5-Substituted (1-Thiolan-2-yl)cytosines as inhibitors of A. aeolicus and E. coli ispe kinases: Very different affinities to similar substrate-binding sites

Article abstract of DOI:10.1002/ejoc.201201454

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are potential new targets for the development of selective drugs for the treatment of important infectious diseases. This pathway is used by major human pathogens, such as Plasmodium falciparum and Mycobacterium tuberculosis, but not by humans. The fourth enzyme in the pathway is the kinase IspE, and we report here the development and biological evaluation of new ligands for this enzyme from Escherichia coli and Aquifex aeolicus species as model systems for the pathogenic enzymes. The study focuses on analysis of the methylerythritol pocket of the 4-diphosphocytidyl-2-C-methyl-D-erythritol binding site. A series of 5-substituted 1-(thiolan-2-yl)cytosines with increasingly polar substituents were synthesized, opting for possible water-replacements in that sub-pocket as well as a high water-solubility of the ligands. In vitro studies showed IC ₅₀ values in the micromolar range against E. coli IspE, but, unexpectedly, no inhibition against A. aeolicus IspE within the measurement range of the biological tests. Copyright

Full text of DOI:10.1002/ejoc.201201454

FULL PAPER
DOI: 10.1002/ejoc.201201454
5-Substituted (1-Thiolan-2-yl)cytosines as Inhibitors of A. aeolicus and E. coli
IspE Kinases: Very Different Affinities to Similar Substrate-Binding Sites
Andri P. Schütz,[a] Sebastian Locher,[a] Bruno Bernet,[a] Boris Illarionov,[b]
Markus Fischer,*[b] Adelbert Bacher,*[b] and François Diederich*[a]
Keywords: Medicinal chemistry / Drug design / Antimicrobial agents / Structure–activity relationships / Inhibitors /
Molecular modeling
The enzymes of the non-mevalonate pathway for isoprenoid
biosynthesis are potential new targets for the development
of selective drugs for the treatment of important infectious
diseases. This pathway is used by major human pathogens,
such as Plasmodium falciparum and Mycobacterium tubercu-
losis, but not by humans. The fourth enzyme in the pathway
is the kinase IspE, and we report here the development and
biological evaluation of new ligands for this enzyme from Es-
cherichia coli and Aquifex aeolicus species as model systems
for the pathogenic enzymes. The study focuses on analysis
of the methylerythritol pocket of the 4-diphosphocytidyl-2-
C-methyl- -erythritol binding site. A series of 5-substituted
D
1-(thiolan-2-yl)cytosines with increasingly polar substituents
were synthesized, opting for possible water-replacements in
that sub-pocket as well as a high water-solubility of the li-
gands. In vitro studies showed IC50 values in the micromolar
range against E. coli IspE, but, unexpectedly, no inhibition
against A. aeolicus IspE within the measurement range of
the biological tests.
In the absence of cocrystal structures of P. falciparum
and, until recently, M. tuberculosis IspE, E. coli and Aquifex
aeolicus IspE were chosen as surrogates for modeling stud-
ies. The cocrystal structure of E. coli IspE with CDP-ME
is depicted in Figure 1, left. A comparison of the amino
acids lining the active sites of the four enzymes under study
suggests a slightly better homology of the A. aeolicus en-
zyme to the two enzymes of the pathogen organisms (see
Figure S1 in the Supporting Information). The main differ-
ence is the change from Phe185 (E. coli) to Tyr (A. aeolicus,
P. falciparum, M. tuberculosis). After completion of this
work, structural data on M. tuberculosis IspE in complex
with CDP-ME was published (Figure S2),[6g] the rather
conserved CDP-ME binding domain of which shows high
structural homology to the CDP-ME sites of E. coli and A.
aeolicus IspE.
In the crystal structure of A. aeolicus IspE in complex
with CDP (PDB code 2V8P),[6f] the presumed ME pocket
is filled by a distinct localized water cluster (Figure 1, right),
which connects to the phenolic hydroxyl group of Tyr175
by a hydrogen bond with an O…O distance of 2.9 Å. This
water cluster is largely replaced by the ME moiety in the
cocrystal structures of CDP-ME in complex with E. coli
(Figure 1, left; PDB code 1OJ4)[6a] and M. tuberculosis
(Figure S2; PDB code 3PYE).[6g] We envisioned that polar
substituents directed by synthetic ligands into this water
cluster in the A. aeolicus enzyme could replace several water
molecules in an energetically favorable way, while at the
same time favorable interactions with the surrounding polar
amino acid side chains of Tyr175, Asp130, Lys9, and Asn11
Introduction
Half of the world’s human population lives in malaria-
endangered regions, causing each year 300–500 million in-
fections and more than one million deaths by the causative
agent Plasmodium falciparum.[1] Despite the use of different
drug combinations[2] in the treatment of malaria, an in-
creasing number of multi-drug-resistant Plasmodium strains
have been reported,[1a,2a,3] clearly showing the urgency for
the development of new antimalarial drugs.[4]
In our preceding paper, which included a detailed intro-
ductory section,[5] we reported the design, synthesis, and
biological evaluation of potential inhibitors against Escher-
ichia coli IspE [4-diphosphocytidyl-2-C-methyl-d-erythritol
(CDP-ME) kinase, EC 2.7.1.148]. IspE is the fourth enzyme
in the non-mevalonate pathway of isoprenoid biosynthesis
used by the parasites P. falciparum and Mycobacterium tu-
berculosis, but not by humans.[6] The earlier work focused
on analysis of the ribose sub-pocket of the CDP-ME bind-
ing site, whereas the present investigation addresses the oc-
cupancy of the methylerythritol (ME) pocket.
[a] Laboratorium für Organische Chemie, ETH Zürich,
Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland
Fax: +41-44-6321109
E-mail: diederich@org.chem.ethz.ch
[b] Hamburg School of Food Science, Institut für
Lebensmittelchemie, Universität Hamburg,
Grindelallee 117, 20146 Hamburg, Germany
Fax: +49-40-428384342
E-mail: markus.fischer@hsfs.uni-hamburg.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201454.
880
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Inhibitors of A. aeolicus and E. coli IspE Kinases
Figure 1. Cocrystal structures of CDP-ME in the substrate pocket of E. coli IspE (PDB code: 1OJ4, 2.01 Å resolution; left) and of CDP
in the active site of A. aeolicus IspE (PDB code: 2V8P, 2.10 Å resolution, right). The unoccupied methylerithritol pocket in A. aeolicus
IspE shows a distinct localized water cluster. Color code: carbon (olive), nitrogen (blue), oxygen (red), sulfur (yellow). Heteroatom
distances are given in Å.
(numbering from Figure 1, right) could be established. ing the propynyl linker, were also developed as two exam-
Gains in binding free enthalpy by replacements of X-ray- ples of ligands with a short distance between the cytosine
localized water molecules by ligand parts in the enzyme
active sites have been demonstrated in other studies from
our group.[7] Interestingly, the ME residue adopts a dif-
ferent orientation in the cocrystal structures of E. coli and
M. tuberculosis IspE with CDP-ME. The different ME con-
formation seen in the latter structure enables formation of
a hydrogen bond to the hydroxyl group of Tyr185 (O···O
distance: 2.8 Å; Figure S2). A similar hydrogen bond is ob-
served between Tyr175 and a water molecule from the water
cluster in the complex of A. aeolicus with CDP (Figure 1,
right), corroborating the necessity for solvation of the lining
polar amino acid side chains by the designed ligands.
