Macrocyclic biphenyl tetraoxazoles: Synthesis, evaluation as G-quadruplex stabilizers and cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2013.05.033

A series of macrocyclic biphenyl tetraoxazoles was synthesized. The latter stages of the synthetic approach allowed for the addition of varied N-protected α-amino acids, which were subsequently deprotected and condensed to provide the desired macrocycles.

Full text of DOI:10.1016/j.bmc.2013.05.033

Bioorganic & Medicinal Chemistry 21 (2013) 4511–4520
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Macrocyclic biphenyl tetraoxazoles: Synthesis, evaluation
as G-quadruplex stabilizers and cytotoxic activity
Gifty A. Blankson a, Daniel S. Pilch b,c, Angela A. Liu b, Leroy F. Liu b,c, Joseph E. Rice a,c, Edmond J. LaVoie a,c,
⇑
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers—The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
b Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA
c The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of macrocyclic biphenyl tetraoxazoles was synthesized. The latter stages of the synthetic
approach allowed for the addition of varied N-protected -amino acids, which were subsequently depro-
Received 24 March 2013
Revised 13 May 2013
Accepted 21 May 2013
Available online 30 May 2013
a
tected and condensed to provide the desired macrocycles. Improved yields could be realized in the mac-
rocyclization step of their synthesis relative to other macrocyclic G-quadruplex stabilizers. These 24-
membered macrocycles were evaluated for their ability to stabilize G-quadruplex DNA and for their rel-
ative cytotoxicity against human tumor cells. These biphenyl tetraoxazoles were not strong ligands for G-
quadruplex DNA relative to other macrocyclic polyoxazoles. This reduced stabilizing potential did corre-
late with their comparatively lower cytotoxic activity as observed in the human tumor cell lines, RPMI
8402 and KB3-1. These studies provide useful insights into the conformational requirements for the
development of selective and more potent G-quadruplex ligands.
Keywords:
G-quadruplex
Biphenyl
Macrocycle
Oxazoles
Ó 2013 Elsevier Ltd. All rights reserved.
Cytotoxicity
G-quadruplex ligand
1. Introduction
stabilization of duplex DNA. Using telomestatin as a template,
the synthetic macrocyclic hexaoxazole HXDV (Fig. 1) was synthe-
Sequences of DNA and RNA rich in guanosine bases are known to
fold into G-quadruplexes.1,2 G-Tetrads (square-planar arrays of four
guanines held together by hydrogen bonds) can be stacked upon
each other to form G-quadruplexes. G-quadruplexes are stabilized
sized and identified as a highly selective G-quadruplex stabilizer
exhibiting no affinity for stabilizing single-stranded, duplex, or tri-
plex DNA.18,19 HXDV induces apoptosis in both telomerase positive
and negative cells, induces M-phase cell cycle arrest, reduces the
expression of the M-phase checkpoint regulator Aurora A, and is
moderately cytotoxic towards several tumor cell lines with an
by p–stacking interactions between the purines as well as by mono-
valent metal cations (usually K+ or Na+) sandwiched between the G-
tetrads.3 G-quadruplexes have been associated with telomeres, the
promoter regions of several oncogenes, and mRNA.4–11 As G-quad-
ruplex stabilizers would be expected to effectively interfere with
processes involving these nucleic acids, they have often been
viewed as a promising new class of anticancer agents.12–15
Mechanistic insight into how G-quadruplex stabilizers princi-
pally function as anticancer agents requires compounds that are
highly selective in stabilizing G-quadruplexes without interacting
with the more common duplex structures of DNA or RNA.16 While
a diverse array of compounds have been reported to stabilize G-
quadruplex DNA17 with some degree of selectivity, most also have
the ability to stabilize the more abundant duplex conformation of
DNA or RNA.
average IC50 value of 0.5 l
M.18,20 METV (Fig. 1) is another 24-mem-
bered macrocycle, which was synthesized using a ring-closing
metathesis cyclization. METV also proved to be a highly selective
G-quadruplex stabilizer and exhibited significantly greater cyto-
toxicity than HXDV against several human tumor cell lines with
IC50 values typically ranging between 20 and 50 nM.21
HXDV and METV have poor aqueous solubility making it difficult
to formulate and assess their antitumor activity in vivo. Among a
variety of analogs related to HXDV that were synthesized and eval-
uated for cytotoxicity, a 2-(N,N-dimethylamino)ethyl derivative,
YAK-1-161 (Fig. 2), was shown to exhibit both improved cytotoxic-
ity and solubility. When evaluated in vivo, YAK-1-161 had a T/C va-
lue of 21.6% and was well tolerated at doses levels 645 mg/kg.22
Efforts to develop additional highly selective G-quaduplex
stabilizers that could be readily synthesized and explored with
regard to their structure-activity led to the design of a series of
24-membered macrocyclic pyridyl polyoxazoles.23 Within this ser-
ies, compounds having a 1,3-bis(aminomethyl)phenyl group link-
ing the ends of a pyridyl tetraoxazole dicarboxylate intermediate
Telomestatin (Fig. 1) is a natural product that was identified as a
G-quadruplex ligand with improved selectivity relative to
⇑
Corresponding author. Tel.: +1 (848) 445 2674.
E-mail address: elavoie@pharmacy.rutgers.edu (E.J. LaVoie).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.033

4512
G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
O
S
N
O
N
O
O
H
O
O
N
H
N
N
N
N
N
O
N
N
N
N
O
N
N
N
O
CH3
O
N
O
N
O
N
O
O
N
O
H
N
H
O
O
N
O
N
N
O
CH3
O
O
O
METV
Telomestatin
HXDV
Figure 1. Structures of telomestatin, HXDV and METV.
bioxazole, Scheme 1. The triisopropylsilyl protected methyl 2-(1-
amino-2-hydroxyethyl)oxazole-4-carboxylate, 3, was prepared as
previously described.18
CH3
O
H3C
N
O
N
O
N
N
O
N
N
Treatment of 3 with the acid chloride prepared from 3-bromo-
benzoic acid and oxalyl chloride provided the benzamide interme-
diate. Removal of the TIPS protecting group, followed by
cyclization with DAST provided the 2,3-dihydro-2-(3-bromophe-
nyl)[20,4]bioxazole, which was aromatized using DBU and bromo-
trichloromethane to provide 4. The second key intermediate 5
was prepared from 4 by reduction of the methyl ester to the
hydroxymethyl derivative, which was converted with DPPA to an
azide. Reduction of this azide with triphenylphosphine and treat-
ment with di-tert-butyl dicarbonate, provided intermediate 5.
Several biphenyl tetraoxazoles were synthesized as outlined in
Scheme 2. Formation of the pinacolatoboranyl derivative of 4 was
accomplished by coupling with bis(pinacolato)diborane. Suzuki-
O
O
O
N
H
N
N
N
N
O
N
O
N
O
N
N
O
O
O
H
H
H
N
O
NR2
1
, R = H
2
, R = CH3
YAK-1-161
Figure 2. Structures of YAK-1-161 and pyridyl polyoxazole macrocycles 1 and 2.
coupling of this intermediate with
5
provided the 3,30-bis-
[4,20]bioxazole]biphenyl derivative, 7. Removal of the Boc-protect-
ing group of 7 provided the aminomethyl derivative, which could
be condensed with various N-Boc or Fmoc protected amino acids
to form the intermediates 8–11. Hydrolysis of the ester and re-
were observed to be among the more cytotoxic. The presence of a
5-(2-aminoethyl) substituent attached to the phenyl moiety as in
compound 1 (Fig. 2) or a 5-[2-(N,N-dimethylamino)ethyl] moiety
as in compound 2 allowed not only for retention of notable cyto-
toxic activity, but also significantly enhanced their ability to be for-
mulated for evaluation in vivo. These analogs had IC50 values of
30–40 nM when assayed against KB3-1 cells and 90–180 nM
against RPMI 8402 cells and strongly stabilize G-quadruplex DNA
with no observable stabilization of duplex DNA.23 Compound 2
was selected for in vivo evaluation against a human breast cancer
xenograft (MDA-MB-435) in athymic nude mice. Results from this
assay indicated that mice treated with the pyridyl polyoxazole
macrocycle had a %T/C value of 27.7% which clearly demonstrated
in vivo efficacy against this breast cancer xenograft.23
moval of the protecting group on the
a-amino substituent pro-
vided the requisite intermediates 12–15 for the formation of the
24-membered biphenyltetraoxazole macrocycles. The macocycli-
zation step proceeded in yields ranging from 31% to 65% for 16–
19. The highest yield obtained was in the formation of 19, the
intermediate required for the preparation of those analogs with
improved drug-like physicochemical properties, 20 and 21. The
N-Boc-(2-amino)ethyl) derivative 19 was smoothly converted to
the primary amine 20, which could be subsequently converted to
the N,N-dimethyl derivative 21.
These data prompted further studies into the development of
other macrocyclic G-quadruplex stabilizers. In the present study,
biphenyl tetraoxazoles were explored for their potential as a new
series of selective G-quadruplex DNA ligands. Modeling results
suggested that these macrocycles would adapt a similar conforma-
tion to YAK-1-161, although not quite as planar, Figure 3.
The synthetic strategy for this series of 24-membered macrocy-
cles was designed to allow for versatility in terms of substituents
that could be appended to the alkyl linkage within this macrocycle.
In addition, it was hoped that improved yields might be realized in
the synthetic step associated with the formation of the core macro-
cyclic structure.
3. Pharmacology
The relative cytotoxic activities of each of the various biphenyl-
tetraoxazole macrocycles 16–21 are summarized in Table 1.
