Synthesis and evaluation of 5-substituted 2′-deoxyuridine monophosphate analogues as inhibitors of flavin-dependent thymidylate synthase in mycobacterium tuberculosis

ARTICLE

pubs.acs.org/jmc

Synthesis and Evaluation of 5-Substituted 2⁰-deoxyuridine

Monophosphate Analogues As Inhibitors of Flavin-Dependent

Thymidylate Synthase in Mycobacterium tuberculosis

Martin K€ogler,^†Bart Vanderhoydonck,^‡Steven De Jonghe,^‡Jef Rozenski,^†Kristien Van Belle,^‡

Jean Herman,^‡Thierry Louat,^‡Anastasia Parchina,^†Carol Sibley,^§Eveline Lescrinier,^†and Piet Herdewijn*^,†

^†Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Minderbroedersstraat 10,

3000 Leuven, Belgium

^‡Katholieke Universiteit Leuven, Interface Valorisation Platform (IVAP), Kapucijnenvoer 33, 3000 Leuven, Belgium

^§Department of Genome Sciences, University of Washington, Seattle, Washington, United States

S Supporting Information

b

ABSTRACT: A series of 5-substituted 2⁰-deoxyuridine mono-

phosphate analogues has been synthesized and evaluated as

potential inhibitors of mycobacterial ThyX, a novel ﬂavin-

dependent thymidylate synthase in Mycobacterium tuberculosis.

A systematic SAR study led to the identiﬁcation of compound

5a, displaying an IC₅₀value against mycobacterial ThyX of

0.91 μM. This derivative lacks activity against the classical myco-

bacterial thymidylate synthase ThyA (IC₅₀> 50 μM) and

represents the ﬁrst example of a selective mycobacterial FDTS

inhibitor.

’ INTRODUCTION

ﬂuoroquinolones, and second-line bacteriostatics. XDR bacteria

are MDR and also resistant to ﬂuoroquinolones and at least one

injectable antibiotic. The XDR-tuberculosis is virtually untrea-

table. The increasing incidences of MDR- and XDR-tuberculosis

urgently demand novel drugs for therapy of tuberculosis. The

side-eﬀects of current drugs, the high costs of second-line drugs,

and the lack of well-established guidelines for the treatment of

MDR-TB and XDR-TB require alternative approaches to target

tuberculosis.^11,12

Traditionally, the only known pathway for de novo synthesis

of thymidylate which is essential for most eubacteria, plants, and

eukaryotic cells was by thymidylate synthase (TS or ThyA).¹³

This enzyme catalyzes the reductive methylation of 2⁰-deoxy-

uridine-5⁰-monophosphate (dUMP) to 2⁰-deoxythymidine-5⁰-

monophosphate using R-N⁵-N¹⁰-methylene-5,6,7,8-tetrahydro-

folate (CH₂THF) as a methylene and hydride donor, resulting in

the formation of 7,8-dihydrofolate (DHF). In the classical ThyA

cycle, formation of thymidylate must be coupled with reduction

of DHF to THF catalyzed by dihydrofolate reductase (DHFR) and

remethylenation by serinehydroxymethyl transferase (SHMT).

However, it has been shown that several microorganisms lack

the genes encoding for ThyA and DHFR but are still fully viable

in thymidine-deﬁcient media. This led to the discovery of the ThyX

gene, which encodes for the ThyX protein, a ﬂavin-dependent

According to the WHO,¹1.7 million people died from

tuberculosis (TB) in 2009 and more than 2 billion people

(constituting one-third of the world’s population) are infected

with TB. Among the 9.4 million new TB cases reported in 2009,

an estimated 1.1ꢀ1.2 million (11ꢀ13%) suﬀer from HIV coin-

fection. Hence, TB, a contagious infectious disease, is regarded as

the leading cause of death due to an infectious agent among

adults worldwide and a major threat to global health.²The

disease establishes mainly in the pulmonary system and is caused

by some mycobacteria of the Mycobacterium tuberculosis complex,

predominantly Mycobacterium tuberculosis.³

Active TB is treated by chemotherapy with so-called ﬁrst-line

drugs.^4ꢀ6From the many trials conducted in the past 50ꢀ

60 years, two standard regimens emerged which are recommended

by the WHO and consist of a two-month daily treatment of either

isoniazid, rifampin, pyrazinamide, and streptomycin or isoniazid,

rifampin, pyrazinamide, and ethambutol, followed by further four

months daily or intermittent doses of isoniazid and rifampin.

Unfortunately, the long treatment times and the side eﬀects⁷of

the drugs (especially hepatotoxicity) lead to a high proportion of

noncompliance patients. This and the rise of the HIV pandemic⁸

in the early 1980s led to the emergence of multidrug resistant⁹

(MDR) and, more recently, even extensively drug resistant¹⁰

(XDR) strains. MDR-TB is deﬁned as resistance to the action of

at least isoniazid and rifampin, and its treatment requires the

inclusion of so-called second-line drugs such as aminoglycosides,

Received: April 19, 2011

Published: June 09, 2011

r

2011 American Chemical Society

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Scheme 1. Current Biochemical Reaction Mechanism Involving Direct Hydride Transfer and 1,3-Hydride Shift

thymidylate synthase (FDTS) which is rare in eukaryotes and

absent in humans.¹⁴ThyX uses CH₂THF only as one-carbon

donor, whereas reduced ﬂavin adenine dinucleotide (FADH₂)

serves as a hydride donor. Members of the ThyX family are

NAD(P)H-oxidases because catalysis requires the presence of

NAD(P)H to furnish suﬃcient amounts of FADH₂via reduction

of FAD. Furthermore, ThyX shows no structural similarity with

ThyA¹⁵and there is no sequence similarity between ThyA and

ThyX proteins, allowing the design of selective ThyX-inhibitors

as a novel approach to target tuberculosis. While some pathogens

solely rely on ThyX, genetic analysis revealed that M. tuberculosis

carries genes encoding for ThyA, ThyX, and DHFR.¹⁴Transpo-

son site hybridization studies have shown that the ThyX gene

is required for mycobacterial growth and survival within

macrophages.^16,17Nonetheless, it remains unclear under which

conditions either gene is activated. Steady-state kinetic analyses

of M. tuberculosis revealed that dTMP-synthesis proceeds with

very low catalytic rates in both enzymes, thereby suggesting that

the true biological substrates still remain to be found.¹⁸These

ﬁndings may be in accordance with the extremely low growth

rates of M. tuberculosis and could be an indicator for down-

regulation of dTMP-production.

In the past few years, several studies to reveal the biochemical

reaction mechanism were documented in literature.^19,20Initially,

an active site catalytic serine was assumed to nucleophilically

attack C-6 of dUMP in a Michael-type reaction to give rise to an

enolate at C-4 and C-5, which in turn attacks the iminium ion of

CH₂THF to form the new CꢀC bond in dTMP. This

dUMPꢀCH₂THF adduct subsequently undergoes β-elimina-

tion to install an exo-methylene bond between C-5 and C-7,

which is reduced by FADH₂in the ﬁnal step to yield dTMP.

However, the following facts led to the supersession of this

mechanism: (i) The active site serine is too far away from the C-6

of dUMP, as shown by Sampathkumar et al., who provided a

crystal structure²¹of Mtb-ThyX in complex with the substrate

analogue 5-Br dUMP and FAD, and only conformational

changes in the enzyme would be able to lead to nucleophilic

catalysis. (ii) No general base is located near this putative serine

which would be required to enhance its otherwise too weak

nucleophilicity. (iii) Point mutation studies in Thermotoga

maritima have shown that the enzyme was still active after

mutation of this serine to either an alanine or cysteine. Recently,

Koehn et al. performed the reaction in 99% D₂O which resulted

in incorporation of deuterium in the 6-position. These new

intriguing ﬁndings led to the suggestion of an alternative reaction

mechanism²²(Scheme 1) which contains the following two

distinctive key steps: (i) a hydride equivalent is transferred

directly from FADH₂to the C-6 of dUMP (part 2, step 2) and

(ii) an unusual 1,3-hydride shift from C-6 to the exo-methylene

C-7 in the last step furnishes dTMP (part 3, step 5). Steps 3 and 4

correspond to the previously proposed mechanism and are

considered as logical on the mechanistic pathway toward dTMP.

An alternative additionꢀelimination mechanism involved in the

last step could be ruled out when the reaction was performed

using 6D-dUMP in H₂O because the formation of nondeuter-

ated dTMP could not be observed.

Although ThyX is an attractive target for antimycobacterial

drug design, the only known inhibitors are 5-F and 5-BrdUMP.²³

However, both are known to act as human ThyA inhibitors

and are therefore of no interest for antibacterial therapy.^24ꢀ28

Up to now, no selective inhibitors of mycobacterial FDTS

are known in literature. In this paper, we describe the synthesis

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Scheme 2. Structures of Synthesized ThyX-Inhibitors

Scheme 3^a

^aReagents and conditions: (a) CH₂CHCN, Pd(PPh₃)₄, (n-Bu)₃N, DMF, 80 °C; (b) boronic acid, Pd(PPh₃)₄, CsF, DMF/H₂O, 60 °C; (c) POCl₃, pyr,

H₂O, CH₃CN/PO(OMe)₃, 0 °C.

of a novel series of C-5 substituted 2⁰-dUMP analogues and

their evaluation as inhibitors of mycobacterial ThyX and ThyA

(Scheme 2).

5-(2-cyanovinyl)-deoxyuridine 8 was synthesized via a Heck

reaction of 7a with acrylonitrile in the presence of a Pd(0) cata-

lyst and a tertiary amine in DMF at elevated temperature.^30ꢀ32

Phosphorylation of the primary hydroxyl group of compound 8

according to the procedure by Sowa and Ouchi³³yielded the

desired monophosphate derivative 1 (Scheme 3).

’ RESULTS AND DISCUSSION

To introduce a phenyl group at position 5 of the uracil moiety,

Pd-catalyzed Suzuki cross-coupling reaction³⁴of 7a has been

performed with suitable boronic acids. Attempts using various

bases (K₂CO₃, Na₂CO₃, NaOH, KF) in an organic solvent/H₂O

mixture predominantly led to dehalogenation. After carefully

Chemistry. To have easy access to C-5 variation of the dUMP

scaffold, 5-iodo-2⁰-deoxyuridine 7a was considered as ideal

starting material. It is commercially available or, alternatively, it

can be prepared from 2⁰-deoxyuridine according to a protocol

described in literature.²⁹

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Scheme 4^a

^aReagents and conditions: (a) 1-dodecyne, Pd(PPh₃)₄, CuI, NEt₃, DMF, rt; (b) POCl₃, proton sponge, PO(OMe)₃, 0 °C.

Scheme 5^a

^aReagents and conditions: (a) Ac₂O, DMAP, NEt₃, CH₃CN, rt; (b) 6-heptynoic acid, Pd(PPh₃)₄, CuI, DIPEA, DMF, rt; (c) n-butylamine, DIC, HOBt,

DCM, 0 °C to rt; (d) NaOMe, MeOH, rt; (e) POCl₃, proton sponge, PO(OMe)₃, 0 °C.

tuning reaction conditions, it was found that successful cross

coupling could be achieved with CsF as base in a mixture of DMF

and water as solvent (Scheme 3). Under these reaction circum-

stances, only small amounts of 2⁰-deoxyuridine were detected.

Phosphorylation of compounds 9aꢀb aﬀorded the 5-aryl-2⁰-

dUMP analogues 2aꢀb.

used as starting material (Scheme 5). For the synthesis of

derivatives 4a and 4b, bearing an N-butyl-6-heptynamide chain,

compounds 11a and 11b were subjected to Pd-catalyzed cross-

coupling with 6-heptynoic acid,⁴³followed by DIC/HOBt

mediated coupling with n-butylamine.⁴⁴Deprotection of the

acetyl groups under alkaline conditions,²⁹followed by phosphor-

ylation, yielded the desired ﬁnal compounds 4a and 4b. On the

other hand, the synthesis of 5-propargylamide derivatives 5aꢀd

and 6aꢀb was performed in a divergent manner. The acetylene

moieties were synthesized through condensation reaction of

propargylamine with suitable acyl chlorides, yielding 15aꢀd as

shown in Scheme 6.⁴⁵

Long-chain acetylenic compounds 3aꢀb have been synthe-

sized via palladium-catalyzed Sonogashira cross-coupling of

suitable acetylenes with unprotected 5-iodo nucleosides as the

key step (Scheme 4).^35,36Compounds 10a and 10b were

accessible from unprotected 5-iodo-2⁰-deoxyuridine 7a and

1-(β-^D-arabinofuranosyl)-5-iodoracil 7b, respectively, by Sono-

gashira cross-coupling with 1-dodecyne with a slightly modiﬁed

workup than the one described in the literature. Yoshikawa

phosphorylation^37,38(POCl₃/proton sponge/trimethyl phos-

phate) ﬁnally furnished compounds 3a and 3b (Scheme 4).

The arabino-based compounds were synthesized from 1-(β-^D-

arabinofuranosyl)-5-iodoracil,³⁹7b, which was prepared by

reaction of 1-(β-^D-arabinofuranosyl)uracil^39,40with I₂in

HNO₃/CHCl₃.

The introduction of the amide-containing terminal acetylenes

15aꢀd at position 5 of the uracil ring was achieved via a

Sonogashira cross-coupling, followed by deacetylation aﬀording

nucleoside analogues 18aꢀd (Scheme 7).

