Simple oxidation of pyrimidinylhydrazones to triazolopyrimidines and their inhibition of Shiga toxin trafficking

Article abstract of DOI:10.1016/j.ejmech.2009.10.007

The oxidative cyclisation of a range of benzothieno[2,3-d]pyrimidine hydrazones (7a-j) to the 1,2,4-triazolo[4,3-c]pyrimidines (8a-j) catalysed by lithium iodide or to the 1,2,4-triazolo[1,5-c]pyrimidines (10a-j) with sodium carbonate is presented. A complementary synthesis of the 1,2,4-triazolo[1,5-c]pyrimidines starting from the amino imine 11 is also reported. The effect of these compounds on Shiga toxin (STx) trafficking in HeLa cells and comparison to the previously reported Exo2 is also detailed.

Full text of DOI:10.1016/j.ejmech.2009.10.007

European Journal of Medicinal Chemistry 45 (2010) 275–283
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Simple oxidation of pyrimidinylhydrazones to triazolopyrimidines and their
inhibition of Shiga toxin trafficking
,
Lucie J. Guetzoyan a, Robert A. Spooner b, J. Michael Lord b, Lynne M. Roberts b, Guy J. Clarkson a
*
a Department of Chemistry, University of Warwick, CV4 7AL, UK
b Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The oxidative cyclisation of a range of benzothieno[2,3-d]pyrimidine hydrazones (7a–j) to the 1,2,4-
triazolo[4,3-c]pyrimidines (8a–j) catalysed by lithium iodide or to the 1,2,4-triazolo[1,5-c]pyrimidines
(10a–j) with sodium carbonate is presented. A complementary synthesis of the 1,2,4-triazolo[1,5-
c]pyrimidines starting from the amino imine 11 is also reported. The effect of these compounds on Shiga
toxin (STx) trafficking in HeLa cells and comparison to the previously reported Exo2 is also detailed
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 23 July 2009
Received in revised form
7 September 2009
Accepted 1 October 2009
Available online 9 October 2009
Keywords:
Pyrimidyl hydrazone
Triazolopyrimidine
Lithium iodide
DMSO
Sodium carbonate
Dimroth rearrangement
Exo2
Shiga toxin
1. Introduction
This paper details our findings from the unexpected oxidative
cyclisation of pyrimidyl hydrazones to give triazolopyrimidines,
Compoundscontaining the 5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidine (1) nucleus have found many applications as adenosine
mimics [1], analgesics [2], anticancer [3], antiviral agents [4], and as
inhibitors of kinases [5] and human epidermal growth factor [6]
(Fig. 1).
and includes a preliminary investigation on the ability of these new
analogues of Exo2 to protect HeLa cells against a challenge by Shiga
toxin.
2. Chemistry
Exo2 (2) is a small molecule inhibitor of exocytosis that func-
tions via the disruption of membrane trafficking [7]. This action also
perturbs retrograde trafficking and as such, it has been used as
a tool to help dissect the entry pathway of lipid binding bacterial
toxins in mammalian cells [8,9]. Indeed, Exo2 has been shown to
have a significant protective effect on HeLa cells against the protein
synthesis inhibiting Shiga toxin (STx). Its effects on organelle
morphology have suggested that its cellular target is involved in
early endosome delivery to the Trans Golgi Network (TGN) and/or
TGN access to the Golgi stack [9]. By blocking delivery into the
Golgi, Shiga toxin cannot reach the endoplasmic reticulum from
where it normally translocates a membrane to reach its ribosome
substrates in the cytosol.
During work on the synthesis of derivatives of Exo2 to discern
structure activity relationships we required the hydrazone phenol
(4). We had planned a simple deallylation of the ether (3) but
concerned by the possible interference of the sulphur with palla-
dium catalysed deallylation, we opted to use sodium iodide in
dimethyl sulphoxide [10]. Instead of the expected deallylation
a new compound was isolated which still contained both the allyl
and n-propyl ethers but lacked the NH and imine hydrogen of the
hydrazone in 1H NMR and had 2 mass units less than the starting
material. This was identified as the 1,2,4-triazolo[4,3-c]pyrimidine
(5) with the aid of correlation spectroscopy which showed NOE
interactions between the pyrimidyl CH and the phenyl CHs ortho to
the hydrazone bond (Scheme 1).
The many literature reports on the synthesis of the tri-
azolopyrimidine fused ring system [11] either start form reaction of
the pyrimidine hydrazine with an acid derivative (orthoformate
* Corresponding author. Tel.: þ44 0 24 765 22255; fax: þ44 0 24 76524112.
E-mail address: guy.clarkson@warwick.ac.uk (G.J. Clarkson).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.007

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L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
This was also corroborated by the discovery that the cyclisation
N
of Exo2 (2) proceeds in DMF alone to give 6 with out salt addition
and that reaction of hydrazone 2 with iodine in DMSO does not give
the expected cyclised product 6 (vida infra). This lead us to reassess
our hypothesis and that may be oxygen and small amounts of base
(DMF is known to breakdown on heating to carbon monoxide and
dimethylamine) were responsible for the cyclisation with the role
of the lithium iodide unclear. Other common amide solvents like
dimethyl acetamide and N-methyl pyrrolidinone which are not
prone to the same degradation as DMF, were not so effective in the
additive free cyclisation of Exo2 (2).
HN N
N
OH
S
N
S
N
OCH3
2
1
Fig. 1. The 5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine core (1) and Exo2 (2).
[12] or activated acids [13]) or via oxidative cyclisation of the
hydrazone with reagents like NBS [14], Pb(OAc)4 [15], FeCl3 [16], or
iodobenzene diacetate [17].
Returning to the effect of salts in DMSO, we next investigated
basic salts.
On treatment of Exo2 (2) with sodium iodide and DMSO we
were pleased to obtain a quantitative conversion to the triazole (6)
(Scheme 2).
Heating Exo2 (2) in DMSO with sodium carbonate gave a new
compound the isomeric triazole (9). The reaction also proceeds
with sodium acetate but is not so effective. The pyrimidine CH of
the 1,2,4-triazolo[1,5-c]pyrimidine now shifts further down field to
9.54 ppm [12b], but the associated 13C signal still resonates around
135 ppm. No NOE effect could be measured between the pyrimidyl
CH and the ortho protons of the hydrazone in accord with the
different orientation of the phenyl group of the isomerised triazole.
The solid state structure of 9 from X-ray crystallography is shown in
Fig. 2 confirming the substitution pattern of the triazole ring
(Scheme 3).
