Novel pseudopeptides incorporating a benzodiazepine-based turn mimetic - Targeting Mycobacterium tuberculosis ribonucleotide reductase

Article abstract of DOI:10.1016/j.bmc.2013.01.020

Peptides mimicking the C-terminus of the small subunit (R2) of Mycobacterium tuberculosis ribonucleotide reductase (RNR) can compete for binding to the large subunit (R1) and thus inhibit RNR activity. Moreover, it has been suggested that the binding of t

Full text of DOI:10.1016/j.bmc.2013.01.020

Bioorganic & Medicinal Chemistry 21 (2013) 1992–2000
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel pseudopeptides incorporating a benzodiazepine-based turn
mimetic—targeting Mycobacterium tuberculosis ribonucleotide
reductase
Johanna Nurbo a, Daniel J. Ericsson b, Ulrika Rosenström a, Daniel Muthas a, Anna M. Jansson b,
Gunnar Lindeberg a, Torsten Unge b, Anders Karlén a,
⇑
a Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
b Department of Cell and Molecular Biology, Structural Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptides mimicking the C-terminus of the small subunit (R2) of Mycobacterium tuberculosis ribonucleo-
tide reductase (RNR) can compete for binding to the large subunit (R1) and thus inhibit RNR activity.
Moreover, it has been suggested that the binding of the R2 C-terminus is very similar in M. tuberculosis
and Salmonella typhimurium. Based on modeling studies of a crystal structure of the holocomplex of the S.
typhimurium enzyme, a benzodiazepine-based turn mimetic was identified and a set of novel compounds
incorporating the benzodiazepine scaffold was synthesized. The compounds were evaluated in a compet-
itive fluorescence polarization assay and in an RNR activity assay. These studies revealed that the com-
pounds incorporating the benzodiazepine scaffold have the ability to compete for the M. tuberculosis
R2 binding site with low-micromolar affinity.
Received 5 October 2012
Revised 8 January 2013
Accepted 11 January 2013
Available online 18 January 2013
Keywords:
Mycobacterium tuberculosis
Ribonucleotide reductase
Peptide inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
Pseudopeptide
1. Introduction
two genes is required for bacterial growth.4 In addition, Mowa
et al.5 have suggested that the product of these genes, the NrdEF2
Tuberculosis is an infection that mainly affects the lungs and is
caused by the pathogen Mycobacterium tuberculosis. In 2010, the
World Health Organization estimated 8.8 million new cases of
tuberculosis worldwide.1 Since the disease remains a global health
crisis with an alarming rise of multi-drug resistant2 and
extensively drug resistant2,3 bacterial strains, the need for new
antitubercular therapeutics is very urgent.
M. tuberculosis ribonucleotide reductase (RNR) has been identi-
fied as a potential drug target.4,5 RNR is responsible for the reduc-
tion of ribonucleotides to the corresponding deoxyribonucleotides
and the bioactive enzyme is composed of two larger (R1) and two
smaller subunits (R2).6 Association of the subunits is essential for
catalytic activity because the substrate binding site is located on
R1, while R2 harbors a radical involved in the reduction. The C-ter-
minus of R2 is crucial for this association and small peptides corre-
sponding to the C-terminal sequence can compete for binding to
R1 and thus inhibit the enzyme.7,8 In M. tuberculosis, R1 and R2
are encoded by the genes nrdE (Rv3051c) and nrdF2 (Rv3048c).4,9
These genes have been identified as essential for optimal bacterial
growth in a transposon site hybridization study.10 It has also been
shown by gene knockout studies, that the combination of these
enzyme, provides the RNR activity required by the bacteria for DNA
synthesis and repair at every stage of infection.
We have previously reported on the evaluation of a set of
N-acetylated peptide inhibitors based on the C-terminal end of
the M. tuberculosis RNR R2 subunit.8 Starting from the heptapep-
tide Ac-Glu1-Asp2-Asp3-Asp4-Trp5-Asp6-Phe7 (1) we could show
that Trp5 and Phe7 are important for inhibition of enzyme activity.
Furthermore, our study indicated that there is room for larger side-
chains, as compared to Trp, in position 5. This was also supported
by inspection of the X-ray structure of Salmonella typhimurium
RNR.11 Interestingly, studies on mammalian RNR have revealed
that Fmoc-protected peptides can work as inhibitors of the en-
zyme.12,13 Encouraged by these results, we could also show that
an N-terminal Fmoc group could be advantageous in potential R2
C-terminal derived peptide inhibitors of M. tuberculosis RNR.14
Incorporation of secondary structure mimetics is an alternative
strategy to transform biologically active peptides into nonpeptides.
This approach has already been employed in the search for inhib-
itors of mammalian RNR.15,16 By studying the S. typhimurium
RNR holocomplex, we were inspired to explore the use of turn
mimetics in our development of less peptidic inhibitors targeting
M. tuberculosis RNR. There are several successful examples of ben-
zodiazepine-based scaffolds as turn mimetics.17–20 In this Letter,
we report on the design and synthesis of novel pseudopeptides
⇑
Corresponding author. Tel.: +46 18 471 4293; fax: +46 18 471 4474
E-mail address: anders.karlen@orgfarm.uu.se (A. Karlén).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.020

J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
1993
comprising a benzodiazepine turn scaffold. The target compounds
are evaluated, with respect to binding affinity for the M. tuberculosis
R1 subunit, using a competitive fluorescence polarization (FP)
assay. Selected compounds were also evaluated using an activity
assay where the reduction of CDP is monitored.
11m also can adopt a similar overall 3D-shape as the C-terminal
fragment 1m, including the orientation of the Trp and Phe side
chains, a conformational analysis of 11m was performed.
The 3D-structure of the peptide fragment 1m, based on the crys-
tal structure of S. typhimurium RNR, is shown in Figure 2A. The con-
formational analysis of model compound 11m was performed using
the OPLS_2005 force field22 and the general Born Solvent Accessible
(GB/SA) water solvation model23 in Macromodel.24 The number of
conformations within 5 kcal/mol of the global energy minimum
found for 11m was 872. These conformations were then compared
to model compound 1m (Fig. 2A) by using 3D-shape screening in
phase 3.1 in the Maestro modeling environment. In this approach,
the phase_shape program searches for the conformations of 11m
that are most similar in 3D shape to 1m. The conformer and align-
ment that gave the highest similarity is shown in Figure 2B. In gen-
eral the overlap between these two structures is very good as seen
for the C-terminal carbonyl carbon and the Phe and Trp side chains,
although the direction of the N-terminal acetyl fragments are ori-
ented somewhat differently. Encouragingly, when the R1 protein
is included it can be seen that 11m also fits nicely in the cavity
(Fig. 2C). The Phe side chain seems to be making contact with
Arg685 in R1, although it should be noted that the density for the
2. Results and discussion
2.1. Molecular modeling
In 2006, Uppsten et al. reported the 4 Å structure of the R1/R2
complex from S. typhimurium (PDB ID: 2BQ1) and it was suggested
that the binding of the last eight residues of the R2 C-terminus to R1
is very similar in S. typhimurium and M. tuberculosis.11 Unfortu-
nately only the side chains of Thr, Trp and Phe in the R2 C-terminal
peptide (Thr-Glu-Asp-Glu-Asp-Trp-Asn-Phe) of the S. typhimurium
R1/R2 complex could be identified from the obtained electron den-
sity and the remaining amino acids were built as Ala in the reported
structure. Furthermore, only the carbon of the R2 C-terminal car-
boxyl group was present in the PDB structure. Nevertheless this
was an excellent starting point for our further peptidomimetic de-
sign efforts, since we had previously shown that both Trp5 and
Phe7 in 1 are important for inhibiting M. tuberculosis RNR. We thus
decided to investigate if it was possible to substitute part of the
C-terminal region of 1 with a turn mimetic. Overlay with different
turn scaffolds previously investigated by our group, revealed that
a benzodiazepine based scaffold, originally developed as a c-turn
mimic, as a potential mimetic.21 In the comparison we used the
model compounds 1m and 11m shown in Figure 1A and B, respec-
tively. Model compound 1m represents the C-terminal fragment of
peptide 1 and 11m the corresponding fragment with the benzodi-
azepine scaffold incorporated. Based on an atom to atom compari-
son of the back-bones of 1m to 11m there is an excellent fit to the
benzodiazepine structure. To investigate if the model compound
H
A
N
O
H
HN
N
HN
O
O
NH
O
1m
H
B
N
O
H
N
HN
N
O
NH
O
O
OH
11m
Figure 2. Comparison of the C-terminal fragment of the S. typhimurium R2 (1m)
and the model compound 11m incorporating the benzodiazepine scaffold. (A)
Conformation of peptide fragment 1m, in the crystal structure of S. typhimurium
RNR. (B) Shape aligned conformer of 11m that gave the highest similarity. (C) Same
as in B but with the surface of the protein included (Connolly surface with color
coding according to cavity-depth).
