Synthesis and G-quadruplex stabilizing properties of a series of oxazole-containing macrocycles

Article abstract of DOI:10.1016/j.bmcl.2006.05.038

The synthesis of 24-membered macrocycles containing four, six, and seven oxazole moieties is described. Selected compounds were evaluated for their ability to specifically bind and stabilize G-quadruplex DNA and for cytotoxic activity. An unexpected oxida

Full text of DOI:10.1016/j.bmcl.2006.05.038

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3891–3895
Synthesis and G-quadruplex stabilizing properties of a series
of oxazole-containing macrocycles
Gurpreet Singh Minhas,a Daniel S. Pilch,b,c John E. Kerrigan,b,c
Edmond J. LaVoiea,c and Joseph E. Ricea,*
aDepartment of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers,
The State University of New Jersey, Piscataway, NJ 08854-8020, USA
bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, Piscataway, NJ 08901, USA
cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 19 April 2006; revised 10 May 2006; accepted 12 May 2006
Available online 2 June 2006
Abstract—The synthesis of 24-membered macrocycles containing four, six, and seven oxazole moieties is described. Selected com-
pounds were evaluated for their ability to specifically bind and stabilize G-quadruplex DNA and for cytotoxic activity. An unex-
pected oxidative cleavage reaction afforded a macrocyclic imide that was also evaluated for G-quadruplex stabilizing and
cytotoxic activity.
Ó 2006 Elsevier Ltd. All rights reserved.
Telomeres are the ends of chromosomes consisting of
repeated DNA sequences and bound proteins. In
humans TTAGGG repeats are present at the ends of
telomeres.1,2 These G-tails can readily adopt the thermo-
dynamically stable G-quadruplex conformation
in vitro,3,4 although it is not known whether G-quadru-
plexes exist in vivo in human cells. It is known however
that deprotection of telomeres due to defects in telomere
binding proteins or shortened telomeres can activate
DNA damage responses including cell cycle arrest,
senescence, and apoptosis.5–11 Telomerase is an enzyme
that maintains telomere length. Telomerase activity has
been found in 85–90% of all human tumors, but not
in normal cells.12 Thus, telomerase may play a role in
the uncontrolled growth of tumor cells. A number of
compounds have been identified that stabilize G-quad-
ruplexes and also inhibit telomerase.13 Telomestatin
template in its G-quadruplex form.15 Telomestatin is
reported to shorten telomere length and induce apopto-
sis in freshly isolated primary acute leukemia cells.16
These results suggest that G-quadruplex stabilizers
may represent a new class of anticancer agents (Fig. 1).
In this report, the synthesis of a series of macrocyclic
compounds that retain the 24-membered ring structure
of telomestatin is described. Each compound contains
4–7 oxazole units and at least one valine residue. Com-
pounds were evaluated for their ability to specifically
bind and stabilize G-quadruplex DNA and also for
cytotoxic activity.
S
H
O
(SOT-095) 1,
a
natural product isolated from
N
CH3
O
N
Streptomyces anulatus 3533-SV4, is the strongest and
most specific inhibitor of telomerase identified to date.14
The inhibition of telomerase in vitro by telomestatin is
presumed to occur by sequestering the telomere
N
N
O
N
N
O
O
CH3
N
O
N
Keywords: Synthesis; Macrocycles; Oxazoles; G-quadruplex;
Cytotoxicity.
O
*
Corresponding author. Tel.: +1 732 445 5382; fax: +1 732 445
6312; e-mail: jrice@rci.rutgers.edu
Figure 1. Structure of telomestatin (1).
