).
Starting from 15 (575 mg, 3.57 mmol) and cyclohexanecarboxaldehyde (431 µL, 3.57 mmol) the title
compound was obtained following the procedure described to get 13a. The residue was purified by
chromatography on silica gel (20% EtOAc in petroleum ether) affording 16 (270 mg, 30%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 8.2 Hz, 1H), 7.54 – 7.20 (m, 4H), 3.82 (s, 3H), 3.16 (m, 1H), 2.35
– 2.12 (m, 2H), 1.99 – 1.83 (m, 2H), 1.75 (m, 4H), 1.42 (m, 2H); ESI-MS m/z: [M + H]+ 258.1.
5.2.20. 4-(((4-Cyclohexyl-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)oxy)methyl)-N-hydroxybenzamide (8).
Starting from 16 (270 mg, 1.05 mmol) methyl 4-(((4-cyclohexyl-1-methyl-2-oxo-1,2-dihydroquinolin-3-
yl)oxy)methyl)benzoate was obtained following the procedure described to get 14a. The residue was purified
by chromatography on silica gel (33% EtOAc in petroleum ether) affording methyl 4-(((4-cyclohexyl-1-
1
methyl-2-oxo-1,2-dihydroquinolin-3-yl)oxy)methyl)benzoate (317 mg, 74%) as a yellow oil. H NMR (300
MHz, CDCl3) δ 8.15 – 8.01 (m, 2H), 7.96 (s, 1H), 7.61 (d, J = 7.9 Hz, 2H), 7.54 – 7.40 (m, 1H), 7.33 (dd, J =
8.6, 1.2 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 5.26 (s, 2H), 3.89 (s, 3H), 3.73 (s, 3H), 3.20 (d, J = 13.8 Hz, 1H),
2.16 – 1.93 (m, 2H), 1.89 – 1.52 (m, 5H), 1.48 – 1.07 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 167.0, 159.1,
142.9, 140.9, 137.3, 129.7, 129.5, 128.6, 127.9, 126.4, 124.6, 122.1, 120.4, 114.6, 72.7, 52.1, 38.4, 30.6,
29.9, 27.2, 26.1; ESI-MS m/z: [M + H]+ 406.1; mp 79.1-82.2 °C.
This intermediate (100 mg, 0.25 mmol) was converted into compound 8 following the procedure previously
described for the synthesis of compound 7a. The residue was purified by chromatography on silica gel (0.1%
NH4OH, 10% MeOH in DCM) affording 8 (100 mg, 98%) as a brown solid. 1H NMR (300 MHz, DMSO-d6)
δ 11.21 (s, 1H), 9.01 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 7.9 Hz, 2H), 7.55 (m, 4H), 7.40 – 7.16 (m,
1H), 5.16 (s, 2H), 3.68 (s, 3H), 3.25 (m, 1H), 2.15 – 1.85 (m, 2H), 1.85 – 0.87 (m, 8H); 13C NMR (75 MHz,
DMSO-d6) δ 164.4, 158.3, 141.0, 140.5, 137.4, 132.6, 129.3, 128.3, 128.1, 127.4, 125.8, 122.7, 119.8, 115.6,
72.4, 37.7, 30.4, 30.1, 27.0, 26.0; ESI-MS m/z: [M + H]+ 407.1. HRMS-ESI m/z: calcd for C24H27N2O4
+
[M+H]+: 407,1965; found: 407.1960; FT-IR (neat) νmax 3314, 3248, 2919, 2849, 1661, 1618, 1585, 1452,
1206, 1106, 1028 cm-1; mp 197.2-200.0 °C, decomposition Anal. (C24H26N2O4) C, H, N.

5.2.21. 4-(Hydroxymethyl) pyridine (18).
To a solution of 17 (2.0 g, 18.67 mmol) in methanol (20 mL), cooled at 0 °C, NaBH4 (706 mg, 18.67 mmol)
was added. The reaction was stirred for 2 h and allowed to reach to 25 °C. After the complete consumption
of starting material, water (20 mL) was added, solvent was evaporated, and the aqueous residue was
extracted with EtOAc (3 x 15 mL). The combined organic layers were filtered over Na2SO4 and concentrated
1
in vacuo to get 18 (1.60 g, 80%) as a colorless oil. H NMR (300 MHz, CDCl3) δ 8.52 (dd, J = 4.6, 1.5 Hz,
2H), 7.40 – 7.19 (m, 2H), 4.74 (s, 2H), 3.20 (s, 1H).
5.2.22. 4-(Bromomethyl)pyridinium bromide (19) [27].
Compound 18 (1.810 g, 16.58 mmol) was dissolved in 48% HBr (16 mL) and stirred at reflux for 4 h. The
water was removed in vacuo to give a thick gum which was treated with ethanol at 5 and then filtered. The
white crystals were collected and washed with cold ethanol to get 4-(hydroxymethyl)pyridinium bromide
(1.9 g). To a suspension of this salt in chloroform (25 mL) PBr3 (457 µL, 4.85 mmol) was added, and the
mixture was stirred at reflux for 4.5 h. After cooling down to 25 °C, the white precipitate was collected and
washed with cold chloroform to give 19 (2.3 g, 93%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.80 (d,
J = 6.4 Hz, 2H), 8.00 (d, J = 6.4 Hz, 2H), 4.61 (s, 2H). The spectroscopic data are consistent with those
reported in literature [27].