Among the tumor lines employed were the human lymphoblas-
toma RPMI 8402, a human epidermoid carcinoma KB3-1 as well
as its variants, KBV-1, that overexpresses the efflux transporter
MDR1, and KBH5.0 that overexpresses BCRP. None of these biphe-
nyl macrocylic tetraoxazoles exhibited cytotoxic activity toward
RPMI 8402 or KB3-1 comparable to other 24-membered macrocy-
cles such as HXDV, YAK-1-161, METV, or 2, which have a much
greater effect on stabilization of G-quadruplex DNA. Against RPMI
8402 cells, only 16 and 19 had IC50 values that were 63.5
Against KB3-1 cells, 16, 17, 20, and 21 exhibited modest cytotoxic-
ity with IC50 values that were 65.0 M. Consistent with a greater
potential to stabilize G-quadruplex DNA, HXDV, YAK-1-161, METV,
and 2 had IC50 values against KB3-1 cells that were one or two or-
ders of magnitude lower. Despite their relative weak cytotoxic
activity to the parent cell line KB3-1, there is evidence to suggest
lM.
2. Chemistry
l
Two key intermediates were selected as the principal sub-
units for the preparation of these biphenyl tetraoxazoles. These
were 4, methyl 2-(3-bromophenyl)bis[4,20]bioxazole-40-carboxyl-
ate and 5, 2-(3-bromophenyl)-40-(N-Boc-aminomethyl)[4,20]-

G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
4513
Figure 3. Overlaid structures of YAK-1-161 (in solid green) and biphenyl tetraoxazole macrocyle 21. ChemDraw3D Pro 12.0 was used for these illustrations and the structures
were minimized using MM2. The minimized structures were comparable to the minimized structures obtained using SPARTAN 06 (equilibrium conformation/MMFF).
OTIPS
O
H2N
N
TIPSO
Br
Br
Br
Br
a
3
COOMe
c,d
O
N
HOOC
N
H
Cl
O
O
b
O
O
N
MeOOC
N
COOMe
Br
Br
Br
Br
g
h,i
f
e
O
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
OH
N3
NHBoc
COOMe
4
5
Scheme 1. Formation of the 2-phenyl[4,20]bioxazoles 4 and 5. Reagents and conditions: (a) (COCl)2, CH2Cl2, rt (quant.); (b) Et3N, 53%; (c) HF/pyridine, 94%; (d) DAST, K2CO3,
79%; (e) DBU, BrCCl3; 88%; (f) LiBH4, THF/EtOH; 89%; (g) DPPA, DBU, THF, 71%; (h) PPh3, THF:H2O (10:1), 96%; (i) Boc2O, Et3N, 82%.
for 16, 20, and 21 that these macrocyclic biphenyl tetraoxazoles
are substrates for MDR1 and BCRP efflux transporters.
to be quite selective for the quadruplex relative to the duplex
form.
Each of the macrocycles 16–21 was evaluated for its ability to
selectively stabilize G-quadruplex versus duplex DNA. As listed
in Table 2, the relative ability of these compounds to stabilize
G-quadruplex and duplex DNA was measured by examining their
effect on the thermal transition temperature (Ttran) of a human
telomeric G-quadruplex-forming DNA sequence, (TTAGGG)4, or
salmon testes (ST) duplex DNA in the presence of either 50 or
150 mM K+. Among these macrocyclic biaryl tetraoxazoles nei-
ther 18, the benzyl-substituted derivative, nor 19, the N-Boc
derivative of the 2-aminoethyl derivative, had a significant im-
The methodology employed in the syntheses of these macrocy-
clic biaryl tetraoxazoles provides for significant versatility in terms
of the varied substitutents that could be attached to this macrocy-
cle by the selection of the appropriately substituted a-amino acid.
In addition, it appears that a considerable improvement in yield
during the macrocyclization of these biaryl tetraoxazoles could
be achieved relative to that observed for several other 24-mem-
bered macrocycles evaluated as G-quadruplex stabilizers. How-
ever, these macrocyclic biphenyl tetraoxazoles lack the potent
stabilizing effect on G-quadruplex DNA observed with 2, HXDV,
YAK-1-161, or METV. While the biphenyl macrocyle 21 structurally
coincides with are large portion of YAK-1-161, it is clear from Fig-
ure 3 that it does not have the same planar character. The biphenyl
macrocycles are also significantly less cytotoxic against the human
tumor cells. It is known that replacement of a phenyl with a pyridyl
moiety within macrocycles designed as G-quaduplex stabilizers
can have a pronounced effect. Studies are in progress, therefore,
to assess the promise of similarly structured macrocyclic 2-phenyl-
pyridyl tetraoxazoles and 2,20-bipyridyl tetraoxazoles.
pact on the melting profile of the quadruplex DNA (DTtran =
1.1–2.8 °C). Within this series of macrocycles, 16 and the isopro-
pyl derivative 17 did stabilize quadruplex DNA, but only to a
limited extent
(DTtran = 4.1–6.4 °C). Among these macrocyclic
biaryl tetraoxazoles, the 2-aminoethyl derivative 20 and the
2-(N,N-dimethylamino)ethyl derivative 21 had a more significant
G-quadruplex stabilizing effect (DTtran = 8.4–14.6 °C) than either
16, 17, 18 or 19 (Fig. 4, Table 2). However, the ability of these
aminoalkyl substituted macrocyclic biphenyl tetraoxazoles to
stabilize G-quadruplex DNA was considerably less relative to
several of the other 24-membered oxazole-containing macrocy-
cles investigated in our laboratory, such as 2, YAK-1-161, and
METV (Fig. 4, Table 2). None of the biaryl tetraoxazoles stabilized
4. Experimental
4.1. Chemistry: general methods
ST duplex DNA to
Ttran values being close to or within the experimental uncer-
tainty. Thus, the DNA stabilizing activities of 20 and 21 appear
a significant degree, with the observed
All reactions, unless otherwise stated, were done under nitro-
gen atmosphere. Reaction monitoring and follow-up were done
D

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G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
O
Br
B
Br
O
O
O
O
O
O
O
O
O
a
b
N
N
N
N
N
+
N
N
N
N
N
O
O
BocHN
CO2CH3
CO2CH3
CO2CH3
NHBoc
4
6
5
7
O
O
O
O
O
O
N
O
O
O
N
N
N
N
N
O
g
e,f
c,d
N
N
O
N
N
N
N
O
O
COOH
H2N
CO2CH3
NH
NH
NH
NH
X
NH
R
R
O
O
R
O
16; R = H
12; R = H
17; R = CH(CH3)2
8; X = Boc; R = H
13; R = CH(CH3)2
14; R = CH2Ph
18; R = CH2Ph
9; X = Boc; R = CH(CH3)2
10; X = Boc; R = CH2Ph
19; R = CH2CH2NHBoc
20; R = CH2CH2NH2
21; R = CH2CH2N(CH3)2
15; R = CH2CH2NHBoc
c
11; X = Fmoc; R = CH2CH2NHBoc
h
Scheme 2. Synthesis of 3,30-bis(2,4-bioxazol]-20yl)-1,10biphenylderivatives. Reagents and conditions: (a) PdCl2ꢁ(dppf)ꢁCH2Cl2, bis(pinacolato)B2, KOAc, toluene, 93%; (b)
PdCl2ꢁ(dppf)ꢁCH2Cl2, Cs2CO3, dioxane:H2O (10:1), 59%; (c) TFA, CH2Cl2, 79%; (d) Boc-amino acid, EDC, HOBt, 2,6-lutidine for 8–10, Fmoc-Dab(Boc)-OH, EDC, HOBt, DIPEA for
11; (e) LiOH, THF:H2O (3:1) for 8–10, 1% piperidine/acetonitrile for 11; (f) TFA, CH2Cl2 for 12, 13, 14, LiOH, THF:H2O (3:1) for 15; (g) EDC, HOBt, 2,6-lutidine; (h) formaldehyde,
NaBH (OAc)3, CH3OH:CH2Cl2 (1:4).
Table 1
4.2. Compound (4)
Relative cytotoxic activities (IC50; l
M)a
4.2.1. Methyl 2-(1-(3-bromobenzamido)-2-
Compd
RPMI 8402
KB3-1 wt
KBV-1 +MDR1
KBH5.0 +BCRP
((triisopropylsilyl)oxy)ethyl)oxazole-4-carboxylate
16
17
2.5
10
2.3
5.0
>10
6.5
>10
10
3-Bromobenzoic acid (1.7 g, 8.4 mmol) was dissolved in dry
dichloromethane (5 mL) and oxalyl chloride (1.5 mL, 16.8 mmol)
was added. A catalytic amount of DMF (two drops) was added
and the reaction stirred at room temperature for 2 h. The solvent
was concentrated under reduced pressure and the residue was
placed under the vacuum to dry. The residue was redissolved in
dichloromethane (5 mL) and methyl 2-(1-amino-2-((triisopropyl-
silyl)oxy)ethyl)oxazole-4-carboxylate (241 mg, 7 mmol) was
added followed by triethylamine (2.9 mL, 21 mmol). The reaction
mixture was allowed to stir overnight at room temperature under
N2. The mixture was then washed with saturated NaHCO3 and or-
ganic layer concentrated and purified by flash chromatography
using a gradient of 0–40% ethyl acetate/hexane to give a colorless
oil (1.83 g, 53% yield); 1H NMR (400 MHz) (CDCl3) d 8.21 (s, 1H),
7.96 (s, 1H), 7.73–7.71 (m, 1H), 7.64–7.62 (m, 1H), 7.28–7.32 (m,
1H), 7.22 (d, 1H, J = 8), 5.51 (m, 1H), 4.29 (m, 1H), 4.14 (m, 1H),
3.89 (s, 3H), 0.87–1.05 (m, 21H); 13C NMR (CDCl3) d 165.5, 163.3,
161.3, 144.1, 135.6, 134.8, 133.4, 130.5, 130.1, 125.6, 122.7, 64.6,
18
19
20
21
>10
3.5
7.0
>10
>10
4.0
>10
>10
>10
>10
>10
5.0
>10
6.0
>10
>10
0.5
0.03
0.04
>10
6.5
3.5
HXDV
YAK-1-161
METV
2
0.41
0.07
0.03
0.18
0.30
0.03
0.04
0.04
3.3
>10
a
Values are mean of at least two experiments.
