Synthesis of new mono and bis amides projected as potential histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.tet.2010.01.061

In our ongoing efforts to discover new potent histone deacetylase (HDAC) inhibitors as promising anticancer candidates, we designed and synthesized a small collection of 3-substituted amines possessing macro heterocyclic skeletons bearing variable-length tails. As a metal binder domain, all the compounds possess an amide function suitable for Zn²⁺ chelation in the enzyme active site. A combination of solution and solid phase techniques were employed to synthesize the compounds and, as the key synthetic step to obtain the rings, a ring closing metathesis (RCM) reaction was adopted. The putative affinity of the compounds for the histone deacetylase-like protein (HDLP) model receptor active site was explored through docking calculations, and we also report preliminary studies on their pharmacological properties.

Full text of DOI:10.1016/j.tet.2010.01.061

Tetrahedron 66 (2010) 2520–2528
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of new mono and bis amides projected as potential histone
deacetylase (HDAC) inhibitors
Stefania Terracciano, Maria Giovanna Chini, Giuseppe Bifulco, Elisabetta D’Amico,
*
Stefania Marzocco, Raffaele Riccio, Ines Bruno
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2009
Received in revised form 9 December 2009
Accepted 18 January 2010
Available online 22 January 2010
In our ongoing efforts to discover new potent histone deacetylase (HDAC) inhibitors as promising an-
ticancer candidates, we designed and synthesized a small collection of 3-substituted amines possessing
macro heterocyclic skeletons bearing variable-length tails. As a metal binder domain, all the compounds
possess an amide function suitable for Zn2þ chelation in the enzyme active site. A combination of so-
lution and solid phase techniques were employed to synthesize the compounds and, as the key synthetic
step to obtain the rings, a ring closing metathesis (RCM) reaction was adopted. The putative affinity of the
compounds for the histone deacetylase-like protein (HDLP) model receptor active site was explored
through docking calculations, and we also report preliminary studies on their pharmacological
properties.
Keywords:
Histone deacetylase
Ring closing metathesis reaction
Anticancer drugs
Ó 2010 Elsevier Ltd. All rights reserved.
Docking studies
1. Introduction
focused our interest on FR235222,3 a natural cyclopeptide pos-
sessing a potent HDAC inhibitory activity and whose total synthesis
The epigenetic modulators1 have emerged as new and attractive
therapeutics as a consequence of their ability to influence tran-
scriptional events. Histone deacetylases (HDACs), in fact, have been
recently highlighted as promising targets in the epigenetic therapy
for the treatment of several disorders including cancer, as they are
responsible, together with histone acetyl transferase (HAT), for the
acetylation level of histone proteins which, in turn, is associated to
the transcriptional state of chromatin.2 The great potential of HDAC
inhibitors as anticancer drugs seems to be related to the tran-
scription and expression of oncogenes, which are silent in cancer
pathology. In the course of our research on bioactive natural
products as useful tools in the drug discovery field, we recently
has been successfully realized by us.4 With the aim of gaining more
information on the nature of the interaction between this structural
class of inhibitors and the biological target, a first collection of
FR235222 cyclopeptidic analogues was efficiently synthesized5 and
their biological profile was also evaluated in order to identify some
structural aspects involved in the activity modulation. At the same
time, through molecular docking calculations, a 3D model of the
interaction between a potential ligand and the active site of HDPL,6
the bacterial homologue of human HDAC, was also proposed, in
order to clarify the influence of some structural elements exploit-
able for a further structural optimization process.7 Basing on the
advances made in receptor mapping knowledge, we decided to
explore the effect on HDAC of entirely non-peptidic compounds,
having, as the binding recognition domain, macro-heterocycles or
substituted amines, and possessing, as metal binders, amide func-
tions, which proved to be very efficient in the case of the azuma-
mides,8 to produce the crucial Zn chelation event. Here, we report
the design, virtual screening, and synthesis of this new collection of
compounds, whose cytotoxic properties are also described.
Abbreviations: 4-aba, 4-allyloxybenzaldehyde; 4-Pea, 4-pentenoic acid; 6-Epa,
6-eptenoic acid; BAP, borane–pyridine complex; Boc, tert-butyloxycarbonyl; DCM,
dichloromethane; DIC, N,N0, diisopropyl-carbodiimide; DIEA, N,N-diisopropyle-
thylamine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethyloxycarbonyl;
Fmoc-6-Ahx-OH, Fmoc-6-aminohexanoic acid; Fmoc-8-Aoc-OH, Fmoc-8-amino-
octanoic acid; Grubbs second gen., Grubbs catalyst second generation; HBTU,
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt,
N-hydroxybenzotriazole; NMM, N-methylmorpholine; Rink Amide MBHA resin, 4-
(20,40-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamico-norleucyl-MBHA
resin; RCM, ring closing metathesis; TFA, trifluoroacetic acid; TFE, 2,2,2-tri-
fluoroethanol; TIS, triisopropylsilane; TMOF, trimethyl orthoformate; TNBS,
2,4,6-trinitrobenzenesulfonic acid; Uda, undecylenic aldehyde.
2. Results and discussion
The present work can be considered a further evolution of our
ongoing project involved in the discovery of new potential HDAC
inhibitors; in this regard, having only experience with compounds
* Corresponding author. Tel.: þ39 089 969743; fax: þ39 089 969702.