Herein, we report the design, synthesis, and biological
activity of 5-substituted, 1-thiolanylated cytosines. A first
series of ligands was patterned after the earlier reported
template with a 3-(cyclopropylsulfonamido)prop-1-yn-1-yl
substituent in the 5-position of the 1-thiolanylated cytosine,
which had been found to be the strongest inhibitor of E.
coli IspE (median inhibitory concentration IC50 = 1.7 Ϯ
0.2 μm, Ki = 0.29 Ϯ 0.10 μm).[6c,6d] We exchanged the ter-
minal cyclopropyl moiety by more polar substituents, tar-
geting the interaction with the hydroxyl group of Tyr175 in
A. aeolicus: i.e., vinylsulfonamide (Ϯ)-1, methyl ether (Ϯ)-
2, oxetane (Ϯ)-3, and furans 4 (see Schemes 1 and 2). Phen-
yl- and pyridinylsulfones (Ϯ)-5 and (Ϯ)-6 (Scheme 3), lack- [Pd(PPh3)4], CuI, DMF, 22 °C; 76% of (Ϯ)-1, 88% of (Ϯ)-2.
Scheme 1. Reagents and conditions: (a) propargylamine, NEt3,
CH2Cl2, 0 °C; 92%; (b) NaOMe, MeOH, 22 °C; 86%. (c) (Ϯ)-14,
Scheme 2. Reagents and conditions: (a) thioacetic acid, PPh3, diisopropyl azodicarboxylate (DIAD), THF, 22 °C; 20% of 17, 26% of (Ϯ)-
18. (b) NCS, aq. HCl, MeCN, 10 °C; propargylamine, NEt3, CH2Cl2, 0 °C; 20% of 19, 56% of (Ϯ)-20. (c) (Ϯ)-14, [Pd(PPh3)4], CuI,
DMF, 22 °C; 60% of (Ϯ)-3, 80% of 4.
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881

M. Fischer, A. Bacher, F. Diederich et al.
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residue and an aromatic substituent. Finally, the ribosyl
moiety in cytosines 7 and 9 and the mannosyl moiety in
analogues 8 and 10 (Scheme 4) were introduced to replace
large parts of the water cluster seen in the ME pocket of A.
aeolicus. All these ligands bind to the cytidine pocket and
Results and Discussion
Design
Molecular modeling was performed with the modeling
the ME/phosphate binding site, while leaving the ATP bind- software MOLOC[8] using the cocrystal structure of E. coli
ing site of the kinase open for its natural substrate. Bio- IspE with bound CDP-ME and the ATP analogue ADPNP
logical studies with these ligands were expected to enhance (adenosine-5Ј-(γ-imino)triphosphate; 2.0 Å resolution; PDB
the understanding of the molecular recognition properties code 1OJ4),[6a] and the cocrystal structure of A. aeolicus
of the different IspE enzymes.
IspE with bound CDP (cytosine-5Ј-diphosphate), ADP
(adenosine-5Ј-diphosphate), and a well-defined water clus-
ter in the CDP-ME pocket (2.1 Å resolution; PDB code
2V8P). The proposed ligand binding modes are supported
by the cocrystal structures of A. aeolicus IspE (PDB codes
2V2Q, 2.4 Å resolution and 2V2V, 2.3 Å resolution) and E.
coli IspE (PDB codes 2VF3, 2.2 Å resolution) with first-
generation inhibitors.[6d,6e]
For inhibitor design with MOLOC, the cocrystallized li-
gands were replaced by the modified derivatives. The en-
zyme structure was kept rigid during the energy minimi-
zation process. The central cytosine scaffold of our target
inhibitors is sandwiched by Phe185 and Tyr25 (E. coli) or
Tyr175 and partially Tyr24 (A. aeolicus), respectively. The
cytosine motif forms three hydrogen bonds with the peptide
backbone and the side chain of His26(Ec)/His25(Aa).[6d]
The thiolanyl moiety at N(1) occupies the ribose sub-
pocket.
We focused our investigations on the different environ-
ment in the E. coli and A. aeolicus ME pockets formed in
the E. coli enzyme by the amino acids Phe185, Leu28, Ile17,
Leu15, Asn12, Lys10, and Asp141, and in the A. aeolicus
enzyme by Tyr175, Ile27, Leu208, Leu14, Asn11, Lys9, and
Asp130, respectively (Figure 1). An increased polarity of
the A. aeolicus ME pocket results from the exchange of
Phe185 (E. coli) by Tyr175. Because previously synthesized
inhibitors[5,6c,6d] were optimized for binding in the less polar
pocket of E. coli IspE, reaching into a distinctly apolar sub-
pocket not used by CDP-ME, they lack a suitable structural
feature to form a hydrogen bond to Tyr175 of A. aeolicus.
The designed inhibitors (Ϯ)-2–4 (see Schemes 1 and 2) with
terminal ether moieties were expected to show enhanced
binding to A. aeolicus IspE by forming a hydrogen bond to
Tyr175. This is depicted in Figure S3 for oxetane (Ϯ)-3.
The sulfonylated cytosines (Ϯ)-5 and (Ϯ)-6 were ex-
pected to adopt a roof-type geometry, given the conforma-
tional preferences of aryl sulfones.[9] Phenylsulfone (Ϯ)-5
was expected to display a higher affinity for the less polar
pocket of E. coli IspE, whereas pyridylsulfone (Ϯ)-6 should
bind better to A. aeolicus IspE by forming a hydrogen bond
to Tyr175.
Scheme 3. Reagents and conditions: (a) TMSOTf, NEt3, toluene/
CH2Cl2, 22 °C; tetramethylene sulfoxide, CH2Cl2, 22 °C (28%); (b)
chloroacetonitrile, 1 m NaOH, THF, 22 °C; mCPBA, CH2Cl2,
22 °C (43%); (c) (Z)-Methyl-N-cyanoformimidate, NaOMe,
MeOH, 22 °C; conc. HCl, 22 °C (30%); (d) 2 m NaOH, 22 °C;
TMSOTf, NEt3, toluene/CH2Cl2, 22 °C; tetramethylene sulfoxide,
CH2Cl2, 22 °C (10%).
The sugar moieties in 7–10 can be accommodated strain-
free into the ME pocket and were expected to replace a
large number of the water molecules of the water cluster
seen in this pocket in the cocomplex of A. aeolicus IspE
with CDP. Additionally, their hydroxyl groups should en-
gage in hydrogen bonds to the surrounding polar side
chains of Tyr175, Lys9, and Asp130, as shown in the mod-
eled complex of riboside 9 in Figure S4.
Scheme 4. Reagents and conditions: (a) nBuLi, hexane/CH2Cl2,
–75 °C (85%); (b) nBuLi, hexane/CH2Cl2, –90 °C (86%); (c)
TsOH·H2O, MeOH, 65 °C (47%); (d) (Ϯ)-14, [Pd(PPh3)4], CuI,
NEt3, DMF, 22 °C; 75% of 7, 40% of 8. (e) 10% Pd/C, H2, MeOH,
22 °C; 95% of 9, 92% of 10.
