Diversity oriented syntheses of fused pyrimidines designed as potential antifolates

Article abstract of DOI:10.1039/b818339b

Diversity oriented syntheses of some furo[2,3-d]pyrimidines and pyrrolo[2,3-d]pyrimidines related to folate, guanine, and diaminopyrimidine- containing drugs have been developed for the preparation of potential anti-infective and anticancer compounds. Ami

Full text of DOI:10.1039/b818339b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Diversity oriented syntheses of fused pyrimidines designed as
potential antifolates†
Colin L. Gibson,a Judith K. Huggan,a Alan Kennedy,a Lionel Kiefer,a Jeong Hwan Lee,a Colin J. Suckling,*a
Carol Clements,b Alan L. Harvey,b William N. Hunterc and Lindsay B. Tullochc
Received 16th October 2008, Accepted 30th January 2009
First published as an Advance Article on the web 6th March 2009
DOI: 10.1039/b818339b
Diversity oriented syntheses of some furo[2,3-d]pyrimidines and pyrrolo[2,3-d]pyrimidines related to
folate, guanine, and diaminopyrimidine-containing drugs have been developed for the preparation of
potential anti-infective and anticancer compounds. Amide couplings and Suzuki couplings on the basic
heterocyclic templates were used, in the latter case yields being especially high using aromatic
trifluoroborates as the coupling partner. A new ring synthesis of 6-aryl-substituted deazaguanines
bearing 2-alkylthio groups has been developed using Michael addition of substituted nitrostyrenes.
Diversity at C-2 has been introduced by oxidation and substitution with a range of amino nucleophiles.
The chemical reactivity of these pyrrolopyrimidines with respect to both electrophilic substitution in
ring synthesis and nucleophilic substitution for diversity is discussed. Several compounds were found to
inhibit pteridine reductases from the protozoan parasites Trypanosoma brucei and Leishmania major at
the micromolar level and to inhibit the growth of Trypanosma brucei brucei in cell culture at higher
concentrations. From these results, significant structural features required for inhibition of this
important drug target enzyme have been identified.
lipophilicity to permit passive transport into cells in the absence
Introduction
of active uptake.
Synthetic fused pyrimidines are a class of compounds with
Fused pyrimidines show an exceptional range of biological
widespread biological activity on account of their relationship to
activity. Pyrido[2,3-d]pyrimidines feature in protein kinase
inhibition,5 small molecule kinase inhibition,6 receptor
antagonism,7 anti-infectives,8 cytotoxic9 and anti-inflammatory
agents.10 Similarly broad in their applications are pyrrolo[2,3-
d]pyrimidines which have been investigated as inhibitors of folate
metabolism and biosynthesis,11 extensively as protein kinase
inhibitors,12 as antivirals,13 in combating multidrug resistance,14
in controlling gastric secretion15 and as receptor antagonists.16
Furo[2,3-d]pyrimidines have been considered as templates for
drug discovery for many years with the inhibition of dihydrofolate
reductase (DHFR) as the primary target.17 Similarly, 5-arylfuro-
pyrimidines that are analogues of trimethoprim have been
prepared and were found to be only weak inhibitors of
DHFR.18 More recently, furopyrimidines have been found to
be active as kinase inhibitors19 and as components of antiviral
compounds.20 Of interest to the compounds described in this
paper, arylthiopyrrolopyrimidines have featured as thymidylate
synthase (TS) inhibitors.21 With the above background in
mind, we have investigated the properties of series of furo[2,3-
d]pyrimidines and pyrrolo[2,3-d]pyrimidines containing aryl
substituents attached to the heterocyclic templates. Of particular
interest is the discovery of new inhibitors of pteridine reductase
(PTR1), an essential enzyme for protozoan parasites of the order
Kinetoplastida.
naturally occurring cofactors such as folate and biopterin deriva-
tives and to the purine bases of DNA. As such, they have found ap-
plications in antibacterial, antiparasitic, antiviral, and anticancer
therapies.1 There is much concern in modern medicinal chemistry
about both the evident and developing resistance of infectious or-
ganisms to existing drugs and the very limited range of compounds
available for the treatment of tropical parasitic infectious diseases
including malaria, trypanosomiasis, and leishmaniasis. Chemistry
has responded to these challenges by developing methodologies for
the synthesis of designed ‘intelligent’ libraries of compounds. In
our work this has led to methodologies for the synthesis of diverse
fused pyrimidines including pteridines, pyrido[2,3-d]pyrimidines,
and pyrrolo[2,3-d]pyrimidines.2–4 Such templates typically contain
many hydrogen bonding sites especially on the pyrimidine ring
and one requirement for new biologically active compounds is a
suitable balance of polarity so that there is sufficient hydrogen
bonding available for binding to the target enzymes but sufficient
aWestCHEM, Department of Pure & Applied Chemistry, University of
Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, Scotland. E-mail:
c.j.suckling@strath.ac.uk; Fax: +44 141 548 5743; Tel: +44 141 548 2271
bStrathclyde Institute for Pharmacy and Biomedical Science, Univer-
sity of Strathclyde 29 Taylor Street, Glasgow, G4, Scotland. E-mail:
a.l.harvey@strath.ac.uk; Tel: +44 141 548 4155
cDivision of Biological Chemistry and Drug Discovery, College of Life
Sciences, University of Dundee, 1 Dow St, Dundee, DD2 1NT. E-mail:
w.n.hunter@dundee.ac.uk; Fax: +44 1382 385764; Tel: +44 1382 385745
Initial design of compound libraries
† Electronic supplementary information (ESI) available: Preparation and
properties of compounds listed in experimental section. CCDC reference
number 705654. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b818339b
In most pterin binding enzymes, there are important hydrogen
bonds between the 2-amino, 3-NH, and 4-oxo groups of the pterin
and the enzyme (Fig. 1). Pyrrolo[2,3-d]pyrimidines, analogous to
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Fig. 1 Strategy for diversity.
pterins, therefore have three sites for diversification in the pyrrole
ring at C5, C6, and N7 and one in the pyrimidine ring at C2.
At C2, a secondary amine substituent, for example, would retain
the ability to hydrogen bond but increase the lipophilicity. The
use of 2-alkylthio substituents at C2 is a convenient approach
to diversity oriented synthesis not only because of the synthetic
benefit but also because relevant biological activity has been found
in fused pyrimidines containing 2-alkylthio groups.21 As will be
described further below, C2 alkylthio substituted pyrimidines have
greatly reduced reactivity towards carbon electrophiles attacking
at C5 compared with 2-amino compounds. This reduced reactivity
has prompted the development of ring syntheses based upon
Michael addition. A further challenge is that in purines themselves,
displacement of C2 alkylthio groups either directly by amino
nucleophiles or by oxidation and substitution of the sulfone so
formed has been reported to fail.22,23
The selection of target compounds was initially based upon
a high resolution molecular model of the active site of pteridine
reductase 1 (PTR1) from the parasite Leishmania major complexed
with several pteridines.24 In Leishmania metabolism PTR1 is able
to catalyse the same reaction as DHFR and thus provides a by-
pass to the inhibition of the biosynthesis of tetrahydrofolate by
conventional inhibitors such as methotrexate.
PTR1 is weakly inhibited by methotrexate and this contributes
to the failure of antifolates as therapies against Leishmania
infections. Since there are potent DHFR inhibitors already in
clinical use, the discovery of a potent PTR1 inhibitor could
lead to an effective combination therapy to completely block the
generation of tetrahydrofolate. The start of this project was the
construction of a model of 2-amino-6-bromo-5-cyanopyrrolo[2,3-
d]-4(3H,7H)-one, 1, in the active site of L. major PTR1 (Fig. 2).24
From this model, a number of hypotheses was derived. The
model suggested that aryl substituents at C5 or C6 of a furopyrim-
idine or pyrrolopyrimidine could occupy one of two hydrophobic
pockets at the active site (Fig. 2).A pocket near C5 was also acces-
sible by 4-substituted-5-arylamides. In addition, substituents at C2
would probably be detrimental to binding to the target enzyme.
By examining both diaminofuropyrimidines and deazaguanines
(pyrrolopyrimidines with 2-amino-4-oxo substituents), both of the
major pyrimidine substitution patterns in antifolates are covered.
Fig. 2 A model for compound 1 in the active site of L. major PTR1.
Atomic positions are colored according to atom type. C atoms of 1 are
black, C of NADPH cyan, C of protein residues grey. N is colored blue, O
red, P yellow, Br orange and S green. Blue dashed lines represent potential
˚
hydrogen bonding interactions, of distance 2.5–3.5 A, formed between 1
with the cofactor and protein. Hydrogen atoms are omitted for the purpose
of clarity. Inset is the chemical structure of 1.
with bromine in acetic acid afforded the 6-bromo derivative 4
(98%), which was the primary precursor for Suzuki coupling.
Hydrolysis of the ethyl ester with lithium hydroxide (84%)
afforded the 5-carboxylic acid 5 which was the precursor of
the arylcarboxamides. Suzuki coupling reactions were carried
out using dichlorobis(triphenylphosphine)palladium(II) in the
presence of aqueous sodium carbonate as base and ethanol as
organic solvent under nitrogen.25 A two phase reaction took place
affording both the aryl coupled esters 6a–f and the corresponding
carboxylic acids, 7a–d and f; the yields of esters were in the range
16–45% and the yields of carboxylic acids in the range 18–31%
and the products were noticeably fluorescent. Couplings of aryl
boronates with oxygen, carbon, and chlorine substituents in the
aryl ring were straightforward but coupling of an aryl boronate
with a 4-thiomethyl substituent was erratic and much lower
yielding. In the case of 4-formylphenylboronate, the ester product
6f (16%) was obtained from the organic phase as the diethyl
acetal whereas the acidic product 7e was obtained as the expected
4-formyl compound. Relevant to binding to the target enzyme
and as expected, the phenyl substituent and furan ring are not
coplanar as the crystal structure 4-methoxyphenyl compound 6c
shows (Fig. 3). The amides 8a–f were prepared using active ester
coupling with benzotriazole and the products were purified by
flash chromatography and aqueous base wash to remove residual
benzotriazole. From these reactions altogether, 20 compounds for
were obtained for evaluation.
