New organic nitrate-containing benzyloxy isonipecotanilide derivatives with vasodilatory and anti-platelet activity

Article abstract of DOI:10.1016/j.ejps.2015.03.004

Abstract A number of new nitric oxide (NO)-precursors were synthesized by grafting nitrate-containing moieties on the structures of the benzyloxy isonipecotanilide derivatives 1 and 2 already reported as moderately potent antiplatelet agents. Various nitrooxy (ONO₂)-alkyl side chains were covalently linked to the piperidine nitrogen of the parent compounds through carbamate and amide linkage, and the synthesis of a benzyl nitrate analog (15) of compound 1 was also achieved. The in vitro vasodilatory activities, as well as platelet anti-aggregatory effects, of the newly synthesized organic nitrates were assessed. The (ONO₂)methyl carbamate-based derivative 5a and the benzyl nitrate analog 15, which on the other hand retain activity as inhibitors of ADP-induced platelet aggregation, exhibited strong NO-mediated vasodilatory effects on pre-contracted rat aorta strips, with EC₅₀ values in the low nanomolar range (13 and 29 nM, respectively). Experiments carried out with the selectively inhibited soluble guanylate cyclase (sGC), which is the key enzyme of the NO-mediated pathway leading to vascular smooth muscle relaxation, confirmed the involvement of NO in the observed vasodilation. The nitrate derivatives proved to be stable in acidic aqueous solution and at pH 7.4. In human serum, unlike 5a, which showed not to undergo enzyme-catalyzed decomposition, the other tested (ONO₂)-alkyl carbamate-based compounds (5b and 5e) and benzyl nitrate 15 underwent a faster degradation. However, their decomposition rates in serum were quite slow (t_1/2 > 2.6 h), which suggests that nitrate moiety is poorly metabolized in blood plasma and that much of the in vitro anti-platelet activity has to be attributed to the intact (ONO₂)-containing molecules.

Full text of DOI:10.1016/j.ejps.2015.03.004

Accepted Manuscript
New organic nitrate-containing benzyloxy isonipecotanilide derivatives with
vasodilatory and anti-platelet activity
Modesto de Candia, Elisabetta Marini, Giorgia Zaetta, Saverio Cellamare,
Antonella Di Stilo, Cosimo D. Altomare
PII:
S0928-0987(15)00090-1
DOI:
http://dx.doi.org/10.1016/j.ejps.2015.03.004
PHASCI 3206
Reference:
European Journal of Pharmaceutical Sciences
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 December 2014
19 February 2015
3 March 2015
Please cite this article as: Candia, M.d., Marini, E., Zaetta, G., Cellamare, S., Stilo, A.D., Altomare, C.D., New
organic nitrate-containing benzyloxy isonipecotanilide derivatives with vasodilatory and anti-platelet activity,
European Journal of Pharmaceutical Sciences (2015), doi: http://dx.doi.org/10.1016/j.ejps.2015.03.004
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New organic nitrate-containing benzyloxy
isonipecotanilide derivatives with vasodilatory and anti-
platelet activity
Modesto de Candiaa, Elisabetta Marinib, Giorgia Zaettaa, Saverio Cellamarea,
Antonella Di Stilob, Cosimo D. Altomarea,*
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, Via
E. Orabona 4, 70125 Bari, Italy
b
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P.
Giuria 9, 10125 Torino, Italy
* Corresponding author. Phone: +39 080 5442781; fax: +39 080 5442230
E-mail address: cosimodamiano.altomare@uniba.it (C.D. Altomare)
1

ABSTRACT
A number of new nitric oxide (NO)-precursors were synthesized by grafting nitrate-containing
moieties on the structures of the benzyloxy isonipecotanilide derivatives 1 and 2 already reported as
moderately potent antiplatelet agents. Various nitrooxy (ONO2)-alkyl side chains were covalently
linked to the piperidine nitrogen of the parent compounds through carbamate and amide linkage,
and the synthesis of a benzyl nitrate analog (15) of compound 1 was also achieved. The in vitro
vasodilatory activities, as well as platelet anti-aggregatory effects, of the newly synthesized organic
nitrates were assessed. The (ONO2)methyl carbamate-based derivative 5a and the benzyl nitrate
analog 15, which on the other hand retain activity as inhibitors of ADP-induced platelet
aggregation, exhibited strong NO-mediated vasodilatory effects on pre-contracted rat aorta strips,
with EC50 values in the low nanomolar range (13 and 29 nM, respectively). Experiments carried out
with the selectively inhibited soluble guanylate cyclase (sGC), which is the key enzyme of the NO-
mediated pathway leading to vascular smooth muscle relaxation, confirmed the involvement of NO
in the observed vasodilation. The nitrate derivatives proved to be stable in acidic aqueous solution
and at pH 7.4. In human serum, unlike 5a, which showed not to undergo enzyme-catalyzed
decomposition, the other tested (ONO2)-alkyl carbamate-based compounds (5b and 5e) and benzyl
nitrate 15 underwent a faster degradation. However, their decomposition rates in serum were quite
slow (t > 2.6 h), which suggests that nitrate moiety is poorly metabolized in blood plasma and that
½
much of the in vitro anti-platelet activity has to be attributed to the intact (ONO2)-containing
molecules.
Keywords: Isonipecotamides; Nitrooxy alkyl carbamate derivatives; Nitric oxide-donors;
Vasodilation; Antiplatelet activity.
2

Abbreviations: AChE, acetylcholinesterase; ADP, adenosine 5’-diphosphate; BuChE,
butyrylcholinesterase; cGMP, cyclic guanosine monophosphate; CHF, congestive heart failure;
CINOD, cyclooxygenase-inhibiting nitric oxide donator; GSH, glutathione; GST, glutathione S-
transferase; GTN, glyceryl trinitrate; ISDN, isosorbide dinitrate; MLC, myosin light chain; MLCK,
MLC kinase; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinaxolin-1-one; PON1,
paraoxonase; PRP, platelet rich plasma; RP-HPLC, reversed phase-high performance liquid
chromatography; sGC, soluble guanylated cyclase.
3

1. Introduction
The organic nitrates are known for over a century as coronary artery medications, since the use of
glyceryl trinitrate (GTN) as anti-anginal drug (Münzel et al., 2014). Administered through
transdermal and sublingual routes, GTN is an essential medication in angina, acute myocardial
infarction, severe hypertension, and coronary artery spasm. Isosorbide dinitrate (ISDN, Fig. 1) is
another long-acting nitrate reported in the World Health Organization’s List of Essential Medicines
(WHO, 2013), used for heart-related chest pain and congestive heart failure (CHF) as adjunct to
other drugs.
H
O
ONO2
O2NO
ONO2
ONO2
O2NO
O
H
Isosorbide dinitrate (ISDN)
Glyceryl trinitrate (GTN)
O
O
ONO2
O
ONO2
N
H
O
N
O
Nicorandil
NO-aspirin
O
O
F
O
ONO2
ONO2
O
O
HTC-3012
HCT-1026
Fig. 1. Organic nitrates used in clinics and some representative experimental NO-donor drugs.
GTN and ISDN, which undergo denitration in vivo with production of the active metabolite nitric
oxide (NO), act primarily via vascular smooth muscle relaxation, with decrease of cardiac output
4

and improvement of myocardial oxygen supply-to-demand ratio without affecting the heart’s
contractions.
The mechanism by which nitrovasodilators liberate NO in the body has not been completely
elucidated. Nonenzymatic pathways involving endogenous sulfhydryl-containing molecules
(Harrison, 2005) and several enzymes, such as the cytosolic glutathione S-tranferase (GST),
xanthine oxidoreductase (XO), the mitochondrial aldehyde dehydrogenase, or the microsomal
cytochrome P450 (CYP), have been proposed as mediators of bioactivation of organic nitrates
(Chen et al., 2002; DiFabio et al., 2003; Keen et al., 1976; Kollau et al., 2005; Kurz et al., 1993;
McDonald and Bennett, 1990; Schroder, 1992; Servent et al., 1989; Taylor et al., 1989). The
released NO activates the soluble guanylate cyclase (sGC) (Follmann et al., 2013), thereby
increasing the formation of cyclic guanosine monophoshate (cGMP), which, in turn, activates the
myosin light chain kinase (MLCK), the enzyme phosphorylating MLC in the presence of ATP,
ultimately preventing the phosphorylation of myosin and resulting in vascular muscle relaxation
(Lucas et al., 2000; Murad, 2006; Sogo et al., 2000).
Endogenous NO, generated from L-arginine by the nitric oxide synthase (NOS) enzymes, has
several different physiological actions targeted at kidney, reproductive apparatus, immunity system,
inflammation, and neurotransmission (Gasco et al., 2005; Scatena et al., 2010). In the
cardiovascular system NO predominates in large conduits, which supports its primarily anti-
atherothrombotic effects (Miller et al., 2008, 2000; Miller and Megson, 2007; Schade et al., 2010).
NO contributes to control the vascular endothelium smooth muscle cells tone and platelets’
adhesion and aggregation (Miller and Megson, 2007; Moncada et al., 1991; Murad, 2006; Scatena
et al., 2010; Schade et al., 2010).
Besides GTN and ISDN, nicorandil (Fig. 1) has been marketed in several countries as a
vasodilatory medication for the treatment of angina pectoris and CHF. As a hybrid between organic
nitrates and K+-ATP channel agonists, it acts through dual mechanism of action, combining the
5