E-mail address: brunoin@unisa.it (I. Bruno).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.061

S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
2521
of a cyclopeptide nature and aimed at further increasing the in vivo
stability of the potential HDAC binders, we decided to reproduce, to
some extent, the molecular shape of our previous synthetic com-
pounds,5 but, instead of the cyclopeptide framework, we utilized
heterocyclic moieties or open chain tertiary amines, bearing a ter-
minal functionalized aliphatic chain of appropriate length. As the
metal binder domain, we selected the amide functionality present
in other potent HDAC inhibitors of natural origin, such as the azu-
mamides.8 Concerning the retrosynthetic plan of our molecules, we
immediately recognized the possibility of applying, the ring closing
metathesis (RCM) reaction,9 as the key step to close the rings,
which has emerged as a powerful tool for the construction of car-
bocyclic and heterocyclic ring systems. On the basis of this as-
sumption, and in consideration of the commercial availability of the
building blocks, we designed seven molecules (Scheme 1), five of
which (1, 2, 3, 6, 7) possessing, as cap group, cyclic structures of
different size, and bearing variable-length functional tails; the
other two (4, 5) mainly represent, to some extent, the open versions
of the previous ones.
The first step was a docking study on the designed molecules to
obtain a prediction of their histone deacetylases inhibitory activity,
through virtual screening process. Prior to the docking calculations,
we performed a conformational search on the cyclic10 compounds
by means of molecular dynamics at different temperatures (400,
600, and 800 K) using the MMFFs11 force field included in the
MacroModel software package.12 On the so-obtained model, QM
optimization of the energies and the geometries was performed in
vacuo at the hybrid DFT B3LYP level, using the 6-31G(d) basis set
(Gaussian 03 Software Package).13 Subsequently, the charges of 1–7
were calculated with the ChelpG method14 at the B3LYP/6-31Gþ(d)
level.
Figure 1. 3D model of the HDLP.
The results obtained (Table 1) showed satisfactory Kd values
for all compounds, even if among them, compounds 6 and 7
showed a little better binding properties for the HDLP receptor
surface.
Concerning all cyclic molecules (1–3, 6, 7), docking studies
suggest strong interactions between the recognition binding do-
main, represented by the heterocyclic framework, and the hy-
drophobic surface of the HDLP active site. For the sake of
simplicity, we will only describe the detailed docking results for
compounds 1 and 7, the latter showing the best calculated affinity
for the target.
Our docking studies indicate that the linker chain and the cap
group of 7 and 1 fill equivalent spaces (hydrophobic pocket D:
H170, A197, L265, F198, and F200). Moreover, the linker chain ex-
erts a set of interactions with the tubular hydrophobic pocket and
the zinc-coordinating amidic group, forming hydrogen bonds with
H32 of H131.
The cap group portion of both compounds is accommodated in
a shallow groove, establishing Van der Waals interactions and hy-
drogen bonds with the receptor counterpart, formed by H170, A197,
F198, and F200 residues. The cap group extends its hydrophobic
contacts thanks to the phenyl ring, which is accommodated in the
deep pocket D (Y264, L265, S266, K267, see Fig. 2); however, the
macrocycle size seems to slightly modulate the activity, as emerged
by directly comparing 1 with 7; this last compound, in fact, in virtue
Docking studies were performed on compounds 1–7 with the
HDLP binding pocket,6 using AutoDock 3.0.5 software,15 which has
been successfully used in the interpretation of the inhibitory ac-
tivity of several HDAC ligands.7,16 In addition to the compounds
optimized as described above, we used as model receptor the HDLP
active site refined at the QM level17 by us in previous studies,16d in
order to improve our calculations with the aim to obtain a good
qualitative accordance between theoretical Kd and biological assays
results. According to the receptor mapping, besides the binding
channel (11 Å) there are four hydrophobic cavities (A–D)16a acting
as the molecular recognition domain (Fig. 1).
Scheme 1. Molecular structures of 1–7 compounds.

2522
S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
Table 1
Calculated (Kd) activities of 1–7 compounds
1
2
3
4
5
6
7
Kd.
1.78ꢀ10ꢁ8
4.09ꢀ10ꢁ8
7.81ꢀ10ꢁ8
2.74ꢀ10ꢁ8
3.65ꢀ10ꢁ7
8.88ꢀ10ꢁ9
1.82ꢀ10ꢁ9
Figure 2. (a) 3D model of the interaction between 1 and the HDLP binding site. The protein is represented by molecular surface and sticks and balls. Compound 1 is depicted by
sticks (by atom type: C green, polar H white, N dark blue, O red). (b) 3D model of the interaction between 7 and the HDLP binding site. The protein is represented by molecular
surface and sticks and balls. Compound 7 is depicted by sticks (by atom type: C sky blue, polar H white, N dark blue, O red).
of its higher dimension of the cycle (13 C atoms vs 15, respectively)
showed to fit better with the enzyme binding domain. In fact, the
suboptimal hydrophobic interactions are responsible for a pre-
dicted increase in the binding affinity to the receptor of about 10-
fold (Kd of 1 1.78ꢀ10ꢁ8 vs Kd of 7 1.82ꢀ10ꢁ9).
but, at the same time, allowed us to make some considerations on
the virtual screening outcome.
For example, the predicted negative response for some of the
designed molecules (in good qualitative accordance with the ex-
perimental data) can be ascribed to unfavorable structural features,
such as the lack of aromatic rings in compound 3, while, the un-
suitable size of the aliphatic chain spacer in 2 could account for the
absence of cytotoxicity, in coherence with its slightly less satisfac-
tory Kd value.
Furthermore, comparing the docking results of 2 and 6 (Fig. 3),
presenting the same cap group but differing for the linker length,
we can suppose that it should be of nine carbon atoms in order to
have the optimal fit with receptor surface.
Figure 3. (a) 3D model of the interaction between 2 and the HDLP binding site. The protein is represented by molecular surface and sticks and balls. Compound 2 is depicted by
sticks (by atom type: C violet, polar H white, N dark blue, O red). (b) 3D model of the interaction between 6 and the HDLP binding site. The protein is represented by molecular
surface and sticks and balls. Compound 6 is depicted by sticks (by atom type: C pink, polar H white, N dark blue, O red).
Finally, compound 3, which presents the same linker as 6, but an
entirely aliphatic cap group (Fig. 4), could not establish strong hy-
drophobic interactions with the protein counterpart, being totally
lacking of aromatic elements with respect to the other cyclic
compounds.
