Synthesis and evaluation of 6-Aza-2′-deoxyuridine monophosphate analogs as inhibitors of thymidylate synthases, and as substrates or inhibitors of thymidine monophosphate kinase in mycobacterium tuberculosis

Article abstract of DOI:10.1002/cbdv.201100285

A series of 5-substituted analogs of 6-aza-2′-deoxyuridine 5′-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt. Copyright

Full text of DOI:10.1002/cbdv.201100285

536
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Synthesis and Evaluation of 6-Aza-2’-deoxyuridine Monophosphate Analogs
as Inhibitors of Thymidylate Synthases, and as Substrates or Inhibitors of
Thymidine Monophosphate Kinase in Mycobacterium tuberculosis
by Martin Kçglera), Roger Bussona), Steven De Jongheb), Jef Rozenskia), Kristien Van Belleb),
Thierry Louatb), He´le`ne Munier-Lehmannc)d), and Piet Herdewijn*a)
a) Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Medicinal
Chemistry, Minderbroedersstraat 10, B-3000 Leuven (phone: þ32-16-337387; fax: þ32-16-337340;
e-mail: piet.herdewijn@rega.kuleuven.be)
b) Katholieke Universiteit Leuven, Interface Valorisation Platform (IVAP), Kapucijnenvoer 33,
B-3000 Leuven
c
´
´
) Institut Pasteur, Unite de Chimie et Biocatalyse, Departement de Biologie Structurale et Chimie, 28
Rue du Dr Roux, F-75724 Paris Cedex 15
d) CNRS, URA 2128, F-75015 Paris
A series of 5-substituted analogs of 6-aza-2’-deoxyuridine 5’-monophosphate, 6-aza-dUMP, has been
synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a
flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replace-
ment of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak
ThyX inhibitory activity (33% inhibition at 50 mm). Introduction of alkyl and aryl groups at C(5) of 1a
resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl
side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at
50 mm). None of the synthesized derivatives displayed any significant inhibitory activity against
mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial
thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt
inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-
dTMP (1b), which still was recognized as a substrate by TMPKmt.
Introduction. – The emergence of multidrug resistant (MDR) and more recently,
extremely drug resistant (XDR) strains of Mycobacterium tuberculosis has compli-
cated the successful implementation of tuberculosis (TB) control programs [1]. MDR-
TB is defined as resistance to the first-line drugs isoniazid and rifampin [2], whereas
XDR-TB is defined as resistance to isoniazid and rifampin, to any fluoroquinolone, and
to at least one of the three injectable antibiotics (capreomycin, kanamycin, and
amikacin), all belonging to the aminoglycoside family [3]. MDR and XDR-TB are
particularly significant in developing countries where HIV-prevalence is high [4]. Co-
infection with HIVand malaria has exacerbated the spread of the TB epidemic in those
countries, due to poor infrastructure and socioeconomic conditions, inadequate
regimens, and insufficient resources [5]. Globally, the number of prevalent cases of
MDR-TB has increased by 64% from 2000 to 2004, and the number of countries that
have observed at least one case of XDR-TB has tripled from 20 in 2007 to 69 by the end
of 2010 [6]. Due to the exclusion of the fluoroquinolones, which are the most potent
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
537
and least toxic second line antibiotics from the treatment regimens, patients being
diagnosed with XDR-TB must be subjected to strict supervision and patient monitoring
for an extended period of time, up to 24 months, and must be treated with less
efficacious and more expensive second line drugs. In view of these recent develop-
ments, the emergence of even totally drug-resistant strains might become reality within
the next decade [7]. It highlights the urgent demand for new antimycobacterial drugs
with novel mode of actions to avoid cross-resistance.
Drug-susceptible TB is treated in a six-month short course (the so-called DOTS
program) with first-line drugs. The standard regimen comprises of a two-month
initiation period with isoniazid, rifampin, pyrazinamide, and ethambutol, followed by a
four-month period of isoniazid and rifampin [8]. Isoniazid and ethambutol target
essential enzymes involved in the biosynthesis of components of the mycobacterial
cell wall [9]. Rifampin binds to the b-subunit of the RNA-polymerase, resulting in
abortive initiation of transcription [10]. Pyrazinamide is enzymatically deaminated to
pyrazinoic acid which is the active agent [11]. Its activity is most probably due to its
capacity in lowering the membrane potential which alters the bacterial uptake of
nutrients.
In the search for novel tuberculosis medication, drug-discovery programs starting
from marketed antibacterials have been implemented. Using the licensed drug
linezolid (I) as starting point, new oxazolidinon derivatives (e.g., PNU-100480; II) have
been prepared with better antimycobacterial activity [12]. Their antibacterial activity is
due to inhibition of protein synthesis by binding to ribosomal RNA and thereby
blocking formation of the initiation complex. New targets that are currently being
approached are ATP synthase [13] (e.g., TMC207; III) and mycobacterial phosphatases
[14]. A library-based search for potent and selective inhibitors of mycobacterial PTPB
resulted in the discovery of I-A09 (IV), a non-competitive inhibitor with an IC50 value
of 1.26 mm, which was able to restore IFN-g induced apoptosis in Raw264.7 macro-
phages expressing mPTPB (Fig. 1).
Bacterial thymidylate synthase (TS) is a promising target for antimicrobial agents,
since the sequences of the TS enzymes are highly conserved among different bacterial
species [15]. The amino acid sequences of TS are, however, also highly conserved
across species, particularly among the residues that form the substrate and cofactor-
binding pockets [16]. Therefore, the synthesis of selective bacterial TS inhibitors is
highly challenging. Recent genomic analysis has revealed that M. tuberculosis carries
the genes for both the classical thymidylate synthase, ThyA, as well as for an alternative
thymidylate synthase, called ThyX [17]. Both enzymes catalyze the reductive
methylation of 2’-deoxyuridine 5’-monophosphate (dUMP) to thymidine 5’-mono-
phosphate, which constitutes an essential step in nucleotide metabolism. While ThyA
uses N5,N10-methylenetetrahydrofolic acid (CH2THF) as both, carbon and hydride
donor [18], ThyX uses CH2THF only as carbon donor, but it depends on the NADPH/
FAD redox system to fulfill the role as hydride donor [19]. The biochemical reaction
mechanism of ThyX [20] involves the transfer of a hydride from reduced flavin adenine
dinucleotide (FADH2, generated via reduction of FAD by NADPH) to C(6) of dUMP,
thereby generating an enolate anion at C(4)/C(5), which, in turn, nucleophilically
attacks the iminium cation of CH2THF. The resulting CH2THFꢀdUMP adduct
subsequently undergoes b-elimination of the H-atom at C(5) of dUMP which generates

538
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Drugs in the clinical pipeline and recently reported selective mycobacterial ThyX inhibitors
an exocyclic methylidene group at C(5). An intramolecular 1,3-hydride shift, finally,
furnishes dTMP (Scheme 1).
ThyA and ThyX show neither structural nor sequential similarity, and substantially
differ in their biochemical reaction mechanism. Therefore, the design of selective ThyX
inhibitors does not have to rely on small structural differences between ThyX and
human ThyA proteins. In addition, transposon site hybridization experiments have
shown that mycobacterial ThyX is an essential gene for growth of the pathogen [21].
In view of these findings, mycobacterial ThyX was selected as a promising target for
the discovery of novel tuberculosis medication. Recently, 5-aryl- and 5-alkynyl-dUMP
derivatives have been developed in our laboratory as selective ThyX inhibitors (Fig. 1)
[22]. To further study the structureꢀactivity relationship (SAR) of dUMP analogs as
mycobacterial ThyX inhibitors, we envisioned to prepare 6-aza-dUMP analogs in which
C(6) of the uracil moiety is replaced by a N-atom. This would preclude the nucleophilic
attack of a hydride from FADH2. In this report, the synthesis and biological evaluation
of 6-aza-dUMP analogs, whose structures are depicted in Fig. 2, are described. These
compounds can also be considered as structural analogs of thymidine monophosphate,
and, therefore, they also have been evaluated as potential inhibitors of thymidine
monophosphate kinase from M. tuberculosis (TMPKmt). TMPKmt is responsible for

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
539
Scheme 1. Biochemical Reaction Mechanism Reported by Koehn et al. [20]
the phosphorylation of thymidine monophosphate to thymidine diphosphate, using
ATP as the preferred phosphate donor. It is considered as an attractive target for the
development of novel tuberculosis medication for the following reasons: i) TMPK acts
at the junction of the de novo and salvage pathways for thymidine triphosphate
metabolism, and is the last specific enzyme for its synthesis [23]; ii) it has been
demonstrated that TMPKmt is an essential enzyme for mycobacterial DNA synthesis

540
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
[24], and iii) biochemical [25] and structural [26] characterizations revealed that
TMPKmt is sufficiently different from its human counterpart, which allows the
synthesis of selective TMPKmt inhibitors. A number of inhibitors of TMPKmt are
known [27]. These include sugar- and base-modified nucleosides and nucleotides [28],
as well as substituted pyrimidine analogs [29]. To the best of our knowledge, 6-aza-
dUMP analogs have never been evaluated as potential inhibitors of TMPKmt.
Fig. 2. Structures of synthesized 6-aza-dUMP analogs
Results and Discussion. – Chemistry. The syntheses of 6-aza-dUMP derivatives 1a–
1d and 2–4 have been carried out via Vorbrꢀggen-type glycosylation [30] of suitable 5-
substituted 6-azauracils with Hofferꢂs chloro sugar 7 [31]. Chloro sugar 7 was prepared
in three steps from 2-deoxy-d-ribose 5 (Scheme 2). Upon treatment of intermediate 6
with saturated HCl in AcOH, diastereomerically pure compound 7 precipitated out
after a few minutes.
Scheme 2
i) HCl/MeOH, r.t. ii) p-TolCl, Pyr, r.t. iii) HCl/AcOH, r.t.
The synthesis of 6-azathymine 10b and 5-iPr-6-azauracil 10c, outlined in Scheme 3,
starts from commercially available keto esters 8b and 8c, respectively. Keto ester 8d,
necessary for the preparation of 5-octyl-6-azauracil 10d, was prepared from diethyl