using aluminum backed Silica G TLC plates with UV254 (Sorbent
Technologies), visualizing with ultraviolet light. Flash column
chromatography was done on a Combi Flash Rf Teledyne ISCO
using hexane, ethyl acetate, dichloromethane, and methanol. The
1H (400 MHz) and 13C (100 MHz) NMR spectra were done in CDCl3,
methanol-d4, or DMSO-d6 and recorded on a Bruker Avance III
(400 MHz) Multinuclear NMR Spectrometer. Data is expressed in
parts per million relative to the residual non-deuterated solvent
signals, spin multiplicities are given as s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet),
m (multiplet), and bs (broad singlet), and coupling constants (J) are
reported in Hertz. Melting points were determined using Mel-temp
II apparatus and are uncorrected. IR data was recorded on a Ther-
mo Nicolet Avatar Model 360 FTIR. HRMS experiments were con-
ducted by Washington University Resource for Biomedical and
Bioorganic Mass Spectrometry Department of Chemistry.
52.1, 50.2, 17.7, 11.7; IR 3324, 1740, 1668 cmꢀ1
.
4.2.2. Methyl 2-(1-(3-bromobenzamido)-2-hydroxyethyl)
oxazole-4-carboxylate
Methyl
2-[(1-(3-bromobenzamido)-2-[(triisopropylsilyl)oxy]
ethyl]oxazole-4-carboxylate (1.83 g, 3.5 mmol) was dissolved in
THF:pyridine (5:1, 6 mL) and cooled to 0 °C under nitrogen. This
was treated dropwise with HF-pyridine (1 mL) and the reaction
was stirred at room temperature for 18 h. On completion of the

G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
4515
Table 2
Relative impacts on the thermal stabilities of d(TTAGGG)4 quadruplex and salmon testes (ST) duplex DNA
Compound
d(TTAGGG)4 quadruplex DNA
ST duplex DNA
50 mM K+a
150 mM K+b
50 mM K+a
150 mM K+b
c
d
c
d
c
d
c
d
Ttran (°C)
D
Ttran (°C)
Ttran (°C)
D
Ttran (°C)
Ttran (°C)
D
Ttran (°C)
Ttran (°C)
D
Ttran (°C)
None
16
17
18
19
20
21
YAK-1-161
METV
2
55.3
60.1
61.7
58.1
57.7
68.2
63.7
92.7
92.2
91.2
—
4.8
6.4
2.8
2.4
12.9
8.4
37.4
36.9
35.9
63.1
67.3
67.2
64.7
64.2
77.7
73.7
92.2
93.2
91.2
—
4.2
4.1
1.6
78.2
78.2
78.2
77.2
78.3
81.1
79.1
78.2
78.2
78.7
—
0
0
86.1
86.7
86.2
85.7
86.5
87.1
86.7
86.7
86.2
86.8
—
0.6
0.1
ꢀ0.4
0.4
1.0
0.6
0.6
0.1
0.7
ꢀ1.0
1.1
0.1
2.9
0.9
0
14.6
10.6
29.1
30.1
28.1
0
0.5
a
b
c
Solution conditions were 10 mM potassium phosphate (pH 7.5) and sufficient KCl (32 mM) to bring the total K+ concentration to 50 mM.
Solution conditions were 10 mM potassium phosphate (pH 7.5) and sufficient KCl (132 mM) to bring the total K+ concentration to 150 mM.
Ttran reflects the thermal transition temperature, with values of Ttran being determined from the minima (for quadruplex DNA) or maxima (for duplex DNA) of first-
derivative UV melting profiles exemplified by those in Figure 4. The uncertainty in the Ttran values is 0.5 °C.
d
D
Ttran reflects the change in the Ttran of the quadruplex or duplex DNA induced by the presence of the compound. The uncertainty in the DTtran values is 1.0 °C.
0.001
0
0.02
0.015
0.01
0.005
0
No Compd.
2
20
21
-0.001
-0.002
-0.003
-0.004
No Compd.
2
20
21
40
50
60
70
80
90 100 110
70
75
80
85
90
95
100
Temperature (°C)
Temperature (°C)
Figure 4. First derivatives of UV melting profiles of a 24mer human telomeric quadruplex DNA sequence, d(TTAGGG)4, and salmon testes (ST) duplex DNA in the absence
(filled circles) and presence of either 2 (filled triangles), 20 (open circles), or 21 (filled diamonds). The UV melting profiles of d(TTAGGG)4 were acquired at 295 nm, while
those of ST DNA were acquired at 260 nm. The concentration of d(TTAGGG)4 was 5 lM in strand, while that of ST DNA was 30 lM in base pair. When present, all compounds
were used at a concentration of 20
l
M in the d(TTAGGG)4 experiments and 15
l
M in the ST DNA experiments. The solution conditions were 10 mM potassium phosphate (pH
7.5) and sufficient KCl (132 mM) to bring the total K+ concentration to 150 mM.
reaction, the reaction mixture was poured slowly into saturated
solution of NaHCO3. This mixture was extracted with chloroform
and the chloroform layer dried with sodium sulfate. The solvent
was concentrated under reduced pressure and the residue azeotr-
oped with toluene to give a white solid (1.2 g, 94% yield);
mp = 168–170 °C; 1H NMR (CD3OD) d 8.75–8.76 (m, 1H), 8.52 (s,
1H), 8.07–8.08 (m, 1H), 7.84–7.87 (m, 1H), 7.70–7.73 (m, 1H),
7.38–7.42 (m, 1H), 5.38–5.41 (m, 1H), 4.04–4.07 (m, 2H), 3.88 (s,
3H); 13C NMR (CD3OD) d 168.3, 164.5, 162.7, 146.1, 136.6, 135.6,
133.3, 131.5, 131.1, 127.2, 123.2, 62.9, 52.3, 51.7.
low solid (894 mg, 79%); mp = 82–84 °C; 1H NMR (CDCl3) d 8.17 (s,
1H), 7.97 (s, 1H), 7.74 (dd, 1H, J = 0.96, 7.76), 7.46–7.48 (m, 1H),
7.13–7.18 (m, 1H), 5.45 (t, 1H, J = 8.44), 4.76 (t, 1H, J = 8.44), 4.67
(t, 1H, J = 8.44), 3.76 (s, 3H); 13C NMR (CDCl3) d 165.0, 163.2,
161.1, 144.8, 134.7, 133.2, 131.3, 129.9, 128.6, 127.0, 122.2, 70.6,
63.7, 52.0; IR 1741 cmꢀ1
.
4.2.4. Methyl 20-(3-bromophenyl)-[2,40-bioxazole]-4-
carboxylate (4)
Methyl
20-(3-bromophenyl)-40,50-dihydro-[2,40-bioxazole]-4-
carboxylate (100 mg, 0.28 mmol) was dissolved in dry dichloro-
methane (5 mL), placed under nitrogen and cooled to 0 °C. DBU
(0.09 mL, 0.6 mmol) was added dropwise and the solution became
brown. BrCCl3 (0.07 mL, 0.72 mmol) was added and the reaction
was stirred at room temperature overnight. On completion of the
reaction, reaction mixture was poured into saturated solution of
ammonium chloride. The aqueous layers were extracted with
dichloromethane and the combined organic layers dried over so-
dium sulfate, concentrated and purified by flash chromatography
using a gradient of 0–100% ethyl acetate/hexane to give a white so-
lid (87 mg, 88%); mp = 175–177 °C, 1H NMR (CDCl3) d 8.41 (s, 1H),
8.32 (s, 1H), 8.26–8.27 (m, 1H), 8.01–8.04 (m, 1H), 7.59–7.62 (m,
4.2.3. Methyl 20-(3-bromophenyl)-40,50-dihydro-[2,40-bioxazole]-
4-carboxylate)
Methyl 2-(1-(3-bromobenzamido)-2-hydroxyethyl)oxazole-4-
carboxylate (1.2 g, 3.2 mmol) was suspended in dry dichlorometh-
ane (10 mL) and placed under nitrogen. After cooling to ꢀ78 °C,
DAST (787 mg, 4.88 mmol) was added and the reaction stirred at
ꢀ78 °C for 3 h. This was followed by addition of potassium carbon-
ate (675 mg, 4.88 mmol) and the mixture was allowed to warm to
room temperature. Upon completion of the reaction, the reaction
mixture was poured carefully into saturated solution of sodium
bicarbonate and extracted with dichloromethane to give a pale yel-

4516
G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
1H), 7.32–7.37 (m, 1H), 3.94 (s, 3H); 13C NMR (CDCl3) d 161.3,
161.2, 155.6, 143.8, 139.7, 134.4, 134.2, 134.1, 130.4, 129.7,
128.1, 125.2, 123.0, 52.2; IR 1745 cmꢀ1 HRMS calculated for
chromatography (0–50% ethyl acetate/hexane) to give a white solid
(122.8 mg, 59%); mp = 145–147 °C; 1H NMR (CDCl3) d 8.30 (s, 1H),
8.23 (s, 1H), 8.02–8.04 (m, 1H), 7.58–7.63 (m, 2H), 7.30–7.34 (m,
1H), 5.19 (br s, 1H), 4.29 (d, 2H, J = 8), 1.44 (s, 9H); 13C NMR (CDCl3)
d 161.2, 155.6, 155.1, 139.6, 138.4, 135.2, 133.9, 131.8, 130.3,
129.7, 128.2, 125.2, 122.9, 79.6, 36.5, 28.3; IR 1679, 1263, 748,
421 cmꢀ1; HRMS calculated for C18H19BrN3O4 (M+H)+, 420.0553;
found, 420.0551.