For the analysis of linear compounds 4 and 5, the small differ-
ence of their Kd values, could be ascribed to their different aromatic
ring content. In fact, the two aromatic rings present in compound 4
were correctly accommodated in the A and D hydrophobic pockets
(see Supplementary data) of the enzyme, increasing the stability of
the drug–receptor complex (Fig. 5).
Prompted by the virtual screening results, we decided to un-
dertake the synthesis of the designed molecules aimed at verifying
the qualitative accordance between the theoretical and the exper-
imental data (see Chemistry section). A cytotoxicity assay was then
Figure 4. 3D model of the interaction between 3 and the HDLP binding site. The
performed on the synthesized molecules and the analysis of the
results, reported in Table 2, was disappointing for some compounds
protein is represented by molecular surface and sticks and balls. Compound 3 is de-
picted by sticks (by atom type: C dark red, polar H white, N dark blue, O red).

S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
2523
was performed through a ring closing metathesis (RCM) reaction,
which represents a suitable approach for the construction of
variable-sized macrocyclic ring systems.22 As suggested by our
previous studies we used second-generation Grubbs’ catalyst,
which was found to be more stable and more reactive in our re-
action conditions. RCM reactions were carried out using microwave
irradiation, which enhanced the reaction rate. The optimal condi-
tions found were: 0.3 mM solutions, 10 mol % of Grubbs catalyst
second generation, DCM as solvent and microwave heating at
300 W for two 40 min periods. The structures of the desired com-
pounds were confirmed by mass spectrometry and analytical
RP-HPLC. The crude products were purified by semipreparative RP-
HPLC on a C18 column and the structures of 1–3 and 6, 7 (20–33%
yield after two purification HPLC steps) were confirmed by MS and
NMR spectroscopic analysis. The 1H and 13C NMR spectra showed
that the RCM reactions lead only to the Z-olefin-stereoisomer for
compounds 2 and 6, while compounds 1, 3, and 7 were obtained as
Figure 5. Compounds 4 and 5 superimposition in the zinc-binding site.
23
a mixture of cis and trans isomers.
Unfortunately, we were
unable to separate the isomers by semipreparative HPLC.
Table 2
Biological (IC50) activities of compounds 1–7 on HEK-293, J774A.1, and WEHI-164. Control cells viability was designated as 100%, and results were expressed as the con-
centration of tested compounds able to induce the 50% of mortality in cells (IC50). Results are expressed as meanꢂSEM from at least three-independent experiments
1
2
3
4
5
6
7
IC50 [M] HEK-293
IC50 [M] WEHI-164
IC50 [M] J774A.1
1.85ꢀ105ꢂ0.04
2.0ꢀ10ꢁ5ꢂ0.05
2.4ꢀ10ꢁ5ꢂ0.18
n.d.
n.d.
n.d.
n.d.
7.49ꢀ10ꢁ5ꢂ0.72
3.2ꢀ10ꢁ4ꢂ0.21
7.38ꢀ10ꢁ5ꢂ0.12
n.d.
n.d.
n.d.
1.4ꢀ10ꢁ5ꢂ0.03
2.4ꢀ10ꢁ5ꢂ0.05
n.d.
1.0ꢀ10ꢁ4ꢂ0.02
2.2ꢀ10ꢁ5ꢂ0.08
2.4ꢀ10ꢁ4ꢂ0.13
3.0ꢀ10ꢁ4ꢂ0.02
n.d.
In contrast, as concerns compounds 6 and 7, the biological re-
sults were in disagreement with the virtual screening response. In
fact, these two compounds, despite displaying the best affinity
properties in docking studies, did not exert the expected higher
potency in antiproliferative assay, probably due to unfavorable
pharmacokinetic parametersdsuch parameters are in fact not
quantifiable in the computer simulation approach.
4. Conclusion
A new collection of potential HDAC inhibitors has been de-
veloped and a partial rationalization of their biological behavior has
been pursued through Molecular docking calculations. Four of the
seven synthesized molecules showed a satisfying level of anti-
proliferative activity and all of them are currently investigating for
their ability to inhibit HDAC enzyme.
3. Chemistry
5. Experimental section
The synthesis of compounds 1–7 is depicted in Schemes 2 and 3.
Attachment of the starting amino acid residue to the previously
deprotected Rink Amide MBHA resin was performed with HOBt and
DIC in DMF for 3 h, and the obtained loading degree was de-
termined by UV spectrophotometric analysis. After the acetylation
step, the Fmoc protecting group was removed by treatment with
a 20% solution of piperidine in N,N-dimethylformamide (DMF).
Alkylation of the primary amine with the aldehydes (5 equiv), on
solid phase, was performed in a two-step procedure in order to
minimize dialkylation18 reaction and employing (MeO)3CH (TMOF)
as both solvent and dehydrating agent.19 Reduction of the in-
termediate imines was achieved by reaction with borane–pyridine
complex (BH3$Py, BAP) at room temperature overnight.20 The re-
action was monitored (after a mini-cleavage step from the resin) by
HPLC analysis and ESI MS; data obtained indicated clean and high
yielding reactions. For the synthesis of compounds 1, 3, 5, and 7 the
coupling of the secondary amines with the appropriate carboxylic
acids was obtained using HOBt/HBTU, with 5 equiv of reagents, in
DIPEA (10 equiv). For the synthesis of the linear precursors of
compounds 2, 4, and 6, instead, a second reductive alkylation step
of the secondary amine function was realized (Scheme 3).
5.1. Molecular mechanics and dynamics calculations
Molecular mechanics/dynamics (MM and MD) calculations
were performed using the Macromodel 8.5 software package11 and
the MMFFs24 force field at several temperatures (400, 600, and
800 K). The solvent effects are simulated using the analytical
Generalized-Born/Surface-Area25 (GB/SA) model mimicking the
presence of H2O. All the structures were minimized using a Polak-
Ribiere Conjugate Gradient (PRCG, 50,000 steps, maximum de-
rivative less than 0.005 kcal/mol).
