Design, synthesisand preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring

Article abstract of DOI:10.1016/j.ejmech.2013.05.017

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and o

Full text of DOI:10.1016/j.ejmech.2013.05.017

Accepted Manuscript
Design, synthesis and preliminary evaluation of a series of histone deacetylase
inhibitors carrying a benzodiazepine ring
L. Guandalini, M. Balliu, C. Cellai, M.V. Martino, A. Nebbioso, C. Mercurio, V. Carafa,
G. Bartolucci, S. Dei, D. Manetti, E. Teodori, S. Scapecchi, L. Altucci, F. Paoletti, M.N.
Romanelli
PII:
S0223-5234(13)00317-6
10.1016/j.ejmech.2013.05.017
EJMECH 6195
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2013
Revised Date: 3 May 2013
Accepted Date: 10 May 2013
Please cite this article as: L. Guandalini, M. Balliu, C. Cellai, M.V. Martino, A. Nebbioso, C. Mercurio,
V. Carafa, G. Bartolucci, S. Dei, D. Manetti, E. Teodori, S. Scapecchi, L. Altucci, F. Paoletti, M.N.
Romanelli, Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors
carrying a benzodiazepine ring, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.05.017.
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ACCEPTED MANUSCRIPT
Highlights
► A series of hybrids between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines has been
synthesized.
► The compounds were evaluated in vitro on recombinant hHDAC1 and hHDAC 6 and on HeLa
nuclear extracts.
► Antiproliferative activity was tested on different cancer cells types.
► Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and preliminary evaluation of a series of histone deacetylase
inhibitors carrying a benzodiazepine ring.
L. Guandalini,a M. Balliu,b C. Cellai,b M. V. Martino,a A. Nebbioso,c C. Mercurio,d V. Carafa,c G.
Bartolucci,a S. Dei, a D. Manetti, a E. Teodori, a S. Scapecchi,a L. Altucci,c F. Paoletti,b M. N.
Romanellia,*
a NEUROFARBA, Department of Neuroscience, Psychology, Drug Research and Child Health,
Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff 6,
50019 Sesto Fiorentino, Italy.
b Department of Experimental Pathology and Oncology, University of Florence, Viale G.B.
Morgagni 50, 50134 Firenze, Italy.
c Department of General Pathology, Second University of Naples, vico L. De Crecchio 7, 80138
Napoli, Italy.
d Genextra Group DAC srl, via Adamello 16, 20139 Milan, Italy.
* corresponding author:
Maria Novella Romanelli, NEUROFARBA, Department of Neuroscience, Psychology, Drug
Research and Child Health, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy.
Tel +39 055 4573691; Fax +39 055 4573671. Email: novella.romanelli@unifi.it
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Abstract
A series of new histone deacetylase inhibitors were designed and synthesized based on
hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds
were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human
recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells
types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50
values similar to those of SAHA. Compound (S)-8 displayed interesting activity against
hematological and solid malignancies.
Keywords
HDAC inhibitors, 1,4-benzodiazepine, antiproliferative activity, apoptosis, enantioselectivity.
List of abbreviations:
AML, acute myeloid leukemia; BDZ, 5-phenyl-1,4-benzodiazepine; BOP-Cl, bis(2-oxo-3-
oxazolidinyl)phosphonic chloride; ER, Eudismic ratio; HDAC, histone deacetylase; SAHA ,
suberoylanilide hydroxamic acid.
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1. Introduction
Histone deacetylases (HDAC) are a family of enzymes playing a crucial role in chromatin
remodeling therefore affecting trascriptional processes. These enzymes can be grouped into four
classes showing different cellular localization; they act by removing amidic functions on the lysine
amino group of histone and non-histone proteins [1].
HDAC inhibition produces anticancer effects in different types of tumors through inhibition of
cell growth and induction of cell differentiation and apoptosis; as HDAC are often overexpressed in
cells from different tumors, these enzymes have become attractive targets for anticancer therapy [2].
The efficacy of HDAC inhibitors in oncology is highlighted by the number of compounds which
entered clinical trials [3]. Only two compounds, namely SAHA (vorinostat) and FK228
(romidepsin) [4,5], have been approved so far for cutaneous T-cell lymphoma, but this class of
compounds is undergoing testing for both hematological malignancies and solid tumors, with some
adverse effect and not always with success [6]. Therefore, the development of novel HDAC
inhibitors endowed with high anticancer properties and low-toxic profile is still a target of basic and
clinical research.
The structure of HDAC inhibitors requires a group, often an hydroxamate moiety, able to chelate
the zinc ion at the bottom of the active site, a recognition moiety (cap) which binds at the surface of
the enzyme, and a suitable spacer which links the two moieties and which can fit the long and
narrow active site; this pharmacophore is shown, as examples, in Trichostatin-A, SAHA or
oxamflatin (Chart 1). In a previous paper [7] we described the synthesis and preliminary biological
evaluation of some HDAC inhibitors carrying a benzodiazepine ring as cap; the design was based
on an hybridization between the structure of SAHA, the first inhibitor approved for clinical use, and
the 5-phenyl-1,4-benzodiazepine (BDZ) moiety, which has been reported to exert cytostatic,
differentiative and apoptotic effects over a wide panel of cancer cell types [8-10]. The most
interesting compound of the series were 1 and (S)-2 (Chart 1); the latter was found able to trigger
apoptosis in various acute myeloid leukemia (AML) cell lines and blasts from patients with
different AML subtypes, as well as in human prostate LNCaP and PC3 cancer cells, showing a low-
toxic profile in vivo [11,12].
These promising results prompted us to perform structural changes aimed to improve the
pharmacological profile of this class of compounds; therefore, the following modifications were
introduced. 1) The achiral compound 1, which proved to be equipotent as (S)-2 in inhibiting NB4
cell growth, was modified on the spacer: the length was changed into 7 or 5 carbon units
(compounds 3 and 4, general formula A), or it was replaced (compound 5) by the same spacer
found in oxamflatin [13], a compound which has previously inspired our design. 2) The importance
of the N1-methyl group was tested by synthesizing (S)-6, the desmethyl analog of (S)-2. 3) The
effect of changing the position of the linker-chelating group on the benzodiazepine ring was
checked by shifting the suberoyl hydroxamate moiety in position 1 (compound 7, general formula
B) or in position 3 (compound 8, general formula C) on the BDZ ring. Since in the course of this
study compound 8 was found endowed with interesting activity on NB4 cells, its enantiomers were
separated in order to study enantioselectivity, while the consequences of varying the length of the
linker (compounds 9-11) were evaluated on the racemate. 4) With the aim of improving solubility
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of 1, compound 12 (general formula A), carrying a basic amino group in position 3, was prepared as
racemate.
2. Results and discussion
2.1. Chemistry
The synthetic pathways are shown in Schemes 1-4. In order to obtain analogs of 1, the iodo
derivative 13 [7] (Scheme 1) was coupled to 6-heptynoic acid under Sonogashira conditions to
afford 15. The same reaction, attempted on 4-pentynoic acid, did not afford the desired acid 16: a
compound was obtained, showing the correct molecular weight but identified, from the NMR
spectrum, as the enol-lactone 17 [14]. Compound 16 was therefore obtained by coupling 13 with
ethyl 4-pentynoate [15], followed by alkaline hydrolysis of the intermediate ester 18. Ethyl pent-4-
yn-2-enoate [16] was prepared according to Wei as E/Z mixture [17], and coupled with 13;
chromatographic separation of the esters 19, followed by alkaline hydrolysis afforded acid 20.
Acids 15, 16 and 20 were then reacted with ethyl chlorocarbonate and hydroxylamine hydrochloride
to afford the desired hydroxamate 3, 4 and 5. In a similar way, (S)-14 [7] was reacted with hexynoic
acid to give (S)-21 which was transformed in the corresponding hydroxamate (S)-6 in the usual
way.
The preparation of 7 is reported in Scheme 2. Initially the synthesis of 8-oxo-8-(2-oxo-5-phenyl-
2,3-dihydro-1H-1,4-benzodiazepin-1-yl)octanoic acid 24a (R = H, X = O) was attempted, through
reaction of 5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-2-one 22 [18] with ethyl 8-chloro-8-
oxooctanoate followed by alkaline hydrolysis of the ester intermediate. However, attempts to purify
ester 24b (R = Et, X = O) failed, since the compound proved to be unstable; moreover, the
decomposition was much faster when the hydrolysis of the ester group was attempted. Therefore,
compound 7 was designed, where the 2-oxo moiety was replaced by CH2. It was prepared by
reacting 5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine 23, obtained by LiAlH4 reduction of 22 [19],
with ethyl hydrogen suberate, followed by alkaline hydrolysis and coupling with hydroxylamine
hydrochloride under the usual conditions.
The synthesis of compounds 8-11 is reported in Scheme 3. Rac-27 [20] was treated with the
suitable dicarboxylic acid as monoesters to give compounds 28-30, which were hydrolyzed to acids
31-33. Reaction with dimethyl-t-butylsilylhydroxylamine and removal of the protecting group gave
hydroxamates 8-10. Reaction of rac-27 with pimelic acid anhydride gave compound 34, which was
transformed into hydroxamate 11 by reaction with ethyl chlorocarbonate and hydroxylamine.
(S)- and (R)-27 were obtained according to Sherrill [21], and transformed into (S) and (R)-8 as
reported for the racemate. Since the Sherrill’s procedure in our hands gave low yields of the
enantiopure amines 27 (see experimental section), and given the promising pharmacological results
shown by (S)-8, (S)-27 was later obtained in good yield from rac-27 by fractional crystallization of
the (S)-camphorsulfonate salt as reported by Reider [22], and converted into (S)-8 as described for
the racemate.
The synthesis of the aminoderivative 12 is reported in Scheme 4. The pathway used to prepare
27 was applied to the synthesis of compound 36, starting from 2-(1H-1,2,3-benzotriazol-1-yl)-2-
{[(benzyloxy)carbonyl]amino}acetic acid 35 and 2-amino-5-iodobenzophenone [21,23]. After
methylation and exchange of the N-protecting group, the N-Boc derivative 37 was transformed into
hydroxamate 38 as reported for analogs 3-6. Treatment with hydrochloric acid gave compound 12.
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2.2. Biological tests
Initially the compounds were tested for their HDAC inhibitory activity on HeLa cell nuclear
extracts and on recombinant HDAC1 using a fluorometric assay; the results are reported in Table 1.
SAHA was used as the reference compound. Compounds 1 and (S)-2, previously characterized by
us, were also tested in this assay. The data on HDAC1 and on HeLa nuclear extracts show that for
compounds with general formula A (Table 1) the activity is only marginally affected by the length
of the spacer, as compounds 3 and 4 showed activity in the same range as 1. On the contrary, the
stereogenic center in position 3 on the BDZ nucleus was found important for activity: as a matter of
fact, compounds (S)-2 and (S)-6 are 2-5 times more potent than compounds 1, 3 and 4, and, as
previously reported [7,11], (S)-2 is more potent than (R)-2. The presence of the methyl group in
position 1 did not affect activity, (S)-6 being equipotent with (S)-2 in both HeLa extract and
HDAC1. The length of the linker between the triple bond and the hydroxamate moiety is not
important for activity, compounds 1, 3 and 4 showing similar potency; on the contrary, the
restriction of flexibility greatly improves activity, since compound 5 showed a potency similar to
(S)-2 and (S)-6 on HDAC1 and even higher on HeLa nuclear extracts. The introduction of a NH2
group in position 3 of the BDZ ring, replacing the methyl group of compound 2, did not improve
activity, although rac-12 shows on HDAC1 a potency similar to (S)-2.
The linker-hydroxamate group was successfully moved in different position on the BDZ ring:
compounds 7 and 8, carrying this moiety, respectively, in position 1 and 3, show potency in the
high nanomolar range. Substitution in position 3 introduces a stereogenic center in the molecule: as
it happens for 2, also for 8 the S isomer is the eutomer. For compounds of general formula C the
variation of the linker’s length affected activity in a different way on HeLa nuclear extracts with
respect to HDAC1: while on HDAC1 the most potent compound is the azelate derivative rac-11, on
HeLa nuclear extracts (S)-8 is 2-5 times more potent than its analogs. These small differences in
structure-activity relationships can be due to the different experimental conditions or to the presence
of additional HDAC isoforms in the HeLa nuclear extracts; however, the two experimental models
gave IC50 values in the same range.
Since compounds 1 and (S)-2 were shown to increase acetylation of α-tubulin, a substrate for
HDAC6, the inhibitory activity of the compounds was tested also by a cell-free assay of this
recombinant isoform (Table 1). In general, the synthesized compounds proved to be less active on
HDAC6 with respect to HDAC1 with some exceptions, namely (S)-8, its lower homolog rac-11,
and (S)-6. Contrary to what happened on HDAC1, the removal of the N1 methyl group of (S)-2, to
give (S)-6, produced a 5-fold increase of activity on HDAC6. The variation of chain length also
influenced the potency of the compounds with general formula C (Chart 1). On one hand, the
removal of one carbon unit from (S)-8 to rac-11 brought a two-fold increase in potency on both
HDAC1 and HDAC6; on the other hand, further shortening (rac-10, 4 methylene units) or the
increase (rac-9, 7 methylene units) of the chain length abolished or, respectively, reduced the
activity on HDAC6. On this isoform, rac-11 is the most potent inhibitor of this class of compounds,
while rac-10 shows the highest HDAC1/HDAC6 selectivity.
The inhibitory activity of the compounds was also measured on HDAC4, a class IIa HDAC
isoform (Table 1). A preliminary test, performed at a fixed concentration (5 ꢀM), showed that the
compounds were definitely less potent on this isoform with respect to HDAC1 and HDAC6, the
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residual enzymatic activity ranging between 65-100%. This finding suggests for all the compounds