C
14H9BrN2O4 (M+H)+, 348.9818; found, 348.9820.
4.3. Compound (5)
4.3.1. (20-(3-Bromophenyl)-[2,40-bioxazol]-4-yl)methanol
Methyl 20-(3-bromophenyl)-[2,40-bioxazole]-4-carboxylate, 4,
(310 mg, 0.89 mmol) was dissolved in 11 mL of THF:EtOH (10:1)
and cooled to 0 °C under nitrogen. Lithium borohydride (61.7 mg,
2.83 mmol) was added and the reaction was warmed to room tem-
perature overnight. The resulting mixture was poured into water
and extracted with ethyl acetate. The combined organic layers
were washed with brine and dried over sodium sulfate. The organic
layer was concentrated and purified by flash chromatography
using a gradient of 0–100% ethyl acetate/hexane to give a white so-
lid (253 mg, 89%); mp = 142–144 °C; 1H NMR (400 MHz, CDCl3) d
8.35–8.36 (m, 1H), 8.31 (s, 1H), 8.08–8.11 (m, 1H), 7.70–7.71 (m,
1H), 7.63–7.66 (m, 1H), 7.36–7.40 (m, 1H), 4.72 (d, 2H, J = 0.84);
13C NMR (CDCl3) d 161.3, 155.4, 141.7, 138.5, 135.1, 134.0, 131.8,
130.4, 129.8, 128.2, 125.3, 123.0, 57.0; IR 3054, 2986, 2305, 1552,
4.4. Compound (6)
4.4.1. Methyl 20-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)-[2,40-bioxazole]-4-carboxylate (6)
Methyl
20-(3-bromophenyl)-[2,40-bioxazole]-4-carboxylate
(4.2.4) (500 mg, 1.43 mmol) in toluene (5 mL) was treated with
bis(pinacolato)diboron (436 mg, 1.72 mmol) and then potassium
acetate (544 mg, 4.29 mmol) was added. The solution was purged
with nitrogen and PdCl2ꢁ(dppf)ꢁCH2Cl2 (35 mg, 0.049) added and
the reaction mixture heated at 95 °C overnight. The reaction was
cooled to room temperature and filtered. The filtrate was concen-
trated under reduced pressure and purified by flash chromatogra-
phy (0–50% ethyl acetate/hexane) to give a white solid (526 mg,
93%) mp = 160–162 °C; 1H NMR (CDCl3) d 8.59 (s, 1H), 8.43 (s,
1H), 8.35 (s, 1H), 8.20–8.23 (m, 1H), 7.93–7.99 (m, 1H), 7.48–
7.52 (m, 1H), 3.96 (s, 3H), 1.37 (s, 12H); 13C NMR (CDCl3) d
162.9, 161.3, 155.9, 143.7, 139.3, 137.4, 134.3, 133.2, 130.9,
129.4, 128.2, 125.7, 84.1, 52.5, 24.8; IR 3052, 2983, 2253, 1742,
1638, 1608, 1439, 1408, 1370, 1326, 1238, 1265, 1144, 1104,
1421, 1265, 895, 735, 705, 447 cmꢀ1
.
4.3.2. 4-(Azidomethyl)-20-(3-bromophenyl)-2,40-bioxazole
(20-(3-Bromophenyl)-[2,40-bioxazol]-4-yl)methanol
(246 mg,
0.77 mmol) and DPPA (0.25 mL, 1.16 mmol) was dissolved in
10 mL of THF. The reaction was cooled to 0 °C under nitrogen. Neat
DBU (0.17 mL, 1.16 mmol) was added and the reaction stirred for
2 h at 0 °C and for 16 h at room temperature. The resulting mixture
was washed with water, 5% HCl and extracted with ethyl acetate.
The organic layer was washed with brine and dried over sodium
sulfate. The organic layer was then concentrated under reduced
pressure and purified by flash chromatography using a gradient
of 0–50% ethyl acetate/hexane to give a white solid (189 mg,
71%); mp = 130–132 °C; 1H NMR (CDCl3) d 8.21–8.25 (m, 1H),
8.21 (s, 1H), 7.97–7.99 (m, 1H), 7.65 (s, 1H), 7.56–7.53 (m, 1H),
7.26–7.30 (m, 1H), 4.32 (s, 2H); 13C NMR (CDCl3) d 161.3, 155.6,
138.8, 137.2, 136.1, 134.0, 131.6, 130.4, 129.8, 128.2, 125.0,
123.0, 46.29; IR 3053, 2986, 2305, 2104, 1552, 1421, 1265, 1115,
1003, 907, 970, 861, 733, 650 cmꢀ1
.
4.5. Compound (7)
4.5.1. Methyl 20-(30-(4-(((t-butoxycarbonyl)amino)methyl)-[2,40-
bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-
carboxylate (7)
A mixture of t-butyl ((20-(3-bromophenyl)-[2,40-bioxazol]-4-yl)
methyl)carbamate (4.3.4) (100 mg, 0.25 mmol), methyl 20-(3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)[2,40-bioxaz-
ole]-4-carboxylate (3.4.1) (113 mg, 0.29 mmol), cesium carbonate
(233 mg, 0.72 mmol) was dissolved in 11 mL of dioxane:water
(10:1) and the solution purged under nitrogen. PdCl2ꢁ(dppf)ꢁCH2Cl2
(24.5 mg, 0.03 mmol) was added and the mixture heated at 90 °C
for 4 h. The reaction mixture was then cooled to room temperature
and filtered. The filtered solution was concentrated and purified by
flash chromatography using a gradient of 0–100% ethyl acetate/
hexane to give a white solid (85.2 mg, 59%); mp = 115–117 °C; 1H
NMR (CDCl3) d 8.46 (s, 2H), 8.42–8.43 (m, 1H), 8.34 (s, 1H), 8.28
(s, 1H), 8.14–8.18 (m, 2H), 7.78–7.81 (m, 2H), 7.64 (s, 1H), 7.57–
7.64 (m, 2H), 5.12 (br s, 1H), 4.31 (d, 2H, J = 5.84), 3.96 (s, 3H),
1.45 (s, 9H); 13C NMR (CDCl3) d 162.8, 161.9, 156.0, 155.8, 143.9,
141.1, 141.0, 140.9, 139.7, 138.4, 135.1, 134.5, 131.9, 131.2,
130.2, 130.0, 129.7, 129.6, 127.3, 127.2, 127.1, 126.4, 126.35,
126.31, 125.8, 125.7. 79.8, 52.4, 36.7, 28.53; IR 3452, 3166, 2981,
2253, 1711, 1638, 1554, 1503, 1438, 1368, 1323, 1249, 1157,
1115, 1044, 1002, 971, 906, 798, 730, 650, 445; HRMS calculated
for C32H28ꢁN5O8 (M+H)+, 610.1932; found, 610.1927.
909, 736, 650 cmꢀ1
.
4.3.3. (20-(3-Bromophenyl)-[2,40-bioxazol]-4-yl)methanamine
4-(Azidomethyl)-20-(3-bromophenyl)-2,40-bioxazole (181 mg,
0.52 mmol) was dissolved in 11 mL of THF:water (10:1) and poly-
mer-bound triphenylphosphine (262 mg, 0.79 mmol) was added
and the reaction mixture stirred at room temperature overnight.
The reaction mixture was then filtered, concentrated under re-
duced pressure, and the residue azeotroped with toluene to give
a white solid (161.4 mg, 96%); mp = 125–127 °C; 1H NMR (CDCl3)
d 8.20 (s, 1H), 8.14 (s, 1H), 7.93–7.95 (m, 1H), 7.49–7.50 (m, 2H),
7.21–7.25 (m, 1H), 3.76 (s, 2H), 2.69 (br s, 2H); 13C NMR (CDCl3)
d 161.1, 155.1, 142.9, 134.4, 133.9, 131.9, 130.3, 129.73, 125.4,
122.9, 120.2, 119.98, 119.93, 37.75; IR 3053, 2985, 2305, 2253,
1714, 1645, 1591, 1552, 1490, 1463, 1430, 1474, 1311, 1265,
1163, 1113, 1054, 1025, 908, 793, 734, 650 cmꢀ1
.
