Novel R-roscovitine NO-donor hybrid compounds as potential pro-resolution of inflammation agents

Article abstract of DOI:10.1016/j.bmc.2013.01.009

Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi-NO-donor hybrids may have additive pro-resolution of inflammation effects.

Full text of DOI:10.1016/j.bmc.2013.01.009

Bioorganic & Medicinal Chemistry 21 (2013) 2107–2116
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel R-roscovitine NO-donor hybrid compounds as potential
pro-resolution of inflammation agents
Gabriele Montanaro a, Massimo Bertinaria a, Barbara Rolando a, Roberta Fruttero a,
,
⇑
Christopher D. Lucas b, David A. Dorward b, Adriano G. Rossi b, Ian L. Megson c, Alberto Gasco a
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P. Giuria 9, 10125 Torino, Italy
b MRC Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh Medical School, Edinburgh EH16 4TJ, United Kingdom
c Free Radical Research Facilities, Department of Diabetes and Cardiovascular Science, The Univeristy of the Highlands & Islands, Inverness IV2 3JH, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel
pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have
been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor
(CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and pos-
sess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution
derivatives, here we describe the design, synthesis and investigation of the biological potential of a small
series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties
(compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with
their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an
increased pro-apoptotic activity when compared with either R-roscovitine or structurally related com-
pounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble
guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that
reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apop-
tosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi–
NO-donor hybrids may have additive pro-resolution of inflammation effects.
Received 30 October 2012
Revised 22 December 2012
Accepted 3 January 2013
Available online 16 January 2013
Keywords:
R-Roscovitine
Multi-target drugs
Apoptosis
Nitric oxide donors
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
non-inflammatory form of cell death, in order that resolution of
inflammation can occur. Apoptotic granulocytes are rapidly re-
Inflammation is the host response to tissue injury or infection,
aimed at restoring damaged tissue to homeostasis. Following injury
a variety of endogenous mediators are released which mediate the
cardinal signs of inflammation: heat, redness, swelling, and pain.
Vasoactive amines, lysozomal enzymes, cytokines, kinins, eicosa-
noids, clotting and fibrinolytic system products, nitric oxide (NO),
and reactive oxygen species (ROS), are some of the important medi-
ators involved. During an episode of acute inflammation a complex
series of events occurs including the recruitment of leukocytes to the
damaged tissue. Granulocytes (which comprise neutrophils, eosino-
phils and basophils) are the first leukocytes to be recruited, followed
by monocyte/macrophage and lymphocyte infiltration into the in-
flamed tissue. The granulocytes are the body’s first line of innate im-
mune defense and have the task of neutralizing the injurious
stimulus.1–3 After removal of the inciting inflammatory stimulus it
is essential that granulocytes die by apoptosis, a programmed and
moved from inflammatory sites by macrophage phagocytosis with
consequent dampening of inflammation.4 Previously it was believed
that, after removal of the cause of theinjury, the resolutionof inflam-
mation was merely a passive process secondary to the catabolism of
pro-inflammatory mediators. More recently, an increasing number
of studies have demonstrated that the resolution of inflammation
is an active process involving several cellular processes including
apoptosis and phagocytic clearance of apoptotic cells that can be
modulated by endogenous specialized pro-resolving mediators
(SPM). These include cyclopentanone prostaglandins, lipoxins,
resolvins, protectins/neuroprotectins, and maresins.3,5–7 When
inflammation resolution fails, the granulocytes can die by necrosis,
an alternative form of cell death that involves rupture of cellular
membrane with consequent release of granulocyte histotoxic con-
tents into the extracellular milieu. Thiscan perpetuateinflammation
leading to chronicity, with dysregulated granulocyte apoptosis
being involved in the pathogenesis of several human inflammatory
diseases.8 The development of pharmacological agents or strategies
capable of selectively increasing granulocyte apoptosis is a potential
new therapeutic strategy to reduce both acute and chronic
⇑
Corresponding author. Tel.: +39 (0)11 6707850; fax: +39 (0)11 6707286.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.009

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G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
Scheme 1. Preparation of derivative 2. Reagents: (i) 4-nitrooxybutirric acid, EDC, DMAP, dry Pyr, 4 h, rt.
Scheme 2. Preparation of derivatives 9a–d. Reagents and conditions: (i) H2SO4, H2O/MeOH, 3 h,
D; (ii) benzylmercaptane, DIPEA, EtOH, 4 h, D; (iii) 2-bromopropane, K2CO3,
DMSO, 12 h, 50 °C; (iv) R-(2)-amino-1-butanol, nBuOH, 5 days, ; (v) mCPBA, CH2Cl2, 3 h, rt; (vi) 12, 14, 16 or 18, Et3N, MeOH, 5 days, 40 °C.
D

G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
2109
inflammation.3,4 The use of small molecule inhibitors of the serine/
threonine cyclin-dependent kinases (CDKs) is one of the most prom-
ising strategies.9 There are at least 13 different CDK isoenzymes so
far identified, and 25 different cyclins, the binding partners neces-
sary for CDK activation. CDKs play essential functions in many bio-
logical processes including cell division, transcription, neuronal
and release of NO. To achieve this we designed new products in
which R-roscovitine is linked through a hard bridge to moieties con-
taining a NO-donor nitrooxy group (compound 9a) or a NO-donor 3-
furoxancarboxamide substructure (compound 9c). Also compound
2 was considered in which R-roscovitine is linked to a nitrooxy
substituted moiety through a vulnerable ester group. It is generally
accepted that the NO-release from nitrooxy derivative occurs by
enzymatic activation, whereas in the case of furoxan derivatives it
occurs under the action of thiol co-factors.22,23 In this report the syn-
thesis of these compounds and the results of a biological study
aimed at exploring their ability to induce neutrophil apoptosis have
been investigated. Also, compounds 9b, 9d which were structurally
related to 9a, 9c, respectively, but devoid of the ability to release NO
(des-NO analogues), were used for comparison.