Figure 1. Structures used in molecular modeling and conformational analysis. (A)
C-terminal fragment of R2, derived from the crystal structure of S. typhimurium RNR
(note that the oxygens in the C-terminal carboxyl group are lacking in the PDB
structure). (B) Model compound, incorporating the benzodiazepine scaffold.

1994
J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
Table 1
Structures, dissociation constants and inhibitory potency values of benzodiazepine-based compounds
NH
O
NH
O
HN
O
HN
H2N
H2N
N
N
OH
OH
I
II
Compound
Sequence
KD2 SDa
(lM)
IC50 SDb
20 10
(lM)
1
Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe
Ac-Glu-Asp-Asp-I-Phe
Ac-Glu-Asp-Asp-Asp-I-Phe
Ac-Asp-I-Phe
Fmoc-Asp-I-Phe
Fmoc-I
8.3 0.3
81 13
11
12
13
14
15
17
27
2
64
9
>150
1.6 0.2
8.1 0.1
24
2
Fmoc-II
26
1
171 67
a
KD2 is the dissociation constant of the interaction between R1 and compound.
IC50 is the inhibitory capacity where the reduction of CDP is monitored.
b
Arg side chain is not well defined in the complex. The side chain of
Trp can be seen pointing down into the hydrophobic cleft.
the presence of N,N-diisopropylethylamine (DIEA). Fmoc-deprotec-
tion was achieved by treatment with 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU) to give the free amine 5. The subsequent intra-
molecular nucleophilic aromatic substitution proceeded in Et3N
and dimethylsulfoxide (DMSO) at 100 °C to deliver the cyclized
product 6. To generate the benzodiazepine 7, the tryptophan indole
was protected using Boc-anhydride and N,N-dimethyl-4-amino-
We thus decided to incorporate this scaffold into peptide 1 to
give pseudopeptide 11 (Table 1). Because of synthetic reasons,
the side chain corresponding to Asp6 was omitted in the turn scaf-
fold. However, this should not have a detrimental effect on the
activity since our earlier studies have shown that exclusion of
the carboxylic acid functionality in this position only results in a
two-fold drop in potency.8 Since the direction of the N-terminal
amino acid entering the turn mimetic in 11m was slightly different
as compared to 1m we decided to also incorporate the longer frag-
ment Glu-Asp-Asp-Asp to give pseudopeptide 12. We also decided
to introduce the benzodiazepine scaffold in shorter analogues of 1
with either an N-acetyl or an Fmoc protecting group (compound 13
and 14, respectively). Compound 14 was prepared because we re-
cently showed that the Fmoc group had a beneficial effect on bind-
ing. The corresponding N-acetyl analogue (13) was prepared as a
reference compound. Furthermore, we have previously shown that
Fmoc-Trp has a binding affinity comparable to that of peptide 1,14
and therefore also prepared compound 15. In addition, the alcohol
analogue 17 was generated.
pyridine (DMAP). Direct incorporation of Fmoc-L-Trp(Boc)-OH
was not compatible with the cyclization reaction protocol. Under
these high-temperature conditions the Boc-protecting group was
cleaved off to some extent. The alcohol 8 was obtained by cleavage
of the TBDMS-protecting group by tetrabutylammonium fluoride
(TBAF). Then, the carboxylic acid 9 was synthesized in a two step
reaction starting with a Swern oxidation to the aldehyde, followed
by additional oxidation using sodium chlorite. Finally, reduction of
the nitro group of 9 was performed by catalytic hydrogenation
and the resulting amine was protected with FmocCl to give the
scaffold 10.
Pseudopeptides 11–14 were prepared manually from H-L-Phe-
2-chlorotrityl resin. The coupling of scaffold 10 to the solid phase
was performed in N,N-dimethylformamide (DMF), using (ben-
zotriazol-1-yl-oxy)tripyrrolidinophosphonium
hexafluorophos-
2.2. Chemistry
phate (PyBOP) in the presence of DIEA. For introduction of the
additional amino acids, the same conditions were used. After com-
pletion of the sequences, the terminal amino group of pseudopep-
tide 11–13 was acetylated by acetic anhydride whereas the Fmoc
group of 14 was retained. The crude pseudopeptides were purified
by preparative RP-HPLC and the overall yields ranged from 14% to
45%.
Also, the deprotected turn mimetic 15 was generated by treat-
ment of 10 with TFA (Scheme 2). Furthermore, the corresponding
alcohol 17 was produced by catalytic hydrogenation of 7 followed
by coupling with FmocCl to give 16 (Scheme 3). The Boc-group of
the indole and the TBDMS-group on the alcohol were both re-
moved by TFA to deliver 17.
The benzodiazepine based scaffold investigated in this study al-
lows introduction of numerous different amino acid equivalents
and can be coupled into a peptide sequence using standard solid
phase peptide synthesis (SPPS) methodology. Hence, the trypto-
phan-based benzodiazepine turn mimetic 10 was synthesized in
solution followed by incorporation using standard SPPS methodol-
ogy to give the pseudopeptides 11–14. Scaffold 10 was generated
essentially following the procedure for the preparation of turn
mimetics described by Rosenström et al.21 and the synthetic route
is outlined in Scheme 1. First, the aldehyde 321 was synthesized
from 2-chloro-3-nitrobenzoic acid (2) by reduction to the alcohol
and a subsequent Swern oxidation. Preparation of the protected
alcohol 4 started with a reductive amination of 3 with 2-aminoeth-
anol. The generated alcohol was then protected using tert-butyl-
dimethylchlorosilane (TBDMSCl). In the following step, 4 was
2.3. Biochemical evaluation
Target compounds 11–15, 17 and reference compound 1 were
tested in an FP assay. In the assay the compounds competed with
a fluorescent probe, incorporating the R2 C-terminal sequence,
for binding to R1.14 The compounds that showed the best binding
coupled with Fmoc-L-Trp-OH using N-[(dimethylamino)-1H-1,2,
3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanamini-
um hexafluorophosphate N-oxide (HATU) as activating reagent in

J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
1995
NO2
NO2
NO2
Cl
N
NO2
Cl
Cl
Cl
O
a
b
c
NH2
HN
H
OH
TBDMS
O
TBDMS
NH
O
O
O
3
2
(53%)
4
(58%)
5 (77%)
N
Boc
NH
N
Boc
e
O
d
O
f
O
HN
HN
O2N
HN
O2N
O2N
N
TBDMS
N
TBDMS
N
O
O
OH
6
7
8
(89%)
(66%)
(77%)
N
Boc
N
Boc
g
h
O
O
HN
O
HN
O
Fmoc NH
O2N
N
N
OH
OH
9
(69%)
10 (83%)
Scheme 1. Reagents and conditions: (a) (i) NaBH4, BF3ꢀEt2O, THF, (ii) oxalyl chloride, DMSO, Et3N, DCM; (b) (i) 2-aminoethanol, HOAc, NaCNBH3, MeOH, (ii) TBDMSCl, DBU,
THF; (c) (i) Fmoc- -Trp-OH, HATU, DIEA, DCM, (ii) DBU, THF; (d) Et3N, DMSO; (e) Boc2O, DMAP, MeCN; (f) TBAF, THF; (g) (i) oxalyl chloride, DMSO, Et3N, DCM, (ii) NaClO2,
NaH2PO4, cyclohexene, t-BuOH, H2O; (h) (i) H2, Pd/C, MeOH, (ii) FmocCl, Na2CO3 (aq), 1,4-dioxane.
L
in improved binding properties (KD2 = 27 lM). Interestingly, incor-
poration of the benzodiazepine turn mimetic in 1 gave a 10-fold
drop in potency for 11 but only a three-fold drop in potency for
12. That 12 has a lower KD2 value than 11 could be a consequence
of an overall better fit of the pseudopeptide 12 to the binding site.
This could be due to extra interactions of the additional amino acid
of 12, improving the binding affinity as compared to 11. Alterna-
tively, there is a poorer match between the native peptide and
the turn mimetic than suggested by modeling and the extra Asp
residue in 12 is needed to place the amino acids Glu-Asp-Asp in
a more favorable position for binding.
NH
a
10
O
O
HN
O
NH
N
O
OH
15 (44%)
Scheme 2. Reagents and conditions: (a) TFA, DCM.