5.2.23. 4-(Bromomethyl)benzaldehyde (21) [37].
To a solution of 4-(bromomethyl)benzonitrile (2.0 g, 10.20 mmol) in dry DCM (40 mL), cooled at -78 °C a 1
M solution of DIBAL-H in DCM (11.2 mL, 11.22 mmol) was slowly added. The reaction was allowed to
reach 0 °C in 1 h, then it was slowly quenched with 1 N HCl (20 mL). The mixture was extracted with DCM
(3 x 20 mL) and the combined organic layers were washed with NaOH (2 x 20 mL), then dried over Na2SO4
and concentrated in vacuo affording 21 (2.03 g, quantitative yield) as a white solid that was used in the next
step without further purification. 1H NMR (300 MHz, CDCl3) δ 10.02 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.56
(d, J = 8.1 Hz, 2H), 4.52 (s, 2H). The spectroscopic data are consistent with those reported in literature [37].
5.2.24. 2-(4-(Bromomethyl)phenyl)-1,3-dioxolane (22) [37].
To a solution of 21 (2.03 g, 10.20 mmol) in toluene, ethylene glycol (1.1 mL, 20.40 mmol) and PTSA (176
mg, 1.02 mmol) were added. The reaction was stirred at 110 °C for 12 h, then a saturated solution of
NaHCO3 (20 mL) was added. The resulting mixture was extracted with EtOAc (3 x 20 mL) and the
combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude was purified by
chromatography on silica gel (20% EtOAc in petroleum ether) affording 22 (1.7 g, 69%) as a colorless oil.
1H NMR (300 MHz, Acetone-d6) δ 7.56 – 7.39 (m, 4H), 5.75 (s, 1H), 4.66 (s, 2H), 4.20 – 3.88 (m, 4H). The
spectroscopic data are consistent with those reported in literature [37].

5.2.25. 1-Benzylindoline-2,3-dione (23a) [26].
To a solution of isatin (300 mg, 2.03 mmol) in dry DMF (2 mL), cooled at 0 °C, a 60% dispersion of NaH in
mineral oil (95 mg, 2.38 mmol) was added and stirred for 5 min at 0 °C. Then, benzyl bromide (266 µL, 2.34
mmol) was added and the reaction was allowed to reach 25 °C and stirred for 12 h. Thereafter, a saturated
solution of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL). The combined
organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified
by chromatography on silica gel (25% EtOAc in petroleum ether) affording 23a (358 mg, 74% yield) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.67 – 7.55 (m, 1H), 7.47 (td, J = 7.8, 1.3 Hz, 1H), 7.41 – 7.24
(m, 5H), 7.08 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.93 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 183.2,
158.3, 150.7, 138.3, 134.5, 129.1, 128.2, 127.4, 125.4, 123.9, 117.7, 111.0, 44.0; ESI-MS m/z: [M + H]+
238.0; mp 129.8-130.8 °C. The spectroscopic data are consistent with those reported in literature [26].
5.2.26. 1-(Pyridin-4-ylmethyl)indoline-2,3-dione (23b).
To a solution of 19 (860 mg, 3.39 mmol) in diethyl ether (10 mL) a 10% solution of NaHCO3 (10 mL) was
added. The organic layer was extracted, dried over Na2SO4, concentrated in vacuo and the residue was
dissolved in dry DMF (2 mL). In another flask, a solution of isatin (500 mg, 3.39 mmol) in dry DMF (2 mL),
cooled at 0 °C, was treated with a 60% dispersion of NaH in mineral oil (163 mg, 4.07 mmol) and stirred at
the same temperature for 30 min. After this time, the previously prepared solution of 19 (free base in dry
DMF) was added and the reaction was allowed to reach 25 °C and stirred for 12 h. Then a saturated solution
of NH4Cl (5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL). The combined organic
layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The crude was
purified by chromatography on silica gel (EtOAc) affording 23b (311 mg, 38% yield) as an orange solid. 1H
NMR (300 MHz, CDCl3) δ 8.50 (d, J = 6.1 Hz, 2H), 7.56 (dd, J = 7.5, 1.4 Hz, 1H), 7.51 – 7.34 (m, 1H), 7.20
(d, J = 6.2 Hz, 2H), 7.06 (t, J = 7.6 Hz, 1H), 6.66 (d, J = 7.9 Hz, 1H), 4.88 (s, 2H); ESI-MS m/z: [M + H]+
239.0.
5.2.27. 1-Benzyl-3-hydroxy-4-phenylquinolin-2(1H)-one (24a).
Starting from benzaldehyde (67 mg, 0.62 mmol) and 23a (150 mg, 0.62 mmol), the title compound was
obtained following the procedure previously described for compound 13a. The crude was purified by
chromatography on silica gel (25% in EtOAc in petroleum ether) giving 24a (78 mg, 38%) as a brown solid.
1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 7.90 – 6.90 (m, 14H), 5.65 (s, 2H); ESI-MS m/z: 328.0.
5.2.28. 3-Hydroxy-4-phenyl-1-(pyridin-4-ylmethyl)quinolin-2(1H)-one (24b).
Starting from benzaldehyde (53 mg, 0.62 mmol) and 23b (120 mg, 0.50 mmol), the title compound was
obtained following the procedure previously described for compound 13a. The crude was purified by
1
chromatography on silica gel (EtOAc) giving 24b (64 mg, 39%) as a brown solid. H NMR (300 MHz,