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
541
oxalate 11 in quantitative yield by a Grignard reaction. Keto esters 8b–8d were treated
with thiosemicarbazide in EtOH, followed by ring closure under aqueous alkaline
conditions, to furnish 2-thio-6-azauracil derivatives 9b–9d, respectively [32]. Con-
version of 9b–9d to the desired 6-azauracil derivatives 10b–10d, respectively, was
performed in alkaline H2O2 at room temperature [32]. 5-Iodo-6-azauracil 10e was
prepared according to a literature procedure by iodination of commercially available 6-
azauracil, albeit in low yield [33].
Scheme 3
i) Thiosemicarbazide, EtOH, 908. ii) NaOH, H2O, reflux. iii) 35% aq. H2O2, 1m NaOH, r.t. iv)
Me(CH2)7MgBr, THF/Et2O, ꢀ608. v) I2, KI, NaOH, H2O, reflux.
Coupling reactions between 6-azauracil moieties 10a–10e and the chloro sugar 7
were performed in the presence of CuI as catalyst according to the method by Freskos
(Scheme 4) [34]. Yields were usually good, however, in contrast to the literature [34],
1
only a modest b/a-selectivity (usually ca. 60%, as determined by H-NMR spectro-
scopy) was observed. The pure b-epimers were obtained either by recrystallization or
silica-gel column chromatography. Addition of TMSCl (1 equiv.) led to a slight
improvement of that ratio to ca. 70% but can generally be omitted. The nucleosides
12a–12d were subsequently subjected to alkaline deprotection of the toluoyl groups
[35], followed by phosphorylation of the primary OH groups [36], to yield the
nucleoside monophosphate derivatives 1a–1d, respectively.
Toluoyl-protected 5-iodo-6-aza-2’-deoxyuridine derivative 12e served as a key
intermediate for the preparation of 5-(fluorophenyl)-6-aza derivative 2, as well as the

542
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 4
i) Hexamethyldisilazane (HMDS), (NH4)2SO4, reflux. ii) Chloro sugar 7, CuI, Me3SiCl (TMSCl),
CHCl3, r.t. iii) 0.1m MeONa, MeOH, r.t. iv) POCl3, proton sponge, PO(OMe)3, 08.
5-(prop-1-ynyl)-6-aza derivative 3 (Scheme 5). Coupling of compound 12e with (4-
fluorophenyl)boronic acid under SuzukiꢀLiebeskind reaction conditions [37] led to the
corresponding toluoyl protected nucleoside derivative. Alkaline deprotection, followed
by phosphorylation of the primary OH group of derivative 14, yielded the 5-(4-
flurophenyl) nucleotide 2. Sonogashira coupling [38] of 12e with N-(prop-2-ynyl)oc-
tanamide [22] under standard reaction conditions with [Pd(PPh3)4] as catalyst gave
unsatisfactory results, due to low conversion and difficult purification of the reaction
mixture. Changing to [Pd2(dba)3] along with the addition of PPh3 led to a remarkable
increase in reactivity. However, the Sonogashira coupling reaction under these
conditions afforded substantial amounts of the corresponding hydrated congener.
The alkynyl derivative 15 and the hydrated side product 16 were separated by flash
chromatography after alkaline deprotection of the toluoyl groups. Standard phosphor-
ylation of derivatives 15 and 16, finally, gave the desired monophosphate 3 along with 4,
respectively.
The correct structure of derivative 4 was established by 1D- and 2D-NMR
spectroscopy. The 13C-NMR spectrum of this derivative in D2O clearly showed two CD2
peaks at d 40.3 and 48.3 ppm, respectively (Fig. 3).
These intriguing finding indicated the presence of two enolizable CH2 H-atoms
adjacent to a C¼O group. The corresponding regioisomer 17 would give rise essentially
to only one CD2 signal, which is indicative for the correct structure in derivative 4.
When the NMR spectra were run in (D6)DMSO as solvent, enolization was impossible,
and two ꢃnormalꢂ CH2 peaks were obtained instead of the CD2 peaks (Fig. 3).
1
Furthermore, the signals of the two CH2 H-atoms were missing in the H-NMR
1
spectrum recorded in D2O, whereas the H-NMR spectrum of regioisomer 17 would
still show the signals of the two CH2 H-atoms b to the C¼O group. The correct structure
of analog 4 was further confirmed by 2D-COSY-NMR spectroscopy (Fig. 4). In
1
(D6)DMSO (in contrast to the spectrum in D2O), the H-NMR spectrum showed
resonances of two additional CH2 groups at d 3.65 and 4.0 ppm, respectively. In the 2D-

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
543
Scheme 5
i) HMDS, (NH4)2SO4, reflux. ii) Chloro sugar 7, CuI, TMSCl, CHCl3, r.t. iii) (4-Fluorophenyl)boronic
acid, Pd(PPh3)4, copper(I) thiophene-2-carboxylate (CuTC), THF, 508. iv) 0.1m MeONa, MeOH, r.t. v)
POCl3, proton sponge, PO(OMe)3, 08. vi) N-(Prop-2-ynyl)octanamide, [Pd2(dba)3], PPh3, CuI, Et3N,
DMF, r.t.
Fig. 3. 13C-NMR Spectrum of compound 4 in D2O (left) and (D6)DMSO (right)
COSY spectrum, no cross-peak between these two CH2 groups was observed, ruling out
the correct structure to be that of the regioisomer 17.

544
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 4. COSY Spectrum of compound 4 in (D6)DMSO
The correct connection of the nucleobase to the 2’-deoxyribose moiety was
determined by 2D-HMBC-NMR spectroscopy by using compound 1c as representative
example. Fig. 5 shows HꢀC(1’) being coupled to C(2) and C(5) of the nucleobase. It
indicates that the sugar moiety is connected to N(1) of the 5-iPr-6-azauracil base. In case
of the corresponding N(3)-regioisomer, an additional HMBC correlation with C(4)
would be expected. This is absent in the HMBC spectrum and confirms the identity of
compound 1c.
Biological Evaluation. Compounds have been evaluated for their inhibitory activity
against mycobacterial ThyX and ThyA. Cloning the ThyX and ThyA gene, protein
expression and purification were performed as described in [22]. Similarly, the
biochemical assays have been performed as reported in [22][39]. Compounds were
evaluated at a concentration of 50 mm in the ThyX as well as in the ThyA assay. In both
assays, 5-F-dUMP was included as reference compound and positive control.
The co-crystal structure [40] of the ternary complex ThyX-FAD-5-BrdUMP
revealed that the O-atoms of the 5’-monophosphate moiety of 5-Br-dUMP are engaged

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
545
Fig. 5. HMBC Spectrum of compound 1c
in crucial interactions with residues in the active site of the ThyX enzyme. Furthermore,
the pyrimidine ring of 5-Br-dUMP and the isoalloxazine ring of FAD are close enough
to interact via H-bonds and p-stacking. Based on this information from X-ray
crystallography studies, the structureꢀactivity relationship (SAR) study at C(5) of the
natural dUMP substrate has been studied already quite extensively. The presence of
small substituents (F, Br) led to potent ThyX inhibitory activities. Furthermore, it has
been demonstrated by our group that introduction of sterically demanding substituents
such as a 4-fluorophenyl moiety (compound VI) and a 3-(octanamido)prop-1-ynyl side
chain (compound V) yielded nucleotide derivatives with potent and selective
mycobacterial ThyX inhibitory activities, with IC50 values of 10 and 0.91 mm,
respectively [22]. The finding that some structural variety is tolerated at C(5) of the
natural uracil base prompted us to prepare a series of 6-aza-dUMP analogs, in which
the substituent at C(5) was varied in a systematic way. Because of the above mentioned
crucial interactions of the phosphate group, all derivatives synthesized in this study
have been evaluated as their corresponding 5’-monophosphate congeners.
The investigation of 6-aza-dUMP derivatives as potential ThyX-inhibitors started
with the synthesis of the parent compound 6-aza-dUMP 1a. This derivative exhibited
weak mycobacterial ThyX inhibitory activity (33% inhibition at 50 mm; Table 1). To
increase activity, small modifications were introduced at C(5) of the 6-azauracil base
moiety, starting with a Me group in the analog 1b. This resulted in a complete loss of
i
inhibitory activity. Similarly, increasing the size of the substituent to an Pr group
(compound 1c) or an octyl chain (compound 1d) led to complete loss of ThyX-
inhibitory activity.

