Antibacterial 5′-O-(N-dipeptidyl)-sulfamoyladenosines

Article abstract of DOI:10.1016/j.bmc.2008.11.054

The aminoacyl-tRNA synthetase (aaRS) class of enzymes is a validated target for antimicrobial development. Aminoacyl analogues of 5′-O-(N-l-aminoacyl)-sulfamoyladenosines are known to be potent inhibitors of aaRS, but whole cell antibacterial activity of

Full text of DOI:10.1016/j.bmc.2008.11.054

Bioorganic & Medicinal Chemistry 17 (2009) 260–269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antibacterial 50-O-(N-dipeptidyl)-sulfamoyladenosines
P. Van de Vijver a, G. H. M. Vondenhoff a, S. Denivelle a, J. Rozenski a, J. Verhaegen b,
A. Van Aerschot a, P. Herdewijn a,
*
a Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroederstraat 10, B-3000 Leuven, Belgium
b Experimental Laboratory Medicine, Universitary Hospital Gasthuisberg, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2008
Revised 30 October 2008
Accepted 3 November 2008
Available online 27 November 2008
The aminoacyl-tRNA synthetase (aaRS) class of enzymes is a validated target for antimicrobial develop-
ment. Aminoacyl analogues of 50-O-(N-
L-aminoacyl)-sulfamoyladenosines are known to be potent inhib-
itors of aaRS, but whole cell antibacterial activity of these compounds is very limited, and poor
penetration into bacteria has been proposed as the main reason for this. Aiming to find derivatives that
better penetrate bacteria, we developed a simple and short method to prepare dipeptidyl-derivatives of
50-O-(N- -aminoacyl)-sulfamoyladenosines, and used this method to prepare 18 50-O-(N-dipeptidyl)-sul-
L
Keywords:
Microcin C
Aminoacyl-tRNA synthetase
Antibacterial
Peptide antibiotic
famoyladenosines. The antibacterial activity of these derivatives and a number of reference compounds
against S. aureus, E. faecalis and E. coli was determined. Several of the new derivatives showed improved
antibacterial activity and an altered spectrum of antibacterial activity.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The largest group of aaRS inhibitors (aaRSi) are the reaction-
intermediate mimics. These compounds are analogues of aa-AMP
Aminoacyl-tRNA synthetases (aaRSs) are the enzymes that cat-
alyze the transfer of amino acids to their cognate tRNA.1 They play
an essential role in protein biosynthesis and are required for both
growth and survival of all cells. The aminoacyl-tRNA synthetase
class of enzymes is clinically validated as target for antimicrobial
drug development, as exemplified by the clinical use of mupirocin,
an inhibitor of bacterial IleRS, in the topical treatment of bacterial
colonizations.2
At the moment, mupirocin is the only aminoacyl-tRNA synthe-
tase inhibitor in clinical use. An inhibitor of LeuRS, AN-2690 (3,
Fig. 1), is currently in clinical development for the topical treat-
ment of onychomycosis,3,4 and an inhibitor of IleRS, icofungipen
(4), reached clinical phase II for the systemic treatment of oropha-
ryngeal candidiasis. While the latter compound showed a good
clinical response in this trial, mycologic eradication rates were
too low to support further development.5
(1, Fig. 1), which is the natural reaction intermediate of these en-
zymes. Mupirocin belongs to this group, as do the twenty 50-O-
(N-L-aminoacyl)-sulfamoyladenosines (aaSA, 2). The latter are
commercially available and show Ki values in the low nanomolar
NH2
N
N
OH
B
O
O
O
O P O
N
N
O
O-
R1
+
F
NH3
HO OH
AN-2690 (3)
aa-AMP (1)
NH2
N
N
O
O
Abbreviations: aa, amino acid; aaRS, aminoacyl-tRNA synthetases (where aa can
be replaced by any amino acid 3-letter code to indicate individual enzymes); aaRSi,
aminoacyl-tRNA synthetases inhibitor; aaSA (were aa can be replaced by any amino
acid 3-letter code), 50-O-(N-aminoacyl)-sulfamoyladenosine; Boc, tert-butoxycar-
bonyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EDCIꢀHCl, N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride; HOBt, N-hydroxybenzotriazole; McC,
Microcin C; –OSu, N-hydroxysuccinimide ester; rt, room temperature; TBDMS, tert-
butyldimethylsilyl; TFA, trifluoroacetic acid; THF, tetrahydrofurane.
N- S O
N
O
N
R1
O
NH2
COOH
+
NH3
icofungipen (4)
HO OH
aaSA (2): R1 = amino acid side chain
* Corresponding author. Tel.: +32 (0) 16337387; fax: +32 (0) 16337340.
E-mail addresses: piet.herdewijn@rega.kuleuven.be, Chantal. Biernaux@rega.ku-
leuven.be (P. Herdewijn).
Figure 1. Structures of universal aaRS intermediate aa-AMP (1), universal aaRS
inhibitor aaSA (2) and investigational aaRS inhibitors AN-2690 (3) and icofungipen
(4).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.054

P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
261
HO
O
COOH
OH
NH2
N
O
O
N
N
P
O
O
O
HN
O-
H
N P O
N
N
N
O
N
OH
O
HO
O
O-
OH
H
OH
N P O
N
O
O-
OH
HO
7
OH
agrocin 84 (5)
NH2
N
NH2
N
N
N
N
N
O
O
H
O
O O
H
Met Arg Thr Gly Asn Ala
N P O
O
N P O
O
N
N
O
O
HN
-O
+H3N
-O
HO OH
HO OH
O
O
+
+
microcin C (6)
8
NH3
NH3
Figure 2. Structures of the Trojan Horse antibiotics agrocin 84 (5) and microcin C (6), and their aaRS inhibiting payloads 7 and 8, respectively.
range against the corresponding isolated aaRSs.6–10 Since the aaSA
compounds differ only little from the natural reaction intermedi-
ates, they do not show noticeable selectivity towards either bacte-
rial or eukaryotic aaRSs. This is a consequence of the use of the
same general reaction intermediate (1) by aaRSs of all kingdoms
of life, despite extensive divergence in the structure and amino
acid sequence of these enzymes. These evolved differences have al-
lowed the development of derivatives of aaSA that do show good
selectivity for bacteria and that are systemically active in a mouse
model of infection.11,12
Even though these aaSAs (2) are nanomolar inhibitors of aaRS
enzymes in vitro, the in vivo antibacterial activity is considerably
less, and poor uptake has been suggested as the main reason for
this.2,13,14 Several natural inhibitors of aaRS enzymes circumvent
this problem by using a dual active uptake/aaRS inhibition mode
of action. Relevant examples of this are the antibiotics agrocin 84
(5, Fig. 2) and microcin C (6).
Agrocin 84 is a‘Trojan Horse’ antibiotic with potent activity
against Agrobacterium tumefaciens, which causes crown gall tumors
in a wide variety of plants. The compound resembles a tumor-de-
rived substrate and is actively internalized by A. tumefaciens. Once
inside the target cell, leucyl-adenylate analogue 7 is liberated and
inhibits LeuRS.15,16 The mode of action of microcin C (6) is similar:
microcin C is produced by strains of Escherichia coli and consists of
an N-formylated heptapeptide attached to a modified adenylate
moiety. The antibiotic is actively taken up by the Yej transporter,
which is present on a number of Enterobacteriaceae. Following
deformylation by peptide deformylase, the peptide moiety is di-
gested sequentially, starting from the N-terminal methionine. This
way, the bacterial peptidases set free aspartyl-adenylate analogue
8, a potent inhibitor of AspRS.17–19
2. Results
2.1. Design and synthesis
The aim of our research was to find derivatives of 50-O-(N-ami-
noacyl)-sulfamoyladenosine (aaSA, 2) that show improved anti-
bacterial activity compared to the parent aaSAs. To this end, a
series of dipeptidyl-analogues, designed to be taken up by peptide
transporters, was prepared.
Ascamycin (9, Fig. 3) is a natural antibiotic that closely resem-
bles 50-O-(N-
L
-alanyl)-sulfamoyladenosine (AlaSA), although it
has a different mechanism of action. In evaluating several amino-
acyl analogues of ascamycin, Ubukata et al. found the -prolyl-
L
L-
prolyl derivative 10 to have a significantly increased whole-cell
antibacterial activity against both Gram-positive and Gram-nega-
tive bacteria, and they suggested increased uptake by peptide
transporters as the reason for this, though this was not further
investigated.20,21
Our starting hypothesis was that the 50-O-(N-dipeptidyl)-sulfa-
moyladenosines, which are structurally similar to 10, would be ta-
NH2
NH2
N
N
N
+
N
H2
N
O
N- S O
O
O
N- S O
O
O
O
O
N
N
N
Cl
N
Cl
O
O
+H3N
N
HO OH
HO OH
9
10
NH2
N
N
N
O
N- S O
O
O O
HN
+H3N Met Arg Thr Gly
Asn Ala
N
O
O
HO OH
O-
11
Figure 3. Structures of ascamycin (9), its L-Pro-L-Pro dipeptidyl derivative 10 and the structure of AspSA derivative 11, a synthetic analogue of microcin C (4).

262
P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
The choice of the carboxy-terminal amino acid was influenced
NH2
N
aa2
by the results of a preliminary assay (Table S1, Supplementary
data), in which all of the 20 analogues of aaSA were tested for their
antibacterial activity against a diverse panel of bacteria, both
Gram-negative and Gram-positive. In this screening, we found that
the aaSA analogues have only limited whole cell antibacterial
activity, with no compound showing antibacterial activity below
N
N
O
N- S O
O
O
N
O
HN
O
R2
R1
aa1
HO OH
+H3N
16
lg/mL. The compounds that showed meaningful activity at
128
l
g/mL in this screening were AlaSA, ArgSA, HisSA, LeuSA, Lys-
R1 = side chain of the aminoterminal amino acid (aa1)
R2 = side chain of the carboxyterminal amino acid (aa2)
SA, MetSA and ProSA. From these, the Ala, Leu, Met and Pro ana-
logues were selected. Additionally, we also prepared analogues
containing Trp, Phe and Asp as the carboxyterminal amino acid.
