Synthesis and evaluation of a focused library of pyridine dicarbonitriles against prion disease

Article abstract of DOI:10.1016/j.ejmech.2007.02.018

We report the preparation and screening of a set of 55 pyridine dicarbonitriles as potential prion disease therapeutics. Use of microwave irradiation in an attempt to improve the synthesis typically led to only small enhancement in yields but gave cleaner reactions facilitating product isolation. The library was analysed for binding to human prion protein (huPrP^C) by surface plasmon resonance and for inhibition of the formation of its partially protease resistant isoform PrP^Sc in mouse brain cells (SMB). A total of 26 compounds were found to bind to huPrP^C whilst 12 showed discernable inhibition of PrP^Sc formation, five displaying EC₅₀s in the range 2.5-9 μM. Two compounds were found to reduce PrP^Sc levels to below 30% relative to an untreated control at 50 nM.

Full text of DOI:10.1016/j.ejmech.2007.02.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 93e106
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of a focused library of pyridine
dicarbonitriles against prion disease
Kai Guo, Roger Mutter, William Heal, Tummala R.K. Reddy, Hannah Cope,
*
Steven Pratt, Mark J. Thompson, Beining Chen
Department of Chemistry, University of Sheffield, Dainton Building, Brook Hill, Sheffield S3 7HF, UK
Received 27 October 2006; received in revised form 26 February 2007; accepted 26 February 2007
Available online 12 March 2007
Abstract
We report the preparation and screening of a set of 55 pyridine dicarbonitriles as potential prion disease therapeutics. Use of microwave
irradiation in an attempt to improve the synthesis typically led to only small enhancement in yields but gave cleaner reactions facilitating product
isolation. The library was analysed for binding to human prion protein (huPrPC) by surface plasmon resonance and for inhibition of the forma-
tion of its partially protease resistant isoform PrPSc in mouse brain cells (SMB). A total of 26 compounds were found to bind to huPrPC whilst 12
showed discernable inhibition of PrPSc formation, five displaying EC50s in the range 2.5e9 mM. Two compounds were found to reduce PrPSc
levels to below 30% relative to an untreated control at 50 nM.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Prion protein; TSE; vCJD; Pyridine dicarbonitriles; Microwave irradiation; SPR
1. Introduction
(GPI)-anchored protein PrPC to a partially protease resistant
isoform denoted PrPSc. No effective therapy currently exists
for the prevention of either infection or disease progression
[6]. As a part of an ongoing medicinal chemistry research pro-
gramme we are seeking to identify small drug-like molecules
that bind to PrPC and stabilise it against this conversion, with
the aim of identifying novel prion disease therapeutics.
In 2000 Perrier et al. reported a number of compounds exhib-
iting inhibition of PrPSc formation in mouse neuroblastoma cells
(Fig. 1) [7]. Using surface plasmon resonance (SPR) we identi-
fied the pyridine dicarbonitrile substructure common to these
actives as a source of PrPC binders, and recently reported the
synthesis and SPR screening of a small library of such pyridine
dicarbonitriles [8]. Here, we report on our attempts to further op-
timise the synthesis, and on the construction of a second more
focused library followed by quantitative biological analysis.
Prion diseases are a family of invariably fatal neurodegen-
erative disorders affecting humans and animals including, cat-
tle, sheep, cervids (deer and elk) and mink [1]. These
maladies, known as transmissible spongiform encephalopa-
thies (TSEs), pose a significant risk to public health due to
transmission both to animals and humans. As such, they
have been the focus of much interest in recent years following
discovery of a new variant of the human TSE CreutzfeldteJa-
cob disease (vCJD), thought to have been triggered by the con-
sumption of contaminated beef products [2e4]. Iatrogenic
transmission has also been reported via contact with contam-
inated neurosurgical instruments, tissue grafts and blood
products [5]. TSEs are associated with a post-translational
conversion of the cell-surface glycosylphosphatidylinositol
Abbreviations: PrPC, normal cellular prion protein or PrP-sen; PrPSc, dis-
ease-causing isoform or PrP-res; SMB, Scrapie-infected mouse brain; SPR,
surface plasmon resonance; vCJD, variant CreutzfeldteJakob disease.
* Corresponding author. Tel.: þ44 114 222 9467; fax: þ44 114 222 9346.
E-mail address: b.chen@sheffield.ac.uk (B. Chen).
2. Synthesis
Reported yields for the synthesis of pyridine dicarboni-
triles, both by us and others, are at best moderate [8,9]. We
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.018

94
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
Cl
S
of product in both cases. Under the best conditions (solvent-free,
MWI at 90 ꢀC) compound 6, derived from 3-chlorothiophenol,
was isolated in a slightly improved 29% yield (compared to
23% for Method A), and in 32% yield when ethanol was used
as solvent.
The solvent-free conditions described above (Method B;
MWI for 1 h, followed by aerobic oxidation for 3 h) were
then applied to the synthesis of some of the compounds of in-
terest from our previous study which had hitherto been pro-
duced in poor yield, or not at all. It was pleasing to note
that in the cases of compounds 6e9, yields were seen to rise
by an average of 15%; however, MWI conditions offered no
advantage for compounds 10 and 11. Di-hydroxyphenyl com-
pound 8 had proven inaccessible under classical conditions
(Method A), so it was gratifying to note that it could now
be isolated successfully. Furaldehyde derivative 12 remained
unobtainable, however (Table 1).
As lead structures 1 and 3 e containing a furyl substituent
at the 4-position e showed cell line activity, further explora-
tion of the synthesis of 4-(2-furyl) substituted dicarbonitriles
was considered important. Abdel-Latif et al. reported the reac-
tion of Knoevenagel adduct 13 with malononitrile, thiol 14
and piperidine was refluxed with ethanol for 4 h to form the
desired 4-furyl compound 1 in 67% isolated yield (Scheme
2) [13]. However, this result could not be reproduced when
the same experiment was attempted in our laboratories. We
found that when 13 was refluxed in ethanol with malononitrile,
various thiols and piperidine, only a complex mixture of com-
pounds was observed from which none of the desired products
could be isolated. The procedure was repeated at room temper-
ature and after several hours a red crystalline side product 15
was observed, isolated in 36% yield and confirmed by TLC as
a major component of the previously obtained mixtures.
This result implied preferential addition of further malono-
nitrile, rather than the thiol, to intermediate 13 and therefore
led us to investigate the order of reagent addition, aimed at
reducing the amount of free malononitrile present during the
reactions. The best conditions were found to be heating
a two-fold excess of thiol with malononitrile and catalytic
amounts of piperidine for 30 min at reflux before addition of
Cl
Cl
NH2
N
S
NH2
CN
N
NH2
CN
S
N
NH2
CN
S
N
NH2
CN
NC
NC
NC
NC
O
O
S
1
2
3
4
Fig. 1. Dicarbonitriles reported as active in mouse ScN2a cells.
therefore sought to improve the chemistry so as to facilitate
further library synthesis. Since the first pioneering publica-
tions in 1986 [10,11], there have been numerous reports of mi-
crowave irradiation (MWI) being used in place of
conventional heating, often resulting in vastly reduced reaction
times and less side reactions [12]. One of the advantages of
synthesis under MWI conditions is that solvent is not required
(but can be used if desired). It was thus decided to investigate
the effect of MWI upon the one-pot synthesis of pyridine di-
carbonitriles to see what advantages might be gained from
the use of this technique.
The one-pot synthesis we described previously[8] involves re-
fluxing a solution of aldehyde, thiol and malononitrile (in a ratio
of 1:1:2) with 10 mol% piperidine in ethanol for 24 h, followed
by stirring in air for a further 3 h to allow oxidation of the 1,4-
dihydropyridine so formed to the final product. In our hands the
one-pot synthesis of 2-amino-4-phenyl-6-phenylsulfanylpyri-
dine-3,5-dicarbonitrile 5ederived from benzaldehyde, thiophe-
nol and malononitrile e resulted in a 43% isolated yield [8]. To
develop the microwave methodology, initial investigations were
carried out on the solvent-free reaction of these same reagents un-
der MWI (Scheme 1).
Using Method B, irradiation at 90 ꢀC for 10 min led to
product 5 in 31% yield, whereas extending the irradiation pe-
riod to 1 h resulted in a higher yield of 51%. Longer reaction
time (2 h) offered no further advantage, resulting in only
a 38% yield. Irradiation at higher temperatures (150 and
200 ꢀC) for 15 min also proved inferior, giving just 38% yield
PhS
NH
CN
Method A or B
CN
PhSH
Ph
+
CH2(CN)2
PhCHO
CN
Ph
Ph
S
Ph
S
NH
Ph
H
N
PhS
NC
NH2
CN
N
NH2
CN
CN
CN
CH2(CN)2
[O]
NC
NC
H
Ph
Ph
5
Scheme 1. Synthesis of pyridine dicarbonitriles. Method A: malononitrile, piperidine, ethanol were refluxed for 24 h, then underwent aerobic oxidation for 3 h.
Method B: malononitrile, piperidine, MWI for 10e120 min at 90 ꢀC, then underwent aerobic oxidation for 3 h.

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
95
Table 1
Comparison between yields obtained by classical and MWI methods
thiophenols featuring a methyl ester moiety in the meta- or
para-position were considered necessary, along with 4-formyl-
benzoic acid methyl ester, as preliminary screening had re-
vealed potency in the products derived from these materials.
Microwave reactions were carried out using a Personal
Chemistry SmithCreatorÔ system. Achieving the minimum
reaction volume required by our microwave device (2.0 mL)
required relatively large quantities of reagents under solvent-
free conditions (a total of six compounds were prepared in
this way). For this reason, 36 of the library members were syn-
thesised in ethanol (16 compounds at 1 mmol, one at 2 mmol
and 19 at 3 mmol in 2.0 mL). Another five compounds were
synthesised with conventional heating, and those 4-(2-furyl)
compounds not available by these methods were synthesised
by the alternative procedure described above (six compounds).
In total, a library of 45 pyridine dicarbonitriles was prepared.
As syntheses using poly-hydroxybenzaldehydes had proven
unproductive, 1,4-benzodioxan-6-carboxaldehyde and pipero-
nal were introduced as oxygen-bearing alternatives, giving
products in reasonable yields in the majority of cases. How-
ever, in the case of the reaction between piperonal and 2-hy-
droxythiophenol the desired product could not be isolated.
Ar
S
N
NH2
CN
NC
Ar'
Compound No.
Ar
Ar0
Phenyl
Yielda [%]
Yieldb [%]
5
Phenyl
43
23
25
0
51
29
41
23c
43
21
21
0
6
3-Cl-ph
3-Cl-ph
Benzyl
Thiophene
4-Cl-ph
7
8
3,4-OH-ph
Thiophene
3-OH-ph
Thiophene
Furyl
9
4-OH-ph
4-OH-ph
Benzyl
27
20
19
0
10
11
12
a
Phenyl
Yield from Method A.
Yield from Method B (MWI mediated).
Piperidine (2.1 equiv).
b
c
adduct 13, maintaining reflux. After a further 30 min the reac-
tion mixture was allowed to cool to room temperature and un-
dergo aerobic oxidation after which the products were
obtained as crystalline solids by careful addition of water, al-
beit in low yields. Though the remaining mother liquors still
contained product, second crops of solids which were col-
lected showed high degrees of impurities and as such were dis-
carded. Nonetheless, using the methodologies outlined here,
a selection of previously elusive pyridine dicarbonitriles de-
rived from furaldehyde was constructed and added to the li-
brary for bioassay.
4. Screening (SPR) methodology
Surface plasmon resonance (SPR) was carried out using
a BIAcore 3000 (BIAcore, Uppsala, Sweden) equipped with
a CM5 sensor chip (carboxymethylated dextran). The method-
ology was as reported previously [8,14]. Interactions were
measured with two forms of prion protein, full length human
(huPrPC) and full length murine (moPrPC). Compounds were
screened at 40 mM in running buffer (10 mM sodium phos-
phate, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v/v)
surfactant P20) containing 6.5% DMSO. DMSO calibration
using buffer samples containing 5.5e7.5% DMSO was carried
out to correct solvent effects. Compounds producing a response
of more than 2.5 response units (RU) were considered to be
binders. Binding affinities are expressed as %RUmax, that is,
as a percentage of the theoretical maximum response for 1:1
proteineligand binding. No data is presented for compounds
either seen to interfere with the chip surface, or for those
that could not be washed off the protein (Table 2).
3. Library synthesis
In our previous studies we commented on an interesting re-
lationship in which relocating the substituent on the phenyl-
thiol group from the meta- to the para-position resulted in
a loss of binding. One of the foci of this second library was
to further investigate this trend, and as such, pairs of meta-
and para-substituted thiophenols were employed. As literature
compounds 1 and 3 possessed a furyl group in the 4-position it
was also appropriate, having overcome the synthetic barrier to
these compounds encountered previously, to enrich the library
with some relevant analogues. Due to the reported cell line ac-
tivity of compound 11 at 24 mM [8], benzyl mercaptan and
5. Screening (SMB cells) methodology
Compounds were screened for inhibition of PrPSc forma-
tion in SMB cells of mesodermal origin, the procedure used
being based upon that reported by Rudyk et al. [15]. A persis-
tently infected mouse cell line (SMB), cloned originally from
scrapie-infected mouse brain but of non-neuronal origin [16],
was used. Cells were grown in tissue culture treated plastic
dishes in Medium 199 (phenol red free), supplemented with
10% newborn calf serum (heat inactivated), 5% foetal calf se-
rum (heat inactivated) and penicillinestreptomycin at
10 mg Lꢁ1 at 37 ꢀC in an atmosphere of 5% CO2 in air at
95% relative humidity. Medium was changed every 3rd or
4th day, and every 7 days confluent cells were passaged using
thiophene-2-carboxaldehyde
were
utilised.
Similarly,
CN
CN
CN
CN
Malononitrile,
H2N
SH
piperidine, EtOH
CN
NH2
+
1 +
Reflux, 4h
O
O
13
14
15
Scheme 2. Synthesis of 4-furyl substituted pyridine dicarbonitriles.