Phosphorylation of derivative 18a was problematic. The

standard method previously used (POCl₃/proton sponge/tri-

methyl phosphate) only gave unsatisfactory results with low

yields and diﬃcult puriﬁcation due to the presence of side

products. However, repeated puriﬁcation (ﬂash chromatogra-

phy and HPLC) led to the isolation of the pure nucleoside

For the introduction of an amide functionality into this long

chain, acetylated 5-iodo nucleosides 11a²⁹and 11b^41,42were

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Scheme 6^a

^aReagents and conditions: (a) acyl chloride, DIPEA, DCM, 0 °C to rt; (b) hexyl isocyanate, DCM, 0 °C to rt; (c) thionyl chloride, reﬂux;

(d) propargylamine, NEt₃, DCM, 0 °C to rt.

Scheme 7^a

^aReagents and conditions: (a) acetylene 15aꢀd, Pd(PPh₃)₄, CuI, DIPEA, DMF, rt; (b) NaOMe, MeOH, rt; (c) POCl₃, proton sponge, PO(OMe)₃,

0 °C; (d) POCl₃, H₂O, pyr, CH₃CN/PO(OMe)₃, 0 °C; (e) dibenzyl phosphate, PPh₃, DIAD, THF, rt; (f) TMSI, CH₃CN, ꢀ10 °C.

monophosphate derivative 5a. For the phosphorylation of deri-

vatives 18bꢀd, the protocol employed by Sowa and Ouchi^33,46

was evaluated in which the addition of H₂O to the reaction

mixture was reported to enhance the reaction rate. Because of the

poor solubility of derivatives 18bꢀd in CH₃CN, a mixture of

acetonitrile and trimethyl phosphate was used as solvent. Un-

fortunately, these reaction conditions (POCl₃, pyridine, H₂O in

CH₃CN/PO(OMe)₃) were not compatible with the acetylenic

group. However, the desired analogues 5b and 5c could be

obtained in 96% and 92% purity, respectively, albeit only after a

second HPLC puriﬁcation step. In the case of the desired

phenacylamide 5d, considerable amounts of the corresponding

keto compound 19, due to hydration of the triple bond, were

1

identiﬁed by NMR spectroscopy (Scheme 7). The H NMR

spectrum shows two distinct singlets at δ = 7.98 and 8.36 ppm,

which were assigned to H-6 in derivatives 5d and 19 in a ratio of

3.26:1, respectively. Furthermore, as expected, the ¹³C NMR

spectrum shows a clear peak at δ = 199.2 ppm for the keto-

carbonyl group, whereas both amide carbonyl peaks of 5d and 19

overlap at δ = 174.2. These ﬁndings were conﬁrmed by mass

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Scheme 8^a

^aReagents and conditions: (a) DMTrCl, DMAP, NEt₃, pyr, rt; (b) Ac₂O, DMAP, NEt₃, CH₃CN, rt; (c) TCA, DCM, rt; (d) acetylene 15d, 16, or 17,

Pd(PPh₃)₄, CuI, DIPEA, DMF, rt; (e) 2-cyanoethyl phosphate, DCC, pyr, rt; (f) 1 M NaOH, 100 °C; (g) 2-cyanoethyl phosphate, DCC, HOBt, pyr, rt;

(h) 2-cyanoethyl phosphate, DCC, HOBt, pyr, 40 °C.

spectrometry. The (ꢀ)-ESI mass spectrum of this mixture shows

two peaks at m/z = 478 and 496, corresponding to the [M ꢀ H^þ]

peak of 5d and 19, respectively.

subsequent phosphorylation step without any further attempt

of puriﬁcation. Addition of HOBt (1 equiv) did not lead to any

signiﬁcant improvement; sulfonamide monophosphate 6a was

obtained in 11% yield. For the synthesis of urea monophosphate

6b, the reaction was carried out at 40 °C together with the

addition of HOBt (1 equiv). Again, no substantial increase in

yield of the desired product (9%) could be achieved (Scheme 8).

Biological Evaluation and StructureꢀActivity Relation-

ship Study. Compounds were evaluated for their inhibitory

activity against mycobacterial ThyX and ThyA. Cloning of the

ThyX gene, protein expression, and purification was done as

published previously.²¹The inhibition of ThyX-catalyzed dTMP

synthesis was measured in a standard radioactive assay^56,57using

purified ThyX-protein and [5-³H]-dUMP as a radiolabeled

substrate. The amount of tritiated water released during the

reaction was measured by liquid scintillation counting. The thyA

gene was expressed in E. coli and the expressed His(6)-tagged

enzymes were purified by nickel-chelate chromatography. The

inhibition of dTMP-synthesis by ThyA was measured in a

standard photometric assay⁵⁸on a purified ThyA-protein. The

extent of inhibition found is based on a decrease of absorption of

DHF (which is formed during the biochemical transformation)

at 340 nm in the presence of an inhibitor (see Experimental

Section).

Attempts to separate the desired acetylenic compound from its

hydrated form by RP-HPLC were unsuccessful, and therefore

alternatives were sought. Compound 18d was coupled with

dibenzyl phosphate under Mitsunobu conditions⁴⁷(PPh₃,

DIAD, THF), furnishing the corresponding nucleoside dibenzyl

phosphate triester 20. To our disappointment, TMSI-mediated

debenzylation⁴⁸equally resulted in an inseparable mixture of 5d

and 19 in a ratio of 2.14:1 according to ¹H NMR spectroscopy.

Finally the problem has been solved by using the method of

Tener.⁴⁹Thus, 5-iodo-3⁰-O-acetyl-2⁰-deoxyuridine 22,^50ꢀ52pre-

pared in three steps from 5-iodo-2⁰-deoxyuridine 7a (tritylation,

acetylation, and detritylation), was coupled with acetylene 15d,

yielding 3⁰-O-acetylated derivative 23. DCC-coupling of 23 with

2-cyanoethyl phosphate (pyridinium salt) in anhydrous pyridine,

followed by deprotection with 1 M NaOH at 100 °C for a few

minutes, ﬁnally gave pure 5d after puriﬁcation, however in only

16% yield (Scheme 8). The same strategy was used for the

synthesis of dUMP derivatives with sulfonamide and urea-

containing acetylene moieties. Reaction of sodium 1-heptane-

sulfonate in neat thionyl chloride,⁵³followed by condensation of

the resulting sulfonyl chloride with propargylamine,⁵⁴furnished

acetylene 16 (Scheme 6). Similarly, condensation of propargy-

lamine with hexyl isocyanate yielded acetylenic urea 17.⁵⁵Cross-

coupling reaction of 22 with 16 proceeded smoothly, aﬀording

compound 24, whereas the corresponding urea derivative 25

could not be obtained in pure form but was used in the

The crystal structure²¹of the ThyX-FAD-5-BrdUMP complex

shows that the substrate analogue is buried completely in a deep

pocket. The oxygen atoms of the phosphate moiety as well as the

nitrogen and oxygen atoms of the pyrimidine ring have crucial

interactions with numerous highly conserved active site residues,

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Table 1. Inhibition of Mtb-ThyX and Mtb-ThyA by Compounds 1ꢀ7

^aValues are means of three independent experiments. ^b5-F dUMP used as positive control. ^c93% purity. ^d92% purity.

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notably arginine, glutamic acid, and aspartic acid. As this 5⁰-O-

monophosphate group is critical for binding of 5-Br-dUMP, all

synthesized nucleosides within this study have been phosphory-

lated, aﬀording the corresponding nucleotides that have been

evaluated as potential mycobacterial ThyX and ThyA inhibitors.

Furthermore, the pyrimidine ring of 5-BrdUMP and the iso-

alloxazine ring of FAD are close enough to interact via hydrogen

bonds and π-stacking. Therefore, the pyrimide ring system of

dUMP has been kept intact in the search for novel mycobacterial

ThyX inhibitors. The C-5 position of dUMP has been subjected

to extensive structural modiﬁcations using human ThyA as drug

target. In this paper, a similar strategy was followed in order to

discover novel mycobacterial ThyX inhibitors based on a dUMP

scaﬀold. Compounds were initially evaluated at a concentration

of 50 μM in the ThyX as well as in the ThyA assay. Compounds

displaying more than 50% inhibition were subjected to do-

seꢀresponse curves in order to determine IC₅₀values. In both

assays, 5-F dUMP was included as reference compound and

positive control.

The SAR study started with the exploration of relatively small

modiﬁcations at the C-5-position of the uracil base. Introduction

of a cyanovinyl moiety in compound 1 confers good inhibitory

activity of mycobacterial ThyX (Table 1, IC₅₀= 7.4 μM),

although no inhibitory eﬀect on mycobacterial ThyA could be

observed.

To probe if ThyX can accommodate sterically more demand-

ing substituents at position 5, aryl groups were introduced via

Suzuki cross-coupling reactions. The 5-(4-ﬂuorophenyl) analo-

gue 2a exhibits good activity against ThyX (IC₅₀= 10 μM),

whereas no activity could be determined against ThyA. On the

other hand, the 5-(3-methoxyphenyl) analogue 2b shows no

activity against both enzymes. It indicates that the nature of the

substitution pattern on the phenyl ring has a large inﬂuence on

the biological activity.

Recently, Johar et al. disclosed arabinonucleoside analogues

with a long alkynyl side chain at the 5-position of the uracil

moiety that exhibited high levels of antimycobacterial activity in

vitro (MIC₉₀= 1ꢀ5 μg/mL).⁵⁹This observation prompted us to

study the eﬀect of a long-chain alkynyl group at C-5 of uridylate

on mycobacterial ThyX and ThyA activity. Indeed, 5-dodecynyl-

dUMP 3a exhibits moderate ThyX inhibition (IC₅₀= 28 μM)

but has no eﬀect on ThyA. The corresponding arabino analogue

3b completely lacks activity against both enzymes.

To further explore the SAR, functional groups were intro-

duced into the alkyl chain of derivatives 3a and 3b. The insertion

of an amide group in the center of the long alkyl chain aﬀorded

compound 4a, which was 3-fold more potent in the ThyX assay

when compared with its long-chain analogue 3a. To our dis-

appointment, again, the corresponding arabino congener 4b was

completely devoid of thymidylate synthase inhibitory activity

(IC₅₀> 50 μM). Consequently, any further eﬀort on the

synthesis of corresponding arabino derivatives was discontinued

and, in subsequent studies, we solely focused on the design and

synthesis of dUMP analogues. Shifting the amide functional

group closer toward the nucleobase furnished propargylamide

N-propargyl phenacylamide derivative 5d led to a decreased

ThyX inhibitory activity, displaying IC₅₀values of 7.3 μM. None

of these derivatives shows any appreciable ThyA inhibition.

A long chain consisting of 12 carbon atoms turned out to be

optimal for ThyX inhibition. Therefore, the total lengths of 12

atoms, as well as the propargylamine moiety, were preserved and

the functional group was modiﬁed in a further round of optimi-

zation. The amide group of compound 5a was replaced by a

sulfonamide moiety and a urea functionality, aﬀording com-

pounds 6a and 6b, respectively. Both analogues show a 5-fold

drop in ThyX inhibition when compared to the amide congener

5a and completely lack activity against ThyA.

’ CONCLUSION

The increasing incidences of resistant M. tuberculosis bacteria

urgently demand novel drugs for therapy of tuberculosis. How-

ever, no new anti-TBC drugs have been brought to the market in

the last 30 years. The last drug with a new mechanism of action

approved for TB was rifampicin (which was already discovered in

1963). Therefore, there is an urgent need for new antimycobac-

terial drugs with new modes of action to potentially avoid cross-

resistance. In this paper, mycobacterial ThyX has been selected as

a promising target for the development of novel antibacterial

drugs. Starting from the natural substrate, dUMP, a SAR study

was carried out in which structural variation at C-5 of the uracil

moiety was introduced. This work led to the discovery of a novel

series of 5-alkynyl dUMP analogues from which the most potent

congener (compound 5a) displays an IC₅₀value of 0.91 μM

against ThyX. The high polarity of this compound makes it very

unlikely that it will be able to cross the cell membrane of

M. tuberculosis and therefore it has not been tested for its anti-

microbial activity. Compound 5a completely lacks activity against

mycobacterial ThyA. To our knowledge, this is the ﬁrst time a

selective mycobacterial FDTS inhibitor is reported. Up to now,

we have no rationale to explain the observed selectivity. Co-

crystallization of compound 5a with the mycobacterial ThyX

enzyme should reveal the binding mode of compound 5a and

might explain the selective mycobacterial ThyX inhibition.

Although compound 5a shows promising activity against the

ThyX enzyme, the presence of the phosphate moiety hampers its

further development. Further medicinal chemistry eﬀorts will

be necessary to improve its drug-like characteristics in order to

have access to compounds which are useful for testing in a

bacterial assay.

’ EXPERIMENTAL SECTION

Chemistry. For all reactions, analytical grade solvents were used.