a IC50 value higher than 5 ꢀM on HDAC4; for this reason, a dose response curve was not
performed.
The ability of the compounds to inhibit cancer cell growth was assessed on K562 (human
chronic myelogenous leukemia), A549 (human alveolar basal epithelial) and HCT-116 (human
colon cancer) cells using an automated assay. IC50 values are reported in Table 2. All the
compounds were able to reduce cell growth with IC50 values in the micromolar range, thus showing
that different tumor cell lines were sensitive to the antiproliferative activity of this class of
substances. Some compounds showed IC50 values in the high nanomolar range, namely (S)-2 and
(S)-6 on HCT116 cells, and 5 and (S)-8 on both HCT116 and K562 cell lines, the latter showing a
potency similar to the reference compound SAHA. Since our first interest was to find compounds
endowed with antileukemic activity, the newly synthesized compounds were tested on human acute
promyelocytic leukemia NB4 cell line as the model. The proapoptotic activity of the compounds
was assessed by means of the Annexin-V test and it is reported as IC50 values (i.e. the drug
concentration necessary to induce apoptosis in 50% of the population compared to untreated
cultures, Table 2). With the exception of 3 and (R)-8, all the new compounds were able to induce
apoptosis in NB4 cells with IC50 values in a low micromolar range, the most active compound being
(S)-8, with an IC50 of 0.86 ꢀM.
In previous papers [7,11] we reported that the enantiomers of compound 2 showed different
potency in several tests. As a matter of fact, the IC50 for antiproliferative activity on NB4 cells was
found 12 times lower for (S)-2 than (R)-2; nevertheless the difference in potency on HDAC1 and
HDAC6 of the two enantiomers as determined by a cell-free assay was less than three-fold. The
cellular tests reported in this paper (see Table 1) also show an eudismic ratio closer to that
previously found in NB4 cells as far as antiproliferative activity is concerned (ER values are 9.5,
8.0 and 9.1 on HCT116, A549 and K562 cell lines, respectively); a similar comparison cannot be
done for the proapoptotic effect, since a IC50 cannot be derived for (R)-2. These findings suggest
that probably other factors, besides the interaction with HDAC, can affect the cellular activity of
(S)-2 and(R)-2. As far as compound 8 is concerned, enantioselectivity is low on HeLa nuclear
extracts, on HDAC1 and also on HCT116, A549 and K562 cell lines, the eudismic ratios being
below 3, but it is higher on HDAC6 (ER = 16). The finding that also for compound 8 the eutomer is
in all of the performed tests the S enantiomer may be just a coincidence, since the two compounds
should adopt an opposite orientation when interacting with the enzyme, thus placing the chiral
center in different position of the protein domain.
On the basis of these results, (S)-8 was selected for further study on NB4 cells. As shown in Fig.
1, the drug was capable of inducing histone H3 and H4 acetylation in a dose-dependent manner as
assessed by Western blot and immunostaining for the acetylated histones after a 6 hour-treatment
with 0.5, 1 and 2 µM concentration (panel A). As it happened for other compounds of this series
[7], histone H3 were more sensitive to the drug: densitometric analysis using tubulin as the
reference protein (see Materials and Methods) shows that a 0.5 µM concentration of (S)-8 was able
to induce 50% acetylation of histone H3 but only 8% of histone H4; at a 1 µM concentration the
percentage of acetylated H3 and H4 were 84% and 51% respectively. By extending the time of
incubation up to 48 hours a marked decrease in cell viability was observed (IC50 0.46 µM, Fig. 1,
panel B) that was accompanied by a significant increase in apoptotic rates in culture. Drug-induced
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apoptosis as measured by cytofluorimetric analysis of Annexin/PI stained cells (Fig. 1. Panel C,
left) was especially evident in cultures treated with 1 and 2 µM drug where the portion of the
population committed to cell death accounted for about 66 and 83%, respectively (see values
reported on the upper right quadrants of dot plots). These findings were confirmed by examining the
May-Grünwald/Giemsa stained cytosmears (panel C, right) showing that in cultures treated with 1
and 2 µM concentration of the drug there was a massive increase in apoptotic bodies.
In conclusion, a new HDAC inhibitor has been found ((S)-8) which displays a good potency in
cell-free assays and an interesting activity against cells from both hematological (NB4 and K562
cell lines) and solid (A549 and HCT-116 cell lines) tumors. These interesting properties may
depend on its ability to inhibit both HDAC1 and HDAC6; as a matter of fact, there are evidences
that simultaneous inhibition of both isoforms can be an effective treatment for AML, as well as for
other malignancies [24, 25]. The different ability of (R) and (S)-8 to inhibit HDAC6 may be a
possible explanation for their distinct effects on NB4 cells, nevertheless the activity of this class of
compounds on HDAC6 (Table 1) does not completely correlate with the pro-apoptotic properties on
NB4 cells (Table 2), highlighting once again that activity in cell-free assay does not necessarily
reflect the real potency of these drugs inside the cells [11]. Further studies on this class of
compounds, as well as the biological characterization of (S)-8 will be reported in due time.
3. Experimental section
3.1. Chemistry. All melting points were taken on a Büchi apparatus and are uncorrected. NMR
spectra were recorded on a Brucker Avance 400 spectrometer (400 MHz for 1H NMR, 100 MHz for
13C). Chromatographic separations were performed on a silica gel column by gravity
chromatography (Kieselgel 40, 0.063- 0.200 mm; Merck) or flash chromatography (Kieselgel 40,
0.040-0.063 mm; Merck). Yields are given after purification, unless differently stated. When
reactions were performed under anhydrous conditions, the mixtures were maintained under
nitrogen. Exact masses of compounds 3-12 were determined with a Thermo Finnigan LTQ Orbitrap
mass spectrometer equipped with an electrospray ionization source (ESI). Analysis were carried out
in positive ion mode observing protonated molecules [M+H]+ using a proper dwell time aquisition
to achieve 60000 units of resolution at Full Width at Half Maximum (FWHM). Elemental
composition of compounds were calculated on the basis of their exact masses, accepting only
results with an attribution error Delta less than 5 ppm and a not integer RDB (double bond/ring
equivalents) value, in order to consider only the protonated species [26]. Compounds were named
following IUPAC rules as applied by Reaxys (version 1.0.9619) software.
3.1.1. 7-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)hept-6-ynoic acid 15
A mixture of 7-iodo-1-methyl-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-2-one 13 [7] (1.01 g,
2.68 mmol), 6-heptynoic acid (0.51 mL, 4.02 mmol), CuI (0.06 g, 0.32 mmol), Pd(PPh3)4 (0.12 g,
0.11 mmol) and anhydrous Et3N (15 mL) were heated at 55 °C for 30 h under nitrogen, then it was
partitioned between a saturated solution of NH4Cl and ethyl acetate. The organic layer was
collected and the solvent was removed under vacuum, then the residue was treated with HCl 2N (30
mL) and washed with EtOAc (3 x 30 mL), then additioned with NaHCO3 until pH 7 and extracted
with EtOAc. Drying (Na2SO4) of the second organic phase and removal of the solvent gave the title
compound in 80% yield. Yellow solid, m.p. 51-52 °C. [1H]-NMR (CDCl3) δ: 1.59-1.66 (m, 2H,
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CH2); 1.72-1.80 (m, 2H, CH2); 2.36-2.44 (m, 4H, 2CH2); 3.41 (s, 3H, NCH3); 3.78 (d, J = 11.4 Hz,
1H, CHH); 4.86 (d, J = 11.4 Hz, 1H, CHH); 7.29 (d, J = 8.8 Hz, 1H, arom.); 7.33 (d, J = 2.0 Hz, 1H,
arom.); 7.42-7.46 (m, 2H, arom.); 7.50-7.53 (m, 1H, arom.); 7.59 (dd, J = 8.8 Hz, 2.0 Hz, 1H,
arom.); 7.63-7.67 (m, 2H, arom.) ppm.
3.1.2. 5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)pent-4-ynoic acid
16
Following the same procedure used for 15, 13 (0.48 g, 1.28 mmol) was treated with 6-pentynoic
acid ethyl ester [15] (0.25 g, 1.95 mmol), CuI (0.03 g, 0.15 mmol), Pd(PPh3)4 (0.06 g, 0.05 mmol)
and anhydrous Et3N (9 mL) at 55 °C for 2 h under nitrogen. Purification with flash chromatography
(CH2Cl2/MeOH 99:01) gave ethyl 5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-
7-yl)pent-4-ynoate 18 in 88% yield. Yellow waxy solid. [1H]-NMR (CDCl3) δ: 1.23 (t, J = 7.2 Hz,
3H, CH2CH3); 2.57 (t, J = 7.0 Hz, 2H, CH2CH2); 2.68 (t, J = 7.0 Hz, 2H, CH2CH2); 3.40 (s, 3H,
NCH3); 3.77 (d, J = 11.0 Hz, 1H, CHH); 4.14 (q, J = 7.2 Hz, 2H, CH2CH3); 4.84 (d, J = 11.0 Hz,
1H, CHH); 7.28 (d, J = 8.4 Hz, 1H, arom.); 7.32 (d, J = 2.0 Hz, 1H, arom.); 7.41-7.45 (m, 2H,
arom.); 7.48-7.52 (m, 1H, arom.); 7.56 (dd, J = 8.8 Hz, 2.0 Hz, 1H, arom.); 7.63-7.65 (m, 2H,
arom.) ppm. When the reaction was performed with 6-pentynoic acid, 17 was obtained; this
compound was not further purified, nor the stereochemistry on the double bond was investigated.
MS (ESI) 347 (M+1). [1H]-NMR (CDCl3) δ: 2.57-2.75 (m, 2H, CH2CO); 2.97-3.02 (m, 2H,
CH2C=C); 3.40 (s, 3H, NCH3); 3.79 (d, J = 11.0 Hz, 1H, CHH); 4.82 (d, J = 11.0 Hz, 1H, CHH);
6.23 (t, J = 2.2 Hz, 1H, C=CH); 7.12 (d, J = 2.0 Hz, 1H, arom.); 7.33 (d, J = 8.4 Hz, 1H, arom.);
7.38-7.49 (m, 4H, arom.); 7.62-7.66 (m, 2H, arom.) ppm.
A solution of 18 (0.42 g, 1.12 mmol), NaOH (0.07 g, 1.70 mmol) in 2 mL of MeOH, 2 mL of THF
and 2 mL of H2O were stirred at RT for 2 hr, then it was treated with sat. aqueous NH4Cl and
extracted with ethyl acetate. Drying (Na2SO4) and removal of the solvent gave the title compound in
93% yield. Rose solid, m.p. 50-51 °C. [1H]-NMR (CDCl3) δ: 2.60-2.70 (m, 4H, CH2CH2); 3.41 (s,
3H, NCH3); 3.78 (d, J = 11.6 Hz, 1H, CHH); 4.90 (d, J = 11.6 Hz, 1H, CHH); 7.31-7.34 (m, 2H,
arom.); 7.15-7.49 (m, 2H, arom.); 7.54-7.58 (m, 1H, arom.); 7.63 (dd, J = 8.8 Hz, 2.0 Hz, 1H,
arom.); 7.69-7.71 (m, 2H, arom.) ppm.
3.1.3. (2E)-5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)pent-2-en-4-
ynoic acid 20
Ethyl pent-4-yn-2-enoate [16] (0.30 g, 2.44 mmol), prepared according to Wei [17] as 4.3:1 E/Z
mixture, was reacted with 13 (0.61 g, 1.62 mmol) CuI (0.04 g, 0.19 mmol), Pd(PPh3)4 (0.07 g, 0.06
mmol) and anhydrous Et3N (10 mL) at 55 °C for 2 h and 30’ under nitrogen, following the same
procedure used for 15. Purification with flash chromatography (cyclohexane/ethyl acetate 7:3) gave
ethyl (2E)-5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)pent-2-en-4-ynoate
19 (0.34 g), the 2-Z isomer 19a (0.06 g) and a mixed fraction (19/19a 2:1, 0.10 g). 19: yellow waxy
solid. [1H]-NMR (CDCl3) δ: 1.30 (t, J = 7.0 Hz, 3H, CH2CH3); 3.43 (s, 3H, NCH3); 3.79 (d, J =
11.2 Hz, 1H, CHH); 4.22 (q, J = 7.0 Hz, 2H, CH2CH3); 4.88 (d, J = 11.2 Hz, 1H, CHH); 6.28 (d, J =
16.0 Hz, 1H, CH=CH); 6.91 (d, J = 16.0 Hz, 1H, CH=CH); 7.35 (d, J = 8.8 Hz, 1H, arom.); 7.41-
7.47 (m, 3H, arom.); 7.50-7.54 (m, 1H, arom.); 7.64-7.68 (m, 3H, arom.) ppm. 19a: Yellow waxy
solid. [1H]-NMR (CDCl3) δ: 1.24 (t, J = 7.2 Hz, 3H, CH2CH3); 3.40 (s, 3H, NCH3); 3.77 (d, J =
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11.0 Hz, 1H, CHH); 4.19 (q, J = 7.2 Hz, 2H, CH2CH3); 4.84 (d, J = 11.0 Hz, 1H, CHH); 6.14 (d, J =
11.6 Hz, 1H, CH=CH); 6.29 (d, J = 11.6 Hz, 1H, CH=CH); 7.33 (d, J = 8.8 Hz, 1H, arom.); 7.38-
7.42 (m, 2H, arom.); 7.45-7.47 (m, 2H, arom.); 7.59-7.61 (m, 2H, arom.); 7.70 (dd, J = 8.4 Hz, 2.0
Hz, 1H, arom.) ppm. 19 was hydrolyzed as reported for the preparation of 16, obtaining the title
compound in 57% overall yields (based on 13). Yellow solid, m.p. 67-68 °C. [1H]-NMR (CDCl3) δ:
3.43 (s, 3H, NCH3); 3.79 (d, J = 11.2 Hz, 1H, CHH); 4.88 (d, J = 11.2 Hz, 1H, CHH); 6.28 (d, J =
16.0 Hz, 1H, CH=CH); 6.97 (d, J = 16.0 Hz, 1H, CH=CH); 7.36 (d, J = 8.8 Hz, 1H, arom.); 7.42-
7.45 (m, 3H, arom.); 7.49-7.53 (m, 1H, arom.); 7.61-7.63 (m, 2H, arom.); 7.67 (dd, J = 8.4 Hz, 2.0
Hz, 1H, arom.) ppm.
3.1.4. N-hydroxy-6-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)hept-6-
ynamide 3
A solution of NH2OH.HCl (0.71 g, 10.18 mmol) in dry MeOH (5 mL) was added to a solution of
KOH (0.57 g, 10.18 mmol) in dry MeOH (5 mL) at 0 °C under nitrogen. The resulting mixture was
stirred for 15’ at 0 °C and filtered in another flask.
Ethyl chloroformate (0.27 mL, 2.88 mmol) and anhydrous Et3N (0.44 mL, 3.14 mmol) were added
to a solution of 15 (0.81 g, 2.00 mmol) in dry THF (5 mL) at 0 °C under nitrogen. The mixture was
stirred for 15’ at 0 °C and filtered directly in the flask containing the previously prepared solution of
NH2OH in MeOH. The resulting mixture was stirred for 30’ at RT under nitrogen, then the solvents
were evaporated. The residue was purified by flash chromatography (first eluent: CH2Cl2/MeOH
95:5, second eluent: CH2Cl2/MeOH 9:1) giving the title compound in 56% yield. Yellow solid, m.p.
80-82 °C. [1H]-NMR (CD3OD) δ: 1.53-1.60 (m, 2H, CH2); 1.69-1.76 (m, 2H, CH2); 2.10 (t, J = 7.2
Hz, 2H, CH2); 2.40 (t, J = 7.2 Hz, 2H, CH2); 3.42 (s, 3H, NCH3); 3.85 (d, J = 11.2 Hz, 1H, CHH);
4.62 (d, J = 11.2 Hz, 1H, CHH); 7.19 (d, J = 2.0 Hz, 1H, arom.); 7.44-7.48 (m, 2H, arom.); 7.50-
7.54 (m, 4H, arom.); 7.63 (dd, J = 8.8 Hz, 2.0 Hz, 1H, arom.) ppm. [13C]-NMR-APT (CD3OD) δ:
19.60 (CH2); 25.95 (CH2); 29.01 (CH2); 33.19 (CH2); 35.25 (NCH3); 56.96 (CH2); 80.05 (C); 92.17
(C); 121.63 (C arom.); 123.06 (CH arom.); 129.31 (C arom.); 129.66 (CH arom.); 130.88 (CH
arom.); 132.51 (CH arom.); 134.62 (CH arom.); 136.35 (CH arom.); 138.89 (C arom.); 144.50 (C
arom.); 171.17 (CN); 172.57 (CO); 173.10 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for
C21H20N3O3: 362.1505, found: 362.1505.
3.1.5. N-hydroxy-5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)pent-4-
ynamide 4
Following the same procedure used for 3, 16 (0.36 g, 1.05 mmol) was treated with ethyl
chloroformate (0.13 mL, 1.39 mmol), Et3N (0.21 mL, 1.52 mmol), NH2OH.HCl (0.34 g, 4.93
mmol) and KOH (0.28 g, 4.93 mmol). Purifications with flash chromatography (first eluent:
CH2Cl2/MeOH/ NH3 95:05:0.5; second eluent: CH2Cl2/MeOH/NH3 90:10:01) gave the title
compound in 36% yield. Pale yellow solid, m.p. 162-163 °C. [1H]-NMR (CD3OD) δ: 2.32 (t, J = 7.2
Hz, 2H, CH2CH2); 2.68 (t, J = 7.2 Hz, 2H, CH2CH2); 3.42 (s, 3H, NCH3); 3.82 (d, J = 11.0 Hz, 1H,
CHH); 4.62 (d, J = 11.0 Hz, 1H, CHH); 7.21 (d, J = 2.0 Hz, 1H, arom.); 7.43-7.46 (m, 2H, arom.);
7.50-7.55 (m, 4H, arom.); 7.63 (dd, J = 8.4 Hz, 2.0 Hz, 1H, arom.) ppm. [13C]-NMR-APT (CD3OD)
δ: 16.47 (CH2); 32.82 (CH2); 35.14 (NCH3); 57.54 (CH2); 80.60 (C); 90.34 (C); 121.15 (C arom.);
9