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Inhibitors of A. aeolicus and E. coli IspE Kinases
Synthesis
Prof. A. Vasella, ETH Zürich[17]) were used for the Sonoga-
shira reaction with 5-iodocytosine (Ϯ)-14 and provided the
The condensation of commercially available 2-chloro- coupled alkynes 7 and 8 in 75 and 40% yield, respectively.
ethanesulfonyl chloride (11) with propargylamine, followed The lower yield of 8 was due to the more difficult purifica-
by elimination of HCl, gave vinylsulfonamide 12[10] in one tion. Palladium-catalyzed hydrogenation yielded the d-ribo-
step in 92% yield (Scheme 1). Nucleophilic addition of configured hexosides 9 and mannopyranosyl-ethanes 10 in
methoxide to 12 yielded the desired methyl ether 13 (86%). almost quantitative yields. Importantly, these hydrogen-
The target compounds (Ϯ)-1 and (Ϯ)-2 were obtained in ations needed to be stopped after one hour because longer
reasonable yield (76 and 88%) from 12 and 13, respectively, reaction times led also to the reduction of the N,S-acetals.
by applying standard Sonogashira cross-coupling condi- The diastereoisomers of both 9 and 10, caused by the thiol-
tions[6d] for the reactions with 5-iodocytosine (Ϯ)-14.
anyl ring, were not separated. In the 1H and 13C NMR
The syntheses of oxetane (Ϯ)-3 and furans 4 started from spectra, signal doubling especially for 10 at 600 MHz (sig-
commercially available 3-hydroxyoxetane (15) and 3-hy- nal doubling of centers at all three rings) provided evidence
droxyfuran [(Ϯ)-16], respectively. Substitution of 15 and for a 1:1 mixture of diastereoisomers.
(Ϯ)-16 with thioacetic acid under Mitsunobu conditions
The d-ribo-configured hex-5-ynoside 7 and hexoside 9
yielded thioesters 17 and (Ϯ)-18[11] in 20 and 26% yield, adopt a northern conformation, as evidenced by their re-
respectively. Following a procedure by Nishiguchi et al.,[12] spective coupling constants (J1,2/J3,4 Յ 0.2 Hz). The
oxidation of 17 and (Ϯ)-18 to the sulfonyl chlorides with N- mannosyl derivatives 8 and 10 adopt the expected 4C1 chair
chlorosuccinimide under acidic conditions and subsequent conformation as revealed by their small J1,2 and J2,3 cou-
condensation with propargylamine to the propargylsulfon- plings (Ͻ 2 and 3.0–3.6 Hz, respectively) and large J3,4 and
amides 19 and (Ϯ)-20 were performed in one step in yields J4,5 couplings (9.3–9.6 Hz).
of 20 and 56%, respectively. Sonogashira couplings with 5-
iodocytosine (Ϯ)-14 yielded the desired oxetane (Ϯ)-3 and
furans 4 (as a mixture of two diastereoisomeric pairs of
enantiomers) in 60 and 80% yield, respectively.
Biological Results
The IC50 values for ligands (Ϯ)-1 to 10 were determined
by enzyme-coupled photometric assays[18] using A. aeolicus
or E. coli IspE, with pyruvate kinase and lactate dehydroge-
nase as auxiliary enzymes (Table 1); the assay is described
in detail in our previous paper.[5]
5-(Phenylsulfonyl)cytosine 21 was prepared according to
a method developed by Pérez et al. (Scheme 2).[13] Because
this sulfonylated cytosine is extremely insoluble in conve-
nient organic solvents, crude 21 was used for thiolanylation
at N(1), giving (Ϯ)-5 in 28% yield.
The synthesis of the corresponding pyridylsulfonyl deriv-
ative (Ϯ)-6 started with commercially available 2-mercap-
topyridine (22). Nucleophilic substitution with chloroaceto-
nitrile and subsequent oxidation to sulfone 23 proceeded
according to a known procedure[14] in 43% yield. Base-cata-
lyzed Knoevenagel condensation, followed by treatment
with concentrated HCl gave crude 24. Cyclization and thiol-
anylation in situ of an intermediate silylated cytosine spe-
cies gave (Ϯ)-6 in 10% yield.
The synthesis of hex-5-ynofuranoside 27 from d-ribose
has been described by two groups using the Corey–Fuchs
reaction either in dioxane/tetrahydrofuran (THF) 3:7 at
–90 °C (72%)[15] or in THF at –78 °C (86%).[16] In our
hands, treatment of a solution of 25 in dichloromethane
with a slight excess of nBuLi at –75 °C led to elimination
of acetone and the formation of hex-3-en-5-ynoside 26
Table 1. Inhibitory activities of (Ϯ)-1–10 against E. coli IspE and
calculated physicochemical properties.
clogP[a]
IC50 [μm]
Ki (calcd.)[b]
(Ϯ)-1
(Ϯ)-2
(Ϯ)-3
4
(Ϯ)-5
(Ϯ)-6
7
8
9
10
1.7 Ϯ 0.8
1.0 Ϯ 0.8
0.5 Ϯ 0.8
0.9 Ϯ 0.8
2.0 Ϯ 0.5
1.9 Ϯ 0.5
2.02 Ϯ 1.06
2.57 Ϯ 1.06
3.08 Ϯ 1.06
1.47 Ϯ 1.09
3 Ϯ 1
1.1
30 Ϯ 3
34 Ϯ 2
50 Ϯ 3
220 Ϯ 10
Ͼ500
11.2
12.8
18.8
82.5
–
Ն500
–
407 Ϯ 31
411 Ϯ 31
Ն500
152.6
154.2
–
[a] clogP values were calculated using ACD/Labs software. [b] Ki
values were calculated from IC50 values and Km = 150 μm (E. coli
IspE) using the Cheng–Prusoff equation.[20]
Interestingly and equally unexpectedly, the inhibitory po-
(85% yield). Lowering the reaction temperature to –90 °C tency of all ligands against A. aeolicus IspE was found to be
and using substoichiometric amounts of nBuLi gave the de- outside the limits of measurement, with IC50 values above
sired hex-5-ynofuranoside 27 in 86% yield. Optimal condi- 500 μm. Water cluster substitution by ligand parts is a very
tions for the selective deisopropylidenation were found by delicate issue, and rules that govern how to gain binding
using catalytic amounts of 4-toluenesulfonic acid in MeOH, free enthalpy from replacement of individual or several mo-
affording a mixture of 28 and its α-anomer (partial anomer- lecules in a cluster by ligand parts are only slowly emerg-
ization). Flash chromatography on silica gel yielded the ing.[19] The water cluster in the ME pocket of A. aeolicus
pure β-anomer 28 in 47% yield. Other conditions led to IspE interacts with polar amino acid side chains, on the left
either incomplete deisopropylidenation or to concomitant with Tyr175 and on the right with Asn11, Lys9, and
glycoside cleavage to give the unprotected hex-5-ynose.
Asp130, making its replacement energetically highly costly
The d-ribo-configured hex-5-ynofuranoside 28 and (Figure 1, right). In the ME pocket of E. coli IspE (Fig-
mannopyranosylacetylene (29) (generously provided by ure 1, left) in contrast, the left side of the pocket has a pro-
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883

M. Fischer, A. Bacher, F. Diederich et al.
FULL PAPER
reported in this article. Additional information can be found in the
Supporting Information.
nounced apolar character (Tyr175 is replaced by Phe) and
therefore the displacement of water molecules from the
cluster occurs with lower energetic costs.