cell physiology, pain signaling, apoptosis, and RNA splicing.10,11
A
variety of CDK inhibitor drugs have been described from different
chemical classes including purines, pyrimidines, flavones, pyrido-
pyrimidines, oxindoles, quinazolines and pyrazoles.12,13 R-Roscovi-
tine 1, a 2,6,9-trisubstituted purine, is one of the most studied. It is
an inhibitor of CDK 1, 2, 5, 7 and 9 isoforms and has been widely
investigated as a potential anticancer drug. The purine portion of
the inhibitor binds to the adenine binding pocket of the enzyme,
even if rotated with respect to the original ATP conformation. The
crystal structure of the CDK2 and R-roscovitine complex is known,
with the drug binding to the ATP binding pocket of the enzyme
through hydrophobic and Van der Waals contacts, as well as hydro-
gen bonds between N(6) and N(7) of the purine ring and Leu83 of the
enzyme. Another hydrogen bond involves O(1) of the aminobutanol
lateral chain that binds to an area occupied by the ribose in the
CDK2/ATP complex.14 Since R-roscovitine is able to enhance neutro-
phil apoptosis it also has a potential application as a pro-resolving
anti-inflammatory agent.9,15 The ability of R-roscovitine to induce
neutrophil apoptosis involves inhibition of CDKs, mainly CDK7 and
CDK9, with consequent down-regulation of the pro-survival protein
Mcl-1.9,11 NO also plays important roles in apoptosis, with it able to
exert either pro-apoptotic or anti-apoptotic effects depending on
the source and concentration of the NO and the cell type involved.16
A number of studies carried out with different NO-donors demon-
strate the capacity of NO to induce apoptosis in neutrophils.17–20
In vivo evidence shows that supplementation of NO may be benefi-
cial in inflammatory diseases with effects mediated by apoptosis of
neutrophils.21 On this basis, we aimed to design, synthesise and
investigate the biological potential of multi-target pro-resolution
of inflammation derivatives based upon combined CDK inhibition
2. Results and discussion
2.1. Chemistry
The pro-drug 2 was easily obtained by coupling 1 with 4-nitro-
oxybutirric acid, under the action of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC), in the presence of catalytic amount of
4-dimethylaminopyridine (DMAP) in pyridine solution (Scheme 1).
All the remaining products were synthesized starting from 6-ben-
zylthio-2-iodo-9-isopropyl-9H-purine 6 (Scheme 2). This interme-
diate was already known in literature, but we prepared it through a
partly modified route. The acetylated ribosyl group present in the
guanosine derivative 3 was removed by the action of sulfuric acid
to afford the already described 6-chloro-2-iodopurine 4. Nucleo-
philic displacement of the chlorine with benzylmercaptane in the
presence of diisopropylethylamine (DIPEA) led to the 2-iodo-6-
benzylthio substituted purine 5. The subsequent reaction with
2-bromopropane in DMSO in the presence of K2CO3 gave the
N(9) regioselective alkylation, with resulting production of 6. This
intermediate, treated in refluxing nBuOH with pure R-(2)-amino-1-
butanol, yielded the expected R-roscovitine thioanalogue 7. This
Scheme 3. Preparation of amino derivatives 12, 14, 16, 18. Reagents and conditions: (i) K2CO3, KI, DMF, CH3CN, 20 h, 50 °C; (ii) TFA 10% CH2Cl2, 3 h, rt; (iii) tBuOꢀK+, dry THF,
4 h, rt ? ; (iv) PPh3, DIAD, dry THF, 12 h, 0 °C ? rt; (v) DBU, CH2Cl2, 12 h, rt.
D

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G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
Figure 1. Induction of neutrophil apoptosis by R-roscovitine and NO-donor R-roscovitine compounds. Representative flow cytometric plot showing forward and side scatter
with neutrophils gated (a), and representative flow cytometric plots of annexin V (x axis)/propidium iodide (y axis) staining after 6 h in culture for control untreated
neutrophils (b), neutrophils cultured with R-roscovitine 3 lM (c) and neutrophils cultured with compound 9a 3 lM (d). For annexin V/PI plots lower left quadrant contains
viable cells (annexin V negative, PI negative), lower right quadrant contains apoptotic cells (annexin V positive, PI negative), upper right quadrant contains necrotic cells (PI
positive). Neutrophil morphology. Representative cyto-centrifuge preparations after staining with Diff-Quick™ (400ꢁ light microscopy) in freshly isolated neutrophils (e),
untreated neutrophils aged for 6 h (f), neutrophils treated with R-roscovitine 3 lM for 6 h (g) and neutrophils treated with compound 9a 3 lM for 6 h (h). Black arrows
indicate apoptotic cells, recognizable by cellular shrinkage and nuclear pyknosis. Contaminating eosinophils (<4%) are also visible, identified by the pink/red staining of their
cytoplasm.
product was oxidized to the corresponding sulfonyl derivative 8
using m-chloroperbenzoic acid (mCPBA). Finally, 8 was treated
with the appropriate benzyl amine derivative (12, 14, 16, 18) in
methanol solution in the presence of Et3N to give the target com-
pounds 9a–d. The preparation of the substituted benzylamines
used in the reaction is outlined in Scheme 3. All the products were
synthesized starting from Boc-protected p-hydroxybenzylamine
10. Compounds 12, 18, were obtained following nucleophilic dis-
placement by 10 of the bromine present in 11 and 17, and subse-
quent Boc-deprotection with trifluoroacetic acid in CH2Cl2. In a
similar manner 14 was prepared using the tosylate 13. Finally
10, treated with 4-hydroxymethyl furoxan-3-carboxyamide (15),
in the presence of PPh3 and diisopropyl azodicarboxylate (DIAD)
in THF solution (Mitsunobu procedure), afforded 16.
2.2. Biological activity
All the NO-donor R-roscovitine derivatives, and the related des-
NO-reference compounds, were tested for their ability to induce
apoptosis in neutrophils isolated from peripheral blood of healthy

G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
2111
Figure 2. R-Roscovitine and NO-donor R-roscovitine compounds induce neutrophil apoptosis in a time-dependent manner. Time course demonstrating the percentage (%) of
viable (solid line, j), apoptotic (dotted line, d) and necrotic (dashed line, N) neutrophils induced by treatment with 10
9a (panel c) and 9c (panel d). Results expressed as mean SEM, n = 3.
lM R-roscovitine (1) (panel a), compounds 2 (panel b),
volunteers. After exposure to the compounds, neutrophil apoptosis
was primarily assessed by flow cytometric analysis of fluorescein
isothiocyanate (FITC)-labeled annexin V and propidium iodide
(PI). This allows discrimination between viable (annexin V and PI
ꢀve), apoptotic (annexin V +ve, PI ꢀve) and necrotic (annexin V
and PI +ve) neutrophils. Representative flow cytometry plots are
shown in Figure 1 for R-roscovitine and compound 9a after 6 h of
culture. All the results obtained by flow cytometry were also con-
firmed by light microscopy, with apoptotic neutrophils demon-
strating cellular shrinkage and nuclear pyknosis (Fig. 1e–h).
