Macrocyclic hexaoxazoles: Influence of aminoalkyl substituents on RNA and DNA G-quadruplex stabilization and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2010.03.086

A series of 24-membered macrocyclic hexaoxazoles containing one or two aminoalkyl substituents was synthesized and evaluated for cytotoxicity and for their ability to selectively stabilize G-quadruplex DNA and RNA. The most cytotoxic analog 4a, with IC₅₀ values of 25 and 130 nM using KB3-1 and RPMI 8402 cells, is efficacious in vivo in athymic nude mice with a human tumor xenograft from the breast cancer cell line MDA-MB-435.

Full text of DOI:10.1016/j.bmcl.2010.03.086

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3150–3154
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Macrocyclic hexaoxazoles: Influence of aminoalkyl substituents on RNA
and DNA G-quadruplex stabilization and cytotoxicity
Mavurapu Satyanarayana a, Young-Ah Kim a, Suzanne G. Rzuczek a, Daniel S. Pilch b,c, Angela A. Liu b,
Leroy F. Liu b,c, Joseph E. Rice a, Edmond J. LaVoie a,c,
*
a Department of Medicinal Chemistry, 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, 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 24-membered macrocyclic hexaoxazoles containing one or two aminoalkyl substituents was
synthesized and evaluated for cytotoxicity and for their ability to selectively stabilize G-quadruplex DNA
and RNA. The most cytotoxic analog 4a, with IC50 values of 25 and 130 nM using KB3-1 and RPMI 8402
cells, is efficacious in vivo in athymic nude mice with a human tumor xenograft from the breast cancer
cell line MDA-MB-435.
Received 28 January 2010
Revised 19 March 2010
Accepted 26 March 2010
Available online 30 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
G-quadruplex DNA
G-quadruplex RNA
Macrocycles
Hexaoxazoles
Antitumor
MDA-MB-435 xenograft
Cytotoxicity
Athymic nude mice
G-quadruplex ligands
Nucleic acid sequences that can form stable G-quadruplexes
have been implicated in a wide range of biological functions. G-
quadruplexes are associated with the regulation of telomere stabil-
ity, regulatory elements in promoters, and viral integration and
recombination.1–3 Several research groups have developed mole-
cules that are focused on targeting and stabilizing G-quadruplex
DNA as anticancer agents. Selective G-quadruplex interactive li-
gands have the potential to act as cancer-specific agents by inhib-
iting the overexpression of oncogenes such as c-Myc, c-Kit, and
KRAS or blocking the 30 extension of telomeric DNA by telome-
rase.1,2 Telomestatin is a macrocyclic polyoxazole isolated from
Streptomyces anulatus 3533-SV4 that can stabilize G-quadruplex
DNA in a highly selective manner.4 Our laboratory has synthesized
several 24-membered macrocyclic polyoxazoles that are highly
selective for G-quadruplexes.5–7 We have recently reported on
the biophysical and pharmacologic properties of these G-quadru-
plex stabilizers.8–10
HXPV (Fig. 1), a 24-membered macrocycle formed by ring-closing
metathesis, has an IC50 of 25 nM.6 Neither HXDV or HXPV have
physicochemical properties that permit facile formulation for
assessment of their potential efficacy as antitumor agents in vivo.
Lysinyl macrocyclic hexaoxazole analogs with improved properties
for formulation were synthesized and identified as selective G-
quadruplex interactive ligands.7 These results were subsequently
confirmed by another laboratory.11 Recently, a lysinyl macrocyclic
O
O
O
O
O
O
N
N
N
N
N
N
N
NH
N
NH
O
O
O
HN
O
N
N
R
N
O
N
O
O
O
HXDV (Fig. 1), a 24-membered macrocycle containing six oxa-
zole moieties and two valine residues, is cytotoxic toward human
O
lymphoblastoma RPMI 8402 cells with an IC50 value of 0.4 l
M.5
HXDV; R = CH(CH3)2
LV; R = (CH2)3CH2NH2
HXPV
* Corresponding author.
E-mail address: elavoie@rci.rutgers.edu (E.J. LaVoie).
Figure 1. Structures of HXDV, HXPV, and HXLV.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.086

M. Satyanarayana et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3150–3154
3151
heptaoxazole with similar cytotoxicity (IC50 = 670 nM) to HXDV
has also been reported.12 The present study examines the influence
of alkylamino chain length within a series of 24-membered hexa-
oxazoles on cytotoxicity with the objective of selecting a suitable
macrocyclic hexaoxazole for in vivo evaluation of antitumor activ-
ity in athymic nude mice with the breast cancer cell line MDA-MB-
435 as the human tumor xenograft.
