NO-donors. Part 171: Synthesis and antimicrobial activity of novel ketoconazole-NO-donor hybrid compounds

Article abstract of DOI:10.1016/j.bmc.2008.05.008

Novel hybrid compounds combining the antifungal drug ketoconazole with a diazen-1-ium-1,2-diolate or an organic nitrate moiety and the corresponding NO-donors without ketoconazole were synthesized and their activities against a broad variety of fungal strains were tested. Hybridization modifies the spectrum of antimicrobial activities and generally, the ketoconazole-NO-donor hybrids are more potent than ketoconazole. The NO-donors alone show insufficient effectiveness.

Full text of DOI:10.1016/j.bmc.2008.05.008

Bioorganic & Medicinal Chemistry 16 (2008) 8294–8300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
NO-donors. Part 171: Synthesis and antimicrobial activity of novel
ketoconazole–NO-donor hybrid compounds
Joerg Konter a, Ute Möllmann b, Jochen Lehmann a,
*
a Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
b Hans Knöll-Institute for Natural Products Research, Beutenbergstr. 11a, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel hybrid compounds combining the antifungal drug ketoconazole with a diazen-1-ium-1,2-diolate or
an organic nitrate moiety and the corresponding NO-donors without ketoconazole were synthesized and
their activities against a broad variety of fungal strains were tested. Hybridization modifies the spectrum
of antimicrobial activities and generally, the ketoconazole–NO-donor hybrids are more potent than keto-
conazole. The NO-donors alone show insufficient effectiveness.
Received 29 January 2008
Revised 29 April 2008
Accepted 5 May 2008
Available online 7 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Ketoconazole
NO-donors
NONOates
Diazeniumdiolates
Nitric oxide
Hybrids
1. Introduction
ture, pH and structure dependent manner with half-lives reaching
from a few seconds to many hours, depending on the compound
Ketoconazole (1a, KT) is a synthetic broad spectrum antifungal
drug that was released in the early 1980s and is used to prevent
and treat mycotic infections systemically, but also externally.
Due to its variable bioavailability, liver toxicity and inhibition of
steroid biosynthesis, the role of KT as an orally administered drug
has been limited to few indications such as candida-oesophagitis of
patients with acquired immune deficiency syndrome or to sup-
press glucocorticoid synthesis in the treatment of Cushing’s dis-
ease, for example.2 Pharmacodynamically, all azole-antimycotics
prevent the fungal synthesis of ergosterole, which is a major con-
stituent in fungal cell membranes, by enzyme inhibition of the
cytochrome P450 dependent 14-a-demethylase.2 The inhibitory ef-
fect of KT against bacterial growth in mycobacteria and streptomy-
cetes is based on the interaction with cytochrome P450 as well.3
Previous studies have demonstrated the antimicrobial activity of
endogenously derived nitric oxide (NO) from the inducible NO
synthaseisoform(iNOS)aswellasfromexogenouslyappliedNO-do-
nors.4–8 Nitrogen bound diazen-1-ium-1,2-diolates (diazeniumdio-
lates, NONOates, ‘solid NO’) represent a commonly used tool in
nitric oxide related research and are NO-donating compounds with
the general structure R1R2N[N(O)NO]À. The predictable and exclu-
sive release of the reactive nitrogen species (RNS) NOÅ in a tempera-
used, are the most mentionable advantages of this class of NO-do-
nor.9–12 In order to prevent the immediate decomposition when dis-
solved in aqueous solution and further modulate the liberation of
NO, NONOates can be derivatized to prodrugs that require enzy-
matic and/or chemical activation to yield in the corresponding NO-
releasing diazeniumdiolate. Recent reports show that not only the
NONOate DETA/NO itself exerts inhibitory effects against Candida
species, but also in synergy when applied in combination with azole
antimycotics such as KT, fluconazole and miconazole.13
Contrary to NONOates, organic nitrates require reduction of the
nitrogen in order to deliver NO. Glutathione reductase, cytochrome
P450, xanthine oxidoreductase and mitochondrial aldehyde dehy-
drogenase were recognized to be mediators in the bioactivation of
organic nitrates.14–18 Furthermore, organic nitrates are supposed to
induce NO-like biological effects without generating free NO radi-
cals and may be addressed as NO-mimetics rather than NO-donors.
Therapeutically, organic nitrates are used for prophylaxis and
acute treatment of angina pectoris, whereas most of the nitrate hy-
brid drugs introduced up to now possess a dihydropyridine or
NSAID structure as a second active principle.19,20
We now report on the synthesis of a series of phenylpipera-
zines—which is a part of the ketoconazole (KT) structure—and
ketoconazole derivatives bearing
a diazen-1-ium-1,2-diolate
or an organic nitrate moiety and the NO-liberation profile
of the NONOate compounds. Furthermore, the NO-donors and
* Corresponding author.
E-mail address: j.lehmann@uni-jena.de (J. Lehmann).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.008

J. Konter et al. / Bioorg. Med. Chem. 16 (2008) 8294–8300
8295
KT–NO-donor hybrid compounds were tested for their in vitro
antimicrobial activity against a series of fungal strains by measur-
ing the minimum inhibitory concentration (MIC).