546
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 1. Inhibition of Mtb-ThyX and Mtb-ThyA by Compounds 1a–1d and 2–4a)
Compound
ThyX Inhibition
ThyA Inhibition
% Inhibition (50 mm)
IC50 [mm]
% Inhibition (50 mm)
IC50 [mm]
5-F-dUMPb)
95.1
33.5
ꢀ9.3
ꢀ42.8
5.7
0.29
>50
>50
>50
>50
>50
>50
>50
100
0.57
>50
>50
>50
>50
>50
>50
>50
1a
1b
1c
1d
2
0.99
8.78
20.04
3.67
8.65
13.18
14.71
ꢀ14.1
3
4
40.9
ꢀ12.6
a) Values are means of three independent experiments. b) Positive control.
As, within the dUMP series, a 4-fluorophenyl group and a long amidopropargyl side
chain at C(5) were found to be optimal for ThyX inhibition, these substituents were
also introduced in the current 6-aza-dUMP series. This led to the synthesis of 6-aza-
dUMP derivatives 2 (bearing a 5-(4-fluorophenyl) group) and 3 (with a long
amidopropargyl side chain). As can be deduced from Table 1, compound 2 lacks any
inhibitory activity, whereas derivative 3 displays only low levels of ThyX inhibitory
activity (40% inhibition at 50 mm). Both analogs are totally devoid of ThyA-inhibitory
activity. These data highlight the fact that replacement of C(6) by a N-atom is
detrimental for ThyX inhibitory activity.
Compounds have also been evaluated on TMPKmt (Table 2), as their structures
were closely related to the natural 5’-monophosphate substrate, namely dTMP. No
activity was detected in the presence of 0.5 mm ATP and with the different compounds
at concentrations from 0.5 up to 1 mm, even with a large excess of TMPKmt (four-times
more than with dTMP), except for 6-aza-dTMP 1b. The latter was found to be a
substrate (Km value 20-fold higher and reaction rate by 21% than that with dTMP). On
the other hand, compounds 1a, 1c, 1d, 2, and 3 were able to inhibit TMPKmt activity,
yet with different potencies. As for the thymidine analogs [41], 6-aza-dUMP 1a was the
weakest inhibitor. The presence of an octyl chain at C(5) of the base moiety (compound
1d) led to the most potent inhibitor in this series, displaying an IC50 value of 400 mm.
Table 2. Inhibitory Activity of Compounds 1a–1d and 2–3 against TMPKmt
Compound
Kampp [mm]a)
% Inhibition (1.5 mm)
IC50 [mm]
>2
dTMP
1a
33
30
1b
1c
1d
2
610
50
95
50
40
1.5
0.4
1.5
3
>1.5
a) Kampp Values were calculated by fitting the experimental data to the MichaelisꢀMenten equation using
the Kaleidagraph software.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
547
Conclusions. – In view of the recent rise in TB incidence, as well as the steady
spread of MDR- and XDR-TB, the targets set by the WHO to halve TB prevalence by
2015 are fully out of reach. These figures highlight an urgent demand for new drugs
acting on novel targets. Due to the structural and sequence dissimilarities of
mycobacterial ThyX and human ThyA, as well as the essential role of ThyX in
nucleotide metabolism in M. tuberculosis, ThyX is a promising target for the discovery
of novel antimycobacterial agents. Here, the synthesis of novel 6-aza-dUMP analogs
with structural variation at C(5) of the base moiety is described. These compounds
were evaluated as potential inhibitors of mycobacterial ThyX and ThyA. Within this
series, only the parent nucleotide 6-aza-dUMP 1a and the 5-substituted 6-aza-dUMP
derivative 3, bearing a amidopropargyl side chain displayed weak ThyX inhibitions
(30–40% inhibition at 50 mm) and completely lacked any activities against ThyA.
Moreover, this series of compounds were found to interact within the dTMP site of
TMPKmt, being either substrate or inhibitor of this enzyme. Therefore, these
compounds are very promising, as they would be able to act simultaneously on
different targets specific of the mycobacterial metabolism. Further input from
structural biology, X-ray crystallographic studies, and modeling are required to
improve their current binding properties.
The authors are grateful to Mr. Luc Baudemprez for recording 500- and 600-MHz NMR spectra, and
to Mrs. Chantal Biernaux for editorial help. This work was partly funded by a collaborative project grant
(NATT project) from the European Commission. We also thank the Institut Pasteur (GPH Tuberculose,
DARRI), the CNRS and INSERM for funding. M. K. is deeply indebted to the IWT (Agentschap voor
Innovatie door Wetenschap en Technologie) for providing a Ph.D. scholarship.
Experimental Part
General. Abbreviations. AcOH, Acetic acid; CH2THF, methylenetetrahydrofolic acid; CuTC,
copper(I) thiophene-2-carboxylate; dba, dibenzylideneacetone; DMSO, dimethyl sulfoxide; DOTS,
directly observed treatment short; dTMP, 2’-deoxythymidine 5’-monophosphate; dUMP, 2’-deoxyuridine
5’-monophosphate; ATP, adenosine 5’-triphosphate; FAD, flavin adenine dinucleotide; HIV, human
immunodeficiency virus; HMDS, 1,1,1,3,3,3-hexamethyldisilazane; HR-MS, high-resolution mass
spectrometry; IC50, half maximal (50%) inhibitory concentration; MDR-TB, multidrug-resistant TB;
mPTPB, mycobacterial protein tyrosine phosphatase B; NADPH, nicotinamide adenine nucleotide
phosphate hydride; PE, petroleum ether; RP-HPLC, reversed-phase high-performance liquid chroma-
tography; SAR, structureꢀactivity relationship; TB, tuberculosis; TEAB, triethylammonium bicarbon-
ate; ThyA, thymidylate synthase; ThyX, flavin-dependent thymidylate synthase; TMPKmt, thymidine
monophosphate kinase from M. tuberculosis; TMSCl, trimethylsilyl chloride; TS, thymidylate synthase;
WHO, World Health Organization; XDR-TB, extensively drug-resistant TB.
For all reactions, anal.-grade solvents were used. Dry MeOH was obtained by distillation over CaH2.
Dry CHCl3, THF, Et2O, and DMF were purchased from commercial suppliers. All moisture-sensitive
reactions were carried out in oven-dried glassware (1208). TLC: Precoated aluminum sheets (Fluka silica
gel/TLC cards, 254 nm). Column chromatography (CC): ICN silica gel (SiO2) 63–200, 60 ꢄ. All final
compounds possessed a purity of at least 95% as determined by anal. RP-HPLC analysis on an XBridge
column (C-18, 5 mm, 4.6ꢁ150 mm) in combination with a Waters 600 HPLC system, a Waters 717 plus
autosampler and a Waters 2996 photodiode array detector from Waters, Milford, Massachusetts, USA.
Prep. HPLC purification: the same instrument with a prep. XBridge column (C-18, 5 mm, 19ꢁ150 mm)
from Waters, Milford, Massachusetts, USA. 1H- and 13C-NMR spectra: Bruker Advance 300 (1H: 300, 13C:
75, and 31P: 121 MHz) or 500 (1H: 500 and 13C: 125 MHz) spectrometer, Me4Si as internal standard for
1H-NMR, and (D6)DMSO (39.52 ppm) or CDCl3 (77.16 ppm) for 13C-NMR spectra; chemical shifts d in

548
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
ppm; coupling constants J in Hz. MS: Finnigan LCQ advantage Max (ion trap) mass spectrometer from
Thermo Finnigan, San Jose, CA, USA; exact mass measurements performed on a quadrupole time-of-
flight mass spectrometer (Q-tof-2, Micromass, Manchester, UK) equipped with a standard electrospray-
ionization (ESI) interface; samples infused in iPrOH/H2O 1:1 at 3 ml/min.
2-Deoxy-a-d-erythro-pentofuranosyl Chloride Bis(4-methylbenzoate) (7). 2-Deoxy-d-ribose (5; 4 g,
29.84 mmol) was dissolved in MeOH (48 ml), and a soln. of 1% HCl in MeOH (8 ml) was added. The
mixture was stirred at r.t. for 25 min, and then the reaction was quenched through addition of solid
NaHCO3 (1.6 g). The resulting suspension was stirred for ca. 5 min, then filtered off, washed with MeOH,
and evaporated in vacuo. The residue was co-evaporated twice with dry pyridine, and subsequently
dissolved in dry pyridine (24 ml) under Ar and cooled to 08. p-Toluoyl chloride (8.8 ml, 65.8 mmol) was
added drop- to portionwise, the ice bath was removed, and the mixture was stirred at r.t. overnight. The
mixture was diluted with H2O (40 ml) at 08 and extracted with CH2Cl2 (3ꢁ40 ml). The org. layer was
washed with sat. NaHCO3 (1ꢁ100 ml), 2n HCl (2ꢁ100 ml), and H2O (1ꢁ100 ml), dried (MgSO4), and
evaporated. The resulting yellow-orange oil was dissolved in AcOH (16 ml) and treated drop- to
portionwise with a sat. soln. of HCl in AcOH (25 ml) at r.t. Towards the end of the addition, 7 (7.07 g,
61% over three steps) readily precipitated, thereby forming a thick milky suspension, which was rapidly
1
filtered off, washed with dry Et2O, and dried in vacuo. H-NMR (500 MHz, CDCl3): 7.99 (d, J¼8.2, 2
arom. H); 7.89 (d, J¼8.2, 2 arom. H); 7.25 (m, 4 arom. H); 6.47 (d, J¼5.1, HꢀC(1)); 5.56 (m, HꢀC(3));
4.86 (m, HꢀC(4)); 4.71–4.57 (ddd, J¼42.6, 12.1, 3.7, CH2(5)); 2.91–2.82 (m, 1 H, CH2(2)); 2.74 (d, J¼
9.0, 1 H, CH2(2)); 2.42 (s, Me); 2.41 (s, Me). 13C-NMR (125 MHz, CDCl3): 166.55; 166.21; 144.44; 144.21;
130.06 (2 C); 129.82 (2 C); 129.39 (2 C); 129.36 (2 C); 126.95; 126.83; 95.47; 84.85; 73.69; 63.64; 44.68;
21.87; 21.83. ESI-MS (pos.): 352.80 ([MꢀCl]þ , C21H21O5þ ; calc. 353.14).
3,4-Dihydro-6-methyl-3-thioxo-1,2,4-triazin-5(2H)-one (9b). To a soln. of ethyl pyruvate (8b; 1.5 ml,
13.5 mmol) in EtOH (30 ml) was added thiosemicarbazide (1.14 g, 12.45 mmol), and the resulting
suspension was stirred at 908 for 30 min. The clear soln. obtained was allowed to cool to r.t. and
concentrated in vacuo. The residue was suspended in H2O (30 ml), NaOH (920 mg, 23 mmol) was added,
and the mixture was refluxed for 30 min. The resulting clear soln. was allowed to cool to r.t. and acidified
to pH 4 with AcOH at 08. The aq. layer was extracted with AcOEt (3ꢁ40 ml), and the org. layer was
dried (MgSO4) and evaporated to afford pure 9b (1.53 g, 86%). White solid. 1H-NMR (300 MHz, CDCl3/
MeOD 6 :1): 2.25 (s, Me). 13C-NMR (75 MHz, CDCl3/MeOD 6 :1): 173.09; 153.52; 148.41; 15.93. ESI-MS
(neg.): 141.80 ([MꢀH]ꢀ , C4H4N3OSꢀ ; calc. 142.01).
3,4-Dihydro-6-(1-methylethyl)-3-thioxo-1,2,4-triazin-5(2H)-one (9c). To a soln. of ethyl dimethyl-
pyruvate (8c; 1.8 ml, 12.35 mmol) in EtOH (28 ml) was added thiosemicarbazide (1.043 g, 11.39 mmol),
and the resulting suspension was stirred at 908 for 30 min. The clear soln. obtained was allowed to cool to
r.t. and concentrated in vacuo. The residue was suspended in H2O (28 ml), NaOH (842 mg, 21.04 mmol)
was added, and the mixture was refluxed for 30 min. The resulting clear soln. was allowed to cool to r.t.
and acidified to pH 4 with AcOH at 08. The aq. layer was extracted with AcOEt (3ꢁ40 ml), and the org.
layer was dried (MgSO4) and evaporated to afford pure 9c (1.86 g, 95%). White solid. 1H-NMR
(300 MHz, CDCl3/MeOD 7:1): 3.17 (m, Me2CH); 1.22 (s, Me); 1.19 (s, Me). 13C-NMR (75 MHz, CDCl3/
MeOD 7:1): 173.44; 155.10; 152.70; 28.66; 19.44 (2 C). ESI-MS (neg.): 169.80 ([MꢀH]ꢀ , C6H8N3OSꢀ
;
calc. 170.04).
3,4-Dihydro-6-octyl-3-thioxo-1,2,4-triazin-5(2H)-one (9d). Diethyl oxalate (11; 1 ml, 7.39 mmol) was
dissolved in dry THF (8 ml) and dry Et2O (8 ml) at 08 under Ar, and the soln. was cooled to ꢀ608.
Octylmagnesium bromide (2m in Et2O, 4.4 ml, 8.8 mmol) was added over 10 min, and the stirred mixture
was allowed to warm to 08 over 21ꢅ2 h. The reaction was quenched by addition of 2n H2SO4 (7 ml), diluted
with H2O, and the phases were separated. The aq. layer was extracted with Et2O (1ꢁ15 ml), and the org.
phase was dried (MgSO4) and evaporated. The resulting yellow oil 8d (1.84 g, quant.) was used in the
next step without further purification. This intermediate was dissolved in EtOH (14 ml), and
thiosemicarbazide (561 mg, 6.16 mmol) was added in one portion. The mixture was stirred at 908 for
30 min, cooled to r.t., and the solvent was evaporated. The residue was taken up in H2O (15 ml), and
NaOH (460 mg, 11.5 mmol) was added. The mixture was refluxed for 30 min, allowed to cool to r.t., and
acidified to pH 4 with AcOH at 08. The suspension was diluted with H2O (25 ml), extracted with AcOEt
(3ꢁ40 ml), and the org. layer was dried (MgSO4) and evaporated. The residue was purified by CC (SiO2;