Time-course experiments of untreated neutrophils showed that
at 6 h the constitutive rate of apoptosis was low (16.8 1.4%).
Time-course experiments of neutrophils treated with the lead
and the selected compounds 2, 9a and 9c highlighted that all the
newly synthesized compounds maintained a R-roscovitine-like
capability to induce apoptosis over this time period (Fig. 2). More-
over, the same experiments ascertained that secondary as opposed
to primary necrosis occurred after 8 h (Fig. 2). This appearance of
secondary necrosis at late time points is due to the absence of
phagocytes, such as macrophages, being present in the highly pure
population of neutrophils used in our experiments. This is well de-
scribed in the literature24 with the temporal distribution of apop-
tosis occurring prior to the onset of necrosis confirming this as
secondary necrosis.
dose–response profile with respect to R-roscovitine, and, in addi-
tion, 9c is significantly more active than its des-NO furazan refer-
ence. The pro-drug 2 also enhanced neutrophil death by inducing
apoptosis in a concentration-dependent manner, but it was a
slightly less potent than R-roscovitine. This behavior could be ex-
plained by the possibility that the product, over the time of the
experiments, acts largely as an intact drug, which should be less
active than the lead, in spite of the presence of NO-donor moiety,
following the masking of the OH group in the lateral chain. Previ-
ous studies with other NO-donor compounds (SIN-1, GEA-3162)
showed that NO-donor triggered neutrophil apoptosis is cGMP-
independent and involves the concurrent generation of superox-
ide anion Oꢀ2 ꢂ that could rapidly react with NO to form the pow-
erful oxidizing agent peroxynitrite (ONOOꢀ).19 Nevertheless, it is
not possible to exclude a partial involvement of NOS and of the
sGC/cGMP pathway in the regulation of neutrophil apoptosis.
Herein, two inhibitors were used with R-roscovitine (1) and com-
pounds 2, 9a and 9c to examine the role of these NO-related
pathways in the induction of apoptosis. Different concentration
of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO-
synthase (NOS), did not modify the behavior of R-roscovitine,
compounds 2, 9a or 9c, suggesting that their pro-apoptotic effect
is not related to iNOS expression or regulation (Fig. 4a). Similarly,
the prior administration of ODQ (10 lM), a well known inhibitor
Analysis of the concentration–response profiles show that all
the new synthesized compounds 2, 9a–d enhanced neutrophil
death by inducing apoptosis in a concentration-dependent man-
ner (Fig. 3), in the range 0.3–20 lM. Both compounds 9a and
9b were significantly more potent than the lead. Moreover, the
of soluble guanylate cyclase (sGC), did not affect the induction of
apoptosis (Fig. 4b), excluding involvement of sGC, and confirmed
the findings previously reported for other classes of NO-donors.
To further investigate the dependency upon NO release for the
increased apoptosis observed with the NO-donor containing
compounds we co-incubated neutrophils with PTIO, a NO scaven-
ger (Fig. 4c). As expected the presence of PTIO did not affect
constitutive neutrophil apoptosis, gliotoxin-induced apoptosis
organic nitrate 9a was markedly more active than its des-NO
model 9b in the 1–10
NO in its additional pro-apoptotic action. Also the furoxan hybrid
compounds 9c and its furazan analogue 9d show better
lM range, suggesting an involvement of
a
or R-roscovitine-induced apoptosis. However PTIO caused a

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G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
Figure 3. R-Roscovitine and NO-donor R-roscovitine compounds induce neutrophil
apoptosis in a concentration-dependent manner. Percentage of neutrophil death
induced by treatment with R-roscovitine (1), compounds 2 and 9a,b (panel a) and
compounds 9c,d (panel b), normalized with respect to the control cells after 6 h of
culture. All non-viable, annexin V positive neutrophils are included as at 6 h the
percentage of necrotic cells are negligible (Fig. 2). Results expressed as mean SEM,
n = 6–35, analysed by one-way ANOVA with Newman–Keuls post hoc test with.
⁄p <0.05, ⁄⁄p <0.01,⁄⁄⁄p <0.001 with respect to R-roscovitine and #p <0.05, ##p <0.01,
###p <0.001 with respect to the corresponding des-NO derivative.
concentration-dependent inhibition of apoptosis when co-incu-
bated with compound 9a. The rate of apoptosis was not different
(p >0.05) between R-roscovitine and compound 9a in the pres-
ence of >1 mM PTIO, suggesting that the additional apoptosis
induced by compound 9a over R-roscovitine was dependent upon
NO.
3. Conclusion
Basing on the known pro-resolution activity of R-roscovitine,
novel NO-donor R-roscovitine multi-target compounds were pre-
pared with a good yield. The biological results indicate that the
new NO-donor R-roscovitine compounds described in the present
work are endowed with an enhanced roscovitine-like capacity of
inducing neutrophil apoptosis, likely involving inhibition of CDKs
as well as release of NO. In particular, derivatives 9a and 9c were
found to be significantly more biologically active than the lead, an
effect reduced in their des-NO donor derivatives 9b and 9d, or by
co-incubating 9a and 9c with an NO scavenger. This suggests a
crucial involvement of the release of NO from the NO-mimetic
compounds in their enhanced biological activity.