vasodilatory property of both nitrates and nicotinamide with its ability to increase K+ conductance
(Edwards and Weston, 1990; Horinaka, 2011).
In the last decades a lot of hybrid nitrates as in vivo NO-donors have been studied for their potential
use in the treatment of a variety of diseases, including pain and inflammation, thrombosis and
restenosis, neurodegenerative diseases, cancer, liver disease, impotence, bronchial asthma and
osteoporosis (Keeble and Moore, 2002). Since some disagreement about whether nitrates really
generate NO at all, some authors prefer to use the term NO-mimetics (Thatcher et al., 2005).
However, among the various pharmacologically relevant families of nitrate-containing agents (Fig.
1), NO-aspirin showed pharmacological effects in cardiovascular, cancer and inflammation models,
and when tested in clinical trials showed little or no gastric toxicity (Cena et al., 2003; Keeble and
Moore, 2002), due to gastro-protective effects of NO (Lazzarato et al., 2009). A new class of
cyclooxygenase-inhibiting nitric oxide donors (CINODs) has been developed with the aim of
achieving greater safety than the existing non-steroidal anti-inflammatory drug (NSAIDs) (Boschi
et al., 2010, 2009). Two promising CINODs are HCT-1026 and HTC-3012 (Fig. 1), i.e., 4-
(ONO2)butyl esters of flurbiprofen and naproxen, respectively. HCT-1026 has been under study for
its therapeutic use in a variety of conditions, including neurodegeneration and inflammation
(Keeble and Moore, 2002; Scatena et al., 2005; Gasparini et al., 2005; Prosperi et al., 2004; Wenk et
al., 2004, 2002; Ronchetti et al., 2009; Idris et al., 2004). HTC-3012, as single (S)-enantiomer, has
been tested in clinical trials for the treatment of osteoarthritis (Geusens, 2009; Zhang et al., 2011).
Other typical examples of NO-donor hybrids of existing drugs have been reported, which include
ACE-inhibitors, statins, calcium antagonists, and phosphodiesterase inhibitors (Martelli et al., 2006;
Napoli and Ignarro, 2003; Serafim et al., 2012).
Some years ago, we have reported a number of moderately potent isonipecotamide-based inhibitors
of adenosine 5’-diphosphate (ADP)-induced human platelet aggregation (de Candia et al., 2003).
Among them, N-(3-(4-fluorobenzyloxy)phenyl)piperidine-4-carboxamide 1 (Fig. 2), with half
maximal inhibitory concentration (IC50) of 68 μM, and the N-(3-[(3’,5’-difluoro-1,1’-biphenyl-4-
6

yl)methoxy]phenyl) analog 2 (IC50 = 27 μM), which proved to be an antiplatelet agent about two-
fold more potent than 1 (de Candia et al., 2009), and a potent factor Xa (fXa)-selective inhibitor (Ki
= 130 nM) as well, were chosen for further optimization through hybridization with the organic
nitrate moiety, the first aim being to possibly strengthen the in vivo antiplatelet activity of the
parent compounds 1 and 2, conferring to them additional NO-mediated vasorelaxing properties.
O
O
O
N
O
N
H
H
NH
F
NH
F
1
2
F
Fig. 2. Fluorinated benzyloxyphenyl piperidine-4-carboxamide derivatives endowed with anti-
thrombotic properties (de Candia et al., 2009). Both compounds proved to inhibit ADP-induced
platelet aggregation (IC50 equals 68 and 27 µM for 1 and 2, respectively), whereas 2 showed
additional nanomolar inhibition potency against blood coagulation factor Xa (Ki = 135 nM).
In this work, we grafted nitrate moieties on the structures of antiplatelet compounds 1 and 2, by
covalently linking various nitrooxy (ONO2)-alkyl side chains to the piperidine nitrogen via
carbamate and amide linkages. A benzyl nitrate analog of compound 1 was also synthesized. The in
vitro vasodilatory and antiplatelet activities of the newly synthesized compounds were evaluated,
and the stability in aqueous solutions and human serum of the most potent compounds was
assessed.
2. Materials and methods
7

Triethylamine (TEA), dichloromethane (DCM), chloroform, ethanol (EtOH), methanol (MeOH),
acetone (Me2CO), ethyl acetate (EtOAc), n-hexane (Hex), acetonitrile (ACN), N,N-
dimethylformamide (DMF), tetrahydrofuran (THF), trifluoroacetic acid (TFA), sodium sulphate
(Na2SO4), sodium bicarbonate (NaHCO3), potassium carbonate (K2CO3), silver nitrate (AgNO3),
deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) and all other
chemicals and reagents were purchased from Sigma-Aldrich (Milan, Italy). Unless otherwise stated,
chemicals and reagents were of analytical grade and were used without further purification.
Melting points were determined by using the capillary method on a Stuart Scientific SMP3
electrothermal apparatus and are not corrected. IR spectra were recorded using KBr disks on a
Perkin-Elmer Spectrum One FT-IR spectrophotometer (Perkin-Elmer Ltd., Buckinghamshire, UK),
1
and the most significant absorption bands expressed in cm-1 are listed. H NMR spectra were
recorded at 300MHz on a Varian Mercury 300 instrument. Chemical shift values are expressed in δ
and the coupling constants J in Hz. Abbreviations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet, dd, doublet of doublets; m, multiplet; br, broad. Signals due to NH and OH protons were
located by deuterium exchange with D2O. Mass spectra were recorded on Agilent GC-MS 689-973.
Chromatographic separations were performed on silica gel 60 for column chromatography (Merck
70-230 mesh, or alternatively 15-40 mesh for flash chromatography). Purity (≥ 95%) of the
pharmacologically tested compounds was established by HPLC and elemental analysis. Elemental
analyses (C, H, N) were performed on Euro EA3000 analyzer (Eurovector, Milan, Italy) in the
Analytical Laboratory Service of the Dipartimento di Farmacia - Scienze del Farmaco, University
of Bari, and the results agreed to within 0.40% of the theoretical values.
2.1 Synthesis
2.1.1. General procedure for preparation of chloroalkyl carbamate derivatives 3a, 3b and 3e
A solution of the appropriate chloroalkyl chloroformate (1.50 mmol) in dry DCM (3 ml) was added
dropwise to a stirred solution of 1.37 mmol of compound 1·HCl (de Candia et al., 2009) and TEA
8

(2.74 mmol) in dry DCM (10 ml). The mixture was stirred at room temperature overnight. Then, the
precipitate was filtered, and the filtrate was evaporated in vacuum. The residue was dissolved in 50
ml of chloroform, and the solution was sequentially washed with 3×20 ml of saturated aqueous
NaHCO3, 1N HCl and brine, dried over anhydrous Na2SO4, filtered and evaporated in vacuum. The
crude product was purified by crystallization or by silica gel flash chromatography to afford the
desired compound.
2.1.1.1
Chloromethyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 3a. Compound 3a was prepared according to the general procedure from compound
1·HCl (500 mg, 1.37 mmol) and chloromethyl chloroformate (0.13 ml, 1.50 mmol). The crude
product was purified by crystallization from EtOH in 54% yield (310 mg) as a pale brown solid; mp
137-138 °C. IR (cm-1): 3264, 1662, 1612, 1445, 1208, 1088, 838, 699. 1H NMR (CDCl3) δ 7.45 (s,
br, 1H), 7.41-7.37 (m, 2H), 7.23 (d, J = 8.0, 1H), 7.18 (s, 1H), 7.06 (t, J = 9.0, 2H), 6.91 (d, J = 7.5,
1H), 6.72 (dd, J = 8.0 and 2.0, 1H), 5.80 (d, J = 13, 2H), 5.01 (s, 2H), 4.28 (d, J = 13, 1H), 4.18 (d, J
= 13, 1H), 2.99 (d, J = 13, 1H), 2.90 (d, J = 13, 1H), 2.50-2.35 (m, 1H), 2.00-1.90 (m, 2H), 1.85-
1.70 (m, 2H).
2.1.1.2.
2-Chloroethyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 3b. Compound 3b was prepared according to the general procedure from compound
1·HCl (500 mg, 1.37 mmol) and 2-chloroethyl chloroformate (0.15 ml, 1.56 mmol). The crude
product was purified by crystallization from EtOH in 78% yield (466 mg) as a brown solid ; mp
132-134 °C. IR (cm-1): 3230, 1660, 1605, 1445, 1205, 1078, 838, 696. 1H NMR (CDCl3) δ 7.46 (t, J
= 2.0, 1H), 7.43-7.36 (m, 2H), 7.25-7.18 (m, 2H), 7.10-7.02 (m, 2H), 6.92 (d, J = 8.0, 1H), 6.72 (dd,
J = 8.0 and 1.5, 1H), 5.01 (s, 2H), 4.34 (t, J = 6, 2H), 4.30-4.15 (m, 2H), 3.69 (t, J = 6.0, 2H), 3.00-
2.85 (m, 2H), 2.45-3.35 (m, 1H), 2.00-1.85 (m, 2H), 1.75-1.20 (m, 2H).
9

