Macrocyclic pyridyl polyoxazoles: Selective RNA and DNA G-quadruplex ligands as antitumor agents

Article abstract of DOI:10.1021/jm1000612

The synthesis of a series of 24-membered pyridine-containing polyoxazole macrocycles is described. Seventeen new macrocycles were evaluated for cytotoxic activity against RPMI 8402, KB-3, and KB-3 cell lines that overexpress the efflux transporters MDR1 (KBV-1) and BCRP (KBH5.0). Macrocycles in which the pyridyl-polyoxazole moiety is linked by a 1,3-bis(aminomethyl)phenyl group with a 5-(2-aminoethyl)- (18) or a 5-(2-dimethylaminoethyl)- substituent (19) displayed the greatest cytotoxic potency. These compounds exhibit exquisite selectivity for stabilizing G-quadruplex DNA with no stabilization of duplex DNA or RNA. Compound 19 stabilizes quadruplex mRNA that encodes the cell-cycle checkpoint protein kinase Aurora A to a greater extent than the quadruplex DNA of a human telomeric sequence. These data may suggest a role for G-quadruplex ligands interacting with mRNA being associated with the biological activity of macrocyclic polyoxazoles. Compound 19 has significant in vivo anticancer activity against a human breast cancer xenograft (MDA-MB-435) in athymic nude mice.

Full text of DOI:10.1021/jm1000612

3632 J. Med. Chem. 2010, 53, 3632–3644
DOI: 10.1021/jm1000612
Macrocyclic Pyridyl Polyoxazoles: Selective RNA and DNA G-Quadruplex Ligands as
Antitumor Agents
Suzanne G. Rzuczek,† Daniel S. Pilch,‡,§ Angela Liu,‡,§ Leroy Liu,‡,§ Edmond J. LaVoie,§,† and Joseph E. Rice*,†
†Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers—The State University of New Jersey, 160 Frelinghuysen Road,
Piscataway, New Jersey 08854-8020, ‡Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854-5635, and §The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901
Received January 15, 2010
The synthesis of a series of 24-membered pyridine-containing polyoxazole macrocycles is described.
Seventeen new macrocycles were evaluated for cytotoxic activity against RPMI 8402, KB-3, and KB-3
cell lines that overexpress the efflux transporters MDR1 (KBV-1) and BCRP (KBH5.0). Macrocycles
in which the pyridyl-polyoxazole moiety is linked by a 1,3-bis(aminomethyl)phenyl group with a 5-(2-
aminoethyl)- (18) or a 5-(2-dimethylaminoethyl)- substituent (19) displayed the greatest cytotoxic
potency. These compounds exhibit exquisite selectivity for stabilizing G-quadruplex DNA with no
stabilization of duplex DNA or RNA. Compound 19 stabilizes quadruplex mRNA that encodes
the cell-cycle checkpoint protein kinase Aurora A to a greater extent than the quadruplex DNA of a
human telomeric sequence. These data may suggest a role for G-quadruplex ligands interacting
with mRNA being associated with the biological activity of macrocyclic polyoxazoles. Compound 19
has significant in vivo anticancer activity against a human breast cancer xenograft (MDA-MB-435) in
athymic nude mice.
Introduction
Recently, the synthesis of a series of 24-membered macro-
cycles incorporating six oxazole rings (two trisoxazole arrays)
joined through an amino acid residue has been reported.7
The lead compound hexaoxazole-divaline (HXDVa) has two
valine residues connecting the tris-oxazole moieties and sta-
bilizes G-quadruplex DNA to an extraordinary degree with
no detectable stabilization of single stranded, duplex, or
triplex DNA (Figure 1).7c HXDV has moderate cytotoxicity
toward tumors cells (average IC50 ∼0.5 μM), which is com-
parable to that reported for telomestatin.8 HXDV induces
robust apoptosis in both telomerase positive and telomerase
negative cells within 16 h, causes M-phase cell cycle arrest, and
reduces the expression of the M-phase checkpoint regulator
Aurora A.9 These effects are not observed for the non-G-
quadruplex stabilizing 24-membered macrocycle TXTLeu
(tetraoxazole-tetraleucine, Figure 1). Formulation of HDXV
for in vivo assessment of its antitumor activity proved difficult
given its physicochemical properties. Efforts were focused on
developing analogues that retain the exquisite selectivity for
G-quadruplexes, exhibit enhanced anticancer activity in cell
cultures, and possess improved physicochemical properties to
permit facile formulation and in vivo administration. Toward
this goal, HXDV analogues with water-solubilizing amino-
alkyl groups having two to four-carbon chains replacing one
or both isopropyl groups were synthesized and evaluated for
their ability to selectively stabilize G-quadruplex DNA and
for theircytotoxicity toward tumor cells.10 Each analoguethat
was prepared in these studies required a lengthy (>20 step)
synthesis that is not readily amenable to the synthesis of
quantities of compound that might be required for extensive
in vivo evaluation against various human tumor xenografts. It
was reasoned that a new structure scaffold was needed that
Compounds that selectively stabilize the G-quadruplex
conformation of nucleic acids represent a unique and novel
class of anticancer agents.1 The discovery of the natural
product telomestatin, which stabilizes G-quadruplex DNA
and inhibits telomerase, prompted research aimed at the
design of similar G-quadruplex interactive agents.2 Telomes-
tatin (Figure 1), a 24-membered macrocycle composed of
seven oxazole ringsandone thiazoline, hasa unique molecular
architecturethatpermitsefficientπ-stacking interactions of its
unsaturated heterocyclic rings with p-orbitals on the guanines
of a nucleic acid when they are arranged as a planar G-quartet
within a quadruplex.3 Studies have suggested that one telo-
mestatin molecule stacks at each terminal G-quartet within a
quadruplex to give a 2:1 complex. Moreover, telomestatin
stabilizes G-quadruplex DNA in a selective manner, with
minimal interaction with duplex DNA.4 A large number of
polycyclic aromatic hydrocarbons and heterocycles have been
designed and synthesized as G-quadruplex stabilizers but
most are less selective than telomestatin.5 The design and
development of compounds that interact with high selectivity
with G-quadruplexes are critical to the elucidation of the
pharmacologicaleffectsof such agentsand for establishingthe
potential clinically utility of this class of anticancer agents.6
*To whom correspondence should be addressed. Phone: 732-445-
5382. Fax: 732-445-6312. E-mail: jrice@rci.rutgers.edu.
aAbbreviations: Boc, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl;
DAST, (diethylamino)sulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-
7-ene; DMF, N,N-dimethylformamide; EDC, N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole; HXDV, hexaoxazole
divaline; TFA, trifluoroacetic acid; TFAA, trifluoroacetic anhydride; TIPS,
triisopropylsilyl.
r
pubs.acs.org/jmc
Published on Web 04/01/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3633
Figure 1. Structures of telomestatin, HXDV, and TXTLeu.
Scheme 1a
a Reagents and conditions: (a) EDC, HOBt, 2,6-lutidine, DMF, 0 °C to rt; (b) HF-pyridine, rt; (c) DAST, CH2Cl2, -78 °C; (d) BrCCl3, DBU,
CH3CN, 0 °C to rt; (e) for RdH, LiOH, THF/H2O, Δ; for RdBr, LiBr, DBU, THF/H2O, Δ.
can be readily prepared on a multigram scale and easily
modified to provide a variety of macrocyclic analogues for
evaluating the effect of structure on selective G-quadruplex
stabilization and antitumor activity. The design and synthesis of
such a scaffold and its elaboration into a series of 24-membered
macrocycles is the subject of this investigation. Cytotoxicity
data is provided for each targeted macrocycle. On the basis
of these data, compound 19 was selected and evaluated for in
vivo efficacy against the human breast tumor xenograft MDA-
MB-435. In addition, the potential of several of these com-
pounds to selectively stabilize G-quadruplexes formed in DNA
and RNA was determined.
BrCCl3/DBU.12 The esters were then hydrolyzed to afford
scaffold 5a in an overall yield of 64%. A second scaffold 5b
was designed to incorporate a 4-bromo substituent on the
pyridine which was intended to provide a point of attachment
for a variety of functionality at that position on the completed
macrocycle. Compound 5b was prepared in 47% overall
yield from 4-bromopyridine-2,6-dicarboxylic acid13 (1b) by
an analogous route as 5a, except that hydrolysis of the ester
groups was achieved by treatment with LiBr and DBU to
avoid displacement of the bromine from the pyridine ring.14
The conversion of 5a,b into 24-membered macrocycles
required them to be condensed with a seven atom bifunctional
linker. Among the linkers initially employed for this purpose
were diethylenetriamine, 1,3-bis(aminomethyl)benzene, and
2,6-bis(aminomethyl)pyridine.15 A dilute solution (2 mM) of
5a or 5b in DMF was treated with EDC, HOBt, and 2,6-
lutidine followed by the linker and stirred at room tempera-
ture for 2 d (Scheme 2). The yields of macrocycles 6a, 6b, and 7
were 30%, 35%, and 22% respectively. Macrolactamization
using diethylenetriamine proved more challenging as exten-
sive polymerization occurred using the standard procedure.
Stirring 5a for 3 h with copper(II) triflate prior to treatment
with EDC, HOBt, and the diamine linker allowed for macro-
lactamization to occur to give macrocycle 8 in 15% yield
after 2 d. Presumably the copper acts as a template to hold the
open ends of 5a in a conformation more favorable for
macrocyclization.
Results and Discussion
Previous results from our laboratories suggested that a
somewhat planar or slightly puckered 24-membered macro-
cycle with a minimum of five heterocyclic rings is optimal for
efficient G-quadruplex stabilization.7 A scaffold having a
symmetrical array of five contiguous heterocyclic rings bear-
ing identical functionality at each end was envisioned to which
a variety of bifunctional linkers could be connected to com-
plete the macrocycle. For ease of synthesis a pyridine having a
bis(oxazole) moiety at the 2- and 6-positions appeared to be a
logical choice. Readily available pyridine-2,6-dicarboxylic
acid (1a) was condensed with oxazole 210a to give bis(amide)
3a in 80% yield (Scheme 1). The TIPS groups were removed
with HF-pyridine complex, and the resulting diol was treated
with DAST to effect cyclodehydration to the bis(oxazoline).11
Dehydrogenation to tetraoxazole 4a was accomplished with
The replacement of one or two oxazole rings by thiazoles
was also investigated (Scheme 3). To that end, treatment of 3a
with 1 equiv of Lawesson’s reagent16 in toluene at 80 °C gave

3634 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rzuczek et al.
monothioamide 9a in good yield. In the presence of excess
Lawesson’s reagent, bis(thioamide) 9b was formed. The con-
version of 9a,b into pyridyl tetraazole derivatives 10a,b in
overall yields of 56% and 79%, respectively was achieved
using an identical sequence as employed for 4a,b. The esters
were hydrolyzed with LiOH and macrocyclization of 10a with
1,3-bis(aminomethyl)benzene afforded 11a in 33% yield.