Figure 4. (a and b) Neutrophil apoptosis induced by NO-donor R-roscovitine
compounds is not affected by inhibition of NO-synthase nor soluble guanylate
cyclase. Percentage of neutrophil apoptosis after 6 h of culture using different
concentrations of
L-NAME (panel a) and 10
lM ODQ (panel b), in the presence of R-
roscovitine (1) and compound 2, 9a and 9c (3
l
M and 10 M). Results expressed as
l
mean SEM, n = 3. (c) PTIO and NO scavenging. Percentage of neutrophil apoptosis
after 6 h of culture using different concentrations of PTIO, in the presence of
gliotoxin (1 lg/mL), R-roscovitine (1), compound 2 and 9a (3 lM). Results as
mean SEM, n = 5–6, analysed by one-way ANOVA with Newman–Keuls post hoc
test ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001, and #p <0.05, ##p <0.01, ###p <0.001 with
respect to R-roscovitine in the same conditions.
abbreviations are used to indicate the peak multiplicity: s = singlet,
d = doublet, t = triplet, m = multiplet. Fx = furoxan ring. Fz = fura-
zan ring. Melting points of unpublished solid derivatives were
measured with a capillary apparatus (Buchi B-540). Flash column
chromatography was performed on silica gel (Merck Kieselgel 60,
230–400 mesh ASTM) using the reported eluents. Thin layer chro-
matography (TLC) was carried out on 5 cm ꢁ 20 cm plates (Fluka)
with a 0.2 mm layer thickness. Purity of final compounds was
4. Experimental
4.1. Reagents and general methods
All the compounds were routinely checked by 1H and 13C
NMR (Bruker Avance 300) at 300 and 75 MHz, respectively, and
mass spectrometry (Finnigan-Mat TSQ-700). The following

G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
2113
P95% as detected by RP-HPLC. RP-HPLC analyses were performed
on a HP1100 chromatograph system (Agilent Technologies, Palo
until the pH reached 4.5. The aqueous phase was extracted with
CH2Cl2 (4 ꢁ 50 mL), Et2O (3 ꢁ 50 ml), dried (Na2SO4), filtered and
evaporated in vacuo to obtain a crude yellow solid. The crude
was purified by flash cromatography eluting with CH2Cl2 gradient
to CH2Cl2/MeOH 5% to obtain 9-H-6-chloro-2-iodopurine (4) pure
enough as an intermediate (960 mg). MS/CI (isobutane): [M+1]+
Alto, CA, USA) on
a Nucleosil 100-5C18 Nautilus column
(250 ꢁ 4.6 mm, m, Macherey–Nagel), eluted with CH3CN/
5
l
H2O + 0.1% TFA 1/1 v/v as mobile phase. Compounds were dis-
solved in the mobile phase and eluted at flow rates of
1.0 mL minꢀ1; the column effluent was monitored at 210, 226,
254 nm referenced against 360 nm. Analysis (C, H, N) of the target
compounds was performed by REDOX (Monza). Iscove’s modified
281, 283. Compound
(80 mL). DIPEA (1.17 mL, 6.84 mmol) and benzylmercaptane
(804 L, 6.84 mmol) were added and the mixture was refluxed un-
4 (3.42 mmol) was dissolved in EtOH
l
Dulbecco’s Modified Eagle’s medium (IMDM), PBS without Ca2+
/
der nitrogen atmosphere for 4 h. The solvent was evaporated in va-
cuo, treated with water (30 mL), extracted with AcOEt (3 ꢁ 50 mL)
and the combined organic phases were washed with brine (40 mL),
dried (Na2SO4), filtered and evaporated under reduced pressure.
The crude product was treated with hexane and collected on a
buchner funnel to obtain the desired product (960 mg) as a yellow
solid (overall yield 31%). An analytical sample was obtained by
flash cromatography eluting with CH2Cl2/MeOH 2% to obtain the
title product as a white solid. MS/CI (isobutane): [M+1]+ 369. 1H
NMR (DMSO-d6): d, 13.63 (s br, 1H, NH), 8.40 (s, 1H, H8), 7.51 (d,
Mg2+ and Hank’s balanced salt solution (HBSS) were obtained from
PAA. Sterile water and saline were from Baxter. Bovine
Serum Albumine (BSA), sterile DMSO, sodium citrate tribasic
dihydrate, PBS 10X, N -nitro-
L-arginine methyl ester hydrochlo-
x
ride (L-NAME), and propidium iodide (PI) were purchased from
Sigma. Dextran 500 and Percoll were from GE Helthcare. 1H-
[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) was from To-
cris Bioscience. Fluorescein isothiocyanate (FITC) labeled Annexin
V was from Roche. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-
oxyl-3-oxide (PTIO) was from Enzo Life Sciences.
2H, HAr 2 ,6 ), 7.44–7.23 (m, 3H, HAr 3 ,4 ,5 ), 4.57 (s, 2H, CH2Ph). 1H
0
0
0
0
0
NMR spectrum is in keeping with that reported in literature.28
4.2. Chemistry
4.2.4. 6-Benzythio-2-iodo-9-isopropyl-9H-purine (6)
4.2.1. (2R)-2-(1-Hydroxybut-2-ylamino)-6-benzylamino-9H-
isopropylpurine (1)
Compound 5 (900 mg, 2.44 mmol) was solubilized in DMSO
(20 mL), K2CO3 (1.01 g, 7.32 mmol) and 2-bromopropane (574 lL,
R-Roscovitine (1) was synthesized as reported in literature.25a
MS/CI (isobutane): [M+1]+ 355. 1H NMR (CDCl3): d, 7.37–7.23 (m,
6H, H8 and HAr), 6.35 (s br, 1H, NH(6)), 5.16 (s br, 1H, CH2OH),
4.94 (d br, 1H, CHNH(2)), 4.76 (s, 2H, CH2Ph), 4.64–4.50 (m, 1H,
CH(CH3)2), 3.91–3.87 (m, 1H, CHNH), 3.82–3.78 (m, 1H, CH2OH),
3.65–3.59 (m, 1H, CH2OH), 1.72–1.49 (m, 8H, CH2CH3 and
CH(CH3)2), 1.00 (t, 3H, CH2CH3). 13C NMR (CDCl3): d, 160.0, 154.9,
152, 138.9, 134.6, 128.5, 127.7, 127.3, 114.7, 68.2, 56.2, 46.4,
44.4, 25.0, 22.6, 22.5, 10.9. Spectral data are in agreement with
those reported in literature.25b
6.1 mmol) were added and the mixture was stirred at 50 °C over-
night. The mixture was treated with water (50 mL), extracted with
AcOEt (3 ꢁ 60 mL), dried (Na2SO4), filtered and evaporated in va-
cuo to obtain a yellow oil. The crude product was purified by flash
cromatography eluting with PE/CH2Cl2 1:1 gradient to CH2Cl2 to
obtain the desired product as a white solid (500 mg, 50% yield).