Table 2
Screening data from a library of pyridine dicarbonitriles
O
O
O
HO
O
O
S
O
O
O
O
O
O
O
MeO
Cl
O
A
B
C
D
E
F
G
29 (29c)
40 (31d)
[–]a
10 (21c)
[34.0, 43.1]
85 8 (10)
16 (42e)
[Non-Binder]
45 15 (7.5)
9 (43b)
[Non-Binder]
86 22 (10)
48 (7f)
[21.9, 19.6]
54 (35d)
[Non-binder]
Toxic
[–]a
HO
SH
1
2
3
4
5
6
7
8
9
24 6 (0.05)
90 8 (10)
82 3 (10)
51 (38c)
[Non-Binder]
36 10 (5); Toxic
(10)
30 (28c)
[–]a
17 (27c)
[8.4, 0.0]h
77 5 (10)
55 (28d)
[–]a
56 (46d)
[–]a
HO
41 (34d)
[250.0, 137.5]
75 11 (10)
Not isolated
SH
73 0 (10)
77 15 (10)
86 10 (10)
4 (23b)
39 (46d)
[–]a
31 (37c)
[19.3, 1.6]
72 7 (10)
35 (13c)
[15.7, 8.9]
EC50 ~ 3 µM
18 (52c)
[10.6, 2.4]
73 4 (10)
3 (14f)
[37.0, 44.8]
73 3 (10)
38 (40d)
[Non-binder]
68 18 (10)
[8.8, 0.0]h
EC50 ~ 3 µM
Cl
SH
67 5 (10)
57 (37c)
[–]a
58 (9f)
[–]a
Cl
7 (41c)
[118.7, 52.3]
71 13 (10)
19 (46e)
[Non-binder]
46 8 (5)
6 (29b)
[123.6, 72.2]
84 13 (10)
52 (34d)
[11.3, 4.3]
42 (39d)
[78.6, 10.5]
96 10 (10)
SH
EC50 ~ 2.5 µM
36 4 (5); Toxic (10)
94 0 (10)
27 (6f)
28 (44d)
[6.0, 0.0] h
23 6 (0.05)
24 (43c)
[Non-binder]
Toxic
25 (41c)
[Non-binder]
70 8 (10)
20 (14e)
[Non-binder]
EC50 ~ 9 µM
26 (17b)
[Non-binder]
90 12 (10)
MeO
O
[Non-binder]a
69 12 (10)
SH
Not isolated
O
32 (41c)
[6.2, 0.0]h
63 25 (1)
59 (41c)
[–]a
60 (46b)
[–]a
53 (43d)
[–]a
21 (52e)
[Non-binder]
EC50 ~ 8 µM
49 (16f)
[26.9, 2.2]
69 1 (10)
43 (50d)
[155.9, 70.4]
75 20 (10)
SH
MeO
123 18 (10)
87 3 (10)
44 8 (0.05)
33 (40c)
[8.6, 0.0]h
92 0 (10)
22 (39c)
[9.3, 0.0]h
107 2 (10)
50 (6f)
[–]a
62 (40d)
[–]a
44 (46d)
[–]a
61 (25e)
[96.5, 112.2]
113 17 (10)
11 (21b)
[55.5, 52.6]
81 16 (10)
SH
111 10 (10)
94 15 (10)
79 1 (10)
34 (43d)
[9.1, 0.0]h
76 14 (10)
23 (42d)
[5.7, 0.0]h
72 0 (10)
OH
46 (30d)
[16.9, 6.1]
65 9 (10)
47 (20d,g
)
[13.6, 4.9]
89 9 (10)
Not isolated
Not isolated
Not isolated
SH
SH
63 (29d)
[27.9, 0.0]h
61 17 (10)
45 (39d)
[Non-binder]
86 3 (10)
aCompound interfered with the chip surface or could not be washed off the protein. bSolvent free conditions. c1 mmol in 2.0 mL EtOH. d3 mmol in 2.0 mL EtOH. eConventional heating.8 fAlternative method.
gCrude yield, <80 pure by HPLC (see experimental section). hBinding observed to huPrPC only and not moPrPC.

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
97
0.05% trypsin and 0.002% EDTA at a split ratio of 4. To assess
the effects of compounds cells were distributed into 96-well
cluster plates at 3 ꢂ 104 cells per well and incubated for
24 h to allow cell attachment. The compounds were diluted
to 400 times the required concentration in DMSO as stock so-
lutions then transferred, at a 20-fold dilution, into Hank’s bal-
anced salt solution. This solution was then transferred at
a further 20-fold dilution into the cell medium. The cells
were incubated with the compound-containing medium for 5
days.
mercaptan (row 7), though no clear relationship between struc-
ture and binding affinity is apparent. Indeed, considering the
library as a whole, no obvious such correlation is evident,
though in some cases direct comparison between different
structural features was hampered by unavailability of data
through synthetic inaccessibility or interaction of compounds
with the chip surface (e.g. comparison of a thiophene or furan
substituent at the 4-position). Nonetheless, given the lack of
any recognisable trends in the binding results it could be pos-
tulated that the data obtained are indicative of non-specific
binding to the proteins.
After 5 days cell viability was assessed by the MTT assay
following the standard protocol supplied with the reagent
(Sigma). For dot blot analyses cells were extracted using lysis
buffer (10 mM TriseHCl [pH 7.6], 100 mM NaCl, 10 mM
EDTA, 0.5% v/v NP40 and 0.5% w/v sodium deoxycholate),
and the content of the well loaded onto a nitrocellulose mem-
brane (0.45 mm) under gentle vacuum at a total cellular protein
concentration of approximately 30e40 mg/well (determined
by the Bradford assay following the protocol supplied with
the reagent e Sigma). The membrane was air dried and sub-
jected to 75 mg mLꢁ1 proteinase K digestion for 1 h at
37 ꢀC. The reaction was stopped with 1 mM phenylmethylsul-
fonyl fluoride (PMSF) in 20 mM Tris-HCl-buffered saline
(TBS), the membrane washed extensively with TBS, and im-
mersed in 1.8 M guanidine thiocyanate in TBS for 10 min at
room temperature. After further washing with TBS the mem-
brane was blocked using 5% fat-free milk powder in phos-
phate buffered saline (PBS), processed with 0.2 mg mLꢁ1
mouse monoclonal anti-PrP 6H4 (Prionics) and developed us-
ing an ECL kit (Amersham Pharmacia Biotech).
Each experiment was carried out in triplicate and an average
value for PrPSc concentration calculated, relative to an un-
treated control, together with a standard deviation. Compounds
were initially screened at 1 and 10 mM and were considered to
be active if PrPSc levels were reduced to less than 70% of that
of the untreated control after 5 days’ exposure. The amounts of
PrPSc remaining as a percentage relative to the control are
given in Table 2. Determination of EC50 values was carried
out by assay over a range of concentrations and calculated
where an acceptable doseeresponse curve was observed. If
such data was inconclusive, the lowest active concentration
supplanted the initial screening result in Table 2.
To investigate this further, simple kinetic experiments were
carried out by measuring binding to huPrPC (%RUmax) as
a function of compound concentration. When applied to the
strongest binders shown in Table 2, however, such measure-
ments were either inconclusive or indicative of non-specific
binding (data not shown); such results are not conclusive, how-
ever. A number of our other screening subjects have been as-
sessed for binding to PrPC using a fluorescence-based assay
[17], taking advantage of the natural fluorophore present in
the protein. This work has proven that some compounds dis-
playing non-specific binding to PrPC under the SPR conditions
do, in actual fact, show specific (i.e. 1:1) binding to the protein
when assessed using the alternative fluorescence assay. Thus,
although the SPR assay is useful as an initial screening tool,
evaluation of proteinesmall molecule interactions appears to
approach the detection limit of the Biacore instrument making
reliable determination of Kd values difficult.
Cell line screening was carried out using SMB cells, a persis-
tently infected murine cell line acting as a host cell for PrPSc
[15,16]. In this cell line, active replication of the PrPSc infec-
tious agent occurs leading to its accumulation in readily detect-
able amounts. Such host cell lines act, to a degree, as a valuable
alternative to animal models of scrapie thereby constituting
a useful initial screening tool in the search for novel prion dis-
ease therapeutics. Cell line screening results are shown to-
gether with the binding data in Table 2 (active compounds
highlighted). It was pleasing to note the compound 4 (entry
D3 in Table 2), reported previously [7] as an anti-prion agent
by Perrier et al., also showed efficacy in our cell line assay.
Though an EC50 of 3 mM was observed (Fig. 2), this compound
did demonstrate some toxicity towards the cells at concentra-
tions not far above the EC50 value (dotted line denotes cell vi-
ability). Two related compounds (35; entry B3, and 52; entry
F4) for which a similar EC50 value could be deduced are also
derived from chlorophenyl substituents at the 4-position
(Fig. 2), but did not suffer from the problems with toxicity ob-
served for 4. Surprisingly, related structure 3 (entry E3), bear-
ing a furan rather than a thiophene substituent at the 4-position,
was found to be inactive under our assay conditions.
6. Results and discussion
Of the 53 compounds shown in Table 2, 26 were binders to
PrPC, 13 were non-binders and 14 interacted with the chip sur-
face or could not be removed from the immobilised protein us-
ing standard regeneration buffer. Binding data is reported in
Table 2. As mentioned above, the purported detrimental effect
of para- vs. meta-substituted thiophenols on binding was one
of the points of investigation for the present library; however,
it can be seen that such a pattern was not observed amongst the
data reported here.
In addition to 35, two other compounds derived from 3-hy-
droxybenzaldehyde, 51 (entry B2) and 57 (entry D2), showed
clear evidence of activity at 5 mM and below but became toxic
to the cells as the concentration approached 10 mM. Nonetheless,
the 3-hydroxyphenyl substituent at the 4-position may serve as
a useful lead for further SAR studies if analogues with lower tox-
icity can be developed whilst retaining cell line activity.
The strongest binders to PrPC were found amongst those
compounds derived from the aldehyde piperonal (column G,
Table 2), or the thiols 3-chlorothiophenol (row 4) and benzyl