2.1.1.3. Chloromethyl 4-[({3-[(3',5'-difluoro-1,1'-biphenyl-4-yl)methoxy]phenyl}amino)carbonyl]
piperidine-1-carboxylate, 3e. Compound 3e was prepared was prepared according to the general
procedure from compound 2·HCl (175 mg, 0.38 mmol) and chloromethyl chloroformate (0.1 ml,
1.14 mmol). The crude product was purified by silica gel flash chromatography (eluent:
Hex/EtOAc, 7:3 v/v) in 89% yield (175 mg) as an orange oil. IR (cm-1): 3333, 2928, 1728, 1660,
1610, 1442, 1206, 1119, 1086, 989, 847, 783, 689. 1H NMR (DMSO-d6) δ 9.93 (s, 1H), 7.75 (d, J =
8.0, 2H), 7.52 (d, J = 8.0, 2H), 7.50-7.40 (m, 3H), 7.30-7.05 (m, 3H), 6.68 (dd, J = 8.0 and 1.5, 1H),
5.88 (d, J = 3.5, 1H), 5.11 (s, 2H), 4.10-3.90 (m, 2H), 2.95 (t, J = 12, 2H), 2.89 (t, J = 12, 2H), 2.60-
2.45 (m, 1H), 1.90-1.70 (m, 2H), 1.60-1.40 (m, 2H).
2.1.2. General procedure for preparation of halogenoalkyl carbamate derivatives 3c and 3d
4-Nitrophenyl chloroformate (3.60 mmol) was added portionwise to a 0 °C cooled solution of the
appropriate halogenoalkyl alcohol (3.60 mmol) and TEA (5.40 mmol) in dry THF (10 ml), and the
mixture was stirred 3 h at 0 °C. Then, compound 1·HCl (3.60 mmol) and TEA (3.60 mmol) were
added, and the mixture was stirred at room temperature overnight. The formed precipitate was
filtered, and the filtrate evaporated in vacuum. The residue was dissolved in 50 ml of chloroform,
and the solution was sequentially washed with 3×20 ml of saturated aqueous NaHCO3, 1N HCl and
brine, dried over anhydrous Na2SO4, filtered and evaporated to dryness. The crude oil product was
purified by silica gel flash chromatography to afford the desired compound.
2.1.2.1.
3-Bromopropyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 3c. Compound 3c was prepared according to the general procedure from compound
1·HCl (1.31 g, 3.60 mmol) and 3-bromo-1-propanol (0.32 ml, 3.60 mmol). The crude product was
purified by silica gel flash chromatography (eluent: Hex/EtOAc, 4:6 v/v) in 50% yield (0.890 g) as
a brown oil. IR (cm-1): 3230, 1656, 1600, 1448, 1210, 1068, 840, 700. 1H NMR (CDCl3) δ 7.48 (s,
1H), 7.46-7.36 (m, 2H), 7.23-7.17 (m, 2H), 7.06 (t, J = 9.0, 2H), 6.91 (d, J = 7.0, 1H), 6.72 (dd, J =
10

8.0 and 2.5, 1H), 5.02 (s, 2H), 4.23 (t, J = 6, 2H), 4.30-4.20 (m, 2H), 3.47 (t, J = 6.5, 2H), 2.95-2.80
(m, 2H), 2.50-2.35 (m, 1H), 2.20 (t, J = 6.5, 2H), 2.00-1.87 (m, 2H), 1.85-1.70 (m, 2H).
2.1.2.2.
4-Chlorobutyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 3d. Compound 3d was prepared according to the general procedure from compound
1·HCl (850 mg, 2.34 mmol) and 4-chloro-1-butanol (0.23 ml, 2.34 mmol). The crude product was
purified by silica gel flash chromatography (eluent: Hex/EtOAc, 7:3 v/v) in 55% yield (570 mg) as
a brown oil. IR (cm-1): 3227, 1650, 1600, 1445, 1208, 1080, 837, 696. 1H NMR (CDCl3) δ 7.45 (s,
1H), 7.42-7.36 (m, 2H), 7.32-7.20 (m, 2H), 7.08 (dd, J = 8.5 and 2.0, 1H), 7.03 (d, J = 2.0, 1H),
6.91 (d, J = 7.0, 1H), 6.72 (d, J = 8.0, 1H), 5.01 (s, 2H), 4.40-4.00 (m, 4H), 3.80 (t, J = 6.0, 2H),
3.58 (t, J = 5.5, 2H), 2.95-2.85 (m, 2H), 2.55-2.45 (m, 1H), 1.98-1.70 (m, 6H).
2.1.3. General procedure for preparation of halogenoalkyl amide derivatives 4a and 4b
A solution of the appropriate bromoacyl halide (2.10 mmol) in dry THF (2 ml) was added dropwise
to a 0 °C cooled solution of compound 1·HCl (1.37 mmol) and TEA (2.74 mmol) in dry THF (20
ml). The mixture was stirred at 0 °C for 1 h and at room temperature overnight. The precipitate was
filtered, and the filtrate evaporated in vacuum. The residue was diluted with EtOAc (50 ml) and the
organic phase was sequentially washed with 3×20 ml of saturated aqueous NaHCO3, 1N HCl and
brine, dried over anhydrous Na2SO4, filtered and evaporated in vacuum. The crude product was
purified by silica gel flash chromatography to afford the desired compound.
2.1.3.1.
1-(Bromoacetyl)-N-{3-[(4-fluorobenzyl)oxy]phenyl}piperidine-4-carboxamide,
4a.
Compound 4a was prepared according to the general procedure from compound 1·HCl (500 mg,
1.37 mmol) and bromoacetyl bromide (0.18 ml, 2.10 mmol). The crude product was purified by
silica gel flash chromatography (eluent: Hex/EtOAc, 1:9 v/v) in 55% yield (340 mg) as a brown oil.
IR (cm-1): 3233, 1652, 1605, 1443, 1198, 1080, 838, 696. 1H NMR (CDCl3) δ 7.43 (t, J = 2.0, 1H),
11

7.45-7.32 (m, 2H), 7.25-7.18 (m, 2H), 7.08-7.00 (m, 2H), 6.90 (d, J = 8.0, 1H), 6.75 (dd, J = 8.0 and
1.5, 1H), 5.00 (s, 2H), 4.30-4.15 (m, 2H), 3.20 (t, J = 6.0, 2H), 3.00-2.85 (m, 2H), 2.45-3.35 (m,
1H), 2.00-1.85 (m, 2H), 1.75-1.20 (m, 2H).
2.1.3.2. 1-(Bromopropanoyl)-N-{3-[(4-fluorobenzyl)oxy]phenyl}piperidine-4-carboxamide, 4b.
Compound 4b was prepared according to the general procedure from compound 1·HCl (500 mg,
1.37 mmol) and 3-bromopropionyl chloride (0.20 ml, 2.10 mmol) in DCM (15 ml). The crude
product was purified by silica gel flash chromatography (eluent: Hex/EtOAc, 1:9 v/v) in 64% yield
(400 mg) as a brown solid; mp 164-165 °C. IR (cm-1): 3275, 1653, 1638, 1433, 1206, 1022, 838,
690. 1H NMR (CDCl3) δ 7.57 (s, br, 1H), 7.44 (s, 1H), 7.41-7.35 (m, 2H), 7.20 (t, J = 8.0, 1H), 7.05
(t, J = 8.0, 2H), 6.93 (t, J = 8.0, 1H), 6.71 (dd, J = 8.0 and 2.0,1H), 5.00 (s, 2H), 4.60 (d, J = 13, 2H),
3.86 (d, J = 13, 2H), 3.64 (t, J = 7.0, 2H), 3.12 (d, J = 12, 1H), 2.92 (t, J = 7.0, 2H), 2.73 (t, J = 13,
1H), 2.55-2.45 (m, 1H), 2.00-1.90 (m, 2H), 1.85-1.65 (m, 2H).
2.1.4. General procedure for preparation of nitric ester derivatives 5a-e and 6a-b
A suspension of the appropriate alkyl halide intermediates (1.0 mmol) and AgNO3 (2.0 mmol) in
dry ACN (10 ml) was heated at reflux until reaction completion (TLC monitoring). After cooling,
the suspension was filtered on Celite, and pad was washed with ACN (50 ml). The combined
filtrates were evaporated in vacuum and the residue was purified by silica gel flash chromatography
and/or crystallization.
2.1.4.1.
(Nitrooxy)methyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 5a. Compound 5a was prepared according to the general procedure from 3a (400 mg,
0.95 mmol) and AgNO3 (323 mg, 1.90 mmol). The crude product was purified by silica gel flash
chromatography (eluent: Hex/EtOAc, 3:7 v/v) in 57% yield (240 mg) as a pale yellow solid, which
was further purified by crystallization from EtOAc/Hex; mp 130-131 °C. IR (cm-1): 3262, 1732,
12