ACCEPTED MANUSCRIPT
122.90 (CH arom.); 129.54 (CH arom.); 129.91(C arom.); 130.65 (2CH arom.); 131.98 (CH arom.);
134.38 (CH arom.); 135.92 (CH arom.); 139.64 (C arom.); 144.52 (C arom.); 170.81 (CO); 171.55
(CN); 172.44 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for C23H24N3O3: 390.1818, found:
390.1814.
3.1.6. (2E)-N-hydroxy-5-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-
yl)pent-2-en-4-ynamide 5
Following the same procedure used for 3, 20 (0.28 g, 0.82 mmol) was treated with ethyl
chloroformate (0.11 mL, 1.10 mmol), Et3N (0.17 mL, 1.19 mmol), NH2OH.HCl (0.27 g, 3.87
mmol) and KOH (0.22 g, 3.87 mmol). Purification by flash chromatography (two separations: first
with CH2Cl2/MeOH 90:10, second with CH2Cl2/MeOH/NH3 90:10:01) gave the title compound in
20% yield. Pale yellow solid, m.p. 125 °C (dec.). [1H]-NMR (CD3OD) δ: 3.43 (s, 3H, NCH3); 3.86
(d, J = 11.0 Hz, 1H, CHH); 4.65 (d, J = 11.0 Hz, 1H, CHH); 6.29 (d, J = 15.6 Hz, 1H, CH=CH);
6.82 (d, J = 15.6 Hz, 1H, CH=CH); 7.33 (d, J = 1.6 Hz, 1H, arom.); 7.44-7.51 (m, 2H, arom.); 7.51-
7.56 (m, 3H, arom.); 7.59 (d, J = 8.8 Hz, 1H, arom.); 7.74 (dd, J = 8.8 Hz, 1.6 Hz, 1H, arom.) ppm.
[13C]-NMR-APT (CD3OD) δ: 35.13 (NCH3); 57.58 (CH2); 88.25 (C); 95.04 (C); 119.63 (C arom.);
120.83 (CH=CH); 123.23 (CH arom.); 129.58 (2CH arom.); 130.12(C arom.); 130.64 (CH arom.);
131.39 (CH=CH); 132.06 (CH arom.); 134.91 (CH arom.); 135.98 (CH arom.); 139.58 (C arom.);
145.55 (C arom.); 164.39 (CO); 171.49 (CN); 172.26 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for
C21H18N3O3: 360.1348, found: 360.1345.
3.1.7. 6-[(3S)-3-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl]hex-5-ynoic
acid (S)-21
Following the same procedure used for 15, 14 (1.12 g g, 2.99 mmol) was treated with 5-hexynoic
acid (0.3 mL, 2.72 mmol), CuI (0.068 g, 0.36 mmol), Pd(PPh3)4 (0.138 g, 0.12 mmol) and
anhydrous Et3N (15 mL) at 55 °C for 41 h under nitrogen athmosphere. Purification by flash
chromatography (CH2Cl2/MeOH 9:1) gave the title compound in 61% yield. White solid, m.p. 73-
75 °C. [1H]-NMR (CDCl3) δ: 1.71 (d, J = 6.4 Hz, 3H, CHCH3); 1.85-1.93 (m, 2H, CH2); 2.44 (t, J =
7.2 Hz, 2H, CH2); 2.48 (t, J = 7.2 Hz, 2H, CH2); 3.73 (q, J = 6.4 Hz, 1H, CHCH3); 7.11 (d, J = 8.4
Hz, 1H, arom.); 7.33 (d, J = 1.6 Hz, 1H, arom.); 7.36-7.45 (m, 3H, arom.); 7.47-7.53 (m, 3H,
arom.); 9.82 (s, bs, 1H, NH) ppm.
3.1.8. N-hydroxy-6-[(3S)-3-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-
yl]hex-5-ynamide (S)-6
Following the same procedure used for 3, (S)-21 (0.53 g, 1.46 mmol) was treated with ethyl
chloroformate (0.19 mL, 1.95 mmol), Et3N (0.29 mL, 2.12 mmol), NH2OH.HCl (0.48 g, 6.88
mmol) and KOH (0.39 g, 6.88 mmol). Two purifications with flash chromatography (eluents:
CH2Cl2/MeOH/NH3 9:1:0.1 and CH2Cl2/MeOH 9:1) gave the title compound in 51% yield. White
solid, m.p. 198-199 °C. [α]20D(DMSO) +288.3°. [1H]-NMR (CD3OD) δ: 1.65 (d, J = 6.4 Hz, 3H,
CHCH3); 1.81-1.88 (m, 2H, CH2); 2.21 (t, J = 7.2 Hz, 2H, CH2); 2.41 (t, J = 7.2 Hz, 2H, CH2); 3.75
(q, J = 6.4 Hz, 1H, CHCH3); 7.19 (d, J = 8.6 Hz, 1H, arom.); 7.22 (d, J = 2.0 Hz, 1H, arom.); 7.42-
7.47 (m, 4H, arom.); 7.48-7.54 (m, 1H, arom.); 7.57 (dd, J = 8.6 Hz, 2.0 Hz, 1H, arom.) ppm. [13C]-
NMR-APT (CD3OD) δ: 17.01 (CH3); 19.45 (CH2); 25.80 (CH2); 32.75 (CH2); 60.01 (CH); 80.76
10