98
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
Fig. 2. Doseeresponse curves for compounds 4, 35 and 52, x-axis values correspond to compound concentration [mM], y-axis values denote percentage of PrPSc
remaining relative to an untreated control. For compound 4, dotted line represents cell viability.
Similarly, three compounds derived from 2,3-dihydrobenzo-
[1,4]dioxine-6-carbaldehyde (column F, Table 2) showed
good cell line activity, thereby identifying this substituent at
the 4-position as an additional useful lead. Despite its similar
structure, none of the compounds derived from piperonal (col-
umn G) proved active, perhaps as a result of the proneness of
the benzo[1,3]dioxolane group towards facile hydrolysis or
metabolisation. In addition to 52 (entry F4, EC50 w 2.5 mM),
derived from the 2,3-dihydrobenzo[1,4]dioxine-6-carbalde-
hyde, compounds 28 (entry F5) and 53 (entry F6) significantly
reduced PrPSc levels at 50 nM. Interestingly, though 28 elimi-
nated most of the PrPSc at lower concentrations, PrPSc levels
recovered at higher concentrations of the drug (Fig. 3); 53
gave a comparable doseeresponse curve, though reduction of
PrPSc levels was not as pronounced at lower concentrations.
The doseeresponse properties shown by these compounds
are not understood, but since both contain a methyl ester sub-
stituent on the thiophenyl group at the 6-position, it may be hy-
pothesised that metabolism through esterase activity results in
loss of efficacy as drug concentration increases.
present case. Compound 20 (entry C5) e also containing
a methyl ester group e gave a similar doseeresponse curve
with an EC50 of 9 mM; and relatedly, 19 (entry C4) and 16 (en-
try C1) displayed evidence for similar activity though a clear
EC50 value could not be deduced in these cases.
In particular, incorporation of a methyl ester group onto the
aromatic ring at the 4-position (column C, Table 2) gave rise to
a striking number of active compounds. As a whole, structures
where a methyl ester group was introduced (at the para-posi-
tion of either the thiol or aldehyde, or the meta-position of the
thiol) showed some promising initial results but their evalua-
tion was hindered by apparent metabolisation of the com-
pounds. Given the activity observed though, structurally
similar analogues which are not as readily metabolised would
constitute an interesting part of any further SAR studies.
Though several cell line active compounds are reported
here, no obvious correlation exists between PrPSc binding af-
finity and such activity. Although, as mentioned above, it can-
not yet be confirmed whether any specific binders to PrPSc are
present among the library under consideration, our observa-
tions do infer that more than one mode of action is present
within the set of active compounds. Despite the considerable
challenge, further studies to ascertain the different modes of
action through which these compounds are acting would
thus prove most valuable.
A similar trend is seen with compound 48 (entry E1),
whose doseeresponse curve is also shown in Fig. 3. In this
case PrPSc levels do not recover as quickly as concentration in-
creases e still only reaching about 50% relative to the control
at 12 mM e perhaps indicating some slower metabolism of the
compound than for the examples described above.
Four other compounds containing methyl ester groups also
showed some efficacy in the cell line assay. Compound 21 (en-
try C6), containing a methyl ester moiety on the substituents at
both the 4- and 6-positions, showed an apparent EC50 of 8 mM
but gave a biphasic doseeresponse curve (Fig. 3). Known as
hormesis [18], this phenomenon is not fully understood, but
is quite likely due to hydrolysis of the ester groups in the
7. Conclusions
It has been postulated [7] that a suitable ligand may stabi-
lise PrPC against conversion to PrPSc. Within our library of 2-
aminopyridine-3,5-dicarbonitriles, no obvious such correlation
between binding and cell line activity was evident, though
some of the active compounds reported were found to be
Fig. 3. Unusual doseeresponse profiles exhibited by compounds 21, 28 and 48, x-axis values correspond to compound concentration [mM], y-axis values denote
percentage of PrPSc remaining relative to an untreated control.

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
99
weak to moderate binders to PrPC, including two 35 and 52
with an EC50 in the range of 3 mM.
8.1.2. Microwave synthesis
SmithCreatorÔ Optimiser EXP reactor (Personal Chemis-
try, Inc.) was used. The machine consists of a continuous fo-
cused microwave power delivery system. Reaction times and
temperatures are operator selectable. Sample temperature is
constantly monitored by IR, pressure by a transducer on the
top of the vial’s septum, and the microwave power is automat-
ically adjusted to maintain programmed temperature profiles.
For the experiments reported here, ‘‘fix hold time’’ was set
to ‘‘on’’ and ‘‘absorption level’’ set to ‘‘normal’’. Reactions
were carried out in Smith Process VialsÔ (2.0e5.0 mL).
Modification of the 4- and 6-positions did not lead to an es-
tablished SAR at the present stage, but did generate some in-
teresting leads which will provide information for directing
further studies. Analysis of the distribution of actives in Table
2 indicates that the substituent at the 4-position of the pyridine
dicarbonitriles may be more influential upon activity than that
at the 6-position. Five of the active compounds contained
a meta- or para-chlorothiophenyl group at the 6-position, mak-
ing these substituents of interest for further investigation. Re-
placement of the chlorine atom with a methyl ester group in
these compounds retained some of, or even improved upon,
their activity, but such potency appeared to be lost through me-
tabolism as drug concentration increased thereby necessitating
the development of more robust structural analogues.
Favoured substituents at the 4-position were 6-(2,3-dihydro-
benzo[1,4]dioxinyl), 3-hydroxyphenyl, and e especially e
para-(carboxymethyl)phenyl. Should the problems of toxicity
and metabolism, respectively, of compounds containing the
latter two groups be overcome by the preparation of suitable
analogues, genuine drug candidates for the treatment of prion
disease may emerge.
8.2. General procedure for solvent-free MWI synthesis
To a mixture of thiol (7.00 mmol), aldehyde (7.00 mmol) and
malononitrile (0.88 mL, 14 mmol) was added piperidine
(0.07 mL, 0.70 mmol). The resultant mixture was heated by mi-
crowave irradiation at 90 ꢀC for 60 min, after which the reaction
mixturewas dissolved in 15.0 mL ethanol. The mixturewas then
exposed to air and stirred at room temperature for 3 h. The pre-
cipitate formed was collected by suction filtration, washed with
n-hexane/ethanol (1:1), and then dried under high vacuum.
We have herein documented two compounds (35 and 52) of
equivalent activity but lower toxicity when compared to the
previously reported [7] active compound 4, thereby achieving
improvement upon this earlier anti-prion compound. Within
our library, a number of structural features have been identi-
fied to assist in the development of more potent compounds,
and three compounds (28, 48 and 53) have been found to ef-
fect significant clearance of PrPSc from infected cells in the
nanomolar concentration range.
8.2.1. 2-Amino-6-(3-chlorophenylsulfanyl)-4-thiophen-2-yl
pyridine-3,5-dicarbonitrile (6)
Yield 29%, yellow powder. Mpt., 197e198 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3372.0, 3091.8, 2360.1, 2203.9, 1611.3,
1574.9, 1542.5, 1506.0, 1458.4, 1429.7, 1408.9, 1314.0,
1256.3, 1239.4, 1123.8, 1080.3, 1064.0, 994.0, 911.6, 866.4,
850.5; dH/ppm (250 MHz, d6-DMSO), 7.25e7.35 (1H, m,
Ar-H), 7.45e7.65 (4H, m, Ar-H), 7.69 (1H, t, J ¼ 2.0, Ar-
H), 7.92 (2H, bs, NH2), 7.97 (1H, d, J ¼ 3.0); dC/ppm
(62.9 MHz, d6-DMSO), 87.5, 93.7, 115.6, 115.9, 128.4,
129.8, 130.3, 131.5, 131.8, 133.1, 134.0, 134.1, 134.6,
151.4, 160.4, 166.6; m/z (EI), 368 (Mþ); found 367.996141
(C17H9ClN4S2 Mþ, requires 367.995718).
8. Experimental
8.1. Chemistry
8.1.1. General procedures
8.2.2. 2-Amino-6-(4-hydroxyphenylsulfanyl)-4-thioph-en-
2-yl pyridine-3,5-dicaronitrile (9)
Melting points were measured using a Bibby-Sterilin SMP10
melting point apparatus. Accurate mass and nominal mass mea-
surements were measured using a Waters-Micromass LCTelec-
trospray mass spectrometer. TLC was performed using
aluminium backed silica gel 60 plates (0.20 mm layer). Flash
column chromatography was carried out using Fluorochem sil-
Yield 43%, yellow crystalline solid. Mpt., 225e226 ꢀC de-
compose; nmax (solid)/cmꢁ1, 3468.7, 3324.3, 3216.1, 3159.3,
3094.2, 2361.9, 2338.2, 2209.4, 1624.9, 1583.4, 1545.4,
1508.4, 1498.0, 1433.9, 1401.8, 1357.8, 1275.4, 1259.7,
1228.3, 1170.0, 1089.0, 1045.6, 1005.7, 876.1, 831.2, 812.5;
dH/ppm (250 MHz, d6-DMSO), 6.87 (2H, d, J ¼ 8.0, Ar-H),
7.29 (1H, t, J ¼ 4.0, Ar-H), 7.37 (2H, d, J ¼ 9.0, Ar-H), 7.57
(1H, d, J ¼ 4.0), 7.79 (2H, bs, NH2), 7.95 (1H, d, J ¼ 5.0,
Ar-H), 9.99 (1H, s, OH); dC/ppm (62.8 MHz, d6-DMSO),
86.4, 92.6, 114.8, 115.2, 115.4, 116.6, 127.9, 130.7, 131.2,
132.8, 137.1, 150.7, 159.1, 159.8, 168.2; m/z (EI), 350
(Mþ); found 350.029786 (C17H10N4OS2 Mþ, requires
350.029605).
˚
ica gel 60 A. All compounds were isolated in >95% purity un-
less otherwise stated. (As determined by HPLC under two sets of
conditions e HPLC 1: Luna 5 m C18 150 ꢂ 4.6 mm, 5e95%
acetonitrile in water [0.1% TFA] over 4 min, 1 mL minꢁ1
,
20 mL injection. Detection was at 256 nm, run time 10 min.
HPLC 2: Altima HP 3 m C18 EPS 150 ꢂ 4.6 mm, 35e98%
acetonitrile in water [0.1% TFA] over 4 min, 1.0 mL minꢁ1
,
20 mL injection. Detection was at 256 nm, run time 11 min.)
All reagents were purchased directly from commercial sources
and used as supplied. Compound 13 was prepared according
to the literature [18], Mpt., 72e73 ꢀC (Literature [19],
72.5e73 ꢀC). Compounds 5, 16, 19, 20, 21, 26 and 61 were
reported previously [8].
8.2.3. 2-Amino-6-benzylsulfanyl-4-thiophen-2-yl-3,5-
dicarbonitrile (11)
Yield 21%, yellow powder. Mpt., 204e205 ꢀC; nmax (solid)/
cmꢁ1, 3435.9, 3320.4, 3212.6, 2200.8, 1994.1, 1623.9, 1537.7,