While the metabolism of 4a/4b in vitro is enzyme related, com-
pounds 5a/5b can be hydrolyzed chemically to the respective
NONOate, formaldehyde and methanol.
The NONOate derivatives 6a/6b, resulting from the acylation of
2a/2b with chloroacetyl chloride followed by the reaction with so-
dium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO), are
reliant on bond cleavage as well in order to produce the NO-liber-
ating NONOate and the hydroxyacetamide derivative. The half-life
of the resulting PYRRO/NO is approximately t1/2 = 3–5 s under
physiologic conditions.24 In contrast to 4a/4b and 5a/5b, this type
of prodrug synthesis strategy advantageously allows the design of
drugs with any kinetic of NO release just by appropriate modifica-
tion on the NONOates structure. For instance, the NO release from
6a/6b can be slowed down easily by changing the amine backbone
linked to the diazeniumdiolate group to a bulky alkylamine or
polyamine.12,23 Additionally, the amide carbonyl function attached
to the phenylpiperazine moiety yields in a more KT-like structure.
The organic nitrates 7a/7b differ from 8a/8b only through the
carbonyl group, introduced by acylation of 2a/2b with carbonyldi-
imidazole (CDI) activated nitratoacetic acid, whereas 8a/8b was
prepared by alkylation of 2a/2b with 2-bromoethyl nitrate. These
two sets of organic nitrates were designed to assess the relevance
of the amide moiety in association with the NO mimetic group.
Furthermore, the nitratopivalinic acid amides 9a/9b were synthe-
sized to recognize the effect of structural changes within the or-
ganic nitrate moiety on the biological activities.
2. Methods
2.1. Chemistry
For the conversion of KT (1a) into a NO-donating hybrid com-
pound, we focused on the phenylpiperazine substructure as
pointed out in Scheme 1. In order to make 1a accessible for deriv-
atization, the acetyl residue was cleaved from the phenylpiper-
azine moiety with NaOH solution. As mentioned above, the time
profile of NO liberation from NO-donors (e.g., NONOates) depends
significantly on their individual chemical structure. To differentiate
whether biologic effects rise from the released NO or from the syn-
ergy of KT and NO, all of the chemical modifications of the deacet-
ylated KT (2a) were performed with phenylpiperazine (2b) as well.
The sodium salt NONOates 3a and 3b were produces by expos-
ing the respective amines to 5 bar of NO under exclusion of oxygen
and water in 1:1 methanol/THF for 3a and dry methanol for 3b.21
We used the NOtizer apparatus as described previously.12,22 The
half-lives (t1/2) of the NONOates were determined both via UV-
spectroscopy monitoring decomposition of the diazeniumdiolate
group and by direct NO measurement by using a nitric oxide sen-
sitive electrode as reported previously.12,23 The half-life of NO re-
lease for 3a/3b was approximately t1/2 = 4.5 min with no
significant difference in kinetics. The alkylation of 3a/3b with bro-
moethane and chlormethyl methylether yielded 4a/4b and 5a/5b,
respectively, which do not release NO until the residues attached
to the O2-atom of the diazen-1-ium-1,2-diolate group are cleaved.
2.2. Biological testing
Antifungal activities of the compounds were studied qualita-
tively by agar diffusion tests according to the literature25,26 and
O
N
N
O
O
H3C
Cl
H
O
H
R
N
i
1a
Cl
2a,2b
N
O-
v
vi
O
vii
viii
ii
N+
R
O
N
O
iii
iv
R
O-
N
O
O-
H3C
4a,4b
O
N+
R
R
N+
N
R
O-
N+
N
R
+ O-
CH3
Na
O2NO
N
O2NO
7a,7b
R
O2NO H3C
3a,3b
O
6a,6b
8a,8b
9a,9b
O
5a,5b
H3C
N
N
O
N
N
2-9a: R =
2-9b: R =
O
Cl
H
N
O
N
Cl
Scheme 1. Reagents and conditions: (i) 20% aq NaOH, 80 °C, 24 h; (ii) 5 bar NO, neq CH3ONa, rt; (iii) bromoethane, 72 h; (iv) chlormethyl methylether, 72 h; (v) 1—
chloroacetyl chloride, 4 °C, 2—PYRRO/NO, rt, 72 h; (vi) CDI activated nitratoacetic acid, rt, 24 h; (vii) 2-bromoethyl nitrate, rt, 72 h; (viii) CDI activated nitratopivalinic acid, rt,
24 h.

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J. Konter et al. / Bioorg. Med. Chem. 16 (2008) 8294–8300
quantitatively by determination of minimal inhibitory concentra-
tions (MIC) according to the NCCLS guidelines using the broth mic-
amine equivalent to structure 2a. Obviously the NO released from
3a is beneficial for the antifungal activity since 3a shows higher
activity than 2a, but not synergistic enough to pursue 1a. Synthe-
sizing diazeniumdiolate-prodrugs 4a and 5a with an ethyl- and
methoxymethyl moiety bound to the O2-atom of the NONOate-
group, respectively, enhances the antifungal properties tremen-
dously compared to the sodium salt NONOate 3a. Both of these
compounds are more active than KT, especially against the tested
Aspergillus strains, but no significant differences were found
against C. albicans. A possible explanation is that this species lacks
the means necessary to metabolize 4a and 5a into its active NO-do-
nor. No difference in efficacy is observed when comparing com-
pound 6a to KT. Cleavage of 6a results, amongst other, in the fast
releasing NONOate PYRRO/NO with t1/2 = 3 s for NO release. Per-
haps the low synergistic effect of this KT–NO-donor hybrid is due
to lower permeability through membranes compared to the
NONOate-prodrugs 4a/5a or maybe due to the very short half-life
of the NO-donor.