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
549
PE/AcOEt 4 :1 containing 1% HCOOH) to yield 9d (1.262 g, 85%) as a white solid, which contained
substantial amounts of impurities and was, therefore, used in the next step without any further
purification.
6-Azathymine (¼6-Methyl-1,2,4-triazine-3,5(2H,4H)-dione; 10b). Compound 9b (716 mg, 5 mmol)
was dissolved in 1m NaOH (15 ml), and the soln. was cooled to 08. Aq. H2O2 (35%; 1.9 ml, 22.27 mmol)
was added dropwise, whereupon the color of the mixture changed from colorless to yellow and finally
back to colorless towards the end of the H2O2 addition. The mixture was stirred at r.t. for 30 min and
subsequently acidified with conc. HCl at 08. The aq. layer was extracted with AcOEt (6ꢁ30 ml), the
combined org. layer was dried (MgSO4) and evaporated, which afforded pure 10b (504 mg, 79%) as a
1
white solid. H-NMR (300 MHz, CDCl3/MeOD 5 :2): 2.20 (s, Me). 13C-NMR (75 MHz, CDCl3/MeOD
5 :2): 157.56; 150.20; 143.68; 15.56. ESI-MS (neg.): 125.50 ([MꢀH]ꢀ , C4H4N3O2ꢀ ; calc. 126.03)
5-(1-Methylethyl)-6-azauracil (¼6-(1-Methylethyl)-1,2,4-triazine-3,5(2H,4H)-dione; 10c). Com-
pound 9c (1.20 g, 7 mmol) was dissolved in 1m NaOH (21 ml), and the soln. was cooled to 08. Aq.
H2O2 (35%; 2.65 ml, 31.06 mmol) was added dropwise, whereupon the color of the mixture changed from
colorless to yellow and finally back to colorless towards the end of the H2O2 addition. The mixture was
stirred at r.t. for 30 min and subsequently acidified with conc. HCl at 08. The aq. layer was extracted with
AcOEt (6ꢁ30 ml), and the combined org. layer was dried (MgSO4) and evaporated to afford pure 10c
1
(830 mg, 76%). White solid. H-NMR (300 MHz, CDCl3/MeOD 6 :1): 3.15 (m, Me2CH); 1.21 (s, Me);
1.18 (s, Me). 13C-NMR (75 MHz, CDCl3/MeOD 6 :1): 156.81; 150.82; 150.22; 28.55; 19.62 (2 C). ESI-MS
(neg.): 153.90 ([MꢀH]ꢀ , C6H8N3O2ꢀ ; calc. 154.06).
5-Octyl-6-azauracil (¼6-Octyl-1,2,4-triazine-3,5(2H,4H)-dione; 10d). Crude 9d (1.02 g, 4.23 mmol)
was suspended in 1m NaOH (13 ml), and 35% aq. H2O2 (1.6 ml, 18.75 mmol) was added dropwise at r.t.
The mixture was stirred at r.t. for 30 min, then acidified with conc. HCl, and diluted with H2O (30 ml).
The aq. layer was extracted with AcOEt (3ꢁ40 ml), and the org. layer was dried (MgSO4) and
evaporated in vacuo. The residue was suspended in petroleum ether, filtered off, and washed with PE to
afford 10d (392 mg, 41%). White solid. 1H-NMR (300 MHz, CDCl3/MeOD 6 :1): 2.57 (t, J¼7.7, CH2(a));
1.61 (m, CH2(b)); 1.27 (m, 5ꢁCH2); 0.87 (t, J¼6.8, Me). 13C-NMR (75 MHz, CDCl3/MeOD 6 :1):
157.24; 150.20; 147.08; 31.76; 29.58; 29.22; 29.14; 29.10; 26.28; 22.56; 13.92. ESI-MS (neg.): 224.16 ([Mꢀ
H]ꢀ , C11H18N3Oꢀ2 ; calc. 224.14).
5-Iodo-6-azauracil (¼6-Iodo-1,2,4-triazine-3,5(2H,4H)-dione; 10e). To a soln. of 10a (3.39 g,
30 mmol) in H2O (105 ml) were added KI (15.9 g, 95.79 mmol), NaOH (4.8 g, 120 mmol), and I2 (15 g,
59.1 mmol) at r.t. The mixture was refluxed for 40 h, then allowed to cool to r.t., and acidified with conc.
HCl at 08. The dark mixture was decolorized with 5% NaHSO3/Na2S2O5 soln. (60 ml) at 08 and
subsequently extracted with AcOEt (3ꢁ150 ml). The org. layer was washed with a soln. containing
150 ml of H2O and 10 ml of 5% NaHSO3/Na2S2O5, dried (MgSO4), and evaporated. The residue was
purified by CC (SiO2; 2% EtOH in CH2Cl2 containing 1% HCOOH) to afford 10e (1.21 g, 17%). Off-
white solid. 1H-NMR (300 MHz, (D6)DMSO): 12.60 (br. s, NH); 12.24 (br. s, NH). 13C-NMR (75 MHz,
(D6)DMSO): 155.08; 148.90; 110.58. ESI-MS (neg.): 237.98 ([MꢀH]ꢀ , C3HIN3O2ꢀ ; calc. 237.91).
3’,5’-Di-O-p-toluoyl-2’-deoxy-6-azauridine (¼2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-erythro-
pentofuranosyl]-1,2,4-triazine-3,5(2H,4H)-dione; 12a). A suspension of 10a (372 mg, 3.3 mmol) and
(NH4)2SO4 (39 mg, 0.3 mmol) in HMDS (20 ml) was refluxed overnight under Ar. The clear soln.
obtained was allowed to cool to r.t., and the solvent was evaporated under exclusion of air and moisture.
To the resulting oil were added 7 (1.17 g, 3 mmol), CuI (573 mg, 3 mmol), and dry CHCl3 (60 ml), and
the mixture was stirred at r.t. for 2 h. The reaction was quenched by addition of sat. NaHCO3 (5 ml), and
the mixture was stirred vigorously for 5 min and evaporated to near dryness. The residue was taken up in
AcOEt (70 ml), and the insoluble material was filtered off. The filtrate was washed with sat. NaHCO3
(1ꢁ60 ml) and brine (1ꢁ60 ml), dried (MgSO4), and evaporated. The residue was purified by CC (SiO2;
1% EtOH in CH2Cl2) to yield 12a as an anomeric mixture. Recrystallization from hot EtOH afforded
pure b-anomer 12a (810 mg, 58%). White solid. 1H-NMR (300 MHz, CDCl3): 9.36 (br. s, NH); 7.95 (m, 4
arom. H); 7.25 (m, 4 arom. H); 6.69 (t, J¼6.4, HꢀC(1’)); 5.72 (m, HꢀC(3’)); 4.66 (m, HꢀC(4’)); 4.56–4.40
(m, CH2(5’)); 3.04–2.93 (m, 1 H, CH2(2’)); 2.55–2.35 (m, 1 H, CH2(2’)); 2.43 (s, Me); 2.41 (s, Me).
13C-NMR (75 MHz, CDCl3): 166.36; 166.16; 155.58; 148.05; 144.51; 144.07; 136.19; 129.95 (2 C); 129.91

