Design, synthesis and structure-activity relationship of novel Relacin analogs as inhibitors of Rel proteins

Article abstract of DOI:10.1016/j.ejmech.2013.10.036

Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term

Full text of DOI:10.1016/j.ejmech.2013.10.036

European Journal of Medicinal Chemistry 70 (2013) 497e504
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationship of novel Relacin
analogs as inhibitors of Rel proteins
,
,
,
*
Ezequiel Wexselblatt a 1, Ilana Kaspy b 1, Gad Glaser b, Joshua Katzhendler a, Eylon Yavin a
a Institute for Drug Research, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel
b Department of Developmental Biology and Cancer Research, Institute for Medical Research IsraeleCanada (IMRIC), The Hebrew University of Jerusalem,
Jerusalem, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response,
responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent
Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell
death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit
in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a
deeper insight on the SAR for a better understanding of their potential interactions and inhibitory ac-
tivity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our
previously reported Relacin.
Received 17 July 2013
Received in revised form
10 October 2013
Accepted 12 October 2013
Available online 22 October 2013
Keywords:
Antibacterial
Stringent Response
(p)ppGpp
RelA
Ó 2013 Elsevier Masson SAS. All rights reserved.
Purine
1. Introduction
abundance of RelA, up to mM levels of (p)ppGpp are rapidly pro-
duced. This is possible since (p)ppGpp synthesis evokes dissocia-
The Stringent Response is a process crucial for bacterial viability
as it activates a series of long term bacterial survival pathways
including the switch into stationary phase of growth, sporulation,
and biofilm formation [1e4]. This response is triggered by the
accumulation of the signal molecules 50-triphosphate-30-diphos-
phate and 50e30-bis-diphosphate, collectively called (p)ppGpp [5]
(Fig. 1A). In Gram negative bacteria (p)ppGpp is mostly synthe-
sized by RelA and hydrolyzed by SpoT, while in Gram positive
bacteria a bifunctional enzyme, Rel/Spo, both synthesizes and hy-
drolyzes (p)ppGpp [6,7].
tion of RelA from the ribosome, allowing the enzyme to “hop” to
another stalled ribosome and repeat the reaction [9].
Recently, the crystal structure of the catalytic N-terminal frag-
ment of the bifunctional RelA homologue from the Gram positive
bacterium Streptococcus equisimilis (Relseq385) was determined
[10]. In contrast to Gram negative bacteria, where RelA and SpoT are
distinct proteins, this fragment displays both the hydrolase and
synthetase active sites. The crystallographic analysis revealed two
conformations for Relseq385 that typify the opposing hydrolase-
OFF/synthetase-ON and hydrolase-ON/synthetase-OFF states. The
crystal structure also suggested a mechanism for the attack of the
RelA is a ribosome-associated (p)ppGpp synthetase activated in
response to amino-acid starvation [8]. During amino-acid depri-
vation the binding of uncharged tRNAs to the ribosomal ‘A’ site
stalls protein synthesis, stimulating a reaction in which RelA
30-OH group of GDP (or GTP) onto the
b-phosphorus atom of ATP.
Most likely, Glu323 of Relseq385 activates the hydroxyl group by
proton abstraction. In addition, Glu323 also is suggested to coor-
dinate the essential Mg2þ ion which is not present in the crystals
and is expected to be bound to the pyrophosphate donor, ATP.
Support for the critical role of Glu323 in the synthetic mechanism
derives from a mutational study showing that a mutant Relseq385
bearing a Glu323Gln substitution exhibited severely defective
synthetase activity [10].
transfers a pyrophosphoryl group comprising the b- and g-phos-
phates of an ATP donor to the 30-hydroxy group of GTP or GDP to
form pppGpp and ppGpp, respectively [8]. Despite the low
* Corresponding author. The Hebrew University of Jerusalem, Faculty of Medi-
cine, The School of Pharmacy, Institute for Drug Research, P.O. Box 12065, Jerusalem
91120, Israel. Tel.: þ972 2 6758692; fax: þ972 2 6757076.
In the past few years, several efforts have been made toward the
development of antibacterial agents based on nucleosides and their
analogs. Such compounds have shown antimicrobial activity by
E-mail address: eylony@ekmd.huji.ac.il (E. Yavin).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.036

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E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
Fig. 1. Structures of (A) (p)ppGpp and (B) Relacin.
inhibiting cell wall biosynthesis [11], DNA ligases [12], riboflavin
synthesis [13], polyamine biosynthesis and quorum sensing [14],
nucleoside phosphorylases [15], and siderophore biosynthesis [16]
among others.
with an excess of carbonyldiimidazole (CDI) in acetonitrile over-
night. After the addition of CDI, the suspension completely dis-
solved and eventually the di-imidazolide derivative precipitated.
After stirring overnight, the precipitate was filtered and re-
suspended in dichloromethane (DCM). The different amino sub-
stituents (Table 1) were dissolved in DCM containing an excess of
diisopropylethylamine (DIEA) and added to the former suspension.
The mixture was stirred at room temperature until the reaction
ended (according to TLC analysis). The final products were obtained
after hydrogenolysis of the benzyl ester group to afford compounds
2aed.