5.2. Docking studies
Autodock 3.0.515 was used for all docking calculations. HDLP
6
(histone deacetylase-like protein) is a metalloprotein, so a non-
bonded model for metallic center according to the nonbonded Zn
parameters of Stote26 (Zinc radius¼1.10 Å, well depth¼0.25 kcal/
mol) was used. In order to have an accurate weight of the elec-
trostatics, we derived the partial charge of Zn¼1.175 and of the
amino acids involved in the catalytic center (A169, H170, D168,
D258) by DFT calculations B3LYP level by the 6-31G(d) basis set and
ChelpG method 14for population analysis (Gaussian 03 Software
Package).13 For what concerns the ligands, the geometries were
optimized (see Supplementary data) at the hybrid DFT B3LYP level
using the 6-31G(d) basis set. Subsequently, the charges of com-
pounds 1–7 were calculated with the ChelpG method14 at the
B3LYP/6-31Gþ(d) level. The above calculated charges were used for
This was performed using the aldehyde (10 equiv) in TMOF and
BAP (10 equiv) at room temperature for 4 days; the dialkylated
products were obtained in quantitative yields.21 The linear pre-
cursors were cleaved from the resin, by a single step treatment with
a TFA/TIS/H20 95:2.5:2.5 solution, according to general procedure e
(81–97% overall yield with greater than 95% purity). Finally, for the
synthesis of compounds 1–3 and 6, 7, the intramolecular cyclization

2524
S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
Scheme 2. Synthetic strategy for compounds 1, 3, 5, and 7.
docking calculations. For all the docking calculations a grid box size
of 66ꢀ64ꢀ48 with spacing of 0.375 Å between the grid points,
centered between Zn2þ and H170 (x¼49.75, y¼5.0, z¼101.491) and
covering the catalytic center surface of HDLP was used. For all the
docked structures, all bonds were treated as active torsional bonds
except the amide bonds. In order to achieve a representative con-
formational space during the docking calculations, six calculations
consisting of 256 runs were performed, obtaining 1536 structures
(256ꢀ6). The Lamarckian genetic algorithm was used for dockings.
An initial population of 450 randomly placed individuals, a maxi-
mum number of 4.0ꢀ106 energy evaluations, and a maximum
number of 3.0ꢀ106 generations were taken into account. A muta-
tion rate of 0.02 and a crossover rate of 0.8 were used. Results
differing by less than 3.5 Å in positional root-mean-square
Scheme 3. Synthetic strategy for compounds 2, 4, and 6.

S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
2525
deviation (RMSD) were clustered together and represented by the
result with the most favorable free energy of binding. All the 3D
models were depicted using the Python software:27 molecular
surfaces are rendered using Maximal Speed Molecular Surface
(MSMS).28
polypropylene ISOSOLUTE syringe on a VAC MASTER system,
washed with DCM (2ꢀ4 mL), DMF (2ꢀ4 mL), and swollen in 4 mL of
DMF for 1 h. A solution of 20% piperidine in DMF (3 mL, 20 min)
was added and then the resin was washed with DMF (3ꢀ4 mL).
A solution of Fmoc-8-Aoc-OH or Fmoc-6-Ahx-OH (5 equiv),
HOBt (5 equiv), and DIC (5 equiv) in 2.5 mL of dry DMF was added
and the mixture was agitated for 3 h under a N2 stream. The mix-
ture was then removed, the resin was washed with DMF 3ꢀ3 mL,
DCM 3ꢀ3 mL, and dried under vacuum.
5.3. General experimental procedures
All the NMR spectra (1H, HMBC, HSQC, TOCSY, COSY, ROESY)
were recorded on a Bruker Avance DRX600 at T¼298 K. The com-
pounds (1–7) were dissolved in 0.5 mL of 99.95% DMSO-d6 (Carlo
5.5.2. (a’) Estimation of the level of first residue attachment. The
loading of the resin was determined by UV quantification of the
Fmoc–piperidine adduct.
Erba, 99.95 Atom % D) (1H,
0.5 mL of 99.8% MeOH-d4 (Aldrich, 99.8þAtom % D). The NMR data
were processed on a Silicon Graphic Indigo 2 workstation using
UXNMR software. Chemical shifts are expressed in parts per million
d
¼2.50 ppm; 13C
¼39.5 ppm) or in
d
The assay was performed on a duplicate samples: 0.4 mL of pi-
peridine and 0.4 mL of DCM were added to two dried samples Fmoc
amino acid-resin in two volumetric flasks of 25 mL. The reaction was
allowed to proceed for 30 min at room temperature and then 1.6 mL
of MeOH was added and the solutions were diluted to 25 mL volume
with DCM. A reference solution was prepared in a 25 mL volumetric
flask using 0.4 mL of piperidine, 1.6 mL of MeOH, and DCM to vol-
ume. The solutions were shaken and the absorbance of the samples
versus the reference solution was measured at 301 nm. The sub-
stitution level (expressed in mmol of amino acid/g of resin) was
calculated from the equation: mmol gꢁ1¼(A301/7800)ꢀ(25 mL gꢁ1 of
(ppm) on the delta (d) scale. Electrospray mass spectrometry (ES-
`
MS) was performed on an LCQ DECA TermoQuest (San Jose, Cal-
ifornia, USA) mass spectrometer. High resolution mass spectra were
acquired on a Q-Tof ULTIMA (Waters, Manchester, UK) calibrated
with [Glu]-Fibrinopeptide B fragments using standard experimen-
tal conditions.
