Synthesis, metal ion binding, and biological evaluation of new anticancer 2-(2′-hydroxyphenyl)benzoxazole analogs of UK-1

Article abstract of DOI:10.1016/j.bmc.2007.11.019

UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A simplified analog of UK-1, 4-carbomethoxy-2-(2′-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1 against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg²⁺ and Zn²⁺. A series of 4-substituted-2-(2′-hydroxyphenyl)benzoxazole analogs of this 'minimal pharmacophore' of UK-1 were prepared. The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in methanol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg²⁺ and Cu²⁺ ions. Although none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(2′-hydroxyphenyl)benzoxazole, some analogs were identified that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs bind Cu²⁺ ions better than Mg²⁺ ions, and the nature of the 4-substituent is important for the Mg²⁺ ion binding ability of these 2-(2′-hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg²⁺ ion binding ability and cytotoxicity; however, within this series of 4-substituted-2-(2′-hydroxyphenyl)benzoxazoles the variations in cytotoxicity do not correlate with either Mg²⁺ or Cu²⁺ ion binding ability. These results, together with recent ESI-MS studies of Cu²⁺-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction with Cu²⁺ or other transition metal ions, rather than Mg²⁺, and that metal ion-mediated DNA binding, rather than metal ion binding affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu²⁺ ions in the cytotoxic action of UK-1 is further supported by the observation that UK-1 in the presence of Cu²⁺ displays enhanced cytotoxicity to MCF-7 and A549 cells when compared to UK-1 alone.

Full text of DOI:10.1016/j.bmc.2007.11.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1775–1783
Synthesis, metal ion binding, and biological evaluation of new
anticancer 2-(20-hydroxyphenyl)benzoxazole analogs of UK-1
Mireya L. McKee and Sean M. Kerwin*
Department of Chemistry and Biochemistry, Division of Medicinal Chemistry, Institute of Cellular and Molecular Biology,
College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA
Received 24 September 2007; revised 2 November 2007; accepted 5 November 2007
Available online 12 November 2007
Abstract—UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A sim-
plified analog of UK-1, 4-carbomethoxy-2-(20-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1
against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg2+ and
Zn2+. A series of 4-substituted-2-(20-hydroxyphenyl)benzoxazole analogs of this ‘minimal pharmacophore’ of UK-1 were prepared.
The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in meth-
anol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg2+ and Cu2+ ions. Although
none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(20-hydroxyphenyl)benzoxazole, some analogs were identified
that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs
bind Cu2+ ions better than Mg2+ ions, and the nature of the 4-substituent is important for the Mg2+ ion binding ability of these 2-(20-
hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg2+ ion
binding ability and cytotoxicity; however, within this series of 4-substituted-2-(20-hydroxyphenyl)benzoxazoles the variations in
cytotoxicity do not correlate with either Mg2+ or Cu2+ ion binding ability. These results, together with recent ESI-MS studies of
Cu2+-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction
with Cu2+ or other transition metal ions, rather than Mg2+, and that metal ion-mediated DNA binding, rather than metal ion bind-
ing affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu2+ ions in the cytotoxic action of UK-1
is further supported by the observation that UK-1 in the presence of Cu2+ displays enhanced cytotoxicity to MCF-7 and A549 cells
when compared to UK-1 alone.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
CO2Me
The bis(benzoxazole) natural product UK-1 (Fig. 1) is
O
N
MeO
O
one of a growing number of structurally related second-
ary metabolites with interesting biological activity.1–3
UK-1 displays a wide spectrum of potent anticancer
activity against leukemia, lymphoma, and certain solid
tumor-derived cell lines; however, this anticancer natural
product does not show antibacterial or antifungal activ-
ity.1,4 Recently, synthetic studies of analogs of UK-1
have been undertaken,4–8 and simplified analogs, such
as the benzoxazole 1 (Fig. 1), have been identified that
possess similar, selective cancer cell cytotoxicity to that
HO
HO
2
2'
4
N
O
N
O
5
6
7
UK-1
1
O
HO
O
MeO
MeO
HO
O
N
N
O
3
Keywords: Antibacterial; Metal ion binding; Cancer cell cytotoxicity;
DNA binding.
2
Figure 1. Structure of UK-1 and 2-(20-hydroxyphenyl)benzoxazole
analogs.
*
Corresponding author. Tel.: +1 512 471 5074; fax: +1 512 232
2696; e-mail: skerwin@mail.utexas.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.019