Dry acetonitrile, pyridine, and MeOH were obtained by distillation over

CaH₂. Dry DCM, DMF, and THF were purchased from commercial

suppliers. All moisture-sensitive reactions were carried out in oven-dried

1

glassware (120 °C). H and ¹³C NMR spectra were recorded with a

Bruker Advance 300 (¹H NMR, 300 MHz; ¹³C NMR, 75 MHz; ³¹P

NMR, 121 MHz) or 500 MHz (¹H NMR, 500 MHz; ¹³C NMR,

125 MHz) spectrometer using tetramethylsilane as internal standard for

¹H NMR spectra and DMSO-d₆(39.52 ppm) or CDCl₃(77.16 ppm) for

¹³C NMR spectra. Abbreviations used are: s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet, br s = broad signal. Chemical shifts are

expressed in parts per million (ppm). Coupling constants are expressed

in hertz (Hz). Mass spectra are obtained with a Finnigan LCQ advan-

tage Max (ion trap) mass spectrophotometer from Thermo Finnigan,

San Jose, CA, USA. Exact mass measurements are performed on a

derivative 5a, which is 1 order of magnitude more potent (IC₅₀

=

0.9 μM) than its congener 4a.

To probe the inﬂuence of the arm length, the total number of

carbon atoms of the alkynyl chain of 5a (12 carbon atom) was

decreased to a total number of 10 (compound 5b) and 8 atoms

(compound 5c), resulting in a 10- to 30-fold decrease of ThyX

inhibition, respectively. Similarly, the presence of a phenyl ring in

4854

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quadrupole time-of-flight mass spectrometer (Q-tof-2, Micromass,

Manchester, UK) equipped with a standard electrospray-ionization

(ESI) interface. Samples were infused in i-PrOH/H₂O (1:1) at 3 μL/min.

Melting points are determined on a Barnstead IA 9200 and are

uncorrected. Precoated aluminum sheets (Fluka Silica gel/TLC-cards,

254 nm) were used for TLC. Column chromatography was performed

on ICN silica gel 63ꢀ200, 60 Å. All final compounds possess a purity of

at least 95% (see Supporting Information) except derivatives 2a and 5c

(93 and 92% purity, respectively), as determined by analytical RP-HPLC

analysis on a XBridge column (C-18, 5 μm, 4.6 mm ꢁ 150 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. Preparative HPLC purification was carried

out on the same instrument using a preparative XBridge column (C-18,

5 μm, 19 mm ꢁ 150 mm) from Waters, Milford, Massachusetts, USA.

5-(2-Cyanovinyl)-2⁰-deoxyuridine (8)^30ꢀ32. 5-Iodo-2⁰-deoxyuri-

dine²⁹7a (250 mg, 0.71 mmol) was dissolved in anhydrous DMF

(10 mL), and the solution was flushed with nitrogen gas. To the solution

were added acrylonitrile (468 μL, 7.1 mmol), tributylamine (185 μL,

0.78 mmol), and tetrakis(triphenylphosphine)palladium(0) (81 mg,

0.07 mmol), and the reaction mixture was heated at 80 °C for 9 h. The

solution was evaporated in vacuo and purified using silica gel column

chromatography (DCM/MeOH = 97:3f95:5) yielding compound 9

(116 mg, 59%). ¹H NMR (300 MHz, DMSO-d₆) δ 11.74 (s, 1H, NH),

8.35 (s, 1H, H-6), 7.23 (d, 1H, J = 16.3 Hz, alkeneH), 6.52 (d, 1H, J = 16.3

Hz, alkene H), 6.09 (t, 1H, J = 6.2 Hz, H-1⁰), 5.27 (s, 1H, OH), 5.13 (s,

1H, OH), 4.25 (m, 1H, H-3⁰), 3.82 (m, 1H, H-4⁰), 3.62 (ddd, 2H, J = 23.3,

12.0, 3.4 Hz, H-5⁰), 2.16 (m, 2H, H-2⁰). ¹³C NMR (75 MHz, DMSO-d₆)

δ 161.65, 149.01, 144.50, 144.35, 119.25, 107.73, 94.27, 87.74, 85.17,

69.68, 60.80, 40.15. HRMS (ESI) calcd for C₁₂H₁₃N₃O₅278.0782 (M ꢀ

H^þ); found 278.0773.

129.51, 120.66, 114.00, 113.72, 113.27, 88.09, 85.11, 70.77, 61.55, 55.51,

40.61. HRMS (ESI) calcd for C₁₆H₁₈N₂O₆333.1092 (M ꢀ H^þ); found

333.1107.

3⁰,5⁰-di-O-Acetyl-2⁰-deoxyuridine-5-heptynoic Acid (12a).

Compound 11a (1.68 g, 3.83 mmol), Pd(PPh₃)₄(433 mg, 0.382 mmol),

and CuI (147 mg, 0.764 mmol) were dissolved in anhydrous DMF

(20 mL) under an Ar atmosphere, and DIPEA (1.32 mL, 7.67 mmol)

and heptynoic acid (1.45 mL, 11.50 mmol) were then added. After being

stirred at rt for 22 h, the solvent was evaporated and coevaporated with

xylene. The residue was taken up in DCM/MeOH = 1:1 (40 mL),

BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the mixture was

stirred at rt for 30 min. After filtration of the resin and evaporation of the

solvents, the crude residue was taken up in EtOAc (70 mL), and the

organic layer was washed with brine (3 ꢁ 70 mL), dried over MgSO₄,

and evaporated. The residue was purified by silica gel column chroma-

tography (heptane/EtOAc = 2:3 containing 1% formic acid), and early

and late fractions were subjected to a second column using the same

eluent to yield compound 12a (783 mg, 47%) as an off-white solid. ¹H

NMR (300 MHz, DMSO-d₆) δ 12.05 (br s, 1H, COOH), 11.65 (br s,

1H, NH), 7.87 (s, 1H, H-6), 6.14 (t, 1H, J = 7.2 Hz, H-1⁰), 5.18 (m, 1H,

H-3⁰), 4.26 (d, 2H, J = 4.3 Hz, H-5⁰), 4.19 (m, 1H, H-4⁰), 2.54ꢀ2.28 (m,

2H, H-2⁰), 2.39 (t, 2H, J = 6.9 Hz, R-CH₂), 2.24 (t, 2H, J = 7.2 Hz,

CH₂COOH), 2.08 (s, 3H, CH₃), 2.06 (s, 3H, CH₃), 1.65ꢀ1.45 (m, 4H,

2 ꢁ CH₂). ¹³C NMR (75 MHz, DMSO-d₆) δ 174.32, 170.05, 161.59,

149.42, 142.36, 99.54, 93.30, 81.46, 73.81, 72.69, 63.56, 36.09, 33.15,

27.63, 23.83, 20.76, 20.55, 18.54. MS (ESI) calcd for C₂₀H₂₃N₂O₉

435.14 (M ꢀ H^þ), found 435.16.

2⁰,3⁰,5⁰-tri-O-Acetyl-1-β-D-arabinofuranosyluracil-5-heptynoic

Acid (12b). Compound 11b (2 g, 4.03 mmol), Pd(PPh₃)₄(455 mg,

0.403 mmol), and CuI (155 mg, 0.8 mmol) were dissolved in anhydrous

DMF (20 mL) under an Ar atmosphere, and DIPEA (1.39 mL, 8.08

mmol) and heptynoic acid (1.52 mL, 12.09 mmol) were then added.

After being stirred at rt overnight, the solvent was evaporated and

coevaporated with xylene. The residue was taken up in DCM/MeOH =

1:1 (40 mL), BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the

mixture was stirred at rt for 30 min. After filtration of the resin and

evaporation of the solvents the crude residue was taken up in EtOAc

(80 mL), and the organic layer was washed with brine (3 ꢁ 80 mL),

dried over MgSO₄, and evaporated. The residue was purified by silica gel

column chromatography (heptane/EtOAc = 2:3 containing 1% formic

acid), and early and late fractions were subjected to a second column

using the same eluent to yield compound 12b (1.04 g, 52%) as an off-

white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.02 (br s, 1H,

COOH), 11.72 (br s, 1H, NH), 7.78 (s, 1H, H-6), 6.23 (d, 1H, J =

5.3 Hz, H-1⁰), 5.41 (m, 1H, H-2⁰), 5.25 (m, 1H, H-3⁰), 4.35 (d, 2H, J =

4.3 Hz, H-5⁰), 4.26 (m, 1H, H-4⁰), 2.38 (t, 2H, J = 6.8 Hz, R-CH₂), 2.24

(t, 2H, J = 7.2 Hz, CH₂COOH), 2.09 (s, 6H, 2 ꢁ CH₃), 1.96 (s, 3H,

CH₃), 1.63ꢀ1.47 (m, 4H, 2 ꢁ CH₂). ¹³C NMR (75 MHz, DMSO-d₆) δ

174.25, 169.99, 169.69, 168.79, 161.34, 148.95, 142.83, 98. 81, 93.28,

82.97, 78.08, 74.90, 74. 36, 72.38, 62.54, 33.13, 27.57, 23.80, 20.56,

20.54, 20.13, 18.51. MS (ESI) calcd (M ꢀ H^þ) C₂₂H₂₅N₂O₁₁, 493.15;

found, 493.14.

General Procedure for the Suzuki Reaction of 5-Iodonu-

cleosides 7aꢀb with Various Boronic Acids. 5-Iodo-2⁰-

deoxyuridine²⁹7a (250 mg, 0.71 mmol) (or 5-iodoracil 1-β-D-arabino-

furanoside 7b, 250 mg, 0.67 mmol) was dissolved in DMF (7.5 mL) and

H₂O (3.75 mL). To the solution were added cesium fluoride (268 mg,

1.76 mmol) and a boronic acid (0.85 mmol), whereupon nitrogen gas

was passed through the solution for 5 min. Tetrakis(triphenylphos-

phine)palladium(0) (81 mg, 0.07 mmol) was added, and the reac-

tion mixture was heated at 60 °C until the reaction was complete

(determined by TLC). The crude mixture was evaporated in vacuo and

purified using silica gel column chromatography (DCM/MeOH =

98:2f90:10), yielding title compounds 9aꢀb.

5-(4-Fluorophenyl)-2⁰-deoxyuridine (9a). This compound

was prepared according to the general procedure described above

starting from 7a using 4-fluorophenylboronic acid (119 mg), yielding

compound 9a (178 mg, 78%). ¹H NMR (300 MHz, CD₃OD) δ 8.31 (s,

1H, H-6), 7.59 (dd, 2H, J = 8.9, 5.4 Hz, ArH), 7.11 (t, 2H, J = 8.9 Hz,

ArH), 6.38 (t, 1H, J = 6.5 Hz, H-1⁰), 4.46 (dd, 1H, J = 8.6, 4.9 Hz, H-3⁰),

3.97 (q, 1H, J = 3.1 Hz, H-4⁰), 3.80 (ddd, 2H, J = 25.7, 11.9, 2.9 Hz,

H-5⁰), 2.34 (m, 2H, H-2⁰). ¹³C NMR (75 MHz, CD₃OD) δ 163.95,

161.93 (d, 1C, J_CF= 185.4 Hz), 150.47, 138.35, 129.87 (d, 2C, J_CF

=

3⁰,5⁰-di-O-Acetyl-2⁰-deoxyuridine-5-(N-butyl)heptynamide

(13a). To a solution of compound 12a (350 mg, 0.802 mmol) and

HOBt (124 mg, 0.924 mmol) in anhydrous DCM (13 mL) under an Ar

atmosphere was added n-butylamine (92 μL, 0.924 mmol), and the

mixture was cooled to 0 °C. DIC (125 μL, 0.807 mmol) was added, and

the reaction mixture was stirred at 0 °C for 1 h and at rt for 5 h. The

solvent was evaporated and the residue taken up in EtOAc (25 mL). The

organic layer was washed with satd NaHCO₃(3 ꢁ 20 mL), dried over

MgSO₄, and evaporated. The residue was purified by silica gel column

chromatography (heptane/EtOAc = 1:3 containing 1% formic acid),

8.0 Hz), 129.14 (d, 1C, J_CF= 3.3 Hz), 114.59 (d, 2C, J_CF= 21.5 Hz),

113.67, 87.63, 85.40, 70.60, 61.08, 40.31. HRMS (ESI) calcd for

C₁₅H₁₅FN₂O₅321.0892 (M ꢀ H^þ); found 321.0894.

5-(3-Methoxyphenyl)-2⁰-deoxyuridine (9b). This compound

was prepared according to the general procedure described above

starting from 7a using 3-methoxyphenylboronic acid (129 mg), yielding

compound 9b (223 mg, 95%). ¹H NMR (300 MHz, CD₃OD) δ 8.31 (s,

1H, H-6), 7.28 (t, 1H, J = 7.9 Hz, ArH), 7.17 (s, 1H, ArH), 7.13 (d, 1H,

J = 7.9 Hz, ArH), 6.89 (dd, 1H, J = 6.9, 2.2 Hz, ArH), 6.37 (t, 1H, J = 6.5

Hz, H-1⁰), 4.46 (m, 1H, H-3⁰), 3.97 (m, 1H, H-4⁰), 3.82 (s, 3H, OCH₃),

3.79 (ddd, 2H, J = 24.3, 12.0, 3.1 Hz, H-5⁰), 2.35 (m, 2H, H-2⁰). ¹³C

NMR (75 MHz, DMSO-d₆) δ 162.47, 159.54, 150.36, 138.53, 135.00,

1

which gave compound 13a (321 mg, 81%) as an off-white solid. H

NMR (300 MHz, DMSO-d₆) δ 11.54 (br s, 1H, NH), 7.84 (s, 1H, H-6),

4855

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7.61 (t, 1H, J = 5.3 Hz, amide NH), 6.14 (t, 1H, J = 7 Hz, H-1⁰), 5.19 (m,

1H, H-3⁰), 4.26 (d, 2H, J = 4.3 Hz, H-5⁰), 4.19 (m, 1H, H-4⁰), 3.03 (m,

2H, CH₂NH), 2.53ꢀ2.28 (m, 2H, H-2⁰), 2.37 (t, 2H, J = 6.9 Hz,

R-CH₂), 2.11ꢀ2.03 (m, 8H, 2 ꢁ CH₃and CH₂CO), 1.65ꢀ1.21 (m, 8H,

4 ꢁ CH₂), 0.87 (t, 3H, J = 7.2 Hz). ¹³C NMR (75 MHz, DMSO-d₆) δ

171.59, 170.02 (2C), 161.60, 149.40, 142.34, 99.55, 93.40, 84.70, 81.43,

73.78, 72.63, 63.55, 38.04, 36.08, 34.82, 31.28, 27.73, 24.64, 20.75, 20.54,

19.56, 18.55, 13.65. MS (ESI) calcd for C₂₄H₃₃N₃O₈492.23 (M þ H^þ),

514.22 (M þ Na^þ), 1005.44 (2M þ Na^þ); found 492.11, 514.10,

1004.86.