In the search for novel strategies to combat multi-drug resistant
bacteria, we have explored the Stringent Response as a potential
target for the development of a new generation of antibiotics
[17,18]. We have recently reported on Relacin [18] (Fig. 1B), a novel
ppGpp analog that prevents the activation of the Stringent
Response by inhibiting (p)ppGpp production. Relacin perturbs the
switch into stationary phase in Gram positive bacteria and leads to
cell death. Furthermore, Relacin inhibits sporulation and biofilm
formation; additional bacterial long term survival strategies used
by such bacteria. In this report we present the synthesis of a series
of symmetrically and asymmetrically substituted analogs of Rela-
cin, designed at gaining a deeper understanding on the importance
of the moieties at positions 50 and 30 on the deoxyribose ring and
their potential interactions within the active site of Rel Proteins.
The inhibitory potential of the prepared compounds was bio-
chemically evaluated on the (p)ppGpp synthetase activity of RelA
and Rel/Spo purified from Escherichia coli (E. coli) and Deinococcus
radiodurans (D. radiodurans), respectively.
As RelA may accommodate different size substituents at the 30
and 50 positions of deoxyguanosine, we decided to explore the
activity of a family of Relacin analogs that are asymmetrical.
Scheme 2 depicts the synthesis of the asymmetrically substituted
compounds. The first step was the formation of an imidazolide
intermediate at position 30 of the suitable DMT-protected 20-
deoxyguanosine (3). This intermediate was prepared adapting the
procedure reported by Korshun et al. [19]. Next, the benzyl esters of
either glycine or glutamic acid were added to a solution of the
freshly prepared imidazolide in DCM containing DIEA. The mixture
was stirred overnight. After acidic workup and column chroma-
tography, the dimethoxytrityl (DMT) group was cleaved from
compounds 4a,b to yield compounds 5a,b and the free hydroxyl
moiety at position 50 was reacted with a slight excess of CDI in DCM.
When all the starting material was consumed, (typically after 15 h)
an excess of the benzyl esters of either glycine or glutamic acid was
added together with DIEA and the mixture was stirred overnight
until the completion of the reaction to afford compounds 6a,b. After
a further chromatographic purification step, the benzyl protecting
groups were cleaved as described for the symmetric analogs
resulting in the final products 7a,b.
2. Results and discussion
2.1. Design of ppGp(p) analogs
Relacin, our model compound, has a di-peptide (Gly-Gly) at both
30 and 50 positions of the deoxyguanosine. This active analog has
the following features: (1) symmetry in the choice of the di-peptide
added to both 30 and 50 positions and, (2) two negative charges (at
physiological pH) from the two acidic C-termini.
Our first goal was to compare the activity of Relacin to other
symmetrically substituted compounds. As shown in Scheme 1, such
analogs were prepared by mixing 2-isobutyryl-20-deoxyguanosine
The potential inhibitors were tested in vitro on purified Rel
proteins from both Gram positive and Gram negative bacteria using
reported methods [18]. The results, summarized in Table 2, show
Scheme 1. Synthetic route for the preparation of symmetric compounds. Reaction conditions: i) a) CDI, acetonitrile, RT, overnight; b) R,R1-NH, DCM, DIEA, RT, 2e20 h, 17e77%; ii)
H2-10% Pd/C, methanol, RT, 3 h, 30 psi, 80e90%.

E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
499
Table 1
Symmetric substituents on the 30 and 50 positions of the deoxyguanosine.
Comp.
R
R1
Comp.
R2
1a
H
2a
1b
1c
H
H
2b
2c
1d
H
H
2d
1e
1f
that addition of basic moieties (1e,f) lowers the inhibitory action of
the compounds. Moreover, compound 1f, with four dimethylamino
groups is less active than compound 1e, bearing only two such
moieties. Replacement of this dimethylamino group by a carboxylic
acid (compound 2a) markedly improved the inhibitory activity
while the addition of a second carboxylic acid (compound 2c) did
not result in a significant change in the inhibitory efficacy in
comparison to 2a. In addition, combining a glycine derivative
moiety with a derivative of glutamic acid as in the asymmetric
compounds (7a,b) showed a similar result regardless of the posi-
tion of the mono or di-acidic substituents.
Out of the 8 new analogs prepared, compound 2d had the
highest inhibitory activity (80.4% at 1 mM, see Table 2) whereas 4
out of the newly prepared compounds (2a, 2c, 7a, 7b) had all about
a 50% inhibitory effect (see Table 2). Therefore, the inhibitory ac-
tivity of the compounds 2a and 2d were further tested and
compared. While compound 2a features two glycine moieties
linked by a carbamate bridge to carbons C30 and C50, compound 2d
presents two glutamyl-glutamic acid moieties at the same posi-
tions. The main differences between these two compounds are in
their bulkiness and in the number of negatively charged sites (at
physiological pH) for potential interactions within the catalytic site
of the enzymes. Compound 2a decreased (p)ppGpp levels in Ba-
cillus subtilis cells in vivo after the onset of the Stringent Response,
by the addition of serine hydroxamate (SHX) (Fig. 2A, gray bars). In
comparison, compound 2d was significantly more active in
reducing (p)ppGpp levels (Fig. 2A, white bars).