MS/CI (isobutane): [M+1]+ 411. 1H NMR (CDCl3): d, 7.93 (s, 1H,
0
0
0
0
0
H8), 7.51 (d, 2H, HAr 2 ,6 ), 7.34–7.21 (m, 3H, HAr 3 ,4 ,5 ), 4.90–4.81
(m, 1H, CH(CH3)2), 4.56 (s, 2H, CH2Ph), 1.61–1.57 (m, 6H,
CH(CH3)2). 13C NMR (CDCl3): d, 161.5, 148.9, 140.0, 137.2, 131.1,
129.5, 128.4, 127.4, 118.2, 47.5, 33.3, 22.7. 1H NMR and 13C NMR
are identical to the spectra reported in literature.28
4.2.2. (2R)-2-[[6-Benzylamino-9-isopropyl-9H-purin-2-
yl]amino]butyl 4-(nitrooxy)-butanoate (2)
Compound 1 (80 mg, 0.225 mmol) was solubilized in dry pyri-
dine (10 mL). 4-Nitrooxybutirric acid26 (74 mg, 0.495 mmol), EDC
(52 mg, 0.27 mmol) and DMAP (cat.) were added and the mixture
was stirred at room temperature for 4 h. The mixture was treated
with HCl 1 N, extracted with CH2Cl2 (3 ꢁ 15 mL) and the combined
organic phases were washed with HCl 1 N (3 ꢁ 10 mL), brine
(20 mL), dried (Na2SO4), filtered and evaporated in vacuo. The
crude residue was purified by flash cromatography eluting with
CH2Cl2 gradient to CH2Cl2/MeOH 1% to obtain the pure product
as a yellow oil (50 mg, yield 48%). MS/CI (isobutane): [M+1]+ 486.
1H NMR (CDCl3): d, 7.36 (s, 1H, H8), 7.33–7.21 (m, 5H, HAr), 6.47
(s br, 1H, NH(6)), 4.80–4.77 (m, 3H, CHNH(2) and CH2Ph), 4.65–
4.55 (m, 1H, CH(CH3)2), 4.44 (t, 2H, CH2ONO2), 4.24–4.16 (m, 3H,
CH2O(CO) and CHNH), 2.41 (t, 2H, CH2(CO)O), 2.04–1.97 (m, 2H,
CH2CH2CH2), 1.68–1.49 (m, 8H, CH2CH3 and CH(CH3)2), 0.97 (t,
3H, CH2CH3). 13C NMR (CDCl3): d, 172.3, 160.0, 154.9, 151.0,
139.4, 134.6, 128.5, 127.6, 127.1, 114.6, 72.0, 66.2, 51.5, 46.2,
44.3, 30.1, 24.9, 22.58, 22.57, 22.2, 10.6. HPLC purity (CH3CN/
H2O + 0.1% TFA, 1/1): 99%. Elemental analysis for C23H31N7O5. Cal-
culated: C, 56.90; H, 6.44; N, 20.19. Found: C, 57.49; H, 6.46; N,
19.99.
4.2.5. (2R)-2-[[6-Benzylthio-9-isopropyl-9H-purin-2-yl]amino]-
butanol (7)
Compound 6 (0.5 g, 1.22 mmol) was suspended in n-butanol
(20 mL). (R)-2-Amino-1-butanol was added (434 mg, 4.88 mmol)
and the reaction was refluxed for 5 days. The solvent was evapo-
rated in vacuo using an oil pump and the crude residue was puri-
fied by flash cromatography eluting with CH2Cl2 gradient to
CH2Cl2/MeOH 1.5% to obtain pure product as a colourless oil
(340 mg, yield 75%). MS/CI (isobutane): [M+1]+ 372. 1H NMR
0
0
(CDCl3): d, 7.67 (s, 1H, H8), 7.43 (d, 2H, HAr 2 ,6 ), 7.29–7.26 (m,
0
0
0
3H, HAr 3 ,4 ,5 ), 5.20 (d br, 1H, CH2OH), 4.70–4.63 (m, 1H, CH(CH3)2),
4.56 (s, 2H, CH2Ph), 4.02–3.90 (m, 1H, CHNH), 3.81 (m, 1H, CH2OH),
3.69 (m, 1H, CH2OH), 1.80–1.53 (m, 8H, CH2CH3 and CH(CH3)2),
1.02 (t, 3H, CH2CH3). 13C NMR (CDCl3): d, 160.9, 158.7, 149.8,
137.6, 137.4, 129.0, 128.4, 127.1, 125.5, 66.8, 55.9, 46.8, 32.7,
24.80, 22.45, 22.39, 10.8.
4.2.6. (2R)-2-[[6-Benzylsulfonyl-9-isopropyl-9H-purin-2-
yl]amino]-butanol (8)
Derivative 7 (170 mg, 0.45 mmol) was solubilized in CH2Cl2
(10 mL), the mixture was cooled at 0 °C and m-CPBA was added
(340 mg, 1.35 mmol). The mixture was stirred at room tempera-
ture for 3 h, then diluted with water. The two layers were divided
and the water phase was extracted with fresh CH2Cl2 (15 mL). The
combined organic phases were washed with NaHCO3 5%
(2 ꢁ 10 mL), water (10 mL), brine (10 mL), dried (Na2SO4), filtered
and evaporated in vacuo. The crude product was purified eluting
with CH2Cl2 gradient to CH2Cl2/MeOH 2% to obtain the title
4.2.3. 6-Benzylthio-2-iodo-9H-purine (5)
9-(20,30,50-Tri-O-acetyl-b-
D-ribofuranosyl)-6-chloro-2-iodopu-
rine 327 (4.42 g, 8.20 mmol) was suspended in water (25 mL) and
EtOH (50 mL), then H2SO4 conc was added (2.5 mL) and the mix-
ture was refluxed for 3 h. The mixture was concentrated in vacuo
to a volume of approximatively 30 mL and NaOH 10% was added

2114
G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
product as a yellow oil (82 mg, yield 45%). Derivative 8 was stored
at ꢀ18 °C or immediately reacted to avoid a possible decomposi-
tion. MS/CI (isobutane): [M+1]+ 404. 1H NMR (CDCl3): d, 7.96
(s, 1H, H8), 7.74–7.26 (m, 5H, HAr), 5.95 (d br, 1H, NH(2)), 4.84 (s,
2H, CH2Ph), 4.79–4.68 (m, 1H, CH(CH3)2), 4.04 (s br, 1H, CHNH),
3.82–3.80 (m, 1H, CH2OH), 3.72–3.66 (m, 1H, CH2OH), 1.76–1.58
(m, 8H, CH2CH3 and CH(CH3)2), 0.99 (t, 3H, CH2CH3). 13C NMR
(CDCl3): d, 158.5, 156.0, 153.3, 142.3, 131.7, 129.6, 129.0, 126.6,
123.3, 64.2, 59.4, 53.5, 47.4, 24.3, 22.2, 22.0, 10.7.