1H, H-3⁰), 3.74 (m, 1H, H-4⁰), 3.61 (m, 2H, H-5⁰), 3.02 (m, 2H,

CH₂NH), 2.36 (t, 2H, J = 6.9 Hz, R-CH₂), 2.07 (t, 2H, J = 7.3 Hz,

CH₂CO), 1.64ꢀ1.20 (m, 8H, 4 ꢁ CH₂), 0.86 (t, 3H, J = 7.2 Hz, CH₃).

¹³C NMR (75 MHz, DMSO-d₆) δ 171.77, 161.94, 149.44, 144.26,

97.45, 92.67, 85.29, 84.72, 75.21, 75.15, 73.06, 60.42, 38.07, 34.86, 31.27,

27.77, 24.65, 19.57, 18.57, 13.67. MS (ESI) calcd for C₂₀H₂₉N₃O₇

424.21 (M þ H^þ), 446.19 (M þ Na^þ), 869.39 (2M þ Na^þ); found

424.06, 446.09, 868.96. HRMS (ESI) calcd for C₂₀H₂₉N₃O₇424.2078

(M þ H^þ), 446.1898 (M þ Na^þ), 462.1642 (M þ K^þ); found

424.2080, 446.1880, 462.1578.

2⁰,3⁰,5⁰-tri-O-Acetyl-1-β-D-arabinofuranosyluracil-5-(N-butyl)-

heptynamide (13b). To a solution of compound 12b (350 mg, 0.712

mmol) and HOBt (110 mg, 0.82 mmol) in anhydrous DCM (12 mL)

under an Ar atmosphere was added n-butylamine (82 μL, 0.82 mmol),

and the mixture was cooled to 0 °C. DIC (117 μL, 0.758 mmol) was

added, and the reaction mixture was stirred at 0 °C for 1 h and at rt for

4 h. The solvent was evaporated and the residue taken up in EtOAc

(25 mL). The organic layer was washed with satd NaHCO₃(3 ꢁ

20 mL), dried over MgSO₄, and evaporated. The residue was purified by

silica gel column chromatography (heptane/EtOAc = 1:3 containing 1%

formic acid), which gave compound 13b (333 mg, 85%) as an off-white

solid. ¹H NMR (300 MHz, DMSO-d₆) δ 11.71 (br s, 1H, NH), 7.77 (s,

1H, H-6), 7.72 (t, 1H, J = 6.0 Hz, amide NH), 6.23 (d, 1H, J = 5.3 Hz,

H-1⁰), 5.41 (t, 1H, J = 4.5 Hz, H-2⁰), 5.26 (t, 1H, J = 4.6 Hz, H-3⁰), 4.35

(m, 2H, H-5⁰), 4.25 (m, 1H, H-4⁰), 3.02 (m, 2H, CH₂NH), 2.37 (t, 2H,

J = 6.9 Hz, R-CH₂), 2.11ꢀ2.03 (m, 8H, 2 ꢁ CH₃and CH₂CO), 1.96 (s,

3H, CH₃), 1.63ꢀ1.20 (m, 8H, 4 ꢁ CH₂), 0.86 (t, 3H, J = 7.1 Hz, CH₃).

¹³C NMR (75 MHz, DMSO-d₆) δ 171.57, 169.97, 169.69, 161.35,

148.94, 142.80, 98.83, 93.40, 82.96, 78.03, 74.86, 74.36, 72.31, 62.53,

38.02, 34.80, 31.27, 27.69, 24.62, 20.53, 20.11, 19.54, 18.52, 13.64. MS

(ESI) calcd for C₂₆H₃₅N₃O₁₀550.24 (M þ H^þ), 572.22 (M þ Na^þ),

1121.45 (2M þ Na^þ); found 550.10, 572.19, 1120.88.

N-(Prop-2-ynyl)heptane-1-sulfonamide (16). Sodium 1-hep-

tanesulfonate (1 g, 4.95 mmol) was refluxed in neat thionyl chloride

overnight under an Ar atmosphere. The mixture was allowed to cool to

rt, and then the volatiles were removed in vacuo and the residue was

dissolved in dry DCM (15 mL) under an Ar atmosphere. A mixture of

NEt₃(705 μL, 5.01 mmol) and propargylamine (315 μL, 4.91 mmol)

were subsequently added dropwise at 0 °C. The ice bath was removed,

and the reaction mixture was stirred at rt overnight, diluted with DCM

(25 mL), washed with H₂O, satd NaHCO₃, and again H₂O (40 mL

each), dried over MgSO₄, and evaporated to yield sulfonamide 16

(535 mg, 50%) as a red oil. This product was used in the Sonogashira

coupling step without further purification. ¹H NMR (300 MHz, CDCl₃)

δ 4.53 (m, 1H, NH), 3.95 (m, 2H, R-CH₂), 3.14 (m, 2H, CH₂SO₂), 2.35

(t, 1H, J = 2.5 Hz, acetylenic H), 1.85 (m, 2H, CH₂CH₂SO₂), 1.48ꢀ1.19

(m, 8H, 4 ꢁ CH₂), 0.89 (t, 3H, J = 6.9 Hz, CH₃) ¹³C NMR (75 MHz,

CDCl₃) δ 79.19, 73.09, 53.73, 32.76, 31.61, 28.87, 28.34, 23.74, 22.66,

14.15. MS (ESI) calcd for C₁₀H₁₉NO₂S 216.11 (M ꢀ H^þ); found

215.70.

1-Hexyl-3-(prop-2-ynyl)urea (17). Propargylamine (500 μL,

7.80 mmol) was dissolved in anhydrous DCM (20 mL) under an Ar

atmosphere, and the resulting yellow solution was cooled to 0 °C. Hexyl

isocyanate (995 mg, 7.82 mmol) dissolved in 10 mL of anhydrous DCM

was added drop- to portionwise, the ice bath was removed, and the

reaction mixture was stirred at rt for 45 min. The solvent was evaporated

to yield compound 17 (1.39 g, 98%) as an off-white solid, which was

used in the following step without further purification. ¹H NMR (300

MHz, CDCl₃) δ 5.51 (br s, 1H, NH), 5.32 (br s, 1H, NH), 3.97 (m, 2H,

R-CH₂), 3.16 (m, 2H, CH₂NH), 2.20 (t, 1H, J = 2.5 Hz, acetylenic H),

1.48 (m, 2H, CH₂CH₂NH), 1.29 (m, 6H, 3 ꢁ CH₂), 0.88 (t, 3H, J = 6.7

Hz, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 158.43, 81.15, 70.91, 40.65,

31.69, 30.33, 30.12, 26.73, 22.71, 14.14. MS (ESI) calcd for C₁₀H₁₈N₂O

183.15 (M þ H^þ); found 183.09.

2⁰-Deoxyuridine-5-(N-butyl)heptynamide (14a). Com-

pound 13a (276 mg, 0.562 mmol) was dissolved in dry MeOH

(8 mL), NaOMe (30%-solution in MeOH, 152 μL, 0.8 mmol) was

added, and the reaction mixture was stirred for 1 h at rt under an Ar

atmosphere. The solution was neutralized with Dowex 50WX-8 resin

(H^þ-form), and the resin was filtered off and washed with MeOH. After

evaporation of the filtrate the residue was purified by silica gel column

chromatography (EtOAc/MeOH = 98:2), which yielded compound

14a (204 mg, 89%) as an off-white solid. ¹H NMR (300 MHz, DMSO-

d₆) δ 11.55 (br s, 1H, NH), 8.15 (s, 1H, H-6), 7.76 (t, 1H, J = 5.4 Hz,

amide NH), 6.11 (t, 1H, J = 6.6 Hz, H-1⁰), 5.24 (d, 1H, J = 4.2 Hz, OH),

5.14 (t, 1H, J = 4.8 Hz, OH), 4.23 (m, 1H, H-3⁰), 3.79 (m, 1H, H-4⁰),

3.59 (m, 2H, H-5⁰), 3.02 (m, 2H, CH₂NH), 2.36 (t, 2H, J = 6.9 Hz,

R-CH₂), 2.13ꢀ2.05 (m, 4H, H-2⁰and CH₂CO), 1.64ꢀ1.19 (m, 8H, 4 ꢁ

CH₂), 0.86 (t, 3H, J = 7.2 Hz, CH₃). ¹³C NMR (75 MHz, DMSO-d₆) δ

171.81, 161.79, 149.48, 142.81, 99.00, 92.99, 87.53, 84.59, 73.00, 70.13,

60.97, 38.08, 34.86, 31.27, 27.74, 24.62, 19.57, 18.59, 13.67. MS (ESI)

calcd for C₂₀H₂₉N₃O₆408.21 (M þ H^þ), 430.20 (M þ Na^þ), 837.40

(2M þ Na^þ); found 408.12, 430.12, 837.04. HRMS (ESI) calcd for

C₂₀H₂₉N₃O₆446.1693 (M þ K^þ); found 446.1674.

N-(3-(5-(2⁰-Deoxyuridine))prop-2-ynyl)octanamide (18a).

Compound 11a (400 mg, 0.913 mmol), Pd(PPh₃)₄(103 mg, 0.091

mmol), and CuI (35 mg, 0.182 mmol) were dissolved in anhydrous

DMF (5 mL) under an Ar atmosphere, and DIPEA (313 μL, 1.82 mmol)

and acetylene 15a (496 mg, 2.74 mmol) were then added. After being

stirred at rt for 12 h, the solvent was evaporated and coevaporated with

xylene. The residue was taken up in DCM/MeOH = 1:1 (20 mL),

BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the mixture was

stirred at rt for 30 min. After filtration of the resin and evaporation of the

solvents, the crude residue was taken up in EtOAc (30 mL) and the

organic layer was washed with brine (3 ꢁ 30 mL), dried over MgSO₄,

and evaporated. The residue was purified by silica gel column chroma-

tography (cyclohexane/EtOAc = 1:1 containing 1% formic acid), and

the obtained intermediate was dissolved in dry MeOH (14 mL).

NaOMe (30%-solution in MeOH, 265 μL, 1.4 mmol) was added, and

the reaction mixture was stirred for 1 h at rt under an Ar atmosphere. The

solution was neutralized with Dowex 50WX-8 resin (H^þ-form), and the resin

was filtered off and washed with MeOH. After evaporation of the filtrate, the

residue was purified by silica gel column chromatography (EtOAc/MeOH =

98:2 containing 0.5% formic acid), which yielded compound 18a (251 mg,

68% over 2 steps) as an off-white solid. ¹H NMR (300 MHz, DMSO-d₆) δ

11.61 (br s, 1H, NH), 8.29 (t, 1H, J = 5.4 Hz, amide NH), 8.16 (s, 1H, H-6),

1-β-D-Arabinofuranosyluracil-5-(N-butyl)heptynamide (14b).

Compound 13b (303 mg, 0.551 mmol) was dissolved in dry MeOH

(8 mL), NaOMe (30%-solution in MeOH, 152 μL, 0.8 mmol) was

added, and the reaction mixture was stirred for 1 h at rt under an Ar

atmosphere. The solution was neutralized with Dowex 50WX-8 resin

(H^þ-form), and the resin was filtered off and washed with MeOH. After

evaporation of the filtrate, the residue was purified by silica gel column

chromatography (EtOAc/MeOH = 95:5), which yielded compound

14b (206 mg, 88%) as an off-white solid. ¹H NMR (300 MHz, DMSO-

d₆) δ 11.55 (br s, 1H, NH), 7.84 (s, 1H, H-6), 7.75 (t, 1H, J = 5.4 Hz,

amide NH), 5.96 (d, 1H, J = 4.4 Hz, H-1⁰), 5.60 (d, 1H, J = 5.1 Hz, OH),

5.47 (br s, 1H, OH), 5.15 (br s, 1H, OH), 4.00 (m, 1H, H-2⁰), 3.90 (m,

4856

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6.11 (t, 1H, J = 6.7 Hz, H-1⁰), 5.25 (d, 1H, J = 3.9 Hz, OH), 5.10 (t, 1H,

J = 4.7 Hz, OH), 4.22 (m, 1H, H-3⁰), 4.06 (d, 2H, J = 5.4 Hz, R-CH₂),

3.79 (m, 1H, H-4⁰), 3.63ꢀ3.53 (m, 2H, H-5⁰), 2.10 (m, 4H, H-2⁰and

CH₂CO), 1.48 (m, 2H, CH₂CH₂CO), 1.24 (m, 8H, 4 ꢁ CH₂), 0.85

(t, 3H, J = 7.0 Hz, CH₃). ¹³C NMR (75 MHz, DMSO-d₆) δ 171.83,

161.59, 149.41, 143.60, 98.15, 89.78, 87.62, 84.70, 74.19, 70.24, 61.03,

35.07, 31.13, 28.60, 28.47, 28.41, 25.11, 22.03, 13.92. MS (ESI) calcd

for C₂₀H₂₉N₃O₆408.21 (M þ H^þ), 430.20 (M þ Na^þ), 837.40

(2M þ Na^þ); found 408.09, 430.05, 836.95. HRMS (ESI) calcd for

C₂₀H₂₉N₃O₆446.1693 (M þ K^þ); found 446.1670.