1358, 1545, 1443, 1291, 1205, 1118, 951, 827. 1H NMR (DMSO-d6) δ 9.90 (s, 1H), 7.75 (d, J = 8.0,
2H), 7.52 (d, J = 8.0, 2H), 7.47-7.40 (m, 4H), 7.28-7.05 (m, 3H), 6.68 (d, J = 8.0, 1H), 6.07 (s, 2H),
5.11 (s, 2H), 3.98 (t, J = 13, 2H), 2.99-2.68 (m, 2H), 2.55-2.45 (m, 1H), 1.851.70 (m, 2H), 1.60-1.45
(m, 2H). MS (ESI) m/z 470 [M + Na]+. Anal. calcd. for C21H22FN3O7: C, 56.37; H, 4.96; N, 9.39%;
found: C, 56.55; H, 4.99; N, 9.43%.
2.1.4.2.
2-(Nitrooxy)ethyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 5b. Compound 5b was prepared according to the general procedure from 3b (500 mg,
1.15 mmol) and AgNO3 (390 mg, 2.30 mmol). The crude product was purified by crystallization
from EtOAc in 75% yield (400 mg) as a brown solid; mp 89-91 °C. IR (cm-1): 3430, 1635, 1442,
1
1384, 1280, 1204, 1040, 860. H NMR (DMSO-d6) δ 9.90 (s, 1H), 7.47 (t, J = 8.5, 2H), 7.39 (s,
1H), 7.25-7.00 (m, 4H), 6.66 (dd, J = 8.0 and 1.5, 1H), 5.02 (s, 2H), 4.76-4.70 (m, 2H), 4.32-4.25
(m, 2H), 4.05-3.90 (m, 2H), 2.95-2.75 (m, 2H), 2.55-2.40 (m, 1H), 1.85-1.70 (m, 2H), 1.60-1.40 (m,
2H). MS (ESI) m/z 484 [M + Na]+. Anal. calcd. for C22H24FN3O7×H2O: C, 55.11; H, 5.47; N,
8.76%; found: C, 55.41; H, 5.27; N, 8.74%.
2.1.4.3. 3-(Nitrooxy)propyl 4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 5c. Compound 5c was prepared according to the general procedure from 3c (845 mg,
1.71 mmol) and AgNO3 (581 mg, 3.42 mmol). The crude product was purified by silica gel flash
chromatography (eluent: Hex/EtOAc, 6:4 v/v) in 61% yield (500 mg) as a pale brown solid, which
was further purified by crystallization from Hex/EtOAc; mp 91-93 °C. IR (cm-1): 3432, 1702, 1622,
1436, 1279, 1207, 1037, 869. 1H NMR (DMSO-d6) δ 9.89 (s, 1H), 7.47 (t, J = 8.5, 2H), 7.38 (d, J =
2.0, 1H), 7.30-7.00 (m, 4H), 6.66 (dd, J = 7.5 and 1.5, 1H), 5.02 (s, 2H), 4.59 (t, J = 6.5, 2H), 4.07
(t, J = 6.5, 2H), 4.00 (t, J = 13, 2H), 2.90-2.75 (m, 2H), 2.55-2.45 (m, 1H), 1.99 (quintet, J = 6.5,
2H), 1.85-1.70 (m, 2H), 1.60-1.40 (m, 2H). MS (ESI) m/z 498 [M + Na]+. Anal. calcd. for
C23H26FN3O7: C, 58.10; H, 5.51; N, 8.84%; found: C, 58.34; H, 5.56; N, 8.58%.
13

2.1.4.4.
4-(Nitrooxy)butyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 5d. Compound 5d was synthesized according to the general procedure from 3d (400
mg, 0.89 mmol) and AgNO3 (303 mg, 1.78 mmol). The crude product was purified by silica gel
flash chromatography (eluent: Hex/EtOAc, 7:3 v/v) in 71% yield (310 mg) as a brown solid, which
was further purified by crystallization from Hex/EtOAc; mp 117-115 °C. IR (cm-1): 3432, 2954,
1716, 1661, 1612, 1523, 1431, 1201, 1155, 1040, 865, 825, 781, 742, 689. 1H NMR (CDCl3) δ 7.45
(s, 1H), 7.42-7.36 (m, 2H), 7.24-7.15 (m, 2H), 7.11-7.00 (m, 2H), 6.92 (d, J = 7, 1H), 6.72 (dd, J =
8.0 and 2.5, 1H), 5.02 (s, 2H), 4.50-4.20 (m, 2H), 4.13 (t, J = 6.0, 2H), 3.58 (t, J = 6.0, 2H), 2.88 (d,
J = 12, 1H), 2.84 (d, J = 12, 1H), 2.50-2.30 (m, 1H), 2.00-1.65 (m, 8H). MS (ESI) m/z 512 [M +
Na]+. Anal. calcd. for C24H28FN3O7: C, 58.89; H, 5.77; N, 8.58%; found: C, 59.02; H, 5.87; N,
8.58%.
2.1.4.5.
(Nitrooxy)methyl
4-[({3-[(3',5'-difluoro-1,1'-biphenyl-4-yl)methoxy]phenyl}amino)
carbonyl]piperidine-1-carboxylate, 5e. Compound 5e was prepared according to the general
procedure from 3e (170 mg, 0.33 mmol) and AgNO3 (70 mg, 0.39 mmol). The crude product was
purified by silica gel flash chromatography (eluent: Hex/EtOAc, 7:3 v/v), in 65% yield (116 mg) as
a pale yellow solid, which was further purified by crystallization from Hex/EtOAc; mp 130-131 °C.
IR (cm-1): 3200, 2930, 1732, 1658, 1598, 1545, 1443, 1384, 1291, 1205, 1118, 1086, 951, 827, 689.
1H NMR (DMSO-d6) δ 9.92 (s, 1H), 7.75 (d, J = 8.0, 2H), 7.52 (d, J = 8.0, 2H), 7.48-7.40 (m, 3H),
7.30-7.05 (m, 3H), 6.68 (dd, J = 8.0 and 1.5, 1H), 6.07 (s, 2H), 5.11 (s, 2H), 4.05-3.95 (m, 2H), 2.94
(t, J = 12, 1H), 2.87 (t, J = 12, 1H), 2.60-2.50 (m, 1H), 1.90-1.70 (m, 2H), 1.60-1.40 (m, 2H). Anal.
calcd. for C27H25F2N3O7: C, 59.89; H, 4.65; N, 7.76%; found: C, 60.02; H, 4.77; N, 7.90%.
2.1.4.6. 2-{4-[({3-[(4-Fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidin-1-yl}-2-oxoethyl nitrate,
6a. Compound 6a was prepared according to the general procedure from 4a (230 mg, 0.5 mmol)
14