Initially, we prepared the 50-O-(N-dipeptidyl)-sulfamoyladeno-
sines by the convergent method developed by Ubukata et al. for
their synthesis of 10.21 In this synthesis, N-Boc-protected dipeptide
is activated at the free carboxyl function and reacted with 20,30-O-
isopropylidene-50-O-sulfamoyl-adenosine in the presence of strong
base. After removal of the protecting groups using aqueous trifluo-
roacetic acid, this yields the desired dipeptidyl-sulfamoyladeno-
sine. However, with this method we found the second,
carboxyterminal amino acid to be prone to racemization (data
not shown). Therefore, we abandoned this method and switched
to a serial approach instead. Initially, we coupled N-Boc protected
amino acid to the respective sugar-protected aaSA using standard
peptide coupling procedures, followed by a two step deprotection
procedure to remove all protecting groups (Scheme 1). This meth-
od was used for the synthesis of PhePheSA (12). For the other 50-O-
(N-dipeptidyl)-sulfamoyladenosines 13–29, this synthesis was fur-
ther modified, and the N-Boc protected amino acid was coupled di-
rectly to unprotected aaSA, followed by a short and simple aqueous
acid deprotection step to remove the N-Boc protecting group
Figure 4. General structure of the 50-O-(N-dipeptidyl)-sulfamoyladenosines 12–29.
For the nature of aa1 and aa2: see Table 1.
ken up into microbial cells by dedicated peptide transporters and
be digested by intracellular peptidases to yield the active aaSAs,
which would in turn inhibit the corresponding aaRS and inhibit
bacterial cell growth. Also, these compounds can be regarded as
truncated or contracted analogues of synthetic microcin C ana-
logue 11, which was shown to act via the same mode of action
as its natural counterpart 6. The mode of action of 11 is comparable
to that of microcin C, and involves active uptake by the Yej trans-
porter, intracellular digestion by peptidases A, B and N and finally
inhibition of AspRS of sensitive Enterobacteriaceae [Kazakov et al.,
in press].
To test this hypothesis, a diverse set of 50-O-(N-dipeptidyl)-sul-
famoyladenosines was prepared (Fig. 4 and Table 1). The amino-
terminal amino acids were chosen to obtain a set of compounds
with sufficient differences in physicochemical parameters such as
hydrophobicity, net charge and size. Hereto, the amino acids ala-
nine, methionine, proline, glutamine, lysine and phenylalanine
were selected.
Table 1
Overview of the antibacterial activities of dipeptidyl-sulfamoyladenosines 12–29 (Fig. 4) and selected reference compounds
a
b
Compd (30 nmol)c
aa1
aa2
Growth inhibition area (mm2)
S. aureus
E. faecalis
E. coli wt
E. coli DABN
LeuSA
AlaSA
MetSA
TrpSA
PheSA
AspSA
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
12
29
Leu
Ala
Met
Trp
Phe
Asp
Leu
Leu
Leu
Leu
Leu
Ala
Ala
Ala
Met
Met
Met
Trp
Trp
Trp
Trp
Trp
Phe
Asp
NA
0
0
26.9 5.4d
25.4 5.0d
46.3 6.8d
26.8 2.6d
66 4.2d
42.3 3.3e
40.4 3.3e
35 3.1e
44.7 11.8d
18.7 5.7e
0
0
0f
0
0
0
0
0
0
0
0
0
0
0
0f
0
0
0
0
0
0
0
0
0
0
0
40.4 3.3d
21.3 6.3e
24.2 7.1d
33.3 5.1d
38.5 0d
52.7 9.9d
28.3 0e
26.8 2.6d
Ala
0
0
0
0
0
0
0
0
0
0
Met
Pro
Lys
Glu
Ala
Met
Pro
Ala
Met
Pro
Ala
Met
Pro
Lys
Glu
Phe
Met
0
0
0
0
0
0f
56.9 6.7d
76.1 9.1e
25.3 2.6d
26.8 2.6d
18.4 2.2e
0
0
0
0
0
0
59 4e
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0f
0f
0
0
0
0
0
0
0
0
0
0
0
0
0
0
83.9 4.6d
0
0
0
0
0
0
0
0
0
0
0
0f
0
0f
22.4 2.4d
18.4 2.2d
12.6 0d
22.4 2.4e
19.6 0e
26.8 2.6f
63.7 7.1d
89.4 4.9d
31.5 2.8e
52.7 9.9e
Mupirocin
>490.93
0
38.5 0d
MRTGNAD-SAc
Asp
0
0
0
46.7 12.2d
Briefly, a lawn of soft Mueller–Hinton agar (0.75% agar) containing bacteria was spread over Mueller–Hinton agar (1.5% agar).
a
Amino-terminal amino acid.
Carboxyterminal amino acid.
b
c
Antibiotic was added as 5 ll drops containing 30 nmol of compound, except for MRTGNAD-SA (1.2 nmol). The plates were incubated at 37 °C for 16–20 h and analyzed.
Values are indicated as area in mm2 SD. Bacterial strains used were Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli and E. coli
D
ABN.
d
Clear halos were observed, indicating full growth inhibition.
Opaque halos were observed, indicating partial growth inhibition.
e
f
Partial growth inhibition halos were visible but were too faint to be accurately measured.

P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
263
pounds in water, for which we have no explanation, work on this
subset of compounds was abandoned.
NH2
N
N
N
O
O
N- S O
2.2. Antibacterial evaluation
N
O
H2N
O
Et3NH+
For the antibacterial evaluation of the compounds, we exposed
bacteria in Mueller–Hinton agar to droplets containing 30 nano-
moles of the potential antibacterial. After overnight incubation,
inhibition zones were determined. All tests were performed with
6 nanomole-doses as well, but here antibacterial activity was very
limited (Table S2, supplementary data). Three bacterial species
were used for these tests: Staphylococcus aureus, Enterococcus fae-
cialis and Escherichia coli. In addition, a mutant E. coli strain lacking
broad specificity peptidases A, B and N was subjected to a selection
of the compounds, to determine if processing by these peptidases
is required for activity of the dipeptidyl-compounds.
Ph
O O
38
TBDMS
TBDMS
(a)
NH2
N
N
O
O
Ph
N- S O
N
O
N
HN
O
Ph
The data presented in Table 1 show differing sensitivities
amongst the strains tested, with the S. aureus strain being clearly
less sensitive to the compounds studied. Comparing activity of
the aaSA parent compounds, inhibition of the S. aureus strain can
be detected with 2/6 compounds. In contrast, the E. faecalis and
E. coli strains are both sensitive to 4/6 and 5/6 compounds, respec-
tively. None of the bacterial strains was inhibited by the tryptopha-
nyl analogue.
R3 NH
O
O O
R1
R2
48: R3 = Boc; R1,R2 = TBDMS
12: R3 = H; R1,R2 = H
b)
Scheme 1. Synthesis of Phe/Phe analogue (12). Reaction conditions: (a) N-Boc-L-
Phe-OH, HOBt, EDCIꢀHCl, Et3N, DMF; (b) (i) TFA/H2O (5:2 v/v), (ii) Et3Nꢀ3HF, THF.
The situation with the dipeptidyl compounds is more diverse:
again, the S. aureus strain is resistant to all but one (MetAla ana-
logue 19), while the E. faecalis strain is, to varying extents, inhib-
ited by 7/18 compounds and the E. coli wt strain by 6/18.
A number of interesting observations can be made when com-
NH2
N
N
O
O
N- S O
N
N
O
H2N
paring inhibition between the wt and
DABN E. coli strains: The mu-
O
Et3NH+
R2
tant strain appears to be more sensitive to some of the parent aaSA
compounds, and for LeuSA and MetSA this difference is significant
(P = 0.0004 and 0.005, respectively). The mutant strain has gained
resistance towards aa/Ala compounds 18–20, indicating that at
least one of the broad specificity peptidases A, B or N is required
for digestion of these compounds to the active derivative.19 In con-
trast, sensitivity against Glu/Trp analogue 28, Phe/Phe analogue 12
and Met/Asp analogue 29 is indistinguishable from the wt strain.
Finally, we also tested these strains against the established anti-
biotic mupirocin and against synthetic McC analogue MRTGNAD-
SA (11, Fig. 3, [Kazakov2008, in press]). The inhibition zone caused
by mupirocin against S. aureus was too large to be accurately mea-
sured. At the same dose, E. faecalis and E. coli were also sensitive,
albeit a lot less than S. aureus. At the low dose tested (1.2 nmol),
MRTGNAD-SA was only active against E. coli. This is in line with
HO OH
40, 43-47
(a)
NH2
N
N
N
O
O
O
N- S O
N
O
R1 HN
R2
Et3NH+
R3 NH
O
HO OH
49-65: R3 = Boc
13-29: R3 = H
(b)
the observation that microcin
C is primarily active against
R1, R2 = amino acid side chains
Enterobacteriaceae.22
Scheme 2. Synthesis of dipeptidyl-sulfamoyladenosines 13–29 of Table 1. Reaction
conditions: (a) N-Boc-
v).
L-amino acid, HOBt, EDCIꢀHCl, Et3N, DMF; (b) TFA/H2O (5:2 v/
3. Discussion
3.1. Variable sensitivity to dipeptidyl analogues
(Scheme 2). We found the latter method to be more time-efficient
while keeping the overall yield unchanged.
When comparing the inhibition zones between different dipep-
tidyl compounds and between dipeptidyl compounds and their
parent compounds, large differences can be observed: in a number
of cases, the addition of a second amino acid to the N-terminal
amino acid of aaSA abolished antibacterial activity. However, in
other cases, the derivative is much more active than the parent
compound: an example from Table 1 is Met/Met analogue 22: this
compound has potent activity against E. faecalis but not against any
of the other strains tested, while its parent compound MetSA is less
active but has a broader spectrum (all three strains are inhibited to
some extent). A second example is Met/Asp analogue 29: com-
pared to the parent compound AspSA, 29 is more active against
E. faecalis but less active against E. coli.
To our surprise, we were not able to prepare analogues with
proline as the carboxyterminal amino acid. We tried to synthesize
both the Ala/Pro (30) and the Pro/Pro (31) analogue via the meth-
ods described above, but were not able to isolate the target com-
pounds. The coupling reactions were successful, and both HRMS
and NMR showed the presence of the desired compounds. How-
ever, extensive decomposition occurred during the subsequent
deprotection reaction. In one case, we did observe the desired mol-
ecule by mass spectrometric analysis of the partly purified com-
pound but we found the compound to decompose to adenosine
during size-exclusion chromatography with H2O/MeOH (3:7 v/v,
data not shown). As this suggested instability of aaProSA com-

264
P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
These results point to a potential for modifying the antibacterial
pling constants are given in Hertz (Hz). The peak patterns are indi-
cated by the following abbreviations: br s = broad singlet,
d = doublet, m = multiple, q = quadruplet, s = singlet, and t = triplet.