4.2.7.4.
isopropyl-9H-purin-6-yl]amino]-benzyl]phenoxy]methyl]fura-
zan-3-carboxamide (9d). White foam (yield 49%). MS/CI (iso-
butane): [M+1]+ 496. 1H NMR (CDCl3 + CD3OD): d, 7.67 (s, 1H, H8),
4-[[4-[[2-[[(1R)-1-(Hydroxymethyl)propyl]amino]-9-
0
0
0
0
7.30 (d, 2H, HAr 3 ,5 ), 6.94 (d, 2H, HAr 2 ,6 ), 5.45 (s, 2H, Fz-CH2OPh),
4.72–4.56 (m, 3H, CH2Ph and CH(CH3)2), 4.00–3.93 (m, 1H, CHNH),
3.67–3.65 (m, 2H, CH2OH), 1.73–1.51 (m, 8H, CH2CH3 and
CH(CH3)2), 0.97 (t, 3H, CH2CH3). 13C NMR (CD3OD): d, 160.6,
160.3, 157.9, 155.5, 153.0, 151.2, 149.1, 135.7, 133.5, 129.6,
115.6, 114.3, 65.6, 60.5, 55.6, 47.3, 44.2, 25.2, 22.6, 22.5, 11.0. HPLC
purity (CH3CN/H2O + 0.1% TFA, 1/1): 95%. Elemental analysis for
4.2.7. General procedure for preparation of 9a–d
Compound 8 (107 mg, 0.265 mmol) was solubilized in EtOH
C
23H29N9O4ꢂ1/4H2O. Calculated: C, 55.24; H, 5.95; N, 25.21. Found:
(10 mL). Triethylamine (111
lL, 0.795 mmol) and the correct ami-
C, 55.52; H, 5.97; N, 25.46.
no derivatives (12, 14, 16 or 18) (1.5 equiv) were added, and the
mixture was stirred at 40 °C for 5 days. The solvent was evaporated
in vacuo, dissolved in CH2Cl2 (30 mL), washed with water
(2 ꢁ 20 mL), dried (Na2SO4), filtered and evaporated under reduced
pressure. The crude residue was purified by flash cromatography
eluting with CH2Cl2/AcOEt 6:4 gradient to 1:9 to obtain pure prod-
ucts. Derivatives 9a–d were stored at ꢀ18 °C.
4.2.8. 3-[4-(Aminomethyl)phenoxy]propyl nitrate (12)
tert-Butyl-N-(4-hydroxybenzyl)carbamate
1029
(730 mg,
3.27 mmol) was solubilized in CH3CN (20 mL), K2CO3 (565 mg,
4.09 mmol), 3-nitrooxypropyl bromide (11)30 (0.5 g, 2.73 mmol),
KI (cat.) and DMF (0.5 mL) were added. The mixture was stirred
at 50 °C for 20 h, evaporated under reduced pressure, treated with
water (20 mL), extracted with CH2Cl2 (3 ꢁ 30 ml) and the com-
bined organic phases were washed with brine (35 mL), dried
(Na2SO4), filtered and evaporated. The crude product was purified
by flash cromatography eluting with PE/iPrOH 1% to obtain tert-bu-
tyl-(4-(3-nitrooxy)propoxybenzyl)carbamate (510 mg). This prod-
uct (1.56 mmol) was then solubilized in CH2Cl2 (15 mL).
Trifluoroacetic acid was added (1.5 mL) and the mixture was stir-
red at room temperature for 3 h. The solvent was evaporated in va-
cuo, cooled at 0 °C and NaHCO3 10% was added. The mixture was
stirred for 10 min, extracted with CH2Cl2 (3 ꢁ 20 ml) and the com-
bined organic phases were washed with water (2 ꢁ 15 mL), brine
(20 mL), dried (Na2SO4), filtered and evaporated. The crude residue
was purified by flash cromatography eluting with CH2Cl2 gradient
to CH2Cl2/MeOH 5% to obtain the pure product as a colourless oil
(160 mg, overall yield 30%). MS/CI (isobutane): [M+1]+ 227. 1H
4.2.7.1. (2R)-2-[[9-Isopropyl-6-[[4-[3-(nitrooxy)propoxy]benzyl]
amino]-9H-purin-2-yl]amino]-butanol (9a).
Pale yellow oil
(yield 40%). MS/CI (isobutane): [M+1]+ 474. 1H NMR (CDCl3): d,
0
0
0
0
7.36 (s, 1H, H8), 7.25 (d, 2H, HAr 2 ,6 ), 6.79 (d, 2H, HAr 3 ,5 ), 6.54
(s br, 1H, NH(6)), 5.20 (s br, 1H, OH), 5.02 (d br, 1H, NH(2)),
4.66–4.60 (m, 4H, CH2Ph and CH2ONO2), 4.58–4.54 (m, 1H,
CH(CH3)2), 4.04 (t, 2H, CH2OPh), 3.92–3.87 (m, 1H, CHNH),
3.82–3.77 (m, 1H, CH2OH), 3.65–3.59 (m, 1H, CH2OH), 3.21–3.13
(m, 2H, CH2CH2CH2), 1.64–1.47 (m, 8H, CH2CH3 and CH(CH3)2),
0.99 (t, 3H, CH2CH3). 13C NMR (CDCl3): d, 160.0, 157.8, 154.8,
150.2, 134.4, 131.5, 129.0, 114.7, 114.4, 70.0, 67.8, 63.5, 56.1,
46.3, 43.7, 26.9, 24.9, 22.7, 22.5, 10.9. HPLC purity (CH3CN/
H2O + 0.1% TFA, 1/1): 98%. Elemental analysis for C22H31N7O5ꢂ1/
2H2O. Calculated: C, 54.76; H, 6.68; N, 20.32. Found: C, 55.14; H,
6.53; N, 20.10.
0
0
0
0
NMR (CD3OD): d, 7.23 (d, 2H, HAr 3 ,5 ), 6.84 (d, 2H, HAr 2 ,6 ), 4.66
(t, 2H, CH2ONO2), 4.04 (t, 2H, CH2OPh), 3.79 (s, 2H, PhCH2), 2.24–
2.15 (m, 2H, CH2CH2CH2). 13C NMR (CD3OD): d, 157.5, 135.2,
128.5, 114.5, 70, 63.6, 45.8, 27. This intermediate was not further
characterized but reacted immediately.