Sodium carbonate has catalysed both the oxidative cyclisation
and Dimroth [21] type rearrangement of (2) to yield the 1,2,4-tri-
azolo[1,5-c]pyrimidine (9). We presume that the 1,2,4-triazolo[4,3-
c]pyrimidine 6 is an intermediate which then undergoes the Dim-
roth type rearrangement. Small quantities of the triazole 6 can be
observed by 1H NMR and thin layer chromatography as the reaction
We examine other salts as possible additives (Table 1). There is
no measurable reaction at room temperature and the oxidative
cyclisation in DMSO is slow with low conversion without additive.
Other halide salts like sodium bromide were far less efficient
than the iodide and sodium chloride was similar to the uncatalysed
reaction in DMSO with a slow cyclisation and gradual decomposi-
tion at elongated reaction times. The other iodide salts tested all
showed excellent ability for the cyclisation with lithium iodide
being the most effective. The addition of excess lithium iodide did
not notably enhance the rate, whereas the use of sub-molar
quantities had a rate limiting effect.
Table 2 details the effect of solvent choice on the oxidative
cyclisation reaction of Exo2 (2) catalysed by lithium iodide. The
most favourable conditions use DMSO or DMF with little reactivity
seen in other common solvents though in several cases this is more
likely due to the low solubility of the starting material and lower
reaction temperatures.
proceeds and the rearrangement of the triazole
6 to its
isomeric1,2,4-triazolo[1,5-c] pyrimidine 9 is in accord with pub-
lished work under similar basic conditions [12,22].
The application of these basic conditions to the range of
hydrazones to catalyse the oxidative cyclisation and rearrangement
was followed by 1H NMR and is reported in Table 4.
Table 3 shows the range of pyrimidinyl hydrazones 7a–j that
were investigated using the lithium iodide in DMSO reaction
conditions. The fluorophenyl hydrazone 7c and the ester hydrazone
7d were difficult to cyclise and gave mixtures with longer reaction
times leading to decomposition. Hydrazones with a hydroxyphenyl
group were the best substrates. The most convenient approach
used lithium iodide in DMF as the triazole product can simply be
filtered off on cooling.
The reported yields are unoptimised. The products are charac-
terised by loss of the NH and imine hydrogen and the shift of the
pyrimidine CH in 1H NMR. The 1H NMR and 13C NMR of the hydra-
zone (w8.3 ppm/152 ppm in the starting hydrazone) shift to around
9.1 ppm [22] and 135 ppm respectively in the cyclised products
(8a–j). Spectra and physical properties also match the known
derivatives 8a and 8b [18]. Additionally, we were fortunate to obtain
suitable crystals for X-ray diffraction of 8b which confirmed the
cyclised structure and the substitution pattern (Fig. 2).
We originally suspected that I2 was the reagent responsible for
the cyclisation and that DMSO was the auxiliary oxidant generating
the I2 from iodide [19]. I2 could act in an analogous manner to the
reaction catalysed by NBS [14] and there is literature precedent for
similar I2 catalysed cyclisations of diaminobenzenes and aldehydes
to benzimidazoles [20]. However, running the reaction under
nitrogen does not yield the cyclisation, with or without lithium
iodide highlighting the requirement for oxygen.
In general, only compounds containing a 4-hydroxy group and
a
thiopyrimidine ring underwent efficient transformation to
the1,2,4-triazolo[1,5-c]pyrimidines (9, 10g, and 10h) but this is not
so clear cut as the ester substituted hydazone 7d is a good substrate
but the hydroxy containing phenylpyrimidine 7i reacts slowly.
Following these reactions by 1H NMR, the initial cyclisation cata-
lysed by sodium carbonate seems to be inefficient in some of these
substrates and other reaction pathways start to predominate rather
than the oxidative cyclisation and isomerisation to the 1,2,4-tri-
azolo[1,5-c]pyrimidine product.
Investigation of the related reaction of amino imine 11 and
aldehydes also furnishes the 1,2,4-triazolo[1,5-c]pyrimidines (9,10a,
10c, 10g, and 10k) shown in Table 5.
Similar cyclisations have been reported but required acetic acid
catalysis [23].
The most practical procedure was simply to reflux equimolar
amounts of the amino imine 11 and the required aldehyde in
methanol. Salt addition is not required and unlike the sodium
carbonate oxidative cyclisation above, there are no constraints on
the starting aldehyde nor the requirement to start from the
N
N
NaI
NaI
HN
N
N
HN
N
N
S
N
OH
OCH2CH2CH3
O
O
DMSO
DMSO
S
N
S
5
4
3
N
OCH2CH2CH3
N
OCH2CH2CH3
Scheme 1. Oxidative cyclisation during attempted deprotection of allyloxy ether 3.

L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
277
N
HN N
N
N
NaI
OH
OCH3
OH
N
S
S
DMSO
N
N
2
OCH3
6
Scheme 2. Sodium iodide catalysed oxidative cyclisation of Exo2.
oxidative cyclisation product 6 compared to DMSO strengthening
the idea that the reaction goes by a radical oxidation.
Table 1
Influence of the salt on the oxidative cyclisation of Exo2.
We finally looked at the reaction of 2 with an equivalent of
iodine in DMSO which does lead to a rapid oxidative cyclisation but
to the isomeric 1,2,4-triazolo[1,5-c]pyrimidine with concomitant
iodinisation to give isomer 13 based on the coupling constant
between the two remaining hydrogens on the electron rich
phenolic ring.
N
N
HN
N
N
N
1 equivalent salt
DMSO 110oC
OH
OCH3
OCH3
OH
S
S
N
N
2
6
Application of these conditions tothe hydrazone 7a also gives the
oxidative cyclisation and rearrangement to the triazole isomer 10a
without the iodination of the less reactive phenyl ring. The genera-
tion of hydrogen iodide must catalyse the Dimroth type rearrange-
ment of the intermediate 1,2,4-triazolo[4,3-c]pyrimidine to the
isomeric 1,2,4-triazolo[1,5-c]pyrimidine ring system in a similar
manner to other acid (Scheme 5) catalysed Dimroth rearrangement
[12,22,23a,25]. This again would indicate that the lithium iodide
catalysed cyclisation does not involve the direct oxidation via
elemental iodine. Iodide may reduce a peroxide intermediate from
arial oxidation of the diamine precursor 12 or there may be a role for
small amounts of iodine to stabilize a radical intermediate in the
oxidation as has recently been described for the addition-frag-
mentation chain transfer processes in living radical polymer
synthesis [26].