For estimation of Fmoc amino acids on the resin, absorbance at
301 nm was recorded on a Shimadzu UV 2101 PC. Analytical and
semipreparative reverse phase HPLC was performed on a Jupiter C-
18 column (250ꢀ4.60 mm,
5
m
,
300 Å, flow rate¼1 mL/min;
resin). The obtained loading degree was: 0.34–0.57 mmol gꢁ1
.
250ꢀ10.00 mm,10
m
, 300 Å, flow rate¼4 mL/min, respectively). The
binary solvent system (A/B) was as follows: 0.1% TFA in water (A)
and 0.1% TFA in acetonitrile (B). The absorbance was detected at
210–240 nm.
5.5.3. (b) Acetylation step. DMF (3 mL), DIEA (5 equiv), and acetic
anhydride (5 equiv) were combined immediately before use, added
to resin, and left to react for 30 min under N2 stream. After reaction,
the resin was washed with DMF 3ꢀ3 mL, DCM 3ꢀ3 mL, and DMF
3ꢀ3 mL(1.5 min each). The reaction was checked using the TNBS test.
All the solvents used for the synthesis were HPLC grade; they
were purchased from Aldrich, Fluka, Carlo Erba. Dry dichloro-
methane was distilled from CaH2. DCM and DMF used for solid-
phase reactions were dried over activated 4 Å molecular sieves.
Rink Amide MBHA resin (polymer matrix: copoly(styrene–1%
DVB);100–200 mesh, loading level: 0.70 mmol/g and 0.64 mmol/g),
HOBt, and HBTU were purchased from Novabiochem. Fmoc-8-Aoc-
OH and Fmoc-6-Ahx-OH were obtained from NeoMPS. Trimethyl
orthoformate, 4-allyloxybenzaldehyde, undecylenic aldehyde,
borane-pyridine complex (BH3$py, BAP), Grubbs catalyst second
generation, 4-pentenoic acid, and 6-eptenoic acid were purchased
from Aldrich. Solid-phase syntheses, using the Fmoc strategy, were
carried out on a polypropylene ISOLUTE SPE column on a VAC
MASTER system, a manual parallel synthesis device purchased from
Stepbio. Reactions were generally run under an argon or nitrogen
atmosphere. All reagents were purchased from commercial sup-
pliers and used as received.
5.5.4. (c) Fmoc deprotection. Piperidine (20%) in DMF (3 mL,
1ꢀ1.5 min), 20% piperidine in DMF (3 mL, 1ꢀ10 min); washings in
DMF 2ꢀ3 mL, DCM 2ꢀ3 mL, and DMF 2ꢀ3 mL (1.5 min each).
5.5.5. (d) Reductive alkylation of resin-bound primary amine. The
resin was preswollen in DMF, TMOF, and the aldehyde (5 equiv) dis-
solved inTMOF (2.5 mL) was added. The reaction mixture was shaken
at room temperature overnight. The resin was filtered and washed
(3ꢀDCM) and dried. The reaction was checked using the TNBS test.
Imine reduction: the resin was suspended in DCM/MeOH/AcOH
(2:2:1), BAP (4 equiv) was added, and the mixture was shaken
overnight. The resin was filtered and then washed with DMF
2ꢀ3 mL, DCM 2ꢀ3 mL, MeOH 2ꢀ3 mL, DCM 2ꢀ3 mL and then
dried. HPLC analysis and ESI MS data gave a clear indication of
completed reductive amination.
5.4. Microwave irradiation experiments
5.5.6. (d0) Reductive alkylation of resin-bound secondary amine. The
resin was preswollen in DMF, and suspended in TMOF (1 mL). Al-
dehyde (10 equiv) was dissolved in DMF/EtOH (3:1) (1 mL) and
added to the resin, followed by the addition of BAP (10 equiv). The
reaction mixture was shaken at room temperature for 4 days, then
the resin was filtered and washed with DMF 2ꢀ3 mL and DCM
2ꢀ3 mL (1.5 min each). HPLC analysis and ESI MS data were used to
monitor the reductive alkylation of secondary amine. Quantitative
yields were obtained in all the cases.
All microwave irradiation experiments were carried out in
a dedicated CEM-Discover Focused Microwave Synthesis apparatus,
operating with continuous irradiation power from 0 to 300 W
utilizing the standard absorbance level of 300 W maximum power.
The reaction was carried out in 100 mL round-bottomed flask
containing a magnetic stirrer and fitted with a reflux condenser.
The DiscoverÔ system also offers controllable ramp time, hold
time (reaction time), and uniform stirring. The temperature was
monitored using the CEM-Discover built-in-vertically-focused IR
temperature sensor. After the irradiation period, the reaction vessel
was cooled rapidly (60–120 s) to ambient temperature by air jet
cooling.
5.5.7. (e) Peptide coupling conditions. The coupling reaction was
promoted by an HOBt/HBTU in DMF coupling protocol.
Carboxylic acid (5 equiv), HOBt (5 equiv), HBTU (5 equiv), and
DIEA (10 equiv) were agitated under N2 in 2.5 mL of DMF for 2 h.
After the coupling step, washings were carried out with DMF (3 mL,
3ꢀ1.5 min) and DCM (3 mL, 3ꢀ1.5 min). The reaction was checked
using the HPLC analysis and ESI MS spectra. The yields obtained
were >90%.
5.5. General procedures for the synthesis of compounds 1–7
5.5.1. (a) Loading of the resin. The Fmoc-Rink amide MBHA resin
(400.0–600.0 mg, loading 0.7 mmol gꢁ1) was placed in a 25 mL

2526
S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
5.5.8. (f) Cleavage. Cleavage from the resin was performed by
a single step treatment with TFA/TIS/H2O 95:2.5:2.5 for 2 h, under
O
7
N
14
11
8
5
3
9
13
12
1
NH2
stirring (10
m
Lꢀ1 mg of resin). Then the resin was filtered off and
6
4
2
27
24
10
25
15
17
the beads were washed with neat cleavage mixture (3 mL,
3ꢀ1.5 min) and DCM (3ꢀ3 mL). The combined filtrates were con-
centrated in vacuo and lyophilized. Yields were >90% in all cases,
with high purity, as determined by HPLC and ESI MS spectra.