The N-acetylated pseudopeptide 13 (KD2 = >150 lM) exhibited a
very poor binding affinity. Most interestingly, when an Fmoc-
in the FP assay and for which material was available were also
evaluated for their inhibitory capacity using the [3H]CDP assay
where the reduction of CDP is monitored.8 Structures, dissociation
constants (KD2 values) and inhibitory potency values (IC50) are pre-
sented in Table 1
Incorporation of the benzodiazepine-based scaffold into the se-
quence of peptide 1 gave pseudopeptide 11 with a KD2 value of
81 lM. Introduction of the additional Asp residue of 12 resulted
protecting group is introduced in the same sequence (14), the
KD2 value is improved to 1.6 lM. The poor binding affinity of 13
is supported by the SAR obtained from the series of N-acetyl
truncated peptides that showed that at least a hexapeptide is
needed for inhibition at micromolar concentrations.8 Similarly,
the beneficial effect of introducing an Fmoc group in short peptides
is seen for compound 14, which has the best binding affinity in
this series.

1996
J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
N
Boc
O
NH
a
b
7
O
O
O
O
HN
HN
NH
NH
N
TBDMS
N
O
OH
17 (71%)
16 (62%)
Scheme 3. Reagents and conditions: (a) (i) H2, Pd/C, EtOH, (ii) FmocCl, Na2CO3 (aq), 1,4-dioxane; (b) TFA, DCM.
Evaluation of compound 15 (KD2 = 8.1
l
M) revealed that the
50 ꢁ 4.6 mm, H2O/MeCN gradient with 25 mM NH4OAc, pH 6.3)
and column 2 (Thermo Hypersil C4, 50 ꢁ 4.6 mm, 5 m, H2O/MeCN
Fmoc-protected benzodiazepine scaffold, lacking the C-terminal
Phe residue, had a KD2 value comparable to peptide 1. Substitution
of the C-terminal carboxylic acid functionality with the corre-
l
gradient with 0.1% TFA). Optical rotation was measured on a Per-
kin–Elmer model 241 polarimeter. NMR spectra were recorded
on a Varian Mercury plus spectrometer (1H at 399.8 MHz, 13C at
100.5 MHz) at ambient temperature unless otherwise stated.
Chemical shifts are reported as d values in ppm and are indirectly
referenced to TMS via the solvent signal (1H: CHCl3 d 7.26, CHD2OD
d 3.31, DMSO-d5 d 2.50; 13C: CDCl3 d 77.2). Exact molecular masses
were determined on a Micromass Q-Tof2 mass spectrometer
equipped with an electrospray ion source. Elemental analyses were
performed by Analytische Laboratorien, Lindlar, Germany.
sponding hydroxylic group, as in compound 17 (KD2 = 26 lM), only
reduces the ability to compete for the R2 binding site by a factor of
three as compared to 15. However, without an X-ray structure it is
very difficult to speculate how compound 15 and 17 bind in the
protein cavity.
Finally, we decided to study the best compounds in Table 1 (for
which material was available) using the activity assay measuring
the reduction of tritium labeled cytidine diphosphate. The IC50 of
the reference compound 1 was seven times better in the present
study as compared to our previously reported value.8 This most
likely reflects the slightly modified assay conditions were the
experimental procedure has been optimized. However, it was
pleasing to learn that the compounds that had affinity for M. tuber-
culosis R1 also were active in the activity assay. Furthermore, there
was a reasonable correlation between the KD2 and IC50 values.
4.1.1. 2-Chloro-3-nitrobenzaldehyde (3)
Compound 3 was synthesized as previously reported21 starting
from 2-chloro-3-nitrobenzoic acid (10 g, 50 mmol) to give 3 (4.9 g,
53%) as beige solid.
4.1.2. 2-(tert-Butyldimethylsilyloxy)-N-(2-chloro-3-
nitrobenzyl)ethanamine (4)
To a solution of 3 (4.7 g, 25 mmol) in MeOH (250 mL) was added
2-aminoethanol (7.6 mL, 127 mmol) and HOAc (2.3 mL, 41 mmol).
After 10 min NaCNBH3 (1.75 g, 28 mmol) was added and the reac-
tion mixture was refluxed for 2.5 h. The mixture was cooled to
room temperature and was acidified to pH 1 by addition of 2.0 M
aqueous HCl. The residue was partly evaporated and was extracted
with diethyl ether. The organic layer was washed with H2O and
brine, dried with MgSO4 and was concentrated by rotary evapora-
tion to give 2-(2-chloro-3-nitro-benzylamino)ethanol21 as a solid.
The unprotected alcohol in MeCN (120 mL) was cooled in an H2O
bath. DBU (4.1 mL, 27 mmol) and TBDMSCl (4.2 g, 27 mmol) were
added and the reaction mixture was stirred at room temperature
for 4 h. H2O and EtOAc were added, the layers were separated
and the aqueous layer was extracted with EtOAc. The organic lay-
ers were washed with H2O and brine, dried with MgSO4 and con-
centrated by rotary evaporation. The crude product was purified
by column chromatography (EtOAc:pentane 1:4) to give 4 (4.6 g,
58%) as yellow solid. 1H NMR (CDCl3): d 7.72 (dm, J = 7.9 Hz, 1H),
7.66 (dd, J = 1.6, 7.9 Hz, 1H), 7.38 (dd, J = 7.9, 7.9 Hz, 1H), 3.99 (s,
2H), 3.78–3.73 (m, 2H), 2.77–2.72 (m, 2H), 2.09 (br s, 1H), 0.89
(s, 9), 0.06 (s, 6H). 13C NMR (CDCl3): d 149.4, 141.4, 132.8, 127.3,
125.7, 123.7, 62.5, 51.3, 51.0, 26.1, 18.5, ꢂ5.1. Anal. Calcd for
3. Conclusions
Based on modeling studies, a benzodiazepine-based turn mi-
metic has been identified, synthesized and incorporated into the
acetylated heptapeptide corresponding to the R2 C-terminus of
M. tuberculosis RNR. The benzodiazepine scaffold successfully re-
placed amino acids of the original sequence and was thus able to
mimic the starting peptide. Smaller benzodiazepine analogues
were also prepared and showed binding affinity to R1 comparable
to the original heptapeptide. The activities of these compounds
were also confirmed in an activity assay. Benzodiazepines are con-
sidered as privileged structures and these compounds should
therefore serve as excellent starting points for the development
of potent nonpeptide inhibitors of M. tuberculosis RNR.
4. Experimental section
4.1. Chemistry: general
Reagents and solvents were obtained commercially and used
without further purification. TLC was performed using aluminium
sheets precoated with silica gel 60 F254 (Merck) with visualization
of spots using UV-detection and/or ninhydrin treatment followed
by heating. Column chromatography was performed using silica
C15H25ClN2O3Si: C, 52.23; H, 7.31; N, 8.12, found: C, 52.52; H,
7.45; N, 8.18.
gel 60 (40–63 lm) (Merck). LC–MS was carried out on a Gilson–
4.1.3. (S)-2-Amino-N-(2-(tert-butyldimethylsilyloxy)ethyl)-N-
(2-chloro-3-nitrobenzyl)-3-(1H-indol-3-yl)propanamide (5)
To a solution of 4 (1.0 g, 2.9 mmol) in DCM (25 mL) was added
Finnigan AQA system in ESI mode using a Chromolith SpeedROD
RP-18e 4.6 ꢁ 50 mm column (Merck) and a CH3CN/H2O linear gra-
dient with 0.05% HCOOH. Preparative RP-HPLC was done using a
Fmoc-L-Trp-OH (1.5 g, 3.5 mmol), HATU (1.3 g, 3.5 mmol) and DIEA
(1.1 mL, 6.4 mmol). The reaction mixture was stirred at room tem-
perature overnight. H2O and EtOAc were added, the layers were
Zorbax SB-C8, 5
inhibitors was determined by analytical RP-HPLC in the following
systems (UV detection at 220 nm): column (ACE C18,
l
m, 21.2 ꢁ 150 mm column. The purity of the
1
5

J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
1997
separated and the aqueous layer was extracted with EtOAc. The or-
ganic layers were washed with H2O and brine, dried with MgSO4,
concentrated by rotary evaporation and purified by column chro-
matography (EtOAc:hexane 1:2). The resulting Fmoc protected
amine was dissolved in dry THF (20 mL) and DBU (0.47 mL,
3.1 mmol) and stirred at room temperature for 1.5 h. The crude
product was purified by column chromatography (2% MeOH in
DCM). Concentration by rotary evaporation gave 5 (1.2 g, 77%) as
J = 16.9 Hz, 1H), 3.89–3.71 (m, 3H), 3.54 (ddd, J = 1.2, 4.8, 15.1 Hz,
1H), 3.52–3.43 (m, 1H), 3.13 (dd, J = 9.2, 15.1 Hz, 1H), 1.68 (s,
9H), 0.88 (s, 9H), 0.025 (s, 3H), 0.019 (s, 3H). 13C NMR (CDCl3): d
168.7, 149.5, 144.0, 135.8, 135.7, 133.1, 129.9, 127.2, 125.2,
124.6, 122.9, 122.6, 118.5, 115.5, 115.3, 115.1, 83.7, 61.9, 54.0,
53.2, 49.7, 28.2, 27.0, 25.9, 18.2, -5.5. Anal. Calcd for C31H42N4O6Si:
C, 62.60; H, 7.12; N, 9.42, found: C, 62.81; H, 7.29; N, 9.26.