100
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
1504.9, 1432.5, 1401.8, 1356.5, 1256.1, 1236.6, 1203.2,
1070.2, 1008.6, 852.6, 833.0; dH/ppm (250 MHz, d6-DMSO),
4.50 (2H, s, CH2), 7.15e7.40 (4H, m, Ar-H), 7.45e7.60
(3H, m, Ar-H), 7.94 (1H, d, J ¼ 2.5, Ar-H), 8.16 (2H, bs,
NH2); dC/ppm (62.8 MHz, d6-DMSO), 32.3, 85.7, 93.0,
115.3, 127.3, 127.9, 128.4, 129.4, 130.8, 131.2, 132.7,
137.5, 150.7, 159.7, 166.7; m/z (EI), 348 (Mþ); found
348.050581 (C18H12N4S2 Mþ, requires 348.050340).
1096.1, 1013.9, 994.8, 954.3, 859.8, 830.1, 804.3; dH/ppm
(250 MHz, d6-DMSO), 7.45e7.75 (8H, m, Ar-H), 7.95 (2H,
bs, NH2); dC/ppm (62.8 MHz, d6-DMSO), 87.4, 93.4, 114.8,
115.1, 128.9, 129.3, 129.8, 131.0, 132.7, 133.5, 133.6,
134.0, 135.4, 157.5, 159.6, 165.4; m/z (EI), 396 (Mþ); found
396.000846 (C19H10Cl2N4S Mþ, requires 396.000324).
8.3.2. 2-Amino-4-(3-hydroxyphenyl)-6-(4-hydroxyphenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (10)
8.2.4. 2-Amino-6-(4-chlorophenylsulfanyl)-4-thiophen-2-yl
pyridine-3,5-dicarbonitrile (4)
After 20 h stirring at room temperature the product was iso-
lated as a yellow powder (21% yield). Mpt., 224e226 ꢀC; nmax
(solid)/cmꢁ1, 3399.8, 3325.5, 3223.0, 2220.1, 1639.9, 1587.9,
1542.3, 1521.3, 1496.0, 1465.6, 1442.2, 1381.3, 1340.5,
1316.1, 1269.5, 1213.3, 1169.8, 1088.0, 1039.7, 1010.7,
997.7, 948.5, 903.5, 871.1, 836.0, 817.1; dH/ppm (250 MHz,
d6-DMSO), 6.75e7.05 (5H, m, Ar-H), 7.25e7.50 (3H, m,
Ar-H), 7.73 (2H, bs, NH2), 9.89 (1H, s, OH), 9.99 (1H, s,
OH); dC/ppm (62.9 MHz, d6-DMSO), 87.0, 93.1, 115.3,
115.5, 115.8, 117.0, 117.7, 119.4, 130.4, 135.6, 137.6,
157.8, 159.0, 159.6, 160.1, 168.0; m/z (EI), 360 (Mþ); found
360.068956 (C19H12N4O2S Mþ, requires 360.068098); Purity:
HPLC 1, 94%; HPLC 2, 95%.
Yield 23%, yellow powder. Mpt., 226e227 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3475.1, 3329.9, 3219.9, 2208.6, 1635.8,
1544.8, 1525.6, 1508.5, 1472.2, 1431.8, 1403.9, 1386.3,
1355.5, 1255.2, 1235.9, 1175.8, 1087.9, 1007.7, 906.2,
835.9, 820.0; dH/ppm (250 MHz, d6-DMSO), 7.30 (1H, t,
J ¼ 4.0, Ar-H), 7.50e7.70 (5H, m, Ar-H), 7.90 (2H, bs,
NH2), 7.97 (1H, d, J ¼ 5.0, Ar-H); dC/ppm (62.8 MHz, d6-
DMSO), 86.9, 93.0, 115.1, 115.4, 126.0, 127.9, 129.5, 130.9,
131.3, 132.7, 134.8, 136.7, 150.9, 159.8, 166.3; m/z (EI),
368 (Mþ); found 367.994946 (C17H9ClN4S2 Mþ, requires
367.995718); Purity: HPLC 1, 98%; HPLC 2, 90%.
8.2.5. 3-(6-Amino-3,5-dicyno-4-thiophen-2-yl pyridin-2-
ylsulfanyl)-benzoic acid methyl ester (60)
8.3.3. 4-[2-Amino-3,5-dicyano-6-(3-hydroxyphenyl
Yield 46%, yellow powder. Mpt., 262e263 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3454.1, 3348.7, 3227.8, 2211.0, 1980.0,
1969.4, 1933.1, 1792.7, 1636.8, 1548.0, 1517.5, 1460.0,
1429.9, 1404.0, 1359.9, 1293.4, 1271.6, 1256.3, 1239.4,
1193.0, 1126.2, 1093.6, 1072.1, 1019.8, 997.7, 957.2, 886.2,
852.1, 811.9; dH/ppm (250 MHz, d6-DMSO), 3.88 (3H, s,
CH3), 7.30 (1H, t, J ¼ 4.5, Ar-H), 7.55e7.75 (2H, m, Ar-H),
7.75e7.95 (3H, m, Ar-H, NH2), 7.97 (1H, d, J ¼ 5.5, Ar-H),
8.05e8.15 (2H, m, Ar-H); dC/ppm (62.8 MHz, d6-DMSO),
52.4, 87.0, 115.1, 115.4, 127.9, 128.0, 130.0, 130.4, 131.4,
132.7, 135.6, 139.7, 151.0, 159.8, 165.5, 166.2; m/z (EI),
392 (Mþ); found 392.039440 (C19H12N4O2S2 Mþ, requires
392.040169).
sulfanyl)-pyridin-4-yl]-benzoic acid methyl ester (17)
Yield 27%, yellow powder. Mpt., 224e226 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3418.1, 3330.1, 3232.0, 2357.1, 2193.0,
5152.6, 1997.6, 1957.9, 1849.7, 1711.0, 1639.9, 1571.2,
1545.1, 1527.0, 1473.9, 1433.2, 1405.2, 1319.4, 1270.0,
1115.4, 1019.8, 994.7, 889.0; dH/ppm (250 MHz, d6-DMSO),
3.90 (3H, s, CH3), 6.85e7.10 (3H, m, Ar-H), 7.29 (1H, t,
J ¼ 4.0, Ar-H), 7.72 (2H, d, J ¼ 4.0, Ar-H), 7.91 (2H, bs,
NH2), 8.14 (2H, d, J ¼ 4.0, Ar-H), 9.81 (1H, s, OH); dC/ppm
(62.9 MHz, d6-DMSO), 52.9, 87.4, 93.7, 115.3, 115.6, 117.5,
121.8, 125.8, 128.0, 129.5, 129.9, 130.8, 131.7, 138.9,
158.1, 158.4, 160.1, 166.1, 166.9; m/z (EI), 402 (Mþ); found
402.078545 (C21H14N4O3S Mþ, requires 402.078662); Purity:
HPLC 1, 97%; HPLC 2, 92%.
8.3. General procedure for MWI synthesis in ethanol
(1 mmol)
8.3.4. 4-[2-Amino-6-(4-chlorophenylsulfanyl)-3,5-dicyano
pyridin-4-yl]-benzoic acid methyl ester (18)
To a stirred solution of aldehyde (1.00 mmol) in ethanol
(2.0 mL) containing thiol (1.00 mmol) and malononitrile
(0.13 mL, 2.00 mmol) piperidine (0.01 mL, 0.10 mmol) was
added. The resultant solution was heated by microwave irradi-
ation at 90 ꢀC for 60 min, after which the reaction mixture was
exposed to air, and stirred for 3 h at room temperature. The
precipitate formed was collected by suction filtration, washed
by n-hexane/ethanol (1:1), and then dried under high vacuum.
After purification by flash column chromatography (n-hex-
ane/ethyl acetate, 2:1), product 18 was isolated as a white
powder (52% yield). Mpt., 246e247 ꢀC, decompose; nmax
(solid)/cmꢁ1
, 3456.8, 3307.8, 3231.3, 2535.7, 2359.8,
2215.5, 2170.0, 2150.0, 2024.4, 1952.1, 1919.9, 1772.0,
1700.1, 1652.6, 1635.9, 1558.1, 1540.4, 1521.0, 1506.7,
1473.0, 1457.5, 1418.3, 1067.2, 1031.4, 977.9, 876.3; dH/
ppm (250 MHz, d6-DMSO), 3.91 (3H, s, CH3), 7.56 (2H, d,
J ¼ 4.5, Ar-H), 7.63 (2H, d, J ¼ 4.5, Ar-H), 7.72 (2H, d,
J ¼ 4.5, Ar-H), 7.95 (2H, bs, NH2), 8.14 (2H, d, J ¼ 4.5, Ar-
H); dC/ppm (62.9 MHz, d6-DMSO), 52.5, 87.0, 114.7, 115.0,
126.0, 129.0, 129.5, 131.3, 134.9, 136.7, 138.4, 157.7,
159.5, 165.6, 165.8, m/z (ES), 421 ([M þ H]þ); found
421.0536 (C21H13ClN4O2S [M þ H]þ, requires 421.0526).
8.3.1. 2-Amino-4-(4-chlorophenyl)-6-(3-chlorophenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (7)
Yield 41%, yellow powder. Mpt., 216e217 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3390.9, 2358.2, 2219.6, 2209.8, 1982.1,
1964.8, 1937.7, 1608.6, 1572.4, 1543.1, 1519.4, 1493.9,
1461.9, 1422.3, 1407.9, 1318.5, 1256.7, 1153.7, 1116.0,

