Synthesis, molecular structure, NMR spectroscopic and computational analysis of a selective adenosine A2A antagonist, ZM 241385

Article abstract of DOI:10.1007/s11224-012-0151-7

Herein, we describe the synthesis of the adenosine A_2A antagonist ZM 241385 (9) starting from commercially available 2-furanhydrazide (1) and including a comprehensive structural characterization of all the intermediates and the final product.

Full text of DOI:10.1007/s11224-012-0151-7

Struct Chem (2013) 24:1241–1251
DOI 10.1007/s11224-012-0151-7
Synthesis, molecular structure, NMR spectroscopic
and computational analysis of a selective adenosine
A2A antagonist, ZM 241385
•
Manuela Jorg Mark Agostino Elizabeth Yuriev
•
•
¨
Frankie S. Mak Neil D. Miller Jonathan M. White
•
•
•
•
Peter J. Scammells Ben Capuano
Received: 30 August 2012 / Accepted: 8 October 2012 / Published online: 27 October 2012
ꢀ Springer Science+Business Media New York 2012
Abstract Herein, we describe the synthesis of the aden-
osine A2A antagonist ZM 241385 (9) starting from com-
mercially available 2-furanhydrazide (1) and including a
comprehensive structural characterization of all the inter-
mediates and the final product. In addition, extensive NMR
analysis, including temperature and concentration-depen-
dent experiments, are reported as well as the first single-
crystal structure of the compound ZM 241385 (9) as the
trihydrate. Furthermore, an extensive structural comparison
of the single-crystal structure with the published protein
bound X-ray structures is reported.
Introduction
The recent interest in the adenosine A2A receptor as a
therapeutic target has significantly increased following
studies that revealed the co-administration of an adenosine
A2A antagonist with a dopamine D2 agonist showed
promising clinical efficacy in the treatment of dyskinesias
associated with Parkinson’s disease therapy [1–3].
ZM 241385 (9) is a potent and selective adenosine A2A
antagonist [4] and the first ligand for which X-ray struc-
tures in complex with the adenosine A2A receptor have
been solved [5, 6]. The molecule exhibits a distinctive
2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-amine
scaffold and has been extensively used as a lead compound
for various structure–activity relationship studies [2, 7, 8].
Nevertheless, limited experimental data and no compre-
hensive structural characterization (including X-ray crystal
structure) or analysis of the NMR spectral characteristics
has been published to date. Furthermore, the comparison of
the two published protein bound X-ray structures showed
some interesting differences which will be discussed later.
Keywords ZM 241385 ꢀ A2A antagonist ꢀ Synthesis ꢀ
Spectroscopic analysis ꢀ Crystal structure
Electronic supplementary material The online version of this
article (doi:10.1007/s11224-012-0151-7) contains supplementary
material, which is available to authorized users.
¨
M. Jorg ꢀ M. Agostino ꢀ E. Yuriev ꢀ P. J. Scammells ꢀ
B. Capuano (&)
Medicinal Chemistry, Monash Institute of Pharmaceutical
Sciences, 381 Royal Parade, Parkville, VIC 3052, Australia
e-mail: ben.capuano@monash.edu
Synthesis
M. Agostino
Western Australian Biomedical Research Institute, Curtin Health
Innovation Research Institute, School of Biomedical Sciences,
Curtin University, GPO Box U1987, Perth, WA 6845, Australia
ZM 241385 (9) was synthesized according to the pathway
illustrated in Scheme 1. The original patent synthesis of
ZM 241385 [4] commenced with the precursor compounds
aminoguanidine nitrate and 2-furonitrile. Our modified
synthesis, although comparable with literature, commenced
with commercially available 2-furanhydrazide (1) and
incorporates relatively inexpensive reagents.
F. S. Mak ꢀ N. D. Miller
GlaxoSmithKline, GSK R&D China, Singapore Research
Centre, 11 Biopolis Way, Helios Bldg #03-01/02, Singapore
138667, Singapore
J. M. White
A mixture of furan-2-carbohydrazide (1), S-meth-
ylisothiourea (2), and sodium hydroxide in water was stirred
at room temperature for 14 h and following work-up gave the
Bio21 Molecular Science & Biotechnology Institute, The
University of Melbourne, 30 Flemington Road, Parkville, VIC
3010, Australia
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Struct Chem (2013) 24:1241–1251
Scheme 1 Chemical synthesis of ZM 241385 (9)
furanoyl hydrazinecarboximidamide intermediate 3 in a
moderate yield of 58 %. The yield obtained was consistent
with that observed in the literature (52 % yield) [9]. The
following step was accomplished by employing one of two
methods published by Dolzhenko et al. [10], depending on
reaction scale. On a scale up to 1 g, intermediate 3 was
suspended in water and the reaction was performed in the
microwave at 140 ꢁC for 1 h before the solvent was removed
under vacuum. The yield for this method was virtually
quantitative (99 %). On a larger scale, the reaction of the
aqueous suspension was performed using conventional
heating at 100 ꢁC for 29 h. The reaction volume was reduced
and the precipitate filtered. Yields up to 80 % were achieved
via the second method. 5-(Furan-2-yl)-1H-1,2,4-triazol-3-
amine (4) was reacted with N-cyanodithioiminocarbonate
(5) to afford 6 in 32 % yield after column chromatography
[4, 11]. The sulfide intermediate 6 was subsequently oxi-
dized with m-chloroperoxybenzoic acid at room temperature
to afford the sulfone 7 in excellent yield (82 %) [4, 11].