Salt
Time
6a
none
NaI
NaBr
NaCl
Na2SO4
KI
LiI
54 h
24 h
48 h
48 h
48 h
48 h
18 h
48 h
25%
>99%
31%b
24%b
24%
>99%
>99%
>99%
NMe4I
a
Conversion determined by NMR.
2 and 6 are identified in the mixture, but also decomposition of the starting
b
material.
hydrazone. Simple filtration gives good yields for both electron rich
and electron deficient benzaldehydes and aliphatic aldehydes.
This reaction is very similar to the cyclisation reaction of 2 to 9
involving the cyclisation of an aldehyde derived intermediate and
arial oxidation.
3. Inhibition of Shiga toxin trafficking
The arial oxidation of 2 to 6 probably goes via a diamino acetal
12 as shown in Scheme 4. There are several other examples of the
arial oxidation of other dinitrogen containing heterocycles [3a,24]
and the benzylic nature and hydroxy substitution would all aid
a radical oxidation. Adding a catalytic amount of AIBN to a salt free
DMSO solution of hydrazone 2 gave an increase in the amount of
The new triazoles represent cyclised Exo2 derivatives that lack
a potentially hydrolysable hydrazone linkage between the thieno-
pyrimidine core and the phenyl substituent. Additionally, the
different triazole isomers allow the presentation of the phenyl ring
and its substituents in different orientations to optimise structure
activity relationships accessing three alternative compounds from
Table 2
Influence of the solvent on LiI catalysed oxidative cyclisation of Exo2.
N
HN
N
N
X equivalent LiI
N
OH
OCH
OCH
OH
N
3
S
S
110oC or reflux
N
N
2
6
3
Solvent
LiI (equiv.)
Time
2a
6a
DMF
DMF
1
0
1
0
1
1
1
1
0
1
28 h
48 h
24 h
54 h
48 h
48 h
48 h
48 h
48 h
48 h
–
–
–
78%b
69%b
>99%
25%
–
DMSO
DMSO
MeOH
EtOAc
acetone
CH3CN
CH3CN
NO2CH3
75%
>99%
83%
>99%
5%c
17%
–
95%c
þ
>99%c
>99%
–
a
Ratio determined by NMR.
Isolated yield.
Precipitate collected and NMR recorded in DMSO(>80% recovery).
b
c

278
L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
the one hydrazone starting material. The 1,2,4-triazolo[4,3-
Table 3
c]pyrimidines analogues 6, 8e, 8g and the 1,2,4-triazolo[1,5-
c]pyrimidines 9 and 10g (the other compounds were either too
insoluble or showed no meaningful activity in this assay) were
tested in parallel for their inherent toxicity towards protein
synthesis and also their ability to confer a protective effect on HeLa
cells from a STx challenge in comparison to Exo2, by modifying
a previously published procedure [9].
Oxidative cyclisation of pyridyl- and pyrimidyl- hydrazones to 1,2,4-triazolo[4,3-
a]pyridines and 1,2,4-triazolo[4,3-a]pyrimidines.
N
N
R
2
HN
N
N
Equiv LiI, DMSO or DMF
110oC, 24Hrs
R
2
N
R
1
R
1
R
N
3
R
3
N
To summarise, the compounds were first assessed for their
inherent cytotoxicity, based on the level of protein synthesis
remaining in chemical-treated HeLa cells when normalised against
protein synthesis levels in cell treated with the DMSO carrier alone
(Fig. 3 Graph A, light grey bars). The procedure was replicated but
this time, the cells were challenged for 1 h with a dose of Shiga
toxin previously determined to promote a 60% drop in protein
synthesis of the control, and the level of remaining protein
synthesis measured (Fig. 3 Graph A, dark grey bars). Most of the
triazoles were substantially less toxic than Exo2 in this regard,
although 10g inhibited protein synthesis significantly more than
Exo2. When challenged with toxin, Exo2-treated cells almost
completely retain their level of protein synthesis under these
conditions (Graph A, dark grey versus light grey bars). The
protective effect (Fig. 2 Graph B) is quantified by comparing protein
synthesis from exposure to just the compound with protein
synthesis after treatment with the compound and a toxin chal-
lenge, and takes into account any effect of the compound alone. All
of the triazoles examined were less effective in this comparison
than Exo2. The loss of an NH hydrogen bond donating group of the
hydrazone linkage and the tethering of the substituted phenyl
group into a triazole ring may significantly alter the orientation of
the phenyl ring from that adopted as a hydrazone in Exo2 and any
advantages from using non-hydrolysable analogues is not revealed
under these conditions.
7a-j
8a-j
R1
R2
R3
H
X
N
Product Conversion Yield
(DMSO) a (DMF)b
7a
7b
7c
8a
8b
8c
–
76%
64%
–
S
S
S
OMe
F
H
H
N
N
–
55%c
CO2Me
7d
H
N
8d
70%c
–
S
7e
7f
H
H
H
N
N
N
8e
8f
–
–
–
24%
52%
65%
S
S
S
OH
OH
4. Conclusions
HO
In conclusion, mild and simple conditions have been discovered
to induce the oxidative cyclisation of pyrimidine hydrazones to
1,2,4-triazolo[4,3-c]pyrimidines by heating in DMSO or DMF.
Lithium iodide is an effective additive to increase the reaction rate
and yield and no further isomerisation takes place under these
conditions. These triazoles undergo rearrangement to the isomeric
1,2,4-triazolo[1,5-c]pyrimidines using sodium carbonate in DMSO
or can be furnished directly from oxidative cyclisation and rear-
rangement of some appropriately substituted starting hydrazones.
A more general complementary procedure as exemplified by the
synthesis of the 1,2,4-triazolo[1,5-c]pyrimidines 9, 10a, 10c, 10g,
and 10k starting from the amino imine 11 was also realised.
Although we have been unable to elucidate a mechanism, the
reactions discussed above allow routes to specific triazolopyr-
imidine isomers from aldehyde precursors by simple arial
oxidation.
These triazoles were tested in a toxin challenge assay in
comparison to Exo2. Although most showed a reduced level of
inhibition of protein synthesis than Exo2, they were less effective in
giving a protective effect against Shiga toxin than Exo2 in this assay.
Whether they protect against alternative toxins that follow the
retrograde pathway was not investigated here. Nevertheless, the
novel compounds described in this report may provide a valuable
platform for future studies.
7g
8g
OH
2
H
N
N
N
C
6
>99%
78%
S
S
OMe
OH
7h
7i
CH3
8h
8i
8j
80%
–
OMe
OH
c
H
32%
–
OMe
OH
7j H–, H–
H
>99%
–
OMe
4.1. Experimental
a
Conversion determined by NMR.