Further, compound 2b with a hexanoic acid substituent similar
in length to Relacin, is a much less effective inhibitor. This indicates
the importance of the amide bond, most likely involved in the
formation of hydrogen bonds at the active site. Thus, compound 2d
bearing two glutamyl-glutamic acid moieties, i.e. six carboxylic
acids in total, was found to be a more effective inhibitor and more
potent than our previously reported Relacin. Compound 2d is more
acidic and voluminous than Relacin, providing more potential in-
teractions including hydrogen bonds and electrostatic salt bridges.
The isobutyryl group at position N2 in the nucleobase was pre-
dicted to form important hydrophobic interactions between Rela-
cin and the active site of Rel/Spo from S. equisimilis [18]. However,
removing it from our inhibitors didn’t clearly point at an increase or
a decrease in the inhibitory activity (data not shown).
Taken together, the data presented in this work will be useful for
the future design of new generations of potential antibacterial
agents aimed at inhibiting the Stringent Response. The effect of
compound 2d on bacterial survival will be the subject of future
work.
3. Experimental
3.1. Materials and instruments
All chemicals and reagents were purchased from SigmaeAldrich
Ltd. and used without further purification. Ultra-dry solvents
stored with molecular sieves under an argon atmosphere were
purchased from Acros Ltd. HPLC solvents were acquired from Bio
Lab Ltd.
We next evaluated the in vitro activity at varying concentrations
of compound 2d as an inhibitor of Rel enzymes from Gram positive
(D. radiodurans) and Gram negative (E. coli) bacteria. Fig. 2B pre-
sents the data resulting in a dose dependent inhibitory pattern for
both enzymes. Notably compound 2d was significantly more
effective inhibiting RelA than Rel/Spo (Fig. 2B), suggesting different
interactions within their active sites.
3.1.1. NMR
NMR data were collected with a Varian VXR-300 MHz spec-
trometer equipped with a 5-mm switchable probe. Data were
processed using the VNMR and MestreNova software. NMR

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E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
Scheme 2. Synthetic route for the preparation of asymmetric compounds. Reaction conditions: i) CDI, DCM, RT, 4 h, 92.7%; ii) H2N-CHR-OBzl, DCM, DIEA, RT, overnight, 53e68%; iii)
5% DCA in DCM, RT, 15 min, 95e98%; iv) a) CDI, DCM, RT, overnight; b) H2N-CHR1-OBzl, DCM, DIEA, RT, overnight, 40e90%; v) H2-10% Pd/C, methanol, 3 h, RT, 30 psi, 79e95%.
abbreviations are as follows: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br (broad).
25 ꢀ 4.6-mm column. Preparative and semi-preparative chroma-
tography fractions were collected on a SpectraSystem SCM 1000
HPLC system equipped with a Gilson 117 UV detector, using Phe-
nomenex RP-C18 25 ꢀ 10-mm and 25 ꢀ 22-mm columns.
3.1.2. High resolution mass spectroscopy (HRMS)
HRMS was carried out with an Orbi-trap MS (Thermo Finnigen)
using nanospray attachment. MRFA (met-arg-phe-ala) peptide was
used as internal standard. Data were processed using bioworks 3.3
package.
3.2. Chemical synthesis
3.2.1. General procedure for the preparation of compounds 1aef
2-Isobutyryl-20-deoxyguanosine (200 mg, 0.6 mmol) was sus-
pended in dry acetonitrile (10 mL) and four equivalents of
3.1.3. Electron spray ionization mass spectroscopy (ESI-MS)
Electrospray ionization mass spectrometry was carried out us-
ing a ThermoQuest Finnigan LCQ-Duo in the positive ion mode.
Data were processed using ThermoQuest Finnigan’s XcaliburÔ
Biomass Calculation and Deconvolution software.
Table 2
Comparison of inhibitory potential (1 mM of inhibitor) on RelA isolated from E. coli.
The most potent analog (2d) is highlighted in bold.
Compound
1e
1f
2a
2b
2c
2d
7a
7b
Relacina
% Inhibition 31.2 12.0 51.1 25.6 50.6 80.4 52.1 50.4 66.3
3.1.4. HPLC
(ꢁSD)
a
9.4
1.7
4.5
5.7
4.3
4.1
2.8
0.1
2.1
Analytical HPLC was performed using a Shimadzu LC-2010 HT
HPLC system equipped with a UV detector using a Merck RP-C18
Data published by Wexselblatt et al. [18].

E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
501
Fig. 2. Inhibition of Rel proteins by compounds 2a and 2d. A) (p)ppGpp synthesis in B. subtilis cells after the addition of serine hydroxamate. White: cells grown with 2 mM of 2d.
Gray: cells grown with 2 mM of 2a. Black: cells grown without 2a. B) Inhibition of Rel proteins by 2d in vitro. White: Rel/Spo from D. radiodurans, gray: RelA from E. coli.
carbonyldiimidazole were added. After stirring overnight at room
temperature, the precipitate was filtered and re-suspended in dry
dichloromethane (10 mL). Four equivalents of the carboxy-
protected R,R0-NH2 and eight equivalents of diisopropylethyl-
amine were added. The suspension was stirred at room tempera-
ture from two to 48 h.