High resolution mass spectra were recorded on a quadrupole
time-of-flight mass spectrometer (Q-Tof-2, Micromass, Manches-
ter, UK) equipped with a standard ESI interface; samples were in-
spectrum of aaSA compounds by the preparation of dipeptidyl
derivatives. This is similar to the results obtained with MRTG-
NAD-SA: here, addition of the MRTGNA hexapeptide to AspSA cre-
ates a derivative that is much more active against E. coli: an
inhibition zone of 46.7 12.2 mm2 is produced by only 1.2 nmol
of MRTGNAD-SA, compared to an inhibition zone of
26.8 2.6 mm2 produced by 30 nmol of parent compound AspSA.
Combined, these results indicate the possibility of generating ami-
noacyl- and oligopeptidyl-derivatives of compounds with known
or expected antibacterial activity to modify the antibacterial effi-
cacy and spectrum of these compounds. It is also important to
evaluate the potential immunosuppressive activity of tRNA-syn-
fused in 2-propanol/H2O (1:1, v/v) at 3 l .
L minꢂ1
For TLC, precoated aluminum sheets were used (Merck, Silica
Gel 60 F254). The spots were visualized by UV light. Column chro-
matography was performed on ICN Silica Gel 60A 60–200. For size
exclusion chromatography, a 2 ꢃ 30 cm column of Sephadex LH-20
was used as the solid phase and MeOH/H2O (7: 3, v/v) as the elu-
ent. Eluent compositions are expressed as v/v.
Several of the final compounds were found to contain low but
varying amounts of the counterions triethylammonium and N-
(chloromethyl)-N,N-diethylethanaminium. To compensate for this
in the biological assays, concentrations of compounds were deter-
mined spectrophotometrically (UV, k ¼ 260 nm) and purified N-
(chloromethyl)-N,N-diethylethanaminium chloride salt was sub-
jected to the biological assays as well.
thetase inhibition, as this side effect is unwanted for antibiotics23
.
In addition, aminoacyl- or dipeptidyl-derivation may also be a
viable strategy to improve physicochemical characteristics of anti-
bacterial drugs. By coupling with more or less polar amino acids, it
is conceivable that oligopeptidyl-derivatives of aaSAs and other
antibacterials can be prepared with improved oral availability,
water solubility or skin penetration with only limited effects on
toxicity.
4.1.2. Preparation of 50-O-(N-
4.1.2.1. 20,30-Di-O-(tert-butyldimethylsilyl)-50-O-[N-(N-Boc-
ptophanyl)-sulfamoyl]adenosine (Et3N salt, 33). To an ice-
cooled solution of 20,30-di-O-(tert-butyldimethylsilyl)-50-O-sulfa-
moyladenosine (32, 4.60 g, 8.00 mmol, 1.0 equiv) and N-Boc-
L
-aminoacyl)-sulfamoyladenosines
3.2. Influence of peptidases A, B and N
L-try-
Digestion by peptidases A, B and N is required for activity of
some but not all dipeptidyl-sulfamoyl-adenosines: No inhibition
zones were found for the aa/Ala compounds 18–20, indicating that
digestion by at least one of the missing peptidases is necessary for
digestion of these compounds, similar to the situation with MRTG-
NAD-SA [Kazakov2008, in press].
In contrast, no difference in sensitivity to Glu/Trp analogue 28,
Phe/Phe analogue 12 and Met/Asp analogue 29 was observed, indi-
cating that these compounds are either digested by enzymes other
than peptidases A, B or N, or that these compounds do not require
intracellular activation.
L-
tryptophan N-hydroxysuccinimide ester (3.53 g, 8.79 mmol,
1.1 equiv) in DMF (30 mL) was added DBU (2.0 g, 13.13 mmol,
1.6 equiv) and the reaction mixture was allowed to come to rt
overnight. Next, the volatiles were removed in vacuo and the res-
idue was purified by silica gel chromatography (Et3N 1%, MeOH
2.5?10% in CH2Cl2) to yield 7.50 g (7.79 mmol, 97%) of 33.
1H NMR (DMSO-d6): d ꢂ0.41 (s, 3H, Si—CH3), ꢂ0.07 (s, 3H,
Si—CH3), 0.12 (s, 3H, Si—CH3), 0.14 (s, 3H, Si—CH3), 0.67 (s, 9H,
Si—C—ðCH3Þ3), 0.92 (s, 9H, Si—C—ðCH3Þ3), 1.15 (t, 9H,
A second finding is the increased sensitivity of
inhibitors LeuSA, MetSA, PheSA and mupirocin. For LeuSA
(P = 0.0004) this difference is significant. A plausible explanation
DABN to aaRS
Et3NHþ—H3, J = 7.3 Hz), 1.32 (s, 9H, Boc—C—ðCH3Þ ), 2.94–3.15
3
(m, 7H, Et3NHþ—H2, Trp-b-HA) 3.26 (dd, 1H, Trp-b-HB, J = 4.2 Hz,
J = 14.3 Hz), 3.97–4.27 (m, 4H, 40-H, 50-H2, Trp-
a-H), 4.36 (d, 1H,
for this increased sensitivity is that the
DABN E. coli strain has a de-
30-H, J = 4.4 Hz), 4.92 (dd, 1H, 20-H, J = 4.4 Hz, J = 7.3 Hz), 5.86 (d,
1H, Trp-amide-NH, J = 7.4 Hz), 5.97 (d, 1H, 10-H, J = 7.3 Hz), 6.87–
7.12 (m, 3H, Trp-2H, Trp-5H, Trp-6H), 7.26 (s, 2H, Ade-NH2), 7.30
(d, 1H, Trp-7-H, J = 8.0 Hz), 7.49 (d, 1H, Trp-4-H, J = 7.8 Hz), 8.14
(s, 1H, 2-H), 8.52 (s, 1H, 8-H), 10.7 (s, 1H, Trp-NH).
creased capacity for using the available peptides present in the
Mueller–Hinton broth, compared to the wt strain. As a result, the
aaRS inhibitors have to compete with lower levels of amino acids
and are more efficient in inhibiting bacterial growth.
13C NMR (DMSO-d6): d ꢂ5.7, 8.7, 17.8, 25.8, 45.8, 66.8, 74.8,
84.1, 110.8, 118.0, 118.9, 123.4, 135.9, 149.9, 154.7, 176.0.
HRMS for C38H59N8O9SSi2 [MꢂH]ꢂ calcd: 859.3664; found:
859.3668.
4. Experimental
4.1. Chemistry
4.1.2.2. 50-O-(N-
34a and Na salt, 34b).
L
-Tryptophanyl)-sulfamoyladenosine (Et3N salt,
Compound 33 (7.40 g, 7.69 mmol,
4.1.1. General information
Reagents and solvents were from commercial suppliers (Acros,
Sigma–Aldrich, Bachem, Novabiochem) and used as provided, un-
less indicated otherwise. DMF and THF were analytical grade and
were stored over molecular sieves (4 Å). All other solvents used
for reactions were analytical grade and used as provided. Reactions
were carried out in oven-dried glassware under a nitrogen atmo-
sphere and stirred at room temperature, unless indicated
otherwise.
1.0 equiv) in TFA/H2O (30 mL, 5:2, v/v) was stirred for 30 min at
rt. Next, the volatiles were evaporated, coevaporated twice with
EtOH and once with EtOH þ Et3N (10 mL), to neutralize any
remaining acid. The residue was dried for 48 h (P2O5, in vacuo,
rt) and Et3N ꢀ 3HF (2.5 mL, 15.3 mmol, 6.0 equiv) was added, fol-
lowed by THF (150 mL, added in portions) and the reaction mixture
was stirred overnight. As TLC analysis (HOAc 1%, MeOH 20% in
CH2Cl2) showed remaining partially protected compound, more
Et3N ꢀ 3HF (2 mL, 12.2 mmol, 4.8 equiv) was added and the reac-
tion was continued for an additional 4 h. Next, Et3N (10 mL) was
added and the volatiles were removed in vacuo. The residual oil
was partly purified on a short silica gel column (Et3N 1%, MeOH
5?30% in CH2Cl2). Fractions containing the title compound were
collected and evaporated, and subjected to silica gel chromatogra-
phy (Et3N 1%, MeOH 10?30% in CH2Cl2) to afford 4.46 g
(7.04 mmol, 92%) of 34a. The compound was used as such for fur-
1H and 13C NMR spectra of the compounds were recorded on a
200 MHz Varian Gemini spectrometer or a Bruker UltraShield
Avance 300 MHz spectrometer. Spectra were recorded in DMSO-
d6, D2O, or CDCl3. The chemical shifts are expressed as d values
in parts per million (ppm), using the residual solvent peaks (chlo-
roform: 1H, 7.26 ppm, 13C, 77.36 ppm; DMSO: 1H, 2.50 ppm, 13C,
39.60 ppm; HOD: 1H, 4.79 ppm) as a reference. For 13C spectra in
D2O, ꢁ0.1% dioxane (67.19 ppm) was added as a reference. Cou-

P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
265
ther syntheses. For analytical purposes and for biological assays, a
small portion of 34a was precipitated as the sodium salt 34b by
adding acetone to a solution of 34a and NaOH (1.2 equiv) in water.
volatiles, the residue was subjected to silica gel chromatogra-
phy (MeOH 20% in CHCl3, v/v). The residue obtained this way
was further purified by size exclusion chromatography to give
133 mg (0.27 mmol, 44%) of 37.
1H NMR (40b, DMSO-d6 þ ðD2OÞ):
d
3.05 (dd, 1H, b-HA,
J = 8.6 Hz, J = 15.1 Hz), 3.68 (dd, 1H,
a
-H, J = 4.5 Hz, J = 8.2 Hz),
1H NMR (DMSO-d6): d 2.93 (dd, 1H, b-HA, J = 7.8, 14.2 Hz), 3.14
4.03–4.24 (m, 5H, 30-H, 40-H, 50-H2), 4.61 (t, 1H, 20-H, J = 5.3 Hz),
5.92 (d, 1H, 10-H, J = 5.8 Hz), 6.99 (m, 1H, Trp-5H), 7.05 (m, 1H,
Trp-6H), 7.23 (d, 1H, Trp-2H, J = 12.6 Hz), 7.35 (d, 1H, Trp-7H,
J = 7.9 Hz), 7.57 (d, 1H, Trp-4-H, J = 7.8 Hz), 8.14 (s, 1H, 2-H), 8.40
(s, 1H, 8-H).