The data for the inhibition of E. coli IspE are listed in
Table 1. Vinylsulfonamide (Ϯ)-1 was found to be the best
(؎)-N-{3-[1-(Thiolan-2-yl)cytosin-5-yl]prop-2-yn-1-yl}vinylsulfon-
amide [(؎)-1]: A solution of 5-iodocytosine (Ϯ)-14 (100 mg,
0.3 mmol), sulfonamide 12 (73 mg, 0.5 mmol) and Et3N (0.3 mL) in
ligand, coming close to the affinity of the previously re- DMF (5 mL) was degassed by freeze-pump-thaw cycles performed
under Ar, treated with [PdCl2(PPh3)2] (21 mg, 0.03 mmol) and CuI
(11 mg, 0.06 mmol), and stirred at 22 °C for 13 h. Concentration
and chromatography (SiO2; CH2Cl2/MeOH, 98:2) gave (Ϯ)-1
(78 mg, 76%) as an off-white solid, m.p. 197–200 °C. 1H NMR
(300 MHz, CDCl3/CD3OD, 4:1): δ = 1.68–1.85 (m, 1 H, 4ЈЈ-Ha),
1.89–2.10 (m, 2 H, 3ЈЈ-Ha, 4ЈЈ-Hb), 2.15–2.31 (m, 1 H, 3ЈЈ-Hb),
2.81–2.92 (m, 1 H, 5ЈЈ-Ha), 3.02–3.16 (m, 1 H, 5ЈЈ-Hb), 3.90 (br. s,
2 H, CH2N), 5.87 (d, J = 10.0 Hz, 1 H, 2-Htrans), 6.13 (br. s, 1 H,
2ЈЈ-H), 6.15 (d, J = 16.5 Hz, 1 H, 2-Hcis), 6.48 (dd, J = 16.5,
10.0 Hz, 1 H, 1-H), 8.04 (s, 1 H, 6Ј-H) ppm. 13C NMR (100 MHz,
ported ligand with a cyclopropyl-sulfonamide residue (IC50
= 1.7 Ϯ 0.2 μm and Ki = 0.29 Ϯ 0.1 μm).[5,6d] The vinyl
group presumably binds into the hydrophobic sub-pocket at
the ME binding site, as previously suggested for the initial
cyclopropylsulfonamide lead.[6c,6d] With increasing polarity
of the sulfonamide substituent, binding to the apolar ME
sub-pocket becomes less favorable and the IC50 values for
ethers (Ϯ)-2 to 4 increase by an order of magnitude.
The 5-sulfonylated cytosines (Ϯ)-5 and (Ϯ)-6 are weak
binders, with only phenylsulfone (Ϯ)-5 showing some mod- [D6]acetone): δ = 28.92 (4ЈЈ-C), 32.79 and 32.80 (NCH2, 5ЈЈ-C),
36.85 (3ЈЈ-C), 64.32 (2ЈЈ-C), 75.49 (5Ј-CϵC), 89.23 (5Ј-C), 90.95
(5Ј-CϵC), 126.03 (CH=CH2), 136.64 (CH=CH2), 145.23 (6Ј-C),
erate activity against E. coli IspE. The sugar containing
compounds 7–11 are weak inhibitors of E. coli IspE, and
show activities close to the upper limit of measurement of
the biological assay.
153.59 (2Ј-C), 163.86 (4Ј-C) ppm. IR (ATR): ν = 3285 (w), 3107
˜
(w), 2923 (w), 2864 (w), 2229 (w), 2126 (w), 1641 (s), 1613 (m),
1426 (s), 1387 (m), 1320 (s), 1255 (m), 1141 (s), 1068 (s), 1020 (m),
971 (m), 926 (m) cm–1. HRMS (EI): m/z (%) = 340.0664 (14)
[M]+. HRMS (EI): calcd. for C13H20N4O3S2+ [M]+ 340.0658; found
340.0664.
Conclusions
A series of inhibitors of the kinase IspE was designed
against A. aeolicus IspE kinase. The compounds feature 1-
(thiolan-2-yl)cytosines to bind to the cytidine pocket at the
active side and bear different substituents (propynyl-an-
chored sulfonamides, arylsulfones, and alkynylated sugars)
at C(5) of the cytosine ring to reach and fill the methyl-
erythritol pocket. In a cocrystal structure of A. aeolicus
with CDP, this pocket is filled with a cluster of localized
water molecules that interact by hydrogen bonding with the
surrounding polar amino acid side chains, in particular
Tyr175. However, enzyme assays showed that the com-
pounds had no activity against A. aeolicus IspE within the
limits of the assay (IC50 Ͼ 500 μm), which suggests a high
energetic penalty for substitution of water molecules in this
cluster by ligand parts. The study thus provides one more
indication of the complexity of water replacement at en-
zyme active sites and illustrates how difficult it is to perform
such replacements with energetic gains. Although not de-
signed against E. coli IspE, a series of ligands bearing ether
functionality showed affinity in the lower micromolar IC50
range against this enzyme; the sugar derivatives were either
only poorly active or inactive. At this stage, E. coli IspE
appears to be a more tractable enzyme than A. aeolicus
IspE for the structure-based development of ligands that
should ultimately inhibit the corresponding enzyme of the
P. falciparum and M. tuberculosis pathogens. Ultimately,
more structural information on the parasite enzymes will
be required for a successful application of structure-based
ligand design.
(؎)-N-{3-[1-(Thiolan-2-yl)cytosin-5-yl]prop-2-yn-1-yl}-2-methoxy-
ethanesulfonamide [(؎)-2]: A solution of 5-iodocytosine (Ϯ)-14
(100 mg, 0.3 mmol), sulfonamide 13 (80 mg, 0.45 mmol), and Et3N
(0.25 mL) in DMF (8 mL) was degassed by freeze-pump-thaw cy-
cles performed under Ar, treated with [PdCl2(PPh3)2] (21 mg,
0.03 mmol) and CuI (11 mg, 0.06 mmol), and stirred at 22 °C for
14 h. Concentration and chromatography (SiO2; CH2Cl2/MeOH,
97:3) gave (Ϯ)-2 (99 mg, 88%) as a light-brown solid. Rf = 0.49
(SiO2; CH2Cl2/MeOHЈ, 97:3), m.p. 187–191 °C. 1H NMR
(500 MHz, CDCl3/CD3OD, 4:1; assignment based on a HSQC
spectrum): δ = 1.70–1.75 (m, 1 H, 4Ј-Ha), 1.85–1.95 (m, 1 H, 3Ј-
Ha), 1.95–2.03 (m, 1 H, 4Ј-Hb), 2.12–2.20 (m, 1 H, 3Ј-Hb), 2.75–
2.85 (m, 1 H, 5Ј-Ha), 3.00–3.10 (m, 1 H, 5Ј-Hb), 3.21 (s, 3 H, OMe),
3.24 (t, J = 5.9 Hz, 2 H, CH2SO2), 3.65 (t, J = 5.9 Hz, 2 H, CH2O),
3.97 (s, 2 H, CH2N), 6.08 (dd, J = 6.3, 3.7 Hz, 1 H, 2Ј-H), 8.01 (s,
1 H, 6-H) ppm. 13C NMR (125 MHz, CDCl3/CD3OD 4:1; assign-
ment based on a HSQC spectrum): δ = 28.11 (4Ј-C), 32.89 (NCH2),
33.16 (5Ј-C), 38.45 (3Ј-C), 52.42 (CH2SO2), 58.62 (OMe), 65.83 (2Ј-
C), 66.34 (CH2O), 74.93 (5-CϵC), 90.35 (5-C), 91.15 (5-CϵC),
145.23 (6-C), 155.16 (2-C), 164.37 (4-C) ppm. IR (ATR): ν = 3379
˜
(w), 3100 (w), 2948 (w), 2883 (w), 2229 (w), 1641 (s), 1620 (s), 1542
(m), 1518 (m), 1491 (s), 1446 (m), 1403 (m), 1328 (s), 1294 (s), 1252
(m), 1233 (m), 1199 (m), 1150 (s), 1103 (m), 1047 (m), 1019 (m),
989 (m), 953 (m), 908 (m), 853 (m), 820 (m) cm–1. HRMS (ESI):
m/z (%) = 395.0820 (100) [M + Na]+. HRMS (ESI): calcd. for
C14H20NaN4O4S2 [M + Na]+ 395.0818; found 395.0820.