At the end of the reaction water was added and the organic
phase was washed three times with 5% aqueous citric acid (20 mL).
After drying and evaporating, the crude material was purified by
column chromatography (compounds 1aed, DCM to MeOH/DCM
1:10) or by RP-HPLC (compounds 1e,f). The desired compounds
were obtained as white powders.
preparation of compound 1d, dipeptide H-Glu(Bzl)-Glu(Bzl)-OBzl
was synthesized as previously reported by Nakata et al. [20].
White powder, 320 mg, 36% (0.22 mmol).
ESI-MS: calc: 1482.54. Found [M þ Hþ]: 1483.43.
1H NMR (DMSO-d6): 8.46 (m, 2H, NH (peptide bond)), 8.26 (m,
2H, H-8 and NH carbamate), 7.64 (d, J ¼ 7.8 Hz, 1H, NH carbamate),
7.33 (m, 30H, Ar), 6.18 (t, J ¼ 15 Hz, 1H, H-10), 5.17 (d, J ¼ 5.8 Hz, H-
30), 5.12 (bs, 12H, CH2ePhenyl), 4.42 (m, 2H, CH), 4.17 (m, 3H, H40
and H50), 3.89 (m, 2H, CH), 2.9 (m, 1H, H-20), 2.7 (m, 1H, CH (iBu)),
2.48 (m, 8H, CH2), 2.4 (m, 1H, H-20), 2.0 (m, 8H, CH2), 1.1 (d,
J ¼ 6.9 Hz, 6H, CH3 (iBu)).
3.2.1.5. (2R,5R)-2-((((2-(Dimethylamino)ethyl)carbamoyl)oxy)
methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetra-
3.2.1.1. Benzyl((((2R,5R)-3-(((2-(benzyloxy)-2-oxoethyl)carbamoyl)
oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahy-
drofuran-2-yl)methoxy)carbonyl)glycinate (1a). White powder,
563 mg, 65% (0.78 mmol).
hydrofuran-3-yl(2-(dimethylamino)ethyl)carbamate
powder, 73 mg, 21% (0.13 mmol).
(1e). White
HRMS: calc [M þ Hþ]: 566.3045. Found: 566.3045.
ESI-MS: calc: 719.70. Found [M þ Hþ]: 720.83.
1H NMR (DMSO-d6): 7.89 (s,1H, H-8), 7.35 (m, 2H, NHCH2), 6.2 (t,
J ¼ 15 Hz, 1H, H-10), 5.11 (d, J ¼ 5.8 Hz, H-30), 4.1 (m, 3H, H40 and
H50), 3.06 (q, J ¼ 18.5 Hz, 5H, H-20 and CH2CH2NH), 2.85 (m, 1H, CH
(iBu)), 2.25 (t, J ¼ 12.9 Hz, 4H, CH2CH2NH), 2.5 (m, 1H, H-20), 2.11 (s,
12H, NCH3), 1.1 (d, J ¼ 6.9 Hz, 6H, CH3 (iBu)); 13C NMR (75 MHz,
1H NMR (DMSO-d6): 8.24 (s, 1H, H-8), 7.87 (m, 2H, NHCH2), 7.33
(m, 10H, Ar), 6.2 (t, J ¼ 15 Hz, 1H, H-10), 5.13 (d, J ¼ 5.7 Hz, 1H, H-30),
5.12 (s, 4H, CH2ePhenyl), 4.17 (s, 3H, H40, H50), 3.84 (t, J ¼ 12.9 Hz,
4H, CH2), 2.9 (m,1H, H-20), 2.7 (m,1H, CH (iBu)), 2.4 (m,1H, H-20),1.1
(d, J ¼ 6.9 Hz, 6H, CH3 (iBu)).
DMSO-d6) d 156.40, 155.78, 135.41, 82.86, 82.71, 75.30, 64.26, 58.95,
58.80, 45.67, 38.95, 36.81, 20.70.
3.2.1.2. Benzyl 6-(((((2R,3S,5R)-3-(((6-(benzyloxy)-6-oxohexyl)car-
bamoyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)
3.2.1.6. (2R,5R)-2-(((Bis(3-(dimethylamino)propyl)carbamoyl)oxy)
methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetra-
tetrahydrofuran-2-yl)methoxy)carbonyl)amino)hexanoate
White powder, 380 mg, 77% (0.46 mmol).
(1b).
hydrofuran-3-yl
bis(3-(dimethylamino)propyl)carbamate
(1f).
ESI-MS: calc: 831.38. Found [M þ Hþ]: 832.0.
Colorless oil, 53 mg, 17% (0.07 mmol).
HRMS: calc: 764.5141. Found: 764.5147.