The synthetic approach used in the preparation of the eighteen
new aminoalkyl substituted hexaoxazole macrocycles listed was
similar to that previously reported for HXLV.7a The three N-Boc
aminoalkyl teroxazoles 14a–c were synthesized as outlined in
Scheme 1. Hydrolysis of the methyl ester of each of these aminoal-
kyl teroxazoles provided the carboxylic acid derivative, 15a–c,
which were each then subsequently condensed as illustrated in
Scheme 2 with the valine teroxazole 16, prepared as previously de-
scribed.5 Macrocyclization of the hexaoxazole intermediate was
accomplished by hydrogenolysis of the N-Cbz group, hydrolysis
of the methyl ester, and lactam formation using HATU and N-meth-
ylmorpholine (NMM).
was accomplished using either BOPCl, DMAP, DIPEA, DMF for 5a
and 5b or BOP, HOBt, DIPEA, NaI, DMF for 5c. Primary amines
2a–c and 6a–c were prepared by treatment of the Boc derivatives
with TFA. Acetylation provided acetamide derivatives 3a–c and
7a–c. Reductive methylation of 2a–c and 6a–c afforded the N,N-
dimethylamine derivatives, 4a–c and 8a–c.
The cytotoxic activities of these various aminoalkyl substituted
macrocyclic hexaoxazoles are summarized in Table 1. The N-Boc
and bis-N-Boc derivatives of these aminoalkyl hexaoxazoles, 1a–c
and 5a–c, have similar cytotoxic activities towards RPMI 8402, with
IC50 values ranging from 0.29 to 1.40
served with the human carcinoma cell line KB3-1 with IC50 values
ranging from 0.23 to 0.45 M. The primary amine derivatives of
lM. Similar activity was ob-
l
substituted hexaoxazoles with a single aminoalkyl side chain, 2a–
c, were less cytotoxic than their N-Boc derivatives, but were more
cytotoxic than their bis-aminoalkyl derivatives 6a–c in both of these
cell lines. Similarly, the N-acetyl derivatives of the hexaoxazole ana-
logs with a single aminoalkyl side chain 3a–c were also more cyto-
toxic than the bis-N-acetyl derivatives, 7a–c. In comparing the
cytotoxicity of the N,N-dimethylaminoalkyl derivative 4a–c with
the bis-N,N-dimethylaminoalkyl derivatives 8a–c, it is evident that
the presence of a second N,N-dimethylaminoalkyl substituent is
associated with a dramatic loss in activity. Among the bis-dimethyl-
aminoethyl analogs, 8a is the most cytotoxic with IC50 values of 4.3
The bis-aminoalkyl substituted hexaoxazole macrocycles were
prepared as outlined in Scheme 3. The N-Cbz group was removed
from 14a–c by hydrogenolysis to provide the primary amine deriv-
atives 17a–c, which were condensed with the carboxylic acid
derivatives 15a–c. The remaining N-Cbz group of the resulting
hexaoxazole was removed by hydrogenolysis and the methyl ester
hydrolyzed with LiOH in THF. Formation of the macrocyclic lactam
and 2.8 lM in RPMI 8402 and KB3-1 cells, respectively. The most
striking difference in cytotoxicity observed among homologs was
NHCbz
CbzNH
NHCbz
TIPSO
O
H
OH
N
a,b
c,d,e
O
H2N
BocHN
BocHN
OMe
n
+
n
O
n
H2N
O
N
O
OH
N
10a n = 1 (99%)
n = 1 (67%)
11a
9a n = 1
OH
10b
11c
n = 2 (99%)
n = 3 (98%)
11b n = 2 (65%)
9b
9c
12
n = 2
n = 3
O
CO2Me
11c
n = 3 (73%)
BocHN
O
N
BocHN
O
O
N
f,g
O
H
c,d
O
N
CO2Me
n
N
N
N
CbzHN
n
NHCbz
O
CO2Me
OH
13a n = 1 (98%)
n = 1 (48%)
n = 2 (52%)
14a
14b
13b n = 2 (97%)
13c
n = 3 (86%)
14c n = 3 (46%)
Scheme 1. Synthesis of aminoalkyl teroxazoles. Reagents and conditions: (a) Boc2O, NaHCO3; (b) Serine Me ester HCl, BOP, Et3N; (c) DAST, K2CO3; (d) DBU, BrCCl3; (e) LiOH,
THF/H2O; (f) EDC, HOBt, 2,6-lutidine; (g) HF/pyridine.