pale yellow solid. ½a D23
ꢃ
= +54° (c = 1.00, DCM). 1H NMR (CDCl3): d
4.1.6. (S)-tert-Butyl 3-((4-(2-hydroxyethyl)-9-nitro-3-oxo-
2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-2-yl)methyl)-1H-
indole-1-carboxylate (8)
(ꢄ5:3 mixture of rotamers) 8.32–8.21 (m, 1H), 7.64–7.58 (m, 2H),
7.40–7.30 (m, 1H), 7.23–6.95 (m, 5H), 4.87 & 4.76 (minor) (d,
J = 16.4 & 18.6 (minor) Hz, 1H), 4.70 (minor) & 4.57 (d, J = 16.4 &
18.6 (minor) Hz, 1H), 4.27–4.22 & 3.68–3.63 (minor) (m, 1H),
3.84–3.36 (m, 3H), 3.17–3.09 (m, 1H), 3.24–3.13 (m, 1H), 3.03 &
2.89 (minor) (dd, J = 7.1, 14.1 & 7.6, 14.1 (minor) Hz, 1H), 1.70 (br
s, 2H), 0.86 (s, 9H), 0.030–(ꢂ0.004) (m, 6H). 13C NMR (CDCl3): d
(ꢄ5:3 mixture of rotamers) 176.7 & 176.1 (minor), 149.2 & 149.1
(minor), 138.3 & 138.2 (minor), 136.5 & 136.4 (minor), 131.6 &
129.7 (minor), 127.54 (minor) & 127.20, 127.52 & 127.33 (minor),
125.1 & 124.4 (minor), 124.0 (minor) & 123.6, 123.3 & 123.2 (min-
or), 122.43 (minor) & 122.39, 119.88 (minor) & 119.86, 118.7 &
118.4 (minor), 111.8 & 111.6 (minor), 111.5 & 111.4 (minor), 61.7
(minor) & 61.6, 52.5 (minor) & 51.8, 50.8 (minor) & 48.1, 50.2 &
49.6 (minor), 32.9 (minor) & 32.4, 26.0 & 25.9 (minor), 18.4 & 18.2
(minor), ꢂ5.3 (minor) & ꢂ5.4. Anal. Calcd for C26H35ClN4O4Si: C,
58.80; H, 6.64; N, 10.55, found: C, 59.02; H, 6.75; Cl, 6.67; N, 10.55.
To a solution of 7 (0.48 g, 8.0 mmol) in THF (15 mL) was added
1.0 M TBAF in THF (1.0 mL). After 20 min, H2O and EtOAc were
added, the layers were separated and the aqueous layer was ex-
tracted with EtOAc. The organic layers were washed with H2O,
brine, dried with Na2SO4 and concentrated by rotary evaporation.
The resulting product was purified by column chromatography
(2% MeOH in DCM) to give the deprotected alcohol 8 (0.34 g,
89%) as orange solid. ½a D23
ꢃ
= ꢂ11° (c = 1.00, DCM). 1H NMR (CDCl3):
d 8.45 (d, J = 2.9 Hz, 1H), 8.22–8.13 (m, 1H), 8.06 (dd, J = 1.6, 8.6 Hz,
1H), 7.70 (s, 1H), 7.59–7.56 (m, 1H), 7.36–7.32 (m, 1H), 7.29–7.24
(m, 1H), 7.13 (dd, J = 1.6, 7.1 Hz, 1H), 6.54 (dd, J = 7.1, 8.6 Hz, 1H),
5.47 (d, J = 16.7 Hz, 1H), 5.23 (ddd, J = 2.9, 4.8, 9.0 Hz, 1H), 3.98
(d, J = 16.7 Hz, 1H), 3.85–3.71 (m, 3H), 3.68–3.59 (m, 1H), 3.54
(ddd, J = 1.1, 4.8, 15.1 Hz, 1H), 3.14 (dd, J = 9.0, 15.1 Hz, 1H), 1.88
(br s, 1H), 1.68 (s, 9H). 13C NMR (CDCl3): d 169.7, 149.5, 143.8,
135.8 (2 signals), 133.2, 129.9, 127.6, 125.3, 124.7, 122.6, 122.5,
118.5, 115.5, 115.4, 114.9, 83.8, 61.8, 54.0, 53.1, 50.4, 28.2, 27.0.
4.1.4. (S)-2-((1H-Indol-3-yl)methyl)-4-(2-(tert-
butyldimethylsilyloxy)ethyl)-9-nitro-4,5-dihydro-1H-
benzo[e][1,4]diazepin-3(2H)-one (6)
4.1.7. (S)-2-(2-((1-(tert-Butoxycarbonyl)-1H-indol-3-yl)methyl)-
9-nitro-3-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-
yl)acetic acid (9)
To a solution of 5 (1.2 g, 2.2 mmol) in DMSO (25 mL) was added
Et3N (0.47 mL, 3.4 mmol). The reaction mixture was stirred at
100 °C for 26 h. H2O and EtOAc were added, the layers were sepa-
rated and the aqueous layer was extracted with EtOAc. The organic
layers were washed with H2O, brine, dried with MgSO4, concen-
trated by rotary evaporation and purified by column chromatogra-
phy (2% MeOH in DCM) to give 6 (0.73 g, 66%) as orange solid.
DMSO (0.10 mL, 1.4 mmol) was added dropwise to a solution of
oxalyl chloride (0.060 mL, 0.70 mmol) in dry DCM (2.0 mL) at
ꢂ78 °C. After 15 min, a solution of the deprotected alcohol 8
(0.23 g, 0.47 mmol) in dry DCM (3.0 mL), was added dropwise. The
reaction mixture was stirred at ꢂ78 °C for 1.5 h. Et3N (0.26 mL,
1.9 mmol) was added and the mixture was allowed to warm to
ꢂ30 °C over 1 h. H2O and DCM were added, the layers were separated
and the aqueous layer was extracted with DCM. The organic layers
were washed with 1.0 M aqueous KHSO4, H2O, saturated aqueous
NaHCO3 and brine, were dried with MgSO4 and concentrated by ro-
tary evaporation. The resulting aldehyde was dissolved in t-BuOH
(5.0 mL) and cyclohexene (0.92 mL, 9.4 mmol). The solution was
cooled on an ice-bath and a cold solution of NaClO2 (0.53 g, 4.7 mmol)
and NaH2PO4ꢀH2O (0.64 g, 4.7 mmol) in H2O (2.5 mL) was added. The
reaction mixture was stirred for 2.5 h and EtOAc was added, the lay-
ers were separated and the aqueous layer was extracted with EtOAc.
The organic layer was washed with H2O, brine, dried with MgSO4 and
concentrated by rotary evaporation. The crude product was purified
by column chromatography (gradient elution, 2% MeOH in DCM to
10% MeOH in DCM), concentrated and redissolved in EtOAc. The or-
ganic solution was washed with 1.0 M aqueous KHSO4, dried with
MgSO4 and concentrated by rotary evaporation to give 9 (0.16 g,
½
a 2D3
ꢃ
= ꢂ149° (c = 1.00, DCM). 1H NMR (CDCl3):
d 8.50 (d,
J = 2.5 Hz, 1H), 8.47–8.41 (m, 1H), 8.00 (dd, J = 1.7, 8.7 Hz, 1H),
7.62–7.58 (m, 1H), 7.45–7.38 (m, 1H), 7.35 (d, J = 2.5 Hz, 1H),
7.23–7.18 (m, 1H), 7.17–7.11 (m, 1H), 7.06 (dd, J = 1.7, 7.1 Hz,
1H), 6.47 (dd, J = 7.1, 8.7 Hz, 1H), 5.38 (d, J = 16.6 Hz, 1H), 5.27
(ddd, J = 2.5, 4.5, 10.0 Hz, 1H), 4.06 (d, J = 16.6 Hz, 1H), 3.90–3.74
(m, 3H), 3.60 (ddd, J = 1.0, 4.5, 15.1 Hz, 1H), 3.52–3.45 (m, 1H),
3.21 (dd, J = 10.0, 15.1 Hz, 1H), 0.91 (s, 9H), 0.058 (s, 3H), 0.056
(s, 3H). 13C NMR (CDCl3): d 169.3, 144.5, 136.8, 135.9, 132.8,
127.2, 127.0, 124.7, 123.0, 122.3, 119.6, 118.4, 115.2, 111.8,
110.1, 62.0, 54.4, 53.2, 49.8, 27.5, 26.0, 18.3, ꢂ5.3.