Surprisingly, similar treatment of 10b failed to give any of
the bis(thiazole) macrocycle 11b. Performing the macrocycli-
zation in a stepwise manner, however, provided a solution to
thisproblem. Thuscondensationof10b withthe commercially
available mono-Boc derivative of 1,3-bis(aminomethyl)-
benzene followed by treatment with TFA afford the mono-
amide in 68% for the two steps. Macrocyclization using EDC
then gave the desired macrocycle 11b in 43% yield.
The ability to prepare macrocycles having water-solubiliz-
ing groups attached to either the pyridine or phenyl ring was a
critical element to allow for the test compounds to be readily
formulated and administered in the assessment of their in vivo
antitumor activity. It was envisioned that a suitably posi-
tioned halide on either the pyridine ring, as in 6b, or on the
phenyl ring could serve as a handle for the attachment of
substituents via transition-metal catalyzed coupling reactions.
To that end, 1,3-bis(aminomethyl)-5-bromobenzene 12 was
prepared17 as an additional linker (Scheme 4). Macrocycliza-
tion of 12 with 5a and 5b afforded compounds 13a and 13b in
58 and 22% yield, respectively. Unfortunately, all attempts to
derivatize 6b, 13a, or 13b by Suzuki-Miyaura coupling with
potassium [2-(benzyloxycarbonylamino)ethyl]trifluoroborate18
led primarily to the debromo compound 6a. It is possible that
the intact macrocycle chelates the palladium catalyst and
prevents it from participating in the coupling reaction.
Scheme 2a
The water-solubilizing substituent could, however, be
attached to the phenyl linker prior to macrocyclization. This
required that the primary amino groups of 12 be protected as
tert-butyl carbamate (Boc) derivatives (Scheme 5). Suzuki-
Miyaura coupling of the resulting bromo compound with the
same trifluoroborate reagent in the presence of PdCl2-
(dppf) CH2Cl2 and Cs2CO3 afforded the desired product
3
Scheme 4a
a Reagents and conditions: (a) 5a or 5b (2 mM in DMF), EDC,
HOBT, 2,6-lutidine, 1,3-bis(aminomethyl)benzene or 2,6-bis(amino-
methyl)pyridine, rt, 2 d; (b) 5a (0.53 mM in DMF), Cu(OTf)2, 2 h, rt
then EDC, HOBt, 2,6-lutidine, diethylenetriamine, rt, 2 d.
a Reagents and conditions: (a) 5a or 5b (2 mM in DMF), EDC,
HOBT, 2,6-lutidine, rt, 2 d.
Scheme 3a
a Reagents and conditions: (a) Lawesson’s reagent (1 equiv for 9a, excess for 9b), toluene, 80 °C; (b) HF-pyridine, rt; (c) DAST, CH2Cl2, -78 °C;
(d) BrCCl3, DBU, CH2Cl2, 0 °C to rt; (e) LiOH, THF/H2O, Δ; (f) (2 mM in DMF), EDC, HOBT, 2,6-lutidine, 1,3-bis(aminomethyl)benzene rt, 2 d.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3635
14a in 79% yield. Removal of the Boc groups with trifluoro-
acetic acid (TFA) and macrocyclization with 5a and 5b
afforded compounds 15a and 15b in identical 42% yields.
Removal of the Cbz group from 15a, however, proved
troublesome under a variety of hydrogenolysis conditions,
affording the primary amine in very low yield. Therefore, 14a
was subjected to hydrogenolysis over Pd(OH)2/C to remove
the Cbz group in quantitative yield. Protection of the amine as
a trifluoroacetamide (TFAA, pyridine) was then followed by
removal of the Boc groups (TFA) and gave diamine 16.
Macrocyclization of 16 with 5a afforded compound 17 in
25% yield. The trifluoroacetamide was removed upon treat-
ment with K2CO3 in MeOH to give 18. A portion of 18 was
reductively methylated using aqueous formaldehyde and
sodium triacetoxyborohydride to give the N,N-dimethylamino
derivative 19.
Scheme 5a
The synthesis of a third scaffold having a basic side-chain
attached to the pyridine ring (Scheme 6) proved necessary due
to our inability to derivatize bromo compound 6b. Chelidonic
acid (4-hydroxypyridine-2,6-dicarboxylic acid) was coupled
with oxazole 2 to form bis(amide) 20. Treatment with N-(3-
bromopropyl)phthalimide and DBU afforded pyridyl ether
21. Removal of the TIPS protecting groups, cyclodehydration
with DAST, and dehydrogenation with BrCCl3/DBU gave
scaffold 22 after removal of the ester groups using LiCl in
refluxing DMF. This was subjected to macrocyclization using
1,3-bis(aminomethyl)benzene, 12, and 14a (after removal of
the Boc groups) to give macrolactams 23a-c. Treatment of
23a with hydrazine hydrate gave primary amine 23d.
Each macrocycle was evaluated for cytotoxic activity against
human lymphoblastoma RPMI 8402, human epidermoid car-
cinoma KB3-1, and KB3-1 cell lines that overexpress the efflux
transporters MDR1 (KBV-1) and BCRP (KBH5.0) (Table 1).
The parent macrocycle 6a, with a 1,3-bis(aminomethyl)phenyl
group linking the ends of scaffold 5a displays modest cytotoxic
potency against RPMI 8402 and KB3-1 (IC50 ∼1μM). Replace-
ment of that linker with either 2,6-bis(aminomethyl)pyridyl, 7a,
or diethylenetriamine, 8, results in a loss of activity. The
replacement of one oxazole ring of 6a with a thiazole (11a)
resulted in a 4-fold increase in activity against KB3-1, but had
no effect on RPMI 8402 cells. Substitution of a second oxazole
by thiazole (11b), however, resulted in a complete loss of
activity. The loss of activity for diethylenetriamine derivative
a Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, rt; (b) CbzNH-
CH2CH2BF3K, PdCl2(dppf) CH2Cl2, Cs2CO3, toluene/H2O, 80 °C;
3
(c) TFA, CH2Cl2, 0 °C; (d) 5a or 5b (2 mM in DMF), EDC, HOBT,
2,6-lutidine, rt, 2 d; (e) 1 atm H2, 20% Pd(OH)2/C, EtOH, rt; (f) TFAA,
pyridine, CH2Cl2, 0 °C; (g) K2CO3, MeOH, Δ; (h) HCHO (aq), NaBH-
(OAc)3, MeOH/CH2Cl2, rt.
Scheme 6a
a Reagents and conditions: (a) 2, EDC, HOBt, 2,6-lutidine, DMF, 0 °C to rt; (b) Br(CH2)3NPhth, DBU, DMF, 60 °C; (c) HF-pyridine, rt; (d) DAST,
CH2Cl2, -78 °C; (e) BrCCl3, DBU, CH2Cl2, 0 °C to rt; (f) LiCl, DMF, Δ; (g) 1,3-bis(aminomethyl)benzene or 12 or 14, (2 mM in DMF), EDC, HOBT,
2,6-lutidine, rt, 2 d; (h) H2NNH2 H2O, EtOH, Δ.
3

3636 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rzuczek et al.
Table 1. Cytotoxicity of Macrocyclic Pyridyl Polyoxazolesa
IC50 (μM)
Within this series, only 23a with an unsubstituted phenyl ring
exhibited moderate activity against KB3-1 cells (0.4 μM) but
was largely inactive (IC50 4.5 μM) against the RPMI 8402
cell line. Changing the nature of the terminal group on the
(3-aminopropyl)oxy to primary amine 23d did not enhance
cytotoxic potency.
HXDV and four selected macrocyclic pyridyl polyoxazoles
(6a, 11b, 18, and 19) were evaluated for their ability to
selectively bind and stabilize quadruplex versus duplex
DNA and RNA in the presence of physiological concentra-
tions of Kþ ions (150 mM). We used salmon testes (ST) DNA
compd RPMI 8402 KB3-1 wt KBV-1 þ MDR1 KBH5.0 þ BCRP
HXDV
6a
0.6
0.4
1.2
8.5
4.5
2.0
0.5
1.5
1.0
1.05
0.3
6b
7a
0.15
>5
>10
1.0
>10
0.3
>10
0.12
>5
>5
>5
8
4.0
0.26
>10
>10
>10
>10
>10
>10
>10
>10 b
>10 b
>10 b
>10
>10
>10
>10
>10
>10
>10
11a
11b
13a
13b
15a
15b
17
>10
0.06
>10
1.0
>10
>10
>10
>10 b
0.14 b
0.55 b
>10
and poly(rA) poly(rU) as representative models of duplex
3
0.12
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 Kþ solution is an intramolecular (3 þ 1)
G-quadruplex in which three strands are oriented in one
direction and the fourth strand is oriented in the opposite
direction.19 In an effort to explore ligand binding interactions
with quadruplex RNA, we used a putative quadruplex-
forming sequence (r[AG4CG2CUG2UCG2AGUG2C]) de-
rived from the 50-untranslated region of the mRNA that
encodes the cell-cycle checkpoint protein kinase Aurora A
(AurA).
1.0
0.6
0.17
0.09
0.18
4.5
0.07 b
0.03 b
0.04 b
0.4
18
19
23a
23b
23c
23d
1.0
>10
5.0
1.0
>10
3.0
>10
>10
>10
a Values represent the average of three experiments. b Values represent
the average of four experiments.
8 might be related to its less rigid structure as compared to 6a,
although the effect of including a strongly basic secondary
amine within the macrocycle cannot be discounted. A substan-
tial difference in lipophilicity exists between thiazole (log P =
0.77) and oxazole (log P = -0.59), which may help to explain
why substitution of a second oxazole by thiazole (compound
11b), results in a loss of activity relative to monothiazole 11a. At
the present time, however, we can offer no explanation for the
results obtained with dipyridyl derivative 7a.