Synthesis
Synthesis of pyrrolo[2,3-d]pyrimidines
Synthesis of furo[2,3-d]pyrimidines. 2,4-Diaminofuro[2,3-
d]pyrimidines are prepared readily when 2,6-diaminopyrimidin-
Palladium-mediated coupling reactions. The first investigated
route to 6-arylpyrrolopyrimidines with scope for C2 diversification
started from the 2-benzylthiopyrimdine precursor 9 (Scheme 2).
Unlike C-substitution at position 5, iodination was achieved in
good yield to give 10. All but one variation of the Sonogashira
coupling with 10 or its derivatives such as the trifluoroacetamide,
11 were unsuccessful; in the successful case using the conditions of
Cacchi,26 it was possible to isolate a mixture of the coupled alkynyl
4(3H)-one
2 reacts with a-bromoketones bearing strong
electron withdrawing substituents such as trifluoromethyl and
ethoxycarbonyl, but not leaving groups, on the acyl carbon atom
(Scheme 1). With a view to accessing diversity at several positions,
ethyl 2,4-diaminofuro[2,3-d]pyrimidine-5-carboxylate
3
was
prepared using ethyl bromopyruvate in 91% yield. Bromination
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Scheme 1 Synthesis of furo[2,3-d]pyrimidines. Reagents and conditions: i Et3N, DMF, 60 ◦C, 20 h; ii Br2, HOAc, 16 h; iii LiOH, aq. EtOH, 4 h, r.t.; iv
(PPh3)2PdCl2 ArB(OH)2, aq. Na2CO3, 60 ◦C, 16 h; v N-methylmorpholine, ArNH2, HBTU, DMF, r.t., 3–18 h.
trifluoroborates were used, very good yields (~90%) were obtained
and this was the method of choice.28
Michael addition by nitrosoalkenes formed in situ. With
2-alkylthiopyrimidines in related studies in the pyrido[2,3-
d]pyrimidine series4,29 we had difficulty in making the essential
C5-bond using electrophilic reagents in contrast to the ready
reactivity of nitrogen (diazonium, nitrosonium) and halogen (iodo,
bromo) electrophiles.30 On the other hand, we had shown that the
Fig. 3 View of the structure of 6c; ellipsoids are drawn at the 50%
reaction of the 2-aminopyrimidine 2 with a-bromo-oximes in the
presence of tertiary bases affords pyrrolo[2,3-d]pyrimidine precur-
sors such as 17 typically in 50% yield presumably via electrophilic
addition to a nitrosoalkene intermediate (Scheme 3). In the case
of the 2-methylthiopyrimidine, 16b, subsequent cyclisation under
acidic conditions afforded the pyrrolo[2,3-d]pyrimidine ester 18
(54%) which was hydrolysed to the carboxylic acid, 19 (88%). The
nitrosoalkene is an example of a soft electrophile and might be
more suitable for reaction at C5 of a 2-alkylthiopyrimidine than
a carbonyl derivative, for example. Under mildly basic conditions,
it would be expected that pyrimidine-2-thiols would be reactive
towards Michael acceptors. However treatment of 16a with ethyl
bromopyruvate oxime in the presence of triethylamine under
standard conditions gave a three component mixture of the C5
monosubsituted product 20, the 2S adduct 21, and the bis-adduct
probability level.
pyrimidine 12 together with its cyclised product, a 6-phenyl-
pyrrolopyrimidine, 13. Sonogashira couplings of sulfur-
containing compounds and guanine derivatives are known to
be problematic27 and the presence of sulfur at C2 in 10 and
11 is likely to exacerbate these problems. Suzuki coupling of
the 6-bromopyrrolopyrimidine 1, which is easily accessible from
the corresponding 6-unsubstituted compound 14 offers an al-
ternative entry into diversity at C6 albeit sacrificing diversity
at C2 (Scheme 2). With the reacting site for coupling further
removed from the potentially palladium-coordinating pyrimidine
substituents, Suzuki coupling was possible in adequate yield
using aryl boronates to give the products 15a–e. However if
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Scheme 2 Palladium catalysed coupling reactions. Reagents and conditions: i NIS, THF, r.t., 20 h; ii Et3N, THF, TFAA, 0 ◦C-r.t., 20 h; iii phenylacetylene,
CuI, PPh3, K2CO3, 1,4-dioxane, 110 ◦C, 12 h; iv Br2, HOAc, r.t., 20 h; v 2-propanol/H2O, PdCl2(PPh3)2, KPhBF3 or boronic acid, 90 ◦C, 24 h.
Scheme 3 Reactivity of 2-thiopyrimidine derivatives. Reagents and conditions: i Et3N, ethylbromopyruvate oxime, DMF, 0 ◦C to r.t., 36–48 h; ii
benzaldehyde, EtOH, HCl, r.t., 3 h; iii LiOH, THF 48–96 h, then 0.1 M HCl.
22 in only 25% yield. The use of the S-alkyl group was therefore
cosolvent. The latter conditions afforded typically 60–90% yields.
The following Nef cyclisation was examined under a variety of
conditions. Using DBU as base under microwave or thermal con-
ditions low yields (16–30%) of the cyclised products were obtained.
Better yields were obtained using titanium(III) chloride (47–71%)
and this method was adopted as standard for the Nef cyclisation
in this series. 5-Substituted pyrrolo[2,3-d]pyrimidines therefore
become readily available through this methodology (Scheme 4)
and for studies of the diversification at C2, the same series of
reactions was carried out using w-nitrostyrene and the 2-methyl-
thiopyrimidine precursor, 16b leading to pyrrolopyrimidine 28.
important in our reactions.31
Michael addition of nitroalkenes. The reactivity of ni-
trosoalkenes together with a precedent of using maleic anhydride
as the Michael acceptor32 and Taylor’s synthesis of Alimta33
suggested that nitroalkenes, which are readily available, might
be appropriate precursors for pyrrolo[2,3-d]pyrimidine synthesis.
The first example studied used nitrocyclohexene and 9 with DBU
as base and surprisingly, a mixture of Michael adduct 23 (12%)
and cyclised product 24 (25%) was formed (Scheme 4). Extension
of this method to nitrostyrenes allowed Michael adducts 25 to
be prepared from which 5-arylpyrrolo[2,3-d]pyrimidines 26a–d
became available. The C5 substitution reaction was carried out
using either di-isopropylethylamine or aqueous benzyltrimethy-
lammonium hydroxide in a two phase system with ethyl acetate as
Diversification at C2 by displacement of alkylthio groups
and their derivatives
Whilst not optimised for individual cases, the reaction sequences
in Schemes 3 and 4 provide access to 5- and 6-substituted
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Scheme 4 Synthesis of pyrrolopyrimidines by Michael addition to nitroalkenes. Reagents and conditions: i DBU, CH3CN, 80 ◦C, 4 d; ii Me3N+CH2Ph
OH-, 70 ◦C, 4 h; iii TiCl3, NH4OAc, NaOMe, MeOH, r.t., 20 h or 3 eq SnCl2.2H2O, EtOAc, 85 ◦C, 24 h. Yields in parentheses refer to the tin(II) chloride
reaction.
pyrrolo[2,3-d]pyrimidines and diversification at C2 requires sub-
stitution of the 2-alkylthio group. This reaction has been widely
used to prepare libraries of monocyclic pyrimidine derivatives
especially those in which the pyrimidine bears an electron
withdrawing substituent such as an ester or trifluoromethyl.34
Substitution can be directly on the alkylthio derivative itself or
more typically on its oxidised derivative, the sulfone, accessible by
oxidation with peracids or peroxides such as dimethyldioxirane.35
Either direct substitution or substitution after oxidation is
successful in the pteridine series; essentially the pyrazine ring
plays the role of an electron withdrawing substituent. In N-alkyl
2- or 4-oxopurines that lack an NH of phenolic acidity, good
yields of alkylamino derivatives have been obtained by di-
rect substitution.36 Similarly, 4-aminopurines are reactive37 and
pyrazino[3,4-d]pyrimidines undergo hydrolysis of a methylthio
group at C4, a carbon atom activated by N6 in the pyrazine
ring.38 However in pyrrolo[2,3-d]pyrimidines, electron withdraw-
ing groups are absent and the pyrrole ring can be regarded
as relatively electron rich. For the compounds prepared in this
paper, without further derivatisation N3 is ionisable with acidity
comparable to that of a phenol and the pKa decreases as a
result of oxidation to the sulfone. In view of these considerations,
the reactivity of 2-alkylthiopyrrolo[2,3-d]pyrimidines and their
oxidised derivatives to nucleophilic substitution at C2 posed a
challenge.
To test these possibilities, pyrrolopyrimidines were oxidised
with m-chloroperbenzoic acid in dimethylformamide solution and
the crude product subjected to substitution with benzylamine
and other liquid amines that were used as solvents in order
to obtain satisfactory yields (Scheme 5). With 18, which has
an electron withdrawing substituent activating substitution at
C2 of the pyrimidine by conjugation, both C2 substitution and
amide formation was found at 100 ◦C for 24 h to give 29
and 30. Amide substitution alone was not observed but it was
considered possible to find reaction conditions that would favour
C2 substitution alone. The substitution reaction was run at
several temperatures between 2 ◦C and 60 ◦C but conversions
were less than 10%. Even with the most extensive conversion at
60 ◦C, the ratio of disubstitution to monosubstitution was not
significantly changed. In general, cleavage of a methylthio group
took place more readily than that of a benzylthio group. 5-Aryl
pyrrolopyrimidines, 26 and 28, would be expected to be less
reactive than 18 because they have no electron withdrawing groups
that might both stabilise them to oxidation during activation of the
Scheme 5 Substitution reactions of 2-alkylthiopyrrolo[2,3-d]pyrimidines. Reagents and conditions: i m-CPBA, DMF, 0–100 ◦C, 4–17 h; amine nucleophile,
100 ◦C, 15 h.