O
N
O
O
O
N
O
N
N
N
O
O
O
b,c,a,d
O
O
N
+
N
HN
BocHN
NH
N
N
NRR'
N
CO2Me
n
n
O
NHCbz
R = Me 14a-c
N
CO2R
NH2
N
O
16
O
a
O
n = 1
R = H 15a
15b
1a n = 1 (23%)
n = 2
n = 3
1b
1c
n = 2 (28%)
n = 3 (31%)
R = H, R'=Boc
R,R'=H
15c
e
f
2a
2b
2c
n = 1 (99%)
n = 2 (99%)
n = 3 (100%)
3a n = 1 (84%)
R=H,R'=Ac
3b
3c
n = 2 (88%)
n = 3 (72%)
g
4a
4b
4c
n = 1 (78%)
n = 2 (86%)
n = 3 (89%)
R,R'=Me
Scheme 2. Synthesis of HXDV derivatives with an aminoalkyl chain. Reagents and conditions: (a) LiOH, THF/H2O; (b) EDC, HOBt, 2,6-lutidine; (c) H2(g), 20% Pd(OH)2/C, THF/
MeOH for 15a and 15b, 1,4-cyclohexadiene, 20% Pd(OH)2/C, D, THF/EtOH for 15c; (d) HATU, NMM, DMF; (e) TFA, CH2Cl2; (f) Ac2O, pyridine; (g) MeOH, HCl, HCHO/H2O, NaBH4.

3152
M. Satyanarayana et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3150–3154
O
R'RN
O
n
N
O
O
N
O
O
O
NH
N
N
O
O
O
N
N
N
b,a,c,d
BocHN
BocHN
+
N
N
N
n
n
NHR
NHCbz
O
CO2Me
CO2H
HN
R = Cbz 14a-c
N
15a n = 1
O
a
15b
15c
N
n = 2
n = 3
17a n = 1
R = H
O
17b
17c
NRR'
n = 2
n = 3
O
n
5a
5b
5c
n = 1 (23%)
n = 2 (23%)
n = 3 (43%)
R = H, R'=Boc
R,R'=H
e
f
6a
6b
6c
n = 1 (95%)
n = 2 (99%)
n = 3 (100%)
7a
7b
7c
n = 1 (49%)
n = 2 (89%)
n = 3 (81%)
R=H,R'=Ac
g
8a
8b
8c
n = 1 (37%)
n = 2 (53%)
n = 3 (64%)
R,R'=Me
Scheme 3. Synthesis of HXDV derivatives with two aminoalkyl chains. Reagents and conditions: (a) H2(g), 20% Pd(OH)2/C, THF/EtOH for 14a and 14b, 1,4-cyclohexadiene,
20% Pd(OH)2/C, , THF/EtOH for 14c; (b) EDC, HOBt, 2,6-lutidine; (c) LiOH, THF/H2O; (d) BOPCl, DMAP, DIPEA, DMF for 5a and 5b, BOP, HOBt, DIPEA, NaI, DMF for 5c; (e) TFA,
D
CH2Cl2; (f) Ac2O, pyridine; (g) MeOH, HCl, HCHO/H2O, NaBH4.
Table 1
respectively. The data summarized in Table 1 indicate that all of
the macrocyclic hexaoxazoles with IC50 values less than 5.0 M in
Relative cytotoxic activities. IC50 values (
l
M)a
l
KB3-1 cells are resistant in KBV-1 cells and are likely MDR1 sub-
strates. In contrast, no notable resistance was observed with these
hexaoxazoles in KBH5.0 cells, with the exception of 4c, suggesting
that these analogs are not substrates for the BCRP efflux transporter.
The in vivo efficacy of 4a as an antitumor agent was evaluated
in athymic nude mice with human tumor xenografts developed
from implanted MDA-MB-435 breast tumor cells.14 This assay
was performed as previously described by our laboratory.15 The
positive control group consisted of mice (8 mice per group) in-
jected ip 3ꢀ weekly with irinotecan at a dose of 20 mg/kg for
31 days. The vehicle control group consisted of seven mice that re-
Compound RPMI 8402 KB3-1 wt
KBV-1 + MDR1 KBH5.0 + BCRP
1a
1b
1c
2a
2b
2c
3a
3b
3c
0.31 0.13
0.29 0.01
0.35 0.07
1.2 0.28
2.5 0.71
4.8 1.7
0.95 0.07
2.5 0.71
0.75 0.21
0.29 0.09
0.23 0.04
0.3 0.00
2.2 1.1
0.8 0.57
3.4 2.5
0.45 0.07
3.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.25 0.07
0.15 0.07
0.33 0.11
1.2 1.1
0.38 0.04
3.0 1.4
0.90 0.14
3.0 0.00
1.9 1.3
0.018 0.011
0.07 0.04
2.7 0.21
0.25 0.07
0.38 0.04
0.33 0.11
>10
0.71 0.38
4a
0.13 0. 08 0.025 0.007 7.5 3.5
4b
4c
5a
5b
5c
6a
0.23 0.04
>10
0.35 0.07
1.4 0.85
0.40 .07
>10
0.30 0.14
3.2 1.8
0.25 0.07
0.45 0.21
0.25 0.7
>10
>10
3.2 1.8.