4.2.7.2.
benzyl]amino]-9H-purin-2-yl]amino]-butanol
yellow oil (yield 42%). MS/CI (isobutane): [M+1]+ 443. 1H NMR
(2R)-2-[[9-Isopropyl-6-[[4-[3-(methoxy)propoxy]
(9b). Pale
0
0
(CDCl3): d, 7.23–7.20 (m, 4H, H8, HAr 2 ,6 and NH(6)), 6.78 (d, 2H,
0
0
H
Ar 3 ,5 ), 5.80 (s br, 1H, OH), 5.26 (d br, 1H, NH(2)), 4.64 (s br, 2H,
4.2.9. 4-(3-Methoxypropoxy)benzylamine (14)
CH2Ph), 4.56–4.47 (m, 1H, CH(CH3)2), 4.00 (t, 2H, CH2OPh), 3.93–
3.91 (m, 1H, CHNH), 3.77–3.73 (m, 1H, CH2OH), 3.64–3.61 (m,
1H, CH2OH), 3.51 (t, 2H, CH2OCH3), 3.31 (s, 3H, OCH3), 2.04–1.96
(m, 2H, CH2CH2CH2), 1.62–1.41 (m, 8H, CH2CH3 and CH(CH3)2),
0.96 (t, 3H, CH2CH3). 13C NMR (CDCl3): d, 159.9, 158.1, 154.8,
150.3, 134.3, 131.2, 129.2, 114.7, 113.9, 69.2, 66.5, 64.7, 58.6,
55.6, 46.2, 43.6, 29.5, 24.7, 22.6, 22.3, 10.9. HPLC purity (CH3CN/
H2O + 0.1% TFA, 1/1): 99%. Elemental analysis for C23H34N6O3. Cal-
culated: C, 62.42; H, 7.74; N, 18.99. Found: C, 62.48; H, 7.78; N,
18.85.
Compound 10 (914 mg, 4.09 mmol) was solubilized in dry THF
(30 mL). tBuOꢀK+ (460 mg, 4.09 mmol) was added and the mixture
was stirred at room temperature for 10 min. 3-Methoxypropyl tos-
ylate (13)31 (1.0 g, 4.09 mmol) was solubilized in dry THF (5 mL)
and added to the mixture that was refluxed for 4 h. The solvent
was evaporated under reduced pressure, and the residue was dis-
solved with water (40 mL), extracted with CH2Cl2 (3 ꢁ 30 mL),
and the combined organic phases were washed with brine
(45 mL), dried (Na2SO4), filtered and evaporated in vacuo. The
crude product was purified by flash cromatography eluting with
PE/iPrOH 4% to obtain tert-butyl-(4-(3-methoxy)propoxyben-
zyl)carbamate (1.10 g), enough pure to be used in the next reaction
step. The product (3.72 mmol) was solubilized in CH2Cl2 (40 mL).
Trifluoroacetic acid was added (4 mL) and the mixture was stirred
at room temperature for 3 h. The solvent was evaporated in vacuo,
and NaOH 10% was added. The aqueous phase was extracted with
CH2Cl2 (3 ꢁ 20 mL) and the combined organic phases were washed
with water (2 ꢁ 15 mL), brine (20 mL), dried (Na2SO4), filtered and
evaporated. The crude residue was purified by flash cromatogra-
phy eluting with CH2Cl2 gradient to CH2Cl2/MeOH 5% to obtain
the title product as a colourless oil (480 mg, overall yield 60%).
4.2.7.3.
isopropyl-9H-purin-6-yl]amino]-benzyl]phenoxy]methyl]furo-
xan-3-carboxamide (9c). Yellow solid (yield 50%). MS/CI
(isobutane): [M+1]+ 512. Mp 136 °C dec. 1H NMR (CD3OD): d,
4-[[4-[[2-[[(1R)-1-(Hydroxymethyl)propyl]amino]-9-
0
0
0
0
7.76 (s, 1H, H8), 7.31 (d, 2H, HAr 3 ,5 ), 6.98 (d, 2H, HAr 2 ,6 ), 5.38
(s, 2H, Fx-CH2OPh), 4.67–4.60 (m, 3H, CH2Ph and CH(CH3)2), 3.94
(m, 1H, CHNH), 3.61 (m, 2H, CH2OH), 1.70–1.52 (m, 8H, CH2CH3
and CH(CH3)2), 0.96 (t, 3H, CH2CH3). 13C NMR (CD3OD): d, 161.2,
158.6, 158.1, 156.6, 156.0, 152.1, 136.4, 134.3, 130.0, 116.1,
114.6, 111.8, 65.5, 62.7, 55.9, 47.9, 44.5, 25.5, 22.7, 22.6, 11.1. HPLC
purity (CH3CN/H2O + 0.1% TFA, 1/1): 95%. Elemental analysis for
1H NMR (CD3OD): d, 7.26 (d, 2H, HAr 2 ,6 ), 6.88 (d, 2H, HAr 3 ,5 ),
4.03–3.97 (m, 2H, CH2OPh), 3.79 (s, 2H, PhCH2), 3.55–3.51 (m,
2H, CH2OCH3), 3.32 (s, 3H, OCH3), 2.03–1.95 (m, 2H, CH2CH2CH2).
0
0
0
0
C
23H29N9O5ꢂ1/4H2O. Calculated: C, 53.53; H, 5.76; N, 24.42. Found:
C, 53.65; H, 5.72; N, 24.10.

G. Montanaro et al. / Bioorg. Med. Chem. 21 (2013) 2107–2116
2115
This intermediate was not further characterized but reacted
immediately.
(55:70) and granulocytes at the lower boundary (70:81). Granulo-
cytes were collected and washed two times with PBS (without
cations).