ACCEPTED MANUSCRIPT
(C); 90.59 (C); 120.29 (C arom.); 122.33 (CH arom.); 128.78 (C arom.); 129.45 (CH arom.); 130.80
(CH arom.); 131.73 (CH arom.); 135.04 (CH arom.); 135.96 (CH arom.); 139.80 (C arom.); 140.28
(C arom.); 171.14 (CN); 172.22 (CO); 173.15 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for
C22H22N3O3: 376.1661, found: 376.1656.
3.1.9. 8-oxo-8-(5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-1-yl)octanoic acid 26
LiAlH4 (0.097 g, 2.55 mmol) was slowly added to a solution of 22 [19] (2.13 g, 9.06 mmol) in dry
THF at 0 °C under nitrogen. The mixture is stirred at RT for 1 h, then quenched with ice and
partitioned between EtOAc and brine. Drying (Na2SO4) of the organic phase, removal of solvent
and purification with flash chromatography (eluent: CH2Cl2/MeOH 95:5) gave 1.22 g (61% yield)
of 5-phenyl-1,3-dihydro-1,4-benzodiazepine 23 together with unreacted 22 (0.72 g). Yellow solid,
m.p. 136-137 °C. [1H]-NMR (CDCl3) δ: 3.80-3.82 (m, 2H, CH2); 3.96-3.98 (m, 2H, CH2); 4.49 (s,
bs, 1H, NH); 6.66-6.70 (m, 1H, arom.); 6.74 (d, J = 8.4 Hz, 1H, arom.); 7.01 (dd, J = 8.0 Hz, 1.2
Hz, 1H, arom.); 7.19-7.23 (m, 1H, arom.); 7.35-7.44 (m, 3H, arom.); 7.53-7.55 (m, 2H, arom.)
ppm.
A mixture of 23 (0.6 g, 2.71 mmol), ethyl hydrogensuberate (0.66 g, 3.26 mmol), WSC (0.51 g,
3.26 mmol) and HOBT (0.44 g, 3.26 mmol) in ethanol-free CHCl3 (20 mL) was stirred at room
temperature for 3 days, then it was treated with 10% NaHCO3 and the organic layer was separated.
Drying (Na2SO4) and removal of the solvent gave a residue which was purified by flash
chromatography (first eluent: CH2Cl2/MeOH/NH3 99:01:0.1, second eluent CH2Cl2/MeOH/NH3
98:02:0.2, third eluent CH2Cl2/MeOH/NH3 90:10:01) giving unreacted starting material (23, 0.44
g) and 25 (0.22 g, 21% yield). Yellow solid, m.p. 85-86 °C. [1H]-NMR (CDCl3) δ: 1.24 (t, J = 7.2
Hz, 3H, CH2CH3); 1.29-1.35 (m, 4H, 2CH2); 1.55-1.65 (m, 4H, 2CH2); 2.22-2.28 (m, 4H, 2CH2);
3.51-3.54 (m, 2H, NCH2); 3.65-3.69 (m, 2H, NCH2); 4.11 (q, J = 7.2 Hz, 2H, CH2CH3); 6.85 (bs,
1H, arom.); 7.04-7.08 (m, 1H, arom.); 7.46-7.50 (m, 2H, arom.); 7.53-7.60 (m, 3H, arom.); 7.67 (d,
J = 7.6 Hz, 2H, arom.) ppm. This compound was hydrolyzed with NaOH (0.032 g, 0.81 mmol) in 1
mL of MeOH, 1 mL of THF and 1 mL of H2O, the title compound was obtained with 91% yield.
Yellow waxy solid. [1H]-NMR (CDCl3) δ: 1.30-1.32 (m, 4H, 2CH2); 1.56-1.63 (m, 4H, 2CH2);
2.21 (t, J = 7.4 Hz, 2H, CH2); 2.28 (t, J = 7.2 Hz, 2H, CH2); 3.47-3.50 (m, 2H, NCH2); 3.59-3.63
(m, 2H, NCH2); 6.56 (bs, 1H, arom.); 6.75-6.79 (m, 1H, arom.); 7.12 (d, J = 8.4 Hz, arom.); 7.44-
7.48 (m, 3H, arom.); 7.51-7.55 (m, 1H, arom.); 7.61-7.63 (m, 2H, arom.) ppm.
3.1.10. N-hydroxy-8-oxo-8-(5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-1-yl)octanamide 7
Following the same procedure used for 3, 26 (0.44 g, 1.16 mmol) was treated with ethyl
chloroformate (0.15 mL, 1.54 mmol), Et3N (0.23 mL, 1.68 mmol), NH2OH.HCl (0.38 g, 5.45
mmol) and KOH (0.31 g, 5.45 mmol). Purification with flash chromatography (eluent:
CH2Cl2/MeOH/NH3 90:10:01) gave the title compound in 75% yield. Yellow solid, m.p. 96-97 °C.
[1H]-NMR (CD3OD) δ: 1.29-1.31 (m, 4H, 2CH2); 1.53-1.63 (m, 4H, 2CH2); 2.04 (t, J = 7.4 Hz, 2H,
CH2); 2.19 (t, J = 7.4 Hz, 2H, CH2); 3.46 (s, 4H, t, 2CH2N); 6.55 (t, J = 7.6 Hz, 1H, arom.); 6.95 (d,
J = 8.8 Hz, 1H, arom.); 7.39-7.43 (m, 2H arom.); 7.46-7.50 (m, 2H, arom.); 7.53-7.56 (m, 3H,
arom.) ppm. [13C]-NMR-APT (CD3OD) δ: 26.51 (CH2); 26.78 (CH2); 29.76 (CH2); 29.82 (CH2);
33.67 (CH2); 37.00 (CH2); 39.64 (CH2); 42.64 (CH2); 112.84 (CH arom.); 115.23 (CH arom.);
11