4.1.5. (S)-tert-Butyl 3-((4-(2-(tert-butyldimethylsilyloxy)ethyl)-
9-nitro-3-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-2-
yl)methyl)-1H-indole-1-carboxylate (7)
The amine 6 (0.56 g, 1.1 mmol) was dissolved in MeCN (35 mL).
Boc2O (0.37 g, 1.7 mmol) and DMAP (0.21 g, 1.7 mmol) were added
and the reaction mixture was stirred at room temperature for 2 h.
H2O and DCM were added, the layers were separated and the aque-
ous layer was extracted with DCM. The organic layers were washed
with saturated aqueous NaHCO3, H2O, 1.0 M aqueous KHSO4 and
brine, dried with MgSO4 and concentrated by rotary evaporation.
The crude product was purified by column chromatography (0.5%
69%) as yellow solid. ½a D23
ꢃ
= ꢂ95° (c = 1.00, DCM). 1H NMR (CDCl3):
d 8.45 (d, J = 2.9 Hz, 1H), 8.23–8.12 (m, 1H), 8.06 (dd, J = 1.7, 8.8 Hz,
1H), 7.70 (s, 1H), 7.61–7.56 (m, 1H), 7.38–7.31 (m, 1H), 7.30–7.24
(m, 1H), 7.14–7.07 (m, 1H), 7.10 (dd, J = 1.7, 7.1 Hz, 1H), 6.55 (dd,
J = 7.1, 8.8 Hz, 1H), 5.54 (d, J = 16.9 Hz, 1H), 5.26 (ddd, J = 2.9, 4.5,
9.2 Hz, 1H), 4.66 (d, J = 17.6 Hz, 1H), 4.04 (d, J = 17.6 Hz, 1H), 3.87
(d, J = 16.9 Hz, 1H), 3.55 (ddd, J = 1.0, 4.5, 15.2 Hz, 1H), 3.15 (dd,
J = 9.2, 15.2 Hz, 1H), 1.68 (s, 9H). 13C NMR (CDCl3): d 172.5, 169.8,
149.5, 143.7, 135.8 (2 signals), 133.4, 129.8, 127.5, 125.3, 124.7,
122.6, 121.8, 118.6, 115.54, 115.50, 114.7, 83.9, 53.7, 52.9, 48.2,
28.2, 26.9. Anal. Calcd for C25H26N4O7: C, 60.72; H, 5.30; N, 11.33,
found: C, 60.84; H, 5.46; N, 11.19.
MeOH in DCM) to give
7 (0.52 g, 77%) as yellow solid.
½
a 2D3
ꢃ
= ꢂ86° (c = 1.03, DCM). 1H NMR (CDCl3): d 8.46 (d, J = 2.9 Hz,
1H), 8.21–8.14 (m, 1H), 8.05 (dd, J = 1.7, 8.7 Hz, 1H), 7.69 (s, 1H),
7.59–7.56 (m, 1H), 7.37–7.30 (m, 1H), 7.29–7.23 (m, 1H), 7.10
(dd, J = 1.7, 7.1 Hz, 1H), 6.52 (dd, J = 7.1, 8.7 Hz, 1H), 5.40 (d,
J = 16.9 Hz, 1H), 5.21 (ddd, J = 2.9, 4.8, 9.2 Hz, 1H), 4.09 8 (d,

1998
J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
4.1.8. (S)-2-(9-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-
((1-(tert-butoxycarbonyl)-1H-indol-3-yl)methyl)-3-oxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)acetic acid (10)
To a solution of 9 (0.10 g, 0.20 mmol) in MeOH (10 mL) was
added Pd/C 10% (0.015 g, 0.014 mmol). The reaction mixture was
stirred under H2 atmosphere for 4 h. The catalyst was filtered off
and the solvent was removed by rotary evaporation. The crude
product was dissolved in 1,4-dioxane (7.0 mL) and was cooled to
0 °C. FmocCl (0.10 g, 0.40 mmol) was added followed by a solution
of Na2CO3 (0.085 g, 8.0 mmol) in H2O (2.0 mL). The reaction mix-
ture was stirred at room temperature over night and then washed
with ether. The aqueous layer was acidified to pH 3 by addition of
5% aqueous citric acid and was extracted with EtOAc. The organic
layers were washed with H2O, brine, dried with MgSO4 and concen-
trated by rotary evaporation to give 10 (0.11 g, 79%) as beige foam.
0.084 g. The pseudopeptide polymer was treated with triethylsilane
(0.075 mL) and 95% aqueous TFA (1.5 mL) for 1.5 h. The polymer
was removed by filtration and washed with TFA (2 ꢁ 0.3 mL). The
combined filtrates were evaporated in a stream of nitrogen to
approximately 0.1 mL and the product was precipitated with
diethyl ether (12 mL). It was collected by centrifugation and
washed with more diethyl ether (3 ꢁ 4 mL) and dried in vacuo to
give 0.034 g. The crude product was purified by preparative RP-
HPLC. Selected fractions were pooled and lyophilized to give
pseudopeptide 11 (0.021 g, 45%). 1H NMR (CD3OD): d 7.65 (dm,
J = 7.9 Hz, 1H), 7.34 (dm, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.24–7.10
(m, 5H), 7.09–7.04 (m, 1H), 7.04–6.99 (m, 1H), 6.96 (dd, J = 1.3,
7.6 Hz, 1H), 6.80 (dd, J = 1.3, 7.6 Hz, 1H), 6.54 (dd, J = 7.6, 7.6 Hz,
1H), 5.17 (d, J = 16.8 Hz, 1H), 4.99 (dd, J = 5.7, 7.9 Hz, 1H), 4.75
(dd, J = 6.7, 6.7 Hz, 1H), 4.65 (dd, J = 6.0, 6.5 Hz, 1H), 4.56 (d,
J = 16.3 Hz, 1H), 4.54 (dd, J = 5.7, 8.2 Hz, 1H), 4.30–4.24 (m, 1H),
3.70 (d, J = 16.8 Hz, 1H), 3.64 (d, J = 16.3 Hz, 1H), 3.43 (dd, J = 5.7,
14.9 Hz, 1H), 3.24 (dd, J = 4.7, 14.7 Hz, 1H), 3.09 (dd, J = 7.9,
14.9 Hz, 1H), 2.91 (dd, J = 8.2, 14.7 Hz, 1H), 2.82–2.73 (m, 3H),
2.67 (dd, J = 6.0, 16.1 Hz, 1H), 2.41–2.33 (m, 2H), 2.12–2.00 (m,
1H), 1.98 (s, 3H), 1.98–1.88 (m, 1H). HRMS calcd for
½
a 2D3
ꢃ
= ꢂ50° (c = 1.01, DCM). 1H NMR (DMSO-d6, 70° C): d 8.40 (br s,
1H), 7.98 (dm, J = 8.3 Hz, 1H), 7.86 (dm, J = 7.7 Hz, 2H), 7.64 (dm,
J = 7.9 Hz, 1H), 7.64–7.58 (m, 2H), 7.58 (s, 1H), 7.42–7.36 (m, 2H),
7.31–7.23 (m, 3H), 7.21–7.16 (m, 1H), 7.04 (dm, J = 7.6 Hz, 1H),
6.88 (dd, J = 1.6, 7.6 Hz, 1H), 6.57 (dd, J = 7.6, 7.6 Hz, 1H), 5.44 (d,
J = 16.5 Hz, 1H), 4.99 (dd, J = 5.6, 7.6 Hz, 1H), 4.93–4.71 (m, 1H),
4.34 (d, J = 17.1 Hz, 1H), 4.27–4.22 (m, 2H), 4.19–4.14 (m, 1H),
4.07 (d, J = 16.5 Hz, 1H), 3.93 (d, J = 17.1 Hz, 1H), 3.29 (dd, J = 5.6,
15.3 Hz, 1H), 3.00 (dd, J = 7.6, 15.3 Hz, 1H), 1.57 (s, 9H).
C
44H49N8O14 (M+H+): 913.3368, found: 913.3378. RP-HPLC purity
(column 1: 96%, column 2: 96%).