O
26
23
16
21 22
O20
18
19
2
5.5.9. (g) RCM under microwave irradiation. Optimized microwave
ring closing metathesis.
1H and 13C NMR data (600 MHz, DMSO-d6): dH: 1.31–1.38 (4H,
m, CH2-4, CH2-5), 1.52 (2H, quintet, J¼7.6 Hz, CH2-3), 2.02 (2H, t,
J¼7.3 Hz, CH2-2), 3.34 (2H, br s, CH2-6), 4.13 (2H, d, J¼13.3 Hz, CH-
16A, CH-19A), 4.66 (2H, d, J¼13.3 Hz, CH-16B, CH-19B), 4.75 (4H, br
s, CH2-8, CH2-27), 5.63 (2H, br s, CH-17, CH-18), 6.47 (2H, d,
J¼8.5 Hz, Ar), 6.54 (2H, d, J¼8.5 Hz, Ar), 6.67 (2H, d, J¼8.5 Hz, Ar),
6.94 (2H, d, J¼8.5 Hz, Ar), 9.32 (2H, br s, NH2).
To a stirred solution (0.3 mM) of the linear precursor in dry
DCM was added 10 mol % of Grubbs catalyst second generation.
The mixture was refluxed at 60 ꢃC and irradiated at a maximum
power (300 W) for 40 min. The reaction mixture was then
degassed with N2 for approximately 1 min to dry off any dissolved
ethene, an additional 10 mol % catalyst was added and the solution
was stirred and subjected to MW radiation (300 W) for a further
30 min. The mixture was treated with DMSO (50 equiv relative to
the catalyst) overnight, finally the solution was concentrated in
vacuo and purified by HPLC.
dC: 26.3–31.1 (C-3-C-5), 35.3 (C-2), 58.0 (C-6), 62.2 (C-8), 62.2 (C-
27), 71.1 (C-19), 72.0 (C-16); 116.4 (Ar), 116.5 (Ar), 129.7 (Ar), 131.1
(Ar), 131.3 (C-17), 131.4 (C-18).
5.8. Compound 3
The crude products were purified by semipreparative reverse
phase HPLC (on a Jupiter C-18 column: 250ꢀ10.00 mm, 10
m, 300 Å,
Compound 3 was synthesized following the general procedure g.
Utilizing 12.5 mg (0.03 mmol) of linear precursor 3e, 10 mol % of
Grubbs catalyst second generation, 0.3 mM solution in dry DCM. The
crude reaction was purified by reverse phase semipreparative HPLC.
RP-HPLC tR¼43.42 min from 15% B to 85% B over 85 min; 2.3 mg
(21% yield, after HPLC purification step) as a slightly yellow oil; ES-
MS, calcd for C22H40N2O2 364.3; found m/z¼365.3 [MþH]þ. HRMS
calcd for C22H41N2O2 [MþH]þ 365.3163; found 365.2938.
flow rate¼4 mL/min) using the following gradient conditions and
characterized by ES-MS and NMR spectra.
5.6. Compound 1
Compound 1 was synthesized following the general procedure g.
Utilizing 19.1 mg (0.05 mmol) of linear precursor 1e, 10 mol % of
Grubbs catalyst second generation, 0.3 mM solution in dry DCM. The
crude reaction was purified by reverse phase semipreparative HPLC.
RP-HPLC tR¼28.20 min from 15% B to 85% B over 60 min; 2.7 mg
(15% yield, after HPLC purification step) as a white solid; ES-MS,
calcd for C21H30N2O3 358.2; found m/z¼359.1 [MþH]þ. HRMS
calcd for C21H31N2O3 [MþH]þ 359.4819; found 359.4899.
21
23
8
6
4
2
9
N
20
NH2
1
22
7
11
5
3
19
O
18
17
10
O
14
12
13
16
15
3
O
16
10
9
11
7
5
3
15
14
N
1
NH2
23
8
O
6
4
2
12
22
1H and 13C NMR data (600 MHz, DMSO-d6): dH: 1.31–1.58 (10H,
m, CH2-3, CH2-7), 2.10 (2H, br s, CH2-2), 2.20 (2H, br s, CH2-11), 2.32
(2H, br s, CH2-12), 3.30 (2H, br s, CH2-8), 1.26–3.31 (18H, m, CH2-15,
CH2-23), 5.44 (2H, br s, CH-13, CH-14), 9.29 (2H, br s, NH2).
dC: 26.0–31.0 (C-3-C-7), 29.0–35.6 (C-15-C-22), 29.9 (C-12), 30.1
(C-11), 36.0 (C-2), 50.7 (C-23), 52.0 (C-8), 129.0 (C-13-C14).
17 O
25
13
21
18
19 20
1
1H and 13C NMR data (600 MHz, MeOH-d4): dH: 1.20–1.70 (10H,
m, CH2-3, CH2-7), 2.25 (2H, m, CH2-2), 2.38 (2H, m, CH2-21), 2.46
(2H, m, CH2-22), 3.17 (1H, t, J¼7.6 Hz, CH-8A), 3.34 (1H, t, J¼7.6 Hz,
CH-8B), 4.47 (2H, br s, CH2-10), 4.55 (2H, br s, CH2-18), 5.57 (1H, br
s, CH-19), 5.80 (1H, br s, CH-20), 6.80 (1H, d, J¼8.5 Hz, Ar), 6.90 (1H,
d, J¼8.5 Hz, Ar), 7.07 (1H, d, J¼8.0 Hz, Ar), 7.15 (1H, d, J¼8.5 Hz, Ar).
dC: 24.0 (C-21), 27.2–30.0 (C-3-C-7), 35.0 (C-22), 38.2 (C-2), 50.2
(C-8), 55.1 (C-10), 70.0 (C-18), 111.7 (Ar), 112.0 (Ar), 134.3 (Ar), 135.0
(Ar), 130.0 (C-19-C-20).