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Table 1 Biological activity of compounds prepared. na = not active. Blank cells in data columns indicate that the compound was not evaluated
in the relevant test. The assays of T. b. brucei were cell based; values in roman font refer to IC50s and values in italics refer to % inhibition at
100 mM
T. brucei
PTR1 Ki mM
L. major
PTR1 Ki mM
T. b. brucei
MIC mM
T. b. brucei %
@ 100 mM
Compound
R2
R5
R6
3
CO2Et
CO2H
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2H
CO2H
CO2H
CO2H
H
H
> 100
> 100
na
na
5
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
8f
Ph
4-Cl-C6H4
4-OMe-C6H4
4-Et-C6H4
4-SMe-C6H4
4-CH(OEt)2-C6H4
Ph
4-Cl-C6H4
4-OMe-C6H4
4-Et-C6H4
4-CHO-C6H4
Ph
H
H
H
H
H
50
> 100
57
50
50
100
25
na
na
na
na
na
77
CO2H
CONHPh
CONH(4-Cl-C6H4)
CONH(4-OMe-C6H4)
CONH(4-Et-C6H4)
CONH(4-SMe-C6H4)
CONH(4-CO2Et-C6H4)
100
100
na
56
49
1
NH2
NH2
NH2
NH2
NH2
BnS
BnS
BnS
BnS
MeS
BnNH
(CH2)4NH
PhNH
CN
CN
CN
CN
CN
4-Br-C6H4
Ph
4-OMe-C6H4
2-CF3-C6H4
Ph
Ph
Ph
Ph
Br
Ph
3.9
> 27
> 27
3.4
16.4
4.2
na
na
na
na
na
75
15a
15b
15c
15d
26a
26b
26c
26d
28
0.71
0.36
0.5
4-OMe-C6H4
4-Et-C6H4
3-CHO-C6H4
0.29
H
H
H
H
H
H
H
H
25
64
55
12
25
31a
31b
31c
50
12.5
thioether and promote nucleophilic substitution. At first, it was
difficult to find reaction conditions for clean oxidation of these
thioethers. With many oxidising agents (oxone at -70 ◦C in aque-
ous methanol to 80 ◦C in acetonitrile, dichlorodioxomolybdate
with hydrogen peroxide, selenium dioxide with hydrogen peroxide,
and sodium tungstate with hydrogen peroxide) the required
sulfone was not obtained. However oxidation in satisfactory
yield was found to be possible using m-chloroperbenzoic acid
in DMF at room temperature. Substitution was then carried
out in neat amines, benzylamine, pyrrolidine and aniline, at
100 ◦C for 15 h. More volatile amines such as diethylamine
and isopropylamine did not give substituted products when used
in ethanol or tetrahydrofuran solution. Using neat, relatively
volatile amines, C2-substituted compounds 31a–c were obtained
in moderate yields (24~55%). This result shows for the first time
that the oxidation-substitution route is practical for the synthesis
of libraries of pyrrolo[2,3-d]pyrimidines and satisfactory in that
primary alkyl, secondary alkyl, and aromatic amines were all
acceptable substrates indicating that a wide diversity of products is
possible.
Biological evaluation
Selected compounds were evaluated in enzyme inhibition assays
against Trypanosoma brucei and Leishmania major and in a
number of cell-based assays investigating antiparasitic and cy-
totoxicity against normal and cancer mammalian cells (Table 1).
With respect to PTR1, none of the furopyrimidines proved to
be significantly active. One pyrimidine precursor (9) was also
found to be inactive although its close relative, 2,4-diamino-
6-benzylsulfanylpyridimine, was found to inhibit PTR1 from
Leishmania major. Several of the pyrrolopyrimidines (15a–15d)
showed significant levels of inhibition for PTR1 at best at
submicromolar concentrations. The active compounds can be
considered to be significant hits, especially bearing in mind
their small size and potential for further structural elaboration.
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They provide a basis for structure-based design for this target.
Recently, a series of quinoxaline containing compounds and some
classical antifolate analogues has been described as inhibitors
of pteridine reductases.39 One folate analogue was an especially
potent inhibitor of PTR1 from L. major (IC50 = 0.037 mM)
but in general, most compounds were active in the 10–100 mM
range. These data and the associated crystal structural infor-
mation also contribute to the basis for design of specific PTR1
inhibitors.
Effective drugs, however, must not only bind to the target
enzyme but also must be able to reach it. Our compounds were
therefore evaluated in a cell based screen against Trypanosma
brucei brucei. That the active compounds in the enzyme assay were
inactive in the cellular assay against T. brucei brucei40 suggests that
poor transport into the cells is a probable explanation. However
two compounds, 31a and 31c in the pyrrolopyrimidine series
were active in the cell based assays and these compounds notably
contain benzene rings, which have a major influence on logP. The
2-amino-5-cyano-6-phenyl pyrrolopyrimidine 15a has a clogP of
1.08 in contrast to the 2-phenylamino-5-phenyl pyrrolopyrimidine
31c (clogP = 4.08) and the 2-benzylamino-5-phenyl pyrrolopyrim-
idine 31a (clogP = 3.55). Some furopyrimidine esters (5a, c–f) were
also hits in the cellular assay but the corresponding carboxylic
acids were inactive, a further illustration of the likely importance
of transport of compounds into cells. It was also notable that
several of the alkylthio pyrrolopyrimidines (26a, b, d) were hits in
the antitrypanosomal screen. However it cannot be inferred that
any of the compounds active in the cellular assay exert their effect
through PTR1.
In view of the universality of folate and guanine metabolism,
the compounds prepared were submitted to a broad range of
screens in house.41 Encouragingly, most were found to be non
toxic to normal mammalian cells (L929 murine fibroblast, H92C
rat heart myoblast, HS27 human foreskin fibroblast) but two
pyrrolopyrimidines, 26d and 31c, showed weak but selective
activity against ZR75 human epithelial breast cancer cells. As
an exception, the furopyrimidine 6f appeared to be toxic to both
normal cells and cancer cell lines.
In summary, hit compounds active against both the parasite
Trypansoma brucei alone and some against the parasite Leish-
mania major have been identified and these compounds showed
in general little mammalian cell toxicity. One exception was
the pyrrolopyrimidine 31c which was active against the human
breast cancer cell line ZR75. The identification of hydrophobic
pockets adjacent to the pterin binding site of PTR1 from the
crystal structure has provided a useful basis for design and in the
pyrrolopyrimidine series, there is a suggestion that a hydrogen
bonding group as in 15b and 15d is also beneficial from the most
active group of compounds, 15a–15d. Compounds in the two series
described here thus appear to have potential further investigation
as antiparasitic compounds.
resonance. IR spectra were determined using a Mattson 1000 FT
spectrometer or a Nicolet Impact 400D FT spectrometer as a KBr
disc. Mass spectra were measured on a JEOL JMS AX505 spec-
trometer at the University of Strathclyde using electrospray (ES),
or chemical ionisation (CI) methods. Accurate mass recorded at
the University of Glasgow on Jeol JMS-7 MStation high resolution
magnetic sector using electron impact (EI) or fast atom bombard-
ment (FAB) ionisation. Melting points, where measurable, were
determined on a Reichert hot stage apparatus and are uncorrected.
Microanalysis is typically unreliable in polyazabicyclic compounds
due to poor combustion even in the presence of a catalyst;
microanalytical data is reported where satisfactory and 400 MHz
1H NMR spectra are included in the Electronic Supplementary
Information†. TLC was carried out on silica (Merck 0.25 mm
60 F254) visualising the plates with either potassium permanganate
solution or UV. Column chromatography was carried out using sil-
ica gel (230–400 mesh; 40–60 mm) according to the method of Still
et al.42All reagents were bought from Aldrich (Gillingham, Dorset,
U.K.).
Furopyrimidines
Ethyl 2,4-diaminofuro[2,3-d]pyrimidine-5-carboxylate 3. 2,4,-
Diaminopyrimidin-4(3H)-one 2 (3.05 g, 24.2 mmol), ethyl bro-
mopyruvate (1.5 eq, 36.3 mmol, 4.5 ml) and dry triethylamine
(1.5 eq, 36.3 mmol, 5 ml) were dissolved in anhydrous DMF
(20 ml) and stirred under nitrogen at 60 ◦C for 20 h until
the reaction was complete as shown by TLC. The solvent was
evaporated under reduced pressure by successive co-evaporation
with toluene and ethanol. The desired furopyrimidine was ob-
tained and recrystallised from ethanol. After being filtered, the
product was dried overnight in a drying pistol. The dark-brown
powder was isolated (2.873 g, 12.9 mmol, 63%). A further crop
was obtained from the evaporated mother liquors by suspending
the powder in a saturated aqueous sodium bicarbonate solution
(50 ml), extraction with ethyl acetate (3 ¥ 70 ml). The organic
layer was dried (Na2SO4), filtered, and evaporated under reduced
pressure to afford a total yield of 91% as a dark-brown pow-
der; mp decomposed from 239–241 ◦C; nmax (KBr)/cm-1 3492,
3367, 3307, 3176, 2975, 2938, 2738, 2677, 2491, 1697, 1614,
1574, 1556, 1474, 1374, 1373, 1170, 1113, 1075, 1037, 754;
dH ((CD3)2SO) 1.30 (3H, t, J = 7.2), 4.32 (2H, q, J = 7.2),
6.31 (2H, br s, NH2), 7.19 (2H, br s, NH2), 8.15 (1H, s); dC
((CD3)2SO) 14.5, 61.5, 88.9, 113.7, 114.8, 159.2, 162.4, 164.4,
170.1; HREIMS: calcd for C9H10O3N4 (M+); 222.0753 found
222.0752.