Isolated yields.
Further heating leads to decomposition of the reaction mixture.
b
c
All reagents and solvents were purchased from Lancaster and
Aldrich and used without further purification. The hydrazone

L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
279
Fig. 2. Solid state structure of 8b, 9 and 10k with thermal ellipsoids drawn at 50% probability.
starting materials were synthesised as detailed elsewhere [9]. NMR
spectra were recorded on a DPX-400 spectrometer at room
temperature (298 K). The spectra were recorded in parts per million
(ppm) and referenced using residual protio solvents relative to
trimethylsilane standard (dH ¼ 0 ppm). 2D COSY, HMQC, HMBC and
NOESYspectra were used to aid with peak assignments. ESI mass
spectra were obtained using a Bruker Esquire 2000 mass spec-
trometer coupled with an Agilent 1100 HPLC (without a column) as
the delivery system. Accurate mass spectra were obtained using
a Bruker micro-TOF ESI attached to a time of flight (TOF) analyzer.
CHN elemental analyses were carried out by Warwick Analytical
Services. Thin Layer Chromatography used in monitoring reaction
progress were performed using silica layer (0.25 mm) coated
alumina plates. Weights were recorded on a balance to 4 decimal
places.
d: 21.7 (CH2), 22.5 (CH2), 24.8 (CH2), 25.2 (CH2), 117.8 (C), 125.9 (C),
128.6 (4 CH), 128.6 (C), 129.2 (C), 129.3 (CH), 130.4 (CH), 137.9 (C),
145.8 (C), 146.3 (C). ES-MS m/z 307.1 (MHþ). HRMS 307.1012, found
307.1015. m.p. 185–186 ꢀC. Anal. Calcd for C17H14N4S: C 66.64, H
4.61, N 18.29; found: C 66.31, H 4.57, N 17.96.
4.1.1.3. 3-(4-methoxyphenyl)-8,9,10,11-tetrahydro[1]benzothieno[3,2-e]
[1,2,4]triazolo[4,3-c]pyrimidine 8b. 1H NMR (400 MHz, DMSO)
d: 1.93
(m, 4H, 2 CH2), 2.93 (m, 2H, CH2), 3.12 (m, 2H, CH2), 3.89 (s, 3H, OCH3),
7.19 (d, J ¼ 8.8 Hz, 1H, CH), 7.93 (d, J ¼ 8.8 Hz,1H, CH), 9.16 (s, 1H, CH).
13C NMR (100 MHz, DMSO)
d: 21.7 (CH2), 22.5 (CH2), 24.8 (CH2), 25.1
(CH2), 55.4 (OCH3),114.7 (CH),117.8 (C),118.1 (C),129.2 (C),130.2 (CH),
134.3(CH)þ,137.8 (C),145.7(C),146.1 (C),148.8 (C),160.8(C). ES-MS m/z
337.1 (MH ). HRMS 337.1118, found 337.1108. m.p. 213–215 ꢀC. Anal.
Calcd for C18H16N4OS: C 64.26, H 4.49, N 16.65, S 9.53; found: C 63.89,
H 4.78, N 16.51, S 9.34.
4.1.1. General procedure for lithium iodide catalysed oxidative
cyclisation of pyrimidyl hydrazones to give the 1,2,4-triazolo-
[4,3-c]pyrimidines (6,8a–j)
4.1.1.4. 3-(3-Hydroxyphenyl)-8,9,10,11-tetrahy-
dro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine 8e. 1H NMR
The hydrazone (5mmole) was dissolved in reagent grade DMF
(15 ml) and one equivalent of lithium iodide was added (1 equiv.,
0.7 g). The mixture was stirred and heated at 110 ꢀC under an air
atmosphere. After the specified time (24 or 48 h), the reaction was
allowed to cool and the precipitate isolated by filtration and
washed with a little methanol and then diethyl ether and dried
under high vacuum.
(300 MHz, DMSO)
2H, CH2), 7.03 (d, J ¼ 7.6 Hz, 1H, CH), 7.36 (s, 1H, CH), 7.43 (m, 2H, 2
CH), 9.18 (s, 1H, CH), 9.92 (brs, 1H, OH). 13C NMR (75 MHz, DMSO)
21.7 (CH2), 22.5 (CH2), 24.8 (CH2), 25.1 (CH2), 115.3 (CH), 117.5 (CH),
117.6 (C),119.0 (CH),126.9 (C),129.2 (C),130.4 (CH),134.3 (CH),137.9
(C), 145.8 (C), 146.3 (C), 148.9 (C), 157.9 (C). m.p. ¼ 252–255 ꢀC. ES-
MS m/z 323.1 (MHþ). HRMS 323.0961, found 323.0959.
d: 1.94 (m, 4H, 2 CH2), 2.94 (m, 2H, CH2), 3.13 (m,
d
:
4.1.1.1. 3-(4-Hydroxy-3-methoxyphenyl)-8,9,10,11-tetrahy-
4.1.1.5. 3-(2-Hydroxyphenyl)-8,9,10,11-tetrahy-
dro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine 6. 1H NMR
dro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine 8f. 1H NMR
(400 MHz, DMSO)
d
: 1.93 (m, 4H, 2 CH2), 3.12 (m, 2H, CH2), 3.35 (m,
(400 MHz, DMSO spectra recorded at 353 K) d: 1.91 (m, 4H, 2 CH2),
2H, CH2), 3.90 (s, 3H, OCH3), 7.02 (d, J ¼ 8.0 Hz, 1H, CH), 7.39 (dd,
2.90 (m, 2H, CH2), 3.10 (m, 2H, CH2), 7.07 (t, J ¼ 8.1 Hz, 1H, CH), 7.13
(d, J ¼ 8.1 Hz, 1H, CH), 7.50 (t, J ¼ 8.1 Hz, 1H, CH), 7.63 (d, J ¼ 8.1 Hz,
1H, CH), 8.84 (s, 1H, CH), 10.57 (brs, 1H, OH). ES-MS m/z 323.1
(MHþ). HRMS 323.0961, found 323.0956. m.p. ¼ decomposes above
260 ꢀC. Anal. Calcd for C17H14N4OS: C 63.33, H 4.38, N 17.38; found:
C 62.97, H 4.39, N 17.17.