5.9. Compound 4
Compound 4 was synthesized following the general procedure.
A portion of the crude cleaved product (15.0 mg) was then purified
as follows: RP-HPLC tR¼25.9 min from 15% B to 85% B over 60 min;
12.5 mg (83% yield, after HPLC purification step) as a yellow oil; ES-
MS, calcd for C26H34N2O3 422.2; found m/z¼423.4 [MþH]þ. HRMS
calcd for C26H35N2O3 [MþH]þ 423.2642; found 423.2722.
5.7. Compound 2
O
10
8
3
7
5
9
11
12
Compound 2 was synthesized following the general procedure g.
Utilizing 17.0 mg (0.04 mmol) of linear precursor 2e, 10 mol % of
Grubbs catalyst second generation, 0.3 mM solution in dry DCM. The
crude reaction was purified by reverse phase semipreparative HPLC.
RP-HPLC tR¼20.6 min from 5% B to 100% B over 60 min;
5.0 mg (32% yield, after HPLC purification step) as a slightly
brown oil; ES-MS, calcd for C24H30N2O3 394.2; found m/z¼395.1
[MþH]þ. HRMS calcd for C24H31N2O3 [MþH]þ 395.2329; found
395.2239.
1
NH2
N
4
2
14
21
6
19
20
15
O
13
22
23
16
17
18
25
26
24
O
27
28
29
4

S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
2527
1H and 13C NMR data (600 MHz, MeOH-d4): dH: 1.32 (2H, quin-
tet, J¼7.5 Hz, CH2-4), 1.52–1.65 (4H, m, CH2-3, CH2-5), 2.14 (2H, t,
J¼7.2 Hz, CH2-2), 3.17 (2H, br s, CH2-6), 4.40 (4H, br s, CH2-8, CH2-
19), 4.63 (4H, br s, CH2-16, CH2-27), 5.29 (2H, br s, CH-18A, CH-29A),
5.42 (1H, br s, CH-18B, CH-29B), 6.05 (2H, m, CH-17, CH-28), 6.72
(2H, d, J¼8.5 Hz, Ar), 6.80 (2H, d, J¼8.5 Hz, Ar), 7.07 (2H, d, J¼8.5 Hz,
Ar), 7.15 (2H, d, J¼8.5 Hz, Ar).
(2H, d, J¼13.0 Hz, CH-18B, CH-21B), 4.75 (4H, br s, CH2-10, CH2-29),
5.66 (2H, br s, CH-19, CH-20), 6.47 (2H, d, J¼8.5 Hz, Ar), 6.54 (2H, d,
J¼8.5 Hz, Ar), 6.67 (2H, d, J¼8.5 Hz, Ar), 6.94 (2H, d, J¼8.5 Hz, Ar),
9.32 (2H, br s, NH2).
dC: 25.1–30.1 (C-3-C-7), 35.3 (C-2), 57.9 (C-8), 58.8 (C-29), 58.9
(C-10), 67.8 (C-18, C-21),116.4 (Ar),116.45 (Ar),129.7 (Ar),131.1 (Ar),
131.4 (C-19, C-20).
dC: 25.6–31.2 (C-3-C-5), 36.2 (C-2), 52.5 (C-6), 62.2 (C-19), 62.2
(C-8), 70.0 (C-16, C-27), 116.0 (Ar), 116.2 (Ar), 130.0 (Ar), 132.4 (Ar),
112.4 (C-18), 112.6 (C-29), 135.4 (C-28), 135.5 (C-17).
5.12. Compound 7
Compound 7 was synthesized following the general procedure g.
Utilizing 35.5 mg (0.08 mmol) of linear precursor 7e, 10 mol % of
Grubbs catalyst second generation, 0.3 mM solution in dry DCM. The
crude reaction was purified by reverse phase semipreparative HPLC.
RP-HPLC tR¼32.30 min from 5% B to 100% B over 65 min; 7.0 mg
(21% yield, after HPLC purification step) as a slightly brown solid;
ES-MS, calcd for C23H34N2O3 386.2; found m/z¼387.2 [MþH]þ.
HRMS calcd for C23H35N2O3 [MþH]þ 387.2642; found 387.2722.
5.10. Compound 5
Compound 5 was synthesized following the general procedure.
A portion of the crude cleaved product (20.0 mg) was then purified
as follows: RP-HPLC tR¼31.5 min from 15% B to 85% B over 60 min;
13.0 mg (65% yield, after HPLC purification step) as a colorless oil;
ES-MS, calcd for C21H30N2O3 358.2; found m/z¼359.4 [MþH]þ.
HRMS calcd for C21H31N2O3 [MþH]þ 359.2329; found 359.2232.
16
O
10
9
11
7
5
3
15
14
O
O
N
21
3
23
5
1
NH2
7
N
25
8
O
6
4
2
12
1
19
10
NH2
17 O
22
20
11
24
22
4
2
13
6
8
18
23
9
19
20
14
12
21
15
13
O
7
16
17
18
1H and 13C NMR data (600 MHz, DMSO-d6): dH: 1.30–1.60 (10H,
m, CH2-3, CH2-7), 1.34–1.66 (4H, m, CH2-22, CH2-23), 2.11 (2H, t,
J¼7.1 Hz, CH2-2), 2.05 (2H, m, CH2-21), 2.25 (2H, br s, CH2-24), 3.33
(2H, br s, CH2-8), 4.09 (2H, br s, CH2-10), 4.59 (2H, br s, CH2-18), 5.70
(2H, br s, CH-19, CH-20), 6.80 (1H, d, J¼8.5 Hz, Ar), 6.89 (1H, d,
J¼8.5 Hz, Ar), 7.07 (1H, d, J¼9.0 Hz, Ar), 7.15 (1H, d, J¼8.6 Hz, Ar).
dC: 26.1–31.0 (C-3-C-7), 28.8 (C-23), 30.0 (C-22), 30.9 (C-24),
36.0 (C-2), 36.0 (C-21), 52.0 (C-8), 61.8 (C-10), 70.9 (C-18),114.6 (Ar),
132.4 (Ar), 134.0 (C19-C-20).