2,4-Diaminofuro[2,3-d]pyrimidine-5-carboxylic acid 5. Ethyl
2,4-diaminofuro[2,3-d]pyrimidine-5-carboxylate
3
(1.00 g,
4.5 mmol) was suspended in a mixture of water and ethanol
(5/1, 20 ml) containing lithium hydroxide monohydrate (3 eq,
13.5 mmol, 0.326 g). The mixture was stirred for 4 h at room
temperature. The resulting solution was acidified (HCl, 1 M) to
pH 4–5. The product was separated as hydrated crystals, and then
was dried overnight in a drying pistol to yield the desired product
as a brown powder (84%, 730 mg, 3.76 mmol); mp decomposed
above 200 ◦C (decomp.); nmax (KBr)/cm-1 3356, 3161, 1709, 1589,
1558, 1433, 1388, 1315, 2242, 1166, 1130, 1113, 1074, 1050, 1004,
810, 756, 702, 696, 671, 593; dH ((CD3)2SO) 6.25 (2H, br s, NH2),
7.16 (2H, br d, NH2), 8.05 (1H, s), 12.54 (1H, br s); dC ((CD3)2SO)
Experimental
Instrumentation and general materials
NMR spectra were recorded on a Bruker Spectrospin spectrometer
operating at 400 MHz for 1H spectra and 100 MHz for 13C
spectra. Chemical shifts are reported as ppm relative to the solvent
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89.0, 114.5, 144.1, 159.0, 162.0, 165.7, 169.7; HRFABMS: calcd
for C7H6O3N4 (M + H+); 194.0440 found 195.0513.
phenyl)furo[2,3-d]pyrimidine-5-carboxylate 6f, details of which
can be found in the Supplementary Information.
Ethyl 2,4-diamino-6-bromofuro[2,3-d]pyrimidine-5-carboxylate
General procedure B for the isolation of carboxylic acids from
Suzuki coupling reactions
4. Ethyl 2,4-diaminofuro[2,3-d]pyrimidine-5-carboxylate
3
(2.00 g, 9.00 mmol) was suspended in glacial acetic acid (30 ml).
Bromine (2 eq, 17.98 mmol, 920 ml) was added to give an orange
solution. The solution was stirred at room temperature for 16 h.
A yellow product precipitated and was filtered off, washed with
acetic acid and dried overnight to afford the title compound
(2.651 mg, 8.81 mmol, 98%); mp which decomposed above
190 ◦C; nmax (KBr)/cm-1 3394, 3305, 3140, 2999, 2936, 1723, 1694,
1678, 1657, 1609, 1537, 1472, 1422, 1360, 1263, 1169, 1120, 1077,
1004, 759, 649, 594, 558; dH ((CD3)2SO) 1.33 (3H, t, J = 7.1),
4.38 (2H, q, J = 7.1), 7.98 (2H, br s, NH2), 8.38 (2H, br s, NH2);
dC ((CD3)2SO) 13.8, 62.3, 90.7, 113.1, 129.8, 152.6, 154.3, 162.4;
HR-FABMS: calcd for C9H9O3N479Br (M + H+); 299.9858 found
300.9944; calcd for C9H9O3N481Br (M + H+); 301.9838 found
302.9924.
The aqueous layer from the extraction of the Suzuki coupling
reaction was filtered. Then, under stirring, the solution was
acidified to pH 4 by the means of a dilute HCl (1 M). The
product was separated as hydrated crystals, and then was dried
overnight.
2,4-Diamino-6-phenylfuro[2,3-d]pyrimidine-5-carboxylic acid
7a. Obtained using the general acidification procedure on the
synthesis of 6a as a grey fluorescent powder (106 mg, 0.48 mmol,
29%); mp which decomposed above 275 ◦C; nmax (KBr)/cm-1
3449, 3311, 3161, 2846, 2672, 1673, 1597, 1550, 1490, 1424,
1390, 1303, 1182, 1052, 1010, 807, 763, 694, 604, 559, 542; dH
((CD3)2SO) 6.35 (2H, br s, NH2), 7.47 (3H, m), 7.60 (2H, br s,
NH2), 7.73 (2H, d, J = 7.7); dC ((CD3)2SO) 91.0, 109.0, 127.9,
129.4, 129.5, 129.5, 152.4, 158.5, 161.2, 165.9; HRFABMS: calcd
for C13H10O3N4 (M + H+); 270.0753 found 271.0835.
General procedure A for the coupling reaction between 4 and
corresponding boronic acid
Similarly prepared were: 2,4-diamino-6-(4-chlorophenyl)furo-
[2,3-d]pyrimidine-5-carboxylic acid 7b, 2,4-diamino-6-(4-metho-
xyphenyl)furo[2,3-d]pyrimidine-5-carboxylic acid 7c, 2,4-dia-
mino6-(4-ethylphenyl)furo[2,3-d]pyrimidine-5-carboxylic acid 7d,
and 2,4-diamino-6-(4-formylphenyl)furo[2,3-d]pyrimidine-5-car-
boxylic acid 7e, details of which can be found in the Supplementary
Information.
Ethyl 2,4-diamino-6-bromofuro[2,3-d]pyrimidine-5-carboxylate 4
(0.5 g, 1.66 mmol) was suspended in ethanol (15 ml). The
corresponding phenylboronic acid (1.4 eq) was added and the
solution was degassed with N2 for 5 minutes. Then, PdCl2(PPh3)2
(5 mol%, 0.084 mmol, 58 mg) was added, and the solution was
degassed. An aqueous Na2CO3 solution (2 M, 15 ml) was added,
and the solution was degassed further. The reaction was heated
at 60 ◦C for an average of 16 hours or until completion of
the reaction as shown by TLC. The reaction mixture was then
cooled to room temperature and extracted with ethyl acetate
(3 ¥ 100 ml). The organic layer was washed successively with
brine (30 ml), water (30 ml), and dried (Na2SO4), filtered and
concentrated under reduced pressure. The resulting powder was
dissolved in methanol, adsorbed on to silica, and purified by
flash chromatography using ethyl acetate/hexane (from 40/60 to
100/0). In some cases, a further recrystallisation was required:
the compound was dissolved in a minimum of ethyl acetate and
recrystallised with the addition of hexane.
General procedure C for the synthesis of amides
2,4-Diaminofuro[2,3-d]pyrimidine-5-carboxylic acid 5 (200 mg,
1.04 mmol) was dissolved in anhydrous DMF (4 ml) under nitro-
gen with warming. After being cooled, NMM (1.2 eq, 1.27 mmol,
140 ml) was added, followed by HBTU (2 eq, 2.08 mmol, 260 ml).
Once it was dissolved, the aromatic amine (2 eq, 2.08 mmol)
dissolved in DMF (200 ml) was added. The reaction was stirred at
room temperature under nitrogen for between 3 and 18 h. DMF
was removed under reduced pressure to afford a powder that was
dissolved in ethyl acetate (80 ml). The organic layer was washed
water (15 ml), and the resulting aqueous layer was extracted with
ethyl acetate (2 ¥ 80 ml). All the organic layers were combined and
washed with brine (20 ml), and water (20 ml), and finally dried
(Na2SO4) and concentrated under reduced pressure. The resulting
powder was dissolved in methanol, adsorbed on to silica, and
purified by silica gel chromatography using ethyl acetate/hexane
(from 80/20 to 100/0). The purified powder was triturated with
aqueous sodium hydroxide solution (1 M), and then filtered to
give the desired compound.
Ethyl 2,4-diamino-6-phenylfuro[2,3-d]pyrimidine-5-carboxylate
6a. Obtained using the general coupling procedure with phenyl-
boronic acid (2 eq, 3.32 mmol, 405 mg) for 16 h as an off-white
fluorescent powder was isolated (223 mg, 0.75 mmol, 45%); mp
174–177 ◦C. Found: C, 60.8; H, 5.0; N, 18.7; C15H14O3N4 requires
C, 60.4; H, 4.7; N, 18.8%. nmax (KBr)/cm-1 3503, 3462, 3380, 3169,
1688, 1647, 1618, 1567, 1460, 1374, 1270, 1240, 1140, 1093, 1066,
779, 764, 696; dH ((CD3)2SO) 1.12 (3H, t, J = 7.1), 4.23 (2H, q, J =
7.1), 6.32 (2H, br s, NH2), 7.25 (2H, br s, NH2), 7.47 (3H, m), 7.72
(2H, dd, J = 7.6, 4.4); dC ((CD3)2SO) 13.5, 61.3, 90.7, 108.2, 127.8,
128.0, 129.4, 129.7, 152.5, 158.8, 161.7, 164.4, 168.0; HRFABMS:
calcd for C15H14O3N4 (M + H+); 298.1066 found 299.1142.
Similarly prepared were ethyl 2,4-diamino-6-(4-chlorophenyl)-
furo[2,3-d]pyrimidine-5-carboxylate 6b, ethyl 2,4-diamino-6-(4-
methoxyphenyl)furo[2,3-d]pyrimidine-5-carboxylate 6c, ethyl 2,
4-diamino-6-(4-ethylphenyl)furo[2,3-d]pyrimidine-5-carboxylate
6d, ethyl 2,4-diamino-6-(4-(methylthio)phenyl)furo[2,3-d]pyrimi-
dine-5-carboxylate 6e, ethyl 2,4-diamino-6-(4-(diethoxymethyl)-
2,4-Diamino-N-phenylfuro[2,3-d]pyrimidine-5-carboxamide 8a.