4.3.4. tert-Butyl [[20-(3-Bromophenyl)-[2,40-bioxazol-4-
yl]methyl]carbamate (5)
4.5.2. Removal of Boc protecting group
Methyl 20-(30-(4-(((t-butoxycarbonyl)amino)methyl)-[2,40-bio-
xazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
(3.5.1) (159.9 mg, 0.26 mmol) was dissolved in anhydrous dichlo-
romethane (2 mL) and cooled to 0 °C. Trifluoroacetic acid (2 mL)
was added and stirred at that temperature for 3 h. The solvents
were removed under reduced pressure and dissolved in dichloro-
methane. This solution was poured into saturated sodium bicar-
bonate solution and the organic layer separated. The organic
(20-(3-Bromophenyl)-[2,40-bioxazol]-4-yl)methanamine
(160 mg, 0.5 mmol) was dissolved in dry dichloromethane (5 mL)
and triethylamine (50 mg, 0.5 mmol) was maintained under nitro-
gen. The solution was cooled to 0 °C and a solution of Boc2O
(152 mg, 0.7 mmol) in dichloromethane (2 mL) was added and stir-
red at room temperature overnight. The solvent was removed un-
der reduced pressure and the residue purified by flash

G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
4517
layer was dried over sodium sulfate and concentrated to give a col-
orless oil; (105 mg, 79%); 1H NMR (CDCl3) d 8.41 (s, 1H), 8.40 (s,
1H), 8.37 (s, 1H), 8.28 (s, 1H), 8.23 (s, 1H), 8.08–8.12 (m, 2H),
7.72–7.76 (m, 2H), 7.52–7.56 (m, 3H), 3.90 (s, 3H), 3.80 (s, 2H);
13C NMR (CDCl3) 162.7, 162.5, 161.3, 155.8, 155.4, 154.0, 143.9,
143.8, 140.9, 140.8, 139.5, 138.2, 134.4, 134.0, 131.9, 131.0,
130.0, 129.8, 129.6, 129.5, 127.2, 127.0, 126.2, 126.1, 125.6, 52.3,
38.1.
(m, 2H), 4.35 (d, 2H, J = 5.28), 3.61 (s, 2H); 13C (DMSO) d 166.0,
162.9, 161.3, 161.2, 157.7, 154.3, 152.8, 144.9, 141.1, 140.3,
140.0, 139.8, 139.5, 138.6, 136.5, 134.3, 131.4, 130.9, 130.4,
130.3, 129.7, 127.0, 126.8, 125.9, 124.3, 115.9, 54.8, 34.6.
4.6.4. (16)
20-(30-(4-((2-Aminoacetamido)methyl)-[2,40-bioxazol]-20-yl)-
[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylic acid (12) as its
trifluoroacetic acid salt (38 mg, 0.06 mmol) was dissolved in dry
DMF (30 mL), EDC (23 mg, 0.12 mmol) and HOBt (16 mg,
0.12 mmol) was added. 2,6-Lutidine (0.04 mL, 0.3 mmol) was
added and reaction stirred at room temperature overnight. The
DMF was removed on Kugelrohr and the residue purified by flash
4.6. Compound (16)
4.6.1. Methyl 20-(30-(4-((2-((t-butoxycarbonyl)amino)
acetamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-
[2,40-bioxazole]-4-carboxylate (8)
chromatography (0–10% MeOH/DCM) to give
a white solid
(6.4 mg, 24%); mp = 350–352 °C (decomposed); 1H NMR (DMSO)
d 9.08 (s, 1H), 8.95 (s, 1H), 8.76–8.77 (m, 2H), 8.65 (s, 1H), 8.61
(s, 1H), 8.46 (m, 1H), 8.12–8.16 (m, 3H), 7.97–8.01 (m, 2H), 7.68–
7.73 (m, 2H), 4.19 (d, 2H, J = 5.48), 3.84 (d, 2H, J = 6.36); 13C NMR
(DMSO) d 168.5, 161.7, 159.8, 154.8, 142.1, 138.5, 138.2, 136.1,
130.8, 130.4, 128.6, 126.9, 126.8, 125.4, 123.5, 45.7; HRMS calcu-
lated for C28H19N6O6 (M+H)+, 535.1361; found, 535.1347.
Boc-protected glycine (31 mg, 0.18 mmol), EDC (37 mg,
0.20 mmol) and HOBt (26 mg, 0.20 mmol) were dissolved in dry
DMF (5 mL) and stirred for 5 min under nitrogen. A solution of
methyl 20-(30-(4-(aminomethyl)-[2,40-bioxazol]-20-yl)-[1,10-biphe-
nyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
(4.5.2)
(54 mg,
0.09 mmol), 2,6-lutidine (0.04 mL, 0.36 mmol) in dry DMF (5 mL)
was added and reaction stirred at room temperature overnight.
On completion of the reaction, the DMF was removed and the mix-
ture purified with flash chromatography (0–5% methanol/dichloro-
methane) to obtain a white solid (41.2 mg, 70%), mp = 132–134 °C;
1H NMR (CDCl3) d 8.46 (s, 1H), 8.42 (s, 1H), 8.41 (s, 1H), 8.40 (s, 1H),
8.35 (s, 1H), 8.12–8.14 (m, 2H), 7.77–7.79 (m, 2H), 7.68 (s, 1H),
7.56–7.60 (m, 2H), 6.97 (t, 1H, J = 5.40), 5.36 (br s, 1H), 4.47 (d,
2H, J = 5.92), 3.96 (s, 3H), 3.37 (d, 2H, J = 5.68), 1.43 (s, 9H); 13C
NMR (CDCl3) d 169.6, 162.6, 162.5, 161.3, 155.8, 155.4, 143.8,
140.8, 140.7, 139.5, 138.6, 138.4, 136.2, 135.6, 134.3, 131.6,
131.0, 129.9, 129.8, 129.6, 129.5, 127.1, 126.9, 126.1, 126.1,
125.5, 124.9, 80.3, 52.2, 44.4, 35.2, 28.2; IR 3625, 3054, 3429,
2986, 2305, 1721, 1678, 1421, 1368, 1265, 1156, 1114, 1018,
970, 896, 737, 705, 439 cmꢀ1; HRMS calculated for C34H31O6N9
(M+H)+, 667.2147; found, 667.2143.
4.7. Compound (17)
4.7.1. Methyl 20-(30-(4-((2-((t-butoxycarbonyl)amino)-3-
methylbutanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-
biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylate (9)
Boc-protected
L-valine (52 mg, 0.24 mmol), EDC (50.5 mg,
0.26 mmol) and HOBt (36 mg, 0.26 mmol) were dissolved in dry
DMF (5 mL) and stirred for 5 min under nitrogen. A solution of
methyl 20-(30-(4-(aminomethyl)-[2,40-bioxazol]-20-yl)-[1,10-biphe-
nyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
(4.5.2)
(72.8 mg,
0.12 mmol), 2,6-lutidine (0.06 mL, 0.48 mmol) in dry DMF (5 mL)
was added and reaction stirred at room temperature overnight.
On completion of the reaction, the DMF was removed and the mix-
ture purified with flash chromatography (0–100% ethyl acetate/
hexane) to obtain a white solid (66.2 mg, 78%); mp = 135–138 °C;
1H NMR (CDCl3) d 8.40 (s, 1H), 8.39 (s, 1H), 8.37 (s, 1H), 8.28 (s,
1H), 8.22 (s, 1H), 8.08–8.12 (m, 2H), 7.73–7.75 (m, 2H), 7.62 (s,
1H), 7.52–7.56 (m, 2H), 6.49 (br s, 1H), 4.95 (br s, 1H), 4.39 (d,
2H, J = 5.48), 3.90 (s, 3H), 3.84–3.86 (m, 1H), 2.08–2.15 (m, 1H),
1.26 (s, 9H), 0.88 (d, 3H), 0.83 (d, 3H); 13C (CDCl3) d 171.9, 162.6,
161.3, 155.9, 155.4, 143.8, 140.8, 140.7, 139.5, 138.7, 138.4,
135.7, 134.3, 131.5, 130.9, 129.9, 129.8, 129.5, 129.4, 127.0,
126.9, 126.2, 126.1, 125.5, 125.0, 118.1, 110.6, 79.9, 60.1, 52.2,
35.2, 30.8, 28.2, 19.3; IR 3054, 2986, 2305, 1724, 1421, 1265,
4.6.2. Lithium 20-(30-(4-((2-((tert-
butoxycarbonyl)amino)acetamido)methyl)-[2,40-bioxazol]-20-
yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
Methyl
20-(30-(4-((2-((tert-butoxycarbonyl)amino)acetam-
ido)methyl)[2,40-bioxazol]-20-yl)[1,10-biphenyl]-3-yl)-[2,40-bioxaz-
ole]-4-carboxylate (40 mg, 0.06 mmol) was dissolved in THF/H2O
(3:1, 2 mL) and treated with lithium hydroxide (3.0 mg,
0.07 mmol) at 0 °C. This was stirred at room temperature for 4 h.
The solvents were concentrated under reduced pressure and dried
over vacuum to yield a white solid (39 mg, 100%) mp = 239–240 °C
(decomposed); 1H NMR (DMSO) d 8.89 (s, 1H), 8.83 (s, 1H), 8.29–
8.30 (m, 3H), 7.95–8.03 (m, 3H), 7.91–7.95 (m, 3H), 7.63–7.67
(m, 2H), 6.92 (t, 1H, J = 5.68), 4.17 (d, 2H, J = 5.28), 3.50 (d, 2H,
J = 5.72), 1.30 (s, 9H); 13C NMR (DMSO) d 169.4, 162.9, 161.3,
161.2, 155.9, 155.4, 154.3, 152.8, 143.0, 140.2, 140.1, 139.9,
139.8, 139.7, 139.5, 138.6, 136.2, 134.3, 131.4, 131.0, 130.2,
129.6, 129.5, 127.0, 126.8, 125.8, 124.3, 78.0, 43.2, 34.6, 28.1.
1156, 895, 737, 705 cmꢀ1
; HRMS calculated for C37H37O9N6
(M+H)+, 709.2617; found, 709.2607.