J1 ¼8.0 Hz, J2 ¼ 2.0 Hz,1H, CH), 7.50 (d, J ¼ 2.0 Hz,1H, CH), 9.20 (s,1H,
CH), 9.69 (brs,1H, OH).13C NMR (100 MHz, DMSO)
d: 21.7 (CH2), 22.5
(CH2), 24.8 (CH2), 25.2 (CH2), 55.8 (OCH3), 112.4 (CH), 115.9 (CH),
116.6 (C), 117.8 (C), 121.8 (CH), 129.2 (C), 134.5 (CH), 137.7 (C), 146.0
(C), 146.1 (C), 148.0 (C), 148.7 (C), 148.8 (C). ES-MS m/z 353.1 (MHþ).
HRMS 353.1067, found 353.1068. Anal. Calcd for C18H16N4O2S:
C 61.35, H 4.58, N 15.90; found: C 61.35, H 4.51, N 15.57.
4.1.1.6. 3-(4-Hydroxyphenyl)-8,9,10,11-tetrahydro[1]benzothieno[3,2-e]
[1,2,4]triazolo[4,3-c]pyrimidine 8g. 1H NMR (400 MHz, DMSO)
d: 1.92
4.1.1.2. 3-Phenyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-e][1,2,4]tri-
(m, 4H, 2 CH2), 2.91 (m, 2H, CH2), 3.10 (m, 2H, CH2), 7.01 (d, J ¼ 8.6 Hz,
azolo[4,3-c]pyrimidine 8a. 1H NMR (400 MHz, DMSO)
d: 1.94
2H, 2 CH), 7.81 (d, J ¼ 8.6 Hz, 2H, 2 CH), 9.14 (s,1H, CH),10.12 (brs,1H,
(m, 4H, 2 CH2), 2.93 (m, 2H, CH2), 3.13 (m, 2H, CH2), 7.66 (m, 3H, 3
OH).13C NMR (100 MHz, DMSO)
d: 21.7 (CH2), 22.5 (CH2), 24.8 (CH2),
25.1 (CH2), 116.0 (CH), 116.4 (C), 117.8 (C), 129.2 (C), 130.2 (CH), 134.3
CH), 8.01 (m, 2H, 2 CH), 9.22 (s, 1H, CH). 13C NMR (100 MHz, DMSO)
OH
HN
N
N
N
Na2CO3
DMSO
OCH3
OH
OCH3
Scheme 3. Oxidative cylisation and rearrangement of Exo2 with sodium carbonate.
N
S
N
S
9
N
2
N

280
L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
Table 4
Oxidative cyclisation of pyridyl- and pyrimidyl- hydrazones to 1,2,4-triazolo[1,5-c]pyridines 8a–j and 1,2,4-triazolo[1,5-c]pyrimidines 10a–j.
R1
R2
R3
H
X
N
Time
2db
Product
Ratio %a
7a
8a
10a
50%
50%
–
7a
7b
8b
20%
–
7b
7c
H
H
N
N
2db
5db
10b
80%
7c
8c
10c
30%
5%
65%
7d
–
8d
–
10d
>90%
7d
H
N
24 hb
7g
–
8g
–
7g
2
H
H
N
N
4d
2d
10g
>99%
2
6
9
–
–
>99%
7h
–
8h
–
10h
>99%
7h
7i
CH3
N
3d
7i
8i
10i
45%
10%
45%
H
H
N
C
4db
4db
7j
8j
10j
45%
10%
45%
7j
H–, H–
a
Ratio determined by NMR.
Further heating leads to decomposition.
b
(CH), 137.7þ(C), 145.9 (C), 146.0 (C), 148.7 (C), 159.4 (C). ES-MS m/z
323.1 (MH ). HRMS 323.0961, found 323.0970. m.p. > 300 ꢀC. Anal.
Calcd forC17H14N4OS:C 63.33, H4.38, N 17.38; found:C 62.88, H4.43,
N 16.98.
CH), 7.07 (dd, J1 ¼8.0 Hz, J2 ¼ 2.0 Hz, 1H, CH), 7.24 (d, J ¼ 2.0 Hz, 1H,
CH), 9.55 (brs, 1H, OH). ES-MS m/z 367.1 (MHþ).
4.1.1.8. 3-(4-Hydroxy-3-methoxyphenyl)-1,2,4-triazolo[4,3-c]pyrimi-
dine, 8j. 1H NMR (400 MHz, DMSO)
d: 3.88 (s, 3H, OCH3), 7.01
4.1.1.7. 3-(4-Hydroxy-3-methoxyphenyl)-5-methyl-8,9,10,11-tetrahy-
(t, J ¼ 7.0 Hz, 1H, CH), 7.04 (d, J ¼ 8.3 Hz, 1H, CH), 7.30 (d, J ¼ 8.3 Hz,
dro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine 8h. 1H NMR
1H, CH), 7.42 (m, 2H, 2 CH), 7.83 (d, J ¼ 7.0 Hz, 1H, CH), 8.55 (d,
(400 MHz, DMSO)
d
: 1.87 (m, 4H, 2 CH2), 2.23 (m, 2H, CH2), 2.86 (m,
J ¼ 7.0 Hz, 1H, CH), 9.72 (s, 1H, CH). 13C NMR (100 MHz, DMSO)
d:
2H, CH2), 3.05 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.92 (d, J ¼ 8.0 Hz, 1H,
55.7 (OCH3), 111.7 (CH), 112.1 (C), 114.3 (CH), 115.4 (CH), 115.9 (CH),

L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
281
Table 5
120.9 (C), 128.5 (C), 135.0 (CH), 137.7(C), 147.8 (C), 148.8 (C), 149.2
(C), 152.6 (C), 164.1 (C). ES-MS m/z 353.1 (MHþ). HRMS 353.1067,
found 353.1069. m.p. ¼ 254–256 ꢀC. Anal. Calcd for C18H16N4O2S:
C 61.35, H 4.58, N 15.90; found: C 59.69, H 4.60, N 15.43.
Synthesis of 1,2,4-triazolo[1,5-c]pyrimidines 2, 10a, 10g and 10k starting from the
amino imine 11
O
NH
R
N
N
NH2
R
H
N
4.1.2.2. 2-Phenyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-e][1,2,4]tri-
N
azolo[1,5-c]pyrimidine 10a. 1H NMR (300 MHz, DMSO)
d: 1.92 (m,
S
MeOH / reflux
S
N
N
4H, 2 CH2), 2.94 (m, 2H, CH2), 3.17 (m, 2H, CH2), 7.57 (m, 3H, 3 CH),
8.27 (m, 3H, 3 CH), 9.67 (s, 1H, CH). ES-MS m/z 307.1 (MHþ). HRMS
307.1012, found 307.1012. Anal. Calcd for C17H14N4S: C 66.64, H 4.61,
N 18.29, S 10.47; found: C 66.48, H 4.60, N 18.00, S 10.26.