Nucleophilic displacement of the methylsulfonyl group with
tyramine (8) [4] furnished the target triazolotriazine (9,
ZM 241385) in a 33 % yield with purity greater than 97 % as
determined by analytical HPLC.
1:1 ratio were actually observed (Figs. 1, 2). Caulkett et al.
[11] observed the same phenomenon and demonstrated that
the splitting was temperature-dependent; at 373 K the
doublet coalesced to one singlet. In the case of compound
6, we performed dilution experiments that showed the
signal shape of the primary amine to be dependent on the
concentration of the sample. Figure 1 shows two broad
singlets (8.7–9.2 ppm) with a sample concentration of
7 mg/mL, one broad singlet at 1 mg/mL and again two
broad singlets at 0.1 mg/mL. Dilution experiments (Fig. 2)
were also performed with molecule 7, but the signals never
coalesced. Interestingly, when the compound [1,2,4]triaz-
olo[1,5-a][1,3,5]triazin-7-amine (10) (Fig. 3) was analyzed
under the same conditions, it did not exhibit any doubling
up of the amino signal at any concentration.
The NMR spectra of ZM 241385 (9) not only exhibited
splitting of the proton signals in the 1H NMR spectrum, but
also splitting of the carbon signals in the 13C NMR spec-
trum. The NH2 and NH groups of ZM 241385 (9) in the
1H NMR spectrum in d6-DMSO each displayed two signals
in a ratio of 1:6 and 1:2, respectively. Figure 4 shows that
the amino signals coalesced at 353 K into one broad singlet
and one broad triplet for the NH2 and NH groups,
respectively. The splitting patterns of the CH2 signals of
the tyramine moiety in the proton spectrum also exhibit
greater resolution at 353 K compared to 298 K.
NMR analysis
Figure 5 shows the amino group resonances in the
1H NMR spectra at different temperatures. A closer look
revealed that the ratio of the two signals did not change, but
rather the two signals moved closer together until they finally
coalesced. At 303 K (red, spectrum 1), the two signals are
clearly observed for each of the two amino groups (NH2 and
NH). By heating the sample in 10 K increments, the signals
slowly moved closer together until they completely coa-
lesced at 343 K, (dark blue, spectrum 5).
Intermediates 6 and 7, as well as the final compound
ZM 241385 (9), showed very interesting and somewhat
unexpected features in their respective NMR spectra. In
this section, a number of NMR experiments are presented
and the respective findings and interpretations are sum-
marized and evaluated. Initially, we expected a broad
singlet for the hydrogens of the NH2 group for compounds
1
6 and 7 in the H NMR, however, two broad singlets in a
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Struct Chem (2013) 24:1241–1251
1243
1
Fig. 1 Section of the H NMR
spectrum of compound 6, which
shows the NH2 signals and the
three furanyl protons at a
3
sample concentration of
7 mg/mL (red, spectrum 1),
1 mg/mL (green, spectrum 2),
and 0.1 mg/mL (blue, spectrum
3) (Color figure online)
2
NH2
N
1
N
N
O
N
S
N
6
9.4
9.0
8.6
8.2
7.8
7.4
7.0
6.6
6.2
f1 (ppm)
1
Fig. 2 Section of the H NMR
spectrum of compound 7 which
shows the NH2 signals and the
three furanyl protons at a
sample concentration of
7 mg/mL (red, spectrum 1),
1 mg/mL (green, spectrum 2),
0.1 mg/mL (blue, spectrum 3),
and 0.01 mg/mL (black,
spectrum 4) (Color figure
online)
4
3
2
1
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
f1 (ppm)
NH2
7 in a 1:1 ratio, however, no anomalies were observed in
the 13C NMR spectrum (Fig. 7).
N
N
N
N
The splitting of the amino signal was not observed for
compound 10, therefore intramolecular interactions such as
hydrogen bonding are unlikely to explain this phenomenon for
intermediates 6 and 7. Tautomerism was excluded due to the
observations that (i) no additional carbon signals were observed,
(ii) no additional cross peaks were present in the 15N-13C
HQMC, and (iii) that it would be very unlikely to observe an
exact 1:1 ratio of the amino signals for both compounds 6 and 7.
The fact that the signals in molecules 6 and 7 appear in exactly a
1:1 ratio, in addition to the observed temperature and concen-
tration dependency, indicates that intermolecular interactions,
such are dimer formation, are likely to be responsible for the
observed doubling up of the amino group resonances.
N
10
Fig. 3 Chemical structure of [1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
amine (10)
Unlike compounds 6 and 7, for which no spectral
anomalies were observed in their respective 13C NMR
spectra, the title compound, ZM 241385 (9), showed dou-
bling up of most carbon signals. This effect was observed
in both d4-methanol and in d6-DMSO. The doubled up
carbon signals also exhibited a temperature-dependence,
whereby they coalesced at 353 K (Fig. 6, blue spectrum).