ACCEPTED MANUSCRIPT
118.74 (C arom.); 129.19 (CH arom.); 129.93 (CH arom.); 132.02 (CH arom.); 136.30 (CH arom.);
136.43 (CH arom.); 141.83 (C arom.); 152.94 (C arom.); 172.93 (CN); 176.72 (CO) ppm. HRMS-
ESI m/z [M+H]+ calcd for C23H28N3O3.H2O: 412.2236, found: 412.2233.
3.1.11. 3-amino-1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (rac-27)20
To a solution of benzyl 2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-ylcarbamate [21]
(1.00 g, 2.60 mmol) in anhydrous DMF (10 mL), kept at 0 °C under N2, NaH (60% dispersion in
mineral oil, 0.10 g, 1 eq) was added in small portions. After 1.5 hr stirring at 0 °C, methyl iodide
(0.17 mL, 1.05 eq) was added at once; the mixture was stirred for additional 1.5 h at the same
temperature, and then poured into a stirred solution of H2O (60 mL) containing aqueous sodium
hydrogen sulphate (2 mL, 1 N). A solid precipitated, which was filtered, dried under vacuum, and
then purified by flash chromatography (CH2Cl2/MeOH/NH3 99:1:0.1 as eluent) giving benzyl 1-
methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4]diazepin-3-ylcarbamate in 91% yield. M.p.
78-80 °C. [1H]-NMR (CDCl3): 3.47 (s, 3H, Me); 5.13 (d, J = 12.4 Hz, 1H, CHH); 5.17(d, J = 12.4
Hz, 1H, CHH); 5.33 (d, J = 8.4 Hz, 1H, CH); 6.72 (d, J = 8.4 Hz, 1H, NH); 7.22-7.28 (m, 1H),
7.28-7.42 (m, 9H), 7.44-7.50 (m, 1H) and 7.58-7.64 (m, 3H) (aromatic protons) ppm. This
compound (0.20 g, 0.50 mmol) was dissolved in 5.7 mL of HBr (33% solution in AcOH) under N2
and the solution was kept stirring at room temperature for 2 hr, then diluted with Et2O (15 mL) to
obtain a suspension. The mixture was filtered and the solid dissolved in H2O. The aqueous solution
was then made alkaline with Na2CO3 and extracted with AcOEt. The organic phase was dried
(Na2SO4), the solvent was removed under vacuum obtaining the title compound in 99% yield. M.p.
62-64 °C. [1H]-NMR (CD3OD): 3.48 (s, 3H, Me); 4.43 (s, 1H, CH); 7.26-7.32 (m, 2H), 7.39-7.46
(m, 2H), 7.47-7.54 (m, 1H), 7.56-7.61 (m, 3H), 7.65-7.70 (m, 1H) (aromatic protons) ppm.
3.1.12. 8-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-ylamino)-8-
oxooctanoic acid rac-31
A mixture of rac-27 [20] (0.54 g, 2.02 mmol), ethyl hydrogensuberate (0.43 g, 2.12 mmol), WSC
(0.38 g, 2.43 mmol) and HOBT (0.33 g, 2.43 mmol) in ethanol-free CHCl3 (20 mL) was stirred at
room temperature for 18 hr, then it was treated with 10% NaHCO3 and the organic layer was
separated. Drying (Na2SO4) and removal of the solvent gave a residue which was purified by flash
chromatography (CH2Cl2/MeOH/NH4OH 99:1:0.1 as eluent). rac-28 was obtained in 66% yield as
a pale yellow solid (m.p. 138-140 °C). [1H]-NMR (CDCl3): 1.25 (t, J = 7.2 Hz, 3H, CCH3); 1.31-
1.45 (m, 4H, 2CH2); 1.60-1.75 (m, 4H, 2CH2); 2.29 (t, J = 7.6 Hz, 2H, CH2); 2.37 (td, J = 7.6, 2.8
Hz, 2H, CH2); 3.47 (s, 3H, NCH3); 4.12 (q, J = 7.2 Hz, 2H, OCH2); 5.54 (d, J = 8.4 Hz, 1H, CH);
7.23 (t, 1H, J = 7.6 Hz, arom.); 7.31 (d, J = 8.0 Hz, 1H, arom.); 7.33-7.41 (m, 4H, arom.); 7.43-7.49
(m, 1H, arom.); 7.56-7.61 (m, 3H, NH and arom.) ppm. This compound (0.58 g, 1.29 mmol) and
NaOH (0.08 g, 1.5 eq) were stirred in a mixture of THF/MeOH/H2O (2 mL each) for 2 h at room
temperature. The mixture was treated with a saturated solution of NH4Cl and extracted with AcOEt.
Drying (Na2SO4) and removal of the solvent gave the title compound in 99% yield as a white solid
(m.p. 140-141 °C). [1H]-NMR (CDCl3): 1.37-1.44 (m, 4H, 2CH2); 1.63-1.75 (m, 4H, 2CH2); 2.34 (t,
J = 7.2 Hz, 2H, CH2); 2.39 (td, J = 7.6, 4.4 Hz, 2H, CH2); 3.48 (s, 3H, NCH3); 5.57 (d, J = 8.0 Hz,
1H, CH); 7.24 (t, 1H, J = 8.0 Hz, arom.); 7.35 (dd, J = 8.0, 1.2 Hz, 1H, arom.); 7.37-7.42 (m, 3H,
arom.); 7.45-7.53 (m, 2H, arom.); 7.57-7.63 (m, 3H, NH and arom.) ppm.
12

ACCEPTED MANUSCRIPT
3.1.13. 9-[(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-ylamino)-9-
oxononanoic acid rac-32
Following the same procedure used for rac-28 starting from rac-27 (0.6 g, 2.26 mmol), methyl
azelate (0.48 g, 2.38 mmol), WSC (0.42 g, 2.72 mmol) and HOBT (0.37 g, 2.72 mmol) in ethanol-
free CHCl3 (20 mL), rac-29 was obtained with 93% yield. Brownish solid, m.p. 116-117 °C. [1H]-
NMR (CDCl3) δ: 1.31-1.37 (m, 6H, 3CH2); 1.58-1.73 (m, 4H, 2CH2); 2.30 (t, J = 7.6 Hz, 2H,
CH2CO); 2.36-2.41 (m, 2H, CH2CO); 3.47 (s, 3H, NCH3); 3.66 (s, 3H, OCH3); 5.56 (d, J = 8.0 Hz,
1H, CH); 7.22-7.26 (m, 1H, arom.); 7.35-7.41 (m, 5H, NH + arom.); 7.46-7.49 (m, 1H, arom.);
7.59-7.61 (m, 3H, arom.) ppm. This compound (0.94 g, 2.09 mmol) was hydrolyzed with NaOH
(0.127 g, 3.18 mmol) as reported for rac-31, obtaining the title compound in 93% yield. White
solid, m.p. 158-159 °C. [1H]-NMR (CDCl3) δ: 1.36-1.41 (m, 6H, 3CH2); 1.61-1.73 (m, 4H, 2CH2);
2.33 (t, J = 7.6 Hz, 2H, CH2CO); 2.38 (td, J = 7.4 Hz, 2.0 Hz, 2H, CH2 CO); 3.48 (s, 3H, NCH3);
5.58 (d, J = 8.6 Hz, 1H, CH); 7.25 (t, J = 7.6 Hz, 1H, arom.); 7.35-7.42 (m, 4H, arom.); 7.46-7.50
(m, 1H, arom.); 7.54 (d, J = 8.6 Hz, 1H, NH); 7.59-7.61 (m, 3H, arom.) ppm.
3.1.14. 6-[(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-ylamino)-6-
oxohexanoic acid rac-33
Following the same procedure used for rac-28, starting from rac-27 (1.2 g, 4.53 mmol), adipic acid
ethyl ester (0.83 g, 4.75 mmol), WSC (0.84 g, 5.43 mmol) and HOBT (0.73 g, 5.43 mmol) in
ethanol-free CHCl3 (20 mL), rac-30 was obtained in 98% yield. Light green solid, m.p. 148-149 °C.
[1H]-NMR (CDCl3) δ: 1.25 (t, J = 7.2 Hz, 3H, CH2CH3); 1.69-1.75 (m, 4H, 2CH2); 2.30-2.36 (m,
2H, CH2); 2.38-2.43 (m, 2H, CH2); 3.47 (s, 3H, NCH3); 4.12 (q, J = 7.2 Hz, 2H, CH2CH3); 5.56 (d,
J = 8.4 Hz, 1H, CH); 7.23-7.27 (m, 1H, arom.); 7.34-7.43 (m, 5H, NH + 4H arom.); 7.46-7.50 (m,
1H, arom.); 7.59-7.63 (m, 3H, arom.) ppm. This compound (1.88 g, 4.46 mmol) was hydrolyzed
with NaOH (0.27 g, 6.79 mmol) in 6 mL of MeOH, 6 mL of THF and 6 mL of H2O, obtaining the
title compound in 92% yield. White solid, m.p. 216-217 °C. [1H]-NMR (CDCl3) δ: 1.73-1.79 (m,
4H, 2CH2); 2.38-2.47 (m, 4H, 2CH2); 3.49 (s, 3H, NCH3); 5.57 (d, J = 8.2 Hz, 1H, CH); 7.24-7.27
(m, 1H, arom.); 7.35-7.42 (m, 4H, arom.); 7.46-7.50 (m, 1H, arom.); 7.56 (d, J = 8.2 Hz, 1H, NH);
7.59-7.64 (m, 3H, arom.) ppm.
3.1.15. 7-[(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-ylamino)-7-
oxoheptanoic acid rac-34
2,8-Oxocanedione (0.54 g, 3.77 mmol), prepared from pimelic acid and acetic anhydride, was
added to a solution of rac-27 (1 g, 3.77 mmol) in dry THF (20 mL), and the mixture was stirred at
RT under nitrogen for 2 h, while the color turned from yellow to green. Water (40 mL) was added,
and the mixture was extracted in EtOAc (3 x 50 mL). The organic phase was then extracted with
NaOH (2 x 30 mL, 10% aqueous solution) and the aqueous phase additioned with HCl (2N aqueous
solution) until pH 2 and extracted with CH2Cl2 (3 x 30 mL). Drying (Na2SO4), removal of solvent
and purification with flash chromatography (eluent: CH2Cl2/MeOH 95:5) gave rac-34 in 48% yield.
White solid, m.p. 170-171 °C. [1H]-NMR (CDCl3) δ: 1.41-1.48 (m, 2H, CH2); 1.64-1.77 (m, 4H,
2CH2); 2.33-2.41 (m, 4H, 2CH2); 3.47 (s, 3H, NCH3); 5.57 (d, J = 8.4 Hz, 1H, CH); 7.22-7.26 (m,
13