Figure 2 shows the UV melting profiles (depicted in their
first derivative forms) of the four nucleic acids described
above in the absence and presence of HXDV and 19. The
ligand-induced changes, if any, in the transition tempera-
tures (Ttran) corresponding to the maxima or minima of
these first-derivative melting profiles are listed in Table 2, as
are the corresponding changes in Ttran values derived from
melting profiles (not shown) conducted in the presence of
6a, 11b, and 18. The presence of neither ligand alters the
Attachment of a single bromine at either the 5-position of the
benzene ring (13a) or the 4-position of the pyridine 6b results in
a 3-fold increase in cytotoxic potency against RPMI 8402 and
about a 10-fold increase in activity against KB3-1 as compared
to unsubstituted benzene derivative 6a. Simultaneous substitu-
tion of bromine on both the benzene and pyridine rings (13b),
however, results in a loss of all activity. This might again be an
effect of the increased lipophilicity of 13b as opposed to 6b and
13a. A similar, although not so dramatic, trend is observed for
the 5-[2-(benzyloxycarbonylamino)ethyl]-1,3-bis(aminomethyl)-
phenyl-linked macrocycles 15a and 15b. Compound 15a, which
is unsubstituted on the pyridine is 5-10 times more active than
15b, which has a bromine on the 4-position of the pyridine ring.
The Cbz-protected 2-aminoethyl derivative 15a is approximately
1 order of magnitude more potent than parent compound 6a. A
change in the amine protecting group to trifluoroacetamide 17
has little effect on activity. Both primary amine 18 and tertiary
amine 19 have potent activity (30-40 nM) against KB3-1 cells
and slightly weaker activity (90-180 nM) against RPMI 8402
cells. It is also noteworthy that while 18 and 19 are both
substrates for MDR1, as evidenced by their total lack of activity
against KBV-1, they retain similar cytotoxicity against KBH5.0
relative to KB3-1, cells indicating that they are not substrates for
the efflux transporter BCRP.
thermal stability of ST duplex DNA or poly(rA) poly(rU)
3
duplex RNA to any significant extent, with any observed
changes in Ttran being within the experimental uncertainty.
This observation is consistent with little or no duplex DNA
or RNA binding on the part of any of the five macrocyclic
polyoxazoles tested here. We previously observed a similar
behavior for HXDV.7,10
In marked contrast to their negligible impacts on the
thermal stabilities of the DNA and RNA target duplexes,
HXDV, 6a, 18, and 19 increase the Ttran value of the hTel
DNA quadruplex by 11.5, 7.5, 21.0, and 20.5 °C, respectively.
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 ligands for the target.20 Our hTel UV
melting results are therefore consistent with the affinities
of HXDV, 6a, 18, and 19 for the target DNA quadruplex
following the hierarchy 18 ≈ 19 > HXDV > 6a. It is perhaps
not surprising that 18 and 19 exhibit a greater apparent
affinity for the hTel quadruplex than HXDV or 6a, because,
unlike HXDV and 6a (which are nonionic), 18 and 19 both
containbasicaminefunctionalities that adoptpositivecharges
at the physiologically relevant pH of 7.5 used in the UV
melting studies. Note that, unlike HXDV, 6a, 18, and 19,
compound 11b exerts no impact on the thermal stability of the
hTel quadruplex. This observation coupled with the duplex
results described above suggests that 11b binds neither the
duplex nor the quadruplex nucleic acid form. The lack of
The effect of attachment of a large group (OCH2CH2-
CH2NPhth) on the pyridine ring at its 4-position was to
substantially lower activity. For example, comparison of
4-bromophenyl derivatives 23b and 13a reveals a 3-16 fold
loss of activity for the substituted pyridyl derivative 23b in
RPMI8402andKB3-1 cells. Similarly, for compoundshaving
a protected 2-aminoethyl substituent on the phenyl ring,
activity drops from 120 nM for 15a to >10 μM with the
attachment of the large substituent on the pyridine ring (23c).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3637
Figure 2. First derivatives of the UV melting profiles of ST duplex DNA (A), hTel quadruplex DNA (B), poly(rA) poly(rU) duplex RNA (C),
3
and AurA quadruplex RNA (D) in the absence (red) and presence of either HXDV (blue) or 19 (green). The UV melting profiles of the
quadruplexes were acquired at 295 nm, while those of the duplexes were acquired at 260 nm. In all experiments, the solution conditions were
10 mM potassium phosphate (pH 7.5) and sufficient KCl (132 mM) to bring the total Kþ concentration to 150 mM.
Table 2. Impact of HXDV and Various Macrocyclic Pyridyl Polyox-
azoles on the Thermal Stabilities of the Duplex and Quadruplex Forms
of DNA and RNA
It is of interest to compare the relative extents to which
HXDV, 6a, 11b, 18, and 19 bind and stabilize the quadruplex
nucleic acid form with their relative cytotoxic potencies.
Toward this end, a comparison of the ΔTtran data in Table 2
change in thermal stability, ΔTtrana (°C)
poly(rA)
poly(rU)
hTel
quadruplex
DNA
AurA
quadruplex
RNA
with the corresponding cytotoxicity (IC50) data in Table 1
reveals a good correlation between cytotoxic potency and
quadruplex stabilizing activity.
3
ST duplex
DNA
compd
duplex RNA
Compounds that stabilize G-quadruplex conformations of
nucleic acids are viewed as a potential novel class of anticancer
agents. Many such compounds have been reported, along
with their ability to stabilize G-quadruplex DNA in the test
tube.5 Cytotoxicity data has also been reported for several of
these G-quadruplex stabilizers.5d,f Such in vitro studies, how-
ever, provide little insight into the ability of G-quadruplex
stabilizers to penetrate into cancer cells and to stop the growth
and destroy cancer cells in vivo. Telomestatin is one of the few
compounds known to stabilize G-quadruplex DNA for which
in vivo anticancer activity in a human tumor xenograft has
been reported. The results showed that telomestatin does
indeed kill such tumor cells in a mouse model.21 In compari-
son with telomestatin, however, the compounds reported in
the present study are more selective for stabilizing G-quad-
ruplex DNA. The decision was made to synthesize the 2-(N,N-
dimethylamino)ethyl substituted analogue 19 on a larger scale
and to evaluate its antitumor activity in vivo against a human
breast cancer xenograft (MDA-MB-435) in athymic nude
mice. The results from the in vivo bioassay of 19 are presented
in Figure 3. An experimental group of seven tumor-bearing
mice was initially treated with 19 on day 10 and subsequently
treated three times a week on alternate days for three weeks at
dosing that escalated each week and averaged 32 mg/kg.
Despite increasing the dosing from 25 to 42 mg/kg, no signi-
ficant change in body weight was observed during the three
HXDV
6a
0
0
0
0
0
0
11.5
7.5
19.5
ND
ND
ND
37.0
0
11b
18
0
0
0.5
0.5
21.0
20.5
19
a ΔTtran 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 2. The uncertainty
in the ΔTtran values is (0.5 °C. ND denotes not determined.
quadruplex binding by 11b may be a consequence of the two
thiazole moieties in this compound.
The temperature-induced hypochromic transition at 295 nm
exhibited by the AurA RNA sequence in the absence of ligand
(see Figure 2D) indicates that this RNA oligomer does indeed
adopt a quadruplex structure in the presence of 150 mM Kþ.
Moreover, the presence of HXDV and 19 enhance the thermal
stability of AurA RNA quadruplex by 19.5 and 37.0 °C,
respectively. These ΔTtran values are significantly greater than
the corresponding values (11.5 °C for HXDV and 20.5 °C for
19) associated with the binding of these compounds to the hTel
DNA quadruplex. Thus, HXDV and 19 bind to the AurA
RNA quadruplex with an even greater affinity than the hTel
DNA quadruplex. Our collective UV melting studies indicate
that macrocyclic polyoxazoles bind the quadruplex nucleic
acid form with a high degree of specificity.

3638 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rzuczek et al.
Figure 3. The test compounds, irinotecan (2), 10 mM citrate ([), and 19 (9) were administered by ip injection to athymic nude mice with
human tumor xenografts established using MDA-MB-435 breast cancer cells. Mice were injected ip 3ꢀ weekly. Negative controls (7 mice) were
injected with 150 μL of 10 mM citrate. The positive control group (8 mice) received irinotecan by ip injection at a dose of 20 mg/kg, 3ꢀ weekly,
for all four weeks. Compound 19 was similarly administered to seven mice, 3ꢀ weekly, at a dose of 25 mg/kg starting at week 2 with increasing
doses of 32 and 42 mg/kg on weeks 3 and 4, respectively. Data are presented as the mean ( SE. The % T/C (average tumor volume of treated as
compared to control group) is 27.7% for 19 and 6.1% for irinotecan.
Table 3. Average Body Weights of Athymic Nude Mice Treated with
Citrate, 19, and Irinotecan
can also be incorporated on the pyridine ring of the scaffold to
allow for the rapid preparation of series of analogues. Of the
average body weight ( standard deviation (g)
17 new macrocycles reported herein, those employing a 1,3-
bis(aminomethyl)phenyl linker with a 5-(2-aminoethyl) 18 or a
5-(2-dimethylaminoethyl) substituent 19 displayed the greater
cytotoxic potency. These compounds exhibited exquisite selec-
tivity for stabilizing G-quadruplex DNA with no stabilization
of duplex DNA or RNA. Compound 19 stabilizes quadruplex
mRNA that encodes the cell-cycle checkpoint protein kinase
Aurora A. These data may suggest a role for G-quadruplex
ligands interacting with mRNA being associated with the
biological activity of macrocyclic polyoxazoles. The anticancer
activity of 19 was evaluated in vivo against a human breast
cancer xenograft (MDA-MB-435) in athymic nude mice.