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.038

3892
G. S. Minhas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3891–3895
Syntheses of compounds having alternating oxazole and
amino acid residues are outlined in Scheme 1. Oxazoles
having an amino acid residue and carboxyl group at the
2- and 4-positions, respectively, were prepared using
established methodology starting from serine methyl es-
ter and the appropriate N-Boc-protected amino
acid.17,18 The resulting oxazoles 3a,b were then divided
into two portions, with one treated with trifluoroacetic
acid to remove the Boc group to give amines 4a,b and
the other hydrolyzed with LiOH to give carboxylic acids
5a,b. The amines were coupled in very high yield with
the carboxylic acids using BOP reagent to give amides
6a,b.19 Once again the products were divided into two
portions with one treated with TFA to give 7a,b and
the other with LiOH to form 8a,b. These were again
coupled with BOP, deprotected, and then the macrocyc-
lization was performed using BOP under high dilution
conditions (<0.01 M solution). The yields of 9a,b were
48% and 51%, respectively.20
Macrocycle 9b was treated with 50% HF in acetonitrile
to remove the TBDPS groups (Scheme 2). Diol 10 was
then cyclized using DAST and dehydrogenated with
BrCCl3 as described above to give macrocyclic hexaox-
azole 11 in 28% yield for the two steps.20
The synthesis of macrocycles containing six and seven
oxazole units is shown in Scheme 3. Amides 12a,b (pre-
pared using the procedure outlined in Scheme 1) were
converted into teroxazoles 13a,b by hydrogenolysis of
the benzyl-protecting group, cyclization of the resulting
alcohol with DAST, and aromatization with BrCCl3.
Treatment of 13a with TFA yielded an amine. Hydroly-
sis of 13b with LiOH afforded a carboxylic acid that was
then coupled with the amine using BOP reagent to give
amide 14. The Boc group was removed with TFA and
the methyl ester hydrolyzed with LiOH to afford an
amino acid that was macrocyclized in 67% yield using
BOP reagent under high dilution conditions. Removal
OH
O
R
O
CO2Me
R
b,c
a
HO
N
CO2Me
N
3a,b
BocHN
H
CO2Me
e
.
NH2 HCl
2a R = i-Pr
BocHN
2b R = CH2OTBDPS
d
R'
O
R
R
O
O
N
O
N
f
N
H
O
N
N
4a,b
H2N
BocHN
CO2Me
CO2H
O
6a,b
5a,b
CO2Me
e
R
NHBoc
R'
O
d
N
H
O
N
N
R'
R'
O
O
O
R
O
O
O
HN
f,d,e,g
NH
N
H
N
R
H
O
O
N
N
N
N
N
N
CO2Me
CO2H
O
H
N
7a,b
8a,b
R
R
O
NH2
NHBoc
R'
9a R,R' = i-Pr (48%)
9b R = i-Pr,R' = CH2OTBDPS (51%)
O
Scheme 1. Reagents: (a) RCH(NHBoc)CO2H, EDC, HOBt, Et3N; (b) DAST, K2CO3; (c) BrCCl3, DBU; (d) TFA, CH2Cl2; (e) LiOH, MeOH/THF;
(f) BOP reagent, Et3N, CH3CN; (g) BOP reagent, DIPEA, DMF, <0.01 M.
O
O
N
O
O
OH
O
N
HN
N
O
NH
O
NH
O
N
a
b,c
N
9b
N
N
HN
O
HN
O
O
O
N
N
O
NH
O
N
O
HO
O
11
10
Scheme 2. Reagents: (a) 50% HF/CH3CN; (b) DAST, K2CO3; (c) BrCCl3, DBU.

G. S. Minhas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3891–3895
3893
OBn
O
O
O
O
O
a,b,c
N
R
H
N
N
N
O
N
R
NHBoc
N
13a,b
CO2Me
NHBoc
12a R = iPr
CO2Me
12b R = CH2OTIPS
d (13a),e (13b),f
O
O
O
O
N
N
N
N
O
O
d,e,f,g
O
NH
N
O
NH
N
CO2Me
BocHN
N
O
N
HN
O
N
O
N
N
O
N
O
OH
OTIPS
O
O
15
14
h,i
b
O
N
O
N
O
H
O
N
N
O
O
O
O
NH
N
N
O
NH
N
N
N
cX
N
O
O
N
O
N
N
O
N
O
H
O
O
16 (41%)
17 (trace)
+
O
O
N
N
O
O
NH
N
N
O
HN
N
O
N
O
O
O
18 (31%)
Scheme 3. Reagents and conditions: (a) H2, Pd(OH)2/C, EtOH; (b) DAST, K2CO3, CH2Cl2, ꢀ78 °C to rt; (c) BrCCl3, DBU, CH2Cl2, 0 °C; (d) 50%
TFA/CH2Cl2; (e) LiOH, MeOH/THF; (f) BOP reagent, Et3N, DMF; (g) HCl, MeOH, CH2Cl2; (h) DMP, CH2Cl2, D; (i) PPh3, I2, Et3N.