550
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(2 C); 129.37 (2 C); 129.25 (2 C); 127.15; 126.67; 86.07; 82.72; 75.04; 64.09; 35.18; 21.85; 21.82. ESI-MS
(pos.): 488.10 ([MþNa]þ , C24H23N3NaO7þ ; calc. 488.14).
3’,5’-Di-O-p-toluoyl-2’-deoxy-6-azathymidine (¼2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-er-
ythro-pentofuranosyl]-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione; 12b). A suspension of 10b (210 mg,
1.65 mmol) and (NH4)2SO4 (20 mg, 0.153 mmol) in HMDS (15 ml) was refluxed overnight under Ar. The
clear soln. obtained was allowed to cool to r.t., and the solvent was evaporated under exclusion of air and
moisture. Compound 7 (585 mg, 1.5 mmol) was added, and the mixture was dissolved in dry CHCl3
(30 ml) under Ar. TMSCl (192 ml, 1.5 mmol) and CuI (287 mg, 1.5 mmol) were then added, and the
mixture was stirred at r.t. for 3 h. The reaction was quenched by addition of sat. NaHCO3 (3 ml), and the
mixture was stirred vigorously for 5 min and evaporated to near dryness. The residue was taken up in
AcOEt (50 ml), and the insoluble material was filtered off. The filtrate was washed with sat. NaHCO3
(1ꢁ40 ml) and brine (1ꢁ40 ml), dried (MgSO4), and evaporated. The residue was purified by CC (SiO2;
3–5% acetone in CH2Cl2) to yield 12b as an anomeric mixture. Recrystallization from hot EtOH
afforded pure b-anomer 12b (525 mg, 73%). White solid. 1H-NMR (300 MHz, (D6)DMSO): 12.15 (br. s,
NH); 7.88 (m, 4 arom. H); 7.33 (m, 4 arom. H); 6.49 (t, J¼6.0, HꢀC(1’)); 5.63 (m, HꢀC(3’)); 4.58–4.37
(m, HꢀC(4’), CH2(5’)); 2.94–2.77 (m, 1 H, CH2(2’)); 2.60–2.20 (m, 1 H, CH2(2’)); 2.40 (s, Me); 2.38 (s,
Me); 2.03 (s, Me). 13C-NMR (75 MHz, (D6)DMSO): 165.39; 165.26; 156.57; 148.73; 143.99; 143.95;
143.74; 129.38, 129.26, 129.22 (8 C); 126.69; 126.49; 85.00; 81.11; 74.79; 64.13; 34.38; 21.15 (2 C); 16.11.
ESI-MS (pos.): 502.10 ([MþNa]þ , C25H25N3NaO7þ ; calc. 502.16).
5-Isopropyl-3’,5’-di-O-p-toluoyl-2’-deoxy-6-azauridine (¼2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-
b-d-erythro-pentofuranosyl]-6-(1-methylethyl)-1,2,4-triazine-3,5(2H,4H)-dione; 12c). A suspension of
10c (341 mg, 2.2 mmol) and (NH4)2SO4 (25 mg, 0.191 mmol) in HMDS (20 ml) was refluxed overnight
under Ar. The clear soln. obtained was allowed to cool to r.t., and the solvent was evaporated under
exclusion of air and moisture. Compound 7 (780 mg, 2 mmol) was added, and the mixture was dissolved
in dry CHCl3 (40 ml) under Ar. TMSCl (256 ml, 2 mmol) and CuI (383 mg, 2 mmol) were then added, and
the mixture was stirred at r.t. for 3 h. The reaction was quenched by addition of sat. NaHCO3 (3 ml), and
the mixture was stirred vigorously for 5 min and evaporated to near dryness. The residue was taken up in
AcOEt (60 ml), and the insoluble material was filtered off. The filtrate was washed with sat. NaHCO3
(1ꢁ50 ml) and brine (1ꢁ50 ml), dried (MgSO4), and evaporated. The residue was purified by CC (SiO2;
1
2% acetone in CH2Cl2) to yield pure b-anomer 12c (434 mg, 43%). White solid. H-NMR (300 MHz,
(D6)DMSO): 12.15 (br. s, NH); 7.86 (m, 4 arom. H); 7.30 (m, 4 arom. H); 6.52 (dd, J¼7.0, 4.7, HꢀC(1’));
5.70 (m, HꢀC(3’)); 4.56–4.35 (m, HꢀC(4’), CH2(5’)); 3.09–2.98 (m, Me2CH); 2.91–2.79 (m, 1 H,
CH2(2’)); 2.60–2.20 (m, 1 H, CH2(2’)); 2.39 (s, Me); 2.35 (s, Me); 1.16 (d, J¼6.9, Me); 1.12 (d, J¼6.9,
Me). 13C-NMR (75 MHz, (D6)DMSO): 165.42; 165.32; 156.0; 150.37; 148.48; 144.0; 143.77; 129.42,
129.30, 129.23 (8 C); 126.61; 126.49; 85.21; 80.98; 74.87; 64.57; 34.51; 28.25; 21.19; 21.15; 19.75 (2 C). ESI-
MS (pos.): 530.0 ([MþNa]þ , C27H29N3NaO7þ ; calc. 530.19).
5-Octyl-3’,5’-di-O-p-toluoyl-2’-deoxy-6-azauridine (¼2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-
erythro-pentofuranosyl]-6-octyl-1,2,4-triazine-3,5(2H,4H)-dione; 12d). A suspension of 10d (248 mg,
1.1 mmol) and (NH4)2SO4 (13 mg, 0.1 mmol) in HMDS (10 ml) was refluxed overnight under Ar. The
clear soln. obtained was allowed to cool to r.t., and the solvent was evaporated under exclusion of air and
moisture. To the resulting oil were added 7 (389 g, 1 mmol), CuI (191 mg, 1 mmol), and dry CHCl3
(20 ml), and the mixture was stirred at r.t. for 4 h. The reaction was quenched by addition of sat. NaHCO3
(5 ml), and the mixture was stirred vigorously for 5 min and evaporated to near dryness. The residue was
taken up in AcOEt (40 ml), and the insoluble material was filtered off. The filtrate was washed with sat.
NaHCO3 (1ꢁ40 ml), and the aq. layer was extracted with AcOEt (1ꢁ40 ml). The combined org. layer
was dried (MgSO4) and evaporated. Upon purification by CC (SiO2; 3% acetone in CH2Cl2), the desired
b-isomer 12d (385 mg, 66%, white solid) eluted first, followed by the a-isomer of 12d (25 mg, 4%, white
solid). 1H-NMR (300 MHz, CDCl3; b-isomer): 8.63 (br. s, NH); 7.93 (m, 4 arom. H); 7.23 (m, 4 arom. H);
6.69 (t, J¼6.4, HꢀC(1’)); 5.70 (m, HꢀC(3’)); 4.53 (m, HꢀC(4’), CH2(5’)); 3.06–2.94 (m, 1 H, CH2(2’));
2.66–2.45 (m, 3 H, CH2(2’), CH2(a)); 2.43 (s, Me); 2.40 (s, Me); 1.60 (m, CH2(b)); 1.26 (m, 5ꢁCH2); 0.86
(t, J¼6.7, Me). 13C-NMR (75 MHz, CDCl3; b-isomer): 166.33; 166.10; 155.60; 148.39; 148.04; 144.45;
143.99; 129.94, 129.36, 129.24 (8 C); 127.12; 126.75; 85.95; 82.28; 75.20; 64.54; 35.07; 31.99; 30.13; 29.39;

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
551
29.37; 29.35; 26.45; 22.77; 21.81 (2 C); 14.23. ESI-MS (pos.): 600.10 ([MþNa]þ , C32H39N3NaO7þ ; calc.
600.29).
5-Iodo-3’,5’-di-O-p-toluoyl-2’-deoxy-6-azauridine (¼2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-
erythro-pentofuranosyl]-6-iodo-1,2,4-triazine-3,5(2H,4H)-dione; 12e). A suspension of 10e (789 mg,
3.3 mmol) and (NH4)2SO4 (39 mg, 0.3 mmol) in HMDS (30 ml) was refluxed overnight under Ar. The
clear soln. obtained was allowed to cool to r.t., and the solvent was evaporated under exclusion of air and
moisture. To the resulting oil were added 7 (1.17 g, 3 mmol), CuI (573 mg, 3 mmol), and dry CHCl3
(60 ml). TMSCl (383 ml, 3 mmol) was added in one portion, and the mixture was stirred at r.t. for 3 h. The
reaction was quenched by addition of sat. NaHCO3 (5 ml), and the mixture was stirred vigorously for
5 min and evaporated to near dryness. The residue was taken up in AcOEt (100 ml), and the insoluble
material was filtered off. The filtrate was washed with sat. NaHCO3 (1ꢁ80 ml) and brine (1ꢁ80 ml),
dried (MgSO4), and evaporated. The residue was purified by CC (SiO2; 3–5% acetone in CH2Cl2) to
yield 12e as an anomeric mixture. Recrystallization from hot EtOH afforded pure b-anomer 12e (798 mg,
45%). White solid. 1H-NMR (300 MHz, (D6)DMSO): 12.43 (br. s, NH); 7.91–7.84 (m, 4 arom. H); 7.36–
7.30 (m, 4 arom. H); 6.44 (dd, J¼6.8, 5.4, HꢀC(1’)); 5.58 (m, HꢀC(3’)); 4.46 (m, HꢀC(4’), CH2(5’));
2.85–2.78 (m, 1 H, CH2(2’)); 2.56–2.49 (m, 1 H, CH2(2’)); 2.39 (s, Me); 2.37 (s, Me). 13C-NMR (75 MHz,
(D6)DMSO): 165.55; 165.37; 154.58; 147.94; 144.08; 143.83; 129.51, 129.44, 129.37 (8 C); 126.70; 126.54;
112.29; 85.66; 81.41; 74.53; 64.39; 34.92; 21.27 (2 C). ESI-MS (pos.): 614.07 ([MþNa]þ
,
C24H22IN3NaOþ7 ; calc. 614.04).
2’-Deoxy-6-azauridine (¼2-(2-Deoxy-b-d-erythro-pentofuranosyl)-1,2,4-triazine-3,5(2H,4H)-dione;
13a). To a suspension of 12a (350 mg, 0.752 mmol) in dry MeOH (10 ml) was added MeONa (30% soln.
in MeOH, 200 ml, 1.05 mmol), and the mixture was stirred at r.t. for 6 h. The resulting soln. was
neutralized with Dowex 50 WX-8 (Hþ form), and the resin was filtered off and washed with MeOH.
After evaporation of the solvent, the residue was purified by CC (SiO2; 10% EtOH in CH2Cl2) to yield
13a (113 mg, 66%). Off-white solid. 1H-NMR (300 MHz, CDCl3/MeOD 5 :2): 7.44 (s, HꢀC(5)); 6.51 (t,
J¼6.0, HꢀC(1’)); 4.48 (m, HꢀC(3’)); 3.94 (m, HꢀC(4’)); 3.75–3.60 (m, CH2(5’)); 2.66–2.50 (m, 1 H,
CH2(2’)); 2.40–2.20 (m, 1 H, CH2(2’)). 13C-NMR (75 MHz, CDCl3/MeOD 5 :2): 156.59; 148.22; 135.77;
87.04; 85.73; 70.99; 62.44; 37.37. HR-ESI-MS (pos.): 252.0607 ([MþNa]þ , C8H11N3NaO5þ ; calc.
252.0591).
2’-Deoxy-6-azathymidine (¼2-(2-Deoxy-b-d-erythro-pentofuranosyl)-6-methyl-1,2,4-triazine-
3,5(2H,4H)-dione; 13b). To a suspension of 12b (400 mg, 0.834 mmol) in dry MeOH (14 ml) was added
MeONa (30% soln. in MeOH, 266 ml, 1.4 mmol), and the mixture was stirred at r.t. for 6 h. The resulting
soln. was neutralized with Dowex 50 WX-8 (Hþ form), and the resin was filtered off and washed with
MeOH. After evaporation of the solvent, the residue was purified by CC (SiO2; 7–10% EtOH in
1
CH2Cl2) to yield 13b (188 mg, 92%). Off-white hygroscopic solid. H-NMR (300 MHz, (D6)DMSO):
12.08 (br. s, NH); 6.31 (dd, J¼7.0, 5.5, HꢀC(1’)); 5.15 (br. s, OH); 4.60 (br. s, OH); 4.27 (m, HꢀC(3’));
3.70 (m, HꢀC(4’)); 3.49–3.43 (dd, J¼11.5, 5.4, 1 H, CH2(5’)); 3.39–3.33 (dd, J¼11.6, 6.2, 1 H, CH2(5’));
2.44–2.37 (m, 1 H, CH2(2’)); 2.08 (s, Me); 2.07–2.0 (m, 1 H, CH2(2’)). 13C-NMR (75 MHz, (D6)DMSO):
156.80; 148.95; 143.54; 87.33; 84.62; 70.76; 62.36; 37.04; 16.39. HR-ESI-MS (pos.): 266.0769 ([MþNa]þ
,
C9H13N3NaOþ5 ; calc. 266.0748).
5-Isopropyl-2’-deoxy-6-azauridine (¼2-(2-Deoxy-b-d-erythro-pentofuranosyl)-6-(1-methylethyl)-
1,2,4-triazine-3,5(2H,4H)-dione; 13c). To a suspension of 12c (434 mg, 0.855 mmol) in dry MeOH
(14 ml) was added MeONa (30% soln. in MeOH, 273 ml, 1.44 mmol), and the mixture was stirred at r.t.
for 6 h. The resulting soln. was neutralized with Dowex 50 WX-8 (Hþ form), and the resin was filtered off
and washed with MeOH. After evaporation of the solvent, the residue was purified by CC (SiO2; 5–7%
MeOH in CH2Cl2) to yield 13c (223 mg, 96%). White solid. 1H-NMR (300 MHz, (D6)DMSO): 12.06 (br.
s, NH); 6.35 (dd, J¼7.2, 4.8, HꢀC(1’)); 5.17 (br. s, OH); 4.62 (br. s, OH); 4.32 (m, HꢀC(3’)); 3.69 (m,
HꢀC(4’)); 3.54–3.44 (m, 1 H, CH2(5’)); 3.43–3.33 (m, 1 H, CH2(5’)); 3.13–2.97 (m, Me2CH); 2.48–2.37
(m, 1 H, CH2(2’)); 2.14–2.03 (m, 1 H, CH2(2’)); 1.13 (d, J¼6.8, Me); 1.12 (d, J¼6.8, Me). 13C-NMR
(75 MHz, (D6)DMSO): 155.99; 149.72; 148.58; 87.31; 84.48; 70.67; 62.28; 37.0; 28.12; 19.87; 19.78. HR-
ESI-MS (pos.): 294.1079 ([MþNa]þ , C11H17N3NaOþ5 ; calc. 294.1061).
5-Octyl-2’-deoxy-6-azauridine (¼2-(2-Deoxy-b-d-erythro-pentofuranosyl)-6-octyl-1,2,4-triazine-
3,5(2H,4H)-dione; 13d). To a suspension of 12d (380 mg, 0.658 mmol) in dry MeOH (12 ml) was added