The synergistic antimicrobial effects of organic nitrates with KT
have not been investigated before. Looking at this class of NO-do-
nors it is difficult to distinguish if the observed activity results from
the synergy of KT with NO, with another nitrogen species or results
from changes in a receptor-related interaction. The organic nitrates
7a and 9a, which are bound to the KT structure as amides, showed
to be more potent than KT alone, especially compound 9a against
the tested Aspergillus strains. It can be recognized clearly that the
spectrum of antifungal activity of the tested KT–NO-donors is dif-
ferent from the spectrum of KT, mainly resulting in a higher po-
tency against Aspergillus species. Unexpectedly, the alkyl nitrate
residue incorporated in compound 8a turned out to reduce the
activity, suggesting that the combination of amide linkage and or-
ganic nitrate is obligatory for antifungal properties.
The NO-donor compounds 2b–9b lacking the KT structure
showed no inhibition of test organism growth in all agar diffusion
tests on a broad spectrum of bacterial and fungal strains at a con-
centration of 0.2 mM. The NONOate DETA/NO has been previously
reported to have a MIC-value of about 2000 lg/mL (12.5 mM) on
Candida species, DPTA/NO even up to 5000 lg/mL (26.2 mM).13,30
Therefore, as expected, the NONOate, NONOate-prodrugs and or-
ganic nitrates derived from phenylpiperazine (2b–9b) showed no
reckonable antimicrobial activity at a concentration of 2000 lM if
applied exclusively. This demonstrates the necessity of the anti-
fungal KT-backbone for synergistic effects with NO-donor moieties.
The difference in antimicrobial activity of the KT–NO-donors in
comparison to KT can be explained through several possible mech-
anisms that can vary in the respective pathogen species. For in-
stance, from a chemical point of view the entry of the hybrids
into microbial cells can differ through properties such as lipophil-
icity or molecular weight of the compounds. Microbial cellular tar-
gets for NO such as proteins, lipids or DNA can be altered or
rodilution method.27 Fifty microliters of
a
8 mM compound
solution in DMSO/methanol (1:10) were serially diluted by factor
two with the culture medium (RPMI 1640 with -glutamine, MOPS
L
and without sodium bicarbonate, LONZA Verviers SPRL, Belgium).
Exceptionally, compounds 3a/3b were diluted in 0.1 M NaOH solu-
tion, only the last dilution step was performed in the given media
and used immediately. Then the wells were inoculated with 50 lL
of test organisms to give a final concentration of 6 Â 103 CFU/mL.
After microtiter plates were incubated at 37 °C (Aspergillus fumiga-
tus, Aspergillus terreus) or at 30 °C (Aspergillus niger, Candida, Sporo-
bolomyces, and Penicillium strains) for 24 h, the MIC-values were
read with a Nepheloscan Ascent 1.4 automatic plate reader (Lab-
systems, Vantaa, Finland) as the lowest dilution of compound
allowing no visible growth.
The MIC for Fusarium oxysporumwere determined by the agar
diffusion method. Fifty microliters of each of the 12 serial twofold
dilutions were filled in agar (malt extract agar from Roth, Kar-
lsruhe, Germany; seeded with 0.5 mL of a pretested mycelial solu-
tion) holes of 9 mm in diameter. After incubation at 30 °C for 24 h
the MIC was read as the lowest concentration giving an inhibition
zone.