(dd, 1H, b-HB, J = 4.9, 14.2 Hz), 3.66 (dd, 1H, a-H, J = 5.0, 7.7 Hz),
3.95–4.21 (m, 4H, 30-H, 40-H, 50-H2), 4.62 (t, 1H, 20-H, J = 5.3 Hz),
5.92 (d, 1H, 10-H, J = 5.8 Hz), 7.15-7.35 (m, 7H, phenyl-H, Ade-
NH2), 8.14 (s, 1H, 2-H), 8.46 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 37.4, 56.3, 67.6, 70.8, 73.5, 82.5, 87.2,
119.0, 126.8, 128.5, 129.6, 136.3, 139.5, 149.6, 152.7, 156.1, 172.2.
HRMS for C19H24N7O7S [M+H]+ calcd: 494.1458; found:
494.14491.
13C NMR (40b, DMSO-d6): d 27.4, 55.7, 67.6, 70.8, 73.6, 82.6,
87.2, 108.2, 111.5, 118.4, 118.6, 119.0, 121.1, 124.6, 127.3, 136.4,
139.5, 149.7, 152.8, 156.1, 172.5.
HRMS for C21H25N8O7S [M+H]+ calcd: 533.1569; found:
533.1569.
The compounds 50-O-(N-
O-(N-
-leucyl)-sulfamoyladenosine (39), 50-O-(N-
famoyladenosine (40), 50-O-(N-
-aspartyl)-adenosine (41) were
L
-alanyl)-sulfamoyladenosine (38), 50-
L
L-methionyl)-sul-
L
4.1.2.3. 20,30-Di-O-(tert-butyldimethylsilyl)-50-O-[N-(N-Boc-
L
-
synthesized by the methods described above for the synthesis of
TrpSA and PheSA. Analytical data of the final compounds were in
agreement with literature values.6,10,23–27
phenylalanyl)-sulfamoyl]adenosine (Et3N salt, 35).
Fol-
lowing the procedure used for the synthesis of 33, 20,30-di-O-
(tert-butyldimethylsilyl)-50-O-sulfamoyladenosine (32, 575
mg, 1.00 mmol, 1.0 equiv) and N-Boc-
L
-phenylalanine N-
4.1.3. Preparation of 50-O-(N-dipeptidyl)-sulfamoyladenosines
hydroxysuccinimide ester (435 mg, 1.20 mmol, 1.2 equiv) were
reacted to give 892 mg (0.96 mmol, 97%) of 35.
12–29
4.1.3.1. 20,30-Di-O-(tert-butyldimethylsilyl)-50-O-[N-[N-(N-Boc-
L
-
1H NMR (DMSO-d6): d ꢂ0.42 (s, 3H, Si—CH3), ꢂ0.08 (s, 3H,
Si—CH3), 0.11 (s, 3H, Si—CH3), 0.13 (s, 3H, Si—CH3), 0.66 (s, 9H,
Si—C ꢂ ðCH3Þ3), 0.92 (s, 9H, Si—C—ðCH3Þ3), 1.15 (t, 9H,
phenylalanyl)-
L-phenylalanyl]-sulfamoyl]adenosine (Et3N salt,
42). A solution of 36 (375 mg, 0.46 mmol, 1.0 equiv), N-Boc-
L-
phenylalanine (179 mg, 0.68 mmol, 1.5 equiv) and HOBt (50 mg,
0.37 mmol, 0.8 equiv) in DMF (5 mL) was cooled to 0 °C and ED-
CIꢀHCl (104 mg, 0.54 mmol, 1.2 equiv) was added. The mixture
was stirred and allowed to come to rt overnight. Next, the volatiles
were removed in vacuo and the residue was purified by silica gel
column chromatography (Et3N 1%, MeOH 5% in CH2Cl2), yielding
470 mg (0.44 mmol, 96%) of 42.
Et3NHþ—H3, J = 7.3 Hz), 1.31 (s, 9H, Boc—C—ðCH3Þ ), 2.67–3.15
3
(m, 8H, Et3NHþ—H3, b-H2), 3.87–4.24 (m, 4H, 40-H, 50-H2, Phe-
a
-
H), 4.34 (d, 1H, 30-H, J = 4.4 Hz), 4.92 (dd, 1H, 20-H, J = 4.4 Hz,
J = 7.3 Hz), 5.96 (d, 1H, 10-H, J = 7.2 Hz), 7.13–7.36 (m, 7H, phe-
nyl-H, Ade-NH2), 8.13 (s, 1H, 2-H), 8.49 (s, 1H, 8-H).
13C NMR (DMSO-d6): d ꢂ5.7, ꢂ4.7, 9.3, 17.4, 17.8, 25.5, 25.8,
28.2, 36.8, 45.7, 57.5, 66.9, 73.4, 74.7, 77.53, 84.2, 86.1, 119.0,
126.0, 128.0, 128.2, 129.4, 129.6, 139.0 and 139.7, 150.1, 152.9,
154.9, 156.2, 175.6.
HRMS for C45H69N8O10SSi2 [M+H]+ calcd: 969.4396; found:
969.4408.
HRMS for C36H60N7O9SSi2 [M+H]+ calcd: 822.3712; found:
822.3717.
4.1.3.2. 50-O-[N-(N -
adenosine (12). Compound 42 (470 mg, 0.44 mmol, 1.0 equiv)
L-Phenylalanyl)-L-phenylalanyl]-sulfamoyl-
was treated with TFA/H2O (5:2, v/v, 5 mL) for 2 h at rt. Next, the
volatiles were evaporated, coevaporated twice with EtOH and once
with EtOH þ Et3N (1 mL). After drying (16 h, in vacuo, P2O5), the
4.1.2.4. 20,30-Di-O-(tert-butyldimethylsilyl)-50-O-[N-(
anyl)-sulfamoyl]adenosine (Et3N salt, 36). Compound 35 was
L-phenylal-
dissolved in TFA/H2O (5:2 v/v, 5 mL) and stirred for 2 h at rt. Next,
the volatiles were evaporated, coevaporated twice with EtOH and
once with EtOH þ Et3N (10 mL), to neutralize any remaining acid.
The residue was purified by silica gel chromatography (Et3N 1%,
MeOH 5?10% in CH2Cl2) to give 389 mg of 36.
residue was dissolved in THF (20 mL) and Et3N ꢀ 3HF (381
lL,
2.34 mmol, 16.0 equiv) was added. The reaction mixture was stir-
red overnight at rt. Next, Et3N (0.5 mL) was added and the volatiles
were removed in vacuo. The residue was purified by silica gel chro-
matography (Et3N 1%, MeOH 5?10% in CH2Cl2) to give 162 mg
(0.21 mmol, 47%) of 12.
1H NMR (DMSO-d6): d ꢂ0.38 (s, 3H, Si—CH3), ꢂ0.09 (s, 3H,
Si—CH3), 0.11 (s, 3H, Si—CH3), 0.13 (s, 3H, Si—CH3), 0.69 (s, 9H,
Si—C—ðCH3Þ ), 0.92 (s, 9H, Si—C—ðCH3Þ ), 1.18 (t, Et3NHþ—H3,
1H NMR (DMSO-d6 þ D2O): d 2.70–2.91 (m, 2H, 2 ꢃ Phe-b-H),
3.00–3.22 (m, 2H, 2 ꢃ Phe-b-H), (m, 1H, Phe1-
a-H), 3.94–4.18
3
3
J = 7.3 Hz), 2.85–3.30 (m, Et3NHþ—H3, b-H2), 3.69 (m, 1H,
a-H),
(m, 4H, 40-H, 50-H2, Phe2- -H), 4.28 (dd, 1H, 30-H, J = 4.7, 8.1 Hz),
a
3.94-4.55 (m, 4H, 40-H, 50-H2, 30-H), 4.92 (dd, 1H, 20-H, J = 4.4 Hz,
J = 7.0 Hz), 5.96 (d, 1H, 10-H, J = 6.8 Hz), 7.14-7.46 (m, 7H, phenyl-
H, Ade-NH2), 8.17 (s, 1H, 2-H), 8.49 (s, 1H, 8-H).
4.59 (t, 1H, 20-H, J = 5.5 Hz), 5.91 (d, 1H, 10-H, J = 6.1 Hz), 7.10–
7.35 (m, 10H, Aryl-H), 8.13 (s, 1H, 2-H), 8.39 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 37.9, 38.3, 53.9, 56.9, 67.4, 71.0, 73.6,
82.8, 86.9, 118.9, 126.0, 126.9, 128.0, 128.5, 129.4, 129.7, 138.7,
139.4, 149.7, 152.7, 156.1, 174.8.
13C NMR (DMSO-d6): d ꢂ5.6, ꢂ5.4, ꢂ4.8, 8.5, 17.4, 17.7, 25.5,
25.7, 37.1, 45.7, 56.2, 67.3, 73.1, 74.6, 83.7, 86.6, 119.1, 126.9,
128.5, 129.7, 36.3, 139.9, 149.8, 152.5, 156.0, 172.0.
HRMS for C31H52N7O7SSi2 [M+H]+ calcd: 722.3187; found:
722.3166.
HRMS for C28H31N8O8S [MꢂH]ꢂ calcd: 639.1986; found:
639.1989.
4.1.3.3. 50-O-[N-[N-(N -Boc-
sine (Et3N salt, 43). A solution of 39 (230 mg, 0.41 mmol,
1.0 equiv), N-Boc- -alanine (85 mg, 0.45 mmol, 1.1 equiv) and
L-alanyl)-L-leucyl]-sulfamoyl]adeno-
4.1.2.5. 50-O-[N-(
salt, 37). Compound 35 (500 mg, 0.61 mmol, 1.0 equiv)
L-Phenylalanyl)-sulfamoyl]adenosine (Et3N
L
was dissolved in TFA/H2O (5:2, v/v, 5 mL) and stirred for 1.5 h
at rt. Next, the volatiles were evaporated, coevaporated twice
with EtOH and once with EtOH þ Et3N (10 mL). After removal
of the volatiles, the residue was dissolved in THF (20 mL) and
HOBt (50 mg, 0.37 mmol, 0.9 equiv) in DMF (5 mL) was cooled to
0 °C and EDCIꢀHCl (86 mg, 0.45 mmol, 1.1 equiv) was added. The
mixture was allowed to come to rt and stirred overnight. Next,
the volatiles were removed in vacuo and the residue was purified
by silica gel column chromatography (Et3N 1%, MeOH 10?20% in
CH2Cl2), yielding 293 g (0.40 mmol, 98%) of 43.