4.7.2. Lithium 20-(30-(4-((2-((t-butoxycarbonyl)amino)-3-
methylbutanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-
biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
Methyl 20-(30-(4-((2-((tert-butoxycarbonyl)amino)-3-methylb-
utanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-
[2,40-bioxazole]-4-carboxylate (31 mg, 0.05 mmol) was dissolved
in THF/H20 (3:1, 2 mL) and treated with lithium hydroxide
(2.5 mg, 0.06 mmol) at 0 °C. This was stirred at room temperature
for 4 h. The solvents were concentrated under reduced pressure
and dried over vacuum to yield a white solid (35 mg, 100%);
mp = 209–210 °C (decomposed); 1H NMR (CD3OD) d 8.35 (s, 2H),
8.22 (s, 1H), 8.03–8.06 (m, 2H), 7.81–7.84 (m, 3H), 7.58–7.67 (m,
4H), 7.21–7.24 (m, 2H), 4.37 (s, 2H), 3.87 (d, 1H), 2.00–2.07 (m,
1H), 1.41 (s, 9H), 0.94 (d, 6H, J = 7.16); 13C (CD3OD) d 174.7,
169.8, 168.1, 166.6, 163.8, 156.7, 155.9, 145.9, 144.6, 144.0,
142.5, 142.0, 141.4, 140.7, 137.6, 132.4, 130.9, 130.8, 128.9,
4.6.3. 20-(30-(4-((2-Aminoacetamido)methyl)-[2,40-bioxazol]-20-
yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylic acid (12)
as its trifluoroacetic acid salt
Lithium
20-(30-(4-((2-((tert-butoxycarbonyl)amino)acetam-
ido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-biox-
azole]-4-carboxylate (39 mg, 0.06 mmol) was suspended in dry
DCM (1 mL) and cooled to 0 °C. Trifluoroacetic acid (1 mL) was
added and the reaction stirred at that temperature for 2 h. The sol-
vent was concentrated under reduced pressure and azeotroped
with toluene to give product (38 mg, 100%); mp = 153–155 °C; 1H
NMR (DMSO) d 9.10 (s, 1H), 8.98 (s, 1H), 8.83–8.86 (m, 2H),
8.39 (d, 2H), 8.11–8.15 (m, 3H), 8.02–8.04 (m, 2H), 7.74–77.79

4518
G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
128.4, 128.3, 127.1, 126.1, 125.9, 124.8, 118.6, 112.5, 80.6, 63.3,
35.6, 30.93, 28.7, 18.5.
126.1, 125.5, 79.0, 60.3, 55.9, 52.3, 38.5, 35.2, 31.4, 28.2; IR 3432,
2253, 1672, 1493, 1385, 1157, 1115, 906, 730, 650, 416 cmꢀ1
;
HRMS calculated for
757.2615.
C
41H37ꢁN6O9 (M+H)+, 757.2617; found,
4.7.3. (S)-20-(30-(4-((2-Amino-3-methylbutanamido)methyl)-
[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-
carboxylic acid (13) as its trifluoroacetic acid salt
4.8.2. Lithium 20-(30-(4-((2-((tert-butoxycarbonyl)amino)-3-
phenylpropanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-
biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylate
Lithium 20-(30-(4-((2-((tert-butoxycarbonyl)amino)-3-methylb-
utanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-
[2,40-bioxazole]-4-carboxylate (35 mg, 0.05 mmol) was suspended
in dry DCM (2 mL) and cooled to 0 °C. Trifluoroacetic acid (2 mL)
was added and the reaction stirred at that temperature for 2 h.
The solvent was concentrated under reduced pressure and azeotr-
oped with toluene to give of the desired product (34 mg, 100%);
mp = 105–107 °C; 1H NMR (CD3OD) d 8.58 (s, 1H), 8.50 (s, 1H),
8.48 (s, 1H), 8.29 (s, 2H), 7.98–8.00 (m, 2H), 7.83 (s, 1H), 7.76–
7.80 (m, 2H), 7.66 (d, 1H, J = 8.24), 7.52–7.56 (m, 2H), 4.30–4.41
(m, 2H), 3.63 (d, 1H, J = 6), 2.11–2.16 (m, 1H), 0.99 (d, 3H,
J = 2.44), 0.97 (d, 3H, J = 2.44); 13C (CD3OD) 169.7, 164.1, 164.0,
161.1, 158.7, 145.9, 142.0, 141.7, 140.8, 139.8, 137.8, 132.4,
131.9, 131.0, 128.3, 128.2, 127.3, 127.1, 126.2, 118.7, 118.5,
117.4, 115.8, 114.6, 113.0, 111.5, 60.0, 36.0, 31.4, 18.8, 18.0.
Methyl 20-(30-(4-((2-((t-butoxycarbonyl)amino)-3-phenylprop-
anamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-
bioxazole]-4-carboxylate (19.7 mg, 0.026 mmol) was dissolved in
THF:H2O (3:1, 1.2 mL) and treated with lithium hydroxide
(1.3 mg, 0.03 mmol) at 0 °C. This was stirred at room temperature
for 4 h. The solvents were concentrated under reduced pressure
and the residue dried under vacuum to yield a white solid
(16 mg, 100%); mp = 235–236 °C; 1H NMR (400 MHz) (DMSO) d
8.89 (s, 1H), 8.84 (s, 1H), 8.28 (d, 2H, J = 1.64), 8.07 (s, 1H), 8.02
(d, 2H, J = 7.76), 7.91 (d, 2H, J = 7.56), 7.80 (s, 1H), 7.63–7.67 (m,
2H), 7.08–7.14 (m, 7H), 4.17 (s, 2H), 4.13 (m, 1H), 2.88 (d, 1H,
J = 4.52), 2.71 (d, 1H, J = 4.52), 1.22 (s, 9H); 13C NMR (100 MHz)
(DMSO) d d171.75, 163.1, 162.5, 161.3, 161.2, 155.8, 154.3, 152.9,
143.8, 140.9, 140.8, 140.2, 139.9, 138.1, 136.0, 135.5, 134.4,
131.3, 130.2, 129.8, 129.6, 129.2, 123.6, 127.9, 126.9, 126.8,
126.1, 125.8, 124.3, 77.9, 55.8, 34.9, 37.4, 28.1.
4.7.4. (17)
(S)-20-(30-(4-((2-Amino-3-methylbutanamido)methyl)-[2,40-
bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylic
acid (13) as its trifluoroacetic acid salt (34 mg, 0.05 mmol) was dis-
solved in dry DMF (25 mL), EDC (19 mg, 0.1 mmol) and HOBt
(13 mg, 0.1 mmol) was added. 2,6-Lutidine (0.03 mL, 0.25 mmol)
was added and reaction stirred at room temperature overnight.
The DMF was removed on Kugelrohr and residue purified by flash
chromatography using a gradient of 0–10% MeOH/DCM) to give a
white solid (8.7 mg, 31%); mp = 290–292 (decomposed) °C; 1H
NMR (400 MHz) (CDCl3) d 8.98 (s, 1H), 8.94 (s, 1H), 8.24 (s, 2H),
8.15 (s, 1H), 7.84–7.91 (m, 5H), 7.59 (s, 1H), 7.50–7.55 (m, 3H),
4.94 (dd, 1H, J = 8, 16), 4.6 (dd, 1H, J = 4, 8), 3.79 (dd, 1H, J = 4, 8),
2.40–2.46 (m, 1H), 0.85 (d, 3H, J = 8), 0.71 (d, 3H, J = 8); 13C NMR
(100 MHz) (CDCl3)d 170.9, 162.9, 162.6, 160.6, 155.9, 155.2,
141.7, 139.0, 138.9, 138.7, 138.5, 138.0, 137.0, 134.9, 131.4,
130.9, 129.5, 129.4, 128.2, 128.0, 127.2, 126.9, 126.1, 126.0,
125.2, 125.1, 57.7, 34.1, 30.79, 19.3, 16.8; IR 3053, 2986, 2305,
2253, 1421, 1265, 909, 734, 705, 650 cmꢀ1; HRMS calculated for
4.8.3. (S)-20-(30-(4-((2-Amino-3-phenylpropanamido)methyl)-
[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazol]-4-
carboxylic acid (14) as its trifluoroacetate salt
Lithium 20-(30-(4-((2-((tert-butoxycarbonyl)amino)-3-phenyl-
propanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-
[2,40-bioxazole]-4-carboxylate (16 mg, 0.02 mmol) was suspended
in dry dichloromethane (1 mL) and cooled to 0 °C. Trifluoroacetic
acid (1 mL) was added and the reaction stirred at that temperature
for 2 h. The solvent was concentrated under reduced pressure and
azeotroped with toluene to give the desired product as a pale yel-
low solid (16 mg, 100%) mp = 135–137 °C; 1H NMR (400 MHz)
(DMSO) d 9.16 (s, 1H), 9.03 (s, 1H), 8.99 (m, 1H), 8.94 (s, 1H),
8.44 (s, 1H), 8.43 (s, 1H), 8.16–8.20 (m, 2H), 8.07–8.09 (m, 2H),
7.97 (s, 1H), 7.79–7.84 (m, 2H), 7.28–7.38 (m, 5H), 4.33–4.35 (m,
2H), 4.05–4.08 (m, 1H), 3.12 (d, 1H, J = 6.25), 3.07 (d, 1H,
J = 7.32); 13C NMR (100 MHz) (CDCl3) d171.7, 163.1, 162.5, 161.3,
157.4, 154.3, 152.9, 143.8, 140.9, 140.8, 140.2, 139.9, 138.1,
136.0, 135.5, 134.4, 131.3, 130.2, 129.4, 129.6, 129.2, 123.6,
127.9, 126.9, 126.8, 126.1, 125.8, 124.3, 55.8, 48.5, 37.4.