Results from this bioassay indicate that 19 has significant in
vivo efficacy as an anticancer agent and administration of
doses of 19 increasing from 25 to 42 mg/kg did not result in
weight loss or other observable adverse effects in mice. The
synthetic methodology outlined will permit more in depth
studies on the structure-activity relationships associated with
the stabilization of various types of G-quadruplexes. The
increased accessibility of test compounds within this series of
G-quadruplex stabilizers relative to macrocyclic hexaoxazoles
will also allow for expanded studies on the pharmacological
properties of these highly selective G-quadruplex stabilizers.
day
citrate
compd 19
irinotecan
17
24
31
35
22.0 ( 1.8
22.5 ( 1.5
23.9 ( 1.0
24.6 ( 1.3b
19.7 ( 1.9
20.2 ( 2.3
20.9 ( 2.4
21.1 ( 1.9
19.5 ( 1.3
19.8 ( 1.4
18.4 ( 2.0a
20.7 ( 1.6
a One cage of four mice was found not to have a water bottle, which is
reflected the drop in average weight on day 31. b One mouse was
euthanized on day 31 because the tumor volume was 2266 mm3, which
exceeded protocol limitations.
weeks of treatment, Table 3. The negative control group, which
was treated with only citrate buffer, had an average tumor
volume >1350 mm3 after 35 days. In contrast, the tumor-
bearing mice treated with 19 had an average tumor volume of
<400 mm3 and had a %T/C value of 27.7%. The positive
control group was treated three times a week with irinotecan
starting on day 0 at a dose of 20 mg/kg and 35 days after initial
treatment had an average tumor volume of <100 mm3. These
results clearly demonstrate the in vivo efficacy of compound 19.
Conclusions
The results presented above describe an efficient synthesis of
pyridyl tetraoxazole platforms that can be performed on a
multigram scale. A key feature of these syntheses is the
symmetry of the central pyridine, which allows for the use of
only a single oxazole building block to rapidly arrive at a
difunctional molecule that is elaborated into a macrocycle in
only one step. A wide variety of functionalized diamine linkers
can be employed for the macrolactamization. Functionality
Experimental Section
Chemistry. All reactions were conducted under an atmo-
sphere of argon in oven-dried glassware unless otherwise noted.
THF was dried by distillation from sodium-benzophenone. Toluene,
CH2Cl2, 2,6-lutidine, Et3N, pyridine, DBU, and CH3CN were
freshly distilled from CaH2. Anhydrous DMF was obtained by

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3639
stirring overnight over anhydrous CuSO4, followed by distillation
under reduced pressure. All starting materials and reagents were
commercially available and were used as received with the exception
of 4-bromopyridine-2,6-dicarboxylic acid13 (1b), 1-bromo-3,5-bis-
(aminomethyl)benzene17 (12), and 2,6-bis(aminomethyl)pyridine,15
which were prepared as described previously. Flash chromato-
graphy was conducted using 230-400 mesh silica gel obtained
from Dynamic Adsorbents, Inc. Melting points were obtained
on a Thomas-Hoover apparatus and are uncorrected. Proton
(400 MHz) and carbon (125 MHz) NMR spectra were recorded
on a Bruker Avance III spectrometer in CDCl3 unless other-
wise noted. Chemical shifts are reported as δ units relative to
internal tetramethylsilane. High resolution mass spectra were
provided by the Washington University Mass Spectrometry
Resource, St. Louis, MO. Each substrate that was evaluated
for biological activity was >95% pure as determined by
HPLC. All HPLC analyses were performed using a Hewlett-
Packard model 1090 system with a YMC CombiScreen C18
column (50 mm ꢀ 4.6 mm). The solvent system was: solvent A
(H2O þ 0.5% TFA); solvent B (CH3CN þ 0.1% TFA).
Analyses were performed using a program of 10% solvent B
for 1 min, a linear gradient of 10-90% solvent B over 10 min,
90% solvent B for 3 min, and then a linear gradient back to
10% solvent B over 5 min with a flow rate of 1.0 mL/min.
Nucleic Acid Molecules. The hTel DNA 24mer (50-TTGGGT-
TAGGGTTAGGGTTAGGGA-30) was obtained in its HPLC-
purified form from Integrated DNA Technologies, Inc.
(Coralville, IA), while the AurA RNA 22mer (50-AGGGGC-
GGCUGGUCGGAGUGGC-30) was obtained in its PAGE-
purified and desalted form from Dharmacon, Inc. (Chicago, IL).
The concentrations of all AurA and hTel solutions were deter-
mined spectrophotometrically using the following extinction
coefficients at 260 nm and 85 °C (ε260-85 °C) [in units of (mol
General Method for the Synthesis of Oxazoles. Method B: 2,6-
Bis[4-[(4-methoxycarbonyl)oxazol-2-yl]oxazol-2-yl]pyridine (4a).
TIPS ether 3a (750 mg, 0.92 mmol) was dissolved in a mixture of
anhydrous THF (20 mL) and pyridine (2 mL) and HF-pyridine
complex (0.5 mL) was added. The reaction was stirred at room
temperature (drying tube) overnight and a white solid precipitated.
A saturated aqueous solution of NaHCO3 was added, whereupon
additional precipitate formed and was filtered. The solids were
washed with water, and the filtrate was then extracted with
CH2Cl2. The organic phase was separated, dried over Na2SO4,
and concentrated in vacuo to a white solid. This was combined with
the filtered solid to give the diol as a white solid; 463 mg (100% yield);
mp 218-220 °C. 1H NMR (DMSO-d6) δ9.58 (d, 2H, J=8),8.84(s,
2H), 8.24 (m, 3H), 5.29 (m, 2H), 4.04 (m, 4H), 3.79 (s, 6H). 13C
NMR (DMSO-d6) δ 163.3, 162.7, 160.9, 148.2, 145.4, 132.1, 125.1,
60.8, 51.6, 50.0. IR (Nujol) 3500, 3364, 3287, 1732 cm-1
.
The diol (463 mg, 0.92 mmol) was suspended in anhydrous
CH2Cl2 (30 mL) and placed under argon. After cooling to
-78 °C, DAST (0.3 mL, 2.3 mmol) was added and the reaction
was stirred for 4 h at that temperature. Solid potassium carbo-
nate (317 mg, 2.3 mmol) was added and the reaction mixture
warmed slightly and was poured into saturated NaHCO3 and
extracted with CH2Cl2. The organic extracts were dried over
Na2SO4 and concentrated under reduced pressure to give the
bis(oxazoline) as a white solid; 428 mg (99.5% yield); mp
1
214-217 °C. H NMR (CD3OD) δ 8.36 (s, 2H), 8.26 (d, 2H,
J = 8), 8.01 (t, 1H, J = 8), 5.67 (t, 2H, J = 9), 4.97 (d, 4H, J =
8), 4.28 (s, 6H). 13C NMR (CD3OD) δ 165.5, 163.1, 161.5, 146.0,
145.1, 138.0, 133.3, 126.9, 71.4, 63.7, 52.1.
The bis(oxazoline) (428 mg, 0.92 mmol) was suspended in
anhydrous CH3CN (40 mL) and placed under argon. After
cooling to 0 °C, the magnetically stirred solution was treated
dropwise with DBU (0.55 mL, 3.68 mmol) and then BrCCl3
(0.44 mL, 4.42 mmol). The reaction was allowed to warm to
room temperature overnight and a white precipitate formed.
The precipitate was filtered and washed with CH2Cl2 to give 4a
strand/L)-1 cm-1]: 237500 ( 4700 for hTel and 236200 ( 3300
3
for AurA. These extinction coefficients were determined by
enzymatic digestion and subsequent colorimetric phosphate
assay using previously established protocols.22 Salmon testes
(ST) duplex DNA, as well as the RNA polynucleotides, poly-
(rA) and poly(rU), were obtained from Sigma (St. Louis, MO)
and used without further purification. Polynucleotide solution
concentrations were determined using the following extinction
1
as a white solid; 341 mg (80% yield); mp >220 °C (dec). H
NMR δ 8.59 (s, 2H), 8.44 (d, 2H, J = 8), 8.37 (s, 2H), 8.08 (t, 1H,
J = 8), 3.98 (s, 6H).
2,6-Bis[4-[(4-methoxycarbonyl)oxazol-2-yl]oxazol-2-yl]-4-bromo-
pyridine (4b). Compound 4b was prepared from 3b using method B.
Thus, 3b (910 mg, 1.02 mmol) afforded 282 mg of 3b (71% overall
yield for the three steps) as a white solid; mp 280-282 °C (dec). 1H
NMR δ 8.63 (s, 2H), 8.60 (s, 2H), 8.37 (s, 2H), 3.83 (s, 6H).
General Method for Ester Hydrolysis to Form Pyridyl Tetra-
oxazole Scaffolds. Method C: 2,6-Bis[4-(4-carboxyoxazol-2-yl)-
oxazol-2-yl]pyridine (5a). Diester 4a (150 mg, 0.32 mmol) was
suspended in THF (50 mL) and H2O (15 mL) and treated with
LiOH (30 mg, 0.71 mmol). The mixture was heated to 80 °C
overnight and was then cooled to room temperature. THF was
removed by rotary evaporator, and the residue was treated with
5% HCl. A white precipitate formed and was filtered and washed
with water to give 5a as a white solid; 140 mg (100% yield); mp
coefficients in units of (mol nucleotide/L)-1 cm-1: ε258-25 °C
9800 for p(rA) and ε260-25 °C = 9350 for p(rU).
=
3
General Method for Amide Formation. Method A: 2,6-Bis-
[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-2-(triisopropylsilyloxy)-
ethyl]amino]carbonyl]pyridine (3a). A solution of 2,6-pyridine
dicarboxylic acid (418 mg, 2.5 mmol), 2 (1.71 g, 5 mmol), EDC
(1.92 g, 10 mmol), and HOBT (1.35 g, 10 mmol) in anhydrous
DMF (100 mL) was treated with 2,6-lutidine (2.9 mL, 25
mmol) at 0 °C under argon. The reaction mixture was allowed
to warm to room temperature overnight and was then con-
centrated under reduced pressure. The residue was taken up in
a mixture of ethyl acetate and water and the layers were
separated. The organic layer was washed sequentially with
5% HCl, water, and brine and then dried over Na2SO4. Upon
removal of the solvent and flash chromatography (15-50%
EtOAc/hexanes) 3a was obtained as a pale-yellow oil; 1.6 g
1
315-318 °C (dec). H NMR (DMSO-d6) δ 9.00 (s, 2H), 8.52
(s, 2H), 8.15 (m, 3H). IR (Nujol) 3418-2719 (br), 1727 cm-1
.