4.2.2. Ac-Glu-Asp-Asp-Asp-I-Phe (12)
4.2. Solid phase peptide synthesis (SPPS)
A solution of Fmoc-L-Asp(OtBu)-OH (0.082 g, 0.20 mmol), Py-
BOP (0.10 g, 0.20 mmol) and DIEA (0.070 mL, 0.40 mmol) in DMF
(1.0 mL) was added to H-Asp(OtBu)-Asp(OtBu)-I-Phe-2-chlorotrityl
resin (0.083 g, 0.050 mmol) in a 2 mL syringe vessel and was re-
acted for 4 h. The resin was washed and deprotected. After washing
Pseudopeptides 11–15 were prepared manually in a disposable
syringe equipped with a porous polyethylene filter using standard
SPPS Fmoc/t-Bu chemistry. The Fmoc group was removed by treat-
ment with 20% piperidine in DMF for 10 + 10 min. Couplings were
performed in DMF, using PyBOP in the presence of DIEA. Amino
acids with the following side chain protection were used: As-
p(Ot-Bu), Glu(Ot-Bu), Trp(Boc). The crude pseudopeptides were
purified by preparative RP-HPLC.
with DMF,
a
solution of Fmoc-L-Glu(OtBu)-OH (0.085 g,
0.20 mmol), PyBOP (0.10 g, 0.20 mmol) and DIEA (0.070 mL,
0.40 mmol) in DMF (1.0 mL) was added and allowed to react for
19 h. The resin was washed and deprotected. After washing with
DMF, DCM and MeOH the resin was dried in vacuo to give
0.093 g. Then, the material was reacted in DMF (1.0 mL) with acetic
anhydride (0.048 mL, 0.50 mmol) in the presence of DIEA
(0.087 mL, 0.50 mmol) for 1 h. After washing with DMF, DCM and
MeOH the resin was dried in vacuo to give 0.094 g. Cleavage from
the resin and isolation of crude 12 (0.037 g) was performed as de-
scribed above for 11. Purification by preparative RP-HPLC gave
pseudopeptide 12 (0.012 g, 23%). 1H NMR (CD3OD): d 7.65 (dm,
J = 7.8 Hz, 1H), 7.35 (dm, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.23–7.11
(m, 5H), 7.09–7.04 (m, 1H), 7.04–6.98 (m, 1H), 6.95 (dm,
J = 7.7 Hz, 1H), 6.79 (dm, J = 7.7 Hz, 1H), 6.54 (dd, J = 7.7, 7.7 Hz,
1H), 5.17 (d, J = 16.9 Hz, 1H), 4.99 (dd, J = 5.5, 7.5 Hz, 1H), 4.73
(dd, J = 6.7, 6.7 Hz, 1H), 4.66–4.60 (m, 2H), 4.59 (d, J = 16.1 Hz,
1H), 4.57 (dd, J = 4.7, 8.2 Hz, 1H), 4.30 (dd, J = 5.6, 7.9 Hz, 1H),
3.71 (d, J = 16.9 Hz, 1H), 3.64 (d, J = 16.1 Hz, 1H), 3.44 (dd, J = 5.5,
14.8 Hz, 1H), 3.24 (dd, J = 4.7, 13.9 Hz, 1H), 3.11 (dd, J = 7.5,
14.8 Hz, 1H), 2.92 (d, J = 8.2, 13.9 Hz, 1H), 2.88–2.77 (m, 4H),
2.77–2.67 (m, 2H), 2.38–2.32 (M, 2H), 2.12–2.02 (m, 1H), 2.00–
1.89 (m, 1H), 1.96 (s, 3H). HRMS calcd for C48H54N9O17 (M+H+):
1028.3638, found: 1028.3627. RP-HPLC purity (column 1: 97%, col-
umn 2: 96%).
4.2.1. Ac-Glu-Asp-Asp-I-Phe (11)
Compound 10 (0.10 g, 0.15 mmol), PyBOP (0.12 mg, 0.23 mmol)
and DIEA (0.052 mL, 0.30 mmol) were dissolved in DMF (1.5 mL).
The mixture was added to H-L-Phe-2-chlorotrityl resin (0.18 g,
0.15 mmol) in a 2 mL syringe vessel and allowed to react for
22 h. The resin was washed with DMF and the Fmoc group was re-
moved. After washing with DMF, DCM and MeOH the resin was
dried in vacuo to give 0.22 g. The material was further reacted in
DMF (1.0 mL) with Fmoc-L-Asp(OtBu)-OH (0.19 g, 0.45 mmol) and
PyBOP (0.23 g, 0.45 mmol) in the presence of DIEA (0.16 mL,
0.90 mmol) for 24 h. The resin was washed and the coupling was
repeated. Thereafter, the resin was washed and deprotected. After
washing with DMF, DCM and MeOH the resin was dried in vacuo to
give 0.23 g.
Two thirds of the material (0.15 g, 0.10 mmol) was transferred
to another syringe and reacted in DMF (1.0 mL) with Fmoc-L-As-
p(OtBu)-OH (0.17 g, 0.40 mmol) and PyBOP (0.21 g, 0.40 mmol) in
the presence of DIEA (0.14 mL, 0.80 mmol) for 17 h. The resin
was washed and deprotected. After washing with DMF, DCM and
MeOH the resin was dried in vacuo to give 0.16 g.
Half of the remaining material (0.80 g, 0.050 mmol) was trans-
ferred to another syringe and reacted in DMF (1.0 mL) with Fmoc-
4.2.3. Ac-Asp-I-Phe (13)
Acetic anhydride (0.048 mL, 0.50 mmol) and DIEA (0.087 mL,
0.50 mmol) in DMF (1.0 mL) were added to H-Asp(OtBu)-I-Phe-2-
chlorotrityl resin (0.076 g, 0.050 mmol) and was reacted in a
2 mL syringe vessel for 1 h. The resin was washed with DMF,
DCM and MeOH and was dried in vacuo to give 0.078 g. Cleavage
from the resin and isolation of crude 13 (0.025 g) was performed
as described above for 11. Purification by preparative RP-HPLC
gave pseudopeptide 13 (0.0076 g, 23%). 1H NMR (CD3OD): d
7.65 (dm, J = 7.8 Hz, 1H), 7.33 (dm, J = 8.0 Hz, 1H), 7.27 (s, 1H),
L-Glu(OtBu)-OH (0.085 g, 0.20 mmol) and PyBOP (0.10 g,
0.20 mmol) in the presence of DIEA (0.070 mL, 0.40 mmol) for
47 h. The resin was washed and deprotected. After washing with
DMF, DCM and MeOH the resin was dried in vacuo to give 0.084 g.
The material was then reacted in DMF (1.0 mL) with acetic anhy-
dride (0.048 mL, 0.50 mmol) in the presence of DIEA (0.087 mL,
0.50 mmol) for 1 h. After washing with DMF, 20% piperidine in
DMF, DMF, DCM and MeOH the resin was dried in vacuo to give

J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
1999
7.23–7.11 (m, 5H), 7.08 (ddd, J = 1.4, 7.1, 8.0 Hz, 1H), 7.02 (ddd,
J = 1.3, 7.1, 7.8 Hz, 1H), 6.99 (dd, J = 1.6, 7.6 Hz, 1H), 6.81 (dd,
J = 1.6, 7.6 Hz, 1H), 6.57 (dd, J = 7.6, 7.6 Hz, 1H), 5.20 (d,
J = 16.6 Hz, 1H), 5.00 (dd, J = 6.0, 7.1 Hz, 1H), 4.74 (dd, J = 6.6,
6.8 Hz, 1H), 4.59 (dd, J = 5.1, 8.5 Hz, 1H), 4.55 (d, J = 16.3 Hz, 1H),
3.74 (d, J = 16.6 Hz, 1H), 3.67 (d, J = 16.3 Hz, 1H), 3.45 (dd, J = 6.0,
15.1 Hz, 1H), 3.22 (dd, J = 5.1, 14.0 Hz, 1H), 3.14 (dd, J = 7.1,
15.1 Hz, 1H), 2.90 (dd, J = 6.8, 17.0 Hz, 1H), 2.72 (dd, J = 6.6,
17.0 Hz, 1H), 1.94 (s, 3H). HRMS calcd for C35H37N6O8 (M+H+):
669.2673, found: 669.2678. RP-HPLC purity (column 1: 96%, col-
umn 2: 96%).