>10
>10
>10
ceived ip injections 3ꢀ weekly of 150
ll of 10 mM citrate for
31 days. Compound 4a was administered by ip injection 3ꢀ weekly
at a concentration of 4.0 mg per mL in 10 mM citrate at doses of 14,
18, 31 mg/kg for weeks 1–3, respectively. For the final 10 days
mice received a dose of 42 mg/kg 3ꢀ per week for an average dose
of 28.8 mg/kg. On day 35, the average tumor volume SE of the
control group was 1396 296 mm3, compared to 85 29 mm3 for
mice treated with irinotecan and 302 27 mm3 for mice treated
with 4a.
>10
6b
6c
>10
n.d.
6.3 3.2
>10
>10
>10
3.1 0.84
>10
7a
>10
>10
>10
>10
7b
7c
8a
8b
8c
HXPV
HXDV
>10
>10
>10
>10
3.3 2.1
4.3 2.5
>10
>10
0.03 0.01
0.54 0.12
4.0 1.7
2.8 0.35
4.2 1.2
4.0 2.6
0.04 0.01
0.35 0.08
>10
>10
>10
>10
3.5 0.67
7.7 2.1
4.5 2.1
0.65 0.35
3.0 0.00
3.6 2.0
0.04 0.02
0.45 0.19
Four selected macrocyclic hexaoxazoles (HXDV, 4a, 4b, and 4c)
were evaluated for their ability to selectively bind and stabilize the
quadruplex versus duplex forms of DNA and RNA in the presence of
physiological concentrations (150 mM) of K+ ions. We used salmon
testes (ST) DNA and poly(rA)ꢁpoly(rU) as representative models of
duplex DNA and RNA, respectively. As a representative model of
quadruplex DNA, we used the human telomeric sequence
d[T2G3(T2AG3)3A], which we denote as hTel. Patel and co-workers
have shown that the structure adopted by d[T2G3(T2AG3)3A] in K+
solution is an intramolecular (3 + 1) G-quadruplex in which three
strands are oriented in one direction and the fourth strand is ori-
ented in the opposite direction.16 In an effort to explore ligand
binding interactions with quadruplex RNA, we used a putative
quadruplex-forming sequence (r[AG4CG2-CUG2UCG2AGUG2C]) de-
rived from the 50-untranslated region of the mRNA that encodes
the cell-cycle checkpoint protein kinase Aurora A (AurA). We se-
lected this RNA sequence for the present studies in light of our
recently reported observations that HXDV treatment of cancer cells
a
Values are means of three experiments standard deviation, n.d. = not
determined.
with those hexaoxazoles with a single dimethylaminoalkyl moiety,
4a–c. In RPMI 8402, the IC50 values ranged from 0.13 to 11 lM.
The dimethylaminoethyl derivative 4a,13 the shortest homolog, is
the most cytotoxic. This clear difference in cytotoxic potency among
homologs was also observed in KB3-1 cells, with IC50 values of
0.025–3.2 lM, with 4a again being the most cytotoxic. The cytotox-
icity of 4a is substantially greater than HXDV and is comparable to
HXPV, which has previously been recognized as being among the
most cytotoxic of these oxazole-containing macrocyclic G-quadru-
plex selective ligands. KBV-1 and KBH5.0 cells are variants of
KB1-1 that overexpress the efflux transporters MDR1 and BCRP,

M. Satyanarayana et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3150–3154
3153
0.02
0.015
0.01
0.005
0
A
B
C
0.0008
0
0.001
0
AurA Quad. RNA
hTel Quad. DNA
ST Dup. DNA
No Drug
HXDV
4a
-0.001
-0.002
-0.003
-0.004
-0.005
-0.006
-0.0008
-0.0016
-0.0024
-0.0032
-0.004
No Drug
HXDV
4a
No Drug
HXDV
4a
30 40 50 60 70 80 90 100
Temperature (°C)
75
80
85
90
95
100
30 40 50 60 70 80 90 100
Temperature (°C)
Temperature (°C)
Figure 2. First derivatives of the UV melting profiles of ST duplex DNA (A), hTel quadruplex DNA (B), and AurA quadruplex RNA (C) in the absence (red) and presence of either
HXDV (blue) or 4a (green). The UV melting profiles of the quadruplexes were acquired at 295 nm, while those of the duplexes were acquired at 260 nm. In the quadruplex
melting studies, the hTel and AurA concentrations were 5
l
M in strand. When present in these quadruplex studies, the drug concentrations were 20
l
M. In the duplex
melting studies, the ST DNA concentrations were 30 lM base pair. When present, the drug concentrations were 15 lM in the ST DNA studies. In all 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.