552
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
MeONa (30% soln. in MeOH, 228 ml, 1.2 mmol), and the mixture was stirred at r.t. for 6 h. The resulting
soln. was neutralized with Dowex 50 WX-8 (Hþ form), and the resin was filtered off and washed with
MeOH. After evaporation of the solvent, the residue was purified by CC (SiO2; 5% EtOH in CH2Cl2) to
yield 13d (195 mg, 87%). Off-white hygroscopic solid. 1H-NMR (300 MHz, CDCl3/MeOD 5 :2): 6.53 (t,
J¼6.3, HꢀC(1’)); 4.50 (m, HꢀC(3’)); 3.96 (m, HꢀC(4’)); 3.77–3.62 (m, CH2(5’)); 2.73–2.50 (m, 3 H,
CH2(2’), CH2(a)); 2.32–2.19 (m, 1 H, CH2(2’)); 1.71–1.52 (m, CH2(b)); 1.28 (m, 5ꢁCH2); 0.89 (t, J¼6.0,
Me). 13C-NMR (75 MHz, CDCl3/MeOD 5 :2): 156.54; 148.91; 147.64; 87.26; 85.73; 71.58; 62.92; 37.69;
31.67; 29.72; 29.12; 29.05 (2 C); 26.10; 22.46; 13.80. HR-ESI-MS (pos.): 364.1863 ([MþNa]þ
,
C16H27N3NaOþ5 ; calc. 364.1843).
5-(4-Fluorophenyl)-2’-deoxy-6-azauridine (¼2-(2-Deoxy-b-d-erythro-pentofuranosyl)-6-(4-fluoro-
phenyl)-1,2,4-triazine-3,5(2H,4H)-dione; 14). Compound 12e (473 mg, 0.8 mmol), (4-fluorophenyl)bor-
onic acid (143 mg, 1.02 mmol), Pd(PPh3)4 (65 mg, 0.056 mmol), and CuTC (195 mg, 1.02 mmol) were
flushed with Ar and suspended in dry THF (9 ml). The mixture was stirred at 508 for 24 h, and then the
solvent was evaporated in vacuo. The residue was taken up in AcOEt (50 ml) and washed with sat.
NaHCO3 (50 ml) and brine (50 ml), dried (MgSO4), and evaporated. The crude mixture was purified by
CC (SiO2; 2% acetone in CH2Cl2), which afforded 376 mg of impure material, which was used for the
deprotection step without further purification. To a suspension of this impure intermediate in dry MeOH
(13 ml) was added MeONa (30% soln. in MeOH, 255 ml, 1.34 mmol), and the mixture was stirred at r.t.
for 6 h. The resulting soln. was neutralized with Dowex 50 WX-8 (Hþ form), and the resin was filtered off
and washed with MeOH. After evaporation of the solvent, the residue was purified by CC (SiO2; 5%
MeOH in CH2Cl2), early and late fractions were combined, evaporated, and subjected to a second
column using the same eluent to yield 14 (106 mg, 41% over two steps). White solid. 1H-NMR (300 MHz,
CDCl3/MeOD 6 :2): 7.99 (m, 2 arom. H); 7.14 (t, J¼8.7, 2 arom. H); 6.61 (t, J¼6.0, HꢀC(1’)); 4.58 (m,
HꢀC(3’)); 3.96 (m, HꢀC(4’)); 3.72–3.60 (ddd, J¼26.2, 11.9, 4.6, CH2(5’)); 2.76–2.63 (m, 1 H, CH2(2’));
2.40–2.26 (m, 1 H, CH2(2’)). 13C-NMR (75 MHz, CDCl3/MeOD 6 :2): 163.89 (d, J(C,F)¼251.2, 1 C);
156.06; 148.64; 141.83; 130.37 (d, J(C,F)¼8.5, 2 C); 127.62 (d, J(C,F)¼3.2, 1 C); 115.27 (d, J(C,F)¼21.7,
2 C); 87.10; 85.92; 71.06; 62.38; 37.68. HR-ESI-MS (pos.): 346.0814 ([MþNa]þ , C14H14FN3NaO5þ ; calc.
346.0810).
N-[3-(2’-Deoxy-6-azauridin-5-yl)prop-2-ynyl]octanamide (¼2-(2-Deoxy-b-d-erythro-pentofurano-
syl)-6-[3-[(1-oxooctyl)amino]prop-1-yn-1-yl]-1,2,4-triazine-3,5(2H,4H)-dione; 15). Compound 12e
(1 g, 1.692 mmol), [Pd2(dba)3] (142 mg, 0.156 mmol), PPh3 (82 mg, 0.312 mmol), and CuI (60 mg,
0.316 mmol) were dissolved in anh. DMF (12 ml) under Ar, and Et3N (432 ml, 3.11 mmol) and N-(prop-2-
ynyl)octanamide [22] (846 mg, 4.66 mmol) were then added. After stirring at r.t. for 2 h, the mixture was
diluted with AcOEt (120 ml), and the insoluble material was filtered off. The filtrate was washed with
brine (2ꢁ120 ml) and H2O (2ꢁ120 ml), dried (MgSO4), and evaporated. The residue was purified by
CC (SiO2; 5% acetone in CH2Cl2 containing 1% HCOOH), followed by a second silica-gel column with
the same eluent. Appropriate fractions were combined, evaporated, and the residue was recrystallized
from hot EtOH, which afforded a crude product mixture (331 mg, 2 crops) as a white solid, which was
subjected to detoluoylation without further purification. To a suspension of this crude intermediate
(331 mg) in dry MeOH (9 ml) was added MeONa (30% soln. in MeOH, 164 ml, 0.863 mmol), and the
mixture was stirred at r.t. for 6 h. The resulting soln. was neutralized with Dowex 50 WX-8 (Hþ form),
and the resin was filtered off and washed with MeOH. After evaporation of the solvent, the residue was
purified by CC (SiO2; 5–7% MeOH in CH2Cl2) to afford two compounds: 15 (73 mg, 11%, 2 steps) and
side-product 16 (87 mg, 12%, two steps) as slightly yellow oils.
Data of 15. 1H-NMR (300 MHz, CDCl3/MeOD 6 :2): 6.50 (dd, J¼6.8, 5.1, HꢀC(1’)); 4.51 (m,
HꢀC(3’)); 4.25 (s, CH2(a)); 3.96 (m, HꢀC(4’)); 3.79–3.70 (dd, J¼11.9, 4.2, 1 H, CH2(5’)); 3.70–3.60 (dd,
J¼11.9, 4.2, 1 H, CH2(5’)); 2.65–2.51 (m, 1 H, CH2(2’)); 2.35–2.20 (m, 1 H, CH2(2’)); 2.23 (t, J¼7.7,
CH2CONH); 1.63 (m, CH2CH2CONH); 1.30 (s, 4ꢁCH2); 0.88 (t, J¼6.7, Me). 13C-NMR (75 MHz,
CDCl3/MeOD 6 :2): 174.19; 155.70; 147.90; 130.55; 93.20; 87.31; 86.18; 73.76; 70.98; 62.49; 37.70; 35.86;
31.42; 29.30; 28.96; 28.71; 25.38; 22.31; 13.62. HR-ESI-MS (pos.): 431.1925 ([MþNa]þ , C19H28N4NaO6þ ;
calc. 431.1901).
Data of N-[3-(2’-Deoxy-6-azauridin-5-yl)-2-oxopropyl]octanamide (¼2-(2-Deoxy-b-d-erythro-pen-
tofuranosyl)-6-[2-oxo-3-[(1-oxooctyl)amino]propyl]-1,2,4-triazine-3,5(2H,4H)-dione; 16). 1H-NMR

