Library design, synthesis, and screening: Pyridine dicarbonitriles as potential prion disease therapeutics

Article abstract of DOI:10.1021/jm050610f

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a family of invariably fatal neurodegenerative disorders, and there are no effective therapeutics currently available. In this paper, we report on the design, synthesis, and screening of a series of pyridine dicarbonitriles as potential novel prion disease therapeutics. A virtual reaction-based library of 1050 compounds was constructed. Docking and evaluation using GOLD scores assisted the initial selection of compounds for synthesis. The selection was augmented with further compounds to increase structural diversity. A total of 45 compounds were synthesized via a one-pot three-component coupling reaction. The mechanism of the three-component coupling reaction was investigated, and it was discovered that chemical oxidation is required for the last step, forming the pyridine ring (aromatization). A total of 19 compounds were identified as binders to one or more forms of prion protein by in vitro screening using surface plasmon resonance (SPR). A selection of compounds were investigated for activity in cells, resulting in the discovery of a new inhibitor of PrP ^Sc formation.

Full text of DOI:10.1021/jm050610f

J. Med. Chem. 2006, 49, 607-615
607
Library Design, Synthesis, and Screening: Pyridine Dicarbonitriles as Potential Prion Disease
Therapeutics
Tummala R. K. Reddy,†,‡ Roger Mutter,† William Heal,† Kai Guo,† Valerie J. Gillet,‡ Steven Pratt,† and Beining Chen†,*
Department of Chemistry, The UniVersity of Sheffield, Sheffield, S3 7HF, UK, and Department of Information Studies, The UniVersity of
Sheffield, Sheffield, S3 7HF, UK
ReceiVed June 28, 2005
Transmissible spongiform encephalopathies (TSEs) or prion diseases are a family of invariably fatal
neurodegenerative disorders, and there are no effective therapeutics currently available. In this paper, we
report on the design, synthesis, and screening of a series of pyridine dicarbonitriles as potential novel prion
disease therapeutics. A virtual reaction-based library of 1050 compounds was constructed. Docking and
evaluation using GOLD scores assisted the initial selection of compounds for synthesis. The selection was
augmented with further compounds to increase structural diversity. A total of 45 compounds were synthesized
via a one-pot three-component coupling reaction. The mechanism of the three-component coupling reaction
was investigated, and it was discovered that chemical oxidation is required for the last step, forming the
pyridine ring (aromatization). A total of 19 compounds were identified as binders to one or more forms of
prion protein by in vitro screening using surface plasmon resonance (SPR). A selection of compounds were
investigated for activity in cells, resulting in the discovery of a new inhibitor of PrPSc formation.
Introduction
Transmissible spongiform encephalopathies (TSEs) or prion
diseases are a family of invariably fatal neurodegenerative
disorders that are characterized by the appearance of large
vacuoles in the cortex and cerebellum. These include scrapie
in sheep and goats, bovine spongiform encephalopathy (BSE)
in cattle, chronic wasting disease in deer and elk, and Creutzfeldt-
Jacob disease (CJD) in humans.1 They have attracted intense
interest in recent years since a new variant of Creutzfeldt-Jacob
disease (vCJD) was discovered in humans, which is thought to
have been triggered by the consumption of contaminated beef
products.2-4 Iatrogenic transmission has also been observed via
contact with contaminated neurosurgical instruments, tissue
grafts, and blood products.5 As yet, no effective therapy exists
for the prevention of either infection or disease progression.6
TSEs are associated with a posttranslational conversion of
the cell-surface glycosylphosphatidylinositol (GPI)-anchored
protein PrPC to its partially protease-resistant isoform PrPSc.7
As part of an ongoing medicinal chemistry project, we are
looking for compounds that bind to human PrPC (huPrPC) and
stabilize it against this conversion with the aim of identifying
novel prion disease therapeutics.
Pyridine dicarbonitrile 1, along with three analogues 2-4
(Figure 1), were reported to inhibit PrPSc accumulation in
scrapie-infected mouse neuroblastoma cells (ScN2a).8 However,
the mode of action for these compounds is not understood. As
part of a larger screening program hunting for affinity binders
for cellular prion protein (PrPC), these compounds were analyzed
in silico and by SPR for binding to huPrPC.
Our modeling studies indicated that all four compounds fitted
well into our proposed binding site,9 and the SPR screening
results showed moderate binding of compound 3 and weak
binding of compound 2 to PrPC.10 The pyridine dicarbonitrile
substructure was therefore chosen as a basic structural scaffold
Figure 1. Pyridine dicarbonitriles reported as active in mouse ScN2a
cells.8
for the design of a reaction-based library. In this paper we report
details on the design, synthesis, and mechanistic investigation
of this novel series of pyridine dicarbonitriles and examine their
binding to or interaction with huPrPC, truncated human PrPC
(t-huPrPC), and murine PrPC (moPrPC).
Results and Discussion
Docking. The hypothetical huPrPC binding pocket used in
our virtual screening was identified from SYBYL-Molcad
surfaces generated from the PrPC NMR structures available from
the Brookhaven protein data bank (1QM3, conformer 15).9
Compounds 1-4 were docked and scored using the genetic
optimization for ligand docking (GOLD) program, which
resulted in docking scores of 62, 72, 69, and 70, respectively.
These scores indicate that compounds 1-4 fit well in the binding
pocket (Figure 2). It can be seen that both nitrile groups are
interacting with the protein. Based on these results, a virtual
reaction-based library of 1050 compounds from 21 arylthiols
and 50 arylaldehydes was generated. They were also docked
and scored, allowing compound selection for synthesis.
Library Design. The development of reaction-based library
design techniques has allowed access to smaller, more focused
libraries and, because the process is based on the actual chemical
steps of a synthesis, reduces problems of synthetic accessibility.
Using this approach, a virtual library of pyridine dicarbonitriles
was prepared.
* Corresponding author. Phone: +44 114 222 9467. Fax: +44 114 222
9346. E-mail: b.chen@shef.ac.uk.
† Department of Chemistry.
‡ Department of Information Studies.
10.1021/jm050610f CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/22/2005

608 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Reddy et al.
Scheme 2. Reported Mechanism for Pyridine Dicarbonitrile
Synthesis5
be by aerobic oxidation. To probe the reaction mechanism, a
stepwise reaction was carried out under nitrogen. The formation
of product as a crystalline solid was normally observed during
the final period at reflux. However, in the absence of air, no
product formation was observed. On admission of air to the
system, product crystallization was observed in the normal way
giving 8 in 36% yield. Interestingly, after carrying out the
reaction under nitrogen, oxidation could also be achieved using
a stoichiometric amount of a 1 M aqueous solution of KMnO4.
This resulted in the formation of 8 in 44% yield.
Figure 2. Binding pose of compound 2 (ball-and-stick) as predicted
by GOLD within the hypothetical binding pocket (green). The four
amino acids involved in hydrogen bonding, tyr-128, agr-164, asp-167
and tyr-169, are shown (blue), and hydrogen bonds are represented by
yellow dotted lines.