1H NMR (DMSO-d6): 8.19 (s, 1H, H-8), 7.33 (m, 12H, 10H-Ar and
2HeNHCH2), 6.2 (t, J ¼ 15 Hz, 1H, H-10), 5.13 (d, J ¼ 5.7 Hz, 1H, H-30),
5.05 (s, 4H, CH2ePhenyl), 4.17 (m, 3H, H40, H50), 2.94 (m, 5H, H-20
and CH2), 2.7 (m, 1H, CH (iBu)), 2.3 (m, 5H, H-20 and CH2), 1.5e1.2
(m, 16H, CH2), 1.1 (d, J ¼ 6.9 Hz, 6H, CH3 (iBu)).
1H NMR (DMSO-d6): 8.03 (s, 1H, H-8), 6.19 (t, J ¼ 15 Hz, 1H, H-10),
5.19 (d, J ¼ 5.8 Hz, H-30), 4.16 (m, 3H, H40 and H50), 3.1 (m,18H, H-20 and
Me2NCH2CH2CH2N), 2.85 (m, 1H, CH (iBu)), 2.25 (m, 8H,
Me2NCH2CH2CH2N), 2.5 (m, 1H, H-20), 2.11 (s, 24H, NCH3), 1.1 (d,
J¼ 6.9 Hz, 6H, CH3 (iBu)); 13C NMR(75 MHz, DMSO-d6)
d180.85,155.88,
3.2.1.3. Dibenzyl((((2R,5R)-3-(((1,5-bis(benzyloxy)-1,5-dioxopentan-
2-yl)carbamoyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-
purin-9-yl)tetrahydrofuran-2-yl)methoxy)carbonyl)glutamate (1c).
White powder, 220 mg, 35% (0.21 mmol).
155.33, 154.84, 149.29, 137.53, 120.85, 83.69, 82.76, 75.39, 64.66, 56.85,
56.76, 45.92, 45.65, 45.54, 45.46, 36.56, 35.50, 26.84, 26.20,19.73,19.47.
3.2.2. General procedure for the preparation of compounds 2aed
Compounds (1aed) were dissolved in methanol. 10% Pd on
activated carbon was added cautiously and the mixture was shaken
for three hours under hydrogen atmosphere at 30 psi. The reaction
mixture was then filtered and evaporated to yield the free car-
boxylic acids as white crispy compounds.
ESI-MS: calc: 1043.39. Found [M þ Hþ]: 1044.16.
1H NMR (DMSO-d6): 8.24 (s, 1H, H-8), 7.91 (m, 2H, NHCH2), 7.33
(m, 20H, Ar), 6.15 (t, J ¼ 15 Hz, 1H, H-10), 5.13e5.04 (m, 9H, H-30 and
CH2ePhenyl), 4.15 (m, 5H, H40, H50 and CH), 2.9 (m, 1H, H-20), 2.7
(m, 1H, CH (iBu)), 2.4 (m, 5H, H-20 and CH2), 2.1e1.8 (m, 4H, CH2), 1.1
(d, J ¼ 6.9 Hz, 6H, CH3 (iBu)).
3.2.2.1. ((((2R,5R)-3-(((Carboxymethyl)carbamoyl)oxy)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)carbonyl)glycine (2a). White powder, 190 mg, 90%
(0.35 mmol).
3.2.1.4. Tribenzyl((((2R,5R)-3-(((glutamylglutamate)carbamoyl)oxy)-
5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydro-
furan-2-yl)methoxy)carbonyl)glutamylglutamate
(1d). For
the

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E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
HRMS: calc [M þ Hþ]: 540.1685. Found: 540.1685.
J ¼ 8.4 Hz, 8H, DMT), 7.09 (s, 1H, Imidazole), 6.77 (t, J ¼ 17.7 Hz, 3H,
DMT), 6.37 (t, J ¼ 14.3 Hz, 1H, H-10), 5.5 (d, J ¼ 5.9 Hz, 1H, H-30), 4.45
(m, 1H, H-40), 3.7 (s, 6H, OCH3), 3.41 (t, J ¼ 18 Hz, 1H, H-50), 3.19 (m,
2H, H-50 and H-20), 2.84e2.7 (m, 2H, H-20 and CH (iBu)), 1.1 (d,
J ¼ 6.74 Hz, 6H, CH3 (iBu)).
1H NMR (DMSO-d6): 8.24 (s, 1H, H-8), 7.64 (m, 2H, NHCH2), 6.2
(t, J ¼ 15 Hz, 1H, H-10), 5.16 (d, J ¼ 5.7 Hz, 1H, H-30), 4.17 (s, 3H, H40,
H50), 3.66 (t, J ¼ 12.3 Hz, 4H, CH2), 2.9 (m, 1H, H-20), 2.7 (m, 1H, CH
(iBu)), 2.4 (m, 1H, H-20), 1.1 (d, J ¼ 6.9 Hz, 6H, CH3 (iBu)); 13C NMR
(75 MHz, DMSO-d6)
d 180.55, 171.88, 156.64, 156.10, 155.24, 149.13,
148.73, 137.67, 120.65, 83.22, 82.94, 75.66, 64.58, 42.62, 36.78,
35.23, 19.28.