+
(؎)-N-{3-[1-(Thiolan-2-yl)cytosin-5-yl]prop-2-yn-1-yl}oxetane-3-
sulfonamide [(؎)-3]: A solution of 5-iodocytosine (Ϯ)-14 (60 mg,
0.19 mmol), sulfonamide 19 (40 mg, 0.23 mmol), and Et3N
(0.15 mL) in DMF (4 mL) was degassed by freeze-pump-thaw cy-
cles performed under Ar, treated with [PdCl2(PPh3)2] (21 mg,
0.03 mmol) and CuI (11 mg, 0.06 mmol), and stirred at 22 °C for
14 h. Concentration and chromatography (SiO2; CH2Cl2/MeOH,
97:3) gave (Ϯ)-3 (42 mg, 60 %) as an off-white solid. Rf = 0.45
(SiO2; CH2Cl2/MeOH, 97:3), m.p. 195–198 °C (decomp.). 1H NMR
(300 MHz, CDCl3/CD3OD 5:1; assignment based on a HSQC
spectrum): δ = 1.67–1.82 (m, 1 H, 4ЈЈ-Ha), 1.88–2.08 (m, 2 H, 3ЈЈ-
Experimental Section
Materials and General Methods:[5] Only the final step in the synthe-
sis and the characterization of the target ligands (Ϯ)-1 to 10 is
884
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 880–887

Inhibitors of A. aeolicus and E. coli IspE Kinases
Ha, 4ЈЈ-Hb), 2.13–2.25 (m, 1 H, 3ЈЈ-Hb), 2.78–2.90 (m, 1 H, 5ЈЈ-Ha),
3.02–3.13 (m, 1 H, 5ЈЈ-Hb), 4.01 (s, 2 H, NCH2), 4.42 (quint., J =
7.0 Hz, 1 H, 3-H), 4.77 (d, J = 7.1 Hz, 4 H, 2-H, 4-H), 6.11 (dd, J
= 6.2, 3.6 Hz, 1 H, 2ЈЈ-H), 8.06 (s, 1 H, 6Ј-H) ppm. 13C NMR
C), 38.77 (3Ј-C), 67.14 (2Ј-C), 106.49 (5-C), 126.66 (2ЈЈ,6ЈЈ-C),
129.64 (3ЈЈ,5ЈЈ-C), 133.92 (4ЈЈ-C), 140.74 (1ЈЈ-C), 148.95 (6-C),
154.73 (2-C), 159.89 (4-C) ppm. IR (ATR): ν = 3445 (w), 2949 (w),
˜
1662 (s), 1643 (s), 1481 (s), 1445 (m), 1394 (w), 1282 (m), 1159 (s),
(100 MHz, CDCl3/CD3OD, 5:1; assignment based on a HSQC 1136 (s), 1075 (m), 1030 (m), 965 (w), 885 (w), 814 (m), 781 (m),
spectrum): δ = 28.20 (4ЈЈ-C), 33.04 (NCH2), 33.29 (5ЈЈ-C), 38.60 759 (s), 717 (s), 686 (s), 663 (m), 641 (m) cm–1. HRMS (ESI): m/z
(3ЈЈ-C), 55.19 (3-C), 66.06 (2ЈЈ-C), 71.42 (2,4-C), 74.97 (5Ј-CϵC), (%) = 338.0628 (100) [M + H]+. HRMS (ESI): calcd. for
90.15 (5Ј-C), 91.27 (5Ј-CϵC), 145.56 (6Ј-C), 155.26 (2Ј-C), 164.41 C14H16N3O3S2 [M + H]+ 338.0628; found 338.0628.
+
(4Ј-C) ppm. IR (ATR): ν = 3394 (w), 3326 (w), 3050 (w), 2956 (w),
˜
(؎)-5-(Pyridin-2-ylsulfonyl)-1-(thiolan-2-yl)cytosine [(؎)-6]: A solu-
tion of 24 (1.0 g, 3.9 mmol) in 2 m NaOH (132 mL, 52.8 mmol)
was stirred at 22 °C for 5 h and treated dropwise with conc. sulfuric
acid (15 mL) until a white, fluffy precipitate formed. The precipi-
tate was filtered off, dried, and added to a solution of TMSOTf
(2.35 mL, 12.8 mmol) and Et3N (0.44 mL, 3.2 mmol) in toluene
(12 mL). The solution was stirred at 22 °C for 5 min, diluted with
CH2Cl2 (6 mL), stirred at 22 °C for 15 min, treated with a solution
2924 (w), 2853 (w), 2228 (w), 1637 (s), 1602 (m), 1533 (m), 1503
(s), 1403 (m), 1343 (m), 1317 (s), 1266 (m), 1227 (m), 1183 (m),
1137 (s), 1081 (s), 983 (m), 909 (m), 779 (s), 664 (s) cm–1. HRMS
(ESI): m/z (%) = 371.0850 (98) [M + H]+, 285.0656 (100) [M +
+
H – C4H6S]+. HRMS (ESI): calcd. for C14H19N4O4S2 [M + H]+
371.0842; found 371.0850; calcd. for C10H13N4O4S+ [M + H –
C4H6S]+ 285.0652; found 285.0656.
(3R/S,2ЈЈR/S)-N-{3-[1-(Thiolan-2-yl)cytosin-5-yl]prop-2-yn-1-yl}- of tetramethylenesulfoxide (1.23 mL, 6.4 mmol) in CH2Cl2
oxolane-3-sulfonamide (4): A solution of 5-iodocytosine (Ϯ)-14
(80 mg, 0.25 mmol), sulfonamide (Ϯ)-20 (40 mg, 0.21 mmol) and
Et3N (0.17 mL) in DMF (4 mL) was degassed by freeze-pump-thaw
cycles performed under Ar, treated with [PdCl2(PPh3)2] (21 mg,
0.03 mmol) and CuI (11 mg, 0.06 mmol), and stirred at 22 °C for
14 h. Concentration and chromatography (SiO2; CH2Cl2/MeOH,
97:3) gave 4 (65 mg, 80%) as a 1:1 mixture of diastereoisomers as
(7.5 mL), and stirred at 22 °C for 48 h. The mixture was diluted
with ice-water and EtOAc. The organic phase was separated,
washed with satd. aq. NaHCO3 and brine, dried with Na2SO4, and
concentrated. Chromatography (SiO2; CH2Cl2/MeOH, 99:1 Ǟ
95:5) gave (Ϯ)-6 (130 mg, 10%) as a light-orange solid, m.p. 205–
207 °C (decomp.). 1H NMR (300 MHz, CDCl3): δ = 1.75–1.91 (m,
1 H, 4Ј-Ha), 2.13–2.25 (m, 2 H, 3Ј-Ha, 4Ј-Hb), 2.27–2.41 (m, 1 H,
an off-white solid. Rf = 0.41 (SiO2; CH2Cl2/MeOH 97:3), m.p. 197– 3Ј-Hb), 2.99 (td, J = 9.8, 6.1 Hz, 1 H, 5Ј-Ha), 3.29 (ddd, J = 10.9,
200 °C (decomp.). 1H NMR (300 MHz, CDCl3/CD3OD, 5:1; as-