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
101
8.3.5. 4-(2-Amino-6-benzylsulfanyl-3,5-dicyanopyridin-4-
yl)-benzoic acid methyl ester (22)
115.0, 115.2, 116.6, 128.9, 130.5, 132.9, 135.3, 137.1,
157.4, 159.2, 159.6, 167.6; m/z (EI), 378 (Mþ); found
378.035525 (C19H11ClN4OS Mþ, required 378.034211).
Yield 39%, yellow powder. Mpt., 219e220 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3451.7, 3330.9, 3220.2, 2213.7, 2143.3,
1981.0, 1724.4, 1628.8, 1568.4, 1528.1, 1462.7, 1432.5,
1422.4, 1407.3, 1316.7, 1261.4, 1190.9, 1115.4, 1105.4,
1029.9, 964.5, 919.2; dH/ppm (250 MHz, d6-DMSO), 3.89
(3H, s, CH3), 4.51 (2H, s, CH2), 7.20e7.40 (3H, m, Ar-H),
7.52 (2H, d, J ¼ 3.5, Ar-H), 7.69 (2H, d, J ¼ 4.0, Ar-H),
8.10 (2H, d, J ¼ 4.0, Ar-H), 8.20 (2H, bs, NH2); dC/ppm
(62.9 MHz, d6-DMSO), 33.7, 52.9, 86.3, 93.4, 115.5, 127.8,
128.9, 129.5, 129.9, 131.7, 138.0, 139.0, 158.0, 159.9,
166.1, 166.8; m/z (EI), 400 (Mþ); found 400.098441
(C22H16N4O2S Mþ, requires 400.099398).
8.3.9. 2-Amino-4-(4-chlorophenyl)-6-(3-hydroxyphenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (30)
Yield 29%, yellow powder. Mpt., 213e214 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3476.6, 3409.6, 3339.8, 3222.4, 2356.4,
2341.8, 2208.6, 2177.1, 1646.2, 1633.2, 1574.1, 1546.5,
1522.7, 1494.8, 1475.8, 1447.6, 1423.7, 1371.1, 1318.5,
1258.2, 1093.9, 1037.4, 995.5, 890.1, 858.0, 833.3, 806.0;
dH/ppm (250 MHz, d6-DMSO), 6.80e7.05 (3H, m, Ar-H),
7.29 (1H, t, J ¼ 8.0, Ar-H), 7.62 (2H, d, J ¼ 4.5, Ar-H), 7.64
(2H, d, J ¼ 4.5, Ar-H), 7.88 (2H, bs, NH2), 9.80 (1H, s,
OH); dC/ppm (62.8 MHz, d6-DMSO), 87.1, 93.4, 114.9,
115.2, 117.0, 121.3, 125.3, 127.6, 128.9, 130.3, 130.4,
132.8, 135.3, 157.5, 157.9, 159.6, 166.4; m/z (EI), 378
(Mþ); found 378.033594 (C19H11ClN4OS Mþ, requires
378.034211).
8.3.6. 4-[6-Amino-4-(4-chlorophenyl)-3,5-dicyanopyrid-in-
2-ylsulfanyl]-benzoic acid methyl ester (24)
After recrystallisation from THF, product 24 was isolated
as a white powder (43% yield). Mpt., 262e263 ꢀC, decom-
pose; nmax (solid)/cmꢁ1, 3459.2, 3403.3, 3283.8, 3232.0,
2364.8, 1636.0, 1558.0, 1540.0, 1521.9, 1274.5, 1180.1,
1059.1, 980.9; dH/ppm (250 MHz, d6-DMSO), 3.89 (3H, s,
CH3), 7.61 (2H, d, J ¼ 4.5, Ar-H), 7.68 (2H, d, J ¼ 4.5, Ar-
H), 7.75 (2H, d, J ¼ 4.0, Ar-H), 7.96 (2H, bs, NH2), 8.03
(2H, d, J ¼ 4.0, Ar-H); dC/ppm (62.8 MHz, d6-DMSO), 52.3,
106.8, 128.9, 129.8, 130.4, 134.3; m/z (ES), 421 ([M þ H]þ);
found 421.0509 (C21H13ClN4SO2 [M þ H]þ, requires
421.0526).
8.3.10. 2-Amino-4-(4-chlorophenyl)-6-(4-chlorophenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (31)
Yield 37%, yellow powder. Mpt., 261e262 ꢀC; nmax (solid)/
cmꢁ1, 3477.9, 3347.4, 3221.7, 2213.8, 2036.2, 1993.3, 1893.9,
1629.4, 1591.8, 1572.0, 1542.1, 1522.6, 1492.2, 1474.0,
1419.9, 1390.0, 1319.2, 1256.8, 1233.5, 1190.2, 1177.0,
1114.0, 1090.3, 1035.4, 1011.8, 953.9, 876.4, 833.0, 821.3;
dH/ppm (250 MHz, d6-DMSO), 7.45e7.75 (8H, m, Ar-H),
7.92 (2H, bs, NH2); dC/ppm (62.8 MHz, d6-DMSO), 87.2,
93.2, 114.8, 115.1, 125.9, 128.9, 129.5, 130.4, 132.7, 134.8,
135.4, 136.7, 157.5, 159.6, 165.7; m/z (EI), 396 (Mþ); found
396.001444 (C19H10Cl2N4S Mþ, requires 396.000324).
8.3.7. 4-[6-Amino-3,5-dicyano-4-(3-hydroxyphenyl)-
pyridin-2-ylsulfanyl]-benzoic acid methyl ester (25)
Yield 41%, yellow powder. Mpt., 269e270 ꢀC; nmax (solid)/
cmꢁ1, 2354.6, 2217.8, 2203.6, 1706.9, 1643.7, 1547.2, 1522.0,
1494.7, 1475.2, 1441.9, 1396.7, 1293.9, 1277.6, 1257.5,
1215.4, 1180.1, 1122.4, 1110.3, 1083.2, 1050.2, 1013.6,
996.5, 951.4, 882.1; dH/ppm (250 MHz, d6-DMSO), 3.89
(3H, s, CH3), 6.85e7.05 (3H, m, Ar-H), 7.76 (2H, d,
J ¼ 4.0, Ar-H), 7.88 (2H, bs, NH2), 8.02 (2H, d, J ¼ 4.5, Ar-
H), 9.91 (1H, s, OH); dC/ppm (62.9 MHz, d6-DMSO), 52.9,
87.9, 94.3, 115.4, 115.5, 115.7, 117.8, 119.4, 126.6, 130.3,
130.5, 130.6, 134.2, 134.8, 135.5, 157.8, 159.3, 160.2,
165.3, 166.3; m/z (EI), 402 (Mþ); found 402.079337
(C21H14N4O3S Mþ, requires 402.078662); Purity: HPLC 1,
92%; HPLC 2, 93%.
8.3.11. 3-[6-Amino-4-(4-chlorophenyl)-3,5-dicyano-2-yl
sulfanyl]-benzoic acid methyl ester (32)
Yield 41%, yellow powder. Mpt., 250e251 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3465.3, 3399.4, 3330.0, 3226.0, 2362.8,
2332.4, 2212.1, 1712.0, 1648.2, 1631.9, 1594.6, 1571.9,
1544.4, 1523.2, 1497.1, 1430.4, 1416.7, 1285.2, 1259.9,
1188.7, 1124.4, 1097.6, 1033.9, 999.8, 977.4, 893.5, 837.9,
805.2; dH/ppm (250 MHz, d6-DMSO), 3.88 (3H, s, CH3),
7.55e7.75 (5H, m, Ar-H), 7.80e8.00 (3H, m, Ar-H, NH2),
8.05e8.15 (2H, m, Ar-H); dC/ppm (62.8 MHz, d6-DMSO),
52.4, 87.3, 93.3, 114.9, 115.2, 127.9, 128.9, 130.0, 130.5,
130.8, 132.8, 135.4, 135.6, 139.6, 157.6, 159.6, 165.5,
165.6; m/z (EI), 420 (Mþ); found 420.043575 (C21H13
ClN4O2S Mþ, requires 420.044775).
8.3.8. 2-Amino-4-(4-chlorophenyl)-6-(4-hydroxyphenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (29)
After stirring at room temperature for 18 h, product 30 was
isolated as a yellow powder (28% yield). Mpt., 252e253 ꢀC;
nmax (solid)/cmꢁ1, 3482.1, 3396.0, 3341.4, 3222.2, 2213.2,
1632.6, 1599.1, 1573.9, 1542.3, 1520.5, 1491.8, 1455.9,
1420.7, 1365.4, 1317.1, 1279.9, 1256.9, 1235.4, 1172.5,
1092.1, 1034.5, 1010.4, 885.8, 831.9, 820.5, 803.9; dH/ppm
(250 MHz, d6-DMSO), 6.87 (2H, d, J ¼ 8.5, Ar-H), 7.37
(2H, d, J ¼ 8.5, Ar-H), 7.59 (2H, d, J ¼ 8.5, Ar-H), 7.66
(2H, d, J ¼ 8.5, Ar-H), 7.81 (2H, bs, NH2), 9.99 (1H, s,
OH); dC/ppm (62.8 MHz, d6-DMSO), 86.7, 92.7, 114.8,
8.3.12. 2-Amino-6-benzylsulfanyl-4-(4-chlorophenyl)-
pyridine-3,5-dicarbonitrile (33)
Yield 40%, yellow powder. Mpt., 203e204 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3435.1, 3329.2, 3217.6, 2210.9, 2130.3,
1625.7, 1596.0, 1570.3, 1527.4, 1492.9, 1459.7, 1403.4,
1318.9, 1261.5, 1238.1, 1091.2, 1018.1, 917.9, 888.5, 833.4,
801.6; dH/ppm (250 MHz, d6-DMSO), 4.51 (2H, s, CH2),
7.20e7.40 (3H, m, Ar-H), 7.40e7.70 (6H, m, Ar-H), 8.18
(2H, bs, NH2); dC/ppm (62.8 MHz, d6-DMSO), 33.2, 86.0,

102
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
93.2, 115.1, 115.2, 127.3, 128.4, 128.8, 129.4, 130.4, 132.8,
135.3, 137.5, 157.3, 159.5, 166.2; m/z (EI), 376 (Mþ); found
376.054921 (C20H13ClN4S Mþ, requires 376.054946).
H), 8.05e8.15 (2H, m, Ar-H), 9.90 (1H, s, OH); dC/ppm
(62.8 MHz, d6-DMSO), 52.4, 87.2, 93.3, 114.9, 115.1, 115.2,
117.3, 118.9, 128.0, 129.9, 130.3, 130.8, 135.0, 135.6, 139.6,
157.4, 158.7, 159.6, 165.5, 165.6; m/z (EI), 402 (Mþ); found
402.078356 (C21H14N4O3S Mþ, requires 402.078662).
8.3.13. 2-Amino-6-(4-chloro-6-phenylsulfanyl)-4-(3-
hydroxyphenyl)-pyridine-3,5-dicarbonitrile (35)
Yield 13%, yellow powder. Mpt., 187e188 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3322.8, 3227.8, 2361.1, 2211.4, 1640.6,
1621.7, 1545.8, 1518.3, 1494.7, 1473.0, 1420.1, 1387.4,
1310.6, 1275.4, 1247.7, 1217.8, 1087.4, 1034.2, 1011.6,
952.5; dH/ppm (250 MHz, d6-DMSO), 6.82e7.05 (3H, m,
Ar-H), 7.36 (1H, t, J ¼ 8.0, Ar-H), 7.55 (2H, d, J ¼ 4.5, Ar-
H), 7.62 (2H, d, J ¼ 4.5, Ar-H), 7.84 (2H, bs, NH2), 9.91
(1H, s, OH); dC/ppm (62.9 MHz, d6-DMSO), 87.5, 93.6,
115.4, 115.5, 115.7, 117.8, 119.3, 126.6, 129.9, 130.5,
135.3, 135.5, 137.2, 157.8, 159.1, 160.1, 166.2; m/z (EI),
378 (Mþ); found 378.033175 (C19H11ClN4OS Mþ, requires
378.034211); Purity: HPLC 1, 93%; HPLC 2, 93%.
8.4. 2-Amino-6-benzylsulfanyl-4-(3,4-dihydroxyphenyl)-
pyridine-3,5-dicarbonitrile (8)
To
a stirred solution of 3,4-dihydroxybenzaldehyde
(2.00 mmol) in ethanol (2.0 mL) containing benzylthiol
(2.00 mmol) and malononitrile (0.25 mL, 4.00 mmol) piperi-
dine (0.42 mL, 4.20 mmol) was added. The solution was
heated by microwave at 90 ꢀC for 60 min, after which the re-
action mixture was dissolved in 4.0 mL ethanol, and stirred in
air for 3 h at room temperature. The precipitate formed was
collected by suction filtration, washed with n-hexane/ethanol
(1:1), and then dried under high vacuum. The product was ob-
tained as a yellow powder. Yield 23%; Mpt., 185e186 ꢀC, de-
compose; nmax (solid)/cmꢁ1, 3460.2, 3343.3, 3208.3, 2208.2,
1621.6, 1602.4, 1519.5, 1493.2, 1398.5, 1348.9, 1317.9,
1281.2, 1268.2, 1248.1, 1120.8, 1027.2, 960.8; dH/ppm
(250 MHz, d6-DMSO), 1.41 (6H, t, J ¼ 6.5, CH2), 2.66 (4H,
t, J ¼ 5.5, CH2), 4.48 (2H, s, CH2), 6.78e6.86 (3H, m, Ar-
H), 7.25e7.34 (3H, m, Ar-H), 7.49e7.53 (2H, m, Ar-H); dC/
ppm (62.8 MHz, d6-DMSO), 24.4, 25.9, 33.2, 46.3, 85.6,
93.0, 115.5, 115.7, 120.1, 127.3, 128.4, 129.4, 137.6, 145.7,
158.5, 159.8, 166.2; m/z (EI), 374 (Mþ), 84 (piperidine); found
374.082130 (C20H14N4O2S Mþ, requires 374.083748); Purity:
HPLC 1, 98%; HPLC 2, 94%.
8.3.14. 2-Amino-4-(3-hydroxyphenyl)-6-(3-hydroxy
phenylsulfanyl)-pyridine-3,5-dicarbonitrile (51)
Yield 38%, yellow powder. Mpt., 222e223 ꢀC; nmax (solid)/
cmꢁ1, 3421.9, 3314.8, 3214.4, 2207.9, 1642.7, 1601.3, 1585.6,
1541.8, 1518.2, 1475.6, 1447.6, 1420.1, 1360.6, 1318.8, 1278.6,
1244.0, 1088.3, 1033.3, 996.1, 950.7, 891.8, 870.1, 845.4; dH/
ppm (250 MHz, d6-DMSO), 6.85e7.05 (6H, m, Ar-H), 7.23e
7.43 (2H, m, Ar-H), 7.81 (2H, bs, NH2), 9.85 (2H, bs, OH);
dC/ppm (62.9 MHz, d6-DMSO), 87.4, 93.9, 115.5, 115.6,
115.8, 117.4, 119.4, 121.8, 125.8, 128.2, 130.4, 130.8, 135.6,
157.8, 158.4, 159.1, 160.2, 166.2; m/z (EI), 360 (Mþ); found
360.067480 (C19H12N4O2S Mþ, requires 360.068098).
8.5. General procedure for MWI synthesis in ethanol
(3 mmol)
8.3.15. 2-Amino-6-(3-chlorophenylsulfanyl)-4-(3-hydroxy
phenyl)-pyridine-3,5-dicarbonitrile (57)
Yield 37%, yellow powder. Mpt., 272e273 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3397.9, 3313.8, 3227.8, 2210.2, 2168.1,
1639.7, 1546.3, 1521.9, 1500.2, 1462.8, 1442.5, 1402.6,
1315.8, 1250.9, 1162.0, 1115.3, 1085.5, 1028.4, 998.2,
950.1, 869.0, 843.4; dH/ppm (250 MHz, d6-DMSO), 6.85e
7.00 (3H, m, Ar-H), 7.36 (1H, t, J ¼ 8.0, Ar-H), 7.45e7.65
(3H, m, Ar-H), 7.69 (1H, s, Ar-H), 7.87 (2H, bs, NH2), 9.91
(1H, s, OH); dC/ppm (62.9 MHz, d6-DMSO), 87.7, 93.9,
115.4, 115.5, 115.7, 117.8, 119.4, 129.9, 130.2, 130.5,
131.5, 134.0, 134.1, 134.5, 135.5, 157.9, 159.2, 160.2,
165.9; m/z (EI), 378 (Mþ); found 378.035064 (C19H11ClN4OS
Mþ, requires 378.034211).
To a stirred solution of aldehyde (3.00 mmol) in ethanol
(2.0 mL) containing thiol (3.00 mmol) and malononitrile
(0.38 mL, 6.00 mmol) piperidine (0.03 mL, 0.30 mmol) was
added. The solution was heated by microwave at 90 ꢀC for
60 min, after which the reaction mixture was dissolved in
7 mL ethanol, exposed to air, and stirred for 3 h at room tem-
perature. The precipitate formed was collected by suction fil-
tration, washed with n-hexane/ethanol (1:1).
8.5.1. 4-[2-Amino-3,5-dicyano-6-(2-hydroxyphenyl-
sulfanyl)-pyridin-4-yl]-benzoic acid methyl ester (23)
Yield 42%, yellow powder. Mpt., 262e263 ꢀC; nmax (solid)/
cmꢁ1, 3396.1, 3316.4, 3227.2, 2226.0, 1715.4, 1643.0, 1574.2,
1545.3, 1524.0, 1444.4, 1435.3, 1361.4, 1323.9, 1293.8,
1261.3, 1194.5, 1122.8, 1113.4, 1029.8, 969.9; dH/ppm
(250 MHz, d6-DMSO), 3.92 (3H, s, CH3), 6.89 (1H, t,
J ¼ 7.5, Ar-H), 7.00 (1H, d, J ¼ 4.5, Ar-H), 7.35 (1H, t,
J ¼ 8.0, Ar-H), 7.45 (1H, d, J ¼ 4.0, Ar-H), 7.73 (2H, d,
J ¼ 4.0, Ar-H), 7.81 (2H, bs, NH2), 8.14 (2H, d, J ¼ 4.0, Ar-
H), 10.13 (1H, s, OH); dC/ppm (62.8 MHz, d6-DMSO), 52.5,
86.3, 93.0, 112.4, 114.9, 115.3, 116.2, 119.7, 129.1, 129.5,
131.2, 131.9, 136.7, 138.6, 157.5, 158.5, 159.5, 165.7,
8.3.16. 3-[6-Amino-3,5-dicyano-4-(3-hydroxyphenyl)-
pyridin-2-ylsulfanyl]-benzoic acid methyl ester (59)
Yield 41%, yellow powder. Mpt., 262e263 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3457.8, 3332.7, 3223.3, 2219.3, 1712.9,
1636.7, 1583.1, 1549.3, 1519.0, 1490.2, 1463.6, 1432.2,
1407.1, 1299.7, 1275.7, 1246.8, 1195.4, 1130.7, 1095.9,
1075.4, 1036.1, 998.3, 951.4, 937.8, 882.9, 858.1, 814.0; dH/
ppm (250 MHz, d6-DMSO), 3.88 (3H, s, CH3), 6.85e7.00
(3H, m, Ar-H), 7.36 (1H, t, J ¼ 8.0, Ar-H), 7.65 (1H, t,
J ¼ 8.0, Ar-H), 7.81 (2H, bs, NH2), 7.88 (1H, d, J ¼ 7.5, Ar-