11
9, 10a, 10c, 10g, 10k
Product
R
Yield
65%
4.1.2.3. 2-4-Fluorophenyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine 10c. 1H NMR (400 MHz, DMSO)
d:
9
1.92 (m, 4H, 2 CH2), 2.92 (m, 2H, CH2), 3.11 (m, 2H, CH2), 7.40
(t, JHH ¼ 3, JHF ¼ 8.8 Hz, 2H, 2 CH), 8.27 (t, JHH ¼ 3, JHF ¼ 8.8 Hz, 2H, 2
CH), 9.62 (s, 1H, CH). 13C NMR (100 MHz, DMSO)
d
: 21.6 (CH2), 22.4
2
(CH2), 24.7 (2 CH2), 116.0 (CH, JCF ¼ 21.8 Hz), 125.2 (C), 126.4 (C),
3
10a
10c
66%
51%
128.5 (C), 129.4 (CH, JCF ¼ 8.9 Hz), 136.9 (CH), 137.2 (C), 138.1 (C),
148.5 (C), 152.7 (C), 162.9 (C). ES-MS m/z 325.0 (MHþ). HRMS
235.0918, found 325.0914.
4.1.2.4. 2-(4-Hydroxyphenyl)-8,9,10,11-tetrahy-
dro[1]benzothieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
NMR (400 MHz, DMSO) : 1.93 (m, 4H, 2 CH2), 2.94 (m, 2H, CH2),
10g. 1H
d
3.15 (m, 2H, CH2), 6.95 (d, J ¼ 8.6 Hz, 2H, 2 CH), 8.10 (d, J ¼ 8.6 Hz,
2H, 2 CH), 9.59 (s, 1H, CH), 10.02 (brs, 1H, OH).
10g
10k
34%
70%
13C NMR (100 MHz, DMSO)
d: 21.7 (CH2), 22.5 (CH2), 24.9 (CH2),
25.0 (CH2), 115.6 (CH), 119.3 (C), 120.6 (C), 128.6 (C), 128.7 (CH),
136.8 (CH), 137.8 (C), 148.9 (C), 152.7 (C), 159.8 (C), 164.1 (C). ES-MS
m/z 365.1 (MNaþ). HRMS 365.1043, found 365.1056. m.p. decom-
poses at 250 ꢀC. Anal. Calcd for C17H14N4OS: C 63.33, H 4.38, N
17.38; found: C 62.93, H 4.45, N 16.98.
4.1.2.5. 2-(2-Methylpropyl)-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine 10k. 1H NMR (400 MHz, DMSO
117.2 (C),121.1 (CH), 124.0 (CH),127.8 (CH),146.3 (C),148.4 (C),149.5
(C). ES-MS m/z 242.0 (MHþ).
spectra recorded at 353 K)
d
: 0.98 (d, J ¼ 6.8 Hz, 6H, 2 CH3), 1.89 (m,
4H, 2 CH2), 2.20 (m, 1H, CH), 2.51 (d, J ¼ 6.8 Hz, 2H, CH2), 2.90 (m,
2H, CH2), 3.04 (m, 2H, CH2), 9.52 (s, 1H, CH).
4.1.2. General procedure for oxidative cyclisation of amino imime 11
with aldehydes to give the 1,2,4-triazolo[1,5-c]pyrimidines
(9,10a,c,g,k)
To a solution of the amino imine 11 [12,22] (0.25 g, 1.13 mmol) in
methanol (10 ml) was added the required aldehyde (1.1 equiv) and
the mixture stirred and heated at reflux overnight. On cooling, the
precipitate was isolated by filtration and the solid washed with cold
methanol and dried under high vacuum to give the yields shown in
Table 5.
13C NMR (100 MHz, DMSO, 353 K)
d: 22.2 (CH2), 22.8 (2 CH3),
23.0 (CH2), 25.3 (CH2), 25.4 (CH2), 27.9 (CH), 37.7 (CH2), 119.9 (C),
129.0 (C), 136.9 (CH), 138.4 (C), 148.9 (C), 153.0 (C), 167.6 (C). ES-MS
m/z 287.1 (MHþ). HRMS 287.1325, found 287.1326. m.p. ¼121–
122 ꢀC. Anal. Calcd for C15H18N4S: C 62.91, H 6.33, N 19.56, S 11.20;
found: C 62.50, H 6.26, N 19.42, S 11.18.
4.1.2.6. 2-(4-Hydroxy3-iodo-5-methoxyphenyl)-8,9,10,11-tetrahy-
dro[1]benzothieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
NMR (400 MHz, DMSO) : 1.94 (m, 4H, 2 CH2), 2.91 (m, 2H, CH2),
13. 1H
d
4.1.2.1. 2-(4-Hydroxy-3-methoxyphenyl)-8,9,10,11-tetrahy-
dro[1]benzothieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 9. 1H NMR
3.08 (m, 2H, CH2), 3.98 (s, 3H, OCH3), 5.10 (brs, OH in water peak),
7.65 (d, J ¼ 1.8 Hz, 1H, CH), 8.11 (d, J ¼ 1.8 Hz, 1H, CH), 9.52 (s, 1H,
(400 MHz, DMSO) d: 1.91 (m, 4H, 2 CH2), 2.90 (m, 2H, CH2), 3.09 (m,
CH). 13C NMR (100 MHz, DMSO)
d: 21.6 (CH2), 22.4 (CH2), 24.8 (2
2H, CH2), 3.90 (s, 3H, OCH3), 6.95 (d, J ¼ 7.8 Hz,1H, CH), 7.70 9 (s,1H,
CH2), 84.5 (C), 109.6 (CH), 119.2 (C), 122.5 (C), 128.5 (C), 129.2 (CH),
137.9 (C), 147.0 (C), 148.6 (C), 152.7 (C), 162.5 (C). ES-MS m/z 479.0
(MHþ).
CH), 7.71 (d, J ¼ 7.8 Hz, 1H, CH), 9.54 (s, 1H, CH), 9.61 (brs, 1H, OH).
13C NMR (100 MHz, DMSO)
d: 21.6 (CH2), 22.4 (CH2), 24.8 (CH2),
24.9 (CH2), 55.6 (OCH3), 110.4 (CH), 115.7 (CH), 119.2 (C), 120.8 (CH),
N
HN N
heat
NH
oxidation
H
OH
OCH3
OH
6
N
S
S
H
N
N
N
2
OCH3
12
Scheme 4. Possible diamino acetal intermediate in the oxidative cyclisation of Exo2.