signment based on a HSQC spectrum): δ = 1.70–1.87 (m, 1 H, 4ЈЈ-
Ha), 1.93–2.13 (m, 2 H, 3ЈЈ-Ha, 4ЈЈ-Hb), 2.16–2.32 (m, 3 H, 4-Ha,
7.5, 3.7 Hz, 1 H, 5Ј-Hb), 6.27 (dd, J = 6.1, 2.8 Hz, 1 H, 2Ј-H), 6.36
and 7.23 (2 br. s, 2 H, NH2), 7.55 (ddd, J = 7.6, 4.7, 1.1 Hz, 1 H,
5ЈЈ-H), 7.98 (td, J = 7.8, 1.7 Hz, 1 H, 4ЈЈ-H), 8.10 (dt, J = 7.9,
4,3ЈЈ-Hb), 2.81–2.95 (m, 1 H, 5ЈЈ-Ha), 3.06–3.19 (m, 1 H, 5ЈЈ-Hb), 1.0 Hz, 1 H, 3ЈЈ-H), 8.68 (ddd, J = 4.7, 1.7, 0.9 Hz, 1 H, 6ЈЈ-H),
3.69–3.85 (m, 2 H, 3-H, 5-Ha), 3.87–3.99 (m, 2 H, 2-Ha, 5-Hb), 8.97 (s, 1 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 27.91 (4Ј-
4.02–4.10 (m, 1 H, 2-Hb), 4.05 (s, 2 H, NCH2), 6.17 (dd, J = 6.2,
3.6 Hz, 1 H, 2ЈЈ-H), 8.08 and 8.10 (2s, 1 H, 6Ј-H) ppm. 13C NMR
(100 MHz ,[D6]acetone; assignment based on a HSQC spectrum):
C), 32.86 (5Ј-C), 37.42 (3Ј-C), 66.31 (2Ј-C), 102.24 (5-C), 121.70
(3ЈЈ-C), 128.05 (5ЈЈ-C), 139.11 (4ЈЈ-C), 149.74 (6-C), 150.32 (6ЈЈ-C),
152.97 (2-C), 156.83 (2ЈЈ-C), 159.13 (4-C) ppm. IR (ATR): ν = 3424
˜
δ = 28.32 (4ЈЈ-C), 29.39 (4-C), 33.28 (NCH2, 5ЈЈ-C), 37.38 (3ЈЈ-C), (w), 3042 (w), 1676 (s), 1640 (m), 1602 (m), 1571 (w), 1495 (s), 1480
60.38 (3-C), 64.84 (2ЈЈ-C), 67.94 (5-C), 68.12 (2-C), 75.98 (5Ј-CϵC), (s), 1427 (w), 1393 (w), 1313 (m), 1280 (s), 1167 (s), 1147 (m), 1121
91.87 (br., 5Ј-C and 5Ј-CϵC), 145.86 (6Ј-C), 154.21 (2Ј-C), 164.56 (m), 1078 (m), 1028 (m), 1011 (m), 990 (m), 966 (w), 881 (w), 808
(4Ј-C) ppm. IR (ATR): ν = 3389 (w), 3295 (w), 3208 (w), 3059 (w), (w), 776 (s), 748 (s), 668 (s), 642 (w) cm–1. HRMS (EI): m/z (%) =
˜
2926 (w), 2854 (w), 2234 (w), 1638 (s), 1603 (m), 1504 (s), 1406
338.0492 (13) [M]+, 253.0390 (100) [M + H – C4H6S]+. HRMS
(m), 1341 (m), 1314 (s), 1296 (m), 1249 (m), 1229 (m), 1182 (m), (EI): calcd. for C13H14N4O3S2 [M]+ 338.0507; found 338.0492;
+
1137 (s), 1075 (s), 970 (m), 881 (m), 779 (s), 728 (m), 605 (s) cm–1. calcd. for C9H9N4O3S+ [M + H – C4H6S]+ 253.0390; found
HRMS (ESI): m/z (%) = 385.0986 (100) [M + H]+, 299.0793 (63)
253.0390.
[M + H – C4H6S]+. HRMS (ESI): calcd. for C15H21N4O4S2 [M +
+
Methyl
(2ЈЈR/S)-5,6-Dideoxy-6-[1-(thiolan-2-yl)cytosin-5-yl]-β-D-
H]+ 385.0999; found 385.0986; calcd. for C11H15N4O4S+ [M + H –
C4H6S]+ 299.0809; found 299.0793.
ribo-hex-5-ynofuranoside [(2ЈЈR/S)-7]: A solution of iodocytosine
(Ϯ)-14 (100 mg, 0.30 mmol), acetylene 28 (79 mg, 0.5 mmol), and
NEt3 (1.2 mL, 0.84 mmol) in DMF (4 mL) was degassed by freeze-
pump-thaw cycles performed under argon, treated with
[PdCl2(PPh3)2] (32 mg, 0.03 mmol) and CuI (13 mg, 0.03 mmol),
and stirred at 22 °C for 14 h. Concentration and chromatography
(SiO2; CH2Cl2/MeOH, 97:3) gave crude (2ЈЈR/S)-7 (85 mg, 75%),
containing ca. 4% of triethyl ammonium salts. Rf = 0.42 (SiO2;
CH2Cl2/MeOH, 97:3), m.p. 68–71 °C. [α]2D2 = –7.2 (c = 0.07,
CH2Cl2/MeOH, 1:1). 1H NMR (300 MHz, CDCl3/CD3OD, 4:1; as-
signment based on a HSQC spectrum): δ = 1.67–1.79 (m, 1 H, 4ЈЈ-
Ha), 1.85–2.03 (m, 2 H, 3ЈЈ-Ha, 4ЈЈ-Hb), 2.11–2.23 (m, 1 H, 3ЈЈ-Hb),
2.76–2.85 (m, 1 H, 5ЈЈ-Ha), 3.00–3.10 (m, 1 H, 5ЈЈ-Hb), 3.26 (s, 3
H, OMe), 3.97 (br. d, J = 4.7 Hz, 1 H, 2-H), 4.27 (t, J ≈ 5.1 Hz, 1
H, 3-H), 4.62 (d, J = 5.4 Hz, 1 H, 4-H), 4.73 (d, J = 1.1 Hz, 1 H,
(؎)-5-(Phenylsulfonyl)-1-(thiolan-2-yl)cytosine [(؎)-5]: A suspen-
sion of TMSOTf (2.94 mL, 16 mmol), NEt3 (0.55 mL, 4 mmol) and
crude 21 (500 mg, 2 mmol) in toluene (15 mL) was stirred at 22 °C
for 5 min, diluted with CH2Cl2 (7.5 mL), and stirred at 22 °C for
15 min until a clear solution was formed. A solution of tetramethyl-
enesulfoxide (1.54 mL, 8 mmol) in CH2Cl2 (7.5 mL) was added by
using a cannula. The solution was stirred at 22 °C for 48 h and
diluted with ice-water and EtOAc. After separation of the layers,
the organic phase was washed with a satd. aq. NaHCO3 and brine,
and dried with Na2SO4. Concentration and chromatography (SiO2;
CH2Cl2/MeOH, 97:3) gave (Ϯ)-5 (185 mg, 28%) as an off-white
solid, m.p. 197–200 °C. 1H NMR (300 MHz, CDCl3; assignment
based on a HSQC spectrum): δ = 1.80–1.90 (m, 1 H, 4Ј-Ha), 2.11–
2.25 (m, 2 H, 3Ј-Ha, 4Ј-Hb), 2.28–2.41 (m, 1 H, 3Ј-Hb), 2.99 (ddd, 1-H), 6.08 (dd, J = 6.2, 3.6 Hz, 1 H, 2ЈЈ-H), 8.03 (s, 1 H, 6Ј-H) ppm.