Interestingly, the 2-(furan-2-yl)-5-phenethoxy-[1,2,4]
triazolo[1,5-a][1,3,5]triazin-7-amine (11), which contains a
phenethoxy moiety instead of the tyramine moiety, also
exhibited doubling up of the amino group signal at position
In the case of the literature compound ZM 241385 (9), the
spectral phenomena are more complex and we propose that
this is due to the two effects occurring simultaneously,
namely, intermolecular interactions, such as dimer formation,
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Struct Chem (2013) 24:1241–1251
Fig. 4 1H NMR spectrum of
ZM 241385 (9) at 298 K (red,
spectrum 1) and 353 K (blue,
spectrum 2) (upper). The lower
left spectrum shows a zoomed
section of the amino protons,
that each show two signals at
298 K and one signal at 353 K.
The lower right spectrum shows
the CH2 signals of the tyramine
moiety at 298 and 353 K (Color
figure online)
NH
NH
2
10.0
NH
8.0
6.0
f1 (ppm)
4.0
2.0
0.0
NH
2
NH
NH2
NH
2
HO
N
N
O
N
N
N
N
H
9
8.6 8.2
7.8
7.4
3.35
3.00
2.65
f1 (ppm)
f1 (ppm)
Fig. 5 1H NMR section of
ZM 241385 (9) showing the
amino protons at different
temperatures (red = 303 K,
spectrum 1; orange = 313 K,
spectrum 2; green = 323 K,
spectrum 3; light blue = 333 K,
spectrum 4; dark blue = 343 K,
spectrum 5). At 7.85 ppm is a
singlet from an aromatic proton
(Color figure online)
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
f1 (ppm)
in conjunction with hindered rotation at the secondary amino
group of the tyramine moiety. A comparison between mol-
ecules 9 and 11 shows that removing the secondary amine on
the ligand in position 2 affects the ratio of the signals, but not
the doubling up effect. This indicates that intermolecular
interactions still occur but the second underlining effect has
been eliminated. Some publications have examined similar
effects caused either by nitrogen inversion or hindered
rotation [12–14]. In the case of ZM 241385 (9), nitrogen
inversion seems to be very unlikely due to the fact that the
X-ray crystal structure of the molecule exhibits primarily sp2
character for the amino group of the tyramine moiety (vide
infra).
X-ray structure of ZM 241385
Crystals of the title compound 9 were grown by a two layer
system of methanol and petroleum spirits. The crystals
were used to generate an X-ray structure for
ZM 241385 (9) that not only proved that the correct mol-
ecule was synthesized, but also gave interesting insight into
the crystal packing. Compound 9 crystallizes with two
independent molecules and six water molecules in the
asymmetric unit as displayed in Fig. 8. The two molecules
form a head-to-tail arrangement stabilized by hydrogen
bonds (Fig. 9) between the hydroxyl groups (O1-H
and O10-H) on each molecule and the triazine nitrogens
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Struct Chem (2013) 24:1241–1251
1245
Fig. 6 13C NMR spectra of
ZM 241385 (9) at 298 K (blue,
spectrum 2) and 353 K (red,
spectrum 1) (upper). The lower
left spectrum shows a zoomed
section of some of the
quaternary carbons at 298 and
353 K. The lower right
spectrum shows the CH2 signals
of the tyramine moiety that each
show two signals at 298 K and a
single resonance at 353 K
(Color figure online)
170
150
130
110
90
70
50
30
f1 (ppm)
NH2
N
HO
N
N
O
N
N
N
H
9
161 158 155 152
f1 (ppm)
43
40
37
34
f1 (ppm)
NH2
N
N
N
O
N
O
N
11
Fig. 7 Structure of 2-(furan-2-yl)-5-phenethoxy-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-amine (11)
(N60 and N6) (see Table 1 for a complete list of hydrogen
bond contacts in the crystal). The six water molecules form
a cluster that helps to further stabilize the p-stacked layers
of the ligand (Figs. 8, 10).
The crystal structure of ZM 241385 (9) revealed slight
puckering of the triazine portion of the heterocycle
(Table 2). Ideally, the torsions within the triazine should all
be 0ꢁ, reflecting the aromaticity of the structure. However,
one of the crystallographically determined structures
revealed differences of up to 5ꢁ from the ideal predicted
geometry.