2,6-Bis[4-(4-carboxyoxazol-2-yl)oxazol-2-yl]-4-bromopyridine
(5b). Compound 4b (54 mg, 0.1 mmol) and LiBr (9 mg, 0.1 mmol)
were suspended in THF (10 mL) and water (0.5 mL), and the
mixture was then placed under argon. To this was added DBU (60
μL, 0.4 mmol), and the mixture was heated at reflux overnight.
After cooling to room temperature, solvents were removed under
reduced pressure and the residue was treated with 1N HCl to give a
white solid. This was filtered and washed with water and then
CH2Cl2 to give 5b as a white solid; 47 mg (100% yield); mp
205-206 °C (dec). 1H NMR (DMSO) δ 9.19 (s, 2H), 8.90 (s, 2H),
1
(80% yield). H NMR δ 8.54 (d, 2H, J = 8), 8.38 (d, 2H, J =
8), 8.24 (s, 2H), 8.04 (t, 1H, J = 7), 5.62 (dt, 2H, J = 6, 9), 4.26 (d,
4H, J = 7), 3.90 (s, 6H), 0.96 (m, 42H). 13C NMR δ 162.6, 162.4,
147.7, 143.2, 138.1, 132.6, 124.9, 122.4, 63.6, 51.2, 49.3, 16.9, 10.9.
2,6-Bis[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-2-(triisopropyl-
silyloxy)ethyl]amino]carbonyl]-4-bromopyridine (3b). Compound
3b was prepared from 2 (2.86 g, 8.36 mmol) according to the
procedure described for the synthesis of 3a (method A) using
1b (1.03 g mg, 4.18 mmol). Bis(amide) 3b was obtained as a
pale-orange oil; 3.47 g (93% yield). 1H NMR δ 8.50 (m, 4H),
8.24 (s, 2H), 5.59 (dt, 2H, J = 3, 6), 4.25 (d, 4H, J = 6), 3.85 (s,
8.48 (s, 2H). IR (thin film) 3390-2516 (br), 1727 cm-1
.
General Method for Macrocyclization. Method D: Pyridine
Tetraoxazole Macrocycle with a 1,3-Bis(aminomethyl)phenyl
Linker (6a). Diacid 5a (28 mg, 0.065 mmol), EDC (50 mg, 0.26
mmol), and HOBT (35 mg, 0.26 mmol) were dissolved in
13
6H), 0.90 (m, 42H). C NMR δ 162.1, 161.5, 156.2, 148.6,
143.3, 135.3, 132.6, 128.3, 63.4, 51.2, 49.4, 16.9, 11.0.

3640 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rzuczek et al.
anhydrous DMF (120 mL) at room temperature under argon.
The diamine linker, in this case 1,3-bis(aminomethyl)benzene
(8.5 μL 0.065 mmol) in DMF (0.5 mL) and 2,6-lutidine (45 μL,
0.39 mmol) were added and the reaction was stirred at room
temperature for 2 d. Solvent was removed under reduced
pressure, and the resulting residue was triturated with water.
A white precipitate formed and was filtered, collected, and
purified by flash chromatography eluting with 1-5% MeOH/
CH2Cl2. Macrocycle 6a was isolated as a white solid; 10 mg
(30%), mp 320-324 °C (dec). 1H NMR (CDCl3 þ CD3OD) δ
8.34 (d, 4H, J = 5), 8.11, (s, 3H), 7.58 (s, 1H), 7.33 (m, 3H), 4.62
(s, 4H). 13C NMR (CDCl3 þ CD3OD) δ 159.8, 159.3, 153.3,
144.1, 140.5, 138.5, 138.0, 136.8, 136.6, 130.7, 129.3, 128.5,
128.1, 121.9, 42.8. HRMS (ESI) m/z calcd for C27H18N7O6
(M þ H) 536.1368; found 536.1343.
2,6-Bis[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-2-(triisopropyl-
silyloxy)ethyl]amino]thiocarbonyl]-pyridine (9b). A solution of
3a (100 mg, 0.122 mmol) in toluene (10 mL) was treated with
Lawesson’s reagent (149 mg, 0.37 mmol) under argon and
heated at 80 °C for 4 h. The reaction mixture was then allowed
to cool to room temperature, and toluene was removed by
rotary evaporator. The residue was purified by flash chromato-
graphy eluting with 5-25% EtOAc/hexanes to afford 9b as a
yellow oil; 91 mg (88% yield). 1H NMR δ 10.26 (d, 2H, J = 8),
8.79 (d, 2H, J = 8), 8.29 (s, 2H), 7.97 (t, 1H, J = 8), 6.18 (dt, 2H,
J = 3, 6), 4.37 (m, 4H), 3.88 (s, 6H), 1.00 (m, 42H). 13 C NMR δ
190.9, 161.5, 160.3, 148.7, 143.5, 137.4, 132.6, 127.1, 62.6, 54.7,
51.2, 16.8, 10.9.
2-[4-[(4-Methoxycarbonyl)oxazol-2-yl]thiazol-2-yl]-6-[4-[(4-
methoxycarbonyl)oxazol-2-yl]oxazol-2-yl]pyridine (10a).Compound
10a was prepared from 9a (138 mg, 0.166 mmol) using method B.
The yield of 10a for the three-step procedure was 57 mg (72%);
4-Bromopyridine Tetraoxazole Macrocycle with a 1,3-Bis-
(aminomethyl)phenyl linker (6b). Using method D, 5b (43 mg,
0.1 mmol) was macrocyclized with 1,3-bis(aminomethyl)benzene
(13 μL, 0.1 mmol). The product, 6b was obtained as a white solid;
18 mg (35%); mp 211-213 °C. 1H NMR (CDCl3 þ CD3OD) δ
8.37 (s, 2H), 8.32 (m, 2H), 8.26 (s, 2H), 7.36 (s, 3H), 4.61 (s, 4H).
13C NMR (CDCl3 þ CD3OD) δ 160.2, 159.7, 154.2, 146.1, 141.5,
139.8, 137.7, 137.4, 132.0, 130.2, 129.7, 129.4, 126.1, 108.0, 43.9.
HRMS (ESI) m/z calcd for C27H17BrN7O6 (M þ H) 614.0424;
found 614.0420.
1
cream-colored solid; mp 305-310 °C (dec). H NMR (CDCl3 þ
CD3OD) δ 8.62 (s, 1H), 8.49 (d, 1H, J = 7), 8.35 (m, 4H), 8.06
(m, 1H), 3.98 (s, 6H).
2,6-Bis[4-[(4-methoxycarbonyl)oxazol-2-yl]thiazol-2-yl]pyridine
(10b). Compound 10b was prepared from 9b (91 mg, 0.11 mmol)
using method B. The yield of 10b for the three-step procedure was
1
28 mg (52%); brown solid; mp 330-333 °C (dec). H NMR
(DMSO-d6) δ 9.10 (s, 2H), 8.80 (d, 2H, J = 8), 8.72 (s, 2H) 8.33 (t,
1H, J = 8), 3.93 (s, 6H).
Pyridine TetraoxazoleMacrocyclewitha 2,6-Bis(aminomethyl)-
pyridyl Linker (7a). Using method D, 5a (40 mg, 0.092 mmol)
was macrocyclized with 2,6-bis(aminomethyl)pyridine15 (13 mg,
0.092 mmol). The product, 7a was obtained as a white solid; 11 mg
(22%); mp 330 °C (dec). 1H NMR (CDCl3 þ CD3OD) δ 8.33 (s,
4H), 8.09 (s, 3H), 7.72 (t, 1H, J = 8), 7.40 (d, 2H, J = 8), 4.76 (s,
4H). 13C NMR (CDCl3 þ CD3OD) δ 159.8, 159.5, 144.3, 140.4,
138.3, 137.8, 137.4, 136.4, 130.8, 122.5, 121.7, 71.2.HRMS (ESI)
m/z calcd for C26H17N8O6 (M þ H) 537.1271; found 537.1252.
General Method for Macrocyclization. Method E: Pyridine
Tetraoxazole Macrocycle with a Bis(2-aminoethyl)amine Linker
(8). A solution of 5a (23 mg, 0.053 mmol) and copper(II) triflate
(19 mg, 0.053 mmol) in anhydrous DMF (100 mL) was stirred
under argon at room temperature for 3 h. At that point, EDC
(41 mg, 0.21 mmol) and HOBt (29 mg, 0.21 mmol) were added
and the color of the solution immediately became bright yellow.
The solution was cooled to 0 °C and 2,6-lutidine (37 μL, 0.32
mmol) was added, followed by a solution of diethylenetriamine
(6 μL, 0.053 mmol) in 0.5 mL of DMF, which was added by
syringe pump over 30 min. The reaction was stirred at room
temperature, and the color gradually changed to pale blue. After
2 d, solvent was removed under reduced pressure and the residue
was triturated with water to give a white precipitate that was
filtered and subjected to flash chromatography eluting with
1-5% MeOH/CH2Cl2 to give 8 as a white solid; 4 mg (15%
yield); mp 276 °C (dec). 1H NMR (CDCl3 þ CD3OD) δ 8.47 (s,
2H), 8.37 (s, 2H), 8.19 (s, 3H), 3.98 (m, 4H) 3.35 (m, 4H). HRMS
(ESI) m/z calcd for C23H19N8O6 (M þ H) 503.1428; found
503.1413.
2-[[[1-(4-(Methoxycarbonyl)oxazol-2-yl)-2-(triisopropylsilyloxy)-
ethyl]amino]thiocarbonyl]-6-[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-
2-(triisopropylsilyloxy)ethyl]amino]carbonyl]pyridine (9a). A solu-
tion of 3a (100 mg, 0.122 mmol) in toluene (10 mL) was treated
with Lawesson’s reagent (50 mg, 0.12 mmol) under argon and
heated at 80 °C for 4 h. The reaction mixture was then allowed to
cool to room temperature, and toluene was removed by rotary
evaporator. The residue was purified by flash chromatography
eluting with 5-25% EtOAc/hexanes to afford 9a as a yellow oil;
54 mg (53% yield). 1H NMR δ 10.46 (d, 1H, J = 8), 8.82, (dd, 1H,
J = 1, 9), 8.62 (d, 1H, J = 8), 8.36 (dd, 1H, J = 1, 8), 8.31 (s, 1H),
8.23 (s, 1H), 8.00 (t, 1H, J=6), 6.19(dt, 1H, J= 3, 5), 5.61 (dt, 1H,
J = 2, 6), 4.38 (d, 2H, J = 5), 4.26 (d, 2H, 6), 3.88 (s, 6H), 0.95 (m,
42H). 13 C NMR δ 190.7, 170.3, 162.7, 162.6, 161.6, 160.4, 146.5,
143.5, 143.2, 137.7, 133.0, 127.5, 124.6, 62.6, 54.7, 51.2, 49.4.