J = 10.4, 9.3, 6.1 Hz, 1 H, 5Ј-Ha), 3.30 (ddd, J = 10.5, 7.1, 3.7 Hz,
1 H, 5Ј-Hb), 6.26 (dd, J = 6.1, 2.9 Hz, 1 H, 2Ј-H), 7.55 (br. dd, J
≈ 8.4, 6.8 Hz, 2 H, 3ЈЈ,5ЈЈ-H), 7.60–7.65 (m, 1 H, 4ЈЈ-H), 7.87 (br.
13C NMR (100 MHz, CDCl3/CD3OD, 4:1; 1:1 mixture of dia-
stereoisomers; assignment based on a HSQC spectrum): δ = 28.15
(4ЈЈ-C), 33.24 (5ЈЈ-C), 38.46 (3ЈЈ-C), 55.33 (OMe), 65.97 (2ЈЈ-C),
dd, J = 6.9, 2.4 Hz, 2 H, 2ЈЈ,6ЈЈ-H), 8.92 (s, 1 H, 6-H) ppm. 13C 73.08/73.10 (4-C), 74.94 (2-C), 76.21 (3-C), 76.75/76.76 (6-C), 90.59
NMR (125 MHz, CDCl3/CD3OD 6:1): δ = 27.81 (4Ј-C), 33.36 (5Ј- (br., 5Ј-C), 93.67/93.69 (5-C), 109.31 (1-C), 145.27 (6Ј-C), 155.26
Eur. J. Org. Chem. 2013, 880–887
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
885

M. Fischer, A. Bacher, F. Diederich et al.
FULL PAPER
(2Ј-C), 164.30 (4Ј-C) ppm. IR (ATR): ν = 3323 (m, br.), 3204 (m),
(m, 2 H, 6-H), 2.81 (br. dt, J = 10.6, 7.0 Hz, 1 H, 5ЈЈ-Ha), 3.05 (dt,
˜
2932 (m), 2225 (w), 1635 (s), 1534 (m), 1494 (s), 1441 (m), 1401 J = 11.6, 5.7 Hz, 1 H, 5ЈЈ-Hb), 3.24 (s, 3 H, OMe), 3.70–3.80 (m, 1
(m), 1297 (m), 1226 (m), 1121 (s), 1097 (s), 1019 (s), 972 (s), 918 H, 4-H), 3.81 (br. d, J = 4.9 Hz, 1 H, 2-H), 3.86 (br. t, J = 5.9 Hz,
(m), 779 (s) cmϪ1. HRMS (ESI): m/z (%) = 376.0939 (29) [M +
Na]+, 354.1115 (100) [M + H]+, 268.0922 (66) [M + H – C4H6S]+.
HRMS (ESI): calcd. for C15H19N3NaO5S+ [M + Na]+ 376.0938;
found 376.0939; calcd. for C15H20N3O5S+ [M + H]+ 354.1118;
1 H, 3-H), 4.65 (br. s, 1 H, 1-H), 6.15 (dd, J = 6.2, 4.1 Hz, 1 H, 2ЈЈ-
H), 7.61/7.62 (2 s, 1 H, 6Ј-H) ppm. 13C NMR (100 MHz, CDCl3/
CD3OD, 4:1; 1:1 mixture of diastereoisomers; assignment based on
a HSQC spectrum): δ = 23.48 (6-C), 28.50 (4ЈЈ-C), 33.24 (5ЈЈ-C),
33.32 (5-C), 38.41 (3ЈЈ-C), 55.16 (OMe), 65.04 (2ЈЈ-C), 74.43 (3-C),
74.80 (2-C), 81.29/81.41 (4-C), 106.04 (br., 5Ј-C), 108.48 (1-C),
found 354.1115; calcd. for C11H14N3O5 [M + H – C4H6S]+
+
268.0928; found 268.0922.
139.94 (6Ј-C), 156.72 (2Ј-C), 164.72 (4Ј-C) ppm. IR (ATR): ν =
˜
(2ЈЈR/S)-3,7-Anhydro-1,2-dideoxy-1-[1-(thiolan-2-yl)cytosin-5-yl]-
D-
3326 (m), 2929 (w), 1643 (s), 1597 (m), 1497 (m), 1441 (w), 1405
(w), 1299 (w), 1229 (w), 1067 (m, br.), 778 (m) cm–1. HRMS (ESI):
m/z (%) = 358.1429 (100) [M + H]+. HRMS (ESI): calcd. for
C15H24N3O5S+ [M + H]+ 358.1431; found 358.1429.
glycero- -galacto-oct-1-ynitol [(2ЈЈR/S)-8]: A solution of iodocytos-
D
ine (Ϯ)-14 (100 mg, 0.30 mmol), 29[19] (160 mg, 0.84 mmol), and
NEt3 (1.2 mL, 0.84 mmol) in DMF (4 mL) was degassed by freeze-
pump-thaw cycles performed under Ar, treated with [PdCl2-
(PPh3)2] (35 mg, 0.03 mmol) and CuI (15 mg, 0.03 mmol), and
stirred at 22 °C for 5 h. Filtration over Celite, concentration, and
chromatography (SiO2; CH2Cl2/MeOH, 9:1) gave (2ЈЈR/S)-8
(46 mg, 40%) as an off-white solid. Rf = 0.15 (SiO2; CH2Cl2/MeOH
9:1), m.p. 99–102 °C. [α]2D2 = +17.6 (c = 0.07, CH2Cl2/MeOH, 1:1).
1H NMR (400 MHz, CD3OD; assignment based on a HSQC spec-
trum): δ = 1.92–2.05 (m, 1 H, 4ЈЈ-Ha), 2.06–2.19 (m, 2 H, 3ЈЈ-Ha,
4ЈЈ-Hb), 2.26–2.41 (m, 1 H, 3ЈЈ-Hb), 2.92–3.04 (m, 1 H, 5ЈЈ-Ha),
3.24–3.36 (m, 2 H, 7-H, 5ЈЈ-Hb), 3.54 (dd, J = 9.4, 3.0 Hz, 1 H, 5-
H), 3.60 (t, J = 9.3 Hz, 1 H, 6-H), 3.70 (dd, J = 11.9, 6.1 Hz, 1 H,
8-Ha), 3.90 (dd, J = 11.8, 1.5 Hz, 1 H, 8-Hb), 3.98 (br. d, J = 2.4 Hz,
1 H, 4-H), 4.61 (br. s, 1 H, 3-H), 6.28 (br. s, 1 H, 2ЈЈ-H), 8.38 (s, 1
H, 6Ј-H) ppm. 1H NMR (500 MHz, [D6]acetone; 1:1 mixture of
diastereoisomers; assignment based on a DQF-COSY and a HSQC
spectrum): δ = 1.91–2.02 (m, 3 H, 3ЈЈ,4ЈЈ-Ha, 4ЈЈ-Hb), 2.12–2.22 (m,
1 H, 3ЈЈ-Hb), 2.82 (dt, J = 10.0, 6.1 Hz, 1 H, 5ЈЈ-Ha), 3.01–3.08 (m,
1 H, 7-H), 3.22 (dt, J = 9.9, 6.2 Hz, 1 H, 5ЈЈ-Hb), 3.29–3.35 (m, 2
H, 5,6-H), 3.37–3.42 (m hidden by solvent peaks, 1 H, 8-Ha), 3.66
(br. dd, J = 11.7, 5.8 Hz, 1 H, 8-Hb), 3.77 (br. s, 1 H, 4-H), 4.40
(s, 1 H, 3-H), 4.55 (t, J = 5.9 Hz, 1 H, 8-OH), 4.81 (br. d, J = 3.5,
Hz, 1 H), 4.88 (br. d, J = 3.7 Hz, 1 H) (5,6-OH), 5.11 (d, J = 3.8
Hz, 1 H, 4-OH), 6.13 (t, J = 5.7 Hz, 1 H, 2ЈЈ-H), 6.76 and 6.78 (2s,
1 H, NH), 7.79 (s, 1 H, NH), 8.14 (s, 1 H, 6Ј-H) ppm. 13C NMR
(100 MHz, CD3OD; assignment based on a HSQC spectrum): δ =
28.24 (4ЈЈ-C), 32.72 (5ЈЈ-C), 37.90 (3ЈЈ-C), 61.49 (8-C), 66.0 (br.,
2ЈЈ-C), 66.88 (6-C), 70.27 (3-C), 71.97 (4-C), 74.20 (5-C), 80.98 (7-
C), 90.68 (5Ј-C), 146.46 (6Ј-C), 152.94 (2Ј-C), 162.51 (4Ј-C) ppm;
signals of CϵC hidden by the noise. 13C NMR (125 MHz, [D6]-
acetone; 1:1 mixture of diastereoisomers; assignment based on a
HSQC spectrum): δ = 28.98/29.02 (4ЈЈ-C), 32.90 (5ЈЈ-C), 37.00/
37.04 (3ЈЈ-C), 61.38 (8-C), 64.36 (2ЈЈ-C), 66.85 (6-C), 70.10/70.12
(3-C), 71.39 (4-C), 74.01 (5-C), 76.73 (1-C), 81.45 (7-C), 89.50 (5Ј-
C), 93.21 (2-C), 145.78 (6Ј-C), 153.93 (2Ј-C), 164.07 (4Ј-C) ppm.