ACCEPTED MANUSCRIPT
1H, arom.); 7.33-7.41 (m, 4H, arom.); 7.45-7.49 (m, 1H, arom.); 7.56-7.62 (m, 4H, NH + 3 arom.)
ppm.
3.1.16. N1-hydroxy-N8-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-
yl)octanediamide (rac-8)
To a solution of rac-31 (0.55 g, 1.29 mmol) and Et3N (0.23 ml, 1.67 mmol) in anhydrous CH2Cl2 (5
mL), BOP-Cl (0.56 g, 2.2 mmol), dimethyl-t-butylhydroxylamine (0.29 g, 1.98 mmol) and Et3N
(0.54 ml, 3.87 mmol) were added, and the mixture was stirred at room temperature for 20 h. The
solvent was removed under vacuum, the residue was partitioned between H2O and AcOEt, the
organic layer was collected, dried (Na2SO4) and the solvent evaporated. The residue was dissolved
in MeOH (10 mL) and heated at 50°C for 20 h; then the solvent was removed leaving a residue
which was purified by flash chromatography (CH2Cl2/MeOH 95:5 as eluent). The title compound
was obtained in 27 % yield as a white solid m.p. 84-85 °C. [1H]-NMR (CD3OD): 1.33-1.48 (m, 4H,
2CH2); 1.58-1.73 (m, 4H, 2CH2); 2.10 (t, J = 7.2 Hz, 2H, CH2); 2.41 (t, J = 7.6 Hz, 2H, CH2); 3.48
(s, 3H, NCH3); 5.39 (s, 1H, CH); 7.29-7.35 (m, 2H), 7.40-7.45 (m, 2H), 7.47-7.53 (m, 1H), 7.54-
7.63 (m, 3H), and 7.66-7.73 (m, 1H) (aromatic protons) ppm. [13C]-NMR-APT (CD3OD) δ: 24.91
(CH2); 25.15 (CH2); 28.05 (CH2); 32.33 (CH2); 35.71 (NCH3); 36.28 (CH2); 67.31 (CH); 121.69
(CH arom.); 124.33 (CH arom.); 128.47 (CH arom.); 128.92 (C arom.); 130.047 (CH arom.);
130.95 (CH arom.); 131.17 (CH arom.); 132.40 (CH arom.); 137.71 (C arom.); 142.97 (C arom.);
168.10 (CN); 168.96 (CO); 171.28 (CO); 173.79 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for
C24H28N4O4: 437.2189, found: 437.2188.
3.1.17. N1-hydroxy-N9-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-
yl)nonanediamide (rac-9)
Following the same procedure used for rac-8, rac-32 (0.83 g, 1.91 mmol) was treated with Et3N
(1.14 mL, 8.20 mmol), BOPCl (0.83 g, 3.24 mmol) and dimethyl-t-butylsilylhydroxylamine (0.43 g,
2.92 mmol) in 20 mL of CH2Cl2 at RT for 26 h, and after work-up the residue was heated in MeOH
(20 mL) at 50 °C for 20 h. Evaporation of the solvent and purification with flash chromatography
(eluent: CH2Cl2/MeOH 95:5) gave the title compound in 31% yield. Grey solid, m.p. 96-98 °C.
[1H]-NMR (CD3OD) δ: 1.31-1.42 (m, 6H, 3CH2); 1.61-1.69 (m, 4H, 2CH2); 2.09 (t, J = 7.4 Hz, 2H,
CH2CO); 2.40 (t, J = 7.6 Hz, 2H, CH2CO); 3.48 (s, 3H, NCH3); 5.39 (s, 1H, CH); 7.30-7.35 (m,
2H, arom.); 7.41-7.45 (m, 2H, arom.); 7.49-7.53 (m, 1H, arom.); 7.57-7.63 (m, 3H, arom.); 7.69-
7.73 (m, 1H, arom.) ppm. [13C]-NMR-APT (CD3OD) δ: 26.61 (CH2); 29.91 (CH2); 29.96 (CH2);
33.72 (CH2); 35.69 (NCH3); 36.77 (CH2); 69.18 (CH); 123.16 (CH arom.); 125.79 (CH arom.);
129.38 (CH arom.); 130.09 (C arom.); 130.77 (CH arom.); 131.31 (CH arom.); 131.96 (CH arom.);
133.51 (CH arom.); 139.26 (C arom.); 144.34 (C arom.); 169.33 (CN); 169.58 (CO); 172.94 (CO);
176.33 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for C25H31N4O4: 451.2345, found: 451.2346.
3.1.18. N1-hydroxy-N6-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-
yl)hexanediamide (rac-10)
Following the same procedure used for rac-8, rac-33 (1.58 g, 4.01 mmol) was treated with Et3N
(2.40 mL, 17.25 mmol), dimethyl-t-butylsilylhydroxylamine (0.90 g, 6.14 mmol) and BOPCl (1.74
14

ACCEPTED MANUSCRIPT
g, 6.83 mmol) in 20 mL of CH2Cl2 at RT for 22 h, and after work-up the residue was heated in
MeOH (30 mL) at 60 °C for 22 h. Evaporation of the solvent and purification with flash
chromatography (eluent: CH2Cl2/MeOH 95:5) gave the title compound in 18% yield. Light green
solid, m.p. 75-76 °C. [1H]-NMR (CD3OD) δ: 1.68-1.70 (m, 4H, 2CH2); 2.12-2.15 (m, 2H, CH2);
2.40-2.42 (m, 2H, CH2); 3.47 (s, 3H, NCH3); 5.39 (s, 1H, CH); 7.29-7.34 (m, 2H, arom.); 7.40-
7.44 (m, 2H, arom.); 7.48-7.52 (m, 1H, arom.); 7.57-7.62 (m, 3H, arom.); 7.68-7.72 (m, 1H, arom.)
ppm. [13C]-NMR-APT (CD3OD) δ: 26.10 (CH2); 26.29 (CH2); 33.52 (CH2); 35.68 (NCH3); 36.34
(CH2); 69.27 (CH); 123.20 (CH arom.); 125.83 (CH arom.); 129.41 (CH arom.); 130.17 (C arom.);
130.81 (CH arom.); 131.36 (CH arom.); 131.98 (CH arom.); 133.55 (CH arom.); 139.32 (C arom.);
144.40 (C arom.); 169.36 (CN); 169.69 (CO); 172.69 (CO); 175.94 (CO) ppm. HRMS-ESI m/z
[M+H]+ calcd for C22H25N4O4: 409.1876, found: 409.1871.
3.1.19. N1-hydroxy-N7-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-
yl)heptanediamide (rac-11)
A solution of NH2OH.HCl (0.16 g, 2.31 mmol) in dry MeOH (5 mL) was added to a solution of
KOH (0.13 g, 2.31 mmol) in dry MeOH (5 mL) at 0 °C under nitrogen. The resulting mixture was
stirred for 15’ at 0 °C and filtered in another flask.
Ethyl chloroformate (0.06 mL, 0.65 mmol) and anhydrous Et3N (0.1 mL, 0.71 mmol) were added to
a solution of rac-34 (0.2 g, 0.49 mmol) in dry THF (5 mL) at 0 °C under nitrogen. The mixture was
stirred for 15’ at 0 °C and filtered directly in the flask containing the previously prepared solution of
NH2OH in MeOH. The resulting mixture was stirred for 30’ at RT under nitrogen, then the solvents
were evaporated. The residue was purified by flash chromatography (first eluent: CH2Cl2/MeOH
95:5, second eluent: CH2Cl2/MeOH 9:1) giving the title compound in 47.2% yield. White solid,
m.p. 89-90 °C. [1H]-NMR (CD3OD) δ: 1.28-1.44 (m, 2H, CH2); 1.61-1.71 (m, 4H, 2CH2); 2.11 (t, J
= 7.4 Hz, 2H, CH2CO); 2.92 (t, J = 7.6 Hz, 2H, CH2CO); 3.46 (s, 3H, NCH3); 5.39 (s, 1H, CH);
7.28-7.33 (m, 2H, arom.); 7.40-7.44 (m, 2H, arom.); 7.48-7.51 (m, 1H, arom.); 7.57-7.61 (m, 3H,
arom.); 7.67-7.71 (m, 1H, arom.) ppm. [13C]-NMR-APT (CD3OD) δ: 26.26 (CH2); 26.40 (CH2);
29.58 (CH2); 33.61 (CH2); 35.67 (NCH3); 36.61 (CH2); 69.26 (CH); 123.21 (CH arom.); 125.84
(CH arom.); 129.40 (CH arom.); 130.22 (C arom.); 130.80 (CH arom.); 131.37 (CH arom.); 131.96
(CH arom.); 133.54 (CH arom.); 139.36 (C arom.); 144.42 (C arom.); 169.40 (CN); 169.73 (CO);
172.88 (CO); 176.22 (CO) ppm. HRMS-ESI m/z [M+H]+ calcd for C23H27N4O4: 423.2032, found:
423.2028.
3.1.20. Preparation of (S)-27 and (R)-27.
3.1.20.1. Method A. 1,3-dihydro-5-phenyl-3(R,S)-{[(R)-α-methylbenzyloxycarbonyl]amino]}-1,4-
benzodia-zepin-2-one was prepared according to Sherrill [21] as diasteromeric mixture, which was
separated by 7 flash chromatographic runs using toluene/ethyl acetate 8:2 as eluent. 1,3-Dihydro-5-
phenyl-(3S)-{[(R)-α-methylbenzyloxycarbonyl]amino]}-1,4-benzodiazepin-2-one (the first eluting
isomer) and its 3R diastereomer were obtained in 31% and 24% yields, respectively (lit.[21] 41%
and 37%, respectively), with ee > 99% (NMR). N1-Methylation and subsequent removal of the
carbamate moiety according to the literature procedure gave (S)-27 and (R)-27 in 75% yields. The
NMR spectra of the enantiomers were identical to that of the racemate. (S)-27: [α] 20D -170.6° (c=1,
CH3OH). (R)-27: [α] 20D +159.0° (c=1, CH3OH).
15

ACCEPTED MANUSCRIPT
3.1.20.2. Method B. Treatment of rac-27 (0.34 g) with 0.5 eq of (S)-camphorsulfonic acid in ethyl
acetate afforded, after seeding, the 3(S)-amine-camphorsulfonate salt. A second crystallization from
acetonitrile, treatment with NaOH and extraction with CH2Cl2 afforded 0.16 g of (S)-27 with
enantiomeric excess greater than 99.5 %. The original seeds of the (3S)-amine-camphorsulfonate
salt were made from the (3S)-amine, obtained through HPLC separation, using the following
conditions: Column: LUX Amylose-2 (Phenomenex); particle size: 5ꢀm; length: 250 mm; diameter:
10 mm. Eluent (isocratic conditions): CH3CN/CH3OH 95:05 with 0.1% diethylamine. Flow rate:
2ml/min at 40°C for 30 min. The enantiomeric excess was assessed using the following conditions:
Column LUX Amylose-2 (Phenomenex); particle size: 3 ꢀm; length: 50 mm; diameter: 4.6 mm.
Eluent (isocratic conditions): NH4OH (10 mM)/CH3CN 65:35. Flow rate: 1 ml/min at 40°C for 10
min.
3.1.21. (S) and (R) N1-hydroxy-N8-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diazepin-3-yl)octanediamide [(S)-8] and [(R)-8]
These compounds were prepared starting from (S)-27 and (R)-27 as described for the racemate. The
NMR spectra of the intermediate and final compounds are identical to those of the racemates. Other
physicochemical properties are the following:
(S)-28: waxy solid, 60% yield. [α]20D -56.0° (c=1, CHCl3).
(R)-28: waxy solid, 68% yield. [α]20D +55.3° (c=1, CHCl3)
(S)-31: white solid, 82% yield. Mp 48-9°C. [α]20D -51.8° (c=1, CHCl3)
(R)-31: white solid, 89% yield. Mp 48-9°C. [α]20D +50.0° (c=1, CHCl3)
(S)-8: white solid, 29% yield. Mp 82-3°C. [α]20D -53.7° (c=1, CH3OH)
(R)-8: white solid, 24% yield. Mp 84-5°C. [α]20D +55.4° (c=1, CH3OH)
3.1.22. Benzyl N-(7-iodo-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamate 36
Anhydrous CH2Cl2 (6 mL), oxalyl chloride (1.39 mL, 15.91 mmol) and anhydrous DMF (0.12 mL)
were added to a solution of 35 [21] (5.19 g, 15.91 mmol) in anhydrous THF (45 mL) at 0 °C under
nitrogen. After 3 h and 30’ at 0 °C, a solution of 2-amino-5-iodobenzophenone[23] (4.67 g, 14.46
mmol) and N-methylmorpholine (3.85 mL, 35.00 mmol) in anhydrous THF (15 mL). The mixture
was allowed to reach room temperature, then was filtered and the solid washed with THF (15 mL).
NH3 was bubbled in the organic solution for 25’, methanol (70 mL) was added and NH3 was kept
bubbling for 20’. The solution was then partitioned between EtOAc (300 mL) and NaOH (1N,
aqueous solution), the aqueous phase was washed with EtOAc and the combined organic extracts
were washed with brine. Drying (Na2SO4) and removal of the solvent gave a residue that was
solved in glacial acetic acid (70 mL) and additioned with ammonium acetate (5.19 g, 67.33 mmol).
The mixture was stirred at room temperature overnight, evaporated and treated with NaHCO3
(aqueous saturated solution). The resulting slurry was filtered, and the solid residue washed with
water and dried under reduced pressure to give the title compound in 37.5 % yield. White solid,
m.p. 270-272 °C (dec.). [1H]-NMR (DMSO) δ: 5.06 (d, J = 8.6 Hz, 1H, CHN); 5.08 (s, 2H, CH2Ph);
7.11 (d, J = 8.4 Hz, 1H, arom.); 7.26-7.39 (m, 5H, arom.); 7.44-7.47 (m, 4H, arom.); 7.50-7.55 (m,
2H, arom.); 7.95 (dd, J = 8.4 Hz, 1.6 Hz, 1H, arom.); 8.50 (d, J = 8.6 Hz, 1H, NHCBZ); 10.95 (s,
1H, ArNH) ppm
16