of the TIPS groups with HCl in MeOH/CH2Cl2 gave
macrocyclic hexaoxazole 15 in 70% yield. A portion of
15 was treated with DAST to give dihydroheptaoxazole
16 in 41% yield. Treatment with BrCCl3/DBU however
failed to give heptaoxazole 17. A number of other re-
agents were evaluated for the formation of 17 including
NiO2, MnO2, NBS, and DDQ, but all were equally
unsuccessful. Instead, 15 was oxidized with Dess–Mar-
tin periodinane (DMP) to a purported aldehyde which
was then treated with triphenylphosphine and iodine21
in the presence of triethylamine to give traces of hepta-
oxazole 17 along with imide 18 in 31% yield from 15.20 It
was determined that 18 formed during the Dess–Martin
oxidation of alcohol 15, which was performed at reflux
in CH2Cl2 due to solubility problems. There is some
precedent for such a transformation. Cabarrocas et al.
reported oxidative cleavage of several b-hydroxyamides
during oxidation with iodoxybenzoic acid (IBX) at
75 °C in DMSO to give similar imide products.22
DMP can contain varying amounts of hydrolysis prod-
ucts including IBX and acetoxyIBX, and it is possible
that such impurities could be responsible for the oxida-
tive cleavage observed in the present study.23 A detailed
investigation of this is currently underway in our labora-
tory and will be reported separately.
The tetraoxazole macrocycle 9a, as well as the hexaoxaz-
ole macrocycles 11 and 18, were evaluated for their abil-
ities to bind and thermally stabilize both duplex and
G-quadruplex DNA. Toward this end we monitored
the UV absorbance of the DNA as a function of temper-
ature in the absence and presence of saturating amounts

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G. S. Minhas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3891–3895
Table 1. Transition temperatures for the melting of ST-DNA and
d(TTAGGG)4 in the absence and presence of oxazole-containing
macrocyclic ligands
of macrocycle. The melting of duplex DNA is associated
with a hyperchromic shift at 260 nm, while the melting
of quadruplex DNA is associated with a hypochromic
shift at 295 nm. Thus, the temperature-dependent
absorption of duplex and quadruplex DNA was moni-
tored at 260 and 295 nm, respectively. Salmon testes
DNA (ST-DNA) was used as a representative model
for duplex DNA, while the quadruplex structure formed
by an oligomer, d(TTAGGG)4, containing four consec-
utive repeats of the 50-TTAGGG-30 sequence that is
multiply repeated in human telomeric DNA was used
as a representative model for G-quadruplex DNA. All
the UV melting studies were conducted at pH 7.5 in
the presence of 50 mM potassium ion.
a
Sample
No. of oxazoles
Ttran (°C)
ST-DNA
NAb
78.5
78.5
78.9
78.5
56.0
56.0
73.5
62.5
ST-DNA + 9a
ST-DNA + 11
ST-DNA + 18
d(TTAGGG)4
d(TTAGGG)4 + 9a
d(TTAGGG)4 + 11
d(TTAGGG)4 + 18
4
6
6
NAb
4
6
6
a DNA transition temperatures (Ttran) reflect the maxima or minima of
the first derivatives of UV melting profiles acquired as described in
the legend to Figure 2. The uncertainty in the Ttran values is 0.5 °C.
b NA, not applicable.
Figure 2 shows the UV melting profiles (depicted in
their first-derivative forms) of ST-DNA and
d(TTAGGG)4 in the absence and presence of 11.
The transition temperatures (Ttran) corresponding to
the maxima or minima of these first-derivative melting
profiles are listed in Table 1, as are the corresponding
Ttran values derived from UV melting profiles (not
shown) conducted in the presence of 9a and 18. Note
that the presence of neither 9a, 11, nor 18 alters the
Ttran value (i.e., the thermal stability) of ST-DNA,
with any observed changes in Ttran being within the
experimental uncertainty. This observation is consis-
tent with little or no duplex DNA binding on the part
of either macrocycle. Control isothermal titration
calorimetry (ITC) experiments (not shown) in which
9a, 11, and 18 were sequentially titrated into solutions
of ST-DNA confirmed this observation.
Not only does the presence of 9a fail to alter the thermal
stability of ST-DNA, it also fails to alter thermal stabil-
ity of d(TTAGGG)4. Thus, the tetraoxazole macrocycle
exhibits little or no binding to either duplex or G-quad-
ruplex DNA.