(2ЈЈR/S)-3,7-Anhydro-1,2-dideoxy-1-[1-(thiolan-2-yl)cytosin-5-yl]-
D-
glycero- -galacto-octitol [(2ЈЈR/S)-10]: A solution of (2ЈЈR/S)-8
D
(15 mg, 0.04 mmol) in DMF (4 mL) was treated with 10% Pd/C
(15 mg). For 1 h, H2 gas was bubbled through the suspension, then
the suspension was stirred at 22 °C for 14 h. Concentration gave
(2ЈЈR/S)-10 (14 mg, 92%) as a white solid. Rf = 0.17 (SiO2; CH2Cl2/
MeOH 85:15), m.p. 88–92 °C. [α]2D2 = –18.8 (c = 0.08, CH2Cl2/
MeOH, 1:1). 1H NMR (600 MHz, CDCl3/CD3OD, 1:1; 1:1 mixture
of diastereoisomers; assignment based on a DQF-COSY and a
HSQC spectrum): δ = 1.32–1.41 (m, 1 H, 4ЈЈ-Ha), 1.67–1.79 (m, 3
H, 2,3ЈЈ-Ha, 4ЈЈ-Hb), 1.83 (dq, J = 12.3, 6.1 Hz, 1 H, 2-Hb), 2.03–
2.10 (m, 1 H, 3ЈЈ-Hb), 2.14–2.22 (m, 1 H, 1-Ha), 2.26 (ddd, J =
15.1, 8.9, 4.5 Hz, 1 H, 1-Hb); 2.69 (dt, J = 10.3, 6.7 Hz, 1 H, 5ЈЈ-
Ha), 2.90–2.98 (m, 2 H, 7-H, 5ЈЈ-Hb), 3.11–3.15 (m, 1 H, 3-H),
3.204 and 3.205 (2 dd, J = 9.8, 3.6 Hz, 1 H, 5-H), 3.30 and 3.32 (2
t, J = 9.6 Hz, 1 H, 6-H), 3.43–3.49 (m, 2 H, 4-H, 8-Ha), 3.61 and
3.62 (2 dd, J = 11.8, 2.5 Hz, 1 H, 8-Hb), 6.01 and 6.03 (2 dd, J =
6.5, 4.3 Hz, 1 H, 2ЈЈ-H), 7.49 and 7.50 (2 s, 1 H, 6Ј-H) ppm. 13C
NMR (150 MHz, CDCl3/CD3OD, 1:1; 1:1 mixture of diastereoiso-
mers; assignment based on a HSQC spectrum): δ = 23.20/23.27 (1-
C), 28.74 (2-C), 29.96/30.23 (4ЈЈ-C), 33.22 (5ЈЈ-C), 38.30 (3ЈЈ-C),
61.78 (8-C), 64.90/65.00 (2ЈЈ-C), 67.53/67.58 (6-C), 71.33/71.39 (4-
C), 75.10 (5-C), 76.36/76.59 (3-C), 80.19/80.32 (7-C), 106.52/106.68
(5Ј-C), 140.04/140.19 (6Ј-C), 158.86 (2Ј-C), 164.78/164.86 (4Ј-
C) ppm. IR (ATR): ν = 3448 (w), 3330 (w), 3180 (w), 3061 (w),
˜
2862 (w), 2232 (w), 1640 (s), 1599 (m), 1496 (s), 1453 (m), 1404
(m), 1360 (w), 1297 (w), 1228 (m), 1128 (m), 1063 (s), 1027 (s), 986
(m), 909 (m), 781 (m), 734 (s), 696 (s) cm–1. HRMS (ESI): m/z (%)
=
388.1526 (100) [M +
H]+. HRMS (ESI): calcd. for
C16H26N3O6S+ [M + H]+ 388.1537; found 388.1526.
Enzyme-Coupled Photometric Assay for IC50 Determination: For
details, see Schütz et al.[5]
Supporting Information (see footnote on the first page of this arti-
cle): Experimental protocols and NMR spectra for all compounds.
IR (ATR): ν = 3326 (s, br.), 2929 (m), 1643 (s), 1597 (m), 1441 (w),
˜
1405 (w), 1299 (w), 1229 (w), 1067 (s), 778 (s) cmϪ1. HRMS (ESI):
m/z (%) = 384.1220 (100) [M + H]+, 298.1022 (26) [M + H –
C4H6S]+. HRMS (ESI): calcd. for C16H22N3O6S+ [M + H]+
Acknowledgments
+
384.1224; found 384.1220; calcd. for C12H16N3O6 [M + H –
C4H6S]+ 298.1034; found 298.1022.
This work was supported by the Swiss National Science Founda-
tion.
Methyl (2ЈЈR/S)-5,6-Dideoxy-6-[1-(thiolan-2-yl)cytosin-5-yl]-β-D-
ribo-hexofuranoside [(2ЈЈR/S)-9]: A solution of crude (2ЈЈR/S)-7
(35 mg, 0.1 mmol) in MeOH/DMF (2:1, 6 mL) was treated with
10% Pd/C (15 mg). During 1 h, H2 gas was bubbled through the
suspension. Filtration and concentration gave (2ЈЈR/S)-9 (34 mg,
95%) as an off-white oil. Rf = 0.17 (SiO2; CH2Cl2/MeOH, 95:5).
[α]2D2 = –7.3 (c = 0.06, CH2Cl2/MeOH, 1:1); M.p. 97–100 °C. 1H
NMR (300 MHz, CDCl3/CD3OD, 5:1; 1:1 mixture of diastereoiso-
mers; assignment based on a HSQC spectrum): δ = 1.55–2.01 (m,
5 H, 5,3ЈЈ,4ЈЈ-Ha, 5,4ЈЈ-Hb), 2.12–2.25 (m, 1 H, 3ЈЈ-Hb), 2.26–2.41
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Received: October 31, 2012
Published Online: January 3, 2013
Eur. J. Org. Chem. 2013, 880–887
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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