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
103
166.9; m/z (ES), 403 ([M þ H]þ); found 403.0853 (C21H14
N4O3S [M þ H]þ, requires 403.0865).
147.4, 149.0, 158.2, 159.7, 165.6; m/z (EI), 406 (Mþ); found
406.029091 (C20H11ClN4O2S Mþ, requires 406.029125).
8.5.2. 4-[6-Amino-3,5-dicyano-4-(2,3-dihydrobenzo
[1,4]dioxin-6-yl)-pyridin-2-ylsulfanyl]-benzoic
acid methyl ester (28)
8.5.6. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-(4-hydroxyphenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (40)
Yield 31%, yellow powder. Mpt., 268e269 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3338.5, 3224.0, 2218.4, 1625.3, 1598.1,
1578.9, 1549.0, 1522.4, 1487.1, 1448.0, 1420.4, 1341.6,
1276.2, 1245.7, 1191.4, 1166.7, 1095.2, 1030.8; dH/ppm
(250 MHz, d6-DMSO), 6.15 (2H, s, CH2), 6.87 (2H, d,
J ¼ 4.5, Ar-H), 7.01e7.17 (3H, m, Ar-H), 7.37 (2H, d,
J ¼ 4.5, Ar-H), 7.72 (2H, bs, NH2), 9.99 (1H, s, OH); dC/
ppm (62.8 MHz, d6-DMSO), 87.3, 93.4, 102.3, 109.0, 109.4,
115.4, 115.7, 115.9, 117.0, 123.5, 127.9, 137.6, 147.8,
149.4, 158.5, 159.6, 160.1, 167.9; m/z (ES), 389 ([M þ H]þ);
found 389.0716 (C20H12N4O3S [M þ H]þ, requires 389.0708).
Yield 44%, yellow powder. Mpt., 257e258 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3410.8, 3326.3, 3228.4, 2215.4, 1706.8,
1643.2, 1549.4, 1529.4, 1507.8, 1435.7, 1276.8, 1251.9,
1198.0, 1110.8, 1063.2, 1013.6; dH/ppm (250 MHz, d6-
DMSO), 3.89 (3H, s, CH3), 4.33 (4H, s, CH2), 7.04e7.11
(3H, m, Ar-H), 7.75 (2H, d, J ¼ 4.0, Ar-H), 7.84 (2H, bs,
NH2), 8.02 (2H, d, J ¼ 4.0, Ar-H); dC/ppm (62.9 MHz, d6-
DMSO), 52.9, 64.5, 64.7, 99.0, 99.3, 115.4, 115.9, 117.9,
118.0, 119.0, 130.3, 134.8, 143.7, 145.8, 154.8, 155.8,
158.5, 160.2; m/z (EI), 444 (Mþ); found 444.089436
(C23H16N4O4S Mþ, requires 444.089277).
8.5.7. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-(3-hydroxy
phenylsulfanyl)-pyridine-3,5-dicarbonitrile (41)
8.5.3. 2-Amino-4-(4-chlorophenyl)-6-(2-hydroxyphenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (34)
Yield 34%, yellow powder. Mpt., 252e253 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3446.3, 3347.8, 3230.3, 2359.5, 2213.5,
1635.9, 1545.8, 1520.9, 1501.3, 1489.6, 1465.7, 1446.0,
1404.1, 1354.4, 1300.2, 1244.6, 1198.5, 1103.3, 1035.5,
994.7, 931.5; dH/ppm (250 MHz, d6-DMSO), 6.16 (2H, s,
CH2), 6.88 (1H, d, J ¼ 4.0, Ar-H), 6.96e7.18 (5H, m, Ar-
H), 7.29 (1H, t, J ¼ 8.0, Ar-H), 7.79 (2H, bs, NH2), 9.79
(1H, s, OH); dC/ppm (62.9 MHz, d6-DMSO), 87.7, 94.1,
109.0, 109.5, 115.6, 115.9, 117.4, 121.8, 123.5, 125.8,
127.8, 128.2, 130.8, 147.8, 149.4, 158.4, 158.7, 160.2,
166.7; m/z (EI), 388 (Mþ); found 388.064023 (C20H12N4O3S
Mþ, requires 388.063012).
Yield 43%, yellow powder. Mpt., 268e269 ꢀC; nmax (solid)/
cmꢁ1, 3432.3, 3337.9, 3239.5, 2362.6, 2213.9, 1642.4, 1595.0,
1573.8, 1544.3, 1516.1, 1496.8, 1466.0, 1422.1, 1326.3, 1295.8,
1262.0, 1236.4, 1177.3, 1151.4, 1124.2, 1093.2, 1024.9; dH/ppm
(250 MHz, d6-DMSO), 6.88 (1H, t, J ¼ 7.5, Ar-H), 6.99 (1H, d,
J ¼ 4.0, Ar-H), 7.34 (1H, t, J ¼ 8.0, Ar-H), 7.44 (1H, d, J ¼ 4.5,
Ar-H), 7.60 (2H, d, J ¼ 4.5, Ar-H), 7.68 (2H, d, J ¼ 4.5, Ar-H),
7.77 (2H, bs, NH2), 10.11 (1H, s, OH); dC/ppm (62.8 MHz, d6-
DMSO), 86.5, 93.2, 112.5, 115.0, 115.4, 116.2, 119.7, 128.9,
130.5, 131.9, 132.9, 135.3, 136.7, 157.3, 158.5, 159.5, 166.9;
m/z (EI), 378 (Mþ); found 378.033438 (C19H11ClN4OS Mþ, re-
quires 378.034211).
8.5.8. 2-Amino-4-benzo[1,3]dioxin-5-yl-6-(3-chlorophenyl
sulfanyl)-pyridine-3,5-dicarbonitrile (42)
8.5.4. 2-Amino-6-(4-chlorophenylsulfanyl)-4-(2,3-dihydro
benzo[1,4]dioxin-6-yl)-pyridine-3,5-dicarbonitrile (38)
Yield 40%, yellow powder. Mpt., 243e244 ꢀC; nmax (solid)/
cmꢁ1, 3477.2, 3331.2, 3221.6, 2222.1, 1635.6, 1587.1, 1546.2,
1504.1, 1473.9, 1433.8, 1386.8, 1311.3, 1287.2, 1251.9,
1177.1, 1131.4, 1090.7, 1066.2; dH/ppm (250 MHz, d6-
DMSO), 4.33 (4H, s, CH2-CH2), 7.03e7.10 (3H, m, Ar-H),
7.55 (2H, d, J ¼ 4.5, Ar-H), 7.62 (2H, d, J ¼ 4.0, Ar-H),
7.81 (2H, bs, NH2); dC/ppm (62.8 MHz, d6-DMSO), 64.1,
64.3, 87.1, 93.3, 115.2, 115.4, 117.4, 117.5, 121.8, 126.1,
126.5, 129.4, 134.8, 136.7, 143.2, 145.3, 157.9, 159.7,
165.7; m/z (EI), 420 (Mþ); found 420.044837
(C21H13ClN4O2S Mþ, requires 420.044775).
Yield 39%, yellow powder. Mpt., 172e173 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3318.1, 3225.8, 2215.7, 1637.8, 1550.1,
1522.2, 1498.5, 1447.1, 1407.6, 1352.2, 1252.4, 1105.5,
1035.8; dH/ppm (250 MHz, d6-DMSO), 6.16 (2H, s, CH2),
7.03e7.17 (3H, m, Ar-H), 7.54e7.56 (3H, m, Ar-H), 7.68
(1H, s, Ar-H), 7.86 (2H, bs, NH2); dC/ppm (62.8 MHz, d6-
DMSO), 87.9, 94.1, 102.3, 109.1, 109.4, 115.6, 115.8, 123.5,
127.7, 129.9, 130.2, 131.5, 134.0, 134.1, 134.5, 147.9,
149.5, 158.7, 160.2, 165.9; m/z (EI), 406 (Mþ); found
406.031081 (C20H11ClN4O2S Mþ, requires 406.029125).
8.5.9. 3-(6-Amino-4-benzo[1,3]dioxol-5-yl-3,5-dicyano
pyridi-2-ylsulfanyl)-benzoic acid methyl ester (43)
8.5.5. 2-Amino-4-benzo[1,3]dioxin-5-yl-6-(4-chloro
phenylsulfanyl)-pyridine-3,5-dicarbonitrile (39)
Yield 50%, yellow powder. Mpt., 245e246 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3433.7, 3318.3, 3226.4, 2360.1, 2214.6,
1728.2, 1703.2, 1638.3, 1550.5, 1522.0, 1497.3, 1447.6,
1352.6, 1297.3, 1253.1, 1129.7, 1037.6; dH/ppm (250 MHz,
d6-DMSO), 3.88 (3H, s, CH3), 6.16 (2H, s, CH2), 7.03e7.18
(3H, m, Ar-H), 7.65 (1H, t, J ¼ 8.0, Ar-H), 7.80 (2H, bs,
NH2), 7.87 (1H, d, J ¼ 4.0, Ar-H), 8.07 (2H, d, J ¼ 3.0, Ar-
H); dC/ppm (62.9 MHz, d6-DMSO), 87.7, 94.0, 102.3, 109.0,
109.4, 115.6, 115.9, 123.5, 127.7, 127.8, 129.9, 130.2,
135.3, 147.8, 149.5, 158.7, 160.2, 166.5; m/z (EI), 430
Yield 46%, yellow powder. Mpt., 251e252 ꢀC, decom-
posed; nmax (solid)/cmꢁ1, 3334.0, 2211.2, 1642.8, 1620.6,
1547.4, 1500.0, 1446.7, 1250.6, 1090.1, 1039.2, 1011.4,
924.0, 814.1; dH/ppm (250 MHz, d6-DMSO), 6.16 (2H, s,
CH2), 7.02e7.17 (3H, m, Ar-H), 7.55 (2H, d, J ¼ 4.5, Ar-
H), 7.62 (2H, d, J ¼ 4.5, Ar-H), 7.83 (2H, bs, NH2); dC/ppm
(62.8 MHz, d6-DMSO), 87.3, 93.4, 101.8, 108.6, 108.9,
115.1, 115.4, 123.1, 126.1, 127.3, 129.5, 134.8, 136.7,