3. Results and discussion
Prescreening in the agar diffusion assay showed significant dif-
ferences in the diameters of inhibition zones for compounds 1a–9a
tested on fungal strains of A. fumigatus, A. terreus, A. niger, Fusarium
oxysporum, Candida albicans, Candida glabrata, Sporobolomyces sal-
monicolor and Penicillium notatum (data not shown). For quantita-
tive evaluation the MIC-values were used. Table 1 displays the MIC
of the KT–NO-donor hybrids 3a–9a compared to KT (1a) and N-
deacetyl-KT (2a) for the different fungal strains. For better compar-
ison, the respective fractional inhibitory concentration (FIC = MIC
compound/ MIC KT) was calculated, values under 1 represent high-
er antifungal activity than KT alone. The N-deacetyl-ketoconazole
2a is a major metabolite of KT and its activity has been tested on
Plasmodium falciparum, Candida albicans and Candida tropicalis pre-
viously.28,29 Literature data reporting no significant differences be-
tween 1a and 2a in activity against these Candida isolates were
confirmed by us for C. glabrata as well. However, major loss of anti-
fungal activity for 2a against Aspergillus isolates and P. notatum can
be recognized in our study. The MIC of 2a is 30- to 60-fold higher
for some of the strains compared to the N-acetylated 1a. The direct
NO-donor derivative of KT, carrying a diazeniumdiolate moiety
(3a), shows similar antifungal potency against all strains tested
compared to 1a. NO-donor 3a exclusively releases the redox-spe-
cies NOÅ with a half-life of approximately t1/2 = 4.5 min at pH 7.4
and 37 °C. Complete NO liberation from 3a yields the secondary
Table 1
Antifungal activities in vitro of ketoconazole and the ketoconazole–NO-donor hybrid compounds 1a–9a: MIC-values (lM)
Compound Aspergillus
Aspergillus
terreus
Aspergillus
niger
Fusarium
oxysporum
Candida
albicans
Candida
glabrata
Sporobolomyces
salmonicolor
Penicillium
notatum
fumigatus
1a = KT
2a
3a
4a
5a
6a
7a
8a
9a
62.5
31.25
62.5
500
25
12.5
50 (4)
1.56
8
2000 (32)
200 (3.2)
12.5 (0.2)
8 (0.13)
125 (2)
25 (0.4)
1000 (16)
3.13 (0.05)
>2000
2000 (64)
100 (3.2)
3.13 (0.1)
8 (0.26)
62.5 (2)
12.5 (0.4)
1000 (32)
1.56 (0.05)
>2000
2000 (32)
100 (1.6)
1.56 (0.025)
4 (0.06)
62.5 (1)
12.5 (0.2)
250 (4)
2000 (4)
200 (0.4)
50 (0.1)
250 (0.5)
1000 (2)
100 (0.2)
1000 (2)
25 (0.05)
>2000
25 (1)
100 (4)
50 (2)
25 (1)
25 (1)
25 (1)
12.5 (0.5)
6.25 (0.25)
>2000
25 (16)
1.25 (0.8)
0.16 (0.1)
2 (1.3)
250 (31.25)
20 (2.5)
1.25 (0.16)
1.56 (0.2)
8 (1)
2.5 (0.31)
62.5 (7.81)
8 (1)
12.5 (1)
3.13 (0.25)
3.13 (0.25)
12.5 (1)
1.56 (0.12)
6.25 (0.5)
3.13 (0.25)
>2000
2 (1.3)
0.16 (0.1)
15.6 (10)
0.6 (0.38)
>2000
1.56 (0.025)
>2000
1–9b
>2000
(FIC = MIC compound/MIC KT).

J. Konter et al. / Bioorg. Med. Chem. 16 (2008) 8294–8300
8297
detoxified by pathogen specific defense mechanisms such as NO-
scavengers like homocysteine or glutathione, detoxifying enzymes
or DNA repair mechanisms.6 Concerning the synergistic effect of KT
with NO, it is also conceivable that NO interacts with the hemopro-
tein CYP450 of the 14-a-demethylase, and hence amplifies the
antimicrobial target of KT.
2a. The solutions were transferred into the reaction chamber of
the NOtizerÒ apparatus and closed tightly. The chamber was evac-
uated and flushed with dry nitrogen for four consecutive times,
then evacuated again. An initial pressure of 5 bar nitric oxide
was applied and the solutions stirred magnetically at 700 rpm.
The pressure was raised back to 5 bar until remaining constant.
The precipitated products after solvent concentration were filtered,
washed with ether and dried in a desiccator under reduced pres-
sure over night.
4. Conclusion
Ketoconazol–NO-donor hybrid drugs represent a useful ap-
proach in antifungal treatment. For KT carrying a diazeniumdiolate
moiety, derivatization to prodrugs is obligatory to ensure stability
of the compounds. Hybrid drugs of KT with an organic nitrate
group are highly active against the tested strains as well, whereas
variation in structure seems to have greater influence on in vitro
efficacy compared to the NONOate-hybrids. This study demon-
strates that it is not regardless of which type of NO-donor is used
to display synergy with KT, but also that the spectrum of suscepti-
bility against different strains can differ from the one of the
antimycotic agent alone. The KT–NO-donor hybrids may advanta-
geously ensure a better drug targeting for NO and other RNS in
comparison to fixed combinations of KT and NO-donor, which will
have different distribution properties in vitro and in vivo. It is also
considerable that the antiinfective properties of these compounds
in vivo could be much greater, since amongst the versatile NO
activities is its important regulatory role in the immune system
or in inflammatory processes that would indirectly contribute
against microbial pathogens.
5.2.3. (iii, iv) General procedure for synthesis of 4a/4b and 5a/5b
A mixture of triturated anhydrous Na2CO3 (2 g) and 3a/3b sus-
pended in 50 mL hexamethylphosphoric triamide (HMPA) was
stirred at 4 °C and an equimolar amount of the respective alkyl
halide added dropwise. After ice bath removal, the mixtures were
stirred at room temperature for 72 h. Insoluble solids were re-
moved by filtration and the solution poured into NaHCO3 satu-
rated water. The products were extracted with ethyl acetate
(2Â 30 mL) and washed with water (5Â 30 mL). The residues
after solvent evaporation were purified by column chromatogra-
phy on silica gel using the given eluent to furnish 4a, 4b, 5a or
5b, respectively.