Et3N ꢀ 3HF (400
lL, 2.45 mmol, 12 equiv) was added and the
mixture was stirred overnight at rt. After evaporation of the

266
P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
HRMS for C24H37N8O10
629.2352.
S
[MꢂH]ꢂ calcd: 629.2354; found:
Leu2-a-H, Pro1-a
-H), 4.60 (t, 1H, 20-H, J = 5.3 Hz), 5.91 (d, 1H, 10-
H, J = 6.0 Hz), 8.14 (s, 1H, 2-H), 8.38 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 21.9, 23.3, 23.7, 24.5, 29.7, 42.0, 45.9,
54.0, 59.1, 67.3, 71.0, 73.6, 82.8, 87.0, 119.0, 139.5, 149.7, 152.7,
156.1, 167.9, 176.1.
4.1.3.4. 50-O-[N-(N-
L-Alanyl)-L-leucyl]-sulfamoyladenosine
(13).
Compound 43 (280 mg, 0.38 mmol, 1.0 equiv) was dis-
solved in ice-cold TFA/H2O 5:2 (5 mL, 5:2, v/v) and the resulting
solution was stirred and allowed to come to rt. After 1.5 h, the
volatiles were removed and the residue was coevaporated twice
with EtOH and once with Et3N (1 mL). The volatiles were re-
moved again and the residue was purified by silica gel chroma-
tography (Et3N 1%, MeOH 10?20% in CH2Cl2) and further
purified by size exclusion chromatography to yield 120 mg
(0.23 mmol, 61%) of 13.
HRMS for C21H32N8O8S [M+H]+ calcd: 557.2142; found:
557.2145.
4.1.3.9. 50-O-[N-[N-(Na; N -Di-Boc-
adenosine (Et3N salt, 46).
L
-lysyl)-
Following the procedure used for
the synthesis of 43, 39 (260 mg, 0.46 mmol, 1.0 equiv) and
Na; N -di-Boc- -lysine (173 mg, 0.50 mmol, 1.1 equiv) were re-
L-leucyl]-sulfamoyl]-
ꢀ
L
ꢀ
acted to yield 310 mg (0.35 mmol, 76%) of 46.
1H NMR (DMSO-d6 þ D2O): d 0.83 (m, 6H, Leu2-d ꢂ H3), 1.28–
HRMS for C32H52N9O12
786.3429.
S
[MꢂH]ꢂ calcd: 786.3456; found:
1.45 (m, 4H, Ala1-H3, Leu2-b-HA), 1.45–1.66 (m, 2H, Leu2-b-HB,
Leu2-
c
-H), 3.79 (q, 1H, Ala1-
a-H, J = 7.0 Hz), 3.93–4.18 (m, 5H,
30-H, 40-H, 50-H2, Leu2-
a
-H), 4.57 (t, 1H, 20-H, J = 5.4 Hz), 5.90 (d,
4.1.3.10. 50-O-[N-(N-
L-Lysyl)-L-leucyl]-sulfamoyladenosine (16).
1H, 10-H, J = 5.9 Hz), 8.14 (s, 1H, 2-H), 8.37 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 17.6, 21.9, 23.3, 24.4, 42.2, 48.3, 54.0,
67.3, 71.0, 73.6, 82.8, 87.0, 119.0, 139.5, 149.7, 152.7, 156.1,
169.0, 176.2.
Following the procedure used for the synthesis of 13, 46 (305 mg,
0.34 mmol, 1.0 equiv) was deprotected. For silica gel chromatogra-
phy Et3N 1%, MeOH 10?70% in CH2Cl2 was used. This way, 66 mg
(0.11 mmol, 32%) of 16 was obtained.
HRMS for C19H31N8O8S [M+H]+ calcd: 531.1985; found:
531.1979.
1H NMR (DMSO-d6 þ D2O): d 0.85 (m, 6H, Leu2-d ꢂ H3), 1.13–
1.68 (m, 9H, Lys1-b-H2, Lys1-c-H2, Lys1-d-H2, Leu2-b-H2, Leu2-c-
H), 2.75 (t, 2H, Lys1-ꢂH2, J = 6.5 Hz), 3.25 (t, 1H, Lys1-
a
-H,
4.1.3.5. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 44).
L
-methionyl)-
Following the procedure used for
L
-leucyl]-sulfamoyl]-
J = 5.8 Hz), 3.96–4.20 (m, 5H, 30-H, 40-H, 50-H2, Leu2-
a-H), 4.58 (t,
1H, 20-H, J = 5.2 Hz), 5.91 (d, 1H, 10-H, J = 5.6 Hz), 8.15 (s, 1H, 2-
H), 8.43 (s, 1H, 8-H).
the synthesis of 43, 39 (250 mg, 0.45 mmol, 1.0 equiv) and N-
Boc- -methionine (125 mg, 0.50 mmol, 1.1 equiv) were reacted
L
13C NMR (DMSO-d6): d 21.6, 22.0, 23.3, 24.5, 26.9, 33.9, 42.4,
48.7, 53.4, 53.8, 67.3, 70.8, 73.7, 82.5, 87.1, 119.0, 139.5, 149.6,
152.7, 156.1, 173.3, 177.4.
to yield 330 mg (0.42 mmol, 93%) of 44.
HRMS for C26H43N8O10S2 [M+H]+ calcd: 691.2543; found:
691.2548.
HRMS for C22H38N9O8S [M+H]+ calcd: 588.2564; found:
588.2564.
4.1.3.6. 50-O-[N-(N-
(14). Following the procedure used for the synthesis of 13, 44
L-Methionyl)-L-leucyl]-sulfamoyladenosine
4.1.3.11. 50-O-[N-[N-(N-Boc-
leucyl]-sulfamoyl]adenosine (Et3N salt; 47).
procedure used for the synthesis of 43, 39 (250 mg, 0.45 mmol,
1.0 equiv) and N-Boc- -glutamic acid -O-tert-butyl ester
L
-Glutamyl-
c
-O-tert-butyl ester)-
L-
(325 mg, 0.41 mmol, 1.0 equiv) was deprotected to yield 162 mg
(0.27 mmol, 67%) of 14.
Following the
1H NMR (DMSO-d6 þ D2O): d 0.83 (m, 6H, Leu2-d ꢂ H3), 1.30–
L
c
1.46 (m, 1H, Leu2-b-HA), 1.46–1.67 (m, 2H, Leu2-b-HB, Leu2-
c-H),
(152 mg, 0.50 mmol, 1.1 equiv) were reacted to yield 347 mg
1.76–2.08 (m, 5H, Met1-S-H3, Met1-b-H2), 2.52 (partly obscured
by DMSO-d6-peak, Met1- -H2), 3.73 (m, 1H, Met1- -H, visible be-
(0.41 mmol, 91%) of 47.
HRMS for C27H41N8O12
701.2560.
c
a
S
[MꢂH]ꢂ calcd: 701.2565; found:
fore addition of D2O), 3.93–4.18 (m, 5H, 30-H, 40-H, 50-H2, Leu2-
a-
H), 4.56 (t, 1H, 20-H, J = 5.5 Hz), 5.90 (d, 1H, 10-H, J = 6.0 Hz), 8.14
(s, 1H, 2-H), 8.37 (s, 1H, 8-H).
4.1.3.12. 50-O-[N-(N-
(Et3N salt, 17). Following the procedure used for the synthesis
L-Glutamyl)-L-leucyl]-sulfamoyladenosine
13C NMR (DMSO-d6): d 14.5, 21.9 and 23.3, 24.4, 28.5, 31.9, 42.1,
52.1, 53.8, 67.3, 71.0, 73.6, 82.8, 86.9, 119.0, 139.5, 149.7, 152.7,
156.1, 168.5, 176.1.
of 13, 47 (342 mg, 0.40 mmol, 1.0 equiv) was deprotected. For sil-
ica gel chromatography Et3N 1%, MeOH 10?50% in CH2Cl2 was
used. This way, 143 mg (0.21 mmol, 52%) of 17 was obtained.
1H NMR (DMSO-d6 þ D2O): d 0.86 (m, 6H, Leu2-d ꢂ H3), 1.15 (t,
HRMS for C21H33N8O8S2 [MꢂH]ꢂ calcd: 589.1863; found:
589.1840.
Et3NHþ—H3, J = 7.3 Hz), 1.19–1.70 (m, 3H, Leu2-b-H2, Leu2-
c
-H),
-H2, J = 7.6 Hz), 2.96
(q, Et3NHþ—H2, J = 7.3 Hz), 3.69 ( before addition of D2O: t, 1H,
4.1.3.7. 50-O-[N-[N-(N -Boc-
sine (Et3N salt, 45). Following the procedure used for the syn-
thesis of 43, 39 (280 mg, 0.50 mmol, 1.0 equiv) and N-Boc-
L
-Prolyl)-
L
-leucyl]-sulfamoyl]adeno-
1.89 (m, 2H, Glu1-b-H2), 2.40 (t, 2H, Glu1-c
L
-
Glu1-a a-
-H, J = 6.1 Hz), 3.92–4.18 (m, 5H, 30-H, 40-H, 50-H2, Leu2-
proline (85 mg, 0.55 mmol, 1.1 equiv) were reacted to yield
H), 4.58 (t, 1H, 20-H, J = 5.1 Hz), 5.90 (d, 1H, 10-H, J = 6.1 Hz), 8.14
(s, 1H, 2-H), 8.38 (s, 1H, 8-H).
381 mg (0.50 mmol, 100%) of 45.
HRMS for C26H39N8O10
655.2488.
S
[MꢂH]ꢂ calcd: 655.2510; found:
13C NMR (DMSO-d6): d 9.0, 21.8, 23.3, 24.4, 27.6, 30.7, 42.1, 45.5,
52.3, 53.8, 67.3, 71.0, 73.7, 82.8, 86.9, 118.9, 139.4, 149.7, 152.7,
156.1, 168.8, 174.5, 176.2.
4.1.3.8. 50-O-[N-(N-
(15). Following the procedure used for the synthesis of
L
-Prolyl)-
L
-leucyl]-sulfamoyladenosine
HRMS for C21H32N8O10
587.1863.