Obtained using the general synthesis procedure with aniline (2 eq,
2.08 mmol, 190 ml) as a yellow-brown powder (168 mg, 0.62 mmol,
68%); mp that sublimed at 212 ◦C and melted at 266–268 ◦C. nmax
(KBr)/cm-1 3473, 3293, 3135, 1662, 1619, 1595, 1548, 1535, 1495,
1464, 1442, 1387, 1315, 1264, 1240, 1116, 1098, 1041, 876, 800,
755, 693; dH ((CD3)2SO) 6.22 (2H, br s, NH2), 7.13 (1H, J = 7.3),
7.37 (2H, m), 7.44 (2H, br s, NH2), 7.67 (2H, d, J = 7.5), 8.32 (1H,
s), 10.21 (1H, br s, NH); dC ((CD3)2SO) 89.3, 117.3, 120.8, 124.1,
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128.7, 138.2, 140.4, 159.1, 161.7, 161.9, 161.4; HR-FABMS: calcd
for C13H11O2N5 (M + H+); 269.0913 found 270.0990.
acetate/hexane (1:1) as eluant. The pyrimidine 12 was obtained as
an orange solid (36%) and the pyrrolopyrimidine 13 as an orange
solid (21%).
Similarly prepared were: 2,4-diamino-N-(4-chlorophenyl)furo-
[2,3-d]pyrimidine-5-carboxamide 8b, 2,4-diamino-N-(4-metho-
xyphenyl)furo[2,3-d]pyrimidine-5-carboxamide 8c, 2,4-diamino-
N-(4-ethylphenyl)furo[2,3-d]pyrimidine-5-carboxamide 8d, 2,4-
diamino-N-(4-(methylthio)phenyl)furo[2,3-d]pyrimidine-5-carb-
oxamide 8e, and ethyl 4-(2,4-diaminofuro[2,3-d]pyrimidine-5-car-
boxamido)benzoate 8f, details of which can be found in the
Supplementary Information.
Characterisation of 12; mp 93–96 ◦C; nmax (KBr)/cm-1 3458,
2923, 1678, 1612, 1568, 1543, 1494, 1463, 1297, 1210, 1141, 1121,
1070, 997, 957, 802, 723, 694, 542; dH ((CD3)2SO) 4.38 (2H, s),
6.73 (2H, br s, NH2), 7.21–7.31 (4H, m), 7.42–7.44 (2H, m),
7.52–7.73 (4H, m), 12.35 (1H, br s, NH); dC ((CD3)2SO) 34.2,
112.9, 116.0, 122.3, 127.4, 128.8, 128.9, 129.3, 129.4, 133.6, 138.0,
159.6, 159.7, 172.9; Characterisation of 13; mp >230 ◦C; nmax
(KBr)/cm-1 3459, 1686, 1635, 1553, 1458, 1295, 1208, 1140, 999,
961, 842, 804, 779, 724, 694; dH ((CD3)2SO) 4.31 (2H, s, CH2),
5.61 (1H, br s, NH), 7.19–7.38 (7H, m), 7.34–7.54 (3H, m); dC
((CD3)2SO) 34.3, 86.2, 116.0, 125.0, 128.1, 128.4, 129.1, 131.6,
132.5, 139.5, 159.7, 162.5, 167.4.
Pyrrolopyrimidines
6-Amino-2-(benzylthio)-5-iodopyrimidin-4(3H)-one 10. To a
50 ml round bottom flask, protected from light by aluminium foil,
was added 6-amino-2-(benzylthio)pyrimidin-4(3H)-one 9 (0.8 g,
3.4 mmol) in THF (10 ml, anhydrous). N-Iodosuccinimide (0.8 g,
3.6 mmol, 1.1 eq) dissolved in THF (10 ml, anhydrous) was
added. The reaction mixture was stirred under nitrogen, at room
temperature for 20 h in the dark. The solvents were removed under
reduced pressure and the residue was dissolved in DCM (50 ml).
The reaction mixture was washed with water, dried (MgSO4) and
concentrated under reduced pressure t◦o give 10 as a brown solid
(2.56 g, 7.1 mmol, 82%); mp 204–206 C; nmax (KBr)/cm-1 3468,
3357, 1617, 1590, 1562, 1524, 1444, 1411, 1271, 1222, 1050, 988,
951, 876, 758, 698, 543, 509; dH ((CD3)2SO) 4.36 (2H, s, CH2), 6.75
(2H, Br s, NH2), 7.22–7.25 (1H, m), 7.30–7.33 (2H, m), 7.46–7.44
(2H, m); dC ((CD3)2SO) 34.4, 53.2, 128.6, 129.4, 130.6, 138.6, 154.0,
163.1, 178.5; HREIMS: calcd for C11H10IN3OS (M+); 359.9668
found 359.9661.
2-Amino-6-bromo-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimi-
dine-5-carbonitrile 1. To a suspension of 2-amino-4,7-dihydro-
4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 1343(1.09 g,
6.24 mmol) in glacial acetic acid (70 ml) was added bromine
(480 ml, 9.40 mmol, 1.5 eq). The mixture was stirred at room
temperature for 20 h then heated to 60 ◦C for 24 h prior to
the solvent being concentrated under reduced pressure. The solid
product was dried in a vacuum desiccator for 12 h to yi◦eld 1 as
a light brown solid (1.48 g, 5.83 mmol, 94%); mp >300 C; nmax
(KBr)/cm-1 3392, 3296, 3101, 2785, 2235, 1691, 1570, 1519, 1448,
1415, 1383, 1284, 1219, 1135, 796; dH ((CD3)2SO) 6.71 (2H, br s
NH2), 10.54 (1H, br s NH), 10.83 (1H, br s, NH); dC ((CD3)2SO)
88.4, 100.0, 110.9, 114.6, 151.6, 153.9, 156.3; HREIMS: calcd for
C7H4BrN5O (M+); 253.9677/255.9657 found 253.9683/255.9661.
2-Amino-4,7-dihydro-4-oxo-6-phenyl-3H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile 15a.
N -(2-(Benzylthio)-1,6-dihydro-5-iodo-6-oxopyrimidin-4-yl)-
2,2,2-trifluoroacetamide 11. To
a solution of 6-amino-2-
(benzylthio)-5-iodopyrimidin-4(3H)-one 9 (0.99 g, 2.7 mmol)
and triethylamine (380 ml, 5.4 mmol, 2 eq) in THF (10 ml),
trifluoroacetic anhydride (1.1 ml, 7.8 mmol, 3 eq) was added at 0 ◦C
dropwise. The solution was stirred at room temperature for 20 h.
Then, the reaction mixture was diluted with ethyl acetate (30 ml),
washed with a saturated sodium hydrogen carbonate solution and
dried (MgSO4) and concentrated under reduced pressure to give
the title compound 11 as a yellow solid (0.84 g, 1.8 mmol, 85%);
mp 93–95 ◦C; nmax (KBr)/cm-1 3461, 3362, 3028, 1735, 1658, 1607,
1567, 1528, 1495, 1452, 1321, 1202, 1161, 1002, 907, 758, 699; dH
((CD3)2SO) 2.56 (1H, s, NHCOCF3), 4.42 (2H, s, CH2), 7.22–7.34
(3H, m, Ph, CH, CH), 7.40–7.46 (2H, m, Ph, CH), 12.39 (1H, br s,
NH); dC ((CD3)2SO) 34.2, 60.1, 127.6, 128.8, 129.5, 137.8, 151.8,
161.3, 165.8, 171.2; LRMS: (M+-1) found 453.93; C13H9F3IN3O2S
requires 454.94.
(a) Using phenyl boronic acid. 2-Amino-6-bromo-4,7-dihydro-
4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 14 (0.16 g,
0.63 mmol), phenylboronic acid (0.16 g, 1.31 mmol, 2.1 eq) and
PdCl2(PPh3)2 (0.023 g, 0.03 mmol, 5 mol%) were suspended in
10% NH4OH (20 ml). The suspension was de-oxygenated using
nitrogen. Under an atmosphere of nitrogen, an aqueous solution
of sodium carbonate (2 M in water, 20 ml) was added and
the mixture was again de-oxygenated for 10 min. The reaction
mixture was stirred at 70 ◦C for 24 h under nitrogen. The reaction
mixture, once cooled to room temperature, was concentrated
under reduced pressure. Ethyl acetate was added to the solid
and then solution was then washed with water and brine. The
organic layers were collected (MgSO4), filtered and evaporated
under reduced pressure. The solid material was then purified
by column chromatography using hexane/ethyl acetate (2:1) as
eluant, increasing the polarity up to 1:1 methanol/ethyl acetate.
The desired product 15a was obtained as a light brown solid
6-Amino-2-(benzylthio)-5-(2-phenylethynyl)pyrimidin-4(3H)-
one 12 and 2-(benzylthio)-6-phenyl-3H-pyrrolo[2,3-d]pyrimidin-
4(7H)-one 13. To a solution of N-(2-(benzylthio)-5-iodo-1,6-
dihydro-6-oxopyrimidin-4-yl)-2,2,2-trifluoroacetamide 10 (0.18 g,
0.43 mmol) in 1,4-dioxane (10 ml), phenylacetylene (52 ml,
0.47 mmol, 1.1 eq), copper iodide (0.016 g, 0.08 mmol, 0.2 eq),
triphenylphosphine (0.04 g, 0.15 mmol, 0.35 eq), and potassium
carbonate (0.12 g, 0.85 mmol, 2 eq) were added. The solution
was stirred at 110 ◦C for 12 h. Once cooled, the reaction mixture
was concentrated under reduced pressure. Purification of crude
products was achieved by column chromatography using ethyl
◦
(0.017 g, 0.07 mmol, 11%); mp >230 C; nmax (KBr)/cm-1 3077,
3042, 1964, 1603, 1494, 1442, 1367, 1348, 1306, 1179, 1086, 1024,
759, 700, 578; dH ((CD3)2SO) 6.50 (2H, br s, NH2), 7.43 (1H, t, J =
7.6), 7.54 (2H, dd, J = 8.2, 7.6), 7.84 (2H, d, J = 8.2), 10.74 (1H,
br s, NH), 12.39 (1H, br s, NH); dC ((CD3)2SO) 82.3, 99.3, 100.2,
116.2, 126.2, 128.9, 129.3, 138.4, 152.2, 154.0, 157.3; HREIMS:
calcd for C13H9N5O (M+); 252.0885 found 252.0880.