In striking contrast to the behavior of 9a, 11 and 18
increase the Ttran value of d(TTAGGG)4 by 17.5 and
6.5 °C, respectively. Thus, both hexaoxazoles bind and
thermally stabilize G-quadruplex DNA, with the extent
of thermal enhancement being greater for 11. ITC exper-
iments are currently underway to assess whether this dif-
ferential extent of thermal enhancement correlates with
a corresponding difference in binding affinity. Viewed
as a whole, our UV melting data indicate that hexaoxaz-
ole macrocycles bind G-quadruplex DNA with a high
degree of specificity, with the G-quadruplex binding
behavior being lost upon reduction of the oxazole num-
ber from six to four. Results from G-quadruplex bind-
ing studies for dihydroheptaoxazole 16 and other
heptaoxazoles will be reported separately. Additional
evidence in support of the importance of six oxazoles
within the macrocyclic structure for specific G-quadru-
plex binding was provided by preliminary computational
studies of 9a, 11, and 18 (Table 2). Hexaoxazoles 11
and 18 have Edock values that are approximately 2-fold
more favorable than that of tetraoxazole 9a. While these
Edock values were determined for a capping interaction
of the macrocycle with the quadruplex, the nature of
the actual interactions between the macrocycles and
quadruplex (intercalation vs capping) remains to be
determined.
Figure 2. First derivatives of the UV melting profiles of ST-DNA and
d(TTAGGG)4 in the absence and presence of 11. Profiles were
acquired on an AVIV model 14DS spectrophotometer using quartz
cuvettes with a 1 cm pathlength. The temperature was raised in 0.5 °C
increments and the samples were allowed to equilibrate for 2 min at
each temperature setting, whereupon absorbances were recorded over
a period of 6 s and averaged. When present, ST-DNA was used at a
base pair concentration of 15 lM, while d(TTAGGG)4 was used at a
strand concentration of 4 lM. Macrocyclic ligands were used at a
concentration of 15 lM in the ST-DNA experiments and 8 lM in the
d(TTAGGG)4 experiments. Solution conditions were 10 mM EPPS
(pH 7.5), 50 mM KCl, and 0.1 mM EDTA. In the ST-DNA experi-
ments, the wavelength (k) was 260 nm, while being 295 nm in the
d(TTAGGG)4 experiments.
Select macrocycles were evaluated for cytotoxic activity.
IC50 values (lM) for 11, 16, and 18 were: 0.4, 0.21, and
0.8, respectively, for the human lymphoblastoma RPMI
8402, and 0.5, 1.5, and 1.1 for the murine leukemia
P388.
In summary, macrocycles containing four, six, and seven
oxazole moieties have been synthesized. Compounds
having at least six oxazole rings bind selectively to

G. S. Minhas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3891–3895
3895
Table 2. Calculated docking energies of macrocyclic ligands with a G-
quadruplex
10. Karlseder, J.; Smogorzewska, A.; de Lange, T. Science
2002, 295, 2446.
a
Edock (kcal/mol)
11. Qi, H.; Li, T.-K.; Nur-E-Kamal, A.; Liu, L. F. J. Biol.
Chem. 2003, 178, 15136.
12. White, L. K.; Wright, W. E.; Shay, J. W. Trends
Biotechnol. 2001, 19, 114.
13. Neidle, S.; Read, M. A. Biopolymers 2001, 56, 195.
14. Shin-ya, K.; Wierzba, K.; Matsuo, K.; Ohtani, T.;
Yamada, Y.; Furihata, K.; Hayakawa, Y.; Seto, H. J.
Am. Chem. Soc. 2001, 123, 1262.
Macrocycle
No. of oxazoles
9a
11
4
6
6
7
7
7
ꢀ4.1
ꢀ8.4
ꢀ9.1
ꢀ9.2
ꢀ9.8
ꢀ9.7
18
16
17
(R)-Telomestatin
a Docking was performed using the Lamarckian Genetic Algorithm24
in Autodock 3.0.5. Edock = EVDW + Eelec + Eint with more negative
values signifying a stronger binding energy. An X-ray crystal struc-
ture of a parallel G-quadruplex derived from human telomeric DNA
(1KF1.pdb) was used as the receptor for these studies.3 Macrocyclic
ligands were constructed and refined using Sybyl (Tripos, Inc.).
15. Kim, M. Y.; Vankayalapati, H.; Shin-ya, K.; Wierzba, K.;
Hurley, L. H. J. Am. Chem. Soc. 2002, 124, 2098.