5.2.4. (v) General procedure for synthesis of 6a/6b
A mixture of triturated anhydrous Na2CO3 (2 g) and 1.5 molar
excess chloroacetyl chloride in 20 mL dichloromethane (DCM)
was stirred at 4 °C and 2a/2b dissolved in 40 mL DCM added drop-
wise and stirred until completion of the reaction (TLC controlled),
respectively. Solids were filtered off and the filtrate was washed
with saturated NaHCO3 solution (1Â 50 mL) and water (3Â
50 mL). The organic phase was dropped to a freshly prepared sus-
pension of Na2CO3 and equimolar amount of sodium 1-(pyrrolidin-
1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) in 50 mL HMPA at
room temperature. A stream of dry nitrogen was passed through
the mixture until the DCM was evaporated and stirred for 72 h
afterwards. The mixture was filtered into 60 mL of NaHCO3 satu-
rated water, extracted with ethyl acetate (2Â 50 mL), washed with
water (5Â 75 mL) and dried over Na2SO4. The residues after solvent
evaporation were purified by column chromatography on silica gel
using the given eluent to furnish 6a or 6b, respectively.
5. Experimental
5.1. General methods and materials
Chemicals (including 2b) were purchased from VWR Interna-
tional Merck KGaA, Darmstadt and Linde AG, Unterschleissheim,
Germany (NO gas 2.5 UN–Nr. 1660) and used without further
treatment unless noted otherwise. Ketoconazole (1a) was pur-
chased from Caesar & Loretz GmbH Hilden, Germany (Art. Nr.
5270-25G). Melting points are uncorrected and were measured
in open capillary tubes, using a Gallenkamp melting point appara-
tus. 1H and 13C NMR spectral data were obtained from a Bruker Ad-
vance 250 spectrometer (250 MHz) and Advance 400 spectrometer
(400 MHz), respectively. Elemental analysis was performed on a
Hereaus Vario EL apparatus and TLC on silica gel F254 plates
(Merck). MS data were determined by GC/MS, using a Hewlett
Packard GCD-Plus (G1800C) apparatus (HP-5MS column; J&W Sci-
entific) or a mass spectrometer type SSQ-710 from Finnigan MAT
(EI and FAB).
5.2.5. (vi, viii) General procedure for synthesis of 7a/7b and 9a/
9b
Compounds 2a/2b (1 mmol) dissolved in 30 mL dichlorometh-
ane were slowly added via a syringe through a septum to a solution
of CDI activated nitratoacetic acid or nitratopivalinic acid
(1.5 mmol) in 20 mL dichloromethane under dry nitrogen atmo-
sphere and stirred for 24 h at room temperature. The solutions
were washed with NaHCO3 saturated water (3Â 40 mL) and the or-
ganic phase evaporated to yield residues of 7a, 7b, 9a or 9b that
were purified through silica gel column chromatography or recrys-
tallization, respectively.
Note that the much simpler acetylation of the secondary nitro-
gen atom with bromoacetyl bromide and subsequent nitration
using silver nitrate in acetonitrile to produce compounds 7a/7b
only worked for the preparation of 7b. The acetal group incorpo-
rated in the KT molecule was cleaved upon workup preventing this
reaction to furnish 7a.
5.2. General procedures for synthesis of compounds in Scheme 1
5.2.1. (i) Deacetylation of 1a to 2a
KT (1a) was purchased from Caesar & Loretz GmbH Hilden, Ger-
many (Art. Nr. 5270-25G) and 8 g (15 mmol) was dissolved in
175 mL methanol under reflux. Afterwards, 20% aqueous NaOH
solution (40 mL) was slowly added and kept under reflux until
completion of the reaction (TLC controlled). Chopped ice was
added to the solution and the precipitated product was filtered
and dried under vacuum to give 2a in quantitative yield.
5.2.6. (vii) General procedure for synthesis of 8a/8b
A given amount of 2a/2b dissolved in dichloromethane (10 mL)
was slowly added to a twofold excess of 2-bromoethyl nitrate and
stirred at 60 °C for 48 h. The organic phase was washed with NaH-
CO3/H2O (3Â 20 mL) and evaporated to give brown fluids that re-
quired column chromatography purification on silica gel to yield
8a/8b.
5.2.2. (ii) General procedure for synthesis of 3a/3b
Equimolar amounts of elementary sodium were dissolved in dry
methanol (75 mL) and the solution filtered and added to 2a
(5 mmol) and 2b (50 mmol), respectively. Additionally, dry tetra-
hydrofuran (75 mL) was only poured to the mixture containing

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J. Konter et al. / Bioorg. Med. Chem. 16 (2008) 8294–8300
5.3. Experimental data for compounds shown in Scheme 1
5H), 3.52 (s, 3H), 3.73–3.77 (dd, J = 8.2, 4.9 Hz, 2H), 3.83–3.95
(dd, J = 6.7, 6.6 Hz, 1H), 4.30–4.39 (quin, J = 5.7 Hz, 1H), 4.39–4.53
(dd, J = 40.7, 14.8 Hz, 2H), 5.24 (s, 2H), 6.73 (d, J = 9.1 Hz, 2H),
6.89 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 13.6 Hz, 2H), 7.29 (d, J = 8.1 Hz,
1H), 7.48 (d, J = 14.7 Hz, 2H), 7.56 (d, J = 8.4 Hz, 1H); 13C NMR:
400 MHz (CDCl3): d 49.3, 51.3, 51.7, 57.0, 67.7, 67.8, 74.7, 97.9,
108.0, 115.3, 118.7, 121.1, 127.2, 128.4, 129.5, 131.3, 133.0,
134.6, 135.9, 138.8, 145.8, 152.4; FAB-MS m/z (%): 255 (40), 257
(33), 307 (100), 432 (20), 446 (28), 459 (33), 487 (43), 518 (50),
593 (24, [M+H]+); Anal. Calcd for C26H30Cl2N6 O6: C, 52.62; H,
5.10; N, 14.16. Found: C, 52.95; H, 5.21; N, 13.82.