ACCEPTED MANUSCRIPT
3.1.23. t-Butyl N-(7-iodo-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-
yl)carbamate 37
To a solution of 36 (3.01 g, 5.89 mmol) in dry DMF (20 mL), kept at 0 °C under N2, NaH (60%
dispersion in mineral oil, 0.25 g, 6.18 mmol) was added in small portions. After 1.5 hr stirring at 0
°C, CH3I (0.38 mL, 6.18 mmol) was added at once; the mixture was stirred for additional 1.5 h at
the same temperature, and then poured into a stirred solution of H2O (60 mL) containing aqueous
sodium hydrogen sulphate (2 mL, 1 N). A solid precipitated, which was filtered, dried under
vacuum, and then purified by flash chromatography (CH2Cl2/MeOH/NH3 99:1:0.1 as eluent) giving
benzyl N-(7-iodo-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamate in
99% yield. Pale yellow solid, m.p. 65-67 °C. [1H]-NMR (CDCl3) δ: 3.43 (s, 3H, NCH3); 5.13 (d, J =
12.0 Hz, 1H, CHHPh); 5.17 (d, J = 12.0 Hz, 1H, CHHPh); 5.30 (d, J = 8.4 Hz, 1H, CHN); 6.66 (d, J
= 8.4 Hz, 1H, NHCBZ); 7.12 (d, J = 8.8 Hz, 1H, arom.); 7.27-7.43 (m, 7H, arom.); 7.48-7.53 (m,
1H, arom.); 7.58-7.60 (m, 2H, arom.); 7.66 (d, J = 2.0 Hz, 1H, arom.); 7.88 (dd, J = 8.8 Hz, 2.0 Hz,
1H, arom.) ppm.
This compound was treated with HBr (33% in AcOH, 5 mL) under N2 and the solution was kept
stirring at room temperature for 2 hr, then diluted with Et2O (15 mL) to obtain a suspension. The
mixture was filtered and the solid dissolved in H2O. The aqueous solution was then made alkaline
with Na2CO3 and extracted with AcOEt. The organic phase was dried (Na2SO4), the solvent was
removed under vacuum obtaining 3-amino-7-iodo-1-methyl-5-phenyl-2,3-dihydro-1H-1,4-
benzodiazepin-2-one in 95% yield. Pale yellow solid, m.p. 69-71 °C. [1H]-NMR (CD3OD) δ: 3.45
(s, 3H, NCH3); 4.44 (s, 1H, CHN); 7.38 (d, J = 8.8 Hz, 1H, arom.); 7.44-7.48 (m, 2H, arom.); 7.51-
7.54 (m, 4H, arom.); 7.98 (dd, J = 8.8 Hz, 2.0 Hz, 1H, arom.) ppm.
This compound was dissolved in anhydrous THF (5 mL) and di-tert-butyl dicarbonate (0.17 g, 0.77
mmol) dissolved in anhydrous THF (5 mL) was added dropwise at 0 °C under nitrogen. The
solution was left stirring at room temperature overnight, then the solvent was evaporated and the
residue partitioned between EtOAc and brine. Drying (Na2SO4) of the organic phase and removal of
the solvent gave a crude that was purified with flash chromatography (cyclohexane/ethyl acetate
6:4) giving the title compound in 84% yield. White solid, m.p. 186-187 °C. [1H]-NMR (CDCl3) δ:
1.47 (s, 9H, tBu); 3.43 (s, 3H, NCH3); 5.27 (d, J = 8.6 Hz, 1H, CHN); 6.41 (d, J = 8.6 Hz, 1H,
NHBOC); 7.11 (d, J = 8.4 Hz, 1H, arom.); 7.39-7.43 (m, 2H, arom.); 7.47-7.51 (m, 1H, arom.);
7.58-7.60 (m, 2H, arom.); 7.64 (d, J = 2.0 Hz, 1H, arom.); 7.86 (dd, J = 8.4 Hz, 2.0 Hz, 1H, arom.)
ppm.
3.1.24. t-Butyl N-{7-[5-(hydroxycarbamoyl)pent-1-yn-1-yl]-1-methyl-2-oxo-5-phenyl-2,3-
dihydro-1H-1,4-benzodiazepin-3-yl}carbamate 38
Following the same procedure used for 15, 37 (0.24 g, 0.49 mmol) was treated with 5-hexynoic acid
(0.10 g, 0.92 mmol), CuI (0.01 g, 0.06 mmol), Pd(PPh3)4 (0.03 g, 0.02 mmol) and anhydrous Et3N
(5 mL) at 55 °C for 3 h under nitrogen. Purification with flash chromatography (CH2Cl2/MeOH
95:0.5)
gave
6-(3-(tert-butoxycarbonylamino)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diaze-pin-7-yl)hex-5-ynoic acid in 99% yield. Yellow solid, m.p. 64-66 °C. [1H]-
NMR (CDCl3) δ: 1.46 (s, 9H, tBu); 1.85-1.92 (m, 2H, CH2); 2.43-2.51 (m, 4H, 2CH2); 3.44 (s, 3H,
NCH3); 5.28 (d, J = 8.6 Hz, 1H, CHN); 6.46 (d, J = 8.6 Hz, 1H, NHBOC); 7.28 (d, J = 8.4 Hz, 1H,
arom.); 7.34 (d, J = 1.6 Hz, 1H, arom.); 7.38-7.44 (m, 2H, arom.); 7.45-7.49 (m, 1H, arom.); 7.55
17

ACCEPTED MANUSCRIPT
(dd, J = 8.8 Hz, 2.0 Hz, 1H, arom.); 7.59-7.61 (m, 2H, arom.) ppm. Following the same procedure
reported for 3, this compound (0.23 g, 0.49 mmol) was treated with ethyl chloroformate (0.06 mL,
0.65 mmol), Et3N (0.10 mL, 0.71 mmol), NH2OH.HCl (0.16 g, 2.29 mmol) and KOH (0.13 g, 2.29
mmol). Purification by flash chromatography (CH2Cl2/MeOH 90:10) gave the title compound in
77% yield. Pale yellow solid, m.p. 80-81 °C. [1H]-NMR (CDCl3) δ: 1.46 (s, 9H, tBu); 1.84-1.87 (m,
2H, CH2); 2.25 (t, J = 7.6 Hz, 2H, CH2); 2.38 (t, J = 7.6 Hz, 2H, CH2); 3.44 (s, 3H, NCH3); 5.27 (d,
J = 8.4 Hz, 1H, CHN); 6.53 (d, J = 8.4 Hz, 1H, NHBOC); 7.28-7.33 (m, 2H, arom.); 7.39-7.42 (m,
2H, arom.); 7.47-7.50 (m, 1H, arom.); 7.56-7.62 (m, 3H, arom.) ppm.
3.1.25. 6-(3-amino-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-7-yl)-N-
hydroxyhex-5-ynamide 12
A freshly prepared solution of HCl in EtOAc (0.28 N, 7.69 mL) was added to a solution of 38 (0.11
g, 0.22 mmol) in EtOAc (1 mL) under nitrogen. The mixture was stirred at room temperature for 24
h and then filtered. The solid residue was washed with EtOAc and purified with flash
chromatography (CH2Cl2/MeOH/NH3 90:10:01) to give the title compound in 58% yield. White
solid, m.p. 67-69 °C. [1H]-NMR (CD3OD) δ: 1.81-1.88 (m, 2H, CH2); 2.21 (t, J = 7.2 Hz, 2H, CH2);
2.42 (t, J = 7.2 Hz, 2H, CH2); 3.46 (s, 3H, NCH3); 4.43 (s, 1H, CHN); 7.25 (d, J = 1.6 Hz, 1H,
arom.); 7.42-7.46 (m, 2H, arom.); 7.49-7.58 (m, 4H, arom.); 7.66 (dd, J = 8.4 Hz, 2.0 Hz, 1H,
arom.) ppm. [13C]-NMR-APT (CD3OD) δ: 19.44 (CH2); 25.71 (CH2); 32.71 (CH2); 35.44 (CH3);
70.95 (CH); 80.51 (C); 91.37 (C); 121.52 (C arom.); 123.19 (CH arom.); 129.49 (2CH arom.);
130.27 (C arom.); 130.73 (2CH arom.); 131.97 (CH arom.); 133.87 (CH arom.); 136.05 (CH
arom.); 139.23 (C arom.); 143.59 (C arom.); 167.85 (CN); 171.32 (CO); 172.13 (CO) ppm. HRMS-
ESI m/z [M+H]+ calcd for C22H23N4O3: 391.1770, found: 391.1765. Treatment with 1 eq. of HCl
(0.28 N in EtOAc) gave the corresponding hydrochloride
3.2. Biological Assays
3.2.1. Reagents
The compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO,
USA) to make stock solutions (100 µM) which were stored at room temperature protected from
light. Aliquots of this solution were diluted with DMSO according to the need and then added
directly to the culture. The amount of DMSO (≤ 0.3%) used as the vehicle did not interfere with
activities of compounds
3.2.2. Histone Deacetylase Assay.
3.2.2.1. HeLa nuclear extracts. The in vitro activity of HDAC inhibitors was assayed using a
BIOMOL Kit AK-500, according to the instructions from the manufacturer (Biomolecular Research
Laborator). The assay is automated and performed by a Tecan Freedom EVO® work station. On
detail, 15 µl of 30 x diluted nuclear fraction of HeLa cells (9 µg/µl), was diluted to 50 µl with the
assay buffer containing the HDAC inhibitor and the substrate (lysine with acetylated amino group
on the side chain) at a concentration of 100 µM.
The samples were incubated for 15 min at room temperature (RT) and then exposed to a developer
(10 min at RT). In this last step a fluorophore was produced, whose fluorescence was measured
18