This experiment was repeated with sampling for analysis by
mass spectroscopy at each stage of the procedure. Molecular
ions from intermediates 9, 10, and 11 were observed to be
present, but product 8 was only discernible on contact with air
(Figure 3). These experiments confirm the presence of the
expected intermediates but also show that chemical oxidation
is required for the final aromatization of the pyridine ring.
As part of the optimization of the synthesis of pyridine
dicarbonitriles, the single-step, one-pot procedure was also
investigated further. Heating a solution of benzaldehyde,
thiophenol, and 2 equiv of malononitrile in ethanol with a
catalytic amount of piperidine at reflux for 3 h resulted in the
formation of 8 in 43% isolated yield. The effects of reaction
time, overall reaction concentration, ratios of reagents, and
reaction temperature were also investigated for their effect on
the yield. It was found that the ratios of 1:1:2 for the aldehyde,
thiol, and malononitrile, respectively, were indeed optimum. It
was also found that both doubling and halving the volume of
solvent used had no discernible effect on the yield. Changing
the solvent from ethanol to ethylene glycol, to allow access to
higher temperatures (130 °C), resulted in a 20% drop in yield.
The most significant result came from variation of the reaction
time. Reducing the reflux period from 3 h to 1 h gave a yield
of 40%, but increasing the reaction to 15 h at reflux raised the
yield to 48%. Consequently, subsequent reactions were allowed
to reflux overnight.
Scheme 1. Reterosynthesis of Pyridine Dicarbonitriles (core
structure numbering shown)
Elghandour et al. have reported a synthesis of pyridine
dicarbonitriles from an aldehyde, a thiol, and 2 equiv of
malononitrile (Scheme 1).11 A restricted substructure search of
the Available Chemicals Directory (ACD) provided a total of
21 arylthiols and 50 arylaldehydes. In combination, these
reagents were used to construct an initial virtual library of 1050
compounds. This library was docked and scored using GOLD
as described above, and analysis of the predicted binding pose,
along with Lipinski’s rule of five,12 was used in the selection
of 45 compounds for synthesis.
Synthesis. When the one-pot, one-step procedure was applied
to our starting materials, the yields were significantly lower than
the 75% reported for 2-amino-4-ethyl-6-phenylsulfanylpyridine-
3,5-dicarbonitrile (Scheme 1).11 For this reason, a simple
variation on the one-step, one-pot procedure was employed for
the investigation of the reaction conditions.
The reported mechanism of pyridine dicarbonitrile formation
(Scheme 2) involves the attack of a malononitrile anion upon
the aldehyde followed by elimination of water to form adduct
5, which is followed by addition of thiol to form 6. Subsequent
addition of a second malononitrile anion provides penultimate
intermediate 7, which is followed by (according to Kambe et
al.13) loss of molecular hydrogen to furnish the product.
Initially, synthesis was therefore carried out by a stepwise
addition of reagents, product 8 being prepared in 34% yield.
For expediency, this procedure was applied to the parallel
synthesis of 36 pyridine dicarbonitriles without further optimiza-
tion (Tables 2 and 3).
In an effort to raise the yield further, several alternative bases
were evaluated. Twenty-four reactions were carried out in
parallel, with ethanol as solvent, and the yields calculated from
HPLC analysis of the crude reaction mixture. Piperidine (used
However, we had doubts about the accuracy of the proposed
loss of molecular hydrogen as the last stage of the reaction as
the mechanism of this process was not clear, and evolution of
gas was not observed during the course of the reaction. It was
postulated that the required loss of two hydrogen atoms may
Figure 3. Intermediates observed by mass spectroscopy.

Pyridine Dicarbonitriles as Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 609
Figure 4. Observed side product.
Table 1. Investigation of Bases in Both Catalytic and Stoichiometric
Amounts
no. of calcd yield [%] isol yield [%]
equiv
entry
basea
of 8b
of 8c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
none
piperidine
piperidine
-
0
34
28
29
27
32
29
0
41
34
42
31
0
0.1
1.1
0.1
1.1
0.1
0.1
1.1
0.1
1.1
0.1
1.1
0.1
1.1
0.1
1.1
0.1
0.1
0.1
0.1
1.1
0.1
1.1
1.1
48
Figure 5. Compounds 14, 15, and X-ray crystal structure conformation
of 14.
morpholine
morpholine
thiomorpholine
pyrrolidine
pyrrolidine
N,N-DIPEA
N,N-DIPEA
Et3N
Et3N
pyridine
pyridine
2,4,6-collidine
2,4,6-collidine
DMAP
The pyridine dicarbonitrile library was prepared using a 24-
well parallel synthesis apparatus. The stepwise procedure was
used initially, as described above. However, once it was found
that the one-step, one-pot procedure offered improved yields,
subsequent syntheses were approached in this way.
It was found that the outcome of the syntheses was influenced
to a greater extent by the aldehyde, rather than the thiol. The
use of 3-methoxy- and 4-methoxybenzaldehydes, 4-formylben-
zoic acid methyl ester, and 4-methanesulfonylbenzaldehyde
consistently gave relatively high yields. The use of both 3-fluoro-
4-hydroxy- and 3-chloro-4-hydroxybenzaldehydes furnished
product in low yields. All of the syntheses attempted with 3,4-
di- and 3,4,5-trihydroxy aldehydes failed to give the desired
products. This was also the case with 2-furaldehyde.
Interestingly, in the attempt to synthesize tert-butyl compound
13, although the desired compound was not formed, penultimate
intermediate 14 was isolated in 32% yield (Figure 5). This
compound was stable in air. Attempts to oxidize 14 under the
KMnO4 conditions described above resulted in dealkylation as
well as aromatization giving pyridine dicarbonitrile 15 in 77%
yield. This was also the case when pyridinium chlorochromate
(PCC) was used, giving 15 in 47% yield. However, oxidation
was achieved by treatment with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) giving 13 in 25% yield.
Screening (SPR). Screening was carried out against three
forms of prion protein: huPrPC, t-huPrPC, and moPrPC. The
interactions between the dicarbonitrile ligands and the prion
proteins were measured using SPR technology. The protein was
covalently bound to a carboxymethylated dextran surface, and
the ligands were dissolved at 40 µM in phosphate buffer
containing 6.5% (v/v) DMSO. The ligand was passed over the
immobilized protein, and real-time association/dissociation was
recorded as sensorgrams. Visual inspection of the sensorgrams
was performed to identify compounds which were interacting
with the surface rather than the protein. These compounds were
removed from the list due to uncertainty over the degree of
binding. Responses of less than 2.5 response units (RU) were
regarded as not binding, and they have been represented as
0%RUmax. The observed equilibrium binding was expressed
as a percentage of maximum binding for a 1:1 interaction
(%RUmax). DMSO correction was performed to account for
small variations between the DMSO concentration of the
running buffer and the sample solution.