S
[MꢂH]ꢂ calcd: 587.1884; found:
13, 45 (375 mg, 0.49 mmol, 1.0 equiv) was deprotected to
yield 184 mg (0.33 mmol, 67%) of 15.
4.1.3.13. 50-O-[N-[N-(N -Boc-
osine (Et3N salt, 48). Following the procedure used for the syn-
thesis of 43, 38 (240 mg, 0.46 mmol, 1.0 equiv) and N-Boc-
L-Alanyl)-L-alanyl]-sulfamoyl]aden-
1H NMR (DMSO-d6 þ D2O): d 0.85 (m, 6H, Leu2-d ꢂ H3), 1.32–
1.48 (m, 1H, Leu2-b-HA), 1.52–1.67 (m, 2H, Leu2-b-HB, Leu2-
c-H),
L-
1.73–1.87 (m, 2H, Pro1-
c
ꢂ H2), 1.87–2.01 (m, 1H, Pro1-b-HA),
alanine (87 mg, 0.46 mmol, 1.0 equiv) were reacted to yield
211 mg (0.31 mmol, 67%) of 48.
2.15–2.32 (m, 1H, Pro1-b-HB), 3.2 (m, partly obscured by residual
Et3NHþ—H2, Pro1-d ꢂ H2), 3.93-4.19 (m, 6H, 30-H, 40-H, 50-H2,
ESI-MS for C21H33N8O10S [M+H]+ calcd: 589.2; found: 589.1.

P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
267
4.1.3.14. 50-O-[N-(N-
(Et3N salt, 18). Following the procedure used for the syn-
L
-Alanyl)-
L
-alanyl]-sulfamoyladenosine
Boc-L-alanine (116 mg, 0.61 mmol, 1.3 equiv) were reacted to
yield 303 mg (0.40 mmol, 87%) of 51.
HRMS for C23H35N8O10S2 [MꢂH]ꢂ calcd: 647.1996; found:
647.1912.
thesis of 13, 48 (205 mg, 0.30 mmol, 1.0 equiv) was deprotec-
ted to yield 118 mg (0.20 mmol, 67%) of 18.
1H NMR (DMSO-d6 þ D2O): d 1.17 (t, Et3NHþ—H3, J = 7.2 Hz),
1.23 (d, 3H, Ala2-b-H3, J = 6.6 Hz), 1.33 (d, 1H, Ala1-b-H3,
J = 6.5 Hz), 3.05 (q, 6H, Et3NHþ—H2, J = 7.2 Hz), 3.77–3.85 (t, 1H,
4.1.3.20. 50-O-[N-(N-
(Et3N salt, 21). Following the procedure used for the synthesis
L-Alanyl)-L-methionyl]-sulfamoyladenosine
Ala1-
a
-H, J = 6.7 Hz), 3.95–4.21(m, 5H, 30-H, 40-H, 50-H2, Ala2-
a
-
of 13, 51 (298 mg, 0.40 mmol, 1.0 equiv) was deprotected to yield
63 mg (0.10 mmol, 24%) of 21.
H), 4.57 (t, 1H, 20-H, J = 5.0 Hz), 5.91 (d, 1H, 10-H, J = 5.8 Hz), 8.14
(s, 1H, 2-H), 8.38 (s, 1H, 8-H).
1H NMR (DMSO-d6 þ D2O): d 1.14 (t, Et3NHþ—H3, J = 7.1 Hz),
1.33 (d, 3H, Ala1-b-H3, J = 6.6 Hz), 1.67–1.88 (m, 1H, Met2-b-HA),
1.88–2.06 (m, 4H, Met2-b-HA, Met-S-CH3), 2.43 (m, 2H, Met2-
13C NMR (DMSO-d6): d 8.7, 17.4, 19.3, 45.5, 48.2, 51.0, 67.3, 71.0,
73.6, 82.8, 87.0, 119.0, 139.5, 149.7, 152.7, 156.1, 168.5, 176.0.
HRMS for C16H24N8O8S [MꢂH]ꢂ calcd: 487.1360; found:
487.1357.
c
-H2) 3.03 (q, Et3NHþ—H2, J = 7.1), 3.78–3.92 (m, 1H, Ala1-
a-H),
3.95–4.24 (m, 5H, 30-H, 40-H, 50-H2, Met2- -H), 4.59 (t, 1H, 20-H,
a
J = 4.7 Hz), 5.91 (d, 1H, 10-H, J = 5.5 Hz), 8.15 (s, 1H, 2-H), 8.38 (s,
4.1.3.15. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 49).
L
-methionyl)-
Following the procedure used for
L-alanyl]-sulfamoyl]-
1H, 8-H).
13C NMR (DMSO-d6): d 9.1, 14.7, 17.5, 29.9, 32.9, 45.7, 48.3, 54.5,
67.4, 70.9, 73.6, 82.7, 87.0, 119.0, 139.4, 149.7, 152.7, 156.1, 169.2,
the synthesis of 43, 38 (270 mg, 0.52 mmol, 1.0 equiv) and N-
Boc- -methionine (130 mg, 0.52 mmol, 1.0 equiv) were reacted
174.8.
L
HRMS for C18H27N8O8S2 [MꢂH]ꢂ calcd: 547.1394; found:
to yield 269 mg (0.36 mmol, 69%) of 49.
HRMS for C23 H35 N8O10 S2 [MꢂH]ꢂ calcd: 647.1918; found: 647.1920.
547.1394.
4.1.3.21. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 52).
synthesis of 43, 40 (250 mg, 0.43 mmol, 1.0 equiv) and N-Boc-L-methi-
L
-methionyl)-vmethionyl]-sulfamoyl]-
Following the procedure used for the
4.1.3.16. 50-O-[N-(N-
(Et3N salt, 19). Following the procedure used for the synthesis
L-Methionyl)-L-alanyl]-sulfamoyladenosine
of 13, 49 (264 mg, 0.35 mmol, 1.0 equiv) was deprotected to yield
177 mg (0.27 mmol, 78%) of 19.
onine (116 mg, 0.51 mmol, 1.2 equiv) were reacted to yield 292 mg
(0.40 mmol, 88%) of 52.
1H NMR (DMSO-d6 þ D2O): d 1.10–1.34 (m, Et3NHþ—H3, Ala2-
HRMS for C25H39N8O10S3 [MꢂH]ꢂ calcd: 707.1952; found:
707.1965.
b-H3), 1.88–2.17 (m, 5H, Met1-S-H3, Met1-b-H2), 2.56 (partly ob-
scured by DMSO-d6-peak, Met1-
J = 7.2 Hz), 3.83 (t, 1H, Met1-
30-H, 40-H, 50-H2, Ala2-
c
ꢂ H2), 3.05 (q, 2 ꢃ Et3NHþ—H2,
a
-H, J = 5.9 Hz), 3.94–4.21 (m, 5H,
4.1.3.22. 50-O-[N-(N-
nosine (22). Following the procedure used for the synthesis of
L-Methionyl)-L-methionyl]-sulfamoylade-
a
-H), 4.58 (m, 1H, 20-H), 5.91 (d, 1H, 10-H,
J = 5.8 Hz), 8.14 (s, 1H, 2-H), 8.38 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 8.6, 14.5, 19.1, 28.3, 31.3, 45.6, 51.0, 51.6,
67.4, 70.9, 73.6, 82.8, 87.0, 119.0, 139.5, 149.7, 152.8, 156.0, 167.2,
176.0.
13, 52 (287 mg, 0.35 mmol, 1.0 equiv) was deprotected to yield
130 mg (0.10 mmol, 21%) of 22.
1H NMR (DMSO-d6 þ D2O): d 1.67–2.09 (m, 10H, 2 ꢃ Met-b-H2,
2 ꢃ Met-S-CH3), 2.32–2.6 (m, partly obscured by DMSO-d6,
HRMS for C18H28N8O8S2 [MꢂH]ꢂ calcd: 547.1394; found:
547.1379.
2 ꢃ Met-
c-H2) 3.72 (t, 1H, Met1-a-H, J = 6.3 Hz), 3.94-4.20 (m,
5H, 30-H, 40-H, 50-H2, Met2-
a
-H), 4.57 (t, 1H, 20-H, J = 5.5 Hz), 5.91
(d, 1H, 10-H, J = 6.0 Hz), 8.15 (s, 1H, 2-H), 8.38 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 14.6, 14.7, 28.6, 29.9, 31.9, 32.7, 52.2,
54.5, 67.3, 70.9, 73.6, 82.7, 86.9, 119.0, 139.4, 149.7, 152.7, 156.1,
168.8, 174.8.
4.1.3.17. 50-O-[N-[N-(N -Boc-
osine (Et3N salt, 50). Following the procedure used for the syn-
thesis of 43, 38 (213 mg, 0.41 mmol, 1.0 equiv) and N-Boc- -proline
L-prolyl)-L-alanyl]-sulfamoyl]aden-
L
(88 mg, 0.41 mmol, 1.0 equiv) were reacted to yield 254 mg
HRMS for C20 H31N8O8S3 [MꢂH]ꢂ calcd: 607.1427; found: 607.1438.
(0.35 mmol, 87%) of 50.
HRMS for C23H33N8O10
613.2035.
S
[MꢂH]ꢂ calcd: 613.2041; found:
4.1.3.23. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 53).
L
-prolyl)-
L-methionyl]-sulfamoyl]-
Following the procedure used for
the synthesis of 43, 40 (265 mg, 0.46 mmol, 1.0 equiv) and N-
Boc- -proline (120 mg, 0.56 mmol, 1.2 equiv) were reacted to
yield 294 mg (0.38 mmol, 83%) of 53.
HRMS for C25H37N8O10S2 [MꢂH]ꢂ calcd: 673.2074; found:
673.2045.
4.1.3.18. 50-O-[N-(N-
(Et3N salt, 20). Following the procedure used for the syn-
L-Prolyl)-L-alanyl]-sulfamoyladenosine
L
thesis of 13, 50 (250 mg, 0.35 mmol, 1.0 equiv) was deprotec-
ted to yield 150 mg (0.24 mmol, 69%) of 20.