stirred under H2 atmosphere for 2 h. The catalyst was filtered off
and the solvent was removed by rotary evaporation. The resulting
amine was dissolved in 1,4-dioxane (3.0 mL) and was cooled to
0 °C. FmocCl (0.035 g, 0.13 mmol) was added followed by a solution
of Na2CO3 (0.028 g, 0.27 mmol) in H2O (1.0 mL). The reaction mix-
ture was stirred at room temperature for 1.5 h and then diethyl
ether was added. The organic layer was separated and was concen-
trated by rotary evaporation. The residue was dissolved in DCM and
was washed with 1.0 M aqueous KHSO4, H2O, and brine. The crude
product was purified by column chromatography (0.5% MeOH in
DCM) and was concentrated by rotary evaporation to give 16
(0.033 g, 62%) as beige foam. ½a D23
ꢃ
= ꢂ62° (c = 1.00, DCM). 1H NMR
4.2.4. Fmoc-Asp-I-Phe (14)
Compound 10 (0.035 g, 0.051 mmol), PyBOP (0.027 mg,
0.051 mmol) and DIEA (0.018 mL, 0.10 mmol) were dissolved in
(DMSO-d6): d 8.62 (br s, 1H), 7.97 (dm, J = 8.4 Hz, 2H), 7.89 (dm,
J = 7.7 Hz, 2H), 7.69 (dm, J = 7.8 Hz, 1H), 7.71–7.61 (m, 2H), 7.58
(s, 1H), 7.43–7.37 (m, 2H), 7.31–7.23 (m, 3H), 7.11–6.96 (m, 1H),
6.86 (dd, J = 1.6, 7.7 Hz, 1H), 6.55 (dd, J = 7.7, 7.7 Hz, 1H), 5.44 (d,
J = 16.6 Hz, 1H), 5.04–5.98 (m, 1H), 4.94–4.85 (m, 1H), 4.34–4.12
(m, 3H), 4.02 (d, J = 16.6 Hz, 1H), 3.68–3.57 (m, 3H), 3.31–3.26 (m,
1H), 3.27 (dd, J = 6.7, 14.8 Hz, 1H), 2.95 (dd, J = 7.0, 14.8 Hz, 1H)
1.55 (s, 9H), 0.81 (s, 9H), -0.052 (s, 6H). Anal. Calcd for C46H54N4O6-
Siꢀ1H2O: C, 68.62; H, 7.01; N, 6.96, found: C, 68.67; H, 6.99; N, 6.84.
DMF (1.5 mL). The mixture was added to H-L-Phe-2-chlorotrityl re-
sin (0.057 g, 0.049 mmol) in a 2 mL syringe vessel and allowed to
react for 24 h. After washing with DMF, DCM and MeOH the resin
was dried in vacuo to give 0.087 g.
Part of the material (0.044 g, 0.025 mmol) was transferred to
another syringe and the resin deprotected. After washing with
DMF, a solution of Fmoc-L-Asp(OtBu)-OH (0.041 g, 0.10 mmol), Py-
BOP (0.052 g, 0.10 mmol) and DIEA (0.035 mL, 0.20 mmol) in DMF
(1.0 mL) was added and allowed to react for 22 h. After washing
with DMF, DCM and MeOH the resin was dried in vacuo to give
0.047 g. Cleavage from the resin and isolation of crude 14
(0.013 g) was performed as described above for 11. Purification
by preparative RP-HPLC gave pseudopeptide 14 (0.0028 g, 14%).
1H NMR (CD3OD): d 7.77–7.72 (m, 2H), 7.62 (dm, J = 7.9 Hz, 1H),
7.57 (dm, J = 7.8 Hz, 1H), 7.52–7.48 (m, 1H), 7.36–7.28 (m, 3H),
7.24–7.11 (m, 8H), 7.08–7.03 (m, 1H), 7.00–6.95 (m, 2H), 6.81
(dd, J = 1.6, 7.7 Hz, 1H), 6.58 (dd, J = 7.7, 7.7 Hz, 1H), 5.23 (dm,
J = 16.5 Hz, 1H), 5.04–4.99 (m, 1H), 4.66 (dd, J = 5.1, 9.0 Hz, 1H),
4.53 (d, J = 16.4 Hz, 1H), 4.53–4.48 (m, 1H), 4.42–4.31 (m, 2H),
4.17–4.12 (m, 1H), 3.73 (dm, J = 16.5 Hz, 1H), 3.71 (dd,
J = 16.4 Hz, 1H), 3.48 (dd, J = 15.0, 6.2 Hz, 1H), 3.22–3.15 (m, 2H),
2.91–2.82 (m, 2H), 2.70 (dd, J = 16.5, 6.9 Hz, 1H). HRMS calcd for
4.2.7. (S)-(9H-Fluoren-9-yl)methyl 2-((1H-indol-3-yl)methyl)-4-
(2-hydroxyethyl)-3-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]dia
zepin-9-ylcarbamate (17)
To a solution of 16 (0.033 g, 0.042 mmol) in DCM (1.0 mL) was
added TFA (0.50 mL) at 0 °C. The reaction mixture was stirred for
1.5 h at room temperature. More DCM was added and the solvent
was evaporated. The residue was dissolved in MeCN and H2O and
was lyophilized. The crude product was purified by preparative
RP-HPLC (MeCN/H2O gradient with 0.1% TFA) to give 17 (0.017 g,
71%) as white solid. 1H NMR (DMSO-d6, 70 °C): d 10.66 (br s, 1H),
8.43 (br s, 1H), 7.87 (dm, J = 7.7 Hz, 2H), 7.66 (dm, J = 7.6 Hz, 2H),
7.60 (dm, J = 7.8 Hz, 1H), 7.44–7.38 (m, 2H), 7.34–7.28 (m, 3H),
7.19 (d, J = 2.5 Hz, 1H), 7.04–6.91 (m, 3H), 6.87 (dd, J = 1.6, 7.7 Hz,
1H), 6.52 (dd, J = 7.7, 7.7 Hz, 1H), 5.36 (d, J = 16.6 Hz, 1H), 4.90
(dd, J = 6.0, 7.8 Hz, 1H), 4.81–4.62 (m, 1H), 4.31–4.26 (m, 2H),
4.23–4.17 (m, 1H), 4.03 (d, J = 16.6 Hz, 1H), 3.61 (ddd, J = 5.5, 6.6,
12.1 Hz, 1H), 3.49–3.44 (m, 2H), 3.40–3.34 (m, 1H), 3.33 (dd,
J = 6.0, 14.8 Hz, 1H), 2.98 (dd, J = 7.8, 14.8 Hz, 1H). HRMS calcd
for C35H33N4O4 (M+H+): 573.2502, found: 573.2498. RP-HPLC pur-
ity (column 1: >99%, column 2: 97%).
C
48H45N6O9 (M+H+): 849.3248, found: 849.3242. RP-HPLC purity
(column 1: >99%, column 2: >99%).
4.2.5. (S)-2-(2-((1H-Indol-3-yl)methyl)-9-(((9H-fluoren-9-yl)me
thoxy)carbonylamino)-3-oxo-2,3-dihydro-1H-benzo[e][1,4]dia
zepin-4(5H)-yl)acetic acid (15)
To a solution of 10 (0.0080 g, 0.012 mmol) in DCM (0.50 mL)
was added TFA (0.50 mL) at 0 °C. The reaction mixture was stirred
for 1.5 h at room temperature. More DCM was added and the sol-
vent was evaporated. The residue was dissolved in MeCN and H2O
and was lyophilized. The crude product was purified by prepara-
tive RP-HPLC (MeCN/H2O gradient with 0.1% TFA) to give 15
(0.0031 g, 44%) as white solid. 1H NMR (DMSO-d6, 70 °C): d 10.66
(br s, 1H), 8.41 (br s, 1H), 7.87 (dm, J = 7.3 Hz, 2H), 7.66 (dm,
J = 7.4 Hz, 2H), 7.59 (dm, J = 7.8 Hz, 1H), 7.44–7.38 (m, 2H), 7.35–
7.27 (m, 3H), 7.19 (d, J = 2.5 Hz, 1H), 7.04–6.96 (m, 2H), 6.96–
6.90 (m, 1H), 6.84 (dm, J = 7.7 Hz, 1H), 6.52 (dd, J = 7.7, 7.7 Hz,
1H), 5.36 (d, J = 16.7 Hz, 1H), 4.93–4.86 (m, 1H), 4.73 (d,
J = 4.7 Hz, 1H), 4.31 (d, J = 16.6 Hz, 1H), 4.31–4.25 (m, 2H), 4.22–
4.16 (m, 1H), 4.01 (d, J = 16.7 Hz, 1H), 3.68 (d, J = 16.6 Hz, 1H),
3.33 (dd, J = 5.5, 14.9 Hz, 1H), 2.98 (dd, J = 7.6, 14.9 Hz, 1H). HRMS
calcd for C35H31N4O5 (M+H+): 587.2294, found: 587.2288. RP-HPLC
purity (column 1: >99%, column 2: >99%).
4.3. Conformational analysis and molecular modeling
Conformational analysis was performed on the model com-
pound 11m. The OLPS_2005 force field22 and the General Born Sol-
vent Accessible (GB/SA) surface area method for water developed
by Still et al.,23 as implemented in the program MacroModel 9.7,24
was used in the calculation. The conformational search was con-
ducted using the Monte Carlo Multiple Minimum (MCMM)25 meth-
od (50,000 steps) in the batchmin program with default settings.