5:2) δ 8.37 (s, 1H, H-6), 6.26 (t, 1H, J = 6.4 Hz, H-1⁰), 4.44 (m, 1H,

H-3⁰), 4.14 (s, 2H, R-CH₂), 3.99 (m, 1H, H-4⁰), 3.89 (dd, 1H, J = 12, 2.6

Hz, H-5⁰), 3.78 (dd, 1H, J = 12, 2.6 Hz, H-5⁰), 2.41ꢀ2.34 (m, 1H, H-2⁰),

2.23ꢀ2.16 (m, 3H, H-2⁰and CH₂CO), 1.66 (m, 2H, CH₂CH₂CO), 0.96

(t, 3H, J = 7.4 Hz, CH₃). ¹³C NMR (75 MHz, CDCl₃:MeOD = 5:2) δ

174.14, 163.09, 149.53, 143.90, 98.82, 88.97, 87.51, 85.70, 73.82, 70.35,

61.19, 40.77, 37.79, 29.50, 18.81, 13.23. MS (ESI) calcd for C₁₆H₂₁N₃O₆

352.15 (M þ H^þ), 374.13 (M þ Na^þ), 725.28 (2M þ Na^þ); found

351.74, 373.68, 724.37. HRMS (ESI) calcd for C₁₆H₂₁N₃O₆390.1067

(M þ K^þ); found 390.1058.

N-(3-(5-(2⁰-Deoxyuridine))prop-2-ynyl)hexanamide (18b).

Compound 11a (400 mg, 0.913 mmol), Pd(PPh₃)₄(103 mg, 0.091

mmol), and CuI (35 mg, 0.182 mmol) were dissolved in anhydrous

DMF (5 mL) under an Ar atmosphere, and DIPEA (313 μL, 1.82 mmol)

and acetylene 15b (420 mg, 2.74 mmol) were then added. After being

stirred at rt for 14 h, the solvent was evaporated and coevaporated with

xylene. The residue was taken up in DCM/MeOH = 1:1 (10 mL),

BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the mixture was

stirred at rt for 30 min. After filtration of the resin and evaporation of the

solvents, the crude residue was taken up in EtOAc (30 mL), and the

organic layer was washed with brine (3 ꢁ 30 mL), dried over MgSO₄,

and evaporated. The residue was purified by silica gel column chroma-

tography (heptane/EtOAc = 2:3 containing 1% formic acid), and the

obtained intermediate was dissolved in dry MeOH (14 mL). NaOMe

(30% solution in MeOH, 265 μL, 1.4 mmol) was added, and the reaction

mixture was stirred for 1 h at rt under an Ar atmosphere. The solution

was neutralized with Dowex 50WX-8 resin (H^þ-form), and the resin was

filtered off and washed with MeOH. After evaporation of the filtrate, the

residue was purified by silica gel column chromatography (EtOAc/

MeOH = 98:2 containing 1% formic acid), which yielded compound

18b (209 mg, 60% over 2 steps) as an off-white solid. ¹H NMR

(300 MHz, CDCl₃:MeOD = 5:2) δ 8.33 (s, 1H, H-6), 6.25 (t, 1H, J =

6.5 Hz, H-1⁰), 4.43 (m, 1H, H-3⁰), 4.13 (br s, 2H, R-CH₂), 3.98 (m, 1H,

H-4⁰), 3.87 (dd, 1H, J = 12, 2.9 Hz, H-5⁰), 3.77 (dd, 1H, J = 12, 2.9 Hz,

H-5⁰), 2.40ꢀ2.34 (m, 1H, H-2⁰), 2.23ꢀ2.14 (m, 3H, H-2⁰and CH₂CO),

1.63 (m, 2H, CH₂CH₂CO), 1.33 (m, 4H, 2 ꢁ CH₂), 0.90 (t, 3H, J = 7

Hz, CH₃). ¹³C NMR (75 MHz, CDCl₃:MeOD = 5:2) δ 174.31, 163.12,

149.54, 143.91, 98.83, 88.98, 87.52, 85.71, 73.85, 70.36, 61.19, 40.79,

35.93, 31.17, 29.53, 25.10, 22.09, 13.48. MS (ESI) calcd for C₁₈H₂₅N₃O₆

380.18 (M þ H^þ), 402.16 (M þ Na^þ), 781.33 (2M þ Na^þ); found

379.72, 401.67, 780.43. HRMS (ESI) calcd for C₁₈H₂₅N₃O₆402.1636

(M þ Na^þ), 418.1380 (M þ K^þ); found 402.1656, 418.1379.

2-Phenyl-N-(3-(5-(2⁰-deoxyuridine))prop-2-ynyl)acetamide

(18d). Compound 11a (400 mg, 0.913 mmol), Pd(PPh₃)₄(103 mg,

0.091 mmol), and CuI (35 mg, 0.182 mmol) were dissolved in anhy-

drous DMF (5 mL) under an Ar atmosphere, and DIPEA (313 μL,

1.82 mmol) and then acetylene 15d (474 mg, 2.74 mmol) were then

added. After being stirred at rt for 14 h, the solvent was evaporated and

coevaporated with xylene. The residue was taken up in DCM:MeOH =

1:1 (10 mL), BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the

mixture was stirred at rt for 30 min. After filtration of the resin and

evaporation of the solvents, the crude residue was taken up in EtOAc

(30 mL), and the organic layer was washed with brine (3 ꢁ 30 mL),

dried over MgSO₄, and evaporated. The residue was purified by silica gel

column chromatography (heptane:EtOAc = 1:2f1:3 each containing

1% formic acid) and the obtained intermediate was dissolved in dry

MeOH (14 mL). NaOMe (30% solution in MeOH, 265 μL, 1.4 mmol)

was added, and the reaction mixture was stirred for 1 h at rt under an Ar

atmosphere. The solution was neutralized with Dowex 50WX-8 resin

(H^þ-form), the resin was filtered off and washed with MeOH. After

evaporation of the filtrate, the residue was purified by silica gel column

chromatography (EtOAc:MeOH = 98:2 containing 1% formic acid),

which yielded compound 18d (231 mg, 63% over 2 steps) as an off-white

solid. ¹H NMR (300 MHz, MeOD) δ 8.29 (s, 1H, H-6), 7.35ꢀ7.19 (m,

5H, ArH), 6.23 (t, 1H, J = 6.6 Hz, H-1⁰), 4.38 (m, 1H, H-3⁰), 4.14 (s, 2H,

R-CH₂), 3.94 (m, 1H, H-4⁰), 3.82ꢀ3.68 (ddd, 2H, J = 24.5, 12, 3.4 Hz,

H-5⁰), 3.52 (s, 2H, CH₂Ph), 2.34ꢀ2.15 (m, 2H, H-2⁰). ¹³C NMR

(75 MHz, MeOD) δ 173.63, 164.53, 151.14, 145.44, 136.64, 130.13

(2C), 129.58 (2C), 127.93, 99.91, 89.92, 89.17, 87.06, 75.25, 72.05,

62.62, 43.63, 41.70, 30.63. MS (ESI) calcd for C₂₀H₂₁N₃O₆400.15

(M þ H^þ), 422.13 (M þ Na^þ), 821.28 (2M þ Na^þ); found 399.77,

421.63, 821.55. HRMS (ESI) calcd for C₂₀H₂₁N₃O₆438.1067 (M þ K^þ);

found 438.1040.

2-Phenyl-N-(3-(5-(5⁰-O-acetyl-2⁰-deoxyuridine))prop-2-ynyl)-

acetamide (23). Compound 22 (200 mg, 0.505 mmol), Pd(PPh₃)₄

(58 mg, 0.051 mmol), and CuI (20 mg, 0.104 mmol) were dissolved in

anhydrous DMF (3 mL) under an Ar atmosphere, and DIPEA (174 μL,

1.01 mmol) and then acetylene 15d (262 mg, 1.51 mmol) were then

added. After being stirred at rt for 16 h, the solvent was evaporated and

coevaporated with xylene. The residue was taken up in DCM/MeOH =

1:1 (10 mL), BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the

mixture was stirred at rt for 30 min. After filtration of the resin and

evaporation of the solvents, the crude residue was purified by a first

column of silica gel (DCM/MeOH = 98:2f97:3 each containing 1%

formic acid), and the obtained product was subjected to a second short

column of silica gel (DCM/MeOH = 95:5 containing 1% formic acid),

N-(3-(5-(2⁰-Deoxyuridine))prop-2-ynyl)butanamide (18c).

Compound 11a (400 mg, 0.913 mmol), Pd(PPh₃)₄(103 mg, 0.091

mmol), and CuI (35 mg, 0.182 mmol) were dissolved in anhydrous

DMF (4 mL) under an Ar atmosphere, and DIPEA (313 μL, 1.82 mmol)

and then acetylene 15c (343 mg, 2.74 mmol, dissolved in 1 mL

anhydrous DMF) were then added. After being stirred at rt for 18 h,

the solvent was evaporated and coevaporated with xylene. The residue

ꢀ

was taken up in DCM/MeOH = 1:1 (10 mL), BioRad XG-X8 (HCO₃

-

form) resin was added, and the mixture was stirred at rt for 30 min. After

filtration of the resin and evaporation of the solvents, the crude residue

was taken up in EtOAc (30 mL), and the organic layer was washed with

brine (3 ꢁ 30 mL), dried over MgSO₄, and evaporated. The residue was

purified by silica gel column chromatography (heptane/EtOAc =

1:2f1:3 each containing 1% formic acid), and the obtained intermedi-

ate was dissolved in dry MeOH (14 mL). NaOMe (30% solution in

MeOH, 265 μL, 1.4 mmol) was added, and the reaction mixture was

stirred for 1 h at rt under an Ar atmosphere. The solution was neutralized

with Dowex 50WX-8 resin (H^þ-form), and the resin was filtered off and

washed with MeOH. After evaporation of the filtrate, the residue was

purified by silica gel column chromatography (EtOAc/MeOH = 98:2

containing 1% formic acid), which yielded compound 18c (193 mg, 60%

over 2 steps) as an off-white solid. ¹H NMR (300 MHz, CDCl₃:MeOD =

1

which yielded compound 23 (118 mg, 53%) as an off-white solid. H

NMR (300 MHz, CDCl₃) δ 9.97 (br s, 1H, NH), 8.23 (s, 1H, H-6), 7.27

(m, 5H, ArH), 6.88 (t, 1H, J = 5 Hz, amide NH), 6.24 (t, 1H, J = 6.9 Hz,

H-1⁰), 5.32 (m, 1H, H-3⁰), 4.12 (m, 3H, R-CH₂and H-4⁰), 3.86 (m, 2H,

H-5⁰), 3.57 (s, 2H, CH₂CO), 2.47 (m, 2H, H-2⁰), 2.08 (s, 3H, CH₃). ¹³C

NMR (75 MHz, CDCl₃) δ 172.14, 170.82, 162.69, 149.58, 144.58,

134.61, 129.54 (2C), 128.95 (2C), 127.40, 99.45, 89.65, 86.08, 85.82,

75.05, 74.26, 62.20, 43.20, 38.32, 30.44, 21.11. MS (ESI) calcd for

C₂₂H₂₃N₃O₇442.16 (M þ H^þ), 464.14 (M þ Na^þ), 905.30 (2M þ

Na^þ); found 441.68, 463.65, 904.37.