Table 2
consistent with this RNA oligomer adopting a quadruplex struc-
Impact of macrocyclic hexaoxazoles on the thermal stabilities of quadruplex DNA and
RNA
ture in the presence of 150 mM K+. In support of this observation,
circular dichroism (CD) studies suggest that the AurA sequence
a
Compound
D
Ttran (°C)
forms
a parallel-stranded RNA structure (see Supplementary
hTel Quadruplex DNA
AurA Quadruplex RNA
Fig. S1). Note that the presence of HXDV and 4a enhances the
thermal stability of the AurA RNA quadruplex by 19.5 and
34.5 °C, respectively (Table 2). These results are indicative of li-
gand-induced stabilization of a quadruplex structure in the AurA
target RNA sequence. In the aggregate, our collective UV melting
studies indicate that macrocyclic hexaoxazoles bind the quadru-
plex nucleic acid form with a high degree of specificity. It should
also be noted that the relative extents to which HXDV, 4a, 4b, and
4c stabilize the quadruplex nucleic acid form correlate reasonably
well with their relative cytotoxic potencies (see Table 1). This
observation supports the hypothesis that a quadruplex structure
(RNA or DNA) could serve as a principle cytotoxic target of the
macrocyclic hexaoxazoles. Similar results have recently been ob-
served with macrocyclic pyridyl polyoxazoles that have been
developed as G-quadruplex ligands.19
HXDV
4a
4b
11.5
21.5
24.0
2.0
19.5
34.5
ND
4c
ND
a
D
Ttran reflects the change in transition temperature (Ttran) of the target nucleic
acid induced by the presence of the compound. Values of Ttran were determined
from the maxima or minima of first-derivative UV melting profiles exemplified by
those shown in Figure 2B and C. The uncertainty in the
denotes not determined.
DTtran values is 0.5 °C. ND
induces down-regulation of AurA protein levels and mitotic ar-
rest.10 These results raise the intriguing possibility that macrocy-
clic hexaoxazoles may bind and stabilize a quadruplex structure
in the mRNA sequence of AurA, thereby interfering with the trans-
lation of the mRNA.
HXDV, 4a, 4b, and 4c exert negligible impacts on the thermal
stabilities of either the ST DNA duplex (Fig. 2A and not shown for
4a–c) or the poly(rA)ꢁpoly(rU) RNA duplex (not shown). This obser-
vation is consistent with little or no duplex DNA or RNA binding on
the part of any of the four macrocyclic hexaoxazoles tested here.
We had observed previously a similar behavior for HXDV.5–8 In
marked contrast to their negligible impacts on the thermal stabil-
ities of the DNA and RNA host duplexes, HXDV, 4a, 4b, and 4c in-
crease the Ttran value of the hTel DNA quadruplex by 11.5, 21.5,
24.0, and 2.0 °C, respectively (Fig. 2B and Table 2).
Acknowledgment
These studies were supported in part by the Department of De-
fense Grant W81XWH06-1-0514.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.086.
These results are not only indicative of binding to hTel, but also
provide information with regard to the relative affinities of the
compounds for the host DNA quadruplex. In this connection, the
relative extents to which ligands stabilize a target nucleic acid
are typically correlated with the relative binding affinities of the li-
gands for the target.17,18 Our hTel UV melting results are therefore
consistent with the affinities of HXDV, 4a, 4b, and 4c for the target
DNA quadruplex following the hierarchy 4b > 4a > HXDV > 4c.
Thus, replacement of a valine linker in HXDV with an N,N-dim-
ethylaminoalkyl functionality appears to enhance affinity for the
hTel DNA quadruplex, but only when the alkyl group is an ethyl
(4a) or a propyl (4b). When the alkyl group is a butyl (4c), however,
affinity for the hTel DNA quadruplex appears to be decreased. Fig-
ure 2C shows the UV melting profiles of the AurA RNA sequence in
the absence and presence of HXDV and 4a.
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