and AgNO3 (170 mg, 1.0 mmol). The crude product was purified by silica gel flash chromatography
(eluent: MeOH/EtOAc, 5:95 v/v) in 56% yield (120 mg) as a white solid, which was further purified
by crystallization from EtOAc; mp 135-136 °C. IR (cm-1): 3306, 1651, 1552, 1446, 1376, 1291,
1
1207, 1037, 955, 856, 798, 785, 689. H NMR (DMSO-d6) δ 9.92 (s, 1H), 7.50-7.43 (m, 2H), 7.39
(s, 1H), 7.20 (t, J = 9.0, 2H), 7.18-7.05 (m, 2H), 6.66 (dd, J = 7.0 and 1.0, 1H), 5.39 (q, J = 13, 2H),
5.02 (s, 2H), 4.80 (s, 2H), 4.30 (d, J = 13, 1H), 3.73 (d, J = 13, 1H), 3.07 (t, J = 12, 1H), 2.69 (t, J =
12, 1H), 2.65-2.50 (m, 1H), 1.80 (d, J = 11, 2H), 1.75-1.55 (m, 1H), 1.55-1.35 (m, 1H). MS (ESI)
m/z 478 [M + Na]+. Anal. calcd. for C22H24FN3O7×H2O: C, 55.11; H, 5.47; N, 8.76%; found: C,
55.41; H, 5.27; N, 8.74%.
2.1.4.7.
2-{4-[({3-[(4-Fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidin-1-yl}-3-oxopropyl
nitrate, 6b. Compound 6b was prepared according to the general procedure from 4b (500 mg, 1.10
mmol) and AgNO3 (372 mg, 2.20 mmol). The crude product was purified by crystallization from
EtOAc in 62% yield (300 mg) as a white solid; mp 154-156 °C. IR (cm-1): 3330, 1683, 1633, 1551,
1
1381, 1285, 1200, 1159, 1041, 974, 857, 783, 690. H NMR (DMSO-d6) δ 9.86 (s, 1H), 7.50-7.45
(m, 2H), 7.38 (s, 1H), 7.19 (t, J = 7.0, 2H), 7.15-7.05 (m, 2H), 6.66 (dd, J = 8.0 and 1.5, 1H), 5.02
(s, 2H), 4.73 (t, J = 6.0, 2H), 4.38 (d, J = 13, 1H), 3.87 (d, J = 13, 1H), 3.03 (t, J = 12, 1H), 2.95-
2.84 (m, 2H), 2.79-2.50 (m, 2H), 1.79 (d, J = 13, 2H), 1.70-1.55 (m, 1H), 1.55-1.35 (m, 1H). MS
(ESI) m/z 468 [M + Na]+. Anal. calcd. for C22H24FN3O7×H2O: C, 55.11; H, 5.47; N, 8.76%; found:
C, 55.41; H, 5.27; N, 8.74%.
2.1.5. Preparation of the nitric ester derivative 9
Compound 9 was synthesized through three main steps, starting with the preparation t-Boc-
protected 2-(methylamino)ethanol, used in the first step, through the following procedure. A
solution of Boc2O (5.80 g, 26.6 mmol) in dry EtOAc (10 ml) was added dropwise to a 0 °C cooled
solution of 2-(methylamino)ethanol (2.14 ml, 26.6 mmol) in dry EtOAc (9 ml). The mixture was
15

stirred at room temperature for 2 h, and then concentrated under reduced pressure. The oil residue
was partitioned between EtOAc (100 ml) and water (300 ml), and the collected organic phases dried
over anhydrous Na2SO4, filtered and evaporated in vacuum to furnish t-butyl 2-
hydroxyethyl(methyl)carbamate in 78% yield (3.66 g) of as a colorless oil. IR (liquid film, cm-1):
1
3444, 2977, 1673, 1394, 1225, 1156, 1074, 877, 774. H NMR (CDCl3) δ 3.72 -3.80 (m 3H), 3.40
(t, J = 5.0, 2H), 2.92 (s, 3H), 1.47 (s, 9H).
2.1.5.1. 2-(Methyl)amino]ethyl 4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl] piperidine-1-
carboxylate, 7. 4-Nitrophenyl chloroformate (498 mg, 2.47 mmol) was added portionwise to a 0 °C
cooled solution of t-butyl 2-hydroxyethyl(methyl)carbamate (433 mg, 2.47 mmol) and TEA (0.86
ml, 6.18 mmol) in dry THF (10 ml), and the mixture was stirred at 0 °C for 3 h. Then, compound
1·HCl (900 mg, 2.47 mmol) was added, and the mixture stirred overnight at room temperature. The
formed precipitate was filtered, and the filtrate evaporated to dryness, leaving a solid residue which
was dissolved in EtOAc (50 ml). The EtOAc solution was sequentially washed with 2×20 ml of
saturated aqueous NaHCO3, 1N HCl and brine, dried over anhydrous Na2SO4, filtered and
evaporated in vacuum. The crude residue which was purified by silica gel flash chromatography
(eluent: Hex/ethyl acetate, 4:6 v/v), providing t-Boc-protected compound 7 as a pale yellow oil in
50% yield (645 mg). IR (cm-1): 3435, 2931, 1697, 1608, 1384, 1204, 1155, 864, 827, 770. 1H NMR
(CDCl3) δ 7.45 (t, J = 2.0, 1H), 7.42-7.36 (m, 2H), 7.22-7.16 (m, 2H), 7.06 (dt, J = 8.5 and 2.0, 2H),
6.92 (dd, J = 8.0 and 1.5, 1H), 6.72 (dd, J = 8.0 and 2.0, 1H), 5.01 (s, 2H), 4.30-4.15 (m, 4H), 3.55-
3.45 (m, 2H), 2.95-2.75 (m, 5H), 2.50-2.35 (m, 1H), 1.95-1.85 (m, 2H), 1.85-1.60 (m, 2H), 1.45 (s,
9H).
A mixture of t-Boc-protected 7 (645 mg, 1.24 mmol) and redistilled TFA (0.55 ml, 6.18 mmol) in
DCM (20 ml) was stirred at room temperature until reaction completion (TLC monitoring), to
produce compound 7 as TFA salt in quantitative yield (535 mg) as a pale brown oil. IR (cm-1):
1
3247, 2945, 1708, 1651, 1606, 1434, 1205, 1009, 837, 721. H NMR (DMSO-d6) δ 9.92 (s, 2H),
16

8.53 (s, br, 1H), 7.48-7.43 (m, 2H), 7.38 (s, 1H), 7.20 (t, J = 8.0, 1H), 7.12-7.00 (m, 2H), 6.67 (dd, J
= 8.0 and 1.5, 2H), 5.02 (s, 2H), 4.19 (t, J = 5.0, 2H), 4.18-4.00 (m, 2H), 3.18 (t, J = 5.0, 2H), 2.90-
2.70 (m, 3H), 2.60 (t, J = 5.0, 2H), 2.55-2.45 (m, 1H), 1.85-1.65 (m, 2H), 1.60-1.40 (m, 2H).
2.1.5.2. 2-[(Bromoacetyl)(methyl)amino]ethyl 4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]
piperidine-1-carboxylate, 8. A solution of bromoacetyl bromide (0.17 ml, 1.93 mmol) in dry DCM
(3 ml) was added dropwise to a 0 °C cooled suspension of 7·TFA (750 mg, 1.75 mmol) and TEA
(0.5 ml, 3.50 mmol) in dry DCM (8 ml). The mixture was stirred overnight at room temperature,
then was filtered and the filtrate diluted with DCM (20 ml). The organic phase was washed with
2×20 ml of saturated aqueous NaHCO3, 1N HCl and brine, dried over anhydrous Na2SO4, filtered
and evaporated to dryness, to give the crude product (407 mg, 48% yield), which was used without
further purification in the subsequent reaction. MS (ESI) m/z: 550 [M + H]+. IR (cm-1): 3321, 2922,
1693, 1646, 1606, 1440, 1204, 1125, 1036, 873, 768.
2.1.5.3.
2-{Methyl[(nitrooxy)acetyl]amino}ethyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)
carbonyl]piperidine-1-carboxylate, 9. Compound 9 was prepared, according to the above described
procedure from 8 (350 mg, 0.64 mmol) and AgNO3 (162 mg, 0.95 mmol). The crude product was
purified by crystallization from EtOAc in 55% yield (195 mg) as a brown solid; mp 120-121 °C. IR
(cm-1): 3433, 1650, 1541, 1439, 1384, 1286, 1045, 852. 1H NMR (DMSO-d6) δ 9.87 (s, 1H), 7.50-
7.40 (m, 2H), 7.39 (s, 1H), 7.25-7.14 (m, 3H), 7.09 (d, J = 8.5, 1H), 6.66 (dd, J = 8.0 and 1.5, 1H),
5.34 (s, 2H), 4.22-4.10 (m, 2H), 4.07 (t, J = 5.0, 2H), 4.05-3.95 (m, 2H), 3.60-3.50 (m, 2H), 2.98 (s,
3H), 2.55-2.45 (m, 1H), 1.65-1.45 (m, 2H), 1.55-1.40 (m, 2H). MS (ESI) m/z 555 [M + Na]+. Anal.
calcd. for C25H29FN4O8: C, 56.39; H, 5.49; N, 10.52%; found: C, 56.58; H, 5.66; N, 10.68%.
2.1.6. General procedure for preparation of hydroxyalkyl carbamate derivatives 12a and 12b
17