31
30
0
15
24
38
1
0
0
0
0
0
0
35
aniline
N-methylaniline
N,N-dimethylaniline
N,N-dimethylaniline
N,N-diethylaniline
K2CO3
NaOH
a N,N-DIPEA ) N,N-diisopropylethylamine, DMAP ) N,N-dimethyl-
aminopyridine. b Calculated from HPLC data. c Isolated yield of 8.
initially), triethylamine, N,N-DIPEA, and DMAP gave the
highest yields (Table 1). It can be seen that, with the exception
of 2,4,6-collidine, the bases performed better in catalytic
amounts. The use of 0.1 equiv of pyrrolidine gave the desired
product, whereas the use of 1.1 equiv led to the formation of
known product14 12 in 52% yield (Figure 4), which may partially
explain why the yields of the desired products were, in most
cases, lower when stoichiometric amounts of base were used.
To investigate the ease of product recovery, reactions with
the four best bases (at 0.1 equiv) were carried out and calculated
yields compared to isolated yields. It was found that the use of
piperidine gave 8 in the highest isolated yield of 48% (Table
1). The disparity between the calculated and isolated yields may
be due to HPLC sample dilution factors. On the basis of these
investigations it was decided to continue using piperidine at
0.1 equiv as catalyst.
No further optimization was attempted in the light of recent
publications. Chang and co-workers reported the stepwise
synthesis of a small library of pyridine dicarbonitriles resulting
in yields ranging from 20 to 50%.15,16 It was therefore presumed
unlikely that our yields could be improved significantly.
However, although our yields are comparable, the one-step, one-
pot procedure offers many advantages.
Library Synthesis. The compounds selected for synthesis
from the virtual library were augmented with a view to
increasing the structural diversity of the library. Pentanethiol,
N-acetylcysteamine, and benzylthiol were included to modify
position 6. Substitution at position 4 was varied further by the
inclusion of 2-thiophenecarboxaldehyde and 2-furaldehyde.
Previously a considerable increase in binding has been
observed with the age of the chip.10,17 For this reason all of the
compounds reported here were screened in two runs, only
allowing one injection per compound, enabling screening of all

610 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Table 2. Synthesis and Screening Resultsa
Reddy et al.
compd
X
Y
yield [%]
huPrPC [%RUmax
]
t-huPrPC [%RUmax
]
moPrPC [%RUmax
]
8
19
2
-
-
-
-
-
-
-
-
-
-
43b
18
-
0
0
0
0
0
11.4
37.6
0
0
0
26.8
0
0
0
0
0
60.6
0
8.8
9.5
33.2
0
0
0
43.1
0
-
53.8
64.6
0
0
-
52.3
0
0
0
0
0
0
18.8
-
8.5
13.3
40.1
0
3-Cl
4-Cl
3-OH
4-OH
c
50
51
52
53
48
49
20
21
22
23
24
25
26
27
28
16
29
30
31
32
37
38
39
33
17
35
36
34
40
10
16
17
11
3
23
47b
34b
20
31b
27
8
70.3
0
8.0
7.7
125.3
0
0
0
34.0
0
-
44.2
53.4
0
0
-
118.7
8.6
0
0
0
0
0
17.2
-
5.9
9.4
37.3
0
3-NH2
4-NH2
3-CO2CH3
4-CO2CH3
-
-
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
3-Cl
3-Cl
3-Cl
3-OCH3
4-OCH3
3-OH
4-SO2CH3
4-CHO
3-F, 4-OH
3-Cl, 4-OH
4-COCH3
4-CO2CH3
4-OH
0
23.1
0
d
d
d
-
28.0
33.4
0
3
6
18
25
25b
47b
40b
46
52
3
14
17
3
2
47
3
0
-
56.5
6.7
0
0
0
0
0
9.2
-
0
6.4
20.0
0
d
d
d
4-Cl
3-OCH3
4-OCH3
4-CO2CH3
4-CO2CH3
4-OH
4-CO2CH3
3-OH
3-Cl, 4-OH
4-CO2CH3
4-CO2CH3
2,6-F
3-Cl
3-CO2CH3
4-CO2CH3
4-CO2CH3
2-F
d
d
d
2-F
4-CO2H
4-NHCOCH3
4-NHCOCH3
4-NHCOCH3
3,5-F
25
a Compounds screened at 40 µM for binding to three forms of PrPC. b Prepared by the one-step, one-pot procedure. c Compound purchased from Maybridge,
UK. d Measurement not possible due to interference with the chip surface.
the compounds within 48 h. Quinacrine was used as a standard
at the start and end of each run to quantify the change of binding
over time.18 The standard deviation for quinacrine over the two
runs was 11.6%, and this value was assumed to represent the
error margin for all the other compounds.
the case of compound 19, no interaction with the proteins was
detected, as was the case for compounds 20 and 21.
Compounds arising from 4-mercaptophenol where Y ) 3-OH,
(3-F, 4-OH) and (3-Cl, 4-OH) (22, 25, and 26) showed good
binding, whereas in the cases of 27 and 28 no binding was
observed. This indicates that where X ) 4-OH substitution at
the 3 and/or 4 position of the aryl aldehyde is required for
binding; however, no clear commonality could be discerned
among the favored functional groups. Compound 29, derived
from 3-chlorothiophenol and 4-chlorobenzaldehyde, bound to
huPrPC strongly, whereas compounds 30, 31, and 32 showed
weak or no binding.
The results are shown in Tables 2 and 3. It was observed
that the chip surface interfered with the binding of four
compounds (16, 17, 18, and 24); therefore, their binding data
has been omitted from the tables as it was deemed unreliable.
The remaining compounds included 19 binders and 24 non-
binders. Most of the binders showed a similar level of interaction
with huPrPC and moPrPC, whereas the binding toward t-huPrPC
was generally lower.
Of the remaining compounds shown in Table 2, compounds
33 and 34 displayed moderate binding, compounds 35 and 36
bound only weakly, and compounds 37, 38, 39, and 40 showed
no binding at all.