Truncated Newton Conjugate Gradient26 (TNCG) minimization with
a maximum of 5000 iterations was used in the conformational
search, with derivative convergence set to 0.05 kJ/mol/Å. Confor-
mations within 5 kcal/mol of the lowest energy minimum were
kept. A subsequent reminimization of all conformations was there-
after performed using a maximum of 5000 iterations of PR Conju-
gate Gradient (PRCG), with the convergence criteria increased to
0.001 kJ/mol/Å. Figure 2 was made in Sybyl-x 2.0 (Tripos Interna-
tional, 1699 South Hanley Rd., St. Louis, MO 63144, USA).
4.2.6. (S)-tert-Butyl 3-((9-(((9H-fluoren-9-yl)methoxy)carbonyla
mino)-4-(2-(tert-butyldimethylsilyloxy)ethyl)-3-oxo-2,3,4,5-tet
rahydro-1H-benzo[e][1,4]diazepin-2-yl)methyl)-1H-indole-1-ca
rboxylate (16)
To a solution of 7 (0.040 g, 0.067 mmol) in EtOH (15 mL) was
added Pd/C 10% (10 mg, 0.0091 mmol). The reaction mixture was
To investigate if model compounds 11m can adopt similar con-
formations as the R2 C-terminal fragment from the X-ray structure
of S. typhimurium RNR, we used shape screening in Phase (v3.1).
The R1/R2 holocomplex from S. typhimurium was used for these
studies (PDB ID: 2BQ1).11 The structure was pre-processed using

2000
J. Nurbo et al. / Bioorg. Med. Chem. 21 (2013) 1992–2000
the Protein Preparation Wizard in Maestro, v8.5 (Schroödinger,
LLC, New York, NY). The PDB structure contains two R1/R2 dimers
and the R1/R2 complex where the last 11 residues of the R2 sub-
unit was present, was used in the modeling (the other R1/R2 com-
plex was deleted). The Ac-Asp-Trp-Asn-Phe fragment from the R2
C-terminus was extracted and the resulting model compound 1m
was used as template in the shape screening. In the volume scoring
all atoms were treated the same.
References and notes
1. In World Health Organization Global Tuberculosis Control, 2011. Available at
http://www.who.int/tb/publications/global_report/en/,
September 2012).
2. In Multidrug and Extensively Drug-Resistant TB (M/XDR-TB): Global Report on
Surveillance and Response, 2010. Available at http://www.who.int/tb/
publications/2010/978924599191/en/index.html (accessed 16 September
2012).
3. Raviglione, M. C.; Smith, I. M. N. Eng. J. Med. 2007, 356, 656.
4. Dawes, S. S.; Warner, D. F.; Tsenova, L.; Timm, J.; McKinney, J. D.; Kaplan, G.;
Rubin, H.; Mizrahi, V. Infect. Immun. 2003, 71, 6124.
5. Mowa, M. B.; Warner, D. F.; Kaplan, G.; Kana, B. D.; Mizrahi, V. J. Bacteriol. 2009,
191, 985.
(accessed
16
4.4. Fluorescence polarization measurements
The target compounds were evaluated using a competitive fluo-
rescence polarization assay as previously described.14 All com-
pounds were dissolved in 100% DMSO to a stock concentration of
5–25 mM and stored at ꢂ20 °C. A peptide probe with the sequence
Dansyl-Gly-Ser-Gly-Glu-Asp-Asp-Asp-Trp-Asp-Phe was used to
evaluate the compounds in a competitive binding model as de-
scribed by Roehrl et al.27,28 Measurements were done on triplicate
reactions on a Perkin Elmer Envision 2104 Multilabel Reader using
340 and 535 nm filters for excitation and emission, respectively.
6. Reichard, P. Science 1993, 260, 1773.
7. Yang, F.; Curran, S. C.; Li, L.-S.; Avarbock, D.; Graf, J. D.; Chua, M.-M.; Lu, G.;
Salem, J.; Rubin, H. J. Bacteriol. 1997, 179, 6408.
8. Nurbo, J.; Roos, A. K.; Muthas, D.; Wahlstrom, E.; Ericsson, D. J.; Lundstedt, T.;
Unge, T.; Karlen, A. J. Pept. Sci. 2007, 13, 822.
9. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.; Harris, D.; Gordon, S.
V.; Eiglmeier, K.; Gas, S.; Barry, C. E.; Tekaia, F.; Badcock, K.; Basham, D.; Brown,
D.; Chillingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.;
Hamlin, N.; Holroyd, S.; Hornby, T.; Jagels, K.; Krogh, A.; McLean, J.; Moule, S.;
Murphy, L.; Oliver, K.; Osborne, J.; Quail, M. A.; Rajandream, M. A.; Rogers, J.;
Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.; Taylor, K.;
Whitehead, S.; Barrell, B. G. Nature 1998, 393, 537.
10. Sassetti, C. M.; Boyd, D. H.; Rubin, E. J. Mol. Microbiol. 2003, 48, 77.
11. Uppsten, M.; Farnegardh, M.; Domkin, V.; Uhlin, U. J. Mol. Biol. 2006, 359, 365.
12. Gao, Y.; Liehr, S.; Cooperman, B. S. Bioorg. Med. Chem. Lett. 2002, 12, 513.
13. Tan, C. H.; Gao, Y.; Kaur, J.; Cooperman, B. S. Bioorg. Med. Chem. Lett. 2004, 14,
5301.
14. Ericsson, D. J.; Nurbo, J.; Muthas, D.; Hertzberg, K.; Lindeberg, G.; Karlén, A.;
Unge, T. J. Pept. Sci. 2010, 16, 159.
15. Fuertes, M. J.; Kaur, J.; Deb, P.; Cooperman, B. S.; Smith, A. B. Bioorg. Med. Chem.
Lett. 2005, 15, 5146.
16. Smith, A. B.; Sasho, S.; Barwis, B. A.; Sprengeler, P.; Barbosa, J.; Hirschmann, R.;
Cooperman, B. S. Bioorg. Med. Chem. Lett. 1998, 8, 3133.
17. Dziadulewicz, E. K.; Brown, M. C.; Dunstan, A. R.; Lee, W.; Said, N. B.; Garratt, P.
J. Bioorg. Med. Chem. Lett. 1999, 9, 463.
18. Lauffer, D. J.; Mullican, M. D. Bioorg. Med. Chem. Lett. 2002, 12, 1225.
19. Lee, J. Y.; Im, I.; Webb, T. R.; McGrath, D.; Song, M. R.; Kim, Y. C. Bioorg. Chem.
2009, 37, 90.
20. Ripka, W. C.; Delucca, G. V.; Bach, A. C.; Pottorf, R. S.; Blaney, J. M. Tetrahedron
1993, 49, 3609.
4.5. RNR activity [3H]CDP assay for IC50 determination
The M. tuberculosis RNR inhibitory properties of compounds 1,
12, 15 and 17 were evaluated in a [3H]CDP assay29 which was per-
formed as reported by Nurbo et al.8 The R1 concentration was 1
lM
and the R2 concentration 0.5 lM, and the buffer composition was
40 mM Hepes pH 7.5, 6 mM MgSO4, 6 mM NaF, 5 mM DTT, 3 mM
ATP, 10 mM CDP, and 3 mM [3H]CDP (888 GBq/mmol). Due to
solubility limitations of the inhibitors experiments were restricted
to the interval 5–50 lM. The compounds were ranked based on
concentration of the compound which caused 50% reduction of
the cpm signal at the given conditions. The IC50 values are an aver-
age of three separate experiments with the standard deviation.
21. Rosenstrom, U.; Skold, C.; Plouffe, B.; Beaudry, H.; Lindeberg, G.; Botros, M.;
Nyberg, F.; Wolf, G.; Karlen, A.; Gallo-Payet, N.; Hallberg, A. J. Med. Chem. 2005,
48, 4009.
Acknowledgments
22. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. J. Phys. Chem. B
2001, 105, 6474.
23. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990,
112, 6127.
24. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
25. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989, 111, 4379.
26. Ponder, J. W.; Richards, F. M. J. Comput. Chem. 1987, 8, 1016.
27. Roehrl, M. H. A.; Kang, S. H.; Aramburu, J.; Wagner, G.; Rao, A.; Hogan, P. G.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7554.
We thank Dr. Aleh Yahorau, Department of Pharmaceutical
Biosciences, Uppsala University, for conducting HRMS analyses.
We also acknowledge the Swedish Foundation for Strategic Re-
search (SSF), the Swedish Research Council (VR) and the EU Sixth
Framework Program NM4TB CT:018 923 for financial support.
Supplementary data
28. Roehrl, M. H. A.; Wang, J. Y.; Wagner, G. Biochemistry 2004, 43, 16056.
29. Engstrom, Y.; Eriksson, S.; Thelander, L.; Akerman, M. Biochemistry 1979, 18,
2941.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.020.