A solution of benzoyl chloride (1.7 ml, 14.7 mmol) in dry THF (10 ml) was added dropwise to a 0
°C cooled solution of ethylene glycol or 1,3-propanediol (16.1 mmol) for 10a or 10b, respectively,
and TEA (4 ml, 29.4 mmol) in dry THF (20 ml). The mixture was stirred at room temperature
overnight. The formed precipitate was filtered, the filtrate was evaporated to dryness and the solid
residue was dissolved in EtOAc (50 ml). The organic phase was sequentially washed with 3×20 ml
of saturated aqueous NaHCO3, 1N HCl and brine, dried over anhydrous Na2SO4, filtered and
evaporated in vacuum, to provide a residue containing the desired crude products 10a and 10b (65%
GC yield), which were used in the subsequent reactions without further purification.
A solution of 4-nitrophenyl chloroformate (950 mg, 4.70 mmol) in dry THF (5 ml) was added
dropwise to a 0 °C cooled solution of crude product 10a or 10b (4.51 mmol) in dry THF (10 ml)
and TEA (1.9 ml, 13.5 mmol), and the mixture was stirred at 0 °C for 2 h. Then, compound 1·HCl
(1.26 g, 3.86 mmol) was added, and the mixture was stirred at room temperature overnight. The
formed precipitate was filtered, the solvent was evaporated in vacuum and the solid residue was
dissolved in EtOAc (50 ml). The organic phase was sequentially washed with 2×20 ml of saturated
aqueous NaHCO3 and brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The crude products 11a and 11b were purified by silica gel flash chromatography.
A mixture of 11a or 11b (1.20 mmol) in THF (10 ml), MeOH (2 ml) and aqueous 2N NaOH
solution (3 ml) was stirred at room temperature for 6h, until reaction completion (TLC monitoring).
The solution was then concentration under reduced pressure, and the oil residue was dissolved in
EtOAc (50 ml) and partitioned with brine. The organic phase was dried over anhydrous Na2SO4,
filtered and evaporated to dryness. The crude product was purified by crystallization or silica gel
flash chromatography.
2.1.6.1. 2-(Benzoyloxy)ethyl 4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 11a. Compound 11a was prepared was prepared according to the general procedure
from crude 2-hydroxyethyl benzoate 10a (750 mg, 4.51 mmol) and compound 1·HCl (1.26 g, 3.86
18

mmol). The crude product was purified by silica gel flash chromatography (eluent: Hex/EtOAc, 1:1
v/v) in 45% yield (1.06 g) as a pale brown solid, which was crystallized from Hex/EtOAc; mp 142-
1
144 °C. IR (cm-1): 3269, 1729, 1698, 1647, 1605, 1276, 1208, 837, 707. H NMR (CDCl3) δ 8.08-
8.02 (m, 2H), 7.56 (t, J = 7.5, 1H), 7.49-7.36 (m, 5H), 7.21 (t, J = 8.0, 1H), 7.16 (s, br, 1H), 7.06 (t,
J = 9.0, 2H), 6.92 (dd, J = 8.0 and 1.5, 1H), 6.72 (dd, J = 8.0 and 1.5, 1H), 5.01 (s, 2H), 4.58-4.53
(m, 2H), 4.46-4.41 (m, 2H), 4.30-4.15 (m, 2H), 2.89 (d, J = 12, 1H), 2.85 (d, J = 12, 1H), 2.45-2.35
(m, 1H), 2.00-1.85 (m, 2H), 1.85-1.65 (m, 2H).
2.1.6.2. 3-(Benzoyloxy)propyl 4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 11b. Compound 11b was prepared was prepared according to the general procedure
from crude 3-hydroxypropyl benzoate (500 mg, 2.68 mmol) and compound 1·HCl (750 mg, 2.28
mmol). The crude product was purified by silica gel flash chromatography (eluent: Hex/EtOAc, 1:1
v/v) in 55% yield (785 mg) as a white solid, which was crystallized from Hex/EtOAc; mp 135-137
°C. IR (cm-1): 3290, 1715, 1703, 1661, 1278, 1202, 1122, 822, 709. 1H NMR (CDCl3) δ 8.03 (d, J =
7.0, 2H), 7.56 (t, J = 7.5, 1H), 7.48-7.35 (m, 5H), 7.23 (s, br, 1H), 7.19 (d, J = 8.0, 1H), 7.06 (t, J =
9.0, 2H), 6.93 (dd, J = 8.0 and 1.0, 1H), 6.71 (dd, J = 8.0 and 2.0, 1H), 5.00 (s, 2H), 4.43 (t, J = 6.0,
2H), 4.28 (t, J = 6.0, 2H), 4.27 (m, 2H), 2.85 (d, J = 12, 1H), 2.81 (d, J = 12, 1H), 2.45-2.30 (m,
1H), 2.14 (quintet, J = 6.0, 2H), 1.95-1.75 (m, 2H), 1.73-1.60 (m, 2H).
2.1.6.3.
2-Hydroxyethyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 12a. Compound 12a was prepared was prepared according to the general procedure
from 11a (635 mg, 1.22 mmol). The crude product was purified by crystallization from EtOH, with
addition of few drops of water, in 55% yield (260 mg) as a pale yellow solid; mp 174-175 °C. IR
1
(cm-1): 3508, 3250, 2922, 2867, 1703, 1654, 1605, 1434, 1229, 1204, 1072, 839, 687. H NMR
(DMSO-d6) δ 9.90 (s, 1H), 7.50-7.43 (m, 2H), 7.39 (t, J = 2.0, 1H), 7.25-7.05 (m, 4H), 6.66 (dd, J =
8.5 and 1.5, 1H), 5.02 (s, 2H), 4.77 (t, J = 5.0, 1H), 4.03 (d, J = 12.5, 2H), 3.98 (t, J = 5.0, 2H), 3.54
19

(q, J = 5.0, 2H), 2.95-2.85 (m, 2H), 2.50-2.40 (m, 1H), 1.80-1.75 (m, 2H), 1.55-1.40 (m, 2H). Anal.
calcd. for C22H25FN2O5: C, 63.45; H, 6.05; N, 6.73%; found: C, 63.63; H, 6.12; N, 6.72%.
2.1.6.4.
3-Hydroxypropyl
4-[({3-[(4-fluorobenzyl)oxy]phenyl}amino)carbonyl]piperidine-1-
carboxylate, 12b. Compound 12b was prepared according to the general procedure from 11b (365
mg, 0.68 mmol). The crude product was purified by silica gel flash chromatography (eluent:
Hex/EtOAc, 3:7 v/v) in 92% yield (270 mg) as a yellow oil. IR (cm-1): 3416, 3316, 2951, 2870,
1
1675, 1606, 1539, 1437, 1225, 1155, 1046, 955, 865, 826, 760. H NMR (CDCl3) δ 7.45 (s, 1H),
7.43-7.37 (m, 2H), 7.21 (t, J = 8.0, 1H), 7.17 (s, br, 1H), 7.06 (t, J = 9.0, 2H), 6.92 (d, J = 8.5, 1H),
6.72 (dd, J = 8.0 and 2.5, 1H), 5.02 (s, 2H), 4.28 (t, J = 6.0, 2H), 4.25-4.10 (m, 4H), 2.95-2.80 (m,
2H), 2.50-2.35 (m, 1H), 2.00-1.55 (m, 7H).
2.1.7. Preparation of benzyl nitrate derivative 15
Compound 15 was prepared through a two-step synthesis, starting from the already reported 1-t-
Boc-protected N-(3-hydroxyphenyl)piperidine-4-carboxamide 13 (de Candia et al., 2009).
2.1.7.1.
Tert-butyl-4-{[(3-{[4-(bromomethyl)benzyl]oxy}phenyl)amino]carbonyl}piperidine-1-
carboxylate, 14. α,α’-Dibromo-p-xylene (298 mg, 1.13 mmol) and anhydrous K2CO3 (195 mg, 1.41
mmol) were added to a solution of compound 13 (300 mg, 0.94 mmol) in Me2CO (15 ml). The
mixture was refluxed until disappearance of 13 (TLC monitoring). After cooling and removal of the
reaction solvent, the residue was partitioned between EtOAc (50 ml) and brine (50 ml). The organic
phase was dried over anhydrous Na2SO4, filtered and evaporated to dryness. The crude product was
purified by silica gel flash chromatography (Hex/EtOAc, 1:1 v/v) in 60% yield (285 mg) as a brown
1
oil. IR (cm-1): 3300, 2973, 2929, 2851, 1694, 1651, 1584, 1421, 1286, 1213, 1163, 1041, 781. H
NMR (CDCl3) δ 7.50-7.40 (m, 5H), 7.11 (s, 1H), 6.92 (d, J = 6.5, 1H), 6.72 (d, J = 6.5, 1H), 5.43 (s,
20