To identify structural motifs important for binding, the
compounds were ordered according to their pattern of substitu-
tion. The compounds shown in Table 2 were built around the
core structure of compound 8, which was found not to be
binding, where the aryl groups of both aldehyde and thiol
precursors are unsubstituted. Compounds arising from func-
tionalized aryl thiols and benzaldehyde showed good binding
where X ) 3-OH and 3-CO2CH3, whereas no interaction was
detected for X ) 4-OH and 4-CO2CH3. However, weaker
binding was observed where X ) 4-Cl, 3-NH2, and 4-NH2. In
The compounds shown in Table 3 depart from the core
structure of 8 in that they originate from an aliphatic thiol and/
or aldehydes bearing a heterocycle, or in the case of 13,
pivalaldehyde. Of the compounds bearing a thiophene at the
4-position only two, compounds 41 and 42, showed significant
interaction with all three forms of PrPC. The para- to meta-
substitution effect, seen with several compounds in Table 2, is

Pyridine Dicarbonitriles as Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 611
Table 3. Synthesis and Screening Results
Compounds screened at 40 µM for binding to three forms of PrPC. a Prepared by the one-step, one-pot procedure. b Compound purchased from Maybridge,
UK. c Measurement not possible due to interference with the chip surface. d After oxidation of 14 with DDQ.
also observed between compounds 4 and 41, with the binding
raised from 0 to 123.6%RUmax respectively. Surprisingly,
compounds 4 and 2 (Table 2) showed weak binding to t-huPrPC
but none to huPrPC. One hypothesis for this anomalous behavior
could be that these compounds are interacting with a binding
region created by the truncation of the protein.
All of the compounds synthesized with benzylthiol showed
binding to huPrPC with the exception of 44. The most
pronounced binding was seen with 45, bearing a 3-OH sub-
stituent on the aryl aldehyde, followed by 42. Compounds 46,
47, and 13 failed to interact with the protein in all three forms,
as did all intermediates and side products isolated (12, 14, 15,
data not shown in tables).
the hypothetical pocket and the group at position 4 is placed
toward the opening.
A good drug candidate is expected to have a fast and strong
association, but a reasonably slow dissociation to maintain a
certain drug concentration (Figure 6A). Both fast association
and dissociation is not ideal (Figure 6B). The binding compari-
son above concerned only association, without taking dissocia-
tion into consideration. To characterize the interactions between
prion protein and the ligands in more detail, the binding data
was further analyzed with respect to R1 and R2 and presented
in Figure 6C.
Most of the data points are concentrated along the y axis,
implying a rapid dissociation. However, compounds 25, 29, 36,
41, 42, and 48, where R1 was >10%, showed a slower rate of
dissociation. Compounds 25, 36, and 42 only showed significant
R2 values when binding to one form of PrPC, whereas
compounds 29, 41, and 48 exhibited higher R2 values upon
From the data presented above, it may be suggested that the
nature of the substituent at the 4-position of the pyridine has
less of an influence on binding than that on the thioether at the
6-position. To some extent this was predicted by the binding
poses generated in silico, where the thioether resides deep inside

612 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Reddy et al.
Figure 6. A: Sensorgram from 3 to illustrate association response 1 (R1) and dissociation response 2 (R2) implying slow dissociation. B: Sensorgram
from 45 to illustrate R1 and a low R2 implying fast dissociation. C: Graph of R1 vs R2 (Compounds with slow dissociation are labeled).
interaction with both forms of human PrPC. Interestingly, all of
hydrogen bonding (4.0 Å), and van der Waals (2.5 Å) parameters
were used as default. A docking sphere was placed within the
hypothetical binding pocket of the PrPC [1QM3 (15)] with a user-
defined origin and a radius of 15 Å.
the latter compounds bear meta substitution on the arylthio
moiety.
In summary, compounds 25, 29, 36, 41, 42, and 48 were
deemed to be the most promising candidates for demonstrating
biological activity, and as such, were subjected to cell-line assay.
Cell-Line Screening. Biological assays were carried out on
SMB cells of mesodermal origin, originally established by the
culture of mouse brain clinically affected by Chandler scrapie
isolate. The procedure used was based upon that reported by
Rudyk et al.19 Full experimental details can be found in the
Supporting Information.
Compounds 25, 29, 36, 41, 42, and 48, together with literature
compound 1, were screened at 24 µM for inhibition of PrPSc
formation. Perrier et al. reported 1 as active in ScN2a cells with
an IC50 of ∼20 µM,8 so it was noted with interest that, in our
SMB cell lines, compound 1, along with 25, 29 and 48, showed
no activity. Compounds 36 and 41 were found to be toxic.
However, at 24 µM, compound 42 proved positive for PrPSc
inhibition. It is interesting to note that this compound had a
favorable R1/R2 ratio as discussed above, and in our assay,
demonstrated a higher potency than 1.
Commercially available structures for the virtual library design
were retrieved from the ACD. SMARTS (SMiles ARbitrary Target
Specification) strings were written consisting of the following
features: any thiophenol or benzaldehyde with any of the following
substituents at either or both the meta- and para-positions, or any
single substituent at either position (Cl, F, OH, NHCH3, COCH3,
NHCOCH3, CO2CH3, CO2H). To avoid steric clashes with the rest
of the molecule, only fluorine was allowed at the ortho-position.
This resulted in a total of 21 arylthiols and 50 arylaldehydes within
chosen cost parameters. In combination, these reagents were used
to construct an initial library of 1050 compounds using Legion 6.8
(SYBYL module). 2-Amino-6-(phenylthio)-4-phenylpyridine-3,5-
dicarbonitrile was defined as a product core structure. Benzaldehyde
and thiophenol were defined as reactant cores for their respective
analogues. Groups that were not part of the core were labeled as
R1, R2, R3, R4, R5. These R groups were extracted as fragment
SLNs. Mapping of the reactants to the product was carried out,
and the library was generated.
SPR Screening. Screening was performed using a BIAcore 3000
(BIAcore, Uppsala, Sweden) equipped with a CM5 sensor chip.10
N-Hydroxysuccinimide (NHS), N-ethyl-N′-(3-diethylaminopropyl)-
carbodiimide hydrochloride (EDC), 1 M ethanolamine, HBS-EP
buffer, surfactant P20, and regeneration solution (10 mM glycine-
HCl, pH 3.0) were purchased from BIAcore. Sodium phosphate,
ethylenediamine tetraacetic acid (EDTA), sodium chloride, sodium
hydroxide, and dimethyl sulfoxide (DMSO) were purchased from
Sigma-Aldrich. HuPrPC, t-huPrPC, and moPrPC were kindly pro-
vided by the Institute for Animal Health (Compton, UK).
HBS-EP buffer was used for immobilization of the protein. The
CM-dextran on the sensor chip was activated by mixing equal
volumes of 100 mM NHS and 400 mM EDC followed by injection
of the mixture over the chip surfaces for 7 min at a flow rate of 5
µL/min. The huPrPC, t-huPrPC, and moPrPC, each at a concentration
of 2 µg/mL, was injected through flow cells 2, 3, and 4 for 7 min.
Flow cell 1 was used as a control channel. The surfaces were
washed thoroughly immediately after the ethanolamine blocking
step with three consecutive injections of a mixture of 25 mM NaOH
and 1 M NaCl at of 8 s intervals. The surface was then equilibrated
with the running buffer for 30 min prior to the injection of sample
solutions. On average, an immobilization response of 3000-4000
RU was achieved for each protein.