1.16 (t, Et3NHþ—H3, J = 7.3 Hz), 1.22 (d,
1H NMR (DMSO-d6 þ D2O): d
Ala2-b-H3, J = 4.7 Hz), 1.75–2.06 (m, 3H, Pro1-b-HA, Pro1-
c
ꢂ H2),
4.1.3.24. 50-O-[N-(N -
(Et3N salt, 23). Following the procedure used for the synthesis
L-Prolyl)-L-methionyl]-sulfamoyladenosine
2.18-2.33 (m, 1H, Pro1-b-HA), 2.94–3.27 (m, Et3NHþ—H2, Pro1-
d ꢂ H2), 3.92–4.22 (m, 7H, 30-H, 40-H, 50-H2, Ala2-
a-H, Pro1-a-H),
of 13, 53 (289 mg, 0.37 mmol, 1.0 equiv) was deprotected to yield
139 mg (0.21 mmol, 56%) of 23.
4.61 (t, 1H, 20-H, J = 5.5 Hz), 5.90 (d, 1H, 10-H, J = 6.1 Hz), 8.14 (s,
1H, 2-H), 8.38 (s, 1H, 8-H).
1H NMR (DMSO-d6 þ D2O): d 1.18 (t, Et3NHþ—H3, J = 7.3 Hz),
13C NMR (DMSO-d6): d 8.6, 19.1, 23.6, 29.6, 45.6, 45.9, 51.2, 59.0,
67.4, 71.0, 73.6, 82.8, 87.0, 119.0, 139.5, 149.7, 152.7, 156.1, 167.3,
175.9.
1.67–2.08 (m, 8H, Pro1-
CH3), 2.16–2.34 (m, 1H, Pro1-b-HB), 2.44 (m, partly obscured by
c
ꢂ H2, Pro1-b-HA, Met2-b-H2, Met2-S-
DMSO- d6-peak, Met2-c-H), 2.92-3.28 (m, partly obscured by
HRMS for C18H26N8O8S [MꢂH]ꢂ calcd: 513.1516; found:
513.1512.
HOD-peak, Et3NHþ—H2, Pro1-d ꢂ H2), 3.93–4.25 (m, 6H, 30-H, 40-
H, 50-H2, Met2- -H), 4.59 (t, 1H, 20-H, J = 5.5 Hz), 5.91
a-H, Pro1-a
(d, 1H, 10-H, J = 6.1 Hz), 8.14 (s, 1H, 2-H), 8.38 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 8.6, 14.7, 23.6, 29.6, 29.9, 32.5, 45.5, 45.8,
54.8, 58.9, 67.4, 70.9, 73.6, 82.7, 87.0, 119.0, 139.4, 149.7, 152.7,
156.1, 167.8, 174.8.
4.1.3.19. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 51).
L
-alanyl)-
Following the procedure used for
L-methionyl]-sulfamoyl]-
the synthesis of 43, 40 (266 mg, 0.46 mmol, 1.0 equiv) and N-

268
P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
HRMS for C20H29N8O8S2 [MꢂH]ꢂ calcd: 573.1550; found:
thesis of 13, 56 (235 mg, 0.28 mmol, 1.0 equiv) was deprotec-
ted to yield 132 mg (0.21 mmol, 75%) of 26.
573.1556.
1H NMR (DMSO-d6 þ D2O): d 1.64–1.79 (m, 2H, Pro1-
c-H2),
4.1.3.25. 50-O-[N-[Na-(N-Boc-
adenosine (Et3N salt, 54).
synthesis of 43, 34a (298 mg, 0.47 mmol, 1.0 equiv) and N-Boc-
L
-alanyl)-
Following the procedure used for the
-ala-
L
-tryptophanyl]-sulfamoyl]-
1.79–1.94 (m, 1H, Pro1-b-HA), 2.08–2.24 (m, 1H, Pro1-b-HB),
2.87–3.07 (m, 3H, Pro1-d ꢂ H2, Trp2-b-HA), 3.26 (dd, partly ob-
scured by D2O-peak, Trp2-b-HB, J = 4.1 Hz, J = 14.4 Hz), 3.89–4.20
L
nine (95 mg, 0.50 mmol, 1.1 equiv) were reacted to yield 180 mg
(m, 5H, 40-H, 50-H2, Pro1- -H), 4.34 (m, 1H, 30-H), 4.62
a-H, Trp2-a
(0.22 mmol, 48%) of 54.
HRMS for C29H36N9O10
702.2316.
(t, 1H, 20-H, J = 5.6 Hz), 5.92 (d, 1H, 10-H, J = 6.2 Hz), 6.92 (m, 1H,
Trp-5H), 7.00 (m, 1H, Trp-6-H), 7.10 (s, 1H, Trp-2-H), 7.30 (d, 1H,
Trp-7H, J = 8.0 Hz), 7.53 (d, 1H, Trp-4-H, J = 7.7 Hz), 8.14 (s, 1H, 2-
H), 8.41 (s, 1H, 8-H).
S
[MꢂH]ꢂ calcd: 702.2306; found:
4.1.3.26. 50-O-[N-[Na-(
adenosine (24). Following the procedure used for the syn-
L
-Alanyl)-
L
-tryptophanyl]-sulfamoyl]-
13C NMR (DMSO-d6): d 23.9, 28.3, 29.7, 46.0, 56.5, 59.2, 67.3,
71.0, 73.7, 82.8, 86.9, 111.0, 111.2, 118.2, 118.4, 119.0, 120.7,
123.3, 127.7, 136.0, 139.4, 149.8, 152.7, 156.1, 168.6, 175.4.
HRMS for C26H30N9O8S [MꢂH]ꢂ calcd: 628.1938; found:
628.1934.
thesis of 13, 54 (175 mg, 0.22 mmol, 1.0 equiv) was deprotec-
ted to yield 44 mg (0.07 mmol, 31%) of 24.
1H NMR (DMSO-d6 þ D2O): d 1.31 (d, 3H, Ala-H3, J = 7.09 Hz),
2.92 (dd, 1H, Trp2-b-HA, J = 8.7 Hz, J = 14.7 Hz), 3.25 (partly ob-
scured by H2O-peak, dd, Trp2-b-HB, J = 4.4 Hz, J = 14.9 Hz), 3.67
4.1.3.31. 50-O-[N-[Na-(Na; N-Di-Boc-
famoyl]adenosine (Et3N salt, 57).
L
-lysyl)-
Following the procedure
L-tryptophanyl]-sul-
(q, 1H, Ala1-
4.18 (m, 1H, Trp2-
a
-H, J = 6.9 Hz), 3.92–4.12 (m, 4H, 40-H, 50-H2), 4.12-
a
-H), 4.34 (m, 1H, 30-H), 4.61 (t, 1H, 20-H,
used for the synthesis of 43, 34a (320 mg, 0.50 mmol, 1.0 equiv)
and Na; N-di-Boc- -lysine (173 mg, 0.50 mmol, 1.0 equiv) were re-
J = 5.6 Hz), 5.93 (d, 1H, 10-H, J = 6.2 Hz), 6.95 (m, 1H, Trp-5-H),
7.03 (m, 1H, Trp-6-H), 7.12 (s, 1H, Trp-2-H), 7.30 (d, 1H, Trp-7H,
J = 8.0 Hz), 7.55 (d, 1H, Trp-4-H, J = 7.7 Hz), 8.14 (s, 1H, 2-H), 8.41
(s, 1H, 8-H).
L
acted to yield 359 mg (0.37 mmol, 75%) of 57.
HRMS for C37H51N10O12
859.3409.
S
[MꢂH]ꢂ calcd: 859.3409; found:
13C NMR (DMSO-d6): d 18.0, 28.4, 48.5, 56.4, 67.3, 71.0, 73.7,
82.8, 86.9, 111.1, 111.2, 118.2, 118.5, 118.9, 120.7, 123.4, 127.7,
136.1, 139.4, 149.8, 152.7, 156.1, 169.7, 175.6.
HRMS for C24H30N9O8S [M+H]+ calcd: 604.1938; found:
604.1932.
4.1.3.32. 50-O-[N -(Na-
sine (27). Following the procedure used for the synthesis of
L-Lysyl)-L-tryptophanyl]-sulfamoyladeno-
16, 57 (355 mg, 0.37 mmol, 1.0 equiv) was deprotected to yield
61 mg (0.09 mmol, 25%) of 27.
1H NMR (DMSO-d6 þ D2O): d 1.09–1.55 (m, 6H, Lys1-b-H2, Lys1-
4.1.3.27. 50-O-[N-[Na-(N -Boc-
famoyl]adenosine (Et3N salt, 55).
used for the synthesis of 43, 34a (230 mg, 0.36 mmol, 1.0 equiv)
and N-Boc- -methionine (90 mg, 0.36 mmol, 1.0 equiv) were re-
L
-methionyl)-
L-tryptophanyl]-sul-
c-H2, Lys1-d-H2), 2.60 (m, 2H, Lys1--H2), 2.96 (dd, 1H, Trp2-b-HA,
Following the procedure
J = 7.4 Hz, J = 14.7 Hz), 3.02-3.10 (m, 1H, Lys1-
b-HB, J = 4.7 Hz, J = 14.6 Hz), 3.94–4.13 (m, 3H, 40-H,50-H2), 4.13–4.19
(m, 1H, Trp2-
-H), 4.31 (m, 1H, 30-H), 4.59 (t, 1H, 20-H, J = 5.5 Hz),
a-H), 3.20 (dd, Trp2-
L
a
acted to yield 193 mg (0.22 mmol, 61%) of 55.
HRMS for C31H40N9O10S2 [MꢂH]ꢂ calcd: 762.2340; found:
762.2331.
5.92 (d, 1H, 10-H, J = 6.0 Hz), 6.92 (m, 1H, Trp2-5-H), 7.01 (m, 1H,
Trp2-6-H), 7.09 (s, 1H, Trp2-2-H), 7.28 (d, 1H, Trp-H, J = 8.1 Hz), 7.49
(d, 1H, Trp-4-H, J = 7.7 Hz), 8.14 (s, 1H, 2-H), 8.43 (s, 1H, 8-H).
13C NMR (DMSO-d6): d 22.0, 28.5, 28.7, 34.5, 54.5, 55.7, 67.3,
70.9, 73.8, 82.7, 86.9, 111.0, 111.1, 118.0, 118.6, 118.9, 120.6,
123.4, 128.0, 135.9, 139.4, 149.7, 152.7, 156.1, 174.2, 176.3.
HRMS for C27H35N10O8S [MꢂH]ꢂ calcd: 659.2360; found:
659.2363.
4.1.3.28. 50-O-[N-[Na-(
adenosine (25). Following the procedure used for the synthesis
L-Methionyl)-L-tryptophanyl]-sulfamoyl]-
of 13, 55 (193 mg, 0.22 mmol, 1.0 equiv) was deprotected to yield
78 mg (0.12 mmol, 55%) of 25.