2H), 5.08 (s, 2H), 4.25-4.15 (m, 2H) 2.79 (t, J = 12, 2H), 2.45-2.30 (m, 1H), 1.95-1.85 (m, 2H),
1.80-1.60 (m, 2H), 1.46 (s, 9H).
2.1.7.2. 4-({3-[(Piperidin-4-ylcarbonyl)amino]phenoxy}methyl)benzyl nitrate hydrochloride, 15.
Compound 15 was synthesized according to the general procedure, as described for 5a, from
compound 14 (350 mg, 0.70 mmol) and AgNO3 (233 mg, 1.37 mmol). The crude N-Boc derivative
of compound 15 was purified by silica gel flash chromatography (Hex/EtOAc, 1:1 v/v) in 65% yield
(220 mg) as a brown oil. IR (cm-1): 3300, 2973, 2930, 2851, 1698, 1661, 1631, 1550, 1421, 1280,
1214, 1163, 1042, 957, 867, 856, 774, 690. 1H NMR (CDCl3) δ 7.48 (s, 1H), 7.46 (d, J = 8.0, 2H),
7.40 (d, J = 8.0, 2H), 7.21 (t, J = 8.0, 1H), 7.15 (s, br, 1H), 6.92 (d, J = 7.0, 1H), 6.71 (dd, J = 8.0
and 2.0, 1H), 5.43 (s, 2H), 5.08 (s, 2H), 4.25-4.10 (m, 2H), 2.78 (t, J = 12, 2H), 2.43-2.30 (m, 1H),
1.95-1.85 (m, 2H), 1.80-1.65 (m, 2H), 1.46 (s, 9H).
A 1.25 M HCl methanolic solution (2.0 ml, 2.25 mmol) was added to a solution of N-Boc-protected
15 (220 mg, 0.45 mmol) in MeOH (20 ml), and the mixture was stirred at room temperature until
reaction completion (TLC monitoring). After solvent evaporation in vacuum and trituration of the
residue with abs. EtOH, the hydrochloride salt of 15 was obtained in quantitative yield (175 mg) as
a pale brown solid, which was crystallized from EtOAc/EtOH; mp 102-104 °C. IR (cm-1): 3399,
2925, 2800, 2709, 1692, 1615, 1544, 1385, 1284, 1208, 1162, 1121, 892, 873. 1H NMR (CDCl3) δ
10.06 (s, 1H), 8.75 (s, br, 1H), 8.47 (s, br, 1H), 7.47 (s, 4H), 7.39 (d, J = 2.0, 1H), 7.18 (t, J = 8.0,
1H), 7.11 (d, J = 8.0, 1H), 6.68 (dd, J = 8.0 and 1.5, 1H), 5.56 (s, 2H), 5.08 (s, 2H), 3.97 (d, J =
12.6, 1H), 3.32 (d, J = 12.6, 1H), 2.88 (t, J = 12.6, 2H), 2.65-2.5 (m, 1H), 2.00-1.85 (m, 2H), 1.85-
1.60 (m, 2H). MS (ESI) m/z 386 [M + H]+. Anal. Calcd. for C24H28FN3O7: C, 58.89; H, 5.77; N,
8.58%; found: C, 59.02; H, 5.87; N, 8.58%.
2.3 Vasodilator activity
21

Male Wistar rats were obtained from Harlan Laboratories (San Pietro al Natisone, Italy) and
individually housed at constant room temperature (25 1 °C) and humidity (60 5 %), with an
artificial 12:12 h light/dark cycle. Thoracic aortas were isolated from rats weighing 180-200 g. As
few animals as possible were used. The purposes and the protocols of our studies have been
approved by Ministero della Salute (Rome, Italy). All the procedures were performed in accordance
with the Ethical Animal Committee of the University of Turin (Italy).
The endothelium was removed and the vessels were helically cut: four to six strips were obtained
from each aorta. The tissues were mounted under 1.0 g tension in organ baths containing 30 ml of
Krebs-bicarbonate buffer with the following composition (mM): NaCl 111.2, KCl 5.0, CaCl2 2.5,
MgSO4 1.2, KH2PO4 1.0, NaHCO3 12.0, glucose 11.1, maintained at 37 °C and gassed with 95%
O2-5% CO2 (pH 7.4). The aorta strips were allowed to equilibrate for 120 min and then contracted
with 1 μM L-phenylephrine. When the response to the agonist reached a plateau, cumulative
concentrations of the test compound were added. Results are expressed as EC50 values; data are the
mean SEM of at least three experiments. The effects of 1 μM ODQ on relaxation were evaluated in
separate series of experiments in which it was added to the organ bath 5 min before contraction.
Responses were recorded by an isometric transducer connected to the MacLab System PowerLab.
Addition of the drug vehicle (1% DMSO) had no appreciable effect on contraction level.
2.4 Inhibition of platelet aggregation
Human blood was obtained from healthy volunteers (25-45 years of age), who had not ingested any
platelet inhibitory drug for at least one week prior to donation. All subjects provided informed
consent, and were treated according to Helsinki protocol for biomedical experimentation.
Blood and blood products were handled in plastic ware, whereas siliconized glass cuvettes and stir
bars were used in the aggregation assay. Platelet rich plasma (PRP) was prepared by centrifugation
of citrated blood at 200×g for 20 min. The transmittance of PPP was taken as 100% aggregation.
22

PRP (500 μl) was added into the aggregometer (Chrono-log 4902D) cuvettes and preincubated at 37
°C for 15 min with the tested compounds (final concentrations in the PRP solutions ranging from 10
μM to 300 µM) or with vehicle (to eliminate the effect of the solvent on the aggregation and release
reaction of platelets; the final concentration of DMSO was fixed at 0.5%, v/v). Then, adenosine 5’-
diphosphate (ADP, 5 μM final concentration), or collagen at submaximal concentration (0.8-1.5
μg/ml) were added to the incubated sample and aggregation was recorded as increased light
transmission under continuous stirring (1000 rpm) at 37 °C for 10 min after addition of the
aggregation inducer. The antiaggregatory activity of the tested compounds is expressed as percent
inhibition of platelet aggregation compared with vehicle control samples. For most of the active
compounds, IC50 values (i.e., the concentration effecting 50% inhibition of aggregation), was
calculated by nonlinear regression analysis (r2 > 0.80); alternatively, percent inhibition at maximal
concentration tested (300 μM) is reported. The number of experiments was 3-5 for IC50 values, and 3
for determination of % inhibition.
2.3 Hydrolysis studies
2.3.1 Hydrolysis in aqueous solutions. To 0.25 ml of a 10 mM stock solution of the compound in
ACN, 1.75 ml of ACN and aqueous buffer solution (0.01 M HCl or 0.04 M phosphate buffer pH 7.4
in 0.15 M KCl) were added to give a final volume of 10 ml. The solution at the final concentration
of 200 μM was thermostated at 37 0.5 °C.
At appropriate time intervals, samples were withdrawn and analyzed by HPLC using 1260 Infinity
Quaternary LC system (Agilent Technologies, Milan, Italy) equipped with autosampler, photodiode
array detector and evaporative light scattering detector, and controlled by Lab Advisory software. A
Phenomenex Kinetex C18 column 5 µm (150 ×3.0 mm i.d.) was used as the stationary phase; the
analytes were eluted with a 23 min gradient from mobile phase A (65% v/v 20 mM ammonium
23

formate aqueous solution in ACN) to mobile phase B (35% v/v 20 mM ammonium formate aqueous
solution in ACN) at a constant flow rate of 0.5 ml/min; injection volume: 10 μl.
Pseudo-first-order rate constants (kobs) for the hydrolysis of the compounds were calculated from
the slopes of the linear plots of log (% remaining compound) against time. Each kinetic experiment
was performed in triplicate.
2.3.2 Stability in human serum. To 1.47 ml of human serum (lyophilized and reconstituted with 4
ml of deionized water), preheated at 37 0.5 °C, 30 μl of a 10 mM stock solution of each compound
in ACN were added and the solution was incubated at 37 0.5 °C (final compound concentration
200 µM). Aliquots (100 μl) of the serum solution were taken at various times and deproteinized by
mixing with 500 μl of cold MeOH. The suspension was vortexed 1 min, centrifuged 10 min at 4350
rpm, and 10 μl of the supernatant were analyzed by HPLC as described above. The % amounts of
the remaining intact compound were plotted against the incubation time.
3. Results and discussion
3.1 Chemistry
The nitrooxy (ONO2)-containing compounds of compounds 1 and 2 were synthesized as outlined in
Scheme 1. Compound 1·HCl was reacted with chloromethyl- and 2-chloroethyl chloroformate, in
the presence of triethylamine (TEA), to provide chloromethyl- and chloroethyl carbamate
derivatives 3a and 3b, respectively, which were then transformed into the corresponding
(ONO2)methyl (5a) and (ONO2)ethyl (5b) derivatives by treatment with AgNO3 in acetonitrile
(ACN) at reflux. Compound 5e, i.e., the (ONO2)methyl carbamate of 2, was prepared using a
similar procedure. Conversely, the synthesis of the intermediate bromopropyl (3c) and chlorobutyl
(3d) carbamates was accomplished by activating 3-bromopropanol and 4-chlorobutanol as
carbonates, by reaction with 4-nitrophenyl chloroformate, and subsequent aminolysis by 1.
24