Pyridine Mono(thiazole) Tris(oxazole) Macrocycle with a 1,3-
Bis(aminomethyl)phenyl Linker (11a). Diester 10a (57 mg, 0.12
mmol) was hydrolyzed to the corresponding diacid in 78%
yield using method C. Macrocyclization of a portion of the
diacid (23 mg, 0.051 mmol) with 1,3-bis(aminomethyl)benzene
(7 μL, 0.051 mmol) using method E afforded 11a as white solid;
9 mg (33% yield); mp 245 °C. 1H NMR δ 8.31 (s, 1H), 8.28 (s,
1H), 8.26 (s, 1H), 8.00 (m, 4H), 7.37 (m, 4H), 4.62, (m, 4H). 13
C
NMR δ 140.0, 139.7, 138.4, 137.9, 136.7, 128.8, 128.3, 127.8,
127.4 123.1, 121.9, 120.6, 113.4, 42.9, 42.3. HRMS (ESI) m/z
calcd for C27H18N7O5S (M þ H) 552.1085; found 552.1086.
Pyridine Bis(thiazole) Bis(oxazole) Macrocycle with a 1,3-
Bis(aminomethyl)phenyl Linker (11b). Diester 10b (52 mg, 0.11
mmol) was hydrolyzed to the corresponding diacid in quantita-
tive yield using method C. A portion of the diacid (21 mg, 0.045
mmol) was dissolved in dry DMF (30 mL) and treated with EDC
(17 mg, 0.09 mmol) and HOBt (12 mg, 0.09 mmol) and cooled to
0 °C under argon. To this was added 2,6-lutidine (26 μL, 0.23
mmol) and 1-(N-Boc-aminomethyl)-3-(aminomethyl)benzene
(11 μL, 0.045 mmol), and the solution was allowed to warm to
room temperature overnight. Solvents were removed under
reduced pressure, and the residue was extracted with EtOAc.
The organic solution was washed with brine and dried over
Na2SO4, filtered, and evaporated to give 34 mg of crude
product. This was dissolved in CH2Cl2 (3 mL), cooled to 0 °C,
and treated with TFA (3 mL). After stirring at 0 °C for 2 h
solvents were removed under reduced pressure to give the free
amine as its TFA salt (25 mg, 68% yield). Macrolactamization
using method D gave product 11b as white solid; 6 mg (43%
1
yield); mp 274-275 °C (dec). H NMR (CDCl3 þ CD3OD) δ
8.38 (m, 4H), 8.42 (s, 2H), 7.97 (m, 2H), 7.30 (m, 3H), 4.63 (d,
4H, J = 6). 13C NMR (CDCl3 þ CD3OD) δ 140.7, 137.4, 136.0,
128.4, 126.6, 126.5, 123.2, 120.5, 42.6. HRMS (ESI) m/z calcd
for C27H18N7O4S2 (M þ H) 568.0863; found 568.0864.
Pyridine Tetraoxazole Macrocyclewitha 1,3-Bis(aminomethyl)-
5-bromophenyl Linker (13a). Using method D, 5a (43 mg, 0.1
mmol) was macrocyclized with 1-bromo-3,5-bis(aminomethyl)-
benzene dihydrochloride 1217 (21 mg, 0.073 mmol). The product,
13a was obtained as a white solid; 26 mg (58%); mp 248-250 °C.
1H NMR δ 8.28 (s, 2H), 8.25(s, 2H), 8.08 (m, 3H), 7.51 (m, 3H),
7.29 (m, 2H), 4.56 (d, 4H, J = 5). 13C NMR δ 159.9, 159.3, 153.0,
144.3, 140.1, 138.5, 138.0, 136.7, 136.6, 130.7, 128.3, 126.0,
122.5, 42.9. HRMS (ESI) m/z calcd for C27H17BrN7O6 (M þ H)
614.0418; found 614.0418.

Article
4-Bromopyridine Tetraoxazole Macrocycle with a 1,3-Bis-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3641
139.4, 138.1, 137.5, 136.7, 134.9, 132.1, 132.0, 130.0, 129.9, 128.5,
128.4, 128.3, 127.9, 127.7, 125.9, 125.9, 121.1, 108.2, 107.8, 66.5,
43.8, 42.2, 35.9. HRMS (ESI) m/z calcd for C37H28BrN8O8 (M þ
H) 791.1213; found 791.1199.
N-(3,5-Bis(aminomethyl)phenethyl-2,2,2-trifluoroacetamide (16).
A solution of 14a (62 mg, 0.13 mmol) in EtOH (10 mL) was treated
with 20% Pd(OH)2/C (10 mg) and stirred under 1 atm H2 (balloon)
for 21 h. The catalyst was filtered and washed with EtOH to afford
the primary phenethylamine as a colorless oil; 50 mg (100% yield).
1H NMR δ 7.02 (m, 3H), 4.94 (s, 2H), 4.27 (d, 4H, J = 8), 2.94 (m,
2H), 2.71 (m, 2H), 1.47 (s, 18H). 13C NMR δ 155.9, 140.7, 139.6,
126.9, 124.3, 79.5, 44.5, 43.4, 39.9, 28.4.
A portion of the amine (37 mg, 0.097 mmol) was dissolved in a
solution of pyridine (3 mL) and trifluoroacetic anhydride (2 mL)
and stirred at room temperature under a drying tube for 19 h.
The reaction mixture was diluted with CH2Cl2 and washed with
5% HCl. The aqueous layer was back-extracted with CH2Cl2
(3 ꢀ 20 mL), and the combined organic layers were washed with
5% HCl and then dried over Na2SO4. After concentration, flash
chromatography (10-40% EtOAc/hexanes) afforded the tri-
fluoroacetamide as a pale-yellow oil; 42 mg (91% yield). 1H
NMR δ 7.06 (s, 1H), 6.98 (s, 2H), 6.82 (s, 1H), 4.96 (s, 2H), 4.25
(d, 4H, J = 8), 3.55 (m, 2H), 2.84 (m, 2H), 1.46 (s, 18H). 13C
NMR δ 157.8, 157.5, 157.1, 156.7, 156.0, 140.1, 138.6, 126.7,
124.9, 117.3, 114.4, 79.7, 44.4, 40.9, 34.8, 28.4.
(aminomethyl)-5-bromophenyl Linker (13b). Using method D,
5b (17 mg, 0.033 mmol) was macrocyclized with 12 (10 mg, 0.033
mmol). The product, 13b was obtained as a white solid; 5 mg
1
(22%); mp 250 °C (dec). H NMR (CDCl3 þ CD3OD) δ 8.28
(s, 2H), 8.22 (s, 2H), 8.15 (s, 2H), 7.39 (s, 3H), 4.46 (s, 4H). 13C
NMR (CDCl3 þ CD3OD) δ 160.3, 159.6, 145.7, 141.6, 139.8,
139.7, 131.7, 131.6, 128.4, 125.9, 122.7, 43.0. HRMS (ESI) m/z
calcd for C27H16Br2N7O6 (M þ H) 691.9529; found 691.9546.
5-[2-(Benzyloxycarbonylamino)ethyl]-1,3-bis[(tert-butyloxy-
carbonylamino)methyl]benzene (14a). A solution of 12 (2.04 g,
9.5 mmol), di-tert-butyl dicarbonate (4.35 g, 19.9 mmol), and
triethylamine (2.9 mL, 20.8 mmol) in anhydrous CH2Cl2 (25 mL)
was stirred overnight under argon. The reaction mixture was
poured into 5% HCl and extracted with CH2Cl2. The organic
layer was dried over Na2SO4, filtered, and concentrated. Flash
chromatography eluting with 10-40% EtOAc/hexanes gave the
di-Boc derivative as a white solid; 2.51 g (63% yield). 1H NMR δ
7.32 (s, 2H), 7.11 (s, 1H), 4.86 (s, 2H), 4.28 (d, 4H, J = 4),
1.46 (18H). 13C NMR δ 154.8, 141.6, 129.2, 124.9, 122.9, 79.9,
44.0, 28.4.
A portion of the di-Boc derivative (209 mg, 0.5 mmol),
potassium 2-(benzyloxycarbonylamino)ethyltrifluoroborate18
(186 mg, 0.76 mmol), Cs2CO3 (429 mg, 1.5 mmol), and PdCl2-
(dppf) CH2Cl2 (21 mg, 0.025 mmol) were dissolved in toluene
3
(15 mL) and water (5 mL) was added. The flask was purged with
argon and heated to 80 °C for 17.5 h. After cooling to room
temperature, the mixture was poured into saturated NH4Cl and
extracted with CH2Cl2. The organic layer was dried over
Na2SO4, concentrated, and purified by flash chromatography
eluting with 10-50% EtOAc/hexanes. The coupled product 14a
was obtained as a colorless oil; 205 mg (79% yield). 1H NMR δ
7.32 (m, 5H), 7.04 (s, 1H), 6.98 (s, 2H), 5.09 (s, 2H), 4.82 (m, 3H),
The Boc groups were removed by dissolving the trifluoro-
acetamide (63 mg, 0.13 mmol) in dry CH2Cl2 (2 mL), cooling to
0 °C under argon, and adding TFA (1 mL). After 1.5 h solvent
was removed under reduced pressure to give 16 as a pale-yellow
oil; 65 mg (100% yield). 1H NMR δ 7.34 (s, 1H), 7.31 (s, 2H),
5.92 (s, 1H), 4.00 (s, 4H), 3.54 (m, 2H), 2.88 (m, 2H). 13C NMR δ
139.9, 137.3, 128.5, 126.0, 47.8, 43.6, 32.8.