C
31H25ꢁN6O6 (M+H)+, 577.1830; found, 577.1821.
4.8. Compound (18)
4.8.1. (S)-Methyl 20-(30-(4-((2-((tert-butoxycarbonyl)amino)-3-
phenylpropanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-
biphenyl]-3-yl)-[2,40-bioxazol]-4-carboxylate (10)
4.8.4. (18)
(S)-20-(30-(4-((2-Amino-3-phenylpropanamido)methyl)-[2,40-
bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxylic
acid, 14, as its trifluoroacetate salt (16 mg, 0.02 mmol) was dis-
solved in dry DMF (11 mL), EDC (8 mg, 0.04 mmol) and HOBt
(6 mg, 0.04 mmol) was added. 2,6-Lutidine (0.02 mL, 0.11 mmol)
was added and reaction stirred at room temperature overnight.
The DMF was removed on Kugelrohr and residue purified by flash
chromatography using a gradient of 0–10% MeOH/DCM to give a
white solid (3.7 mg, 27%); mp = 240–243 °C (decomposed); 1H
NMR (400 MHz) (CDCl3) 9.00 (s, 1H), 8.95 (s, 1H), 8.21 (s, 1H),
8.19 (s, 1H), 8.10 (s, 1H), 7.86–7.93 (m, 4H),7.68 (d, 1H, J = 10.2),
7.62 (s, 1H), 7.51–7.55 (m, 2H), 7.36–7.39 (m, 1H), 6.95–7.09 (m,
5H), 5.16–5.22 (m, 1H), 4.85–4.91 (m, 1H), 3.81 (dd, 1H, J = 3.32,
18.44), 3.43 (dd, 1H, J = 3.76, 14.64), 2.81–2.88 (m, 1H); 13C NMR
(100 MHz) (CDCl3) d 170.6, 162.9, 162.6, 160.4, 155.8, 155.1,
141.4, 139.0, 138.8, 138.7, 138.4, 138.0, 136.8, 136.6, 135.1,
131.6, 130.8, 129.6, 129.5, 128.9, 128.3, 128.1, 128.0, 127.3,
126.9, 126.5, 126.0, 125.9, 125.2, 53.5, 38.3, 34.5; IR 3054, 2986,
Boc-protected L-phenylalanine (53 mg, 0.2 mmol), EDC (76 mg,
0.4 mmol) and HOBt (53 mg, 0.4 mmol) were dissolved in dry
DMF (5 mL) and stirred for 5 min under nitrogen. A solution of
methyl 20-(30-(4-(aminomethyl)-[2,40-bioxazol]-20-yl)-[1,10-biphe-
nyl]-3-yl)-[2,40-bioxazole]-4-carboxylate (4.5.2) (30 mg, 0.05
mmol), 2,6-lutidine (0.07 mL, 0.64 mmol) in dry DMF (5 mL) was
added and reaction stirred at room temperature overnight. On
completion of the reaction, the DMF was removed and the mixture
purified with flash chromatography (0–100% ethyl acetate/hexane)
to obtain a white solid (19.7 mg, 52%); mp = 153–156 °C; 1H NMR
(CDCl3) d 8.39 (s, 1H), 8.37–8.38 (m, 1H), 8.35–8.36 (m, 1H), 8.27
(s, 1H), 8.20 (s, 1H), 8.06–8.10 (m, 2H), 7.94 (s, 1H), 7.71–7.74
(m, 2H), 7.50–7.55 (m, 2H), 7.47 (s, 1H), 7.07–7.17 (m, 5H), 6.41
(t, 1H, J = 5.64), 4.98 (br s, 1H), 4.30 (d, 2H, J = 5.72), 3.89 (s, 3H),
2.99 (d, 2H, J = 6.78), 1.32 (s, 9H); 13C NMR (CDCl3) d 171.2,
162.6, 162.5, 161.3, 161.2, 155.8, 155.3, 154.3, 143.8, 140.9,
140.8, 139.5, 138.3, 136.5, 135.5, 134.4, 131.7, 131.0, 130.0,
129.9, 129.8, 129.5, 129.2, 128.6, 127.1, 127.0, 126.9, 126.2,
2305, 1421 cmꢀ1
625.1830; found, 625.1828.
;
HRMS calculated for C35H24ꢁN6O6 (M+H)+,

G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
4519
4.9. Compound (19)
(s, 9H); 13C (400 MHz) (DMSO) d 175.0, 162.6, 162.5, 161.3,
156.4, 155.8, 155.4, 145.2, 141.3, 140.6, 139.5, 138.9, 137.0,
135.6, 134.4, 131.7, 130.5, 130.0, 129.8, 129.6, 129.5, 127.1,
126.9, 126.3, 126.1, 124.5, 79.4, 50.7, 37.1, 36.4, 31.9, 28.5.
4.9.1. Methyl 20-(30-(4-((2-((((9H-Fluoren-9-yl)methoxy)
carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanamido)
methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-
bioxazol]-4-carboxylate (11)
4.9.4. (19)
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-((t-
butoxycarbonyl)amino)butanoic acid (39 mg, 0.09 mmol), EDC
(17.8 mg, 0.09 mmol) and HOBt (17 mg, 0.10 mmol) were dissolved
in dry DMF (2 mL) and stirred for 5 min under nitrogen. A solution of
methyl 20-(30-(4-(aminomethyl)-[2,40-bioxazol]-20-yl)-[1,10-biphe-
nyl]-3-yl)-[2,40-bioxazole]-4-carboxylate (4.5.2) (44.8 mg, 0.074 mmol),
diisopropylethylamine (0.02 mL, 0.08 mmol) in dry DMF (5 mL) was
added and reaction stirred at room temperature overnight. On
completion of the reaction, the DMF was removed and the mixture
purified with flash chromatography (0–100% ethyl acetate/hexane)
to obtain a white solid (54.4 mg, 78%); mp = 185–187 °C; 1H NMR
(400 MHz) (CDCl3) d 8.41 (s, 1H), 8.35 (s, 2H), 8.28 (s, 1H), 8.20 (s,
1H), 8.06–8.08 (m, 2H), 7.72–7.74 (m, 2H), 7.62–7.66 (m, 3H),
7.50–7.55 (m, 4H), 7.22–7.30 (m, 5H), 6.12 (br s, 1H), 5.23 (br s,
1H), 4.38 (s, 2H), 4.31 (d, 2H, J = 6.84), 4.11–4.18 (m, 2H), 3.89
(s, 3H), 3.28–3.34 (m, 1H), 2.92–2.95 (m, 1H), 1.85–1.90 (m, 1H),
1.58–1.61 (m, 1H), 1.35 (s, 9H); 13C (100 MHz) (CDCl3) d 171.6,
162.6, 162.5, 161.4, 156.3, 155.8, 155.42, 143.87, 143.6, 141.2,
140.9, 140.7, 139.6, 138.6, 138.6, 138.4, 135.6, 134.3, 131.5, 130.9,
130.0, 129.8, 129.6, 129.5, 127.6, 127.1, 127.0, 127.0, 126.9, 126.1,
125.5, 125.0, 119.9, 79.7, 67.00, 52.3, 47.1, 35.6, 28.3, 28.2; IR
3054, 2986, 2685, 2305, 1684, 1637 cmꢀ1; HRMS calculated for
(S)-20-(30-(4-((2-Amino-4-((t-butoxycarbonyl)amino)butanami-
do)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-[2,40-biox-
azole]-4-carboxylate, (4.9.3), (25.3 mg, 0.036 mmol) was dissolved
in dry DMF (18 mL), EDC (14 mg, 0.073 mmol) and HOBt (9.8 mg,
0.073 mmol) was added 2,6-lutidine (0.017 mL, 0.145 mmol) and
the reaction stirred at room temperature overnight. The DMF
was removed on Kugelrohr and residue purified by flash chroma-
tography using a gradient of 0–10% MeOH/DCM)to give a white so-
lid (16 mg, 65%); mp = 200–202 °C (decomposed); 1H NMR
(400 MHz) (CDCl3) d 8.99 (s, 1H0, 8.91 (s, 1H), 8.23 (s, 2H), 8.16
(s, 1H), 7.84–7.90 (m, 4H), 7.60 (s, 1H), 7.50–7.58 (m, 4H), 4.91–
4.95 (m, 2H), 4.77–4.83 (m 1H), 3.83–3.87 (m, 1H), 3.27–3.30 (m,
1H), 2.83–2.89 (m, 1H), 2.28–2.31 (m, 1H), 1.60–1.66 (m, 1H),
1.31 (s, 9H); 13C (100 MHz) (CDCl3) d 170.8, 162.9, 162.5, 160.9,
155.9, 155.7, 155.1, 141.7, 139.1, 138.6, 138.4, 138.2, 138.1,
136.4, 135.3, 131.3, 130.7, 129.5, 129.4, 128.2, 127.9, 127.2,
126.7, 125.0, 125.9, 125.3, 125.2, 79.0, 55.1, 50.3, 34.6, 28.3, 28.6;
IR 1681 cmꢀ1
678.2307; found, 678.2293.
;
HRMS calculated for C35H31ꢁN7O8 (M+H)+,
4.10. Compound (20)
C
51H46O11N7 (M+H)+, 932.3250; found, 932.3225.