4.2.10. 4-[[4-(Aminomethyl)phenoxy]methyl]furoxan-3-
carboxamide (16)
Compound 10 (500 mg, 2.23 mmol) was solubilized in dry THF
(25 mL). CAS 1609 (15) (534 mg, 3.34 mmol), produced by Cassel-
la–Hoechst Co., and PPh3 (1.17 g, 4.46 mmol) were added. The mix-
Neutrophils (>96% purity, with 1–4% contaminating eosino-
phils) were re-suspended at 5 ꢁ 106/mL in IMDM containing pen-
icillin
(100 U/mL)
and
streptomycin
(100
l
g/mL)
and
supplemented with 0.1% w/v bovine serum albumin (BSA).32 Cells
were cultured in flat-bottomed flexible 96-well Falcon polypropyl-
ture was cooled at 0 °C, DIAD (877
lL, 4.46 mmol) was solubilized
ene plates (final volume 150 lL each well) in a 5% CO2 atmosphere
in dry THF (2 mL) and added dropwise. The mixture was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was solubilized in CH2Cl2
(20 mL), cooled at 0 °C, added with trifluoroacetic acid (2 mL)
and stirred at room temperature for 3 h. The solvent was evapo-
rated and the residue treated with water (45 mL) and extracted
with AcOEt (2 ꢁ 50 mL). The water phase was added with Na2CO3
10% and newly extracted with AcOEt (4 ꢁ 40 mL). The combined
organic phases were dried (K2CO3), filtered and evaporated in va-
cuo. The crude product was purified by flash chromatography elut-
ing with CH2Cl2/MeOH 5% to obtain pure product as a yellow solid
(400 mg, yield 67%). MS/CI (isobutane): [M+1]+ 265. Mp 145 °C dec.
at 37 °C and treated in duplicate with the corresponding com-
pounds, added immediately prior of the addition of cells. In the
experiments with the inhibitors L-NAME and ODQ, the cells were
pre-incubated for 15 min in the presence of the inhibitor prior to
the addition of the other tested compounds, to avoid a possible
interference of endogenous NOS and sGC.33
4.3.2. Preparation of the drugs
All tested compounds were prepared in a 50 mM stock solution
in sterile DMSO and diluted daily in IMDM as necessary. L-NAME
and PTIO solution was prepared directly in IMDM. ODQ was pre-
pared in a NaOH 0.01% 10 mM stock solution prior to the correct
dilution in IMDM. The percentage of DMSO in the final cell culture
was within 0.04–0.12%.
1H NMR (CD3OD): d, 7.29 (d, 2H, HAr 3 ,5 ), 7.01 (d, 2H, HAr 2 ,6 ), 5.40
(s, 2H, Fx-CH2OPh), 3.75 (s, 2H, CH2Ph). This intermediate was not
further characterized but reacted immediately.
0
0
0
0
4.3.3. Assessment of neutrophil apoptosis
4.2.11. 4-[(4-(Aminomethyl)phenoxy)methyl]furazan-3-
carboxamide (18)
Neutrophil apoptosis was primarily assessed by flow cytometry
using a FACScan (Becton Dickinson) as previously described.24 An-
nexin V was diluted 1/500 in the binding buffer (2.5 mL of 1 M
Compound 10 (500 mg, 2.23 mmol) was solubilized in dry
CH2Cl2 (50 mL), DBU (490
l
L, 3.34 mmol) and 4-(bromomethyl)-
CaCl2 in 500 mL HBSS) and 280 lL added to 20 lL of cells
1,2,5-oxadiazole-3-carboxamide (17) (460 mg, 2.23 mmol) were
added. The mixture was stirred at room temperature overnight,
cooled at 0 °C, added with trifluoroacetic acid (5 mL) and stirred
at room temperature for 1 h. The solvent was evaporated under re-
duced pressure and the residue treated with water (40 mL) and ex-
tracted with AcOEt (2 ꢁ 50 mL). The water phase was added with
Na2CO3 10% and extracted with fresh AcOEt (4 ꢁ 40 mL). The com-
bined organic phases were washed with brine (60 mL), dried
(Na2SO4), filtered and evaporated in vacuo. The crude residue
was purified by flash cromatography eluting with CH2Cl2/MeOH
5% to obtain pure product as a pale yellow oil (350 mg, yield
63%). MS/CI (isobutane): [M+1]+ 249. 1H NMR (CD3OD + d6-DMSO):
(5 ꢁ 106/mL). Samples were then incubated on ice for 10 min pro-
tecting from light. Prior to analysis on the flow cytometer, 1 lL of
PI (1 mg/mL) per sample was added. Data from the flow cytometry
experiments were collected with Cell Quest Software and analysed
with Flow Jo (Tree Star Inc., version 7.6). Statistical analysis was
performed using Prism 5 (GraphPad Software, Inc., version 5.00).
Apoptosis was also confirmed by cyto-centrifuging 80 lL of
5 ꢁ 106/mL cells (300 rpm, 3 min), fixing the cells on the slide for
2 min in methanol and staining with Diff-Quick™ (Gamidor).
Apoptotic morphology was assessed at 40ꢁ and 100ꢁ objective
on a light microscope using standard criteria.
0
0
0
0
d, 7.27 (d, 2H, HAr 3 ,5 ), 7.00 (d, 2H, HAr 2 ,6 ), 5.49 (s, 2H, Fz-CH2OPh),
4.58 (s br, 2H, CH2NH2), 3.72 (s, 2H, CH2Ph). This intermediate was
not further characterized but reacted immediately.
Acknowledgment
This work was supported in part by a Grant of the University of
Torino 2008.
4.3. Neutrophil culture and assessment of apoptosis
References and notes
4.3.1. Cells isolation and culture
Neutrophils were isolated from peripheral blood of healthy
volunteers as described;15 ethics approval was obtained from
the Lothian Research Ethics Committee (approval no. 08/S1103/
38). Blood (40 mL aliquots) was collected into 4 mL sodium cit-
rate 3.8% (3.8 g in 100 mL sterile water), mixed by gentle inver-
sion and centrifugation (350g, 20 min). Platelet rich plasma
(PRP) was removed and 6 mL of dextran 6% (6 g in 100 mL 0.9%
NaCl) was added, together with pre-warmed sterile saline up to
50 mL. After 30 min sedimentation, the upper layer (containing
leukocytes) was separated and washed with saline. Percoll™
(27 mL) was made isotonic with PBS (without cations) 10ꢁ
(3 mL) and different Percoll™ solutions in PBS (81%, 70% and
55%) were then prepared. A discontinuous gradient was made
up, carefully overlayering 3 mL of each Percoll™ solutions and
resuspending the leukocyte pellets in the 55% layer. Leukocytes
were then divided into the different populations and separated
from the residual erythrocytes by centrifugation (720g, 20 min):
mononuclear cells were retained at the upper gradient boundary
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