104
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
(Mþ); found 430.073462 (C22H14N4O4S Mþ, requires
430.073577).
159.7, 166.9; m/z (EI), 402 (Mþ); found 402.078829
(C21H14SN4O4 Mþ, requires 402.078662).
8.5.14. 2-Amino-6-(3-chlorophenylsulfanyl)-4-(2,3-dihydro
benzo[1,4]dioxin-6-yl)-pyridine-3,5-dicarbonitrile (52)
8.5.10. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-benzylsulfanyl-
pyridine-3,5-dicarbonitrile (44)
Yield 34%, yellow powder. Mpt., 200e201 ꢀC; nmax (solid)/
cmꢁ1, 3330.5, 2208.9, 2150.2, 2072.2, 2042.0, 1991.5, 1617.9,
1583.9, 1545.4, 1507.5, 1460.5, 1429.8, 1411.4, 1324.0,
1294.4, 1251.8, 1198.7, 1124.1, 1069.6, 1040.9; dH/ppm
(250 MHz, d6-DMSO), 4.33 (4H, s, CH2-CH2), 7.02e7.11
(3H, m, Ar-H), 7.51e7.60 (3H, m, Ar-H), 7.68 (1H, t,
J ¼ 1.5, Ar-H), 7.84 (2H, bs, NH2); dC/ppm (62.8 MHz, d6-
DMSO), 64.1, 64.3, 87.3, 93.5, 115.2, 115.4, 117.4, 117.5,
121.8, 126.5, 129.5, 129.7, 131.0, 133.5, 133.6, 134.0,
143.2, 145.3, 157.9, 159.8, 165.5; m/z (EI), 420 (Mþ); found
420.043819 (C21H13ClN4O2S Mþ, requires 420.044775).
Yield 46%, yellow powder. Mpt., 221e222 ꢀC; nmax (solid)/
cmꢁ1, 3471.1, 3329.4, 3213.3, 2217.7, 1616.1, 1547.1, 1526.6,
1497.5, 1485.7, 1440.9, 1407.5, 1349.6, 1311.7, 1248.3,
1233.0, 1194.2, 1123.7, 1106.2, 1030.8; dH/ppm (250 MHz,
d6-DMSO), 4.49 (2H, s, CH2), 6.14 (2H, s, CH2), 6.99e7.14
(3H, m, Ar-H), 7.22e7.35 (3H, m, Ar-H), 7.51 (2H, d,
J ¼ 3.5, Ar-H), 8.11 (2H, bs, NH2); dC/ppm (62.9 MHz, d6-
DMSO), 86.6, 93.8, 108.9, 109.4, 115.8, 123.5, 127.7, 127.8,
128.9, 129.9, 138.1, 147.8, 149.4, 158.5, 160.0, 166.6; m/z
(EI), 386 (Mþ); found 386.083988 (C21H14N4O2S Mþ, re-
quires 386.083748).
8.5.15. 3-[6-Amino-3,5-dicyano-4-(2,3-dihydrobenzo[1,4]
dioxin-6-yl)-pyridin-2-ylsulfanyl]-benzoic acid methyl
ester (53)
8.5.11. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-phenylsulfanyl-
pyridine-3,5-dicarbonitrile (45)
Yield 39%, yellow powder. Mpt., 268e269 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3450.9, 3332.9, 3229.4, 2214.3, 1633.9,
1551.6, 1526.0, 1489.9, 1450.0, 1408.4, 1346.5, 1310.0,
1246.5, 1173.8, 1035.6, 925.5; dH/ppm (250 MHz, d6-
DMSO), 6.16 (2H, s, CH2), 7.03e7.18 (3H, m, Ar-H),
7.50e7.61 (5H, m, Ar-H), 7.79 (2H, bs, NH2); dC/ppm
(62.9 MHz, d6-DMSO), 87.7, 94.0, 102.3, 109.0, 109.4,
115.6, 115.9, 123.5, 127.7, 127.8, 129.9, 130.2, 135.3,
147.8, 149.5, 158.7, 160.2, 166.5; m/z (EI), 372 (Mþ); found
372.068906 (C20H12N4O2S Mþ, requires 372.068098).
Yield 43%, yellow powder. Mpt., 251e252 ꢀC, decompose;
nmax (solid)/cmꢁ1, 333.1, 3231.7, 2197.6, 1730.0, 1708.5,
1649.1, 1585.6, 1548.3, 1504.9, 1434.4, 1310.1, 1251.0,
1125.8, 1067.3, 915.9, 887.3; dH/ppm (250 MHz, d6-DMSO),
3.88 (3H, s, CH3), 4.33 (4H, s, CH2), 7.04 (2H, s, Ar-H),
7.11 (1H, s, Ar-H), 7.65 (1H, t, J ¼ 8.0, Ar-H), 7.79 (2H, bs,
NH2), 7.88 (1H, d, J ¼ 4.0, Ar-H), 8.07 (2H, d, J ¼ 3.0, Ar-
H); dC/ppm (62.8 MHz, d6-DMSO), 52.4, 64.1, 64.3, 87.2,
93.4, 115.2, 115.4, 117.4, 117.5, 121.8, 126.5, 128.1, 130.0,
130.3, 130.8, 135.6, 139.6, 143.2, 145.3, 157.9, 159.7,
165.5, 165.6; m/z (EI), 444 (Mþ); found 444.090396
(C23H16N4O4S Mþ, requires 444.089277).
8.5.12. 2-Amino-6-(2-hydroxyphenylsulfanyl)-4-thio-phen-
2-yl-pyridine-3,5-dicarbonitrile (46)
Yield 30%, yellow powder. Mpt., 212e213 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3439.2, 3341.7, 3236.0, 2211.1, 1638.3,
1543.7, 1512.8, 1468.1, 1433.9, 1404.1, 1325.9, 1298.5,
1260.2, 1234.0, 1100.0; dH/ppm (250 MHz, d6-DMSO), 6.88
(1H, t, J ¼ 7.5, Ar-H), 6.98 (1H, d, J ¼ 4.0, Ar-H), 7.28e
7.37 (2H, m, Ar-H), 7.43 (1H, d, J ¼ 3.5, Ar-H), 7.58 (1H,
d, J ¼ 2.0, Ar-H), 7.76 (2H, bs, NH2), 7.96 (1H, d, J ¼ 2.5,
Ar-H), 10.10 (1H, s, OH); dC/ppm (62.8 MHz, d6-DMSO),
86.3, 93.1, 112.5, 115.3, 115.6, 116.2, 119.7, 127.9, 130.7,
131.2, 131.9, 132.8, 136.7, 150.7, 158.5, 159.7, 167.4; m/z
(EI), 350 (Mþ); found 350.030999 (C17H10N4OS2 Mþ, re-
quires 350.029605).
8.5.16. 2-Amino-4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-6-(4-
hydroxyphenylsulfanyl)-pyridine-3,5-dicarbonitrile (54)
Yield 35%, yellow powder. Mpt., 221e222 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3352.5, 3290.3, 3180.1, 2213.7, 1629.5,
1581.7, 1532.9, 1497.8, 1453.8, 1428.1, 1286.6, 1249.7,
1201.0, 1169.6, 1131.3, 1057.9; dH/ppm (250 MHz, d6-
DMSO), 4.32 (4H, s, CH2-CH2), 6.86 (2H, d, J ¼ 4.0, Ar-
H), 7.01e7.09 (3H, m, Ar-H), 7.37 (2H, d, J ¼ 4.5, Ar-H),
7.70 (2H, bs, NH2), 9.99 (1H, s, OH); dC/ppm (62.8 MHz,
d6-DMSO), 64.1, 64.3, 86.6, 92.7, 114.9, 115.3, 115.5,
116.5, 117.4, 117.5, 121.8, 126.6, 137.1, 143.2, 145.2,
157.7, 159.1, 159.7, 167.6; m/z (ES), 403 ([M þ H]þ); found
403.0879 (C21H14N4O3S [M þ H]þ, requires 403.0865).
8.5.13. 2-Amino-4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-6-(2-
hydroxyphenylsulfanyl)-pyridine-3,5-dicarbonitrile (47)
8.5.17. 2-Amino-6-(3-hydroxyphenylsulfanyl)-4-thiophen-2-
yl-pyridine-3,5-dicarbonitrile (55)
Yield 20%, yellow powder. Mpt., 150e151 ꢀC; nmax (solid)/
cmꢁ1, 3417.2, 3334.6, 3239.8, 2213.4, 1648.8, 1586.7, 1546.3,
1505.9, 1470.6, 1432.1, 1309.8, 1286.0, 1254.1, 1178.6, 1128.9,
1068.4; dH/ppm (250 MHz, d6-DMSO), 4.33 (4H, s, CH2-CH2),
6.88 (1H, t, J ¼ 7.5, Ar-H), 6.97e7.10 (4H, m, Ar-H), 7.34 (1H,
t, J ¼ 7.5, Ar-H), 7.43 (1H, d, J ¼ 4.0, Ar-H), 7.66 (2H, bs, NH2),
10.09 (1H, s, OH); dC/ppm (62.8 MHz, d6-DMSO), 64.1, 64.3,
66.6, 86.4, 93.3, 112.6, 115.3, 115.7, 116.2, 117.4, 117.5,
119.7, 121.8, 126.6, 131.8, 136.7, 143.2, 145.2, 157.6, 158.5,
Yield 28%, yellow powder. Mpt., 232e233 ꢀC; nmax (solid)/
cmꢁ1, 3379.2, 3316.3, 3220.2, 2209.0, 1645.7, 1541.0, 1507.5,
1441.2, 1247.4, 1223.4, 1078.3, 1012.7, 995.5, 885.1, 851.9;
dH/ppm (250 MHz, d6-DMSO), 6.87e7.02 (3H, m, Ar-H);
7.26e7.31 (2H, m, Ar-H); 7.58 (1H, d, J ¼ 2.0, Ar-H); 7.86
(2H, bs, NH2); 7.96 (1H, d, J ¼ 2.5, Ar-H); 9.80 (1H, s,
OH); dC/ppm (62.8 MHz, d6-DMSO), 86.8, 115.1, 115.4,
117.0, 121.3, 125.3, 127.6, 127.9, 130.3, 130.8, 131.3,