(b) Using potassium trifluoro(phenyl)borate. 2-Propanol (5 ml)
was mixed with water (2.5 ml) under nitrogen. Into this stirred
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solution were added compound 14 (0.1 g, 0.39 mmol) and
PdCl2(PPh3)2 (14 mg, 0.02 mmol). After 5 min, potassium
trifluoro(phenyl)borate (86 mg, 0.47 mmol) and t-butylamine
(86 mg, 1.17 mmol) were added. The resulting mixture was
stirred in an oil bath at 85 ◦C for 4 d, methanol (20 ml) was
added, and concentrated. The residue was purified by column
chromatography (silica gel/methanol:ethyl acetate = 1:1) to give
the product 15a as a yellow solid (0.9 g, 0.36 mmol, 92%);
Analytical data were identical to those reported above.
Similarly prepared were: 2-amino-4,7-dihydro-6-(4-methoxy-
phenyl)-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 15b,
2-amino-6-(4-ethylphenyl)-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile 15c, and 2-amino-4,7-dihydro-6-(3,4-
methylenedioxyphenyl)-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-
carbonitrile 15d, 2-amino-6-(3-formyl)-4,7-dihydro-4-oxo-3H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile 15e details of which can
be found in the Supplementary Information.
fanyl-6-oxo-1,6-dihydropyrimidin-5-yl)-2-hydroxyimino-propio-
nic acid ethyl ester 17 (0.09 g, 0.3 mmol) was suspended in
a mixture of ethanol (20 ml), water (20 ml) and concentrated
hydrochloric acid (4 drops). The solution was stirred for 10 min
prior to benzaldehyde (4 ml) being added and the mixture being
heated to reflux under nitrogen until the reaction was complete
as shown by TLC. The solvent was removed under reduced
pressure using co-evaporation with toluene/ethanol (1:1, 10 ml)
to remove residual water. The residue was suspended in ethyl
acetate/diethyl ether (1:1, 15 ml) and filtered to afford the title
compound 18 as a yellow solid (0.04 g, 0.16 mmol, 54%); mp
>230 ◦C; nmax (KBr)/cm-1 3349, 1719, 1639, 1577, 1458, 1240,
695; dH ((CD3)2SO) 1.13 (t, 3H, J = 7.1), 2.56 (s, 3H), 4.27 (q, 2H,
J = 7.1), 7.13 (s, 1H), 12.29 (s, 1H, NH), 12.59 (s, 1H, NH).
2-Methylsulfanyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
dine-6-carboxylic acid 19. 2-Methylsulfanyl-4-oxo-4,7-dihydro-
3H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ethyl ester 18
(0.030 g, 0.12 mmol), lithium hydroxide (0.02 mg, 0.84 mmol),
in water (5 ml) and THF (1 ml) were stirred together at room
temperature under nitrogen. The reaction was left stirring for 4
d after which the solvents were removed by evaporation under
reduced pressure. Water (10 ml) was added and the pH adjusted
to 1–2 using hydrochloric acid solution (1 M). The solvents were
removed by evaporation under reduced pressure, leaving a solid
product 19 as a white solid (0.02 g, 0.1 mmol, 88%); mp >230 ◦C;
nmax (KBr)/cm-1 3437, 3257, 3193, 2956, 2431, 1659, 1563, 1496,
1424, 1385, 1243, 1168, 1139, 983, 771, 671, 531; dH ((CD3)2SO)
2.57 (3H, s), 6.91 (1H, s), 12.17 (1H, s, NH), 12.43 (1H, s, NH),
12.68 (1H, br s); HREIMS: calcd for C8H7N3O3S (M+); 225.0208
found 225.0211.
3-(4-Amino-2-methylsulfanyl-6-oxo-1,6-dihydropyrimidin-5-yl)-
2-hydroxyiminopropionic acid ethyl ester 17.
(a) Thermal method. 6-Amino-2-methylsulfanyl-3H-pyrim-
idin-4-one 16b (1.02 g, 6.5 mmol) was dissolved in dry DMF
(15 ml). Triethylamine (0.65 g, 6.5 mmol) was added and the
mixture was stirred under nitrogen at room temperature for 1 h.
A solution of ethyl bromopyruvate oxime (1.51 g, 7.2 mmol) in
dry DMF (15 ml) was added over a period of 4 h. Stirring was
continued for a further 2 d. The solution was evaporated under
reduced pressure and the residue was dissolved in a solution of
ethyl acetate and methanol (4:1, 10 ml). The resulting solution
was absorbed onto a silica gel chromatography column and eluted
with 100% ethyl acetate, increasing the polarity up to 4:1 ethyl
acetate/methanol solution. The desired product was crystallised
from ethyl acetate and hexane, and dried at 0.1 mm Hg/40 ◦C to
give a pale yellow solid (0.43 g, 1.5 mmol, 21%); mp 123–148 ◦C
(slow decomposition); nmax (KBr)/cm-1 3434, 3349, 3193, 3050,
2972, 2851, 1725, 1621, 1551, 1425, 1313, 1238, 1209, 1141, 1021,
1021, 971, 858, 772, 706, 564; dH ((CD3)2SO) 1.21 (3H, t, J = 7.1),
2.40 (3H, s), 3.64 (2H, s), 4.13 (2H, q, J = 7.1), 6.00 (2H, s, NH2),
11.91 (1H, br s, NOH), 12.10 (1H, br s, NH); dC ((CD3)2SO) 12.8,
14.3, 19.6, 60.9, 88.4, 150.2, 150.7, 160.2, 164.2, 167.1; HREIMS:
calcd for C10H14N4O4S (M+); 286.07358 found 286.07359.
6-Amino-2-(benzylthio)-5-(2-nitrocyclohexyl)pyrimidin-4(3H)-
one 23 and 2-benzylsulfanyl-3,5,6,7,8,9-hexahydro-1,3,9-triaza-
fluoren-4-one 24. 6-Amino-2-(benzylthio)pyrimidin-4(3H)-one
3 (0.29 g, 1.23 mmol) was suspended in acetonitrile (20 ml). To
this was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (374 ml,
0.38 g, 2.50 mmol, 2.0 eq.) and the mixture became a clear solution.
Nitrocyclohexene (180 ml, 0.2 g, 1.60 mmol, 1.3 eq.) was added
and the mixture was stirred at 80 ◦C for 4 d. The mixture was then
concentrated under reduced pressure and the crude material was
purified by column chromatography using ethyl acetate/hexane
(1:2) as eluant. 23 was obtained as a light brown solid (0.055 g,
0.15 mmol, 12%); mp 152–150 ◦C; nmax (KBr)/cm-1 3494, 3359,
3049, 2927, 2862, 1614, 1576, 1454, 1405, 1376, 1312, 1241, 1208,
1104, 1070, 767, 697; dH ((CD3)2SO) 1.61–1.63 (2H, m), 1.72–
1.74 (2H, m), 2.09–2.11 (1H, m), 2.14–2.17 (2H, m), 2.25–2.28
(2H, m), 4.34 (2H, s), 6.96 (2H, br s, NH2), 7.20–7.31 (3H,
m, Ph), 7.42–7.44 (2H, m), 10.65 (1H, s, NH); dC ((CD3)2SO)
20.6, 22.0, 22.2, 25.8, 27.9, 33.9, 97.8, 110.9, 126.8, 128.3, 128.9,
138.6, 157.7, 163.4, 166.1; MS (electrospray) M+ + 1 = 361. The
hexahydrotriazafluorenone 24 was obt◦ained as an off-white solid
(0.096 g, 0.31 mmol, 25%); mp >230 C; nmax (KBr)/cm-1 3414,
3130, 2930, 2849, 1647, 1546, 1477, 1380, 1334, 1246, 1192, 1133,
990, 772, 694; dH ((CD3)2SO) 1.68–1.81 (4H, m), 2.48–2.51 (2H,
m), 2.61–2.63 (2H, m), 4.39 (2H, s), 7.23–7.33 (3H, m,), 7.42–7.44
(2H, m), 11.33 (1H, s, NH), 11.83 (1H, br s, NH); dC ((CD3)2SO)
21.8, 22.0, 22.7, 33.5, 102.6, 111.2, 127.2, 127.7, 128.4, 129.0,
137.5, 147.4, 151.4, 159.0; HREIMS: calcd for C17H17N3OS (M+);
311.1092 found 311.1094.
(b)
Microwave
method. 6-Amino-2-methylsulfanyl-3H-
pyrimidin-4-one 16b (0.5 g, 3.2 mmol) was dissolved in dry DMF
(4 ml) in a 10 ml microwave tube. Triethylamine (0.32 g, 3.2 mmol)
and a solution of ethyl bromopyruvate oxime (0.7 g, 3.5 mmol) in
dry DMF (2 ml) was added. The tube was placed in a microwave
and irradiated at 50 ◦C at 18 psi for 30 min. The solution was
evaporated under reduced pressure and the residue was dissolved
in a solution of ethyl acetate and methanol (4:1, 10 ml). The
resulting solution was absorbed onto a silica gel chromatography
column and eluted with 100% ethyl acetate, increasing the
polarity up to 4:1 ethyl acetate/methanol solution. The desired
product was crystallised from ethyl acetate and hexane, and dried
at 0.1 mm Hg/40 ◦C to yield the desired product 17 (0.46 g,
1.6 mmol, 36%) as a pale yellow solid. Analytical data were
identical to those reported above.