3.2.3. General procedure for the preparation of compounds 4a,b
Compound (3) was dissolved in dry DCM. 2 equivalents of either
H-Gly-OBzl or H-Glu(OBzl)-OBzl and 4 equivalents of diisopropy-
lethylamine were added. After stirring overnight, the mixture was
washed with citric acid, dried, evaporated and purified by column
chromatography (DCM to MeOH/DCM 1:10) yielding white
powders.
3.2.2.2. 6-(((((2R,5R)-3-(((5-Carboxypentyl)carbamoyl)oxy)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)carbonyl)amino)hexanoic acid (2b). White powder,
250 mg, 88% (0.38 mmol).
HRMS: calc [M þ Hþ]: 652.2937. Found: 652.2940.
1H NMR (DMSO-d6): 8.21 (s,1H, H-8), 7.33 (m, 2H, NHCH2), 6.1 (t,
J ¼ 15 Hz, 1H, H-10), 5.14 (d, J ¼ 5.7 Hz, 1H, H-30), 4.16 (m, 3H, H40,
H50), 2.94 (m, 5H, H-20 and CH2), 2.7 (m, 1H, CH (iBu)), 2.42 (m, 1H,
H-20), 2.3 (m, 4H, CH2), 1.5e1.2 (m, 16H, CH2), 1.1 (d, J ¼ 6.9 Hz, 6H,
3.2.3.1. Benzyl
((((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)
methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-
9-yl)tetrahydrofuran-3-yl)oxy)carbonyl)glycinate (4a). White pow-
der, 350 mg, 68% (0.4 mmol).
CH3 (iBu)); 13C NMR (75 MHz, DMSO-d6)
d
180.51, 174.86, 156.22,
ESI-MS: calc: 830.88. Found [M þ Hþ]: 831.71.
155.70, 155.24, 149.08, 148.67, 137.67, 135.55, 122.10, 120.71, 83.34,
83.09, 75.12, 64.13, 36.89, 35.24, 34.08, 29.54, 26.26, 24.65, 19.28.
1H NMR (DMSO-d6): 8.13 (s, 1H, H-8), 7.81 (t, J ¼ 12.36 Hz, 1H,
NHCH), 7.33 (m, 7H, Ar and DMT), 7.18 (d, J ¼ 8.4 Hz, 8H, DMT), 6.77
(t, J ¼ 17.7 Hz, 3H, DMT), 6.25 (t, J ¼ 13.6 Hz, 1H, H-10), 5.16e5.12
(s þ bs, 3H, CH2ePhenyl and H30), 4.1 (m, 1H, H-40), 3.83 (d,
J ¼ 6.12 Hz, 2H, CH2), 3.7 (s, 6H, OCH3), 3.15e2.95 (m, 2H, H-50), 2.74
(m, 1H, CH (iBu)), 1.1 (d, J ¼ 6.74 Hz, 6H, CH3 (iBu)).
3.2.2.3. ((((2R,5R)-3-(((1,3-Dicarboxypropyl)carbamoyl)oxy)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)carbonyl)glutamic acid (2c). White powder, 95 mg, 90%
(0.14 mmol).
HRMS: calc [M þ Hþ]: 684.2107. Found: 684.2108.
3.2.3.2. Dibenzyl((((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)
methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-
1H NMR (DMSO-d6): 8.25 (s, 1H, H-8), 7.65 (m, 2H, NHCH), 6.22
(t, J ¼ 15 Hz, 1H, H-10), 5.13 (d, J ¼ 5.3 Hz, 1H, H-30), 4.15 (m, 3H, H40,
H50), 3.97 (m, 2H, CH), 2.9 (m,1H, H-20), 2.7 (m,1H, CH (iBu)), 2.2 (m,
5H, H-20 and CH2), 2.0e1.7 (m, 4H, CH2), 1.1 (d, J ¼ 6.9 Hz, 6H, CH3
9-yl)tetrahydrofuran-3-yl)oxy)carbonyl)-
L
-glutamate
(4b).
White powder, 3.6 g, 53% (3.6 mmol).
ESI-MS: calc: 992.4. Found [M þ Hþ]: 993.20.
(iBu)); 13C NMR (75 MHz, DMSO-d6)
d
180.55, 174.15, 173.83, 156.28,
1H NMR (DMSO-d6): 8.11 (s, 1H, H-8), 7.90 (d, J ¼ 7.31 Hz, 1H,
NHCH), 7.36 (d, J ¼ 7.4 Hz, 2H, DMT), 7.33 (m, 10H, Ar), 7.16 (d,
J ¼ 8.4 Hz, 8H, DMT), 6.77 (t, J ¼ 17.7 Hz, 3H, DMT), 6.22 (t,
J ¼ 14.3 Hz, 1H, H-10), 5.14 (s, 2H, CH2ePhenyl), 5.1 (d, J ¼ 4.4 Hz, 1H,
H-30), 5.06 (s, 2H, CH2ePhenyl), 4.24e4.1 (m, 2H, H-40 and
NHCHCH2), 3.7 (s, 6H, OCH3), 3.15e2.95 (m, 2H, H-50), 2.72 (m, 1H,
CH (iBu)), 2.45e1.8 (m, 6H, H-20 and CHCH2CH2), 1.1 (d, J ¼ 6.74 Hz,
6H, CH3 (iBu)).