Conclusions
In conclusion, the synthesis of pyridine dicarbonitriles has
been thoroughly investigated leading to an improved under-
standing of the reaction mechanism. Although exploration of
the reaction conditions did not lead to very high yields, a library
of 45 pyridine dicarbonitriles was synthesized and screened for
binding to three forms of PrPC. A total of 19 compounds were
found to bind to huPrPC, and 6 displayed the slow dissociation
characteristics expected in drug candidates. One compound, 42,
showed a higher activity than literature compound 1. The
pyridine dicarbonitriles therefore represent a class of compounds
worthy of further investigation as potential prion disease
therapeutics.
Experimental Section
Modeling. All the ligands were prepared for GOLD docking
using SYBYL 6.9 and used as MOL2 files. Energy minimization
was achieved by using steepest descent and conjugate gradient
methods prior to docking. Molecular docking studies were per-
formed using GOLD 2.1 in 10 independent genetic algorithm (GA)
runs, and for each of these a maximum number of 100000 GA
operations were performed on a single population of 100 individu-
als. Crossover, mutation, migration (95, 95 and 10 respectively),
Screening was run at 25 °C with a flow rate of 30 µL/min using
phosphate buffer as a running buffer (10 mM sodium phosphate,
pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant
P20) containing 6.5% DMSO. DMSO calibration using buffer

Pyridine Dicarbonitriles as Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 613
samples containing 5.5-7.5% DMSO was carried out to correct
for solvent effects.
2-Amino-6-phenylsulfanyl-4-thiophen-2-yl-pyridine-3,5-dicar-
bonitrile (18). Product 18 was isolated as a dark yellow powder
(18% yield). Mp 207-208 °C; m/z (ES), 335 ([M + H]+); found
335.0412 (C17H11N4S2 [M + H]+, requires 335.0425).
2-Amino-6-(3-chlorophenylsulfanyl)-4-phenylpyridine-3,5-di-
carbonitrile (19). Product 19 was isolated as a light brown powder
(18% yield). Mp 212-213 °C; m/z (ES), 363 ([M + H]+); found
363.0489 (C19H12ClN4S [M + H]+, requires 363.0471).
2-Amino-4-(3-hydroxyphenyl)-6-(4-hydroxyphenylsulfanyl)-
pyridine-3,5-dicarbonitrile (22). After ice treatment, no precipitate
was observed. The resultant mixture was extracted thoroughly with
chloroform. The combined organic layers were dried over MgSO4
and concentrated under reduced pressure. Purification of the crude
product by flash column chromatography (THF, Rf ) 0.73) gave
22 as a brown powder (17% yield). Mp 292-293 °C decomp; m/z
(ES); 361 ([M + H]+); found 361.0742 (C19H13N4O2S [M + H]+
requires 361.0759).
2-Amino-4-(4-formylphenyl)-6-(4-hydroxyphenylsulfanyl)py-
ridine-3,5-dicarbonitrile (24). After ice treatment, followed by
flash column chromatography by gradient elusion (petroleum ether/
EtOAc, 2:1 to 1:1, Rf ) 0.2) product 24 was isolated, as a reddish-
yellow powder (27% yield). Mp 275-276 °C decomp; m/z (ES),
371 ([M - H]-); found 371.0599 (C20H11N4O2S [M - H]- requires
371.0603).
2-Amino-4-(3-fluoro-4-hydroxyphenyl)-6-(4-hydroxyphenyl-
sulfanyl)pyridine-3,5-dicarbonitrile (25). After ice treatment,
followed by flash column chromatography (EtOAc, Rf ) 0.6),
product 25 was isolated as a yellow powder (8% yield). Mp > 300
°C; m/z (ES), 379 ([M + H]+); found 379.0663 (C19H12FN4O2S
[M + H]+, requires 379.0665).
2-Amino-4-(3-chloro-4-hydroxyphenyl)-6-(4-hydroxyphenyl-
sulfanyl)pyridine-3,5-dicarbonitrile (26). After ice treatment
followed by flash column chromatography (dichloromethane/
methanol, 95:5, Rf ) 0.1), product 26 was isolated as a yellow
powder (3% yield). Mp 274-275 °C decomp; m/z (ES), 395 ([M
+ H]+); found 395.0367 (C19H12N4ClO2S [M + H]+ requires
395.0370).
4-(4-Acetylphenyl)-2-amino-6-(4-hydroxyphenylsulfanyl)py-
ridine-3,5-dicarbonitrile (27). Isolated by flash column chroma-
tography (petroleum ether/EtOAc, 1:1, Rf ) 0.3) gave product 27
as a white powder (6% yield). Mp 261-263 °C decomp; m/z (ES),
387 ([M + H]+); found 387.0900 (C21H15N4O2S [M + H]+, requires
387.0916).
4-[2-Amino-3,5-dicyano-6-(4-hydroxyphenylsulfanyl)pyridin-
4-yl]benzoic acid methyl ester (28). Product 28 was isolated as a
white powder (18% yield). Mp 299 °C decomp; m/z (ES), 425 ([M
+ Na]+); found 425.0676 (C21H14N4NaO3S [M + Na]+, requires
425.0684).
Each analytical cycle consisted of an injection of a 40 µM
solution of sample in running buffer for 1 min (association phase)
followed by running buffer for 3 min (dissociation phase). Two
subsequent surface regenerations were carried out at a flow rate of
35 µL/min. First, a 30 s injection of 25 mM NaOH/1 M NaCl with
0.0005% SDS (pH 8.5), followed by a 35 s injection of 10 mM
glycine-HCl (pH 3.0). A typical run consisted of 10 buffer
injections, followed by a DMSO calibration block in triplicate. A
single injection of quinacrine was followed by 19 samples, each a
single analytical cycle. A second DMSO calibration block was
followed by a further 19 analytical cycles, with another quinacrine
injection and a third DMSO calibration block to finish. The binding
was expressed as %RUmax to rank the binding affinity of com-
pounds, which can be calculated by the following equation:
RU of compound × MW protein
%RUmax
)
× 100
RU immoblised protein × MW compound
Synthesis. All reagents were purchased directly from commercial
sources and used as supplied. Melting points were measured using
a Bibby-Sterilin SMP10 melting point apparatus and are uncor-
rected. Accurate mass and nominal mass measurements were
measured using a Waters-Micromass LCT electrospray mass
spectrometer. TLC was performed using aluminum backed silica
gel 60 plates (0.20 mm layer). Flash column chromatography was
carried out using Fluorochem silica gel 60 Å. HPLC was carried
out using a Phenomonex Luna ODS (150 mm × 4.6 mm, 5 µm
particle size) column using acetonitrile/water (5% to 100% organic
over 20 min at 1 mL/min). Detection was at 254 nm. All compounds
were isolated in >95% purity unless otherwise stated (see Sup-
porting Information).