282
L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
I
OH
I2
HN N
N
OCH3
OH
DMSO
S
N
N
N
S
N
OCH3
13
2
N
Scheme 5. Iodine catalysed oxidation and rearrangement of Exo2.
4.2. Toxin challenge assay
Ka) ¼ 0.077 mmꢃ1, U ¼ 787.75(3) Å3, Z 2, Dcal ¼ 1.418 g cmꢃ3, Flack
0.01(5) for 1688 Friedel pairs, 21436 reflections measured, 3945
HeLa cells were pretreated for 30 min with growth medium con-
taining 50 M Exo2 ortriazoledilutedfroma 50 mMstockinDMSOor
unique [Rint ¼ 0.0362], R [I > 2
s
(I)] ¼ 0.0467, wR [I > 2 (I)] ¼ 0.0808,
s
m
GooF ¼ 1.016, CCDC 741336.
with vehicle DMSO alone, and were then challenged or not for 1 h
with 50 ng/ml STx in growth medium containing Exo2, compound or
DMSO as appropriate. Under these conditions, trial experiments had
established that this dose of toxin reduced protein synthesis ability of
HeLa cells to 40% of that of non-toxin-treated controls. The remaining
ability of the cells to manufacture protein was then assessed by
incubating the cells for 30 min in PBS containing [35S]-labelled
methionine and cysteine, and measuring incorporation of these into
acid-precipitable material [9]. The error bars are ꢁ1.S.D.
Crystal data for (9) C18H16N4O2S, M ¼ 352.42, yellow block,
0.25 ꢂ 0.20 ꢂ 0.10 mm, Monoclinic, P2(1)/c (No 14),
b
¼ 90.3577(17)ꢀ,
a ¼ 5.85370(10), b ¼ 12.5466(2), c ¼ 21.7465(4) Å, T ¼ 296(2)K,
m(Mo-
Ka) ¼ 0.077 mmꢃ1, U ¼ 1597.12(5) Å3, Z 4, Dcal ¼ 1.466 g cmꢃ3, 18182
reflections
[I > 2
741337.
measured,
2928
unique
[Rint ¼ 0.0435],
R
s
(I)] ¼ 0.0659, wR [I > 2 (I)] ¼ 0.1182, GooF ¼ 1.073, CCDC
s
Crystal data for (10k) C15H18N4S, M ¼ 286.39, yellow block,
0.40 ꢂ 0.10 ꢂ 0.10 mm, Monoclinic, P2(1)/n (No 14),
b
¼ 102.517(2)ꢀ,
a ¼ 13.2799(3), b ¼ 5.24816(9), c ¼ 20.1671(4) Å, T ¼ 100(2)K,
m(Mo-
4.3. Crystallography
Ka) ¼ 0.077 mmꢃ1, U ¼ 1372.14(5) Å3, Z 4, Dcal ¼ 1.386 g cmꢃ3, 16734
reflections
[I > 2
741338.
measured,
4653
unique
[Rint ¼ 0.0353],
R
Suitable crystals of 8b, 9 and 10k were grown by slow diffusion
of methanol into a DMSO solution of the compound in an nmr tube.
The crystals were mounted with glue or oil and the data
recorded using an Oxford Diffraction Gemini four-circle system
with Ruby CCD area detector.
s
(I)] ¼ 0.0390, wR [I > 2 (I)] ¼ 0.0947, GooF ¼ 0.972, CCDC
s
All non hydrogen atoms were refine anisotropically. Hydrogen
atoms were placed at calculated positions except the OH in 9 which
was located in a difference map. It’s position was allowed to refine
freely but given thermal parameters equal to 1.5 times the oxygen
to which it was attached.
The data was solved and refined using the SHELXTL suite of
programs [27].
The supplementary crystallographic data for this paper can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif quoting the
appropriate CCDC number.
Acknowledgements
This work was generously supported by a research grant from
the Biotechnology and Biological Sciences Research Council
(BBSRC) (BB/E012450/1). The Oxford Diffraction Gemini XRD
system was obtained through the Science City Advanced Materials
Crystal data for (8b) C18H16N4OS, M ¼ 336.42, colourless rod,
0.50 ꢂ 0.08 ꢂ 0.06 mm, Monoclinic, P2(1) (No 4),
b
¼ 98.953(2)ꢀ,
a ¼ 5.3889(1), b ¼ 18.1190(3), c ¼ 8.1673(2) Å, T ¼ 296(2)K,
m(Mo-
Fig. 3. Biological activity of the triazolopyrimidines in Shiga Toxin Assay.

L.J. Guetzoyan et al. / European Journal of Medicinal Chemistry 45 (2010) 275–283
283
[15] T. Nagamatsu, H. Yamasaki, T. Akiyama, S. Hara, K. Mori, H. Kusakabe,
Synthesis 4 (1999) 655–663.
[16] A.F. Khattab, F.A. El-Essawy, J. Chem. Res. (2005) 736–740.
[17] (a) R. Kumar, R.R. Nair, S.S. Dhiman, J. Sharma, O. Prakash, Eur. J. Med. Chem. 44
(2000) 2260–2264;
project: Creating and Characterising Next Generation Advanced
Materials, with support from Advantage West Midlands (AWM) and
part funded by the European Regional Development Fund (ERDF)
(b) A.K. Sadana, Y. Mirza, K.R. Aneja, O. Prakash, Eur. J. Med. Chem. 38 (2003)
533–536;
References
(c) O. Prakash, V. Bhardwaj, R. Kumar, P. Tyagi, K.R. Aneja, Eur. J. Med. Chem. 39
(2004) 1073–1077;
(d) O. Prakash, R. Kumar, D. Sharma, R. Naithani, R. Kumar, Heteroat. Chem. 17
(2006) 653–655;
(e) O. Prakash, R. Kumar, R. Kumar, P. Tyagi, R.C. Kuhad, Eur. J. Med. Chem. 42
(2007) 868–872;
(f) B.S. Joe, H.Y. Son, Y.-H. Song, Heterocycles 75 (2008) 3091–3097;
(g) R. Kumar, R.R. Nair, S.S. Dhiman, J. Sharma, O. Prakash, Eur. J. Med. Chem.
44 (2009) 2260–2264.
[1] M.R. Prasad, A.R. Rao, P.S. Rao, K.S. Rajan, S. Meena, K. Madhavi, Eur. J. Med.