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
553
(300 MHz, CDCl3/MeOD 6 :2): 6.49 (dd, J¼6.8, 4.9, HꢀC(1’)); 4.48 (m, HꢀC(3’)); 4.22 (d, J¼1.6,
COCH2NH); 3.92 (m, HꢀC(4’)); 3.79–3.55 (m, CH2(5’)); 2.66–2.52 (m, 1 H, CH2(2’)); 2.34–2.19 (m,
1 H, CH2(2’)); 2.27 (t, J¼7.6, NHCOCH2); 1.63 (m, NHCOCH2CH2); 1.29 (m, 4ꢁCH2); 0.88 (t, J¼6.7,
Me). 13C-NMR (75 MHz, CDCl3/MeOD 6 :2): 201.43; 174.85; 156.21; 148.71; 141.26; 86.94; 85.69; 70.88;
62.28; 48.88; 37.48; 35.76; 31.41; 29.82; 28.94; 28.71; 25.40; 22.34; 13.65. HR-ESI-MS (pos.): 427.2186
([MþH]þ , C19H31N4Oþ7 ; calc. 427.2187).
General Procedure for the Phosphorylation of 5-Substituted Derivatives 13a–13d and 14–16 (GP).
To a soln. of an unprotected 5-substituted nucleoside derivative (1 equiv.) and proton sponge (1.5–
3 equiv.) in PO(OMe)3 was added POCl3 (1.5–3 equiv.) in one portion at 08, and the mixture was stirred
at 08 for 3 h. The mixture was poured into ice/H2O and brought to pH 8–9 with NH4OH or 1m NaOH,
then the volatiles were removed in vacuo, and the residue was purified by CC (SiO2; gradient of iPrOH/
H2O/NH4OH). The phosphates isolated after lyophilization were further purified by prep. RP-HPLC (C-
18, 5 mm, 19ꢁ150 mm; gradient of MeCN and TEAB (30 mm)) to yield anal. pure compounds. Next, the
phosphates were subjected to ion exchange (Dowex 50 WX-8, Naþ ) and lyophilized to afford the
corresponding disodium salts.
2’-Deoxy-6-azauridine 5’-Monophosphate (¼2-(2-Deoxy-5-O-phosphono-b-d-erythro-pentofurano-
syl)-1,2,4-triazine-3,5(2H,4H)-dione Sodium Salt (1:2); 1a). Prepared according to GP: compound 13a
(94 mg, 0.411 mmol), POCl3 (76 ml, 0.82 mmol), and proton sponge (177 mg, 0.82 mmol) in 2.5 ml of
PO(OMe)3. The mixture was stirred at 08 for 3 h and worked up as described above, including CC (SiO2;
iPrOH/NH4OH/H2O 75 :15 :10!70 :20 :5!65 :25 :5) and RP-HPLC (30 mm aq. TEAB/MeCN 99 :1!
1
98 :2, 16 ml/min) to yield 1a (59 mg, 40%). White solid. H-NMR (500 MHz, D2O): 7.54 (s, HꢀC(5));
6.51 (dd, J¼6.9, 5.4, HꢀC(1’)); 4.60 (m, HꢀC(3’)); 4.09 (m, HꢀC(4’)); 3.93–3.85 (m, 1 H, CH2(5’)); 3.85–
3.78 (m, 1 H, CH2(5’)); 2.75–2.66 (m, 1 H, CH2(2’)); 2.33–2.24 (m, 1 H, CH2(2’)). 13C-NMR (125 MHz,
D2O): 162.59; 152.96; 136.22; 85.51; 85.01 (d, J(C,P)¼8.0, 1 C); 71.0; 64.17 (d, J(C,P)¼4.7, 1 C); 35.64.
31P-NMR (121 MHz, D2O): 2.88. HR-ESI-MS (neg.): 308.0280 ([MꢀH]ꢀ , C8H11N3O8Pꢀ ; calc.
308.0289).
2’-Deoxy-6-azathymidine 5’-Monophosphate (¼2-(2-Deoxy-5-O-phosphono-b-d-erythro-pentofura-
nosyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione Sodium Salt (1:2); 1b). Prepared according to GP:
compound 13b (114.2 mg, 0.469 mmol), POCl3 (88 ml, 0.94 mmol), and proton sponge (205 mg,
0.95 mmol) in 3 ml of PO(OMe)3. The mixture was stirred at 08 for 3 h and worked up as described
above, including CC (SiO2; iPrOH/NH4OH/H2O 85 :10 :5!80 :10 :10!75 :15 :10) and RP-HPLC
1
(30 mm aq. TEAB/MeCN 97:3!80 :20, 16 ml/min), to yield 1b (91 mg, 53%). White solid. H-NMR
(500 MHz, D2O): 6.51 (dd, J¼7.0, 5.8, HꢀC(1’)); 4.62 (m, HꢀC(3’)); 4.07 (m, HꢀC(4’)); 3.94–3.88 (m,
1 H, CH2(5’)); 3.85–3.79 (m, 1 H, CH2(5’)); 2.77–2.69 (m, 1 H, CH2(2’)); 2.27–2.17 (m, 1 H, CH2(2’));
2.19 (s, Me). 13C-NMR (125 MHz, D2O): 167.67; 158.16; 145.11; 85.64; 85.18 (d, J(C,P)¼7.7, 1 C); 71.68;
64.50 (d, J(C,P)¼4.6, 1 C); 36.01; 16.48. 31P-NMR (121 MHz, D2O): 3.93. HR-ESI-MS (neg.): 322.0448
([MꢀH]ꢀ , C9H13N3O8Pꢀ ; calc. 322.0446).
5-Isopropyl-2’-deoxy-6-azauridine 5’-Monophosphate (¼2-(2-Deoxy-5-O-phosphono-b-d-erythro-
pentofuranosyl)-6-(1-methylethyl)-1,2,4-triazine-3,5(2H,4H)-dione Sodium Salt (1:2); 1c). Prepared
according to GP: compound 13c (108.5 mg, 0.4 mmol), POCl3 (75 ml, 0.8 mmol), and proton sponge
(175 mg, 0.81 mmol) in 2.5 ml of PO(OMe)3. The mixture was stirred at 08 for 3 h and worked up as
i
described above, including CC (SiO2; PrOH/NH4OH/H2O 85 :10 :5!80 :10 :10!75 :15 :10) and RP-
HPLC (30 mm aq. TEAB/MeCN 95 :5!90 :10!85 :15, 16 ml/min), to give 1c (27.1 mg, 17%). White
solid. 1H-NMR (500 MHz, D2O): 6.48 (dd, J¼7.4, 4.1, HꢀC(1’)); 4.60 (m, HꢀC(3’)); 4.07 (m, HꢀC(4’));
3.97–3.91 (m, 1 H, CH2(5’)); 3.84–3.77 (m, 1 H, CH2(5’)); 3.09 (m, Me2CH); 2.74–2.68 (m, 1 H,
CH2(2’)); 2.33–2.26 (m, 1 H, CH2(2’)); 1.18 (d, J¼6.9, Me); 1.17 (d, J¼6.0, Me). 13C-NMR (125 MHz,
D2O): 158.18; 152.06; 150.35; 85.56; 85.30 (d, J(C,P)¼7.5, 1 C); 71.45; 65.20 (d, J(C,P)¼4.5, 1 C); 36.14;
28.66; 19.19; 19.13. 31P-NMR (121 MHz, D2O): 2.01. HR-ESI-MS (neg.): 350.0742 ([MꢀH]ꢀ
,
C11H17N3O8Pꢀ ; calc. 350.0759).
5-Octyl-2’-deoxy-6-azauridine 5’-Monophosphate (¼2-(2-Deoxy-5-O-phosphono-b-d-erythro-pen-
tofuranosyl)-6-octyl-1,2,4-triazine-3,5(2H,4H)-dione sodium salt (1:2); 1d). Prepared according to GP:
compound 13d (154 mg, 0.451 mmol), POCl3 (84 ml, 0.9 mmol), and proton sponge (177 mg, 0.9 mmol) in
3 ml of PO(OMe)3. The mixture was stirred at 08 for 3 h and worked up as described above, including CC

554
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
i
(SiO2; PrOH/NH4OH/H2O 80 :10 :10!75 :15 :10) and RP-HPLC (30 mm aq. TEAB/MeCN 80 :20!
1
75 :25, 16 ml/min), to yield 1d (98 mg, 47%). White solid. H-NMR (500 MHz, D2O): 6.45 (dd, J¼7.4,
4.0, HꢀC(1’)); 4.55 (m, HꢀC(3’)); 4.07 (m, HꢀC(4’)); 4.0–3.92 (m, 1 H, CH2(5’)); 3.84–3.76 (m, 1 H,
CH2(5’)); 2.70–2.50 (m, 3 H, CH2(2’) and CH2(a)); 2.34–2.26 (m, 1 H, CH2(2’)); 1.69–1.53 (m, CH2(b));
1.35–1.14 (m, 5 CH2); 0.80 (t, J¼6.9, Me). 13C-NMR (125 MHz, D2O): 157.91; 149.82; 147.81; 85.31; 84.96
(d, J(C,P)¼7.5, 1 C); 70.07; 65.09 (d, J(C,P)¼5.1, 1 C); 35.95; 30.88; 28.71; 28.22; 28.18; 27.88; 24.79;
21.74; 13.12. 31P-NMR (121 MHz, D2O): 1.37. HR-ESI-MS (neg.): 420.1537 ([MꢀH]ꢀ , C16H27N3O8Pꢀ
;
calc. 420.1541).
5-(4-Fluorophenyl)-2’-deoxy-6-azauridine 5’-Monophosphate (¼2-(2-Deoxy-5-O-phosphono-b-d-
erythro-pentofuranosyl)-6-(4-fluorophenyl)-1,2,4-triazine-3,5(2H,4H)-dione Sodium Salt (1:2); 2). Pre-
pared according to GP: compound 14 (88.7 mg, 0.274 mmol), POCl3 (51 ml, 0.55 mmol), and proton
sponge (123 mg, 0.56 mmol) in 1.8 ml of PO(OMe)3. The mixture was stirred at 08 for 3 h and worked up
i
as described above, including CC (SiO2; PrOH/NH4OH/H2O 85 :10 :5!80 :10 :10!75 :15 :10) and
RP-HPLC (30 mm aq. TEAB/MeCN 92 :8!80 :20, 16 ml/min), to give 2 (49 mg, 40%). White solid.
1H-NMR (500 MHz, D2O): 7.84 (m, 2 arom. H); 7.24 (m, 2 arom. H); 6.57 (dd, J¼7.2, 5.0, HꢀC(1’)); 4.58
(m, HꢀC(3’)); 4.09 (m, HꢀC(4’)); 3.90–3.83 (m, 1 H, CH2(5’)); 3.82–3.75 (m, 1 H, CH2(5’)); 2.82–2.74
(m, 1 H, CH2(2’)); 2.35–2.27 (m, 1 H, CH2(2’)). 13C-NMR (150 MHz, D2O): 163.24 (d, J(C,F)¼247.0,
1 C); 160.19; 156.59; 143.54; 130.71 (d, J(C,F)¼8.5, 2 C); 129.08; 115.22 (d, J(C,F)¼21.9, 2 C); 85.78;
85.17 (d, J(C,P)¼7.1, 1 C); 71.52; 64.60 (d, J(C,P)¼3.8, 1 C); 35.95. 31P-NMR (121 MHz, D2O): 1.37.
HR-ESI-MS (neg.): 402.0493 ([MꢀH]ꢀ , C14H14FN3O8Pꢀ ; calc. 402.0508.
N-[3-(2’-Deoxy-5’-O-phosphono-6-azauridin-5-yl)prop-2-ynyl]octanamide (¼2-(2-Deoxy-5-O-
phosphono-b-d-erythro-pentofuranosyl)-6-[3-[(1-oxooctyl)amino]prop-1-yn-1-yl]-1,2,4-triazine-
3,5(2H,4H)-dione Sodium Salt (1:2); 3). Prepared according GP: compound 15 (71.3 mg, 0.175 mmol),
POCl3 (37 ml, 0.395 mmol), and proton sponge (89 mg, 0.41 mmol) in 1.5 ml of PO(OMe)3. The mixture
i
was stirred at 08 for 3 h and worked up as described above, including CC (SiO2; PrOH/NH4OH/H2O
85 :10 :5!80 :10 :10!75 :15 :10) and RP-HPLC (30 mm aq. TEAB/MeCN 85 :15!80 :20!65 :35,
16 ml/min), to afford 3 (16 mg, 17%). White solid. 1H-NMR (500 MHz, D2O): 6.51 (t, J¼6.0, HꢀC(1’));
4.59 (m, HꢀC(3’)); 4.23 (s, CH2(a)); 4.10 (m, HꢀC(4’)); 3.98–3.91 (m, 1 H, CH2(5’)); 3.88–3.80 (m, 1 H,
CH2(5’)); 2.75–2.66 (m, 1 H, CH2(2’)); 2.35–2.25 (m, 1 H, CH2(2’)); 2.30 (t, J¼7.1, CH2CO); 1.63 (m,
CH2CH2CO); 1.30 (br. s, 2 CH2); 1.23 (br. s, 2 CH2); 0.81 (t, J¼6.8, Me). 13C-NMR (150 MHz, D2O):
177.40; 163.93; 154.79; 131.01; 91.79; 86.25; 85.28 (d, J(C,P)¼7.7, 1 C); 74.86; 71.34; 65.04 (d, J(C,P)¼4.6,
1 C); 36.16; 35.56; 31.04; 29.41; 28.05; 27.93; 25.27; 21.88; 13.38. 31P-NMR (121 MHz, D2O): 2.14. HR-
ESI-MS (neg.): 487.1594 ([MꢀH]ꢀ , C19H28N4O9Pꢀ ; calc. 487.1599.
N-[3-(2’-Deoxy-5’-O-phoshono-6-azauridine-5-yl)-2-oxopropyl]octanamide (¼2-(2-Deoxy-5-O-
phosphono-b-d-erythro-pentofuranosyl)-6-[2-oxo-3-[(1-oxooctyl)amino]propyl]-1,2,4-triazine-
3,5(2H,4H)-dione Sodium Salt (1 :2); 4). Prepared according to GP: compound 16 (85.9 mg,
0.201 mmol), POCl3 (38 ml, 0.406 mmol), and proton sponge (89 mg, 0.41 mmol) in 1.5 ml of PO(OMe)3.
i
The mixture was stirred at 08 for 3 h and worked up as described above, including CC (SiO2; PrOH/
NH4OH/H2O 85 :10 :5!80 :10 :10!75 :15 :10) and RP-HPLC (30 mm aq. TEAB/MeCN 85 :15!
80 :20!75 :25, 16 ml/min), to yield 4 (24.6 mg, 22%). White solid. 1H-NMR (600 MHz, D2O): 6.49 (dd,
J¼7.2, 4.7, HꢀC(1’)); 4.54 (m, HꢀC(3’)); 4.07 (m, HꢀC(4’)); 3.94–3.97 (m, 1 H, CH2(5’)); 3.83–3.76 (m,
1 H, CH2(5’)); 2.70–2.64 (m, 1 H, CH2(2’)); 2.32–2.23 (m, 1 H, CH2(2’)); 2.28 (t, J¼7.4, NHCOCH2);
1.57 (m, NHCOCH2CH2); 1.31–1.18 (m, 4ꢁCH2); 0.81 (t, J¼6.9, Me). 13C-NMR (150 MHz, D2O):
204.97; 177.82; 158.31; 150.62; 141.96; 85.89; 85.32 (d, J(C,P)¼7.9, 1 C); 71.26; 65.0 (d, J(C,P)¼4.2, 1 C);
48.32 (br. m, CD2); 40.32 (br. m, CD2); 36.19; 35.34; 30.88; 28.05; 27.99; 25.17; 21.87; 13.31. 31P-NMR
(121 MHz, D2O): 1.25. HR-ESI-MS (neg.): 505.1695 ([MꢀH]ꢀ , C19H30N4O10Pꢀ ; calc. 505.1705.
ThyX and ThyA Assays. Cloning, expression, and purification of mycobacterial ThyX and ThyA, as
well as the inhibition assays, were performed according to protocols described in [22].
TMPKmt Assays. Expression and purification of recombinant TMPKmt as well as TMPKmt activity
determination were described in [25].