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Journal of Medicinal Chemistry

ARTICLE

N-(3-(5-(3⁰-O-Acetyl-2⁰-deoxyuridine))prop-2-ynyl)heptane-

1-sulfonamide (24). Compound 22 (325 mg, 0.82 mmol), Pd-

(PPh₃)₄(91 mg, 0.082 mmol), and CuI (32 mg, 0.164 mmol) were

dissolved in anhydrous DMF (4 mL) under an Ar atmosphere, and

DIPEA (283 μL, 1.64 mmol) and then acetylene 16 (535 mg,

2.46 mmol) dissolved in anhydrous DMF (1 mL) were added. After being

stirred at rt overnight, the solvent was evaporated and coevaporated with

xylene. The residue was taken up in DCM/MeOH = 1:1 (10 mL),

BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the mixture was

stirred at rt for 30 min. After filtration of the resin and evaporation of the

solvents, the crude residue was purified by a first column of silica gel

(heptane/EtOAc = 1:1 containing 1% formic acid) and the obtained

product was subjected to a second short column of silica gel (DCM/

acetone =85:15 containing 1% formic acid), which yielded compound

24 (225 mg, 57%) as an off-white solid. ¹H NMR (300 MHz, DMSO-d₆)

δ 11.70 (br s, 1H, NH), 8.24 (s, 1H, H-6), 7.64 (br s, 1H, amide NH),

6.14 (t, 1H, J = 7.1 Hz, H-1⁰), 5.29 (br s, 1H, OH), 5.22 (m, 1H, H-3⁰),

4.04 (br m, 1H, H-4⁰), 3.98 (d, 2H, J = 5.2 Hz, R-CH₂), 3.64 (d, 2H, J =

2.5 Hz, H-5⁰), 3.13 (m, 2H, CH₂SO₂), 2.29 (m, 2H, H-2⁰), 2.06 (s, 3H,

CH₃), 1.65 (m, 2H, CH₂CH₂SO₂), 1.49ꢀ1.12 (m, 8H, 4 ꢁ CH₂), 0.82

(t, 3H, J = 7.0 Hz, CH₃) ¹³C NMR (75 MHz, DMSO-d₆) δ 169.98,

161.44, 149.38, 143.49, 98.21, 88.97, 85.07, 84.57, 75.80, 74.62, 61.16,

52.15, 37.30, 32.38, 30.97, 28.26, 27.47, 23.20, 21.92, 20.80, 13.85 MS

(ESI) calcd for C₂₁H₃₁N₃O₈S 486.19 (M þ H^þ), 508.17 (M þ Na^þ);

found 486.3, 508.2.

chromatography using a gradient of 2-propanol/water/NH₄OH. The

phosphates isolated after lyophilization were further purified by pre-

parative RP-HPLC (C18, 5 μM, 19 mm ꢁ 150 mm) using a gradient

of acetonitrile and TEAB (4ꢀ30 mM), yielding analytically pure

compounds. Next, the phosphates were subjected to ion exchange

(Dowex 50 WX-8, Na^þ) and lyophilized, yielding the corresponding

disodium salts.

5-(2-Cyanovinyl)-2⁰deoxyuridine-5⁰-monophosphate (1).

This compound was prepared according to the general procedure A de-

scribed above. The reaction mixture was worked up as described above

including silica gel column chromatography (2-propanol/NH₄OH/

H₂O = 90:5:5f75:15:15) and RP-HPLC (10 mM aq TEAB/CH₃CN =

95:5f80:20, 16 mL/min), yielding title compound 1 (49 mg, 49%) as

a white solid. ¹H NMR (500 MHz, D₂O) δ 8.15 (s, 1H, H-6), 7.35 (d,

1H, J = 16.4 Hz, vinylic H), 6.52 (d, 1H, J = 16.4 Hz, vinylic H), 6.30 (t,

1H, J = 6.8 Hz, H-1⁰), 4.53 (m, 1H, H-3⁰), 4.12 (q, 1H, J = 3.6 Hz, H-4⁰),

3.92 (d, 2H, J = 4.4 Hz, H-5⁰), 2.35 (m, 2H, H-2⁰). ¹³C NMR (125 MHz,

D₂O) δ 161.82, 156.10, 146.12, 143.99, 120.61, 109.91, 94.10, 86.08 (d,

1C, J_CP= 8.3 Hz), 85.71, 71.03, 63.49, 39.17. ³¹P NMR (121 MHz,

D₂O) δ 3.97. MS (ESI) calcd for C₁₂H₁₄N₃O₈P 358.0446 (M ꢀ H^þ);

found 358.0431.

5-(4-Fluorophenyl)-2⁰-deoxyuridine-5⁰-monophosphate

(2a). This compound was prepared according to the general procedure

A described above. The reaction mixture was worked up as described

above, including silica gel column chromatography (2-propanol/

NH₄OH/H₂O = 90:5:5f75:15:15) and RP-HPLC (10 mM aq

TEAB:CH₃CN = 95:5f80:20, 16 mL/min), yielding title compound

2a (53 mg, 24%) as a white solid. ¹H NMR (500 MHz, D₂O) δ 7.84 (s,

1H, H-6), 7.45 (dd, 2H, J = 8.6, 5.5 Hz, ArH), 7.14 (t, 2H, J = 8.9 Hz,

ArH), 6.33 (t, 1H, J = 7.0 Hz, H-1⁰), 4.52 (m, 1H, H-3⁰), 4.16 (m, 1H,

H-4⁰), 4.04 (m, 2H, H-5⁰), 2.39 (m, 2H, H-2⁰). ¹³C NMR (125 MHz,

D₂O) δ 164.74, 162.33 (d, 1C, J_CF= 244.5 Hz), 151.19, 138.48, 130.60

1-Hexyl-(3-(5-(3⁰-O-acetyl-2⁰-deoxyuridine))prop-2-ynyl)-

urea (25). Compound 22 (300 mg, 0.756 mmol), Pd(PPh₃)₄(84 mg,

0.075 mmol), and CuI (29 mg, 0.151 mmol) were dissolved in

anhydrous DMF (5 mL) under an Ar atmosphere, and DIPEA (261

μL, 1.52 mmol) and then acetylene 17 (415 mg, 2.27 mmol) were added.

After being stirred at rt overnight, the solvent was evaporated and

coevaporated with xylene. The residue was taken up in DCM/MeOH =

1:1 (10 mL), BioRad XG-X8 (HCO₃^ꢀ-form) resin was added, and the

mixture was stirred at rt for 30 min. After filtration of the resin and

evaporation of the solvents, the crude residue was purified by a first

column of silica gel (heptane/EtOAc = 1:2f1:3f1:4 each containing

1% formic acid), and the obtained product was subjected to a second

short column of silica gel (DCM/acetone = 80:20f75:25 each contain-

ing 1% formic acid), which yielded compound 25 (259 mg) as an oil

containing substantial amounts of impurities and was therefore used in

the phosphorylation step without any further purification.

(d, 2C, J_CF= 8.4 Hz), 127.76 (d, 1C, J_CF= 2.2 Hz), 115.31 (d, 2C, J_CF

=

21.8 Hz), 115.11, 85.55 (d, 1C, J_CP= 12.0 Hz), 85.45, 70.95, 64.83 (d,

1C, J_CP= 4.5 Hz), 38.76. ³¹P NMR (121 MHz, D₂O) δ 0.015. MS (ESI)

calcd for C₁₅H₁₆FN₂O₈P 401.0555 (M ꢀ H^þ); found 401.0569.

5-(3-Methoxyphenyl)-2⁰-deoxyuridine-5⁰-monophosphate

(2b). This compound was prepared according to the general procedure

A described above. The reaction mixture was worked up as described

above, including silica gel column chromatography (2-propanol/

NH₄OH/H₂O = 90:5:5f75:15:15) and RP-HPLC (10 mM aq

TEAB/CH₃CN = 95:5f 80:20, 16 mL/min), yielding title compound

2b (102 mg, 37%) as a white solid. ¹H NMR (300 MHz, D₂O) δ 7.65 (s,

1H, H-6), 7.26 (t, 1H, J = 8.2 Hz, ArH), 6.94 (m, 2H, ArH), 6.87 (dd,

1H, J = 8.2, 1.6 Hz, ArH), 6.16 (t, 1H, J = 6.8 Hz, H-1⁰), 4.43 (m, 1H,

H-3⁰), 4.09 (m, 1H, H-4⁰), 4.03 (m, 2H, H-5⁰), 3.73 (s, 3H, OCH₃), 2.29

(m, 2H, H-2⁰). ¹³C NMR (75 MHz, D₂O) δ 163.93, 158.35, 150.65,

138.23, 132.91, 129.59, 120.90, 114.86, 113.78, 113.46, 85.60, 85.06 (d,

General Procedure A^33,46for the Phosphorylation of

5-Substituted Derivatives 8, 9aꢀb, 18bꢀc. An unprotected

5-substituted derivative (1 equiv) was dissolved in PO(OMe)₃/CH₃CN =

1:1, and dry pyridine (4.8 equiv) and H₂O (2.8 equiv) were added and

the resulting solution was cooled to 0 °C. POCl₃(4.4 equiv) was added

in one portion, and the reaction mixture was stirred at 0 °C for 3 h. The

reaction mixture was poured into ice/H₂O and brought to pH 8ꢀ9 with

NH₄OH, and then the volatiles were removed in vacuo and the residue

was purified by silica gel column chromatography using a gradient of

2-propanol/water/NH₄OH. The phosphates isolated after lyophilization

were further purified by preparative RP-HPLC (C18, 5 μM, 19 mm ꢁ

150 mm) using a gradient of acetonitrile and TEAB (4ꢀ10 mM),

yielding analytically pure compounds. Next, the phosphates were

subjected to ion exchange (Dowex 50 WX-8, Na^þ) and lyophilized,

yielding the corresponding disodium salts.

1C, J_CP= 8.2 Hz), 70.63, 65.00 (d, 1C, J_CP= 4.9 Hz), 55.02, 38.55. ³¹

P

NMR (121 MHz, D₂O) δ 0.014. MS (ESI) calcd for C₁₆H₁₉N₂O₉P

413.0755 (M ꢀ H^þ); found 413.0731.

5-Dodecynyl-2⁰-deoxyuridine-5⁰-monophosphate (3a). This

compound was prepared according to the general procedure B described

above. The following amounts of starting material and reagents were

employed: compound 10a (500 mg, 1.29 mmol), POCl₃(181 μL, 1.94

mmol), and proton sponge (416 mg, 1.94 mmol) in 5 mL of PO(OMe)₃.

The reaction mixture was stirred at 0 °C for 2 h and worked up as described

above, including silica gel column chromatography (2-propanol/NH₄OH/

H₂O = 77.5:15:2.5f70:20:5) and RP-HPLC (4 mM aq TEAB/CH₃CN =

73:27f60:40, 16 mL/min), yielding title compound 3a (159 mg, 24%) as a

white solid; mp >151 °C (dec). ¹H NMR (300 MHz, DMSO-d₆) δ 7.77 (s,

1H, H-6), 6.08 (t, 1H, J = 6.9 Hz, H-1⁰), 4.27 (m, 1H, H-3⁰), 3.88 (m, 1H,

H-4⁰), 3.79 (m, 2H, H-5⁰), 2.35 (t, 2H, J = 7.1 Hz, R-CH₂), 2.21ꢀ1.99

(m, 2H, H-2⁰), 1.48 (m, 2H, β-CH₂), 1.24 (m, 14H, 7 ꢁ CH₂), 0.85

General Procedure B^37,38for the Phosphorylation of

5-Substituted Derivatives 10aꢀb, 14aꢀb, 18a. To a solution

of an unprotected 5-substituted derivative (1 equiv) and proton sponge

(1.5ꢀ3 equiv) in trimethyl phosphate was added POCl₃(1.5ꢀ3 equiv)

in one portion at 0 °C, and the reaction mixture was stirred at 0 °C for

2ꢀ3 h. The reaction mixture was poured into ice/H₂O and brought to

pH 8ꢀ9 with NH₄OH or 1 M NaOH, and then the volatiles were

removed in vacuo and the residue was purified by silica gel column

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Journal of Medicinal Chemistry

ARTICLE

(t, 3H, J= 6.7 Hz, CH₃). ¹³C NMR (75 MHz, DMSO-d₆) δ161.72, 149.47,

142.25, 99.40, 93.68, 86.10 (d, 1C, J_CP= 6.3 Hz), 84.43, 72.54, 70.74, 64.11

(d, 1C, J_CP= 4.8 Hz), 39.40, 31.30, 28.98, 28.71, 28.56, 28.38, 28.19, 22.09,

18.88, 13.95. ³¹P NMR (121 MHz, DMSO-d₆) δ 0.40. HRMS (ESI) calcd

for C₂₁H₃₂N₂O₈P 471.1902 (M ꢀ H^þ); found 471.1884.

63.12 (d, 1C, J_CP= 4.4 Hz), 38.64, 34.90, 30.03, 26.79, 24.53, 18.97,

18.06, 12.55. ³¹P NMR (121 MHz, DMSO-d₆) δ 1.61. HRMS (ESI)

calcd for C₂₀H₃₀N₃O₁₀P 502.1596 (M ꢀ H^þ); found 502.1570.

N-(3-(5-(2⁰-Deoxyuridine-5⁰-monophosphate))prop-2-ynyl)-

octanamide (5a). This compound was prepared according to the

general procedure B described above. The following amounts of starting

material and reagents were employed: compound 18a (147.2 mg, 0.361

mmol), POCl₃(101 μL, 1.08 mmol), and proton sponge (235 mg, 1.09

mmol) in 2.5 mL of PO(OMe)₃. The reaction mixture was stirred at 0 °C

for 3 h and worked up as described above, including silica gel column

chromatography (2-propanol/NH₄OH/H₂O = 85:10:5f80:10:10f

75:15:10) and RP-HPLC (30 mM aq TEAB/CH₃CN = 85:15f

80:20f75:25, 16 mL/min), yielding title compound 5a (16 mg, 8%) as

a white solid; mp >171 °C (dec). ¹H NMR (500 MHz, D₂O) δ 8.12 (s,

1H, H-6), 6.23 (t, 1H, J = 6.7 Hz, H-1⁰), 4.51 (m, 1H, H-3⁰), 4.13 (br m,

3H, R-CH₂and H-4⁰), 3.94 (t, 2H, J = 5 Hz, H-5⁰), 2.40ꢀ2.28 (br m, 2H,

H-2⁰), 2.24 (t, 2H, J = 7.3 Hz, CH₂CO), 1.57 (m, 2H, CH₂CH₂CO),

1.29ꢀ1.11 (br m, 8H, 4 ꢁ CH₂), 0.78 (t, 3H, J = 7.0 Hz, CH₃). ¹³C NMR

(125 MHz, D₂O) δ 176.97, 164.33, 150.20, 144.51, 98.73, 89.65, 85.61 (d,

1C, J_CP= 8.5 Hz), 85.34, 72.99, 70.49, 63.52 (d, 1C, J_CP= 4.4 Hz), 38.27,

35.18, 30.64, 29.09, 27.68, 27.58, 24.89, 21.49, 12.97. ³¹P NMR (121

MHz, D₂O) δ 2.68. HRMS (ESI) calcd for C₂₀H₃₀N₃O₉P 486.1647

(M ꢀ H^þ); found 486.1655.