Stepwise One-Pot Procedure: 2-Amino-4-phenyl-6-phenyl-
sulfanylpyridine-3,5-dicarbonitrile (8). To a stirred solution of
benzaldehyde (0.51 mL, 5.00 mmol) in ethanol (10 mL) was added
a solution of malononitrile (0.33 g in 2.0 mL of ethanol, 5.00 mmol)
and 3 drops of piperidine. The pale yellow solution was then
refluxed for 75 min. To this was then added thiophenol (0.51 mL,
5.00 mmol), and the mixture was refluxed for a further 45 min.
Another 1 equiv of malononitrile (0.33 g in 2.0 mL of ethanol,
5.00 mmol) was added, and the reaction mixture was refluxed for
a further 60 min. The reaction mixture was then exposed to air
overnight. The yellow crystalline precipitate formed was collected
by suction filtration, washed with n-hexane/ethanol (9:1), followed
by n-hexane, and then dried in vacuo. Product 8 was obtained as a
yellow crystalline solid (34% yield). Mp 227-228 °C (Lit.,13 223-
224 °C); m/z (ES); 329 ([M + H]+); found 329.0845 (C19H13N4S
[M + H]+ requires 329.0861).
2-Amino-4-tert-butyl-6-phenylsulfanyl-1,4-dihydropyridine-
3,5-dicarbonitrile (14). As precipitate was not observed on
standing, and the reaction mixture was poured onto crushed ice
(50 mL), neutralized with a few drops of concentrated HCl, and
allowed to stand for 2 h. No solids were observed. Extraction of
the aqueous phase with CHCl3 followed by flash column chroma-
tography (EtOAc/n-hexane, 6:4, Rf ) 0.3) gave product 14 as a
yellow powder (32% yield). Mp 198-199 °C; m/z (ES), 311 ([M
+ H]+); found 311.1323 (C17H19N4S [M + H]+, requires 311.1330).
2-Amino-4-(4-hydroxyphenyl)-6-(4-hydroxyphenylsulfanyl)-
pyridine-3,5-dicarbonitrile (16). After ice treatment followed by
flash column chromatography (EtOAc, Rf ) 0.7), product 16 was
isolated as a yellow powder (25% yield). Mp >300 °C; m/z (ES),
359 ([M - H]-); found 359.0604 (C19H11N4O2S [M - H]- requires
359.0603).
2-Amino-4-(3-chloro-4-hydroxyphenyl)-6-(2-fluorophenylsul-
fanyl)pyridine-3,5-dicarbonitrile (17). After ice treatment followed
by flash column chromatography (petroleum ether/EtOAc, 2:1, Rf
) 0.6) followed by recrystallization from EtOAc/n-hexane, product
17 was isolated as a yellow powder (3% yield). Mp 261-263 °C;
m/z (ES), 397 ([M + H]+); found 397.0313 (C19H11ClFN4OS [M
+ H]+, requires 397.0326).
4-[2-Amino-6-(3-chlorophenylsulfanyl)-3,5-dicyanopyridin-4-
yl]benzoic acid methyl ester (32). Product 32 was isolated as a
dark yellow powder (48% yield). Mp 247-248 °C; m/z (ES), 421
([M + H]+); found 421.0526 (C21H14ClN4S [M + H]+, requires
421.0543).
2-Amino-6-(2-fluorophenylsulfanyl)-4-(3-hydroxyphenyl)py-
ridine-3,5-dicarbonitrile (33). After ice treatment followed by flash
column chromatography (EtOAc, Rf ) 0.6), product 33 was isolated
as a white powder (17% yield). Mp 232-233 °C; m/z (ES), 363
([M + H]+); found 363.0732 (C19H12FN4OS [M + H]+, requires
363.0716).
N-{4-[6-Amino-3,5-dicyano-4-(2,6-difluorophenyl)pyridin-2-
ylsulfanyl]phenyl}-acetamide (34). Isolation by flash column
chromatography (petroleum ether/EtOAc, 1:5, Rf ) 0.3) gave
product 34 as a yellow powder (3% yield). Mp 261-263 °C; m/z
(ES), 444 ([M + Na]+); found 444.0720 (C21H13F2N5NaOS [M +
Na]+, requires 444.0707).
2-Amino-6-(4-carboxyphenylsulfanyl)-4-(4-methylesterphenyl)-
pyridine-3,5-dicarbonitrile (35). The product was isolated by
filtration and recrystallization from methanol/dichloromethane
followed by flash column chromatography (petroleum ether/THF,

614 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Reddy et al.
1:3, Rf ) 0.3) gave product 35 as a brown powder (2% yield). Mp
288-290 °C decomp; m/z (ES); 431 ([M + H]+); found 431.0830
(C22H15N4O4S [M + H]+ requires 431.0814).
4-[2-(4-Acetylaminophenylsulfanyl)-6-amino-3,5-dicyanopy-
ridin-4-yl]benzoic acid methyl ester (36). Product 36 was isolated
as a pale yellow powder (47% yield). Mp 271-273 °C; m/z (ES);
444 ([M + H]+); found 444.1145 (C23H18N5O3S [M + H]+ requires
444.1130).
4-[6-Amino-3,5-dicyano-4-(3-methyl ester)pyridin-2-ylsulfa-
nyl]benzoic acid methyl ester (37). Product 37 was isolated as a
dark brown powder (52% yield). Mp 260-261 °C; m/z (ES), 467
([M + Na]+); found 467.0806 (C23H16N4NaO4S [M + Na]+,
requires 467.0790).
4-[6-Amino-3,5-dicyano-4-(4-hydroxyphenyl)pyridin-2-ylsul-
fanyl]benzoic acid methyl ester (38). Product 38 was isolated as
a white powder (3% yield). Mp > 300 °C; m/z (ES), 403 ([M +
H]+); found 403.0884 (C21H15N4O3S [M + H]+, requires 403.0865).
2-Amino-6-(4-methylesterphenylsulfanyl)-4-(4-methylester-
phenyl)pyridine-3,5-dicarbonitrile (39). Product 39 was isolated
as a light yellow powder (14% yield). Mp 294-295 °C; m/z (ES),
445 ([M + H]+); found 445.0956 (C23H17N4O4S [M + H]+, requires
445.0971).
N-{4-[6-Amino-3,5-dicyano-4-(3,5-difluorophenyl)pyridin-2-
ylsulfanyl]phenyl}acetamide (40). Product 40 was isolated as a
yellow powder (25% yield). m/z (ES), 444 ([M + Na]+); found
444.0699 (C21H13F2N5NaOS [M + Na]+, requires 444.0707).
2-Amino-6-(3-chlorophenylsulfanyl)-4-thiophen-2-ylpyridine-
3,5-dicarbonitrile (41). Product 41 was isolated as a brown powder
(23% yield). Mp 204-205 °C; m/z (ES), 369 ([M + H]+); found
369.0050 (C17H10ClN4S2 [M + H]+, requires 369.0035).
2-Amino-6-benzylsulfanyl-4-thiophen-2-ylpyridine-3,5-dicar-
bonitrile (42). Product 42 was isolated as a dark brown crystalline
solid (19% yield). Mp 202-204 °C; m/z (ES), 347 ([M + H]+);
found 347.0410 (C18H13N4S2 [M + H]+, requires 347.0425).