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M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
of UK-1.6–8 Studies of UK-1 and these synthetic analogs
have implicated metal ion complexation as an important
factor in the activity of these agents.6–10 UK-1 binds a
variety of di- and tri-valent metal ions, and binds to dou-
ble-stranded DNA better in the presence of metal ions
than in their absence.6,9,10 Active anticancer analogs of
UK-1, such as 1, also display this affinity for metal ions
and metal-mediated DNA binding ability.6,10 In contrast,
analogs such as 2 neither bind DNA in a metal ion-med-
iated fashion nor display cancer cell cytotoxicity.5,6 How-
ever, the exact role of metal ion coordination in the
mechanism of action of UK-1 and analogs remains to
be determined. There is a growing interest in the role of
metal ions and their complexes in drug design,11–14 par-
ticularly in metal complex–DNA interactions.15–21 Stud-
ies of UK-1 and its analogs may offer important insights,
provided by nature, into how metal ion complexation can
be harnessed in the design of selective cytotoxins.
ing and cytotoxicity of different 4-substituents of 1 while
retaining the benzoxazole core and 2-(20-hydroxy-
phenyl) substituent were prepared. In particular, the ef-
fect of homologation of the 4-methyl ester and its
replacement with an amide was addressed. Given the
evidence that metal ion complexation is important for
cytotoxicity in these UK-1 derivatives, additional poten-
tial metal ion coordination sites were also introduced in
some of these amide and ester analogs.
The synthesis of these analogs was accomplished
through straightforward adaptation of the previously re-
ported synthesis of UK-1 and 1.5,6 The key intermediate,
benzyl-protected carboxylic acid 4, was esterified (6, 11)
or coupled with a primary amine (8–10) (Scheme 1). The
methylamide 7 was prepared from the methyl ester 5
(Scheme 1). The resulting esters and amides were sub-
jected to hydrogenolysis to remove the benzyl phenol
protecting group to afford the 2-(20-hydroxy-
phenyl)benzoxazoles 12–17 in 63–91% yield.
The current work focuses on the synthesis and biological
evaluation of new benzoxazole analogs of 1. These ana-
logs were modified at the C4 position, retaining the 2-
(20-hydroxyphenyl)benzoxazole functionality of UK-1
and 1 that is thought to be important for chelating metal
ions. The cytotoxicity and metal ion binding ability of
these benzoxazoles were determined. The results indicate
that metal ion binding per se is not a predictor of cyto-
toxicity in this series. However, the ability of these com-
pounds to undergo metal ion-mediated DNA binding
may be related to their cytotoxicity, particularly in the
case of Cu2+ or other transition metals.
In addition to providing further sites for metal ion coor-
dination, the incorporation of polar groups at the C4
position in some of the compounds of this series also
favorably impacted their solubility in aqueous buffers.
As previously reported, UK-11 and compound 16 have
low aqueous solubilities. Compounds 15–17 have in-
creased water solubility, relative to 1. Compound 17
with a tetraethyleneglycol side chain was the most solu-
ble in this series, and the only one which remained in
solution in aqueous buffers at concentrations up to
100 lM.
2. Results and discussion
2.1.1. Cytotoxicity. The cytotoxicity of the new 2-(20-
hydroxyphenyl)benzoxazole analogs 12–17 was tested