(A)
1
O
O
(i)
(iii)
O
N
O
N
H
H
N
O
ONO2
n
N
O
X
n
F
F
O
O
3a-d
5a-d
3a → 5a 3b → 5b 3c → 5c 3d → 5d
X
n
Cl
1
Cl
2
Br
3
Cl
4
O
O
(i)
(iii)
O
N
O
N
H
2
H
F
N
O
Cl
F
N
O
ONO2
O
O
3e
5e
F
F
(B)
1
O
O
(ii)
(iii)
O
N
H
O
N
H
N
N
F
ONO2
F
Br
n
n
O
O
6a, n = 1
6b, n = 2
4a, n = 1
4b, n = 2
Scheme 1. Synthesis of (ONO2)alkyl carbamate (A) and amide (B) derivatives of 3-substituted-
benzyloxy isonipecotanilides 1 and 2. Reagents and conditions: (i) a) ClCOOCH2Cl (for 3a and 3e),
ClCOOCH2CH2Cl (for 3b), TEA, dry DCM, r.t., overnight, 54-78%; b) 4-
nitrophenylchloroformate, TEA, Br(CH2)3OH (for 3c), Cl(CH2)4OH (for 3d), dry THF, 0 °C, 3 h,
then 1, r.t., overnight, 50-55%. (ii) BrCH2COBr (for 4a), BrCH2CH2COBr (for 4b), TEA, dry THF,
0 °C, 1 h, then r.t., overnight, 55-64%. (iii) AgNO3, CH3CN, reflux, 16-20 h, 55-75%.
25

N-t-Boc-N’-methyl-2-aminoethanol was used to prepare the intermediate product 7, which after t-
Boc-deprotection and subsequent reaction with bromoacetyl bromide yielded compound 8, which
was finally refluxed with AgNO3 in ACN to give the corresponding (ONO2)-substituted compound
9. The (ONO2)-containing amide derivatives 6a,b were synthesized by reacting compound 1 with
the appropriate bromoacyl bromides and subsequent nitrooxylation of the intermediate bromides
4a,b (Scheme 2).
O
O
N
(i)
H
Boc
OH
1
+
N
O
N
F
N
H
O
7
(ii)
O
O
(iii)
O
N
O
O
N
H
O
H
N
O
Br
N
O
ONO2
F
N
F
N
O
O
8
9
Scheme 2. Synthesis of the (ONO2)-containing compound 9. Reagents and conditions: (i) 4-
nitrophenylchloroformate, TEA, dry THF, 0 °C, 3 h, then 1, r.t., overnight; TFA, DCM, r.t., 50%.
(ii) BrCH2COBr, TEA, dry DCM, r.t., overnight, 48%. (iii) AgNO3, CH3CN, reflux, 16-20 h, 55-
75%.
To prove the effectiveness of our (ONO2)-containing derivatives as NO-releasing agents, the ethyl
and propyl alcohols 12a and 12b, as hypothetical de-nitrated metabolites arising from
biotransformation pathways involving hydrolytic cleavage of the nitric ester functions in 5b and 5c,
respectively, were synthesized as outlined in Scheme 3. The mono benzoate esters 10a-b, once
activated as carbonates by reaction with 4-nitrophenyl chloroformate, were transformed through
26

aminolysis by 1 into the ester products 11a,b, which were then hydrolyzed in basic conditions to
provide the respective hydroxyl derivatives 12a,b.
O
O
(i)
OH
( )n
(ii)
HO
O
O
N
O
( )n
OH
H
N
O
( )n
F
O
O
11a, n = 1
11b, n = 2
10a, n = 1
10b, n = 2
O
(iii)
O
N
H
N
O
( )n
F
OH
O
12a, n = 1
12b, n = 2
Scheme 3. Synthesis of hydroxyalkyl carbamate derivatives of 4-fluorobenzyloxy isonipecotanilide
1. Reagents and conditions: (i) Benzoyl chloride, TEA, dry THF, r.t., overnight, 55-65%; (ii) 4-
nitrophenyl chloroformate, TEA, dry THF, r.t., 3 h; then 1, r.t., overnight, 45-55%; (iii) 2N NaOH,
THF/MeOH 5:1 v/v, r.t., 12-16 h, 55-92%.
Finally, the benzyl nitrate derivative 15 was synthesized as outlined in Scheme 4. Monobenzylation
of the phenol OH in compound 13 (de Candia et al., 2009) by reaction with α,α’-dibromo-p-xylene
furnished the bromobenzyl derivative 14, which was then transformed into the organic nitrate 15.
O
O
O
(i)
(ii), (iii)
O
N
O
N
HO
N
H
H
H
O2NO
NH
.
Br
N
N
Boc
Boc
HCl
13
14
15
27

Scheme 4. Synthesis of the 4-benzyl nitrate derivative 15. Reagents and conditions: (i) α,α-
Dibromo-p-xylene, K2CO3, dry acetone, reflux, 6 h, 60%; (ii) AgNO3, ACN, reflux, 6 h, 65%; (iii)
HCl gas, CHCl3, quantitative yield.
3.2 Vasodilator and antiplatelet activities
All the newly synthesized nitric ester derivatives (5a-e, 6a-b, 9 and 15), along with the parent
compound 1 were assayed to evaluate both the vasodilator and antiplatelet activities; vasorelaxing
properties of the alcohol products 12a and 12b, hypothetically arising from the cleavage of the
nitrate function correspondingly in 5b and 5c, were studied as well. The vasodilator activity was
evaluated on isolated rat aortic strips, precontracted with L-phenylephrine. All the tested compounds
showed concentration-dependent vasorelaxing effects, and their potencies, expressed as EC50
values, are summarized in Table 1.
The (ONO2)-containing derivatives showed good vasodilatory activity with EC50 values ranging
from 0.01 to 5 µM. The (ONO2)-methyl carbamate 5a and the benzyl nitrate derivative 15 proved to
be the most potent compounds, with EC50 values falling in the low nanomolar range (13 and 29 nM,
respectively). The carbamate derivatives 5a and 5b are several times more potent than the
corresponding amide derivatives 6a and 6b. Within the small carbamate series (5a-d) the
vasodilator potency is inversely correlated with the length of the alkyl spacer between the ONO2
and >NCOO moieties (pEC50/nCH2 points lie on a straight line with slope = -0.84 and r2 = 0.994),
suggesting that the closer the secondary carbamate group, the greater its electron-withdrawing
inductive effect on the vasorelaxing activity of the (ONO2)alkyl piperidine-1-carbamate derivatives.
No substantial change in vasodilatory potency was observed when the NO-donor side-chain was
elongated, as we did in compound 9, via a link between the terminal 2-(ONO2)acetamido fragment
and the internal ethyl carbamate group.
28

Co-incubation of the aortic strips with the selective inhibitor of sGC, 1H-[1,2,4]oxadiazolo[4,3-
a]quinaxolin-1-one (ODQ), resulted in a rightward shift of the dose-response curves and higher
EC50 values for all the tested nitrate compounds (Fig. 3). The observed decrease of potency
provided support to NO-mediated activation of sGC, which is the key enzyme modulating smooth
muscle relaxation (and platelet aggregation as well) through increase of cGMP concentration.
Interestingly, also the (ONO2)-lacking compounds 1, 12a and 12b, displayed vasodilatory effects,
with EC50 values around 20 µM.
Table 1
In vitro vasodilator and anti-platelet activity of ONO2-substituted compounds and some related
alcohol derivatives of fluorinated benzyloxyphenyl piperidine-4-carboxamides.
Vasodilator activity a
EC50, µM SEM
[+1 µM ODQ]
Platelet aggregation inhibition b
ADP, 5 µM
Cmpd
Collagen, 0.8-1.5 µg/mL
IC50, µM (CL95%)
IC50, µM (CL95%)
[% inhib. SEM at 300 µM] [% inhib. SEM at 300 µM]
1
17
4
73 (68-79)
87 (82-92)
[46 6]
5a
5b
5c
5d
5e
6a
6b
9
0.013 0.002 [1.3 0.2]
0.11 0.04 [4.5 0.4]
0.50 0.08 [3.1 0.8]
5.0 2 [> 100]
117 (93-147)
188 (150-236)
180 (129-251)
234 (198-277)
108 (81-146)
[22 11]
[11 7]
[7.0 4.0]
[14 5]
0.25 0.06 [> 100]
0.42 0.06 [17 2]
0.49 0.10 [8.5 0.6]
0.11 0.02 [14 1]
[41 11]
[12 5]
[4.0 3.0]
[20 13]
[13 4]
[16 8]
12a
12b
24
23
5
1
15
0.029 0.006 [9.3 1.0]
55 (43-70)
194 (182-205)
a
Concentration of tested compound which reduces by 50% contraction of the rat thoracic aortic
strips, previously treated with 1 μM L-phenylephrine, with and without addition of 1 µM ODQ, as
29