2-Methylsulfanyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimid-
ine-6-carboxylic acid ethyl ester 18. 3-(4-Amino-2-methylsul-
1838 | Org. Biomol. Chem., 2009, 7, 1829–1842
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6-Amino-2-(benzylsulfanyl)-5-[1-(4-bromophenyl)-2-nitroethyl]-
4(3H)-pyrimidinone 25a. To mixture of 6-amino-2-
sulfate, and concentrated. The residue was purified by column
chromatography (silica gel/ethyl acetate:n-hexane = 1:1 to ethyl
acetate 100%) to give the product 25a as a yellow solid (23 mg,
0.056 mmol, 25%); mp 270–272 ◦C. nmax (KBr)/cm-1 3251, 2819,
1649, 1431, 1212, 1073, 1010, 970, 782, 729; dH ((CD3)2SO) 4.44
(2H, s), 7.39 (1H, d, J = 2.5), 7.46–7.51 (4H, m), 12.08 (1H, s),
12.15 (1H, s); dC ((CD3)2SO) 33.5, 101.2, 118.1, 118.7, 118.8, 127.3
(2C), 128.5, 129.1, 129.7, 130.2, 133.5, 137.3 (2C), 149.5, 153.8,
159.1; HREIMS: calcd for C19H1479BrN3OS (M+); 411.0041 found
411.0042.
Similarly prepared were: 2-(benzylsulfanyl)-5-phenyl-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 26b, 2-(benzylsul-
fanyl)-5-(4-methoxyphenyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyri-
midin-4-one 26c, and 2-(benzylsulfanyl)-5-[2-(trifluoromethyl)-
phenyl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 26d, de-
tails of which can be found in the Supplementary Information.
a
(benzylsulfanyl)-4(3H)-pyrimidinone 3 (0.4 g, 1.72 mmol) in water
(10 ml) was added N,N,N-trimethyl(phenyl)methanaminium
hydroxide (40% solution in water, 1 ml, 2.6 mmol). The
mixture was stirred for 5 min and then 1-bromo-4-[(E)-2-
nitroethenyl]benzene (0.39 g, 1.72 mmol) and ethyl acetate (10 ml)
were added. The resulting mixture was stirred in an oil bath
at 70 ◦C for 4 h, whereafter ethyl acetate (30 ml) was added,
and the organic layer was separated, dried, and concentrated
under reduced pressure. The residue was purified by column
chromatography (silica gel/ethyl acetate:n-hexane = 1:3 to 1:1)
to give the product 25a as a brown solid (0.57 g, 1.23 mmol,
◦
72%), mp 93–95 C. Found: C, 65.63; H, 4.29; N, 10.84; S, 8.38;
C19H17BrN4O3S requires C, 66.10; H, 4.71; N, 11.56; S, 8.82%. nmax
(KBr)/cm-1 3489, 3389, 1610, 1545 (CNO2), 1488, 1420, 1374,
1222, 1073, 975, 698; dH ((CD3)2SO) 4.33 (2H, s), 4.60 (1H, t, J =
7.4), 5.29–5.46 (2H, m), 6.79 (2H, br s, NH2), 7.22–7.45 (9H, m),
11.81 (1H, br s); dC ((CD3)2SO) 38.5 (2C), 74.8, 90.9, 127.2, 128.4,
129.1, 130.0, 130.9, 137.6, 139.7, 158.2, 160.0, 162.2; HREIMS:
calcd for C19H1781BrN4O3S (M); 463.0264, found 463.0271.
Similarly prepared were: 6-amino-2-(benzylsulfanyl)-5-(2-nitro-
1-phenylethyl)-4(3H)-pyrimidinone 25b, 6-amino-2-(benzylsul-
fanyl)-5-[1-(4-methoxyphenyl)-2-nitroethyl]-4(3H)-pyrimidinone
25c, and 6-amino-2-(benzylsulfanyl)-5-{2-nitro-1-[2-(trifluoro-
methyl)phenyl]ethyl}-4(3H)-pyrimidinone 25d, the details of
which can be found in the Supplementary Information.
6-Amino-2-(methylsulfanyl)-5-(2-nitro-1-phenylethyl)-4(3H)-
pyrimidinone 27. To
a
mixture of 6-amino-2-(methyl-
sulfanyl)-4(3H)-pyrimidinone 16b (2 g, 12.7 mmol) in water
(40 ml) was added N,N,N-trimethyl(phenyl)methanaminium
hydroxide (40% solution in water, 6 ml). The mixture was stirred
for 10 min at 60 ◦C and then [(E)-2-nitroethenyl]benzene (2 g,
13.4 mmol) and ethyl acetate (40 ml) were added. The resulting
◦
mixture was stirred in an oil bath at 60 C for 3 h. The organic
layer was separated, washed with brine (20 ml), dried, and
concentrated under reduced pressure. The residue was purified
by column chromatography (silica gel/ethyl acetate:n-hexane =
2:1 to 1:1) to give the product 27 as a purple solid (1.0 g,
3.3 mmol, 26%); mp 107–110 ◦C; nmax (KBr)/cm-1 3478, 3389,
3214, 1613, 1574, 1546 (C-NO2), 1419, 1377, 1220, 971, 757, 699;
dH ((CD3)2SO) 2.40 (3H, s), 4.60 (1H, t, J = 7.7), 5.25–5.52 (2H,
m), 6.04 (2H, s, NH2), 7.16–7.47 (5H, m, Ph), 11.78 (1H, s, CH);
dC ((CD3)2SO) 12.4, 38.9, 77.1, 91.1, 126.6, 128.1, 127.7, 140.2,
159.0, 160.1, 162.1; HRFABMS: found 329.0693 (M + Na+);
C13H14N4O3SNa+ requires 329.0684.
2-(Benzylsulfanyl)-5-(4-bromophenyl)-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one 26a.
(a) Titanium(III) chloride reaction. 6-Amino-2-(benzylsulfan-
yl)-5-[1-(4-bromophenyl)-2-nitroethyl]-4(3H)-pyrimidinone 25a
(0.33 g, 0.72 mmol) was dissolved in methanol (10 ml) and
treated with one equivalent of sodium methoxide (38.9 mg). A
titanium(III) chloride solution was prepared separately by adding
an aqueous solution of ammonium acetate (3.3 g, 43 mmol)
in water (5 ml) to titanium(III) chloride (5.6 ml, 4.3 mmol,
10% solution in hydrochloric acid) under nitrogen. The prepared
titanium(III) chloride solution was then added carefully to the
anionic solution under nitrogen with vigorous stirring. The colour
changed slowly from brown to yellow. The mixture was stirred
overnight at room temperature then poured into ethyl acetate
(20 ml) and separated into two phases. The aqueous phase was
extracted with ethyl acetate (30 ml ¥ 3). The organic extracts
were combined, washed with 5% sodium bicarbonate (20 ml)
and with brine (20 ml), then dried with anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue
was recrystallised from ethyl acetate and n-hexane to afford the
product 26a as a yellow solid (0.14 g, 0.34 mmol, 47%). The
physical and spectroscopic data were identical to those recorded
below.
2-(Methylsulfanyl)-5-phenyl-3,7-dihydro-4H -pyrrolo[2,3-d]-
pyrimidin-4-one 28. To a mixture of 6-amino-2-(methylsulfanyl)-
4(3H)-pyrimidinone 27 (2 g, 12.7 mmol) in water (40 ml) was
added N,N,N-trimethyl(phenyl)methanaminium hydroxide (40%
solution in water, 6 ml). The mixture was stirred for 10 min at
60 ◦C and then [(E)-2-nitroethenyl]benzene (2 g, 13.4 mmol) and
ethyl acetate (40 ml) were added. The resulting mixture was stirred
in an oil bath at 60 ◦C for 3 h. The precipitate was collected
by filtration, washed with water (20 ml) and n-hexane (20 ml),
to afford the required product 28 as a light purple solid (0.92 g,
3.6 mmol, 28%); mp >260 ◦C. nmax (KBr)/cm-1 3417, 3170, 3050,
2906, 2829, 1655, 1584, 1523, 1429, 1294, 1207, 1095, 970, 752, 692;
dH ((CD3)2SO) 2.52 (3H, s), 7.15–7.19 (1H, m), 7.27 (1H, d, J =
2.2), 7.29–7.32 (2H, m), 7.91 (2H, m), 11.97 (1H, s), 12.10 (1H,
s); dC ((CD3)2SO) 12.8, 101.0, 117.5, 119.9, 125.7, 127.7, 127.9,
134.2, 149.6, 154.7, 159.0; HREIMS: calcd for C13H11N3OS (M+);
258.0701 found 258.0699.