155.78, 155.25, 149.12, 148.74, 137.81, 125.34, 120.63, 83.16, 82.94,
75.57, 67.46, 64.65, 53.50, 36.94, 35.23, 30.88, 30.60, 29.44, 26.70,
26.59, 25.58, 19.30.
3.2.2.4. ((((2R,5R)-3-(((Glutamylglutamic acid)carbamoyl)oxy)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)carbonyl)glutamylglutamic acid (2d). White powder,
102 mg, 83% (0.1 mmol).
HRMS: calc [M þ Hþ]: 942.2959. Found: 942.2921.
3.2.4. General procedure for the preparation of compounds 5a,b
Compounds (4a,b) were dissolved in 5% dichloroacetic acid in
DCM and stirred for 15 min. After evaporation, the crude material
was purified by column chromatography (DCM to MeOH/DCM
1:10) yielding white foams.
1H NMR (DMSO-d6): 8.46 (m, 2H, NH (peptide bond)), 8.26 (m,
2H, H-8 and NH carbamate), 7.64 (d, J ¼ 7.8 Hz, 1H, NH carbamate),
6.18 (t, J ¼ 15 Hz, 1H, H-10), 5.17 (d, J ¼ 5.8 Hz, H-30), 4.42 (m, 2H,
CH), 4.17 (m, 3H, H40 and H50), 3.89 (m, 2H, CH), 2.9 (m, 1H, H-20),
2.7 (m, 1H, CH (iBu)), 2.48 (m, 8H, CH2), 2.4 (m, 1H, H-20), 2.0 (m, 8H,
CH2), 1.1 (d, J ¼ 6.9 Hz, 6H, CH3 (iBu)); 13C NMR (75 MHz, DMSO-d6)
3.2.4.1. Benzyl((((2R,3S,5R)-2-(hydroxymethyl)-5-(2-isobutyramido-
6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)
carbonyl)glycinate (5a). White foam, 250 mg, 95% (0.47 mmol).
ESI-MS: calc: 528.51. Found [M þ Hþ]: 529.3.
d
180.62, 177.85, 174.36, 174.29, 174.23, 173.51, 172.95, 171.77, 168.27,
155.26, 149.11, 148.72, 137.82, 120.61, 83.18, 61.92, 55.80, 54.23,
53.67, 51.84, 51.74, 35.19, 30.75, 30.68, 29.79, 29.58, 28.61, 27.83,
26.92, 25.51, 19.30.
1H NMR (DMSO-d6): 8.26 (s, 1H, H-8), 7.80 (t, J ¼ 12.36 Hz, 1H,
NHCH), 7.35 (m, 5H, Ar), 6.18 (t, J ¼ 13.6 Hz, 1H, H-10), 5.21e5.12
(s þ bs, 4H, CH2ePhenyl, OH50 and H30), 4.1 (m, 1H, H-40), 3.83 (d,
J ¼ 6.12 Hz, 2H, CH2), 3.57 (t, J ¼ 8.5 Hz, 2H, H-50), 2.74 (m, 2H, H20
and CH (iBu)), 2.38 (m, 1H, H-20), 1.1 (d, J ¼ 6.74 Hz, 6H, CH3 (iBu)).
3.2.2.5. (2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)
methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetra-
hydrofuran-3-yl-1H-imidazole-1-carboxylate (3).
2-Isobutyryl-50-DMT-20-deoxyguanosine (10.6 g, 16.5 mmol) was
dissolved in 140 mL dry DCM and carbonyldiimidazole (13.4 g,
82.5 mmol) was added. The mixture was stirred at room temper-
ature for four hours and washed with water. After drying and
evaporating the organic phase, the desired compound was ob-
tained as a yellowish foam. No further purification was required.
Yield: 11.2 g, 92.7% (15.3 mmol).
3.2.4.2. Dibenzyl((((2R,3S,5R)-2-(hydroxymethyl)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)carbonyl)- -glutamate (5b). White foam, 1.5 g, 98%
L
(2.2 mmol).
ESI-MS: calc: 690.26. Found [M þ Hþ]: 691.20.
1H NMR (DMSO-d6): 8.25 (s, 1H, H-8), 7.88 (d, J ¼ 7.31 Hz, 1H,
NHCH), 7.33 (m, 10H, Ar), 6.14 (t, J ¼ 14.3 Hz, 1H, H-10), 5.17e5.07
(m, 6H, CH2ePhenyl, OH50 and H30), 4.14 (m, 1H, NHCHCH2), 4.01
(m, 1H, H-40), 3.57 (m, 2H, H-50), 2.74 (m, 2H, H20 and CH (iBu)), 2.32
ESI-MS: calc: 733.29. Found [M þ Hþ]: 734.04.