Fig. 8 Crystal packing of six ZM 241385 (9) molecules including
water molecules (red dots) (Color figure online)
task due to problems such as low thermostability and low
receptor expression levels [15]. In 2008, Jaakola et al. [5]
published the first X-ray crystal structure (PDB 3EML)
of the A2A receptor in complex with the A2A antagonist
ZM 241385 (9), which revealed a unique ligand binding
pocket (Fig. 11b). This X-ray structure was generated using
Comparison of ZM 241385 crystal structures
´
a T4 lysozyme (T4L) fusion strategy. More recently, Dore
et al. [6] published a second receptor bound X-ray structure
(PDB 3PWH) of ZM 241385 (9) using a thermostabilized
adenosine A2A receptor (Fig. 11c). The two X-ray structures
show subtle differences in their respective ligand interac-
tions with the receptor, namely (i) the ligand is placed
slightly deeper into the binding site in 3EML compared to
3PWH, and (ii) the conformation of tyrosine residues (Tyr9
and Tyr271 in the PDB sequences) near the phenol moiety of
The science of producing X-ray structures of receptors in
complex with specific ligands has significantly improved
the understanding of structure–function relationships and
progressed the structure-based drug design to a new
dimension. Nevertheless, obtaining ligand-bound X-ray
structures in complex with G protein-coupled receptors,
including the adenosine A2A receptor, is still a challenging
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Struct Chem (2013) 24:1241–1251
Fig. 9 Thermal ellipsoid plot
for compound 9. Ellipsoids are
at the 20 % probability level
Table 1 Hydrogen bonds for two molecules of ZM 241385 (9)
˚
Table 2 Comparison of torsion angles within the triazine portion of
the ZM 241385 heterocycle
[A and ꢁ]
NH2
φ2
φ3
D–HꢀꢀꢀA
d(D–H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA) \(DHA)
HO
N
O
N
N
φ1
φ4
N(4)–H(4A)…O(5)
O(10)–H(10B)…N(7)
O(1)–H(1B)…N(70)
N(40)–H(40A)…O(3)
N(1)–H(1A)…O(4)
O(3)–H(3A)…N(60)
O(7)–H(7D)…O(6)
N(4)–H(4B)…N(20)#1
N(10)–H(10A)…O(5)#2
N(40)–H(40B)…N(2)#2
O(5)–H(5A)…O(10)#3
O(4)–H(4D)…O(3)#1
O(4)–H(4C)…O(7)#4
O(8)–H(8D)…O(6)#5
O(8)–H(8C)…N(3)#6
0.82(3)
0.90(4)
0.96(4)
0.82(3)
0.89(3)
1.00(4)
1.094(17)
0.82(3)
0.87(3)
0.87(3)
0.77(3)
0.93(4)
0.88(4)
1.02(4)
1.02(4)
2.19(3)
1.81(4)
1.75(4)
2.49(3)
2.21(3)
1.87(4)
1.831(19)
2.13(3)
2.09(3)
2.20(3)
2.04(3)
1.86(4)
1.95(4)
1.82(4)
2.17(5)
2.851(3)
2.689(2)
2.697(2)
3.307(3)
3.026(3)
2.826(3)
2.922(4)
2.942(3)
2.927(2)
3.048(3)
2.808(3)
2.776(3)
2.807(3)
2.832(4)
3.158(3)
137(2)
165(3)
168(3)
175(2)
153(3)
160(3)
175(5)
172(3)
163(2)
163(2)
175(3)
168(3)
163(3)
173(5)
162(6)
N
N
H
N
φ6 φ5
Source
U1
U2
U3
U4
U5
U6
PDB 3EML
PDB 3PWH
1.28 -0.88
0.38
0.08 0.38 -0.98
-0.18 -0.18
0.18 -0.18 0.18 -0.18
ZM 241385
structure 1
-0.78
6.08 -5.28 -1.48 6.48 -5.68
ZM 241385
structure 2
1.88 -1.38
0.48 0.38 0.18 -1.28
structures of the ligand in complex with the adenosine A2A
receptor [5, 6] with that of the unbound crystal structure of
ZM 241385 (9) presented here (two molecules per unit
cell).
Symmetry transformations used to generate equivalent atoms: #1 x, y ? 1,
z ? 1; #2 x, y-1, z-1; #3 -x ? 3, -y ? 2, -z ? 3; #4 -x ? 1, -y ? 1,
#5 #6
-z ? 1; -x ? 1, -y, -z; x-1, y-1, z-1
The overlay of the four ZM 241385 (9) structures
revealed that the tyramine portion of the ligand can adopt
different conformations depending on the structural context
(Fig. 11a; Table 3). Most interestingly, the adenosine A2A
receptor appears to be able to recognize two strikingly
different conformations of this portion of the tyramine
ZM 241385 (9) changes dramatically between the two
structures (Fig. 11b, c). Consequently, the adenosine
receptor has space to accommodate the inherent flexibility of
the tyramine moiety.