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
REFERENCES
555
[1] S. E. Haydel, Pharmaceuticals 2010, 3, 2268.
[2] B. Petrini, S. Hoffner, Int. J. Antimicrob. Agents 1999, 13, 93.
[3] S. E. Dorman, R. E. Chaisson, Nat. Med. 2007, 13, 295.
[4] J. M. FitzGerald, S. Houston, Can. Med. Ass. J. 1999, 161, 47.
[5] R. Rappuoli, A. Aderem, Nature 2011, 473, 463
[6] ꢃWHO Progress Report 2011ꢂ, World Health Organization, Geneva, 2011.
[7] A. A. Velayati, M. R. Masjedi, P. Farnia, P. Tabarsi, J. Ghanavi, A. H. ZiaZarifi, S. E. Hoffner,
CHEST 2009, 136, 420.
[8] C. Saltini, Resp. Med. 2006, 100, 2085.
[9] E. K. Schroeder, O. N. de Souza, D. S. Santos, J. S. Blanchard, L. A. Basso, Curr. Pharm. Biotechnol.
2002, 3, 197.
[10] L. P. Miller, J. T. Crawford, T. M. Shinnick, Antimicrob. Agents Chemother. 1994, 38, 805.
[11] Y. Zhang, M. M. Wade, A. Scorpio, H. Zhang, Z. Sun, J. Antimicrob. Chemother. 2003, 52, 790.
[12] K. N. Williams, C. K. Stover, T. Zhu, R. Tasneen, S. Tyagi, J. H. Grosset, E. Nuermberger,
Antimicrob. Agents Chemother. 2009, 53, 1314.
[13] K. Andries, P. Verhasselt, J. Guillemont, H. W. Gçhlmann, J.-M. Neefs, H. Winkler, J. Van Gestel, P.
Timmerman, M. Zhu, E. Lee, P. Williams, D. de Chaffoy, E. Huitric, S. Hoffner, E. Cambau, C.
Truffot-Pernot, N. Lounis, V. Jarlier, Science 2005, 307, 223.
[14] B. Zhou, Y. He, X. Zhang, J. Xu, Y. Luo, Y. Wang, S. G. Franzblau, Z. Yang, R. J. Chan, Y. Liu, J.
Zheng, Z.-Y. Zhang, Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4573.
[15] D. Tondi, A. Venturelli, S. Ferrari, S. Ghelli, M. P. Costi, J. Med. Chem. 2005, 48, 913.
[16] K. M. Perry, E. B. Faumann, J. S. Finer-Moore, W. R. Montfort, G. F. Maley, F. Maley, R. M. Stroud,
Proteins 1990, 8, 315.
[17] H. Myllykallio, G. Lipowski, D. Leduc, J. Filee, P. Forterre, U. Liebl, Science 2002, 297, 105.
[18] C. W. Carreras, D. V. Santi, Annu. Rev. Biochem. 1995, 64, 721.
[19] N. Agrawal, S. A. Lesley, P. Kuhn, A. Kohen, Biochemistry 2004, 43, 10295.
[20] E. M. Koehn, T. Fleischmann, J. A. Conrad, B. A. Palfey, S. A. Lesley, I. I. Mathews, A. Kohen,
Nature 2009, 458, 919; E. M. Koehn, A. Kohen, Arch. Biochem. Biophys. 2010, 493, 96.
[21] J. Rengarajan, B. R. Bloom, E. J. Rubin, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8327.
[22] M. Kçgler, B. Vanderhoydonck, S. De Jonghe, J. Rozenski, K. Van Belle, J. Herman, T. Louat, A.
Parchina, C. Sibley, E. Lescrinier, P. Herdewijn, J. Med. Chem. 2011, 54, 4847.
[23] E. P. Anderson, in ꢃThe Enzymesꢂ, Ed. P. D. Boyer, Academic, New York, 1973, Vol. 8, p. 49.
[24] R. A. Sclafani, W. L. Fangman, Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 5821.
[25] H. Munier-Lehmann, A. Chaffotte, S. Pochet, G. Labesse, Protein Sci. 2001, 10, 1195; S. Pochet, L.
Dugue, D. Douguet, G. Labesse, H. Munier-Lehmann, ChemBioChem 2002, 3, 108.
ˆ
[26] L. Li de la Sierra, H. Munier-Lehmann, A. M. Gilles, O. Barzu, M. Delarue, J. Mol. Biol. 2001, 311,
87.
[27] S. Van Calenbergh, S. Pochet, H. Munier-Lehmann, Curr. Top. Med. Chem., in press, doi:.
[28] V. Vanheusden, P. Van Rompaey, H. Munier-Lehmann, S. Pochet, P. Herdewijn, S. Van Calenbergh,
Bioorg. Med. Chem. Lett. 2003, 13, 3045; V. Vanheusden, H. Munier-Lehmann, M. Froeyen, L.
´
Dugue, A. Heyerick, D. De Keukeleire, S. Pochet, R. Busson, P. Herdewijn, S. Van Calenbergh, J.
Med. Chem. 2003, 46, 3811; I. Van Daele, H. Munier-Lehmann, M. Froeyen, J. Balzarini, S.
Van Calenbergh, J. Med. Chem. 2007, 50, 5281.
[29] C. Gasse, D. Douguet, V. Huteau, G. Marchal, H. Munier-Lehmann, S. Pochet, Bioorg. Med. Chem.
2008, 16, 6075; O. Failiar, H. Munier-Lehmann, A. Negri, F. Gago, D. Douguet, L. Rigouts, A.
`
´
´
Hernandez, M. Camarasa, M. Perez-Perez, ChemMedChem 2008, 3, 1083.
[30] U. Niedballa, H. Vorbrꢁggen, Angew. Chem., Int. Ed. 1970, 9, 461; H. Vorbrꢁggen, K. Krolikiewicz,
U. Niedballa, Liebigs Ann. Chem. 1975, 988; ꢃ Handbook of Nucleoside Synthesisꢂ, Eds. H.
Vorbrꢁggen, C. Ruh-Pohlenz, Wiley-VCH, Weinheim, 2001.
[31] M. Hoffer, Chem. Ber. 1960, 93, 2777; V. Rolland, M. Kotera, J. Lhomme, Synth. Commun. 1997, 27,
3505.

556
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
[32] I. E. Dupont Passelaigue, J. F. Patoiseau, D. Junquero, Y. Rival, A. Delhon, Fr. Demande 2005,
131 pp.
[33] P. K. Chang, J. Org. Chem. 1961, 26, 1118.
[34] J. N. Freskos, Nucleosides Nucleotides 1989, 8, 549.
[35] J. Asakura, M. J. Robins, J. Org. Chem. 1990, 55, 4928.
[36] M. Yoshikawa, T. Kato, T. Takenish, Bull. Chem. Soc. Jpn. 1969, 42, 3505.
[37] C. Savarin, L. S. Liebeskind, Org. Lett. 2001, 3, 2149.
[38] K. Sonogashira, J. Organomet. Chem. 2002, 653, 46.
[39] J. H. Hunter, R. Gujjar, C. K. T. Pang, P. K. Rathod, PLoS One 2008, 3, e2237; S.-C. Kan, J.-S. Liu,
H.-Y. Hu, C.-M. Chang, W.-D. Lin, W.-C. Wang, W.-H. Hsu, Biochim. Biophys. Acta 2010, 1804,
1751.
[40] P. Sampathkumar, S. Turley, J. E. Ulmer, H. G. Rhie, C. H. Sibley, W. G. J. Hol, J. Mol. Biol. 2005,
352, 1091.
´
[41] S. Pochet, L. Dugue, G. Labesse, M. Delepierre, H. Munier-Lehmann, ChemBioChem 2003, 4, 742.
Received August 29, 2011