5.3.1. 1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine (2a)
White powder (99% yield); mp 173 °C; 1H NMR: 400 MHz
(CDCl3): d 3.03 (s, 8H), 3.32–3.36 (dd, J = 9.6, 6.7 Hz, 1H), 3.72–
3.77 (dd, J = 8.2, 4.9 Hz, 2H), 3.86–3.90 (dd, J = 6.7, 6.6 Hz, 1H),
4.31–4.37 (quin, J = 5.7 Hz, 1H), 4.39–4.53 (dd, J = 40.7, 14.8 Hz,
2H), 6.76 (d, J = 9.1 Hz, 2H), 6.88 (d, J = 9.1 Hz, 2H), 6.97 (d,
J = 13.6 Hz, 2H), 7.25 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 14.7 Hz, 2H),
7.57 (d, J = 8.4 Hz, 1H); 13C NMR: 400 MHz (CDCl3): d 46.3, 51.3,
51.8, 67.6, 67.8, 74.9, 108.0, 115.3, 118.1, 121.1, 127.2, 128.6,
129.5, 131.3, 133.0, 134.7, 135.9, 138.8, 146.8, 152.3; EI-MS m/z
(%): 83 (15), 173 (20), 183 (39), 432 (28), 445 (61), 460 (100),
471 (32), 489 (16, [M+H]+) Anal. Calcd for C24H26Cl2N4O3: C,
58.90; H, 5.35; N, 11.45. Found: C, 58.90; H, 5.36; N, 11.38.
5.3.7. O2-Methoxymethyl-1-(phenylpiperazin-1-yl)diazen-1-
ium-1,2-diolate (5b)
Yellow solid (61% yield); mp 187 °C; 1H NMR: 250 MHz (CDCl3):
d 3.36 (mc, 4H), 3.51 (s, 3H), 3.64 (mc, 4H), 5.24 (s, 2H), 6.89–6.97
(m, 3H), 7.25–7.29 (m, 2H); 13C NMR: 250 MHz (CDCl3): d 48.3,
51.0, 57.0, 97.9, 117.2, 121.2, 129.3, 150.3; Anal. Calcd for
5.3.2. Sodium 1-(4-{[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)-4-(diazen-1-
ium-1,2-diolat)piperazine (3a)
C12H18N4O3: C, 54.12; H, 6.81; N, 21.04. Found: C, 54.25; H, 6.78;
N, 20.86.
Crude white solid (82% yield); mp 165–170 °C dec; 1H NMR:
400 MHz (DMSO-d6): d 3.05 (m, 4H), 3.28–3.41 (m, 5H), 3.72–
3.77 (dd, J = 8.2, 4.9 Hz, 2H), 3.86–3.90 (dd, J = 6.7, 6.6 Hz, 1H),
4.31–4.37 (quin, J = 5.7 Hz, 1H), 4.39–4.53 (dd, J = 40.7, 14.8 Hz,
2H), 6.76 (d, J = 9.1 Hz, 2H), 6.88 (d, J = 9.1 Hz, 2H), 6.97 (d,
J = 13.6 Hz, 2H), 7.25 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 14.7 Hz, 2H),
7.57 (d, J = 8.4 Hz, 1H); Anal. Calcd for C24H25Cl2N6NaO5: C,
50.45; H, 4.41; N, 14.71. Found: C, 50.54; H, 4.06; N, 14.37.
5.3.8. O2-(1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine-4-
carboxymethyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate
(6a)
Column purification on silica gel with ethyl acetate:acetoni-
trile (1:1) then methanol washout to give orange solid (38%
yield); mp 237 °C; 1H NMR: 400 MHz (CDCl3): d 1.93 (m, 4H),
3.02–3.13 (m, 8H), 3.32–3.36 (m, 1H), 3.55 (mc, 4H), 3.72–3.77
(m, 2H), 3.86–3.90 (dd, J = 6.7, 6.6 Hz, 1H), 4.31–4.37 (quin,
J = 5.7 Hz, 1H), 4.39–4.53 (dd, J = 40.7, 14.8 Hz, 2H), 4.80 (s,
2H), 6.76 (d, J = 9.1 Hz, 2H), 6.90 (m, 2H), 6.97 (m, 2H), 7.25
(d, J = 8.1 Hz, 1H), 7.49 (d, J = 14.7 Hz, 2H), 7.57 (d, J = 8.3 Hz,
1H); 13C NMR: 400 MHz (CDCl3): d 22.9, 36.8, 51.3, 51.7, 53.5,
67.6, 67.7, 71.4, 74.8, 108.0, 115.3, 118.8, 121.2, 127.2, 128.4,
129.5, 131.3, 133.0, 134.6, 135.9, 138.8, 145.6, 152.9, 166.7;
FAB-MS m/z (%): 202 (48), 217 (55), 235 (50), 255 (100), 432
(38), 446 (51), 459 (70), 471 (63), 489 (64), 517 (39), 547 (82),
600 (69), 660 (42, M++H), 682 (19, [M+Na]+), 698 (17, M++K);
Anal. Calcd for C30H35Cl2N7 O6: C, 54.55; H, 5.34; N, 14.84.