´
moiety; this was previously noted by Dore et al. [6]. The
Herein, we extensively compare the ZM 241385 (9)
coordinates obtained from the two available crystal
two structures from the unbound crystal structure of
ZM 241385 (9) adopted slightly different conformations of
Fig. 10 Partial packing
diagram of compound 9,
showing the cluster of water
molecules which helps to
stabilize the crystal by forming
hydrogen bond between layers
of the ligand
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Struct Chem (2013) 24:1241–1251
1247
Fig. 11 a Overlay of four ZM 241385 structures. Yellow carbons—
ZM 241385 structure 1; teal carbons—ZM 241385 structure 2; gray
carbons—ZM 241385 in complex with A2A receptor (PDB 3EML);
violet carbons—ZM 241385 in complex with A2A receptor (PDB
3PWH). b ZM 241385 (gray) in complex with A2A receptor/lysozyme
fusion (pink) (PDB 3EML). c ZM 241385 (violet) in complex with
thermostabilized A2A receptor (yellow) (PDB 3PWH). Transmem-
brane helices 2 and 3 removed from figures of complex structures for
clarity. Blue and red colors represent oxygen and nitrogen atoms,
respectively (Color figure online)
Table 3 Comparison of torsion angles for the tyramine portion of
ZM 241385 (9)
NH2
τ1
τ2
τ3
τ4
HO
N
N
O
C2
C1
N2
C9
N
C8
C6
C7
N1
τ4
N
τ3
τ1/τ2
Source
s1/s2
s3
s4
PDB 3EML
PDB 3PWH
-43.2ꢁ/135.7ꢁ
-165.7ꢁ/14.0ꢁ
-70.3ꢁ/110.7ꢁ
-68.8ꢁ/112.7ꢁ
-176.1ꢁ
140.4ꢁ
-171.1ꢁ
159.3ꢁ
91.6ꢁ
107.8ꢁ
-167.9ꢁ
89.5ꢁ
ZM 241385 structure 1
ZM 241385 structure 2
the tyramine portion of the ligand, which resided in
between the two extremes of the structures obtained by
´
Dore et al. [6] and Jaakola et al. [5]. As shown in Fig. 11b,
c, the conformation of Glu169 also changes between the
two crystal structures, resulting in hydrogen bonds in dif-
ferent locations of ZM 241385. Phe168 engages in p-
stacking with the triazolotriazine heterocycle in both cases.
Vacuum and explicit aqueous solution conformational
searches
Fig. 12 Conformations of ZM 241385 in different environments. Top
vacuum (modeled); middle explicit aqueous solution (modeled);
bottom solid state (experimental)
Variations in the conformations of the bound ZM 241385
(9) and their comparison to the conformations of the
unbound compound point to its extensive flexibility. In
order to further address this issue, we have extended the
structural analysis of ZM 241385 (9) and carried out con-
formational searches for this compound in vacuo and in
explicit aqueous solution.
Compound ZM 241385 (9) contains a flexible linker,
connecting two rigid moieties; a substituted benzene ring
and a heterocyclic triazolotriazine ring system. The furan
and triazolotriazine rings, the linker itself, as well as the
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Struct Chem (2013) 24:1241–1251
hydroxyl substituent on the aromatic ring make this mol-
ecule fairly hydrophilic. Thus, due to these properties and
to the flexibility of the linker, the two ring systems have the
ability to interact via intramolecular van der Waals (vdW)
and hydrogen bonding interactions, thus potentiating a
range of accessible conformations, depending on the
environment (Fig. 12).
Experimental
General information
All reactions were stirred magnetically in oven-dried glass-
ware. Anhydrous solvents were transferred via oven-dried
syringe or cannula. Technical grade solvents used for
extraction and column chromatography were distilled before
use. Absolute solvents were used without further purifica-
tion. Starting materials and reagents 1, 2, 5, 8 and m-CPBA
were purchased from Aldrich or AK Scientific in the highest
available grade and used without further purification. Com-
pound 10 was purchased from Chembridge Support. Com-
pounds 3, 4, 6, 7, 9, and 11 were synthesized in our
laboratories. Analytical thin layer chromatography (TLC)
plates from Merck were used for reaction control (silica gel
60 on aluminum sheets). Silica gel 60 (Fluka) was used for
silica gel flash chromatography. Microwave reactions were
performed using a Biotage Initiator 2.0. Proton nuclear
magnetic resonance (1H NMR) spectra and carbon-13
nuclear magnetic resonance (13C NMR) spectra were
recorded on Bruker spectrometers Avance 400 (400 MHz
for 1H and 101 MHz for 13C) at ambient temperature (if not
stated differently) in the solvents indicated and referenced to
tetramethylsilane (TMS). Spectra measured at 353 K were
referenced to d6-dimethyl sulfoxide (DMSO). Chemical
shifts (d) are reported in parts per million (ppm). Coupling
constants (J) are reported in Hertz (Hz). The following
abbreviations are used: s (singlet), br s (broad singlet), d,
(doublet), t (triplet), q (quartet), and m (multiplet). 13C NMR
spectra were routinely run with broadband decoupling. Low
resolution electrospray mass spectra (LRMS) using elec-
trospray ionization (ESI) were obtained on a Micromass
Platform II spectrometer. Unless otherwise stated, cone
voltage was 20 eV. High resolution mass spectra (HRMS)
were obtained on a Waters LCT Premier XE (TOF) spec-
trometer fitted with an electrospray ion source. Mass signals
are given in mass units per charge (m/z). The fragments and
intensities are written in brackets. Liquid chromatography
mass spectra (LCMS) were measured on an Agilent 6100
Series Single Quad LC/MS, Agilent 1200 Series HPLC.
(Pump: 1200 Series G1311A Quaternary pump, Autosam-
pler: 1200 Series G1329A Thermostatted Autosampler,
Detector: 1200 Series G1314B Variable Wavelength
Detector). Gradient takes 4 min to get to 100 % ACN;
maintain for 3 min and a further 3 min to get back to the
original 5 % ACN. Melting points were measured with a
MP50 Melting Point System from Mettler Toledo.