16. Nakajima, A.; Tauchi, T.; Sashida, G.; Sumi, M.; Abe, K.;
Yamamoto, K.; Ohyashiki, J. H.; Ohyashiki, K. Leukemia
2003, 17, 560.
17. Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams,
D. R. Org. Lett. 2000, 2, 1165.
18. Williams, D. R.; Lowder, P. D.; Gu, Y.-G.; Brooks, D. A.
Tetrahedron Lett. 1997, 38, 331.
Gasteiger–Huckel charges were used on the ligands25 and Kollman
¨
charges were used on the G-quadruplex.26
19. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron
Lett. 1975, 1219.
G-quadruplex DNA but not to duplex DNA. These
compounds also exhibit moderate levels of cytotoxic
activity against human lymphoblastoma and murine
leukemia.
20. The structures of all compounds were elucidated using 1H
(200 MHz) and 13C NMR (50 MHz) in CDCl3. Results are
reported as ppm downfield from internal TMS. Com-
1
pound 9a: H NMR d 8.15 (s, 4H), 7.27 (d, 4H, J = 9.4),
5.26 (m, 4H), 2.34 (m, 4H), 1.08 (d, 12H, J = 6.6), 0.98 (d,
12H, J = 6.6); 13C NMR d 163.8, 159.8, 141.6, 135.8, 51.7,
32.9, 19.0, 18.7; HRMS (M+Li+) calcd for C32H40N8O8
(Li), 671.3129; found, 671.3148.
Acknowledgments
This study was supported by NIH Grants CA098127
(E.J.L.) and CA097123(D.S.P.) and American Cancer
Society Grant RSG-99-153-04-CDD (D.S.P.). Mass
spectrometry was provided by the Washington Universi-
ty Mass Spectrometry Resource with support from the
NIH National Center for Research Resources (Grant
No. P41RR0954). The authors thank Dr. Michael A.
Grayson of Washington University for helpful discus-
sions relating to elucidation of the structure of com-
pound 18 and Angela Liu of UMDNJ for performing
the cytotoxicity assays.
1
Compound 11: H NMR d 8.51 (d, 2H, J = 8), 8.20 (m,
6H), 5.30 (dd, 2H, J = 8.5), 2.40 (m, 2H), 1.04 (d, 6H,
J = 7), 0.98 (d, 6H, J = 7); 13C NMR d 163.7, 159.1, 155.3,
153.8, 140.0, 138.2, 137.6, 136.2, 130.2, 128.8, 52.3, 33.2,
17.7, 17.5; HRMS (M+H+) calcd for C28H24N8O8(H+),
601.1795; found, 601.1808.
1
Compound 16: H NMR d 8.66 (d, 1H, J = 9), 8.21, 8.18,
8.16, 8.15, 8.04 (s, 6H), 5.74 (dd, 1H, J = 5,9), 5.33 (dd,
1H, J = 5,9), 4.74–4.58 (m, 2H), 2.38 (heptet, 1H, J = 7),
1.07 (d, 3H, J = 7), 1.00 (d, 3H, J = 7); 13C NMR d 163.8,
159.8, 159.1, 159.0, 155.0, 154.9, 153.9, 140.2, 139.5, 138.4,
137.7, 137.6, 137.0, 136.3, 130.4, 130.3, 130.0, 129.1, 128.7,
70.3, 62.5, 52.1, 33.0, 17.6, 17.4; HRMS (M+Li+) calcd for
C26H18N8O8(Li), 577.1408; found, 577.1389.
Compound 18: 1H NMR d 11.17 (s, 1H), 8.50 (d, 1H,
J = 8), 8.47, 8.45 (s, 2H), 8.27, 8.26, 8.25, 8.245 (s, 4H),
5.33 (dd, 1H, J = 8,4), 2.42 (m, 1H), 1.04 (d, 3H, J = 7),
0.93 (d, 3H, J = 7); 13C NMR d 163.5, 158.9, 156.7, 155.6,
154.3, 153.4, 142.9, 141.5, 140.2, 138.6, 138.4, 138.0, 136.3,
135.2, 130.5, 130.2, 130.0, 128.7, 52.3, 32.9, 17.6, 17.0;
HRMS (M+H+) calcd for C25H16N8O9(H+), 573.1118;
found, 573.1091.
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