Chem. 43 (2008) 614–620.
[2] V.H. Bhaskar, P.D. Gokulan, Orient. J. Chem. 23 (2007) 999–1004.
[3] (a) L.D. Jennings, S.L. Kincaid, Y.D. Wang, G. Krishnamurthy, C.F. Beyer,
J.P. McGinnis, M. Miranda, C.M. Discafani, S.K. Rabindran, Bioorg. Med. Chem.
Lett. 15 (2005) 4731–4735;
(b) H. Han, J. Roberts, O. Lou, W.A. Muller, N. Nathan, C. Nathan, J. Leukoc Biol.
79 (2006) 147–154.
[4] (a) A.E. Rashad, M.A. Ali, Nucleosides Nucleotides 25 (2006) 17–28;
(b) C.S. Thakur, B.K. Jha, B. Dong, J. Das Gupta, K.M. Silverman, H. Mao,
H. Sawai, A.O. Nakamura, A.K. Banerjee, A. Gudkov, R.H. Silverman, Proc. Natl.
Acad. Sci. U.S.A. 104 (2007) 9585–9590.
[5] (a) Y. Dai, Y. Guo, R.R. Frey, Z. Ji, M.L. Curtin, A.A. Ahmed, D.H. Albert, L. Arnold,
S.S. Arries, T. Barlozzari, J.L. Bauch, J.J. Bouska, P.F. Bousquet, G.A. Cunha,
K.B. Glaser, J. Guo, J. Li, P.A. Marcotte, K.C. Marsh, M.D. Moskey, L.J. Pease,
K.D. Stewart, V.S. Stoll, P. Tapang, N. Wishart, S.K. Davidsen, M.R. Michaelides,
J. Med. Chem. 48 (2005) 6066–6083;
(b) M.S. Phoujdar, M.K. Kathiravan, J.B. Bariwal, A.K. Shah, K.S. Jain, Tetrahe-
dron Lett. 49 (2008) 1269–1273.
[6] R. Gundla, R. Kazemi, R. Sanam, R. Muttineni, J.A.R.P. Sarma, R. Dayam,
N. Neamati, J. Med. Chem. 51 (2008) 3367–3377.
[7] J.C. Yarrow, Y. Feng, Z.E. Perlman, T. Kirchhausen, T.J. Mitchison, Comb. Chem.
High Throughput Screening 6 (2003) 279–286.
[8] Y. Feng, A.P. Jadhav, C. Rodighiero, Y. Fujinaga, T. Kirchhausen, W.I. Lencer,
EMBO Rep. 5 (2004) 596–601.
[9] R.A. Spooner, P. Watson, D.C. Smith, F. Boal, M. Amessou, L. Johannes,
G.J. Clarkson, J.M. Lord, D.J. Stephens, L.M. Roberts, Biochem. J. 414 (2008)
471–484.
[10] M. Nagaraju, A. Krishnaiah, H.B. Mereyala, Synth. Commun. 35 (2007)
2467–2472.
[11] For reviews on the synthesis of pyrimidine triazoles see (a) M.A.E. Shaban,
A.E.A. Morgaan, Adv. Heterocycl. Chem. 75 (1999) 131–176;
(b) M.A.E. Shaban, A.E.A. Morgaan, Adv. Heterocycl. Chem. 75 (1999)
243–281.
[12] For examples of cyclisation with orthoformates see (a) A.E. Rashad,
O.A. Heikal, A.O.H. El-Nezhawy, F.M.E. Abdel-Megeid, Heteroat. Chem. 16
(2005) 226–234;
(b) D.J. Brown, T. Nagamatsu, Aust. J. Chem. 31 (1978) 2505–2515.
[13] For an example of cyclisation with activated acid derivatives see Y. Wang,
K. Sarris, D.R. Sauer, S.W. Djuric. Tetrahedron Lett. 48 (2007) 2237–2240.
[14] H. Chen, Z. Shang, J. Chang, Synth. Commun. 36 (2006) 445–450.
[18] V.J. Ram, H.K. Pandey, A.J. Vlietinck, J. Heterocycl. Chem. 18 (1981)
1277–1280.
[19] For the oxidation of sodium iodide by sulfoxides see (a) J.H. Krueger, Inorg.
Chem. 5 (1966) 132–136;
(b) G. Modena, G. Scorrano, D. Landini, F. Montanari, Tetrahedron Lett. 28
(1966) 3309–3313.
[20] C. Lin, P.-T. Lai, S.K.-S. Liao, W.-T. Hung, W.-B. Yang, J.-M. Fang, J. Org. Chem. 73
(2008) 3848–3853;
See also the sulphur catalysed oxidation of aminomethylpyridines to imida-
zopyridines in F. Shibahara, R. Sugiura, E. Yamaguchi, A. Kitagawa, T. Murai. J.
Org. Chem. 74 (2009) 3566–3568.
[21] For reviews on the Dimroth rearrangement in triazolopyrimidine systems see
(a) E.S.H. El Ashry, Y. El Kilany, N. Rashed, Adv. Heterocycl. Chem. 75 (1999)
79–167;
(b) See reference [11].
´
[22] E.V. Vorobev, M.E. Kletskii, V.V. Krasnikov, V.V. Mezheritskii, D.V. Steglenko,
Russ. Chem. Bull. 55 (2006) 2247–2255.
[23] For similar cyclisations of amino imines with aldehydes but done in DMF/
HOAc see (a) N.A. Hassan, M.I. Hegab, A.E. Rashad, A.A. Fahmy, F.M.E. Abdel-
Megeid, Nucleosides Nucleotides 26 (2007) 379–390;
(b) E.K. Ahmed, M.A. Ameen, F.F. Abdel-latif, Phosphorus, Sulfur Silicon Relat.
Elem. 181 (2006) 497–510.
[24] For examples of the arial oxidation of other diamino heterocycles see (a)
M. Curini, F. Epifano, F. Montanari, O. Rosati, S. Taccone, Syn. Lett. 10 (2004)
1832–1834.
[25] C.J. Shishoo, M.B. Devani, G.V. Ullas, S. Ananthan, V.S. Bhadti, J. Heterocycl.
Chem. 18 (1981) 43–46.
[26] J. Tonnar, P. Lacroix-Desmazes, Angew. Chem. Int. Ed. Engl. 47 (2009)
1294–1297.
[27] (a) G.M. Sheldrick, SHELXL97. Program for the Refinement of Crystal Struc-
tures. University of Go¨ttingen, Go¨ttingen, Germany, 1997;
(b) G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112–122.