1H NMR (DMSO-d6): d 1.10 (t, Et3N—H3, J = 7.2 Hz), 1.73–2.05
(m, 5H, Met2-b-H2, Met2-S-CH3), 2.83–3.04 (m, Et3NHþ—H2,
Trp2-b-HA), 3.26 (dd, 1H, Trp2-b-HB, J = 4.4 Hz, J = 14.6 Hz), 3.60–
4.1.3.33. 50-O-[N-[Na-(N -Boc-
ptophanyl]-sulfamoyl]adenosine (Et3N salt, 58).
the procedure used for the synthesis of 43, 34a (360 mg,
0.57 mmol, 1.0 equiv) and N-Boc- -glutamic acid -O-tert-butyl
L
-glutamyl-
c-O-tert-butyl)-
L-try-
Following
3.66 (visible after addition of D2O, m, 1H, Met1-
a-H), 3.95–4.13
(m, 3H, 40-H, 50-H2), 4.17 (m, 1H, Trp2- -H), 4.35 (m, 1H, 30-H),
a
L
c
4.61 (m, 1H, 20-H), 5.92 (d, 1H, 10-H, J = 6.2 Hz), 6.94 (t, 1H, Trp-
5H, J = 7.5 Hz), 7.03 (t, 1H, Trp-6H, J = 7.0 Hz), 7.12 (d, 1H, Trp-
2H, J = 2.1 Hz), 7.29 (d, 1H, Trp-7H, J = 8.1 Hz), 7.53 (d, 1H, Trp-4-
H, J = 7.8 Hz), 8.14 (s, 1H, 2-H), 8.41 (s, 1H, 8-H).
ester (172 mg, 0.57 mmol, 1.0 equiv) were reacted to yield
359 mg (0.39 mmol, 69%) of 58.
HRMS for C35H46N9O12
816.2987.
S
[MꢂH]ꢂ calcd: 816.2987; found:
13C NMR (DMSO-d6): d 9.3, 14.5, 28.4, 28.5, 32.0, 52.2, 56.4, 67.3,
71.0, 73.7, 82.8, 86.9, 111.1, 111.2, 118.2, 118.4, 118.9, 120.7,
123.4, 127.7, 136.1, 139.4, 149.7, 152.7, 156.1, 168.8, 175.5.
HRMS for C26H33N9O8S2 [MꢂH]ꢂ calcd: 662.1816; found:
662.1810.
4.1.3.34. 50-O-[Na-(N -
enosine (Et3N salt, 28).
L
-Glutamyl)-
L-tryptophanyl]-sulfamoylad-
Following the procedure used for the
synthesis of 17, 58 (339 mg, 0.37 mmol, 1.0 equiv) was deprotec-
ted to yield 189 mg (0.23 mmol, 61%) of 28.
1H NMR (DMSO-d6): d 1.14 (t, Et3N—H3, J = 7.3 Hz), 1.74–2.03
4.1.3.29. 50-O-[N-[Na-(N-Boc-
adenosine (Et3N salt, 56).
synthesis of 43, 34a (270 mg, 0.43 mmol, 1.0 equiv) and N-Boc-
L
-prolyl)-
Following the procedure used for the
-pro-
L
-tryptophanyl]-sulfamoyl]-
(m, 2H, Glu1-b-H2), 2.38 (t, 1H, Glu1-
Et3N—H2, Trp2-b-HA), 3.26 (dd, 1H, Trp2-b-HB, J = 4.2 Hz,
14.6 Hz), 3.57 (t, 1H, Glu1-
-H, J = 6.0 Hz), 3.95–4.13 (m, 3H, 40-
H, 50-H2), 4.13–4.21 (m, 1H, Trp2- -H), 4.35 (dt, 1H, 30-H,
c-H2, J = 7.5), 2.78–3.03 (m,
L
a
line (92 mg, 0.43 mmol, 1.0 equiv) were reacted to yield 240 mg
(0.29 mmol, 67%) of 56. HRMS for C31H38N9O10S [MꢂH]ꢂ calcd:
728.2463; found: 728.2474.
a
J = 4.3 Hz, 8.4 Hz), 4.61 (t, 1H, 20-H, J = 5.6 Hz), 5.92 (d, 1H, 10-H,
J = 6.2 Hz), 6.94 and 7.03 (2 ꢃ m, 2H, Trp-5-H and Trp-6-H), 7.13
(d, 1H, Trp-2-H, J = 2.2 Hz), 7.26 (br s, 2H, Ade-NH2), 7.30 (m, 1H,
Trp-7-H, J = 7.9 Hz), 7.55 (d, 1H, Trp-4-H, J = 7.7 Hz), 8.13 (s, 1H,
2-H), 8.41 (s, 1H, 8-H), 10.75 (d, 1H, indole-NH, J = 2.1 Hz).
4.1.3.30. 50-O-[N-[Na-(
adenosine (26). Following the procedure used for the syn-
L-Prolyl)-L-tryptophanyl]-sulfamoyl]-

P. Van de Vijver et al. / Bioorg. Med. Chem. 17 (2009) 260–269
269
13C NMR (DMSO-d6): d 9.1, 27.8, 28.4, 30.9, 45.5, 52.4, 56.3, 67.4,
Acknowledgment
71.0, 73.8, 82.9, 86.9, 111.1, 111.2, 118.2, 118.4, 118.9, 120.7,
123.4, 127.7, 136.1, 139.4, 149.8, 152.7, 156.1, 169.3, 174.6, 175.6.
We would like to thank Stephanie Vandenwaeyenberg for assis-
tance with mass spectrometric analyses, Professor Dr. Konstantin
HRMS for C26H30N9O10
660.1833.
S
[MꢂH]ꢂ calcd: 660.1837; found:
Severinov and Dr. Kirill Datsenko for providing the
DABN E. coli
strain, and Dr. Brian Sproat (Integrated DNA Technologies, Leuven)
4.1.3.35. 50-O-[N-[N-(N-Boc-
adenosine (Et3N salt, 59).
L
-Methionyl)-
L-aspartyl]-sulfamoyl]
for providing the 50-O-(N-
L-aminoacyl)-sulfamoyladenosine
Following the procedure used for the
compounds.
synthesis of 43, 41 (80 mg, 0.17 mmol, 1.0 equiv) and N-Boc-L-
methionine (48 mg, 0.19 mmol, 1.1 equiv) were reacted and puri-
fied by silica gel chromatography (Et3N 1%, MeOH 5?30% in
CH2Cl2) to yield 130 mg (0.16 mmol, 96%) of 29.
HRMS for C24H37N8O12S2 [M+H]+ calcd: 693.1972; found:
693.19536.
Supplementary data
The following supplementary data are available: Table S1,
summarizing the results from the initial antibacterial screening
of all 20 analogues of 50-O-(N-
L
-aminoacyl)-sulfamoyladenosine.
Table S2, summarizing the results from the antibacterial evalua-
tion of the new 50-O-(N-
-dipeptidyl)-sulfamoyladenosine 12–29
4.1.3.36. 50-O-[N-(N-
sine (Et3N salt, 29).
L
-Methionyl)-
L-aspartyl]-sulfamoyladeno-
L
Compound 59 (130 mg, 0.16 mmol,
(Fig. 4) and selected reference compounds with lower doses of
compounds (6 nmol). Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2008.11.054.
1.0 equiv) was dissolved in 5 mL TFA/H2O (5:2, v/v) and stirred
for 1.5 h at rt. Next, the volatiles were evaporated and coevaporat-
ed twice with EtOH and once with Et3N (0.5 mL). After evaporation
of the volatiles, the residue was purified by silica gel chromatogra-
phy (Et3N 1%, MeOH 20?60% in CHCl3) and further purified by ion
exchange chromatography (DEAE-cellulose column, triethylam-
monium bicarbonate 0?0.6 M in water) to yield 45 mg
(0.065 mmol, 41%) of 29.
References and notes
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1H NMR (DMSO-d6): d 1.03 (t, 9H, Et3Nþ—H3, J = 7.2 Hz), 1.67–
2.08 (m, 5H, Met1-b-H2, Met1-S-H3), 2.40–2.61 (obscured by
DMSO-d6 peak, b-H2), 2.61–2.80 (m, b-H2, Et3NHþ—H2), 2.86 (q,
6H, Et3N-H2, J = 7.2 Hz), 3.48 (t, 1H, Met1-
a-H, J = 6.0 Hz), 3.96–
4.12 (m, 3H, 40-H, 50-H2), 4.12–4.19 (m, 1H, 30-H), 4.35 (dd, 1H,
Asp2-a
-H, J = 7.2 Hz, 13.1 Hz), 4.58 (t, 1H, 20-H, J = 5.4 Hz), 5.91
(d, 1H, 10-H, J = 6.1 Hz), 7.26 (br s, 2H, Ade-NH2, D2O-exchange-
able), 8.15 (s, 1H, Ade-2-H), 8.20 (d, 1H, Asp-NH, J = 8.0 Hz), 8.39
(s, 1H, Ade-8-H).
13C NMR (DMSO-d6): d 11.3, 14.6, 29.5, 34.2, 38.6, 45.7, 51.9,
53.6, 67.3, 70.9, 73.7, 82.7, 86.8, 118.9, 139.3, 149.7 152.7, 156.1,
172.7, 172.8, 174.7.
HRMS for C19H27N8O10S2 [MꢂH]ꢂ calcd 591.1292; found
591.1281.
Basic data analysis was done in Microsoft Excel 2003 (Microsoft
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P-Values 60.05 were considered statistically significant. INSTANT
JCHEM was used for structure database management, search and
prediction, INSTANT JCHEM 2.3, 2008, CHEMAXON (http://www.chema-
xon.com). Chemdraw was used for chemical structure and reaction
drawing, and for calculating exact masses (Chemdraw 11, Cam-
bridgeSoft Corporation, Cambridge, MA, USA).
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4.2. Biological evaluation
Liquid Mueller–Hinton Agar (0.75%) was mixed with bacterial
suspension (29:1 v/v) at rt. Plates were prepared by adding 4 mL
of this suspension to petri plates containing 7 mL of solidified
Mueller–Hinton Agar (1.5%). Immediately after solidification of
the agar, 5 lL drops containing investigational compound in
H2O milliQ were added and allowed to dry. After incubation for
16 h at 37 °C, the inhibition zones were measured. These evalua-
tions were performed in triplicate for each compound–concentra-
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