The lowest energy structure obtained from the confor-
mational search of ZM 241385 (9) in vacuo featured the
phenol portion of the ligand stacking with the heterocycle
(Fig. 12 [top]). In solution, two main forces determine the
preferred conformation. On one hand, intramolecular vdW
and hydrogen bonding interactions favor formation of
folded conformations, so as to minimize contact with water
for the hydrophobic parts of the molecule (e.g., aromatic
ring). On the other hand, the polarizable groups (e.g.,
hydroxyl) ‘‘push’’ the molecule into bulk solvent, thus
aiding the extended form of the compound. While both
conformations have been observed in the conformational
search carried out in an explicit aqueous environment, the
lowest energy conformation was found in the extended
state (Fig. 12 [middle]). This conformation is very similar
to that in the solid state, except for the torsion angles in the
highly flexible linker. The extended conformation of
the solid state (Fig. 12 [bottom]) is likely to be a result of
the intermolecular interactions in the crystal lattice (dis-
cussed above). We have previously observed similar
environment-dependent conformational behavior for other
molecules containing rigid hydrophobic elements deco-
rated with polarizable groups, and connected by flexible
linkers [16–18].
Conclusion
ZM 241385 (9) has been successfully synthesized in an
overall yield of 5 %. A complete characterization of
ZM 241385 (9) and its intermediates (3–6) has been per-
formed that exhibited some interesting NMR spectral
characteristics that have not been reported to date, namely,
the doubling up of proton and carbon resonances. Our
experiments indicate that these effects are due to the
intermolecular interactions, such as dimer formation in
combination with hindered rotation. The first single mol-
ecule X-ray structure presented in this article confirmed the
synthesis of ZM 241385 (9) and provided the basis for the
structural comparison with the published protein-bound
X-ray structure. The overlay of the ZM 241385 (9) coor-
dinates revealed that the tyramine portion of the molecule
can easily adopt different conformations due to its inherent
flexibility. Conformational analyses in vacuum and in
explicit aqueous solution allowed an insight into the effect
of environment on the conformation.
X-ray structure
Intensity data for compound 9 was collected on an Oxford
Diffraction SuperNova CCD diffractometer using Cu Ka
123

Struct Chem (2013) 24:1241–1251
1249
Synthesis
radiation. The temperature during data collection was
maintained at 130.0(1) K using an Oxford Cryostream
cooling device. The structure was solved by direct methods
and difference Fourier synthesis [19]. Thermal ellipsoid
plot was generated using the program ORTEP-3 [20],
integrated within the WINGX [21] suite of programs.
Crystal data for compound 9. (C16H15N7O2)0.3(H2O),
M = 391.40, T = 130.0 K, k = 1.54180, triclinic, space
group P-1, a = 10.851(2), b = 13.349(3), c = 15.215(3)
N00-(Furan-2-ylcarbonyl)carbonohydrazonic diamide (3)
Furoic acid hydrazide (1) (6.00 g, 47.5 mmol) and S-
methylisothiosulfate hemisulfate (2) (33.1 g, 238 mmol)
were added to a solution of sodium hydroxide (3.04 g,
76.1 mmol) in water (150 mL). The clear solution was
stirred at room temperature for 24 h and the resultant
precipitate was filtered and washed with water and diethyl
ether and afterward dried under vacuum. Product 3 (4.60 g,
58 %) was obtained as a white solid, mp: 181–184 ꢁC ([9]
213 ꢁC). 1H NMR (400 MHz, D2O) d 7.50 (m, 1H, CH–O),
6.87 (m, 1H, CH=C), 6.48 (m, 1H, CH=CH–CH).
13C NMR (101 MHz, D2O) d 158.5 (C), 155.8 (C), 148.4
(C), 144.4 (CH), 112.4 (CH), 111.5 (CH). LRMS: m/z
(ESI 20 V) 169.2 (MH?, 100).
˚
A, a = 107.694(19)ꢁ, b = 101.122(15)ꢁ, c = 108.736
3
(18)ꢁ.
V
1882.4(6) A , Z = 4, Dc = 1.381 Mg M-3
,
˚
l(Cu Ka) 0.889 mm-1
,
F(000) = 824 crystal size
0.36 9 0.32 9 0.04 mm3, 12,553 reflections measured,
6,770 independent reflections [R(int) = 0.0353], the final
R was 0.0539 [I [ 2r(I)] and wR(F2) was 0.1594 (all data).
Overlay of X-ray structures
5-(Furan-2-yl)-1H-1,2,4-triazol-3-amine (4)
The four X-ray structures were overlaid in Maestro 9.2,
using only the heterocyclic portion for the superposition.
Torsion angles were measured using Maestro 9.2.