(b) Tin(II) chloride reaction. To a mixture of 6-amino-2-
(benzylsulfanyl)-5-[1-(4-bromophenyl)-2-nitroethyl]-4(3H )-
pyrimidinone 25a (0.1 g, 0.22 mmol) in ethyl acetate (15 ml)
was added tin(II) chloride dihydrate (0.15 g, 0.65 mmol). The
resulting mixture was stirred in an oil bath at 85 ◦C for 24 hours
then poured into ethyl acetate (20 ml) and washed with saturated
aqueous sodium bicarbonate (20 ml), 1% hydrochloric acid (10 ml)
and with brine (20 ml), then dried with anhydrous magnesium
2-(Benzylamino)-5-phenyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyri-
midin-4-one 31a. To
a
mixture of 2-(methylsulfanyl)-5-
phenyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 28 (0.2 g,
0.78 mmol) in dimethylformamide (5 ml) was added 3-
chlorobenzenecarboperoxoic acid (77%, 0.52 g, 2.3 mmol). The
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resulting mixture was stirred for 4 h at 3 ◦C. Dimethylformamide
was evaporated under vacuum. The resulting solid was washed
with ether (50 ml), to afford the crude sulfone as a light pink solid
(0.18 g, 80%, confirmed by LCMS: M + 1 = 290). The crude
sulfone intermediate was h◦eated with benzylamine (1 ml) in a
sealed tube for 15 h at 100 C. Benzylamine was removed under
vacuum and the residue was purified by column chromatography
(silica gel/ethyl acetate:n-hexane = 2:1 to ethyl acetate 100%)
to afford the required product 31a as a yellow solid (88 mg,
0.28 mmol, 36%); mp 278–280 ◦C. nmax (KBr)/cm-1 3412, 3197,
2785, 1654, 1477, 1292, 1100, 749, 694, 655; dH ((CD3)2SO) 4.49
(2H, J = 5.8), 6.51 (1H, t, J = 5.8), 6.99 (1H, d, J = 2.4), 7.11–
7.34 (8H, m), 7.91–7.93 (2H, m), 10.30 (1H, br s), 11.34 (1H, br s);
dC ((CD3)2SO) 43.7, 97.3, 115.2, 119.7, 126.9, 125.2, 126.3, 127.0,
127.4, 127.8, 139.3, 134.8, 151.5, 152.1, 159.0; HREIMS: calcd for
C19H16N4O (M+); 316.1324 found 316.1326.
product by HPLC (C18 Luna column eluting with a water/0.1%
trifluoroacetic acid and acetonitrile/0.1% trifluoroacetic acid
gradient) to afford the required product as a white solid 30 (24 mg,
0.064 mmol, 10%); mp >250 ◦C. nmax (KBr)/cm-1 3422, 1613, 1544,
1451, 1292, 1245, 1215, 784, 749, 696; dH ((CD3)2SO) 4.40 (2H, d),
J = 5.9), 4.47 (2H, d, J = 5.8), 6.65 (1H, t, J = 5.8), 7.01 (1H,
J = 2.3), 7.21–7.39 (10H, m, Ph), 8.53 (1H, J = 6.0), 10.37 (1H,
s), 11.54 (1H, s); dC ((CD3)2SO) 42.0, 43.8, 101.2, 105.0, 124.4,
126.7, 126.9, 127.2, 127.3, 128.2, 128.3, 139.1, 139.8, 151.5, 152.4,
158.9, 160.3; HREIMS: calcd for C16H16N4O3 (M+); 374.1618
found 374.1617.
Biological assay
Pteridine reductase was purified as described.24 The enzyme
(15 mg/ml, 50 mM Tris.HCl, 250 mM NaCl, 20% (v/v) glycerol,
pH 7.5) was flash frozen in liquid N2 and stored at -20 ◦C
until required. Potential inhibitors were dissolved at 100 mM
in DMSO, while the substrate dihydrobiopterin was dissolved to
10 mM in 0.1 M NaOH. Inhibition assays were performed by
adding increasing concentrations of inhibitor with the DMSO
concentration kept below the level affecting enzyme activity (1%).
NADPH oxidation was followed, at 340 nm, in continuous assays
executed in a Beckman DU640 spectrophotometer. IC50 and Ki
values were determined from dose-response plots using SigmaPlot
(Systat Software Inc).
Cell based assays for trypanocidal activity were carried out
against the blood stream form of Trypanosoma brucei brucei.41
Mammalian cell toxicity was investigated using the normal cell
lines HS27 (human fibroblasts), L929 (murine fibroblasts) and
H9C2 (murine myoblasts), and the cancer cell lines A2780 (human
ovarian carcinoma) and SHSY5Y (human neuroblastoma) using
the Alamar blue microplate method.44 The assays were conducted
in 96 well microplates with incubation at 37 ◦C under a humidified
5% CO2 atmosphere for 24 h for mammalian cells and 48 h for
trypanosomes. Fluorescence was detected using a Wallac Victor
2 multilabel plate reader (excitation 560 nm, emission 590 nm)
giving % of control values. Minimum inhibitory concentration
values (MICs) were obtained for compounds showing less than
20% of control values against T. b. brucei.
Similarly prepared were: 5-phenyl-2-(1-pyrrolidinyl)-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 31b, and 2-anilino-
5-phenyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 31c, the
details of which can be found in the Supplementary Information.
Ethyl
2-(benzylamino)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidine-6-carboxylate 29. To a mixture of 2-methylsulfanyl-4-
oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid
ethyl ester X (0.16 g, 0.63 mmol) in dimethylformamide (5 ml)
was added 3-chlorobenzenecarboperoxoic acid (77%, 0.42 g,
1.9 mmol). The resulting mixture was stirred for 4 h at room
temperature. Dimethylformamide was evaporated under reduced
pressure. The resulting solid was washed with ether (50 ml), to
afford the crude sulfone as a brown solid (0.13 g, 73%, confirmed
by MS (ES): M + 1 = 286). The crude sulfone intermediate
was heated with benzylamine (1 ml) in a sealed tube for 15 h
at 100 ◦C. Benzylamine was removed under vacuum and the
residue was purified by column chromatography (silica gel/ethyl
acetate 100%) and separate from the di-substituted product by
HPLC to afford the required product 29 as a white solid (24 mg,
0.077 mmol, 12%); mp >250 ◦C. Found: C, 61.9; H, 5.2; N, 17.6;
C16H16N4O3 requires C, 61.5; H, 5.2; N, 17.9%. nmax (KBr)/cm-1
3422, 3120, 2978, 1662, 1615, 1552, 1522, 1410, 1303, 1240, 1196,
1026, 776, 696, 548; dH ((CD3)2SO) 1.27 (3H, t, J = 7.1), 4.17
(2H, q, J = 7.1), 4.49 (2H, d, J = 5.8), 6.79 (1H, t, J = 5.8), 6.91
(1H, d, J = 2.2), 7.22–7.34 (5H, m), 10.49 (1H, s), 11.91 (1H, s);
dC ((CD3)2SO) 14.2, 43.7, 59.7, 101.9, 109.7, 120.3, 127.0, 127.2,
128.4, 139.0, 152.4, 153.0, 158.8, 160.3; HR-FABMS: calcd for
C16H16N4O3 (M + H+); 313.1301 found 313.1311.
Crystal data for 6c.0.8MeOH
¯
C16.8H19.2N4O4.8, Mr = 353.96, triclinic, space group P1, a =
˚
7.1471(3), b = 10.1809(4), c = 12.3149(5) A, a = 81.282(3),
◦
3
˚
b = 77.534(2), g = 81.436(3) , V = 858.56(6) A , Z = 2, l =
N-Benzyl-2-(benzylamino)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-6-carboxamide 30. To a mixture of 2-methylsul-
fanyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxy-
lic acid ethyl ester X (0.16 g, 0.63 mmol) in dimethylformamide
(5 ml) was added 3-chlorobenzenecarboperoxoic acid (77%, 0.42 g,
1.9 mmol). The resulting mixture was stirred for 4 h at room
temperature. Dimethylformamide was evaporated under reduced
pressure. The resulting solid was washed with ether (50 ml), to
afford the crude sulfone as a brown solid (0.13 g, 73%, confirmed
by MS (ES): M + 1 = 286). The crude sulfone intermediate
was heated with benzylamine (1 ml) in a sealed tube for 15 h
at 100 ◦C. Benzylamine was removed under reduced pressure
and the residue was purified by column chromatography (silica
gel/ethyl acetate 100%) and separated from the mono-substituted
-1
˚
0.71073 A, m = 0.102 mm , T = 123 K; Data quality was impaired
by generic twinning. Methanol solvate was treated as disordered
over two sites with occupancies of 0.5 and 0.3. 15464 reflections,
3900 unique, Rint 0.040. Final refinement45 to convergence on F2
gave R = 0.0528 (F, 2695 obs. data only) and Rw = 0.1447 (F2, all
data), GOF = 1.039. CCDC reference number 705654.†
Notes and references
1 G. H. Hitchings and J. A. Burchall, Adv. Enzymol., 1965, 27, 417;
C. W. Carreras and D. V. Santi, Annu. Rev. Biochem., 1995, 64, 721;
J. N. McCormick, in Comprehensive Medicinal Chemistry, vol 2, ed.
P. G. Sammes, Pergamon, Oxford, 1990, pp. 272–290; F. Rebeille, D.
Macherel, J. M. Mouillon, J. Garin and R. Douce, EMBO J., 1997,
16, 947; G. M. Brown and J. M. Williamson, in Cellular and Molecular
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Biology, vol 1, ed. F. C. Neidhardt, American Society of Microbiology,
1997; C. A. Nichol, G. K. Smith and S. S. Gross, Annu. Rev. Biochem.,
1985, 54, 729–764; D. B. Bolstad, E. S. D. Bolstad, D. L. Wright and
A. C. Anderson, Expert Opinion on Therapeutic Patents, 2008, 18,
143.
2 C. L. Gibson, S. La, Rosa, K. Ohta, P. H. Boyle, F. Leurquin, A.
Lemac¸on and C. J. Suckling, Tetrahedron, 2004, 60, 943.
3 C. L. Gibson, S. La, Rosa and C. J. Suckling, Org. Biomol. Chem., 2003,
1, 1909.
4 C. L. Gibson, J. K. Huggan, L. Kiefer and C. J. Suckling, Chemistry
and Biology of Pteridines and Folates, G. Jansen, and G. J. Peters, (eds),
SPS Publications, Heilbronn, 2007, p. 269.
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