1-β-D-Arabinofuranosyl-5-dodecynyluracil-5⁰-monopho-

sphate (3b). This compound was prepared according to the general

procedure B described above. The following amounts of starting

material and reagents were employed: compound 10b (500 mg, 1.22

mmol), POCl₃(171 μL, 1.83 mmol), and proton sponge (392 mg, 1.83

mmol) in 5 mL of PO(OMe)₃. The reaction mixture was stirred at 0 °C

for 2 h and worked up as described above, including silica gel column

chromatography (2-propanol/NH₄OH/H₂O = 74:17.5:3.5f70:20:5,

16 mL/min) and RP-HPLC (4 mM aq TEAB/CH₃CN = 75:25f

60:40), yielding title compound 3b (214 mg, 33%) as a white solid; mp

1

>159 °C (dec). H NMR (300 MHz, DMSO-d₆) δ 11.59 (br s, 1H,

NH), 7.57 (s, 1H, H-6), 5.94 (d, 1H, J = 3.6 Hz, H-1⁰), 4.71 (br s, 2H,

2⁰ꢀOH and 3⁰ꢀOH), 4.04 (m, 1H, H-2⁰), 3.92 (br m, 3H, H-3⁰and

H-5⁰), 3.80 (m, 1H, H-4⁰), 2.35 (t, 2H, J = 7.1 Hz, R-CH₂), 1.48 (m, 2H,

β-CH₂), 1.24 (m, 14H, 7 ꢁ CH₂), 0.85 (t, 3H, J = 6.7 Hz, CH₃). ¹³

C

NMR (75 MHz, DMSO-d₆) δ 161.90, 149.40, 144.05, 97.47, 93.20,

85.73, 84.07, 76.34, 74.87, 72.72, 63.91, 31.32, 28.99, 28.72, 28.57, 28.42,

28.25, 22.11, 18.83, 13.97. ³¹P NMR (121 MHz, DMSO-d₆) δ 1.70.

HRMS (ESI) calcd for C₂₁H₃₂N₂O₉P 487.1851 (M ꢀ H^þ); found

487.1847.

N-(3-(5-(2⁰-Deoxyuridine-5⁰-monophosphate))prop-2-ynyl)-

hexanamide (5b). This compound was prepared according to the

general procedure A described above. The reaction mixture was worked

up as described above, including silica gel column chromatography (2-

propanol/NH₄OH/H₂O = 70:20:5) and RP-HPLC (4 mM aq TEAB/

CH₃CN = 88:12f75:25, 16 mL/min), followed by a second run

(30 mM aq TEAB/CH₃CN = 87:13, isocratic, 7 mL/min), yielding

title compound 5b (15.7 mg, 7%) as a white solid; mp >161 °C (dec). ¹H

NMR (500 MHz, D₂O) δ 8.17 (s, 1H, H-6), 6.28 (t, 1H, J = 6.7 Hz,

H-1⁰), 4.56 (m, 1H, H-3⁰), 4.18 (br m, 1H, H-4⁰), 4.17 (s, 2H, R-CH2),

4.05 (m, 2H, H-5⁰), 2.47ꢀ2.34 (br m, 2H, H-2⁰), 2.29 (t, 2H, J = 7.4 Hz,

CH₂CO), 1.61 (m, 2H, CH₂CH₂CO), 1.35ꢀ1.20 (br m, 4H, 2 ꢁ CH₂),

0.86 (t, 3H, J = 7.0 Hz, CH₃). ¹³C NMR (125 MHz, D₂O) δ 177.29,

164.62, 150.52, 144.86, 99.07, 89.89, 85.86 (d, 1C, J_CP=8.4 Hz), 73.47,

70.79, 64.29 (d, 1C, J_CP= 4.3 Hz), 38.93, 35.59, 30.42, 29.55, 24.93,

21.66, 13.20. ³¹P NMR (121 MHz, D₂O) δ 1.23. HRMS (ESI) calcd for

C₁₈H₂₆N₃O₉P 458.1334 (M ꢀ H^þ); found 458.1338.

2⁰-Deoxyuridine-5-(N-butyl)heptynamide-5⁰-monopho-

sphate (4a). This compound was prepared according to the general

procedure B described above. The following amounts of starting

material and reagents were employed: compound 14a (154 mg, 0.378

mmol), POCl₃(53 μL, 0.567 mmol), and proton sponge (121 mg, 0.567

mmol) in 1.5 mL of PO(OMe)₃. The reaction mixture was stirred at

0 °C for 3 h and worked up as described above, including silica gel

column chromatography (2-propanol/NH₄OH/H₂O = 77.5:15:2.5f

74:17:3.5) and RP-HPLC (4 mM aq TEAB/CH₃CN = 85:15, isocratic,

16 mL/min), yielding title compound 4a (52 mg, 26%) as a white solid;

mp >192 °C (dec). ¹H NMR (500 MHz, DMSO-d₆) δ 11.58 (br s, 1H,

NH), 8.20 (br s, 1H, amide NH), 7.85 (s, 1H, H-6), 6.10 (t, 1H, J = 6.9

Hz, H-1⁰), 4.29 (s, 1H, H-3⁰), 3.88 (m, 1H, H-4⁰), 3.80 (m, 2H, H-5⁰),

3.01 (m, 2H, CH₂N), 2.36 (t, 2H, J = 6.9 Hz, R-CH₂), 2.19ꢀ2.03 (m,

2H, H-2⁰), 2.09 (t, 2H, J = 7.4 Hz, CH₂CO), 1.61 (m, 2H, CH₂), 1.47

(m, 2H, CH₂), 1.36 (m, 2H, CH₂), 1.24 (m, 2H, CH₂), 0.84 (t, 3H, J =

7.3 Hz, CH₃). ¹³C NMR (125 MHz, DMSO-d₆) δ 172.02, 161.84,

149.58, 142.57, 99.49, 93.52, 86.35 (d, 1C, J_CP= 6.7 Hz), 84.52, 72.81,

70.85, 64.15, 40.51, 38.16, 35.08, 31.38, 27.69, 24.93, 19.70, 18.68, 13.80.

³¹P NMR (121 MHz, DMSO-d₆) δ 1.41. HRMS (ESI) calcd for

C₂₀H₂₉N₃O₉P 486.1647 (M ꢀ H^þ); found 486.1630.

N-(3-(5-(2⁰-Deoxyuridine-5⁰-monophosphate))prop-2-ynyl)-

butanamide (5c). This compound was prepared according to the

general procedure A described above. The reaction mixture was worked

up as described above, including silica gel column chromatography (2-

propanol/NH₄OH/H₂O = 70:20:5) and RP-HPLC (10 mM aq TEAB/

CH₃CN = 94:6f80:20, 16 mL/min), followed by a second run (30 mM

aq TEAB/CH₃CN = 92:8, isocratic, 7 mL/min), yielding title com-

pound 5c (53 mg, 28%) as a white solid; mp >168 °C (dec). ¹H NMR

(500 MHz, D₂O) δ 8.12 (s, 1H, H-6), 6.27 (t, 1H, J = 6.8 Hz, H-1⁰), 4.55

(m, 1H, H-3⁰), 4.19 (s, 1H, R-CH2), 4.16 (m, 1H, H-4⁰), 4.01ꢀ3.90 (m,

2H, H-5⁰), 2.45ꢀ2.34 (br m, 2H, H-2⁰), 2.27 (t, 2H, J = 7.4 Hz,

1-β-D-Arabinofuranosyluracil-5-(N-butyl)heptynamide-5⁰-

monophosphate (4b). This compound was prepared according to

the general procedure B described above. The following amounts of

starting material and reagents were employed: compound 14b (160 mg,

0.378 mmol), POCl₃(53 μL, 0.567 mmol), and proton sponge (121 mg,

0.567 mmol) in 1.5 mL of PO(OMe)₃. The reaction mixture was stirred

at 0 °C for 3 h and worked up as described above, including silica gel

column chromatography (2-propanol/NH₄OH/H₂O = 74:17.5:3.5f

70:20:5) and RP-HPLC (4 mM aq TEAB/CH₃CN = 90:10, isocratic,

16 mL/min), yielding title compound 4b (56 mg, 27%) as a white solid;

mp >186 °C (dec). ¹H NMR (500 MHz, D₂O) δ 7.81 (s, 1H, H-6), 6.13

(d, 1H, J = 4.7 Hz, H-1⁰), 4.30 (dd, 1H, J = 4.6, 3.6 Hz, H-2⁰), 4.17 (dd,

1H, J = 4.7, 3.6 Hz, H-3⁰), 4.06ꢀ3.95 (m, 3H, H-4⁰and H-5⁰), 3.14 (t,

2H, J = 6.9 Hz, CH₂N), 2.40 (t, 2H, J = 7.1 Hz, R-CH₂), 2.25 (t, 2H, J =

7.3 Hz, CH₂CO), 1.73ꢀ1.66 (m, 2H, CH₂), 1.58ꢀ1.51 (m, 2H, CH₂),

1.46ꢀ1.39 (m, 2H, CH₂), 1.31ꢀ1.23 (m, 2H, CH₂), 0.85 (t, 3H, J = 7.3

Hz, CH₃). ¹³C NMR (125 MHz, D₂O) δ 176.29, 174.03, 157.28, 143.57,

98.72, 93.66, 85.35, 81.81 (d, 1C, J_CP= 7.8 Hz), 75.79, 74.86, 73.46,

CH₂CO), 1.62 (m, 2H, CH₂CH₂CO), 0.92 (t, 3H, J = 7.4 Hz, CH₃). ¹³

C

NMR (125 MHz, D₂O) δ 177.04, 160.24, 151.57, 144.77, 99.08, 89.73,

86.04 (d, 1C, J_CP= 8.2 Hz), 85.65, 73.84, 70.97, 63.70 (d, 1C, J_CP= 4.5

Hz), 38.50, 37.50, 29.55, 18.84, 12.70. ³¹P NMR (121 MHz, D₂O) δ

4.01. HRMS (ESI) calcd for C₁₆H₂₂N₃O₉P 430.1021 (M ꢀ H^þ); found

430.1017.

2-Phenyl-N-(3-(5-(2⁰-deoxyuridine-5⁰-monophosphate))-

prop-2-ynyl)acetamide (5d). Compound 23 (110 mg, 0.25 mmol)

and 2-cyanoethyl phosphate (pyridinium salt, 0.1 M stock solution in

H₂O/pyridine; 5 mL, 0.5 mmol) were evaporated and coevaporated

with dry pyridine (2 ꢁ 2 mL) and then dissolved in dry pyridine (3 mL)

under an Ar atmosphere. DCC (309 mg, 1.5 mmol) was then added, and

the reaction mixture was stirred at rt for 2 1/2 days, after which time the

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Journal of Medicinal Chemistry

ARTICLE

solvent was evaporated. The residue was taken up 1 M NaOH (2 mL)

and stirred at 100 °C for 5 min. The mixture was allowed to cool down to

rt, and the pH was adjusted to ≈8ꢀ9 (dropwise addition of AcOH) and

the volatiles were removed in vacuo. The residue was subjected to a

column of silica gel (2-PrOH/NH₄OH/H₂O = 90:5:5f80:10:10) and

to RP-HPLC (C-18, 10 mM aq TEAB/CH₃CN = 90:10, isocratic,

16 mL/min). Appropriate fractions were pooled and lyophilized, and the

obtained product was converted into its disodium salt by passage

through a column of Dowex 50-WX8 (Na^þ-form) and elution with

H₂O. After evaporation of the solvent and lyophilization, compound 5d

compound 6b (26 mg, 9%) was obtained as a white solid; mp >169 °C

(dec). ¹H NMR (500 MHz, D₂O) δ 8.19 (s, 1H, H-6), 6.29 (t, 1H, J =

6.8 Hz, H-1⁰), 4.57 (m, 1H, H-3⁰), 4.17 (m, 1H, H-4⁰), 4.09 (d, 2H, J =

1.8 Hz, R-CH₂), 3.97 (m, 2H, H-5⁰), 3.13 (td, 2H, J = 6.8, 1.3 Hz,

CH₂NH), 2.45ꢀ2.36 (m, 2H, H-2⁰), 1.49 (m, 2H, CH₂), 1.34ꢀ1.24 (m,

6H, 3 ꢁ CH₂), 0.85 (t, 3H, J = 7.1 Hz, CH₃). ¹³C NMR (125 MHz,

D₂O) δ 164.60, 160.07, 150.41, 144.52, 98.90, 91.11, 85.76 (d, 1C, J_CP

=

8.8 Hz), 85.28, 72.98, 70.50, 63.33 (d, 1C, J_CP= 3.8 Hz), 39.56, 38.28,

30.45, 30.01, 28.75, 25.26, 21.60, 12.92. ³¹P NMR (121 MHz, D₂O) δ

3.43. HRMS (ESI) calcd for C₁₉H₂₉N₄O₉P 487.1599 (M ꢀ H^þ); found

487.1585.