Method (A) N00-(Furan-2-ylcarbonyl)carbonohydrazonic
diamide (3) (1.00 g, 5.95 mmol) was suspended in water
(20 mL). The reaction mixture was stirred in the micro-
wave at 140 ꢁC for 1 h (pressure 6 bar) then cooled to
room temperature and the solvent was removed under
vacuum. The product 4 was obtained as a beige solid
Conformational search of ZM 241385 in vacuo
Conformational search was carried out using Macromodel
9.9 in vacuo. The OPLS_2005 force field was used to
parameterize the structure. The Polak–Ribiere conjugate
gradient method was used to minimize prospective struc-
tures. A maximum of 1,000 iterations with a convergence
1
(880 mg, 99 %). H NMR (400 MHz, d6-DMSO) d 12.02
(s, br, 1H, NH), 7.69 (dd, J = 1.7, 0.7 Hz, 1H, CH–O),
6.69 (d, J = 3.2 Hz, 1H, CH=C), 6.54 (dd, J = 3.3,
1.8 Hz, 1H, CH=CH–CH), 6.01 (br s, 2H, NH2). 13C NMR
(101 MHz, d6-DMSO) d 157.6 (C), 151.7 (C), 147.3 (C),
142.7 (CH), 111.3 (CH), 107.7 (CH). LRMS: m/z (ESI
20 V) 151.2 (MH?, 100).
Method (B) N00-(Furan-2-ylcarbonyl)carbonohydrazonic
diamide (3) (4.16 g, 24.7 mmol) was suspended in water
(85 mL). The reaction mixture was stirred under conventional
heating at reflux for 29 h then cooled to room temperature and
the solvent was removed under vacuum. The residue was
suspended in water (20 mL) and filtered. The filter cake was
washed with water and dried under high vacuum. The product
4 (2.96 g, 80 %) was obtained as a pinkish white solid, mp:
198–204 ꢁC ([9] 230 ꢁC). 1H and 13C NMR and mass spectral
data are in accordance with above characterization.
˚
threshold of 0.1 kcal/mol A was used for each minimiza-
tion. The Torsional Sampling method was used to perform
the conformational search. Automatic Setup was initially
used to identify the search variables, which were subse-
quently edited. Specifically, torsions within the heterocy-
clic and furan rings that were automatically selected by
Automatic Setup were removed from the search, and all
defined ring closures and torsion check parameters were
removed. Extended torsion sampling was used, and mirror
image conformations were not retained. A maximum of
14,000 conformational search steps were set, correspond-
ing to 2,000 steps per rotatable bond. No limit was speci-
fied on the number of structures saved by the search. An
energy window of 6 kcal/mol (25.1 kJ/mol) was specified
for saving structures.
2-(Furan-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-a]
[1,3,5]triazin-7-amine (6)
5-(Furan-2-yl)-1H-1,2,4-triazol-3-amine (4) (50.0 mg,
333 lmol) and N-cyanodithioiminocarbonate (5) (48.7 mg,
333 lmol) were mixed and heated at 170 ꢁC for 1 h under
a nitrogen atmosphere. The reaction mixture was cooled to
room temperature, absorbed on Celite and purified by
Explicit solvent conformational search of ZM 241385
An explicit solvent conformational search of ZM 241385
was performed using HyperChem 7.52, following a pre-
viously established procedure [17].
123

1250
Struct Chem (2013) 24:1241–1251
column chromatography (dichloromethane: ethyl acetate
95:5 ? 85:15). The product 6 (27.0 mg, 32 %) was
obtained as a white solid, mp: 237–240 ꢁC ([4, 11]
238–240 ꢁC). 1H NMR (400 MHz, d6-DMSO) d 8.97 (br s,
1H, NH), 8.78 (br s, 1H, NH), 7.94 (dd, J = 1.8, 0.8 Hz,
1H, O–CH), 7.17 (dd, J = 3.4, 0.8 Hz, 1H, C–CH), 6.72
(dd, J = 3.4, 1.8 Hz, 1H, CH = CH–CH), 2.52 (s, 3H, S–
CH3). 13C NMR (101 MHz, d6-DMSO) 173.3 (C), 157.2
(C), 156.2 (C), 149.6 (C), 145.5 (C), 145.2 (CH), 112.5
(CH), 112.1 (CH), 13.6 (CH3). LCMS: m/z (ESI 20 V)
249.1 (MH?, 100).
(C), 159.0 (C), 155.9 (C), 155.4 (C), 150.1 (C), 146.1 (C),
144.1 (CH), 129.1 (CH), 115.0 (CH), 111.4 (CH), 111.2
(CH), 42.4 (CH2), 33.9 (CH2). HRMS (C16H15N7O2): Calcd.
338.1360 [M ? H]?, Found 338.1353. HPLC: tR 7.34 min,
98 % (214 nm), 97 % (254 nm).
[1,2,4]Triazolo[1,5-a][1,3,5]triazin-7-amine (10)
1H NMR (400 MHz, d6-DMSO) d 8.95 (br s, 2H, NH2),
8.53 (s, 1H, CH–N–C–NH2), 8.35 (s, 1H, CH). 13C NMR
(101 MHz, d6-DMSO) d 159.0 (CH), 156.8 (C), 154.7
(CH), 151.8 (C). LCMS: m/z (ESI 20 V) 137.2 (MH?, 100).