ACCEPTED MANUSCRIPT
using an excitation wavelength of 355 nm and an emission at 460 nm. All determinations were
carried out in triplicate and the IC50 was calculated using GraphPad Software
3.2.2.2. Fluorometric human recombinant HDAC1, 4 and 6 Assays. The HDAC Fluorescent
Activity Assay for HDAC1, 4 and 6 is based on the Fluor de Lys Substrate and Developer
combination (BioMol AK-500) and has been carried out according to supplier’s instructions and as
previously reported. First, the Fluor de Lys Substrate, which comprises an acetylated lysine side
chain, has been incubated with purified recombinant HDAC1 or 6 enzymes in presence or absence
of the inhibitors. Briefly, for HDAC1, and HDAC6, 100 ng of recombinant proteins have been used
per assay, respectively. Full length HDAC1 and 6 with C-terminal His tag were expressed using
baculovirus expression systems. Deacetylation sensitizes the substrates that, in the second step,
treatment with the developer produces a fluorophore. Fluorescence has been quantified with a
TECAN Infinite M200 station and IC50 values has been obtained using GraphPad program.
Inhibition of HDAC4 has been measured using a fixed (5ꢀM) concentration of the compounds,
finding a residual enzymatic activity not below 65%. For this reason a dose response curve was not
performed. On this isoform, the IC50 value for SAHA resulted 0.55 ꢀM [27].
3.2.3. Cell Growth Inhibition Assay
The antiproliferative activity of HDAC inhibitors was evaluated using the CellTiter- Glo®,
according the instruction from the manufacture (Promega). The assay is automated and performed
by a Tecan Freedom EVO® work station. On detail, K562, A549 and HCT-116 cells (from the
ATTC), in exponential growth, were seeded on 96-well plates, 24 hours later the cells were
incubated for 72 hours with different concentrations of the inhibitors. After drug treatment, a
volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium was added. The
content was mixed for 2 min to induce cell lysis. The luminescence was recorded after further 10
min at RT in order to obtain a stabilized luminescent signal. All determinations were carried out in
triplicate and the IC50 was calculated using GraphPad Software
3.2.4. Experiments with human acute promyelocytic NB4 cells
3.2.4.1. Culture conditions and determination of IC50. The all-trans-retinoic acid (ATRA)-sensitive
human acute promyelcytic cell line NB4 [28] was used as the experimental model. Cells were
maintained in RPMI 1640 (Bio-Whittaker Europe, Verviers, Belgium) supplemented with 10% fetal
calf serum (FCS, EuroClone, Life Science Division, Milan, Italy) at 37°C in a humidified
atmosphere of 5% CO2. Cells were counted in a Bürker chamber and viability was assessed by the
trypan blue exclusion test. Cell number in culture was used to determine the IC50 value, i.e. the drug
concentration capable of decreasing cell density by 50% as compared to control.
3.2.4.2. Cell lysate preparation, SDS-PAGE and Western blot. Harvested cells were resuspended in
a lysis buffer [1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 40 mM Tris–HCl pH 8.0,
150 mM NaCl] and sonicated (Microson XL-2000, Misonix, Farmingdale, NY, USA) as described
previously. Acetylated isoforms of histone H3 and H4, and α-tubulin (used as the loading control)
were determined following 12.5% SDS-PAGE separation of extracts and western blot; then,
nitrocellulose membranes were probed using primary rabbit polyclonal antibodies against the
acetyl-H3 or -H4 histone (Upstate Biotechnology, Lake Placid, NY, USA) and suitable peroxidase-
conjugated secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) for immunostaining.
19

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The ECL procedure on Hyperfilm ECL (Amersham Biosciences, Buckinghamshire, UK) was used
for development. The intensity of each stained band on the gel was then quantified by densitometric
analysis with the aid of ImageJ software, using tubulin as the reference protein; values of treated
versus untreated samples were from a typical experiment.
3.2.4.3. Determination of apoptosis. Apoptotic cells in the populations were measured with a
FACScan flow cytometer (Becton-Dickinson) by the Annexin-V-Fluos Apoptosis detection kit
(Roche Molecular Biochemicals, Mannheim, Germany). Usually, 0.5x106 cells were used for
labeling with Annexin-V-Fluos and propidium iodide (PI) in binding buffer according to the
manufacturer’s instructions. Moreover, apoptosis was also assessed by examining May-
Grünwald/Giemsa stained cytosmears (magnification: x200) with the aid of a microscope (Nikon
Eclipse, mod. 50i) equipped with a digital camera DS-5M USB2 (Nikon Instruments, Florence,
Italy).
3.2.5. Statistical analysis.
All experiments were independently done at least 3 times and data were statistically analyzed by
Student’s t test.
4. Acknowledgments
This work was supported by grants from MUR (PRIN 2006 to F.P., # 200606139) and the
University of Florence (ex 60%), Ente Cassa di Risparmio di Firenze (726/2005) and AIL
(Associazione Italiana contro le Leucemie Linfomi e Mieloma, Firenze).
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Chart 1
O
O
O
H
N
OH
OH
N
N
H
H
O
H3C
CH3 CH3
N
SAHA (Vorinostat)
O
Trichostatin-A (TSA)
CH3
Me
N
PhO2S
NH
R
N
1: R = H
(S)-2: R = Me
H
N
OH
O
O
NHOH
( )6
Oxamflatim
R1
O
NHOH
Me
O
O
O
N
N
N
O
Y
N
1
3
R
NH
O
( )n
7
N
N
NHOH
X
O
NHOH
A
B
C
3: X = (CH2)2, R=H, R1=Me
4: X = (CH2)4, R=H, R1=Me
5: X = CH=CH, R=H, R1=Me
(S)-6: X = (CH2)3, R=Me, R1=H
8: n = 6
9: n = 7
10: n = 4
11: n = 5
7: Y = CH2
rac-12: X = (CH2)3, R=NH2 , R1=Me
23

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Table 1. HDAC inhibition of compounds 1-12 on HeLa nuclear extracts and on recombinant
HDAC1 and HDAC6 enzymes.a
HeLa b
HDAC1 c
HDAC6 c
N
formula
R1
X
R2
n
%
IC50
(µM)
inhibition
at 1µM
IC50 (µM)
IC50 (µM)
A
A
A
A
A
A
A
B
C
C
C
C
C
A
CH3
CH3
CH3
CH3
CH3
(CH2)3
(CH2)3
(CH2)3
(CH2)2
(CH2)4
H
-
-
39
21
63
22
36
82
65
82
73
88
57
70
73
nt
-
-
2.15d ± 0.041 3.35d ± 0.057
2.18d ± 0.043 7.08d ± 0.037
1
(R)-2
(S)-2
3
CH3
CH3
-
0.36 0.75d ± 0.029 2.70d ± 0.038
H
-
-
-
2.75 ± 0.046
1.90 ± 0.039
>100
H
-
5.90 ± 0.043
7.72 ± 0.054
0.46 ± 0.047
1.12 ± 0.038
6.50 ± 0.047
0.40 ± 0.051
9.08 ± 0.036
>100
4
CH3 CH=CH
H
-
0.16 0.44 ± 0.027
0.34 0.49 ± 0.042
0.18 0.25 ± 0.036
0.31 0.70 ± 0.044
0.12 0.55 ± 0.047
0.60 0.48 ± 0.024
0.38 0.42 ± 0.046
0.32 0.27 ± 0.044
5
(S)-6
7
H
(CH2)3
CH3
-
-
-
-
-
(R)-8
(S)-8
rac-9
rac-10
rac-11
rac-12
SAHA
-
-
-
6
6
7
4
5
-
-
-
-
-
-
-
-
-
-
-
-
-
0.18 ± 0.036
>100
CH3
(CH2)3
NH2
nt
1.09 ± 0.049
85
0.13 0.31 ± 0.022 0.038 ± 0.0037
a
b
All determinations were carried out in triplicate; values represent the mean. HeLa nuclear
extracts; in this automated assay IC50 values have been calculated only for compounds whose
d
activity at 1 µM was higher than 50%. SD is < 25%. c Human recombinant enzymes. From ref.
11.
24

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Table 2. Antiproliferative activity of compounds 1-11 on cell lines. a
IC50 (µM)
N
formula R1
X
R2
n
HCT 116 b A549
K562
NB4
1.95
> 5d
2.06
> 5d
4.09
2.60
3.04
3.99
b
b
c
A
A
A
A
A
A
A
B
C
C
C
C
C
CH3 (CH2)3
H
-
-
1
(R)-2
(S)-2
3
2.62
7.05
0.74
6.54
1.86
0.46
0.97
1.44
1.07
0.47
3.93
2.01
1.44
0.76
5.95
11.83
1.47
10.28
2.69
1.30
2.82
2.54
2.69
1.44
7.95
5.44
4.42
1.08
4.97
11.59
1.27
8.22
2.67
0.78
1.40
1.67
1.72
0.72
4.59
3.28
1.84
0.64
CH3 (CH2)3 CH3
CH3 (CH2)3 CH3
-
CH3 (CH2)2
CH3 (CH2)4
H
H
-
-
4
CH3 CH=CH H
-
5
(S)-6
7
H
-
(CH2)3 CH3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(R)-8
(S)-8
rac-9
rac-10
rac-11
SAHA
-
6
6
7
4
5
> 5d,e
0.86e
4.00
1.91
2.56
0.98
-
-
-
-
b
a All determinations were carried out in triplicate; values represent the mean. SD are < 25%.
c
Quantification of ATP by luminescence after incubation for 72 hr. Annexin test of apoptosis
after incubation for 48 hrs. d Highest tested concentration; at this dose, the percentage of apoptotic
cells was lower than 50%. e The IC50 of rac-8 was 1.2 ꢀM.
25

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Scheme 1
H
O
N
H
O
N
c
(CH2)3-COOH
(H2C)3
Me
Me
N
N
14
on (S)-
6
(S)-
Ph
Ph
(H2C)3
21
(S)-
COOH
CONHOH
R1
Me
Me
O
O
N
O
N
a
N
c
R
N
13
on
I
N
N
Ph
Ph
X
Ph
X
1
COOR2
13
(S)-14
: R=H, R = Me
CONHOH
1
: R =Me, R = H
2
15
18
: R = H, X = (CH2)4
3:
X = (CH2)4
: X = (CH2)2
5: X = CH=CH
2
4
: R = Et, X = (CH2)2
b
19: R2 = Et, X = CH=CH
13
on
(CH2)2
b
2
16
20
: R = H, X = (CH2)2
: R = H, X = CH=CH
COOH
2
Me
O
N
O
O
N
C
H
Ph
17
a) HC C-X-COOR; b) NaOH; c) ClCOOEt, NH2OH.HCl
26

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Scheme 2
O
(CH2)6COOR
O
(CH2)6CONHOH
H
X
X
N
N
N
a or b
d
N
N
N
Ph
22
Ph
Ph
: X = O
23: X = H2
24a
: R = H, X = O
24b: R = Et, X = O
7
25
: R = Et,X = H2
; R = H, X = H2
c
26
24b
25
); b) ethyl hydrogensuberate (for ); c) NaOH;
a) ClCO(CH2)6COOEt (for
d) ClCOOEt, NH2OH.HCl
27