N-[4-(6-Amino-3,5-dicyano-4-thiophen-2-ylpyridin-2-ylsulfa-
nyl)phenyl]acetamide (43). Isolated by flash column chromatog-
raphy (petroleum ether/EtOAc, 1:3, Rf ) 0.2) gave product 43 as
a yellow powder (9% yield). Mp 261-263 °C decomp; m/z (ES),
390 ([M - H]-); found 390.0497 (C19H12N5OS2 [M - H]-, requires
390.0483).
2-Amino-6-(4-hydroxyphenylsulfanyl)-4-phenylpyridine-3,5-
dicarbonitrile (52). After ice treatment followed by dichlo-
romethane wash gave product 52 as a white powder (16% yield).
Mp 254-256 °C; m/z (ES), 345 ([M + H]+); found 345.0803
(C19H13N4OS [M + H]+, requires 345.0810).
2-Amino-6-(3-aminophenylsulfanyl)-4-phenylpyridine-3,5-di-
carbonitrile (53). Isolated by flash column chromatography
(petroleum ether/EtOAc, 1:1, Rf ) 0.1) gave product 53 as a
whitish-yellow powder (11% yield). Mp 230-232 °C; m/z (ES),
344 ([M + H]+); found 344.0966 (C19H14N5S [M + H]+, requires
344.0970).
2-Amino-6-(4-aminophenylsulfanyl)-4-phenylpyridine-3,5-di-
carbonitrile (54). After ice treatment, recrystallization from
methanol, gave product 54 as a yellow powder (17% yield). Mp
162-164 °C; m/z (ES), 344 ([M + H]+); found 344.0956
(C19H14N5S [M + H]+, requires 344.0970).
2-Amino-6-(4-aminophenylsulfanyl)-4-thiophen-2-ylpyridine-
3,5-dicarbonitrile (55). After ice treatment, product 55 was isolated
as a dark brown powder (9% yield). Mp 220-221 °C decomp; m/z
(ES), 350 ([M + H]+); found 350.0538 (C17H12N5S2 [M + H]+,
requires 350.0534).
2-Amino-6-(3-aminophenylsulfanyl)-4-thiophen-2-ylpyridine-
3,5-dicarbonitrile (56). Product 56 was isolated as a dark reddish
brown powder (22% yield). Mp 237-238 °C; m/z (ES), 350 ([M
+ H]+); found 350.0522 (C17H12N5S2 [M + H]+, requires 350.0534).
4-(6-Amino-3,5-dicyano-4-thiophen-2-ylpyridin-2-ylsulfanyl)-
benzoic acid methyl ester (57). Product 57 was isolated as a yellow
powder (17% yield). Mp 260-261 °C; m/z (ES), 393 ([M + H]+);
found 393.0492 (C19H13N4O2S2 [M + H]+, requires 393.0480).
2-Amino-6-benzylsulfanyl-4-(4-fluorophenyl)pyridine-3,5-di-
carbonitrile (58). Product 58 was isolated as a yellow powder (43%
yield). Mp 188-189 °C; m/z (ES), 361 ([M + H]+); found 361.0939
(C20H14FN4S [M + H]+, requires 361.0923).
One Step, One-Pot Procedure: 2-Amino-4-phenyl-6-phenyl-
sulfanylpyridine-3,5-dicarbonitrile (8). To a stirred solution of
benzaldehyde (0.51 mL, 5.00 mmol) in ethanol (10 mL) containing
3 drops of piperidine was added a solution of malononitrile (0.66
g in 4.0 mL of ethanol, 10.00 mmol) and thiophenol (0.51 mL,
5.00 mmol). The reaction mixture was heated at reflux for 18 h,
after which the reaction mixture was exposed to air for 3 h. The
yellow crystalline precipitate formed was collected by suction
filtration, washed with n-hexane/ethanol (9:1) and with n-hexane,
and then dried in vacuo. Product 8 was obtained as a yellow
crystalline solid (705 mg, 43% yield).
2-Amino-6-benzylsulfanyl-4-phenylpyridine-3,5-dicarboni-
trile (44). Product 44 was isolated as yellow crystalline solid (19%
yield). Mp 212-214 °C; m/z (ES), 341 ([M - H]-); found 341.0864
(C20H13N4S [M - H]-, requires 341.0861).
2-Amino-4-(3-methoxyphenyl)-6-phenylsulfanylpyridine-3,5-
dicarbonitrile (20). Product 20 was isolated as a white powder
(47% yield). Mp 278-279 °C; m/z (ES), 359 ([M + H]+); found
359.0982 (C20H15N4OS [M + H]+, requires 359.0967).
2-Amino-4-(4-methoxyphenyl)-6-phenylsulfanylpyridine-3,5-
dicarbonitrile (21). Product 21 was isolated as a pale yellow
powder (34% yield). Mp 251-253 °C; m/z (ES), 359 ([M + H]+);
found 359.0981 (C20H15N4OS [M + H]+, requires 359.0967).
2-Amino-6-(4-hydroxyphenylsulfanyl)-4-(4-methanesulfonyl-
phenyl)pyridine-3,5-dicarbonitrile (23). Product 23 was isolated
as a yellow powder (31% yield). Mp 203-204 °C; m/z (ES), 423
([M + H]+); found 423.0591 (C20H15N4O3S2 [M + H]+ requires
423.0586).
2-Amino-4-(4-chlorophenyl)-6-(3-chlorophenylsulfanyl)pyri-
dine-3,5-dicarbonitrile (29). Product 29 was isolated as a white
powder (25% yield). Mp 217-220 °C; m/z (ES), 395 ([M - H]-);
found 394.9937 (C19H9Cl2N4S [M - H]-, requires 394.9925).
2-Amino-6-(3-chlorophenylsulfanyl)-4-(3-methoxyphenyl)py-
ridine-3,5-dicarbonitrile (30). Product 30 was isolated as a white
powder (47% yield). Mp 211-212 °C; m/z (ES), 393 ([M + H]+);
found 393.0592 (C20H14ClN4OS [M + H]+, requires 393.0577).
2-Amino-6-(3-chlorophenylsulfanyl)-4-(4-methoxyphenyl)py-
ridine-3,5-dicarbonitrile (31). Product 31 was isolated as a pale
yellow powder (40% yield). Mp 192-194 °C; m/z (ES), 393 ([M
+ H]+); found 393.0592 (C20H14ClN4OS [M + H]+, requires
393.0577).
2-Amino-6-benzylsulfanyl-4-(3-hydroxyphenyl)pyridine-3,5-
dicarbonitrile (45). After ice treatment followed by flash column
chromatography (EtOAc, Rf ) 0.6), product 45 was isolated as a
yellow powder (25% yield). Mp 190-192 °C; m/z (ES), 359 ([M
+ H]+); found 359.0981 (C20H15N4OS [M + H]+, requires
359.0967).