4.10.1. (20)
Compound 19 (4.9.4) (14 mg, 0.02 mmol) was suspended in dry
DCM (1 mL) and cooled to 0 °C. Trifluoroacetic acid (1 mL) was
added and the reaction stirred at that temperature for 2 h. The sol-
vent was concentrated under reduced pressure and washed with
saturated NaHCO3. The organic layer was dried over sodium sulfate
and concentrated to give a white solid (12.4 mg, 100%); mp = 210–
212 °C; 1H NMR (CD3OD) d 8.61 (s, 1H), 8.57 (s, 1H), 8.56 (s, 1H),
8.48 (s, 1H), 8.41 (s, 1H), 7.78–7.87 (m, 6H), 7.48–7.53 (m, 3H),
4.50–4.53 (m, 2H), 3.90–3.94 (m, 1H), 2.89–2.95 (m, 2H), 1.88–
1.92 (m, 2H); 13C NMR (400 MHz) (CD3OD) d 170.8, 162.9, 162.5,
160.9, 155.7, 155.1, 141.7, 139.1, 138.6, 138.4, 138.2, 138.1,
136.4, 135.3, 131.3, 130.7, 129.5, 129.4, 128.2, 127.9, 127.2,
126.7, 125.0, 125.9, 125.3, 125.2, 55.1, 50.3, 34.6, 28.5; HRMS cal-
culated for C30H23O6N7 (M+H)+, 578.1783; found, 578.1783.
4.9.2. Methyl 20-(30-(4-((2-amino-4-((t-butoxycarbonyl)
amino)butanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-
biphenyl]-3-yl)-[2,40-bioxazol]-4-carboxylate
Methyl 20-(30-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-4-((t-butoxycarbonyl)amino) butanamido) methyl)[2,40-
bioxazol]-20-yl)[1,10-biphenyl]-3-yl)-[2,40-bioxazole]-4-carboxyl-
ate (22 mg, 0.024 mmol) was stirred in a solution of 1% piperidine
in acetonitrile (4 mL) for 3 h at room temperature. The solvent was
removed under reduced pressure and purified by flash chromatog-
raphy (0–10% CH3OH/CH2Cl2) to yield a white solid (15.3 mg, 91%);
mp = 113–115 °C 1H NMR (400 MHz) (CDCl3) d 8.40 (s, 1H), 8.37 (s,
1H), 8.35 (s, 1H), 8.28 (s, 1H), 8.23 (s, 1H), 8.07–8.08 (m, 2H). 7.98
(br s, 1H), 7.71–7.74 (m, 2H), 7.62 (s, 1H), 7.51–7.58 (m, 2H), 4.93
(br s, 1H), 4.38 (d, 2H, J = 4), 3.89 (s, 3H), 3.23–3.27 (m, 1H), 3.15–
3.18 (m, 1H), 1.93–1.98 (m, 1H), 1.60–1.66 (m, 1H), 1.35 (s, 9H);
13C NMR (100 MHz) (CDCl3) d 175.0, 162.6, 162.5, 161.3, 156.4,
155.8, 155.4, 143.8, 140.9, 140.8, 139.5, 138.9, 138.3, 135.6,
134.4, 131.7, 131.0, 130.0, 129.8, 129.6, 129.5, 127.1, 126.9,
126.2, 126.1, 125.5, 79.4, 52.8, 52.3, 37.1, 35.8, 35.1, 28.3.
4.11. Compound (21)
4.11.1. (21)
Compound 20 (4.10.1) (8.6 mg, 0.015 mmol) was dissolved in
20% MeOH in dichloromethane (3 mL) and a solution of 37% aque-
ous formaldehyde (1 mL) was added under nitrogen. The reaction
mixture was allowed to stir at room temperature for 10 min and
sodium triacetoxyborohydride (46.6 mg, 0.22 mmol) was then
added. The reaction was stirred at room temperature overnight.
The reaction mixture was then poured into saturated sodium
bicarbonate solution and extracted with dichloromethane. The or-
ganic extracts were dried over sodium sulfate, concentrated and
purified using a gradient of 1–10% MeOH/CH2Cl2 with 0.1% NH4OH
to give a white solid (4.2 mg, 47%); mp = 218–220 °C (decom-
posed); 1H NMR (400 MHz) (CDCl3) 8.91 (s, 1H), 8.88 (s, 1H), 8.22
(s, 1H), 8.21 (s, 1H), 8.16 (s, 1H), 7.86–7.92 (m, 5H), 7.62 (s, 1H),
7.52–7.56 (m, 4H), 4.79–4.88 (m, 2H), 3.86–3.88 (m, 2H), 2.24–
2.35 (m, 1H), 2.19 (s, 6H), 2.03–2.05 (m, 1H), 1.92–1.97 (m, 1H),
1.91–1.92 (m, 1H); 13C NMR (100 MHz) (CDCl3) d 170.8, 162.6,
162.5, 160.9, 155.7, 155.1, 141.0, 139.1, 139.0, 138.6, 138.4,
138.1, 136.4, 135.3, 131.3, 131.0, 129.5, 129.4, 128.2, 127.3,
127.2, 126.7, 125.9, 125.7, 125.4, 125.1, 55.7, 44.6, 34.4, 28.2;
4.9.3. (S)-20-(30-(4-((2-Amino-4-((tert-butoxycarbonyl)amino)
butanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-
yl)-[2,40-bioxazol]-4-carboxylate (15)
Methyl 20-(30-(4-((2-amino-4-((tert-butoxycarbonyl)amino)
butanamido)methyl)-[2,40-bioxazol]-20-yl)-[1,10-biphenyl]-3-yl)-
[2,40-bioxazole]-4-carboxylate (25 mg, 0.037 mmol) was dissolved
in THF:H2O (3:1, 2 mL) and treated with lithium hydroxide
(2.0 mg, 0.05 mmol) at 0 °C. This was stirred at room temperature
for 4 h. The solvents were removed under reduced pressure and the
residue dried under vacuum, 1 M HCl solution was added and a
white solid precipitate formed. The solid was filtered and then
azeotroped with toluene to give a white solid (25 mg, 100%);
mp = 203–205 °C; 1H NMR (400 MHz) (DMSO) d 9.09–9.12 (m,
2H), 8.97 (s, 1H), 8.85 (s, 1H), 8.35–8.36 (m, 2H), 8.21 (s, 1H),
8.08–8.12 (m, 2H), 7.99–8.01 (m, 2H), 7.71–7.76 (m, 2H), 4.49
(s, 2 h), 3.80 (m, 1H), 3.02–3.07 (m, 2H), 1.86–1.87 (m, 2H), 1.37

4520
G. A. Blankson et al. / Bioorg. Med. Chem. 21 (2013) 4511–4520
HRMS calculated for C30H23ꢁN7O6 (M+H)+, 578.1783; found,
Acknowledgments
578.1783.
The Bruker Avance III 400 MHz NMR spectrometer used for this
study was purchased with funds from NCRR Grant No.
1S10RR23698-1A1. Mass spectrometry was provided by the Wash-
ington University Mass Spectrometry Resource with support from
the NIH National Center for Research Resources Grant No.
P41RR0954.
4.12. Cytotoxicity assays
The cytotoxicity was determined using the MTT-microtiter
plate tetrazolinium cytotoxicity assay (MTA). The KB3-1 cell line
and its multidrug-resistant variant KBV-1 were obtained from
K.V. Chin (The Cancer Institute of New Jersey, New Brunswick,
NJ, USA).24 The KBH5.0 cell line as noted previously was derived
from KB3-1 by stepwise selection against Hoechst 33342.25 The
cytotoxicity assay was performed using 96-well microtiter plates.
Cells were grown in suspension at 37 °C in 5% CO2 and maintained
by regular passage in RPMI medium supplemented with 10% heat
References and notes
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inactivated fetal bovine serum, L-glutamine (2 mM), penicillin
(100 U/mL), and Streptomycin (0.1 mg/mL). For determination of
IC50, cells were exposed continuously for FOUR days to varying
concentrations of drug, and MTT assays were performed at the
end of the fourth day. Each assay was performed with a control
that did not contain any drug. All assays were performed at least
twice in six replicate wells.
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4.13. UV melting studies
UV melting experiments were conducted on an AVIV Model
14DS Spectrophotometer (Aviv Biomedical, Lakewood, NJ, USA)
equipped with a thermoelectrically controlled cell holder. Quartz
cells with a pathlength of 1.0 cm were used for all the tempera-
ture-dependent absorbance studies. UV melting profiles were ac-
quired at either 260 nm (for duplex) or 295 nm (for quadruplex)
with a 5 second averaging time. The temperature was raised in
0.5 °C increments, and the samples were allowed to equilibrate
for 1 min at each temperature setting. In the quadruplex melting
studies, the concentration of d(TTAGGG)4 was 5
(120 M in nucleotide). When present in these quadruplex studies,
all test compounds were used at a concentration of 20 M. In the
duplex melting studies, the salmon testes (ST) DNA concentration
was 15 M in base pair (30 M in nucleotide), and all test com-
pounds, when present, were used at concentration of 15 M. The
lM in strand
l
l
l
l
24. Gervasoni, J. E., Jr.; Fields, S. Z.; Krishna, S.; Baker, M. A.; Rosado, M.;
Thuraisamy, K.; Hindenburg, A. A.; Taub, R. N. Cancer Res. 1991, 51, 4955.
25. Li, T.-K.; Houghton, P. J.; Desai, S. D.; Daroui, P.; Liu, A. A.; Hars, E. S.;
Ruchelman, A. L.; LaVoie, E. J.; Liu, L. F. Cancer Res. 2003, 63, 8400.
l
solution conditions for all the UV melting studies were 10 mM
potassium phosphate (pH 7.5) and sufficient KCl (132 mM) to bring
the total K+ concentration to 150 mM. Prior to their use in the UV
melting experiments, all d(TTAGGG)4 solutions were preheated at
90 °C for 5 min and slowly cooled to room temperature over a per-
iod of 4 h.