Found: C, 54.17; H, 5.43; N, 14.78.
5.3.3. Sodium 1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-
diolate (3b)
White powder (86% yield); mp 148 °C dec; 1H NMR: 250 MHz
(NaOD): d 3.10–3.31 (m, 8H), 6.82–6.94 (m, 3H), 7.15–7.21 (m,
2H); Anal. Calcd for C10H13N4NaO2: C, 49.18; H, 5.37; N, 22.94.
Found: C, 49.22; H, 5.37; N, 22.88.
5.3.4. 1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)-4-(O2-ethyl-
diazen-1-ium-1,2-diolat)piperazine (4a)
From 1.4 mmol 3a a brown solid after column purification with
ethanol:acetone (1:1) as eluent (62% yield); mp 181 °C; 1H NMR:
250 MHz (CDCl3): d 1.39 (t, J = 7.0, 3H) 3.07 (m, 4H), 3.26–3.41
(m, 5H), 3.72–3.77 (dd, J = 8.2, 4.9 Hz, 2H), 3.86–3.90 (dd, J = 6.7,
6.6 Hz, 1H), 4.25–4.53 (m, 5H), 6.74 (d, J = 9.1 Hz, 2H), 6.88 (d,
J = 9.1 Hz, 2H), 6.97 (d, J = 13.6 Hz, 2H), 7.25 (d, J = 8.1 Hz, 1H),
7.48 (d, J = 14.7 Hz, 2H), 7.59 (d, J = 8.4 Hz, 1H); 13C NMR:
250 MHz (CDCl3): d 14.4, 46.3, 51.3, 51.8, 67.6, 67.8, 69.8, 74.9,
108.0, 115.3, 118.6, 121.1, 127.2, 128.6, 129.3, 131.7, 133.0,
134.7, 135.9, 138.8, 145.8, 152.9; Anal. Calcd for C26H30Cl2N6O5:
C, 54.08; H, 5.24; N, 14.55. Found: C, 53.98; H, 5.23; N, 14.22.
5.3.9. O2-[(1-Phenylpiperazine-4-carboxymethyl)-1-
(pyrrolidin-1-yl)]diazen-1-ium-1,2-diolate (6b)
Column purification with ethanol/acetone (1:1) as eluent gave
orange oil (58% yield); 1H NMR: 250 MHz (CDCl3): d 1.80 (m, 4H),
2.58 (m, 4H), 3.16 (mc, 4H), 3.37 (s, 2H), 3.76 (mc, 4H), 6.87–6.95
(m, 3H), 7.25–7.31 (m, 2H); 13C NMR: 250 MHz (CDCl3): d 23.8,
41.7, 43.4, 49.4, 49.9, 53.9, 58.5, 116.5, 120.4, 129.3, 151.0, 168.6;
Anal. Calcd for C16H23N5O3: C, 57.64; H, 6.95; N, 21.01. Found: C,
57.53; H, 6.99; N, 21.17.
5.3.5. O2-Ethyl-1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-
diolate (4b)
5.3.10. 1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine-4-
carboxymethylnitrate (7a)
Brown plates (72% yield); mp 198 °C; 1H NMR: 250 MHz
(CDCl3): d 1.40 (t, J = 7.0 Hz, 3H), 3.37 (t, J = 5.0 Hz, 4H), 3.58 (t,
J = 5.1 Hz, 4H), 3.31 (q, J = 7.1 Hz, 2H), 6.88–6.96 (m, 3H), 7.26–
7.33 (m, 2H); 13C NMR: 250 MHz (CDCl3): d 14.5, 48.8, 51.3, 69.8,
117.2, 120.6, 129.3, 150.3; Anal. Calcd for C12H18N4O2: C, 57.58;
H, 7.25; N, 22.38. Found: C, 57.69; H, 7.28; N, 22.22.
Crude light orange solid after column purification with
methanol on silica gel (86% yield); mp > 250 °C; 1H NMR:
250 MHz (CDCl3): d 3.03 (m, 4H), 3.27 (m, 5H), 3.47 (s, 2H),
3.72–3.78 (m, 2H), 3.83–3.90 (dd, J = 6.6, 6.6 Hz, 1H), 4.30–
4.57 (m, 3H), 6.76 (d, J = 9.0 Hz, 2H), 6.88–6.98 (m, 4H), 7.25
(mc, 1H), 7.48 (d, J = 14.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H); 13C
NMR: 250 MHz (CDCl3): d 39.6, 50.1, 50.7, 51.2, 51.7, 67.5,
67.6, 74.7, 108.0, 115.3, 119.2, 121.2, 127.2, 128.5, 129.5,
131.4, 133.0, 134.6, 135.9, 138.8, 145.1, 153.3; Anal. Calcd for
5.3.6. 1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)-4-(O2-
methoxymethyl-diazen-1-ium-1,2-diolat)piperazine (5a)
From 1.0 mmol 3a a beige solid after column purification with
dichloromethane:methanol (9:1) as eluent (47% yield); mp
186 °C; 1H NMR: 400 MHz (CDCl3): d 3.05 (m, 4H), 3.28–3.41 (m,
C26H27Cl2N5 O7: C, 52.71; H, 4.59; N, 11.82. Found: C, 53.01;
H, 4.53; N, 11.68.