3-(6-Amino-3,5-dicyano-4-phenylpyridin-2-ylsulfanyl)-ben-
zoic acid methyl ester (48). After ice treatment, recrystallization
from methanol followed by washing with dichloromethane gave
product 48 as a white powder (3% yield). Mp 247-248 °C; m/z
(ES), 387 ([M + H]+); found 387.0919 (C21H15N4O2S [M + H]+,
requires 387.0916).
4-(6-Amino-3,5-dicyano-4-phenylpyridin-2-ylsulfanyl)-ben-
zoic acid methyl ester (49). Product 49 was isolated as a white
powder (23% yield). Mp 297-298 °C; m/z (ES), 387 ([M + H]+);
found 387.0912 (C21H15N4O2S [M + H]+, requires 387.0916).
2-Amino-6-(4-hydroxyphenylsulfanyl)-4-thiophen-2-ylpyridine-
3,5-dicarbonitrile (50). After ice treatment product 50 was isolated
as a yellow powder (27% yield). Mp 236-238 °C; m/z (ES), 351
([M + H]+); found 351.0391 (C17H11N4OS2 [M + H]+, requires
351.0374).
2-Amino-6-(3-hydroxyphenylsulfanyl)-4-phenylpyridine-3,5-
dicarbonitrile (51). After ice treatment product 51 was isolated as
a yellow powder (10% yield, 93% purity). Mp 228-229 °C; m/z
(ES), 345 ([M + H]+); found 345.0808 (C19H13N4OS [M + H]+,
requires 345.0810).

Pyridine Dicarbonitriles as Prion Disease Therapeutics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 615
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having features in common with the infectious form. EMBO J. 2001,
20, 1547-1554.
(18) Vogtherr, M.; Grimme, S.; Elshorst, B.; Jacobs, D. M.; Fiebig, K.;
Griesinger, C.; Zahn, R. Antimalarial drug quinacrine binds to
C-terminal helix of cellular prion protein. J. Med. Chem. 2003, 46,
3563-3564.
(19) Rudyk, H.; Vasiljevic, S.; Hennion, R. M.; Birkett, C. R.; Hope, J.;
Gilbert, I. H. Screening Congo Red and its analogues for their ability
to prevent the formation of PrP-res in scrapie-infected cells. J. Gen.
Virol. 2000, 81, 1155-1164.
N-[2-(6-Amino-3,5-dicyano-4-phenylpyridin-2-ylsulfanyl)-
ethyl]acetamide (46). Product 46 was isolated as a white powder
(34% yield). Mp 224-226 °C; m/z (ES), 338 ([M + H]+); found
360.0895 (C17H15N5NaOS [M + Na]+ requires 360.0895).
4-(2-Amino-3,5-dicyano-6-pentylsulfanylpyridin-4-yl)-benzo-
ic acid methyl ester (47). Product 47 was isolated as a yellow
solid (20% yield). Mp 197-199 °C; m/z (ES), 381 ([M + H]+);
found 381.1395 (C20H21N4O2S [M + H]+ requires 381.1385).
2-Amino-6-phenylsulfanylpyridine-3,5-dicarbonitrile (15)
(KMnO4 procedure). To a stirred solution of 14 (50.0 mg, 0.15
mmol) in ethanol (5.0 mL) was added a solution of KMnO4 (23.7
mg, 0.15 mmol) in ethanol (15 mL) dropwise, and the reaction
was carried out overnight at room temperature. TLC showed the
presence of starting material, so the reaction was therefore heated
at reflux for 3 h, after which full conversion was achieved. The
reaction mixture was allowed to cool to room temperature and
poured into 50 mL of ice-water, and product 15 was isolated as a
white powder (77% yield). Mp 202-204 °C, m/z (ES), 251 ([M -
H]-); found 251.0380 (C13H7N4S [M - H]-, requires 251.0391).
2-Amino-6-phenylsulfanylpyridine-3,5-dicarbonitrile (15) (PCC
procedure20). Silica gel 60 Å (2.88 g) was added to a solution of
pyridinium chlorochromate (PCC) (206.9 mg, 0.96 mmol) in
acetone (4.0 mL). The solvent was evaporated under reduced
pressure, and the resulting solid was dried at 75 °C overnight.
Compound 14 (100 mg, 0.32 mmol) was added to a stirred
suspension of silica gel supported PCC (206.9 mg, 0.96 mmol) in
dichloromethane at room temperature. After 25 min, the reaction
was found to be complete by TLC. The solid was filtered, and the
solution was concentrated under reduced pressure to give product
15 as a white powder (47% yield).
2-Amino-4-tert-butyl-6-phenylsulfanylpyridine-3,5-dicarboni-
trile (13). To a stirred solution of 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) (109.0 mg, 0.48 mmol) in dichloromethane
(13 mL) was added 14 (100.0 mg, 0.32 mmol), and the reaction
was stirred overnight at room temperature. The reaction mixture
was evaporated to dryness, and the resultant solids were extracted
with 20 mL of n-hexane/EtOAc (6:4). After the removal of all
volatiles under reduced pressure, the crude product was purified
by flash column chromatography (n-hexane/EtOAc, 6:4, Rf ) 0.6).
Product 13 was obtained as a brown powder (25% yield). Mp 157-
159 °C, m/z (ES), 307 ([M - H]-); found 307.1005 (C17H15N4S
[M - H]-, requires 307.1017).
Acknowledgment. We thank the Department of Health for
their generous funding (Contract No. DH007/0102). We would
also like to thank J. Louth for carrying out the Biacore screening,
R. Hanson for the HPLC analysis, H. Adams for the X-ray
crystallography service, A. Gill (IAH, Compton, UK) for
supplying the PrPC, and C. R. Birkett at the TSE Resourse
Centre (IAH, Compton, UK) for the SMB cells.
Supporting Information Available: Experimental details of
1
the cell-line assays, IR and H and 13C NMR spectroscopic data,
X-ray crystallographic data for compounds 8 and 14, and assessment
of compound purity by HPLC. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Prusiner, S. B. Prions. Proc. Nat. Acad. Sci. U.S.A. 1998, 95, 13363-
13383.
(2) Will, R. G.; Ironside, J. W.; Zeidler, M.; Cousens, S. N.; Estibeiro,
K.; Alperovitch, A.; Poser, S.; Pocchiari, M.; Hofman, A.; Smith, P.
G., A new variant of Creutzfeldt-Jakob disease in the UK. Lancet
1996, 347, 921-925.
(20) Vandeneynde, J. J.; Mayence, A.; Maquestiau, A. A Novel Applica-
tion Of The Oxidizing Properties Of Pyridinium Chlorochromate -
Aromatization Of Hantzsch 1,4-Dihydropyridines. Tetrahedron 1992,
48, 463-468.
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