Pyridine Tetraoxazole Macrocycle with a 5-[2-(Trifluoro-
acetamido)ethyl]-1,3-bis(aminomethyl)phenyl Linker (17). Using
method D, 5a (44 mg, 0.1 mmol) was macrocyclized with 16
(51 mg, 0.1 mmol). The product, 17, was obtained as a white
solid; 21 mg (31% yield); mp 290 °C (dec). 1H NMR (CDCl3 þ
CD3OD) δ 8.38 (s, 2H), 8.37 (s, 2H), 8.12 (s, 2H) 7.60 (s, 1H),
7.47 (s, 1H), 7.19 (s, 2H), 4.60 (s, 4H), 4.07 (m, 2H), 3.40 (m, 2H).
13C NMR (CDCl3 þ CD3OD) δ 160.9, 154.1, 145.0, 141.6,
140.0, 139.5, 138.2, 137.6, 131.6, 129.7, 128.6, 122.9, 43.7, 38.8,
34.1. HRMS (ESI) m/z calcd for C31H22F3N8O7 (M þ H)
675.1564; found 675.1546.
Pyridine Tetraoxazole Macrocycle with a 5-(2-Aminoethyl)-
1,3-bis(aminomethyl)phenyl Linker (18). A suspension of 17
(15 mg, 0.02 mmol) and K2CO3 (17 mg, 0.12 mmol) in MeOH
(10 mL) was heated at reflux for 4.75 h. After cooling to room
temperature, solvent was removed under reduced pressure and
the residue was partitioned between water and CH2Cl2. The
organic layer was separated and dried over Na2SO4, filtered, and
evaporated to give 18 as a white solid; 10.5 mg (81% yield); mp
277-280 °C (dec). 1H NMR (CDCl3 þ CD3OD) δ 8.42 (s, 2H),
8.40 (s, 2H), 8.13 (s, 3H), 7.45 (s, 1H), 7.19 (s, 2H), 4.60 (s, 4H),
2.92 (m, 2H), 2.75 (m, 2H). 13C NMR (CDCl3 þ CD3OD) δ
161.0, 160.5, 154.4, 145.1, 141.7, 140.9, 139.7, 139.2, 138.1,
137.6, 131.7, 129.5, 129.2, 128.9, 123.0, 43.8, 43.0, 39.4. HRMS
(ESI) m/z calcd for C29H23N8O6 (M þ H) 579.1741; found
579.1723.
4.26 (d, 4H, J = 4), 3.42 (m, 2H), 2.78 (m, 2H), 1.46 (s, 18H). 13
C
NMR δ 156.3, 155.9, 139.8, 139.5, 136.6, 128.5, 128.1, 126.8,
124.6, 79.6, 66.7, 44.5, 42.1, 36.0, 28.4.
5-[2-(Benzyloxycarbonylamino)ethyl]-1,3-bis(aminomethyl)-
benzene (14b). A solution of 14a (205 mg, 0.4 mmol) in CH2Cl2
(1 mL) and anisole (1 mL) and cooled to 0 °C. TFA (1 mL) was
added and the solution was stirred at 0 °C for 3.75 h. The
solution was evaporated under reduced pressure, and residual
anisole was removed azeotropically with benzene. The pro-
duct was dissolved in MeOH and stirred for several minutes
with IRA-400 resin. The resin was filtered and washed several
times with MeOH. Upon evaporation of the solvent, diamine
1
14b was obtained as a white solid; 125 mg (100% yield). H
NMR δ 7.34 (m, 5H), 7.13 (s, 1H), 7.01 (s, 2H), 5.10 (s, 2H),
4.80 (s, 1H), 3.84 (s, 4H), 3.46 (m, 2H), 2.81 (m, 2H), 1.48 (br s,
4H). 13C NMR δ 156.3, 144.0, 136.6, 128.5, 128.2, 126.0,
124.1, 66.7, 46.4, 42.2, 36.1.
Pyridine Tetraoxazole Macrocycle with a 5-[2-(Benzyloxy-
carbonylamino)ethyl]-1,3-bis(aminomethyl)phenyl linker (15a).
Using method D, 5a (50 mg, 0.11 mmol) was macrocyclized
with 14b (36 mg, 0.11 mmol). The product, 15a was obtained as a
white solid; 33 mg (42%); mp 196-198 °C. 1H NMR (CDCl3 þ
CD3OD) δ 8.41 (d, 2H, J = 4), 8.34 (s, 2H), 8.32 (s, 2H), 8.10 (s,
3H), 7.42 (s, 1H), 7.30 (m, 5H), 7.18 (s, 2H), 5.37 (s, 1H), 5.04 (s,
2H), 4.59 (d, 4H, J = 4), 3.41 (m, 4H). 13C NMR (CDCl3 þ
CD3OD) δ 160.9, 160.4, 156.8, 156.7, 154.3, 145.1, 141.5, 140.4,
139.5, 139.0, 138.1, 137.6, 136.8, 131.7, 129.6, 128.5, 128.2,
128.0, 127.9, 122.9, 66.6, 42.2, 35.9. HRMS (ESI) m/z calcd
for C37H29N8O8 (M þ H) 713.2108; found 713.2091.
Pyridine Tetraoxazole Macrocycle with
a 5-[2-(N,N-
Dimethylamino)ethyl]-1,3-bis(aminomethyl)phenyl Linker (19).
Primary amine 18 (6 mg, 0.01 mmol) was dissolved in 1:4
MeOH/CH2Cl2 (5 mL) and placed under argon. A solution of
37% aqueous formaldehyde (0.25 mL) was added, and the
reaction mixture was allowed to stir at room temperature for
10 min. At that point, sodium triacetoxyborohydride (15 mg,
0.07 mmol) was added and stirring continued for 16 h. The
reaction was not complete by TLC, and additional formaldehyde
solution (0.25 mL) and NaBH(OAc)3 (15 mg) was added. After
6 h, TLC showed the reaction to be complete and it was poured into
saturated NaHCO3 and extracted with CH2Cl2. After concentrating,
4-Bromopyridine Tetraoxazole Macrocycle with a 5-[2-(Benzyl-
oxycarbonylamino)ethyl]-1,3-bis(aminomethyl)phenyl Linker (15b).
Using method D, 5b (20 mg, 0.039 mmol) was macrocyclized with
14b (12 mg, 0.039 mmol). The product, 15b, was obtained as a
white solid; 13 mg (42%); mp 204-205 °C. 1H NMR δ 8.24 (m,
5H), 7.65 (s, 2H), 7.56 (m, 2H), 7.3 (m, 5H), 7.17 (s, 2H), 5.06 (s,
2H), 4.91 (s, 1H), 4.56 (s, 2H), 3.44 (m, 2H), 2.80 (m, 2H). 13C
NMR δ 159.6, 159.4, 159.4, 156.3, 154.0, 153.9, 147.6, 146.2, 140.8,

3642 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rzuczek et al.
flash chromatography (1-15% MeOH/CH2Cl2 afforded 19 as a
white solid; 6 mg (100% yield); 295 °C (dec). 1H NMR (CDCl3 þ
CD3OD) δ8.34(s,2H),8.33(s,2H),8.10(s,3H),7.41(s,1H),7.19(s,
2H), 4.59 (s, 4H), 2.79 (m, 2H), 2.61 (m, 2H), 2.30 (s, 6H). 13CNMR
(CDCl3 þ CD3OD) δ 160.8, 160.3, 154.2, 145.1, 141.5, 139.4, 139.0,
137.9, 131.7, 129.4, 128.1, 122.8, 45.0, 43.8. HRMS (ESI) m/z calcd
for C31H27N8O6 (M þ H) 607.2054; found 607.2029.
4-[(3-Phthalimidopropyl)oxy]pyridine Tetraoxazole Macrocycle
with a 5-[2-(Benzyloxycarbonylamino)ethyl-1,3-bis(aminomethyl)-
5-bromophenyl Linker (23c). Using method D, 22 (21 mg, 0.033
mmol) was macrocyclized with 14b (15 mg, 0.048 mmol). The
product, 23c, was obtained as a white solid; 11 mg (40%); mp
167-170 °C. 1H NMR δ 8.24 (s, 2H), 8.23 (s, 2H), 7.85 (dd, 2H,
J = 3,5), 7.72 (dd, 2H, J = 3,5), 7.56 (m, 2H), 7.47 (s, 2H), 7.27
(m, 5H), 7.17 (s, 3H), 5.04 (s, 2H), 4.95 (m, 1H), 4.56 (d, 4H, J =
4), 4.27 (t, 2H, J = 6), 3.97 (t, 2H, J = 6), 3.45 (m, 2H), 2.81 (m,
2H), 2.32 (dt, 2H, J = 6, 12). 13C NMR δ 168.4, 166.8, 160.5,
159,7, 156.3, 154.2, 146.7, 140.6, 138.8, 138.1, 137.4, 134.1, 132.1,
131.8, 130.0, 129.1, 128.4, 128.3, 128.2, 127.9, 125.3, 123.4, 109.4,
66.7, 43.8, 35.0, 28.0. HRMS (ESI) m/z calcd for C48H38N9O11
(M þ H) 916.2691; found 916.2669.
2,6-Bis[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-2-(triisopropyl-
silyloxy)ethyl]amino]carbonyl]-4-hydroxypyridine (20). Using
method A, chelidonic acid (230 mg, 1.26 mmol) was condensed
with 2 (860 mg, 2.51 mmol). The product, 20 was obtained as a
yellow oil; 900 mg (86% yield). 1H NMR δ 10.62 (s, 1H), 8.70
(d, 2H, J = 9), 8.25 (s, 2H), 7.82 (s, 2H), 5.57 (m, 2H), 4.24 (d,
13
4H, J = 6), 3.90 (s, 6H), 0.96 (m, 42H). C NMR δ 165.9,
163.0, 162.0, 160.4, 149.2, 143.2, 132.4, 112.6, 63.5, 51.2, 49.2,
16.8, 10.9.