J. Konter et al. / Bioorg. Med. Chem. 16 (2008) 8294–8300
8299
5.3.11. 1-Phenylpiperazine-4-carboxymethylnitrate (7b)
Red crystals after recrystallization with ethanol/ether (74%
yield); mp 243 °C; 1H NMR: 250 MHz (CDCl3):
3.21 (m,
4H), 3.70 (mc, 4H), 5.07 (s, 2H), 6.91–6.97 (m, 3H), 7.26–
7.34 (m, 2H); 13C NMR: 250 MHz (CDCl3): d 42.0, 44.5, 49.4,
67.7, 116.9, 121.0, 129.3, 150.6, 162.9; Anal. Calcd for
5.4. NO release measurements
d
NO release was measured using the Apollo 4000 free radical
analyzer system with an ISO-NOP nitric oxide selective sensor built
in a multi-port measurement chamber by World Precision Instru-
ments Inc., USA. The measurement chamber was tempered with
C12H15N3O4: C, 54.33; H, 5.70; N, 15.84. Found: C, 54.01; H,
a Lauda M3 circulating thermostat at 37 °C and stirred at
5.56; N, 15.56.
800 rpm. All experiments were started after the initial background
signal was below 7000 pA at a channel range of 10 nA for the elec-
trode. Calibration was conducted daily with 2.0 mL of 0.1 M KI/
0.1 M H2SO4 solution in the chamber and an increasing amount
of 50 lM NaNO2 solution to produce nitric oxide by reduction.
The nitrite solution was applied with an Eppendorf pipette (10,
20, 30, 40, 60 lL) resulting in 249, 493, 728, 952 and 1388 nM solu-
tions of NO, respectively. The dissociation of the diazeniumdiolates
was measured by injecting 20 lL of a 50 lM NONOate/0.01 M
NaOH solution into 2.0 mL of phosphate buffer, pH 6.4, resulting
in a 495 nM solution of NONOate. Since 1 mol NONOate produces
2 mol NO, the redox current flow of a 990 nM NO solution was
measured. Note that the nitric oxide sensor measurement is not
accumulative and only measures the arising current at the given
time. For this reason a phosphate buffer pH 6.4 was used to ensure
a sharper signal.12
5.3.12. 1-(4-{[2-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine-4-
ethylnitrate (8a)
Yellow plates after column purification with methanol on sil-
ica gel (62% yield); mp > 250 °C; 1H NMR: 400 MHz (CDCl3): d
2.64–2.81 (m, 6H), 3.22 (mc, 4H), 3.35 (mc, 1H), 3.72–3.91 (m,
3H), 4.30–4.37 (quin, J = 5.7 Hz, 1H), 4.42–4.65 (m, 4H), 6.76–
6.91 (m, 4H), 6.97 (d, J = 13.6 Hz, 2H), 7.25–7.53 (m, 4H); 13C
NMR: 400 MHz (CDCl3): d 49.1, 52.9, 53.4, 57.7, 67.6, 67.8,
70.4, 74.6, 108.0, 115.3, 117.9, 121.0, 127.2, 128.6, 129.5,
131.3, 133.2, 134.7, 136.0, 138.8, 146.7, 152.3; Anal. Calcd for
C26H29Cl2N5 O6: C, 53.99; H, 5.05; N, 12.11. Found: C, 54.02; H,
4.78; N, 12.14.
5.3.13. 1-Phenylpiperazine-4-ethylnitrate (8b)
Yellow solid after column chromatography first with metha-
nol, then with dichloromethane/n-hexane (2:8) as eluent (55%
yield); mp > 250 °C; 1H NMR: 250 MHz (CDCl3): d 2.65–2.79 (m,
6H), 3.50 (mc, 4H), 4.62 (t, J = 6.2 Hz, 2H), 6.85–6.95 (m, 3H),
7.25–7.30 (m, 2H); 13C NMR: 250 MHz (CDCl3): d 49.0, 52.9,
55.0, 70.7, 116.2, 119.9, 129.0, 151.1; Anal. Calcd for
Acknowledgements
We thank Uta Wohlfeld and Gabi Schwinger for the antimicro-
bial/antifungal testing and excellent technical assistance.
References and notes
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7.09; N, 16.63.
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5.3.15. 1-Phenyl-4-(2,2-dimethyl-3-
oxopropylnitrate)piperazine (9b)
Colourless crystals after recrystallization from dichlorometh-
ane/n-hexane (1:1) (92% yield); mp > 250 °C; 1H NMR: 250 MHz
(CDCl3): d 1.42 (s, 6H), 3.19 (t, J = 5.0 Hz, 4H), 3.80 (t, J = 5.0 Hz,
4H), 4.60 (s, 2H), 6.89–6.95 (m, 3H), 7.26–7.33 (m, 2H); 13C NMR:
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5.3.16. Intermediate compounds for synthesis of 6a/6b, 7a/7b,
8a/8b and 9a/9b
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