1

(21 mg, 16%) was obtained as a white solid; mp >174 °C (dec). H

NMR (500 MHz, D₂O) δ 7.98 (s, 1H, H-6), 7.36 (m, 5H, ArH), 6.28 (t,

1H, J = 6.9 Hz, H-1⁰), 4.51 (m, 1H, H-3⁰), 4.16 (s, 2H, R-CH₂), 4.11 (m,

1H, H-4⁰), 3.92 (m, 2H, H-5⁰), 3.64 (s, 2H, CH₂Ph), 2.34 (m, 2H, H-2⁰).

¹³C NMR (125 MHz, D₂O) δ 174.21, 172.60, 156.60, 143.64, 134.47,

128.92 (2C), 128.61 (2C), 126.99, 98.74, 88.01, 85.33 (d, 1C, J_CP= 8.2

Hz), 85.13, 75.78, 70.77, 63.44 (d, 1C, J_CP= 4.5 Hz), 41.91, 38.25, 29.63.

³¹P NMR (121 MHz, D₂O) δ 3.99. HRMS (ESI) calcd for

C₂₀H₂₂N₃O₉P 478.1021 (M ꢀ H^þ); found 478.1020.

Cloning, Expression, and Purification of Mycobacterial

ThyX. This was done according to protocols described.²¹

ThyX-Inhibition Assay. The tritium-release assay was adapted

from Hunter et al.⁵⁶Briefly, the reaction was performed in a final volume

of 25 μL, in a 96-well flat bottom plate, including 1.5 μg of M.

tuberculosis ThyX, 20 μM homemade mTHF, 10 μM FAD and

500 μM NADPH, and compounds if required. Reaction was initiated by

addition of 10 μL of 2 μM [5-³H] dUMP (13 Ci/mmol). After a 10 min

reaction period, at room temperature, the reaction was terminated by the

addition of 20 μL of a stop solution (a 3:1 ratio of 2 N TCA:4.3 mM

dUMP). To remove unreacted substrates, 150 μL of 10% (w/v)

activated charcoal in water was added to the reaction mixture. The plate

was incubated on ice for 15 min and then centrifuged at 3800g at 4 °C for

10 min. A 100 μL aliquot of the supernatant was assayed by liquid

scintillation counting to determine the amount of tritium-containing

water produced by the reaction. Percentages of activity compared to

control points were calculated and IC₅₀values determined.

Cloning, Expression, and Purification of Mycobacterial

ThyA. Cloning. PCR reactions were performed using a BioRad MJ

Mini Personal Thermal Cycler. Primers were purchased from Euro-

gentech: 5⁰-CTTGACCATATGGGCCATCATCATCATCATCACG-

GCACGCC ATACGAGGACCTGCTG, 5⁰-CTAGTCGGATCCT-

CATACCGCGACTGGAGC cDNA containing ThyA gene was used

as a template. PCR reactions were carried out using Ex Taq polymerase

and standard reaction conditions (Ta 55 °C). Reactions were purified

using QIAquick PCR purification kit (Qiagen). NdeI and BamHI

restrictions enzymes (BioLabs) were used to create sticky ends on the

ThyA PCR-fragment, and manufacturer’s standard reaction conditions

were used. Pet3a vector was also cut with NdeI and BamHI restriction

enzymes and treated with alkaline phosphatase. Afterward, pet3a vector

was purified from 0.8% agarose gel using QIAEXII Gel extraction kit

(Qiagen). Ligation reaction (T4 DNA ligase, BioLabs) was performed

according to manufacturer’s instructions. TG1 E. coli RbCl chemical

competent cells were transformed with the ligation mixture, grown

during 1 h at 37 °C and 250 rpm in 900 μL LB-medium and then plated

on the LB agar plates and grown overnight at 37 °C. Several colonies

were picked up for colony PCR and also overnight cultures in LB

medium were set up. Pet3a plasmid of a positive colony was purified

from an overnight culture using QIAprep Spin Miniprep kit (Qiagen),

and the concentration was measured using NANODROP 1000.

Expression. BL21 (DE3) pLysS E. coli RbCl chemical competent cells

were transformed with pet3a vector containing the ThyA insert, grown

during 1 h at 37 °C and 250 rpm in 900 μL LB-medium, then plated on

the LB-agar plates and grown overnight at 37 °C. Afterward, a colony

was picked up into an overnight culture. Bacteria from the overnight

N-(3-(5-(2⁰-Deoxyuridine-5⁰-monophosphate))prop-2-ynyl)-

heptane-1-sulfonamide (6a). Compound 24 (121 mg, 0.25 mmol),

HOBt (38 mg, 0.25 mmol), and 2-cyanoethyl phosphate (pyridinium salt,

0.1 M stock solution in H₂O/pyridine; 6.25 mL, 0.625 mmol) were

evaporated and coevaporated with dry pyridine (3 ꢁ 2 mL), then dissolved

in dry pyridine (3 mL) under an Ar-atmosphere. DCC (309 mg, 1.5 mmol)

was then added, and the reaction mixture was stirred at rt for 2 days, after

which time the solvent was evaporated. The residue was taken up 1 M

NaOH (2 mL) and stirred at 100 °C for 5 min. The mixture was allowed to

cool down to rt and the pH was adjusted to ≈8ꢀ9 (dropwise addition of

AcOH) and the volatiles were removed in vacuo. The residue was subjected

to a column of silica gel (2-PrOH/NH₄OH/H₂O = 90:5:5f75:15:10)

and to RP-HPLC (C-18, 10 mM aq TEAB/CH₃CN = 80:20, isocratic,

16 mL/min). Appropriate fractions were pooled and lyophilized, and

the obtained product was converted into its disodium salt by passage

through a column of Dowex 50-WX8 (Na^þ-form) and elution with

H₂O. After evaporation of the solvent and lyophilization, compound

6a (15 mg, 11%) was obtained as a white solid; mp >175 °C (dec). ¹H

NMR (500 MHz, D₂O) δ 8.19 (s, 1H, H-6), 6.21 (t, 1H, J = 6.8 Hz,

H-1⁰), 4.50 (m, 1H, H-3⁰), 4.11 (m, 1H, H-4⁰), 4.08 (s, 2H, R-CH₂),

3.93 (m, 2H, H-5⁰), 3.28 (m, 2H, CH₂SO₂), 2.39ꢀ2.27 (m, 2H,

H-2⁰), 1.72 (m, 2H, CH₂), 1.28 (m, 2H, CH₂), 1.19ꢀ1.09 (m, 6H,

3 ꢁ CH₂), 0.74 (t, 3H, J = 6.9 Hz, CH₃). ¹³C NMR (125 MHz, D₂O)

δ 164.14, 150.14, 144.77, 98.51, 89.36, 85.70 (d, 1C, J_CP= 7.5 Hz),

85.36, 74.63, 70.41, 63.41, 51.66, 38.43, 32.16, 30.56, 27.62,

27.06, 22.52, 21.52, 12.86. ³¹P NMR (121 MHz, D₂O) δ 2.72.

HRMS (ESI) calcd for C₁₉H₃₀N₃O₁₀PS 522.1317 (M ꢀ H^þ); found

522.1310.

1-Hexyl-(3-(5-(2⁰-deoxyuridine-5⁰-monophosphate))prop-

2-ynyl)urea (6b). Crude 25 (228 mg, 0.51 mmol), HOBt (77 mg, 0.51

mmol), and 2-cyanoethyl phosphate (pyridinium salt, 0.1 M stock

solution in H₂O/pyridine; 15 mL, 1.5 mmol) were evaporated and

coevaporated with dry pyridine (3 ꢁ 5 mL) and then dissolved in dry

pyridine (6 mL) under an Ar atmosphere. DCC (625 mg, 3.04 mmol)

was then added, and the reaction mixture was stirred at 40ꢀ45 °C for

2 days, after which time the solvent was evaporated. The residue was taken

up 1 M NaOH (7 mL) and stirred at 100 °C for 5 min. The mixture was

allowed to cool down to rt and the pH was adjusted to ≈8ꢀ9 (dropwise

addition of AcOH) and the volatiles were removed in vacuo. The residue

was subjected to a column of silica gel (2-PrOH/NH₄OH/H₂O =

90:5:5f75:15:10) and to RP-HPLC (C-18, 10 mM aq TEAB/CH₃CN =

85:15, isocratic, 16 mL/min). Appropriate fractions were pooled and

lyophilized, and the obtained product was converted into its disodium

salt by passage through a column of Dowex 50-WX8 (Na^þ-form) and

elution with H₂O. After evaporation of the solvent and lyophilization,

culture were grown in 1 L LB medium at 37 °C and 250 rpm until OD₆₀₀

=

0.6ꢀ0.8 and then induced with 200 μL 1 M IPTG solution and grown

overnight at 22 °C and 250 rpm. Bacteria were spun down for 10 min at

10000 rpm and 4 °C. The obtained bacterial pellet was stored at ꢀ20 °C.

Purification. The bacterial pellet was resuspended in 10 mL of lysis

buffer containing 5 mg of lysozyme, 1 mM DTT, and protease inhibitor

cocktail. After five times, French Press lysate was spun down for 20 min.

ThyA protein was purified from supernatants using His6-based affinity

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Journal of Medicinal Chemistry

ARTICLE

chromatography (NTA magnetic beads, Qiagen) according to the

manufacturer’s instructions.

TS, thymidylate synthase; WHO, World Health Organization;

XDR, extensively drug resistant

ThyA-Inhibition Assay. ThyA activity was adapted from Kan

et al.⁵⁸In brief, the reaction mixture (100 μL) contained 1 μg of ThyA

enzyme, 50 mM Tris-HCl (pH 7.5), 500 μM homemade mTHF, 10 mM

MgCl₂, and compound if required. Reactions, performed at room

temperature in a 96-well flat bottom plate, were initiated by addition

of 20 μL of 2 mM dUMP. The increase in absorbance at 340 nm was

measured directly after dUMP addition and after 30 min, using a

spectrophotometer (envision, Perkin elmer). Percentages of activity

compared to control points (no enzyme, no inhibitor) were calculated

and IC₅₀values determined.

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’ ASSOCIATED CONTENT

S

Supporting Information. Experimental procedures for

b

the synthesis of compounds 10aꢀb, 11aꢀb, 15aꢀd, 21ꢀ22,

and purity of synthesized derivatives 1ꢀ2, 3aꢀb, 4aꢀb, 5aꢀd,

and 6aꢀb. This material is available free of charge via the Internet

at http://pubs.acs.org.

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’ AUTHOR INFORMATION

Corresponding Author

*Phone: þ32 16 337387. Fax: þ32 16 337340. E-mail: Piet.

Herdewijn@rega.kuleuven.be.

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’ ACKNOWLEDGMENT

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We are grateful to Prof. Roger Busson for his kind assistance

and his expertise regarding the interpretation of complex product

mixtures by NMR spectroscopy and Luc Baudemprez for run-

ning 500 and 600 MHz NMR spectra. This work was partly

funded by a collaborative project grant (NATT-project) from the

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nologie) for providing a Ph.D. scholarship.

’ ABBREVIATIONS USED

AcOH, acetic acid; Ac₂O, acetic anhydride; CAN, cerium(IV)-

ammonium nitrate; CH₂THF, methylenetetrahydrofolic acid; DCC,

dicyclohexylcarbodiimide; DCM, dichloromethane; DHF, dihy-

drofolate; DHFR, dhydrofolate reductase; DIAD, diisopropyl

azodicarboxylate; DIC, diisopropycarbodiimide; DIPEA, diiso-

propylethylamine; DMAP, 4-dimethylaminopyridine; DMF, di-

methylformamide; DMSO, dimethylsulfoxide; DMTrCl, 4,4⁰-

dimethoxytrityl chloride; dTMP, 2⁰-deoxythymidine-5⁰-mono-

phosphate; dUMP, 2⁰-deoxyuridine-5⁰-monophosphate; EDTA,

ethylenediamine tetraacetic acid; EtOAc, ethyl acetate; EtOH,

ethanol; FAD, ﬂavin adenine dinucleotide; FDTS, ﬂavin-depen-

dent thymidylate synthase; HIV, human immunodeﬁciency

virus; HOBt, hydroxybenzotriazole; IC₅₀, half maximal (50%)

inhibitory concentration; MDR-TB, multi drug-resistant TB; MeOH,

methanol; MIC₉₀, minimum inhibitory concentration 90%; NADPH,

nicotinamide adenine nucleotide phosphate hydride; NEt₃, triethyla-

mine; RP-HPLC, reversed-phase high performance liquid chro-

matography; rt, room temperature; SAR, structureꢀactivity

relationship; SHMT, serine hydroxymethyl transferase; TB, tuber-

culosis; TCA, trichloroacetic acid; TEAB, triethylammonium bicar-

bonate; THF, tetrahydrofuran; ThyA, thymidylate synthase; ThyX,

ﬂavin-dependent thymidylate synthase; TMSI, trimethylsilyl iodide;

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Synthesis and evaluation of 5-substituted 2′-deoxyuridine monophosphate analogues as inhibitors of flavin-dependent thymidylate synthase in mycobacterium tuberculosis

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