2-(Furan-2-yl)-5-(methylsulfonyl)-[1,2,4]triazolo[1,5-a]
[1,3,5]triazin-7-amine (7) [4]
2-(Furan-2-yl)-5-phenethoxy-[1,2,4]triazolo[1,5-a]
[1,3,5]triazin-7-amine (11) [4]
A solution of meta-chloroperoxybenzoic acid (1.06 g,
4.48 mmol) in dichloromethane (10 mL) was added drop-
wise at -5 ꢁC to a suspension of 2-(furan-2-yl)-5-(methyl-
thio)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-amine (6) (278 mg,
1.12 mmol) in dichloromethane (17 mL). The reaction was
stirred at room temperature for 22 h before the solvent was
removed under vacuum. The crude material was suspended
in ethanol (5 mL) and stirred at room temperature for
30 min. The solid was collected by filtration, washed with
ethanol and dried to give the title compound 7 (230 mg,
82 %) as a yellowish-white solid, mp: 168 ꢁC (dec).
1H NMR (400 MHz, d6-DMSO) d 9.81 (s, 1H, NH), 9.48
(s, 1H, NH), 7.99 (dd, J = 1.7, 0.8 Hz, 1H, O–CH), 7.27
(dd, J = 3.4, 0.7 Hz, 1H, C–CH), 6.76 (dd, J = 3.4, 1.8 Hz,
1H, CH = CH–CH), 3.37 (s, 3H, S–CH3). 13C NMR
(101 MHz, d6-DMSO) 165.3 (C), 157.3 (C), 156.8 (C),
152.2 (C), 145.8 (CH), 145.0 (C), 113.4 (CH), 112.3 (CH),
38.9 (CH3). LCMS: m/z (ESI 20 V) 281.0 (MH?, 100).
1H NMR (400 MHz, d6-DMSO) d 8.95–8.60 (2 9 br s,
ratio 1:1, 2H, NH2), 7.91 (m, 1H, HFuran), 7.32 (m, 4H,
HAr), 7.24 (m, 1H, HAr), 7.14 (dd, J = 3.4, 0.5 Hz, 1H,
H
Furan), 6.71 (dd, J = 3.4, 1.8 Hz, 1H, HFuran), 4.52 (t,
J = 6.8 Hz, 2H, CH2–O), 3.05 (t, J = 6.8 Hz, 2H,
CH2CH2–O). 13C NMR (101 MHz, d6-DMSO) d 164.9 (C),
158.9 (C), 156.6 (C), 151.6 (C), 145.6 (C), 145.1 (CH), 138.1
(C), 128.9 (CH), 128.3 (CH), 126.3 (CH), 112.3 (CH), 112.0
(CH), 67.9 (CH2), 34.4 (CH2). HRMS (C16H14N6O2): Calcd.
323.1251 [M ? H]?, found 323.1521. mp: 201–204 ꢁC.
HPLC: tR 9.14 min, [99.5 % (214 nm), [99.5 % (254 nm).
Accessory publication
Crystallographic data (excluding structure factors) for the
structure reported in this article has been deposited with the
Cambridge Crystallographic Data Centre CCDC 901689.
Copies of the data can be obtained free of charge on appli-
cation to the CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (Fax: ?44 1223/336033; deposit@ccdc.cam.ac.uk).
1H and 13C NMR spectra for all synthesized compounds,
including HPLC, HRMS, and X-ray structure details of
ZM 241385 (9) are documented in the supplementary
information.
4-(2-((7-Amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]
[1,3,5]triazin-5-yl)amino)ethyl)phenol (9) (ZM 241385)
Tyramine (8) (490 mg, 3.75 mmol) was added to a suspen-
sion of 2-(furan-2-yl)-5-(methylsulfonyl)-[1,2,4]triazol-
o[1,5-a][1,3,5]triazin-7-amine (7) (250 mg, 892 lmol) in
acetonitrile (25 mL). The reaction mixture was stirred
overnight at room temperature. After 22 h the solvent was
evaporated, the residue was adsorbed on Celite and purified
by column chromatography (dichloromethane: methanol
25:75). The collected fractions with product were evaporated
to dryness and recrystallized from ethyl acetate. The title
compound 9 (100 mg, 33 %) was obtained as a white solid,
mp: 229–231 ꢁC ([4] 225–227 ꢁC). 1H NMR (400 MHz, d6-
DMSO, 353 K) d 8.91 (s, 1H, OH), 7.92 (br s, 2H, NH2), 7.79
(m, 1H, HFuran), 7.14 (br t, 1H, NH), 7.04 (m, 3H, 2 9 HAr,
Acknowledgments M.J. is a recipient of the Australian Post-
graduate Award Industry (APAI). We thank GlaxoSmithKline, GSK
R&D China, Singapore for financial support and Dr. Jason Dang
(Monash University) for assistance in obtaining NMR and HRMS
data. We also thank Associate Professor Martin Scanlon, Dr Jamie
Simpson and Dr James Swarbrick from Monash University for
insightful discussions on the NMR aspects in this paper.
References
H
Furan), 6.71 (m, 2H, 2 9 HAr), 6.64 (dd, J = 3.3, 1.8 Hz,
1H, HFuran), 3.49 (m, 2H, CH2-CH2-NH), 2.78 (m, 2H,
CH2–CH2–NH). 13C NMR (101 MHz, d6-DMSO) d 161.1
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