4-[(3-Aminopropyl)oxy]pyridine Tetraoxazole Macrocycle with
a 1,3-Bis(aminomethyl)phenyl Linker (23d). Hydrazine mono-
hydrate (0.5 mL, 10.3 mmol) was added to a solution of 23a
(24 mg, 0.033 mmol) in EtOH (10 mL). The solution was heated
at reflux for 3 h, cooled to room temperature, and concentrated
under reduced pressure. The residue was dissolved in CH2Cl2,
washed with 10% NaOH, and dried over Na2SO4. Evaporation
of the solvent afforded 23d as a white solid; 20 mg (100% yield);
mp 237-239 °C (dec). 1H NMR (CDCl3 þ CD3OD) δ 8.32 (m,
4H), 7.57 (s, 3H), 7.36 (m, 3H), 4.63 (s, 4H), 4.27 (m, 2H), 2.94 (t,
2H J = 6), 2.04 (dt, 2H, J = 6.12). 13C NMR (CDCl3 þ CD3OD)
δ 167.4, 160.9, 160.3, 154.3, 146.4, 141.4, 139.4, 137.8, 137.5,
131.6, 130.2, 129.5, 129.2, 109.6, 66.7, 43.8, 38.4, 32.0. HRMS
(ESI) m/z calcd for C30H25N8O7 (M þ H) 609.1846; found
609.1825.
Cytotoxicity Assays. Cytotoxicity was determined using the
MTT-microtiter plate tetrazolinium assay (MTA). The human
lymphoblast RPMI 8402 cell line was provided by Dr. Toshiwo
Andoh (Aichi Cancer Center Research Institute, Nagoya,
Japan).23 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).24 The KBH5.0 cell line 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 inactivated fetal bovine serum,
L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin
(0.1 mg/mL). For determination of IC50 values, 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.
2,6-Bis[[[1-(4-(methoxycarbonyl)oxazol-2-yl)-2-(triisopropyl-
silyloxy)ethyl]amino]carbonyl]-4-[(3-phthalimidopropyl)oxy]-
pyridine (21). A solution of 20 (642 mg, 0.77 mmol) and N-(3-
bromopropyl)phthalimide (227 mg, 0.85 mmol) in anhydrous
DMF (10 mL) was treated with DBU (0.13 mL, 0.85 mmol) and
warmed to 60 °C for 6 h. After cooling to room temperature, the
reaction mixture was poured into water and extracted with
EtOAc. The organic phase was washed with 5% HCl and then
brine and then dried over Na2SO4. Flash chromatography
(1-4% MeOH/CH2Cl2) afforded 21 as a colorless oil; 727 mg
(92% yield). 1H NMR δ 8.61 (d, 2H, J = 9), 8.24 (s, 2H), 7.84 (m,
2H), 7.73 (m, 4H), 5.59 (m, 2H), 4.23 (m, 6H), 3.93 (m, 8H), 2.25
(m, 2H), 1.03 (m, 42H). 13 C NMR δ 167.4, 166.4, 162.5, 162.3,
160.5, 149.5, 143.3, 133.2, 132.6, 131.2, 122.5, 111.1, 65.7, 63.5,
51.2, 49.3, 34.2, 27.0, 16.9, 10.9.
2,6-Bis[4-(4-carboxyoxazol-2-yl)oxazol-2-yl]-4-[(3-phthalimido-
propyl)oxy]pyridine (22). Using method B, 21 (1.05 g, 1.03 mmol)
was deprotected, cyclodehydrated, and converted into the pyridyl
tetraoxazole diester, a white solid; 470 mg (68% yield for the three
steps).; 1H NMR (DMSO-d6) δ 9.16 (s, 2H), 9.02 (s, 2H), 7.82 (m,
4H), 7.61 (s, 2H), 4.37 (t, 2H, J= 6), 3.81 (t, 2H, J=6),2.14(m,2H).
A portion of this material (150 mg, 0.23 mmol) was dissolved
in DMF (5 mL) and treated with LiCl (95 mg, 2.3 mmol) at
reflux overnight. After cooling to room temperature, DMF was
removed under reduced pressure. Addition of 1N HCl to the
residue formed a precipitate that was filtered and washed with
water to give 22 as a white solid; 144 mg (100% yield); mp
147-150 °C. 1H NMR (DMSO-d6) δ 9.14 (s, 2H) 8.90 (s, 2H),
7.82 (m, 4H), 7.62 (s, 2H), 4.37 (t, 2H, J = 6), 3.81 (t, 2H, J = 6),
2.14 (m, 2H).
4-[(3-Phthalimidopropyl)oxy]pyridine Tetraoxazole Macrocycle
with a 1,3-Bis(aminomethyl)phenyl linker (23a). Using method D,
22 (45 mg, 0.071 mmol) was macrocyclized with 1,3-bis(amino-
methyl)benzene (9 μL, 0.071 mmol). The product, 23a was
obtained as a white solid; 18 mg (35%); mp 249-252 °C (dec).
1H NMR δ 8.24 (s, 2H), 8.23 (s, 2H), 7.85 (dd, 2H, J = 3, 5), 7.72
(dd, 2H, J = 3, 5), 7.49 (m, 4H), 7.37 (m, 4H), 4.59 (d, 4H, J = 5),
Temperature-Dependent Spectrophotometry. Temperature-
dependent absorption experiments were conducted on an AVIV
model 14DS Spectrophotometer (Aviv Biomedical, Lakewood,
NJ) equipped with a thermoelectrically controlled cell holder.
Quartz cells with a path length of 1.0 cm were used for all the
absorbance studies. Temperature-dependent absorption pro-
files were acquired at either 260 nm (for ST duplex DNA and
4.27 (t, 2H, J = 6), 3.97 (t, 2H, J = 6), 2.30 (dt, 2H, J = 6, 12). 13
C
NMR δ 168.4, 166.7, 160.5, 159.7, 154.3, 146.8, 140.5, 138.8,
137.8, 137.5, 134.1, 132.1, 131.9, 129.9, 129.9, 129.5, 123.4, 109.4,
66.7, 43.9, 35.1, 28.0. HRMS (ESI) m/z calcd for C38H27N8O9
(M þ H) 739.1896; found 739.1891.
poly(rA) poly(rU) duplex RNA) or 295 nm (for hTel quad-
3
ruplex DNA and AurA quadruplex RNA) with a 7 s averaging
time. The temperature was raised in 0.5 °C increments, and the
samples were allowed to equilibrate for 1 min at each tempera-
ture setting. In the quadruplex melting studies, the hTel and
AurA concentrations were 5 μM in strand. When present in
these quadruplex studies, the drug concentrations were 20 μM.
In the duplex melting studies, the ST DNA concentrations were
4-[(3-Phthalimidopropyl)oxy]pyridine Tetraoxazole Macrocycle
with a 1,3-Bis(aminomethyl)-5-bromophenyl Linker (23b). Using
method D, 22 (22 mg, 0.034 mmol) was macrocyclized with 12
(10 mg, 0.034 mmol). The product, 23b was obtained as a white
solid; 8.3 mg (30%); mp 289-290 °C (dec). 1H NMR (CDCl3 þ
CD3OD) δ 8.33 (s, 4H), 7.86 (dd, 2H, J = 3, 5), 7.75 (dd, 2H,
J = 3,5), 7.51 (m, 5H), 4.58 (s, 4H), 4.29 (t, 2H, J = 6), 3.98 (t, 2H,
J = 7), 2.31 (dt, 2H, J = 6, 12). 13C NMR (CDCl3 þ CD3OD) δ
168.6, 167.2, 160.9, 160.5, 154.3, 146.4, 141.6, 139.9, 139.3, 137.5,
134.4, 132.1, 131.9, 131.6, 128.7, 123.5, 122.8, 109.5, 66.9, 43.2,
35.1, 28.0. HRMS (ESI) m/z calcd for C38H26BrN8O9 (M þ H)
817.1006; found 817.0986.
30 μM base pair, while the poly(rA) poly(rU) concentrations
3
were 15 μM base pair. When present, the drug concentrations
were 15 μM in the ST DNA studies and 7.5 μM in the poly(rA)
3
poly(rU) studies. The buffer for all the UV melting experiments
contained 10 mM potassium phosphate (pH 7.5) and sufficient
KCl (132 mM) to bring the total Kþ concentration to 150 mM.
Prior to their use in the UV melting experiments, all nucleic acid

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3643
solutions were preheated at 90 °C for 5 min and slowly cooled to
room temperature over a period of 4 h.
by substituted acridine derivatives. Bioorg. Med. Chem. Lett. 1999, 9,
2463–2468. (e) Koeppel, F.; Riou, J.-F.; Laoui, A.; Mailliet, P.;
ꢀ ꢁ
Arimondo, P. B.; Labit, D.; Petitgenet, O.; Helene, C.; Mergny, J.-L.
Human Tumor Xenograft Assay. Bioassays were performed
using female NCR/NU NU mice of approximately 9 weeks of
age as obtained from Taconic Farms, Inc. (Germantown, NY).
Mice were housed 3-4 per cage in laminar flow HEPA filtered
microisolator caging (Allentown Caging Equipment Co., Allen-
town, NJ). Mice were fed Purina autoclavable breeder chow no.
5021 and given drinking water, purified by reverse-osmosis, ad
libitum. Five days after arrival within the animal facility, the
mice were inoculated on the right flank with 1.5 ꢀ 106 MDA-
MB-435 tumor cells in 0.1 mL of RPMI 1640 Media by sc
injection (25 gauge needle ꢀ 5/8”). The MDA-MB-435 cells
were grown in 75 cm2 flasks using RPMI 1640 media and 10%
fetal bovine serum. Tumors were of sufficient size at 19-20 days
after inoculation. Tumor-bearing mice were evenly matched in
each experimental group based on tumor volume. Mice were
injected ip 3ꢀ weekly on alternate days. Negative controls
consisted of seven mice that received 150 μL of 10 mM citrate.
The positive control group consisted of eight mice that received
irinotecan (CPT-11, Camptosar) by ip injection at a dose of
20 mg/kg, 3ꢀ weekly, for all four weeks. Compound 19 was
similarly administered to seven mice, 3ꢀ weekly, at doses of 0,
25, 32, and 42 mg/kg for weeks 1-4, respectively. Tumor volume
was calculated by measuring the tumor with a microcaliper. The
length (l) is the maximum two-dimensional distance of the
tumor, and the width (w) is the maximum distance perpendicular
to this length measured in mm. Tumor volume was calculated
using the formula (lw2)/2. Body weights and tumor volumes of
individual mice were determined at a minimum of twice a week.
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Acknowledgment. Financial support for this research was
provided by Department of Defense grant W81XWH06-1-
0514. The Bruker Avance III 400 NMR spectrometer used
for this study was purchased with funds from NCRR grant no.
1S10RR23698-1A1. Mass spectrometry was provided by the
Washington University Mass Spectrometry Resource with
support from the NIH National Center for Research Resources
(grant no. P41RR0954).
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