K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
105
132.7, 150.9, 157.9, 159.9, 166.9; m/z (ES), 351 (Mþ); found
351.0388 (C17H10N4S2O [M þ H]þ, requires 351.0374); Pu-
rity, HPLC 1, 76%; HPLC 2, 79%.
was allowed to stand for 2 h. This was repeated until formation
of solids was observed. Solids were isolated as above.
8.6.1. 4-(6-Amino-3,5-dicyano-4-furan-2-ylpyridin-2-
ylsulfanyl)-benzoic acid methyl ester (27)
8.5.18. 2-Amino-4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-6-(3-
hydroxyphenylsulfanyl)-pyridine-3,5-dicarbonitrile (56)
Yield 46%, yellow powder. Mpt., 236e237 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3438.1, 3330.1, 3218.1, 2212.5, 1629.2,
1581.9, 1545.6, 1502.8, 1432.0, 1305.7, 1285.0, 1249.8,
1199.9, 1126.8, 1065.7, 995.5, 919.0, 887.4; dH/ppm
(250 MHz, d6-DMSO), 4.33 (4H, s, CH2-CH2), 6.88 (1H, d,
J ¼ 4.5, Ar-H), 6.96e7.11 (5H, m, Ar-H), 7.28 (1H, t, J ¼ 7.5,
Ar-H), 7.77 (2H, bs, NH2), 9.79 (1H, s, OH); dC/ppm
(62.8 MHz, d6-DMSO), 64.1, 64.3, 87.0, 93.5, 115.2, 115.5,
116.9, 117.4, 117.5, 121.3, 121.8, 125.3, 126.6, 127.8, 130.3,
143.2, 145.2, 157.9, 159.8, 166.3; m/z (EI), 402 (Mþ); found
402.07932 (C21H14N4O3S Mþ, requires 402.078662).
Yield 6%, pale orange solid. Mpt., 267e268 ꢀC decom-
pose; nmax (Solid)/cmꢁ1, 3401, 3326, 3227, 2211, 1706,
1645, 1538, 1517, 1267, 1117, 1012, 758; dH/ppm
(250 MHz, d6-DMSO), 3.89 (3H, s, CH3), 6.85 (1H, dd,
J ¼ 4.0, 2.0, Ar-H), 7.43 (1H, d, J ¼ 3.5, Ar-H), 7.74 (2H, d,
J ¼ 8.5, Ar-H), 7.89 (2H, bs, NH2), 8.01 (2H, d, J ¼ 8.5, Ar-
H), 8.13 (1H, d, J ¼ 1.0, Ar-H); dC/ppm (62.8 MHz, d6-
DMSO), 52.6, 83.2, 113.1, 115.6, 115.8, 116.8, 126.4, 130.0,
130.3, 133.9, 134.6, 144.3, 145.2, 155.3, 160.4, 165.9,
166.3; m/z (EI), 376 (Mþ); found 376.0612 (C19H12N4O3S
Mþ, requires 376.0630); Purity, HPLC 1, 93%; HPLC 2, 93%.
8.6.2. 2-Amino-6-(4-chlorophenylsulfanyl)-4-furan-2-yl-
pyridine-3,5-dicarbonitrile (3)
8.5.19. 2-Amino-6-benzylsulfanyl-4-(2,3-dihydrobenzo
Yield 14%, pale yellow solid. Mpt., 276e278 ꢀC, decom-
pose; nmax (Solid)/cmꢁ1, 3480, 3319, 3212, 2212, 1624,
1571, 1541, 1513, 1470, 1268, 1240, 1010, 828, 760; dH/
ppm (250 MHz, d6-DMSO), 6.85 (1H, dd, J ¼ 3.5, 1.5, Ar-
H), 7.42 (1H, dd, J ¼ 3.5, 0.6, Ar-H), 7.53 (2H, dt, J ¼ 8.5,
2.0, Ar-H), 7.61 (2H, dt, J ¼ 9.0, 2.0, Ar-H), 7.84 (2H, bs,
NH2), 8.12 (1H, dd, J ¼ 2.0, 0.5, Ar-H); dC/ppm (62.8 MHz,
d6-DMSO), 82.9, 89.4, 113.1, 115.7, 115.9, 116.7, 126.3,
129.6, 134.9, 136.9, 138.1, 144.2, 145.2, 160.4, 167.1; m/z
(ES), 353 ([M þ H]þ); found 353.0249 (C17H10ClN4OS
[M þ H]þ, requires 353.0264).
[1,4]dioxin-6-yl)-pyridine-3,5-dicarbonitrile (62)
Yield 40%, yellow powder. Mpt., 195e196 ꢀC; nmax (solid)/
cmꢁ1, 3442.0, 3326.6, 3221.0, 2360.1, 2208.4, 1629.9, 1584.8,
1543.2, 1505.9, 1458.6, 1431.1, 1287.6, 1250.5, 1199.4, 1130.2,
1067.1; dH/ppm (250 MHz, d6-DMSO), 4.30 (4H, s, CH2-CH2),
4.49 (2H, s, CH2), 6.99e7.07 (3H, m, Ar-H), 7.25e7.34 (3H, m,
Ar-H), 7.51 (2H, d, J ¼ 4.0, Ar-H), 8.10 (2H, bs, NH2); dC/ppm
(62.8 MHz, d6-DMSO), 64.1, 64.3, 85.9, 93.2, 115.4, 117.3,
117.5, 121.8, 126.6, 127.3, 128.4, 129.4, 137.6, 143.2, 145.2,
157.7, 159.6, 166.2; m/z (ES), 401 ([M þ H]þ); found
401.1075 (C22H16N4O2S [M þ H]þ, requires 401.1072).
8.6.3. 2-Amino-4-furan-2-yl-6-(4-hydroxyphenylsulfanyl)-
pyridine-3,5-dicarbonitrile (48)
8.5.20. 2-Amino-4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-6-
phenylsulfanyl-pyridine-3,5-dicarbonitrile (63)
After addition of water (7 ꢂ 1.0 mL). Yield 7%, yellow
solid. Mpt., 240e241 ꢀC; nmax (Solid)/cmꢁ1, 3332, 3230,
2217, 1630, 1536, 1508, 1476, 1412, 1268, 1227, 1168,
1024, 833, 765; dH/ppm (250 MHz, d6-DMSO), 6.82e6.90
(3H, m, Ar-H), 7.32e7.42 (3H, m, Ar-H), 7.74 (2H, bs,
NH2), 8.1 (1H, dd, J ¼ 1.5, 0.5, Ar-H), 9.99 (1H, bs, OH);
dC/ppm (62.8 MHz, d6-DMSO), 82.5, 89.0, 113.0, 115.1,
115.8, 115.9, 116.5, 116.7, 137.3, 144.1, 145.3, 159.3,
160.4, 168.9; m/z (ES), 333 ([M ꢁ H]ꢁ); found 333.0460
(C17H9N4O2S [M ꢁ H]ꢁ, requires 333.0446).
Yield 29%, yellow powder. Mpt., 227e228 ꢀC; nmax (solid)/
cmꢁ1, 3444.4, 3340.7, 3228.6, 2360.7, 2217.7, 1640.8, 1582.2,
1545.5, 1504.2, 1433.2, 1308.6, 1285.6, 1249.8, 1129.8,
1066.6; dH/ppm (250 MHz, d6-DMSO), 4.33 (4H, s, CH2-
CH2), 7.03e7.10 (3H, m, Ar-H), 7.50e7.59 (5H, m, Ar-H),
7.76 (2H, bs, NH2); dC/ppm (62.8 MHz, d6-DMSO), 64.1,
64.3, 87.0, 93.4, 115.2, 115.5, 117.4, 117.5, 121.8, 126.6,
127.2, 129.4, 129.7, 134.8, 143.2, 145.3, 157.9, 159.8,
166.1; m/z (ES), 387 ([M þ H]þ); found 387.0897
(C21H14N4O4S [M þ H]þ, requires 387.0916).
8.6.4. 3-(6-Amino-3,5-dicyano-4-furan-2-ylpyridin-2-
ylsulfanyl)-benzoic acid methyl ester (49)
8.6. General procedure for the synthesis of 4-furan-2-yl-
pyridine-3,5-dicarbonitriles
Yield 16%, pale orange solid. Mpt., 269e271 ꢀC decom-
pose; nmax (solid)/cmꢁ1, 3468, 3358, 2213, 1724, 1630,
1522, 1440, 1266, 1127, 1022, 768, 742; dH/ppm (250 MHz,
d6-DMSO), 3.87 (3H, s, CH3), 6.80e6.90 (1H, m, Ar-H),
7.38e7.46 (1H, m, Ar-H), 7.60e7.70 (1H, m, Ar-H), 7.76e
7.92 (3H, m, Ar-H þ NH2), 8.02e8.10 (2H, m, Ar-H),
8.11e8.16 (1H, m, Ar-H); dC/ppm (62.8 MHz, d6-DMSO),
52.6, 83.0, 89.5, 113.1, 115.7, 115.9, 116.7, 118.7, 128.2,
130.1, 130.5, 130.9, 135.8, 139.9, 144.2, 145.2, 160.4,
165.6, 186.3; m/z (EI), 376 (Mþ); found 376.0612
(C19H12N4O3S Mþ, requires 376.0630).
Malononitrile (0.132 g, 2.0 mmol), thiol (4.0 mmol) and pi-
peridine (1 drop) were dissolved in ethanol (8 mL) and refluxed
for 30 min. 2-Furan-2-ylmethylenemalononitrile 13 (0.288 g,
2.0 mmol) was added as a solid to the hot reaction mixture
and reflux was maintained for further 30 min. After allowing
the reaction mixture to cool down it was exposed to air over
night. The solids formed were collected, washed twice with eth-
anol/water (10 mL, 1:1) and dried under high vacuum. If no
solids were formed water (1 mL) was added and the mixture

106
K. Guo et al. / European Journal of Medicinal Chemistry 43 (2008) 93e106
8.6.5. 2-Amino-6-benzylsulfanyl-4-furan-2-yl-pyridine-3,5-
dicarbonitrile (50)
References
Yield 6%, yellow solid. Mpt., 219e220 ꢀC, decompose;
nmax (solid)/cmꢁ1, 3444, 3324, 3211, 2208, 1622, 1537,
1515, 1414, 1240, 1025, 757, 698; dH/ppm (250 MHz, d6-
DMSO), 4.48 (2H, s, CH2), 6.82 (1H, dd, J ¼ 3.5, 2.0, Ar-
H), 7.20e7.40 (4H, m, Ar-H), 7.46e7.54 (2H, m, Ar-H),
8.09 (1H, dd, J ¼ 2.0, 0.5, Ar-H), 8.11 (2H, bs, NH2); dC/
ppm (62.8 MHz, d6-DMSO), 33.5, 81.8, 89.4, 104.2, 113.0,
115.9, 116.5, 127.5, 128.6, 129.5, 137.6, 144.0, 145.3,
146.7, 160.3, 167.5; m/z (ES), 333 ([M þ H]þ); found
333.0820 (C18H13N4OS [M þ H]þ, requires 333.0810).
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[2] R.G. Will, J.W. Ironside, M. Zeidler, S.N. Cousens, K. Estilbeiro,
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8.6.6. 2-Amino-6-(3-chlorophenylsulfanyl)-4-furan-2-yl-
pyridine-3,5-dicarbonitrile (58)
After addition of water (3 ꢂ 1.0 mL). Yield 9%, off white
solid. Mpt., 198e200 ꢀC; nmax (Solid)/cmꢁ1, 3468, 3312, 3210,
2206, 1622, 1535, 1511, 1459, 1397, 1266, 1023, 775, 757; dH/
ppm (250 MHz, d6-DMSO), 6.85 (1H, dd, J ¼ 3.5, 2.0, Ar-H),
7.42 (1H, d, J ¼ 3.5, Ar-H), 7.46e7.60 (3H, m, Ar-H), 7.67
(1H, t, J ¼ 1.5, Ar-H), 7.87 (2H, bs, NH2), 8.13 (1H, d, J ¼ 1.5,
Ar-H); dC/ppm (62.8 MHz, d6-DMSO), 83.0, 89.6, 113.0,
115.6, 115.8, 116.7, 129.6, 129.9, 131.1, 133.7, 134.3, 143.9,
144.2, 145.2, 146.8, 160.4, 166.9; m/z (ES), 353 ([M þ H]þ);
found 353.0271 (C17H10ClN4OS [M þ H]þ, requires 353.0264).
[9] L.C.W. Chang, J. Kunzel, T. Mulder-Krieger, R.F. Spanjersberg,
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We thank the Department of Health for their generous funding
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Appendix. Supplementary data
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Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.ejmech.2007.02.018.