5
1H and 13C NMR data (600 MHz, MeOH-d4): dH: 1.23–1.67 (6H,
m, CH2-3, CH2-5), 2.16 (2H, t, J¼7.2 Hz, CH2-2), 2.20 (2H, m, CH2-20),
2.43 (2H, m, CH2-21), 3.08 (1H, br s, CH-6A), 3.16 (1H, br s, CH-6B),
4.37 (2H, s, CH2-8), 4.57 (2H, br s, CH2-16), 5.00 (2H, br s, CH2-23),
5.30 (1H, br s, CH-18A), 5.45 (1H, br s, CH-18B), 5.75 (2H, m, CH2-
23), 6.02 (1H, m, CH-17), 6.69 (1H, d, J¼8.6 Hz, Ar), 6.95 (1H, d,
J¼9.0 Hz, Ar), 7.09 (1H, d, J¼8.6 Hz, Ar), 7.15 (1H, d, J¼8.6 Hz, Ar).
dC: 27.3–30.0 (C-3-C-5), 30.2 (C-21), 33.0 (C-20), 38.2 (C-2), 50.2
(C-6), 58.4 (C-8), 74.0 (C-16),113.0 (Ar),138.0 (Ar),111.4 (C-18),113.0
(C-23), 134.4 (C-17), 135.1 (C-22).
5.13. Biological tests
5.13.1. Cells. J774.A1, murine macrophage cell line were grown in
adhesion on Petri dishes and maintained with Dulbecco’s modified
Eagle’s medium (DMEM) at 37 ꢃC in DMEM supplemented with 10%
fetal born serum (FBS), 25 mM HEPES, 2 mM glutamine, 100 u/mL
5.11. Compound 6
Compound 6 was synthesized following the general procedure g.
Utilizing 12.5 mg (0.03 mmol) of linear precursor 6e, 10 mol % of
Grubbs catalyst second generation, 0.3 mM solution in dry DCM. The
crude reaction was purified by reverse phase semipreparative HPLC.
RP-HPLC tR¼20.20 min from 5% B to 100% B over 65 min; 3.0 mg
(22% yield, after HPLC purification step) as a colorless oil; ES-MS,
calcd for C26H34N2O3 422.2; found m/z¼423.4 [MþH]þ. HRMS
calcd for C26H35N2O3 [MþH]þ 423.2642; found 423.2339.
penicillin, and 100 mg/mL streptomycin. WEHI-164, murine fibro-
sarcoma cell line were maintained in adhesion on Petri dishes with
DMEM supplemented with 10% heat-inactivated FBS, 25 mM
HEPES, 100 u/mL penicillin, and 100 mg/mL streptomycin. HEK-293,
human epithelial kidney cell line were maintained and grown in
adhesion on Petri dishes with DMEM supplemented with 10% FCS,
25 mM HEPES, 100 u/mL penicillin, and 100
All reagents for cell culture were from Euroclone (Paignton Devon,
UK); [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium
mg/mL streptomycin.
O
bromide] (MTT) was from Sigma Chemicals (Milan, Italy).
9
12
10
7
5
3
11
13
14
N
1
NH2
5.13.2. Antiproliferative assay29. J774.A1, WEHI-164, and HEK-293
(3.5ꢀ104 cells) were plated on 96-well plates and allowed to ad-
here at 37 ꢃC in a 5% CO2 atmosphere for 2 h. Thereafter, the me-
dium was replaced with fresh medium and serial dilution of each
test compound was added and then the cells incubated for 72 h.
Cell viability was assessed through an MTT conversion assay.
29
26
8
6
4
2
16
17
27
28
O
15
25
18
19
24
23
O22
20
21
6
Briefly, 25
cubated for an additional 3 h. Thereafter, cells were lysed and the
dark blue crystals solubilized with 100 L of a solution containing
mL of MTT (5 mg/mL) was added and the cells were in-
1H and 13C NMR data (600 MHz, DMSO-d6): dH: 1.40–1.52 (8H,
m, CH2-4, CH2-7), 1.67 (2H, m, CH2-3), 2.24 (2H, t, J¼7.2 Hz, CH2-2),
3.45 (2H, br s, CH2-8), 4.15 (2H, d, J¼13.0 Hz, CH-18A, CH-21A), 4.60
m
50% (v:v) N,N-dimethylformamide, 20% (w:v) SDS with an adjusted
pH of 4.5.29 The optical density (OD) of each well was measured

2528
S. Terracciano et al. / Tetrahedron 66 (2010) 2520–2528
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Fores-
man, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov,
B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.;
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, Revision E.01; Gaussian: Wallingford, CT, 2004.
with a microplate spectrophotometer (Titertek Multiskan MCC/
340) equipped with a 620 nm filter. The viability of each cell line in
response to treatment with tested compounds was calculated as: %
dead cells¼100ꢁ(OD treated/OD control)ꢀ100. Results were
expressed as the concentration of tested compounds able to induce
the 50% of mortality in cells (IC50).
Acknowledgements
Financial support by the University of Salerno and by Ministero
14. Breneman, C. M.; Wiberg, K. B. J. Comput. Chem. 1990, 11, 361.
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Olson, A. J. J. Comp. Chem. 1998, 19, 1639.
`
dell’Istruzione, dell’Universita e della Ricerca (MIUR), PRIN-06 are
gratefully acknowledged.
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Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.01.061.
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sidered protonated at physiological pH.
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