1H NMR (DMSO-d6): 8.34 (s, 1H, Imidazole), 8.18 (s, 1H, H-8),
7.69 (s, 1H, Imidazole), 7.31 (d, J ¼ 7.4 Hz, 2H, DMT), 7.18 (d,

E. Wexselblatt et al. / European Journal of Medicinal Chemistry 70 (2013) 497e504
503
(m, 1H, H-20), 2.1e1.8 (m, 4H, CHCH2CH2), 1.1 (d, J ¼ 6.74 Hz, 6H, CH3
13C NMR (75 MHz, DMSO-d6)
156.36, 155.67, 154.98, 149.14, 148.77, 137.91, 120.64, 83.22, 82.82,
75.34, 64.27, 54.56, 44.20, 36.99, 35.22, 32.56, 28.48, 19.31.
d
180.61, 174.99, 173.68, 172.10, 171.86,
(iBu)).
3.2.5. General procedure for the preparation of compounds 6a,b
Compounds (5a,b) were dissolved in dry DCM and 2 equivalents
of carbonyldiimidazole were added. After stirring at room tem-
perature for 16 h, 4 equivalents of either H-Gly-OBzl or H-
Glu(OBzl)-OBzl and 4 equivalents of diisopropylethylamine were
added. After stirring overnight, the mixture was washed with citric
acid, dried, evaporated and purified by column chromatography
(DCM to MeOH/DCM 1:10) yielding white powders.
3.3. Inhibition tests
The potential inhibitors were tested in vitro using reported
methods [18].
Acknowledgments
EY acknowledges the David R. Bloom Center for Pharmacy and
the Grass Center for Drug Design and Synthesis of Novel Thera-
peutics for financial support.
3.2.5.1. Dibenzyl((((2R,3S,5R)-3-(((2-(benzyloxy)-2-oxoethyl)carba-
moyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)
tetrahydrofuran-2-yl)methoxy)carbonyl)glutamate
756 mg, 37.3% (0.86 mmol).
(6a).
ESI-MS: calc: 881.32. Found [M þ Hþ]: 882.04.
Appendix A. Supplementary data
1H NMR (DMSO-d6): 8.24 (s, 1H, H-8), 7.90 (d, J ¼ 7.31 Hz, 1H,
NHCH), 7.84 (t, J ¼ 12.36 Hz, 1H, NHCH), 7.33 (m, 15H, Ar), 6.16 (t,
J ¼ 13.6 Hz, 1H, H-10), 5.16e5.09 (m, 7H, CH2ePhenyl, and H30), 4.1
(m, 4H, H-40, H50 and NHCHCH2), 3.83 (d, J ¼ 6.12 Hz, 2H, CH2), 2.9
(m, 1H, H-20), 2.74 (m, 1H, CH (iBu)), 2.38 (m, 1H, H-20), 2.1e1.8 (m,
4H, CHCH2CH2), 1.1 (d, J ¼ 6.74 Hz, 6H, CH3 (iBu)).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.036.
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moyl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-
9-yl)tetrahydrofuran-3-yl)oxy)carbonyl)glutamate (6b). 2.6 g, 90%
(2.95 mmol).
ESI-MS: calc: 881.32. Found [M þ Hþ]: 881.94.
1H NMR (DMSO-d6): 8.25 (s, 1H, H-8), 7.9e7.8 (m, 2H, NHCH),
7.33 (m, 15H, Ar), 6.18 (t, J ¼ 13.6 Hz, 1H, H-10), 5.15e5.05 (m, 7H,
CH2ePhenyl, and H30), 4.1 (m, 4H, H-40, H50 and NHCHCH2), 3.85 (d,
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CH3 (iBu)).
3.2.6. General procedure for the preparation of compounds 7a,b
These compounds were prepared following the procedure used
for the preparation of compounds (2aed), using compounds (6a,b)
as starting materials.
3.2.6.1. ((((2R,5R)-3-(((Carboxymethyl)carbamoyl)oxy)-5-(2-
isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)carbonyl)glutamic acid (7a). White powder, 250 mg,
79% (0.41 mmol).
HRMS: calc [M þ Hþ]: 612.1896. Found: 612.1899.
1H NMR (DMSO-d6): 8.24 (s, 1H, H-8), 7.7 (m, 2H, NHCH), 6.23 (t,
J ¼ 13.6 Hz, 1H, H-10), 5.16 (d, J ¼ 5.65 Hz, 1H, H30), 4.2e4.01 (m, 4H,
H-40, H50 and NHCHCH2), 3.68 (d, J ¼ 6.12 Hz, 2H, CH2), 2.93 (m, 1H,
H-20), 2.77 (m,1H, CH (iBu)), 2.45 (m,1H, H-20), 2.31 (m, 2H, CHCH2),
2.05e1.75 (2*m, 4H, CHCH2CH2), 1.1 (d, J ¼ 6.74 Hz, 6H, CH3 (iBu));
13C NMR (75 MHz, DMSO-d6)
d 180.54, 174.13, 173.82, 171.87, 171.67,
156.65, 155.80, 155.23, 149.13, 148.73, 137.70, 120.65, 83.20, 83.01,
75.64, 64.62, 53.48, 42.53, 36.87, 35.24, 30.59, 26.56, 19.29.
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H-20), 2.77 (m,1H, CH (iBu)), 2.45 (m,1H, H-20), 2.31 (m, 2H, CHCH2),
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