using the AlamarBlue assay in the breast cancer cell
line MCF-7 and the lung cancer cell line A549 (See Ta-
ble 1). For comparison, UK-1, the isomeric 2-(20-
hydroxyphenyl)benzoxazole 3, and the benzoxazole car-
boxylic acid 2 (Fig. 1) were also tested. As expected
based on previous reports,6 the anticancer activity of
compound 1 is somewhat less than that of UK-1,
whereas the benzoxazole carboxylic acid 2 is not cyto-
toxic at the highest concentration tested (50 lM). Benz-
oxazole 3, which is regioisomeric to 1, is less cytotoxic
than 1, particularly against the A549 cell line. Similar
results had been observed previously for 3 in the PC-
3 and HT-29 cell lines, in which it is several fold less
cytotoxic than 110.
2.1. Analog design and synthesis
It was previously determined that compound 1 was the
minimum pharmacophore structure that retained the
selective cytotoxicity of UK-1.6 The key structural ele-
ments that affect metal binding and cytotoxicity were
addressed by comparing 1 with its 7-substituted benzox-
azole analog 3 (Fig. 1). Compound 3 differs from 1 so-
lely in the disposition of the benzoxazole N and O
atoms, but it is less cytotoxic than 1.10 In addition, the
complexation of Mg2+ by 1 and 3, as determined by
1H NMR and ESI-mass spectra, was quite distinct.10
There was also minimal DNA–metal mediated binding
of 3 compared to 1 as observed by ESI-MS.10 These data
highlighted the importance of the arrangement between
the phenolic hydroxyl group, the benzoxazole nitrogen
atom, and the ester for metal coordination, DNA bind-
ing, and cytotoxicity. A similar observation was made
by Wang and co-workers by comparing a different series
of UK-1 analogs and their inhibition of topoisomerase
II.7 Their research suggested that the analogs required
a structural motif comprised of an isosceles triangular
arrangement between the oxygen atom of carbonyl
group, the heterocyclic nitrogen atoms, and the phenolic
hydroxyl for enzyme inhibition.
Although none of the new benzoxazoles reported here
are superior to UK-1 or to 1 in their cytotoxicity to these
cancer cell lines, there are subtle variations in activity
that can be understood in structural terms. Replacing
the methyl ester group of 1 with a methyl amide, as in
13, leads to comparable cytotoxicity. However, as the
size of the amide group increases from the N-butyl ana-
log 14 to the N-(6-hydroxyhexyl) analog 15 to the N-
(hydroxyethoxyethyl) analog 16, the cytotoxicity de-
creases. While it is also true that the ethyl ester 12 is less
active than the methyl ester 1, the tetraethyleneglycol es-
ter 17 has a similar cytotoxic profile to 1, retaining activ-
ity against both cell lines.
Keeping these considerations in mind, a new series of
compounds that explore the influence on metal ion bind-

M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
1777
HO
O
BnO
N
O
R
R
O
O
BnO
N
HO
4
N
O
d.
a., b., or c.
or
O
O
MeO
6. R = OEt (69 %)
12. R = OEt (96 %)
BnO
7. R = NHMe (49 %)
8. R = NHBu (45 %)
9. R = NH(CH2)6OH (45 %)
13. R = NHMe (85 %)
N
14. R = NHBu (91 %)
15. R = NH(CH2)6OH (90 %)
16. R = NH(CH2)2O(CH2)2OH (63 %)
17. R = (OCH2CH2)4OH (85 %)
O
10. R = NH(CH2)2O(CH2)2OH (86 %)
11. R = (OCH2CH2)4OH (51 %)
5
Scheme 1. Synthesis of 2-(20-hydroxyphenyl)benzoxazole analogs of UK-1. Reagents: (a) SOCl2, EtOH (from 4); (b) MeAl(Cl)NHMe, benzene (from
5); (c) CDI, CH2Cl2 (from 4); (d) H2 (20 psi), Pd/C, EtOH.
association constants of all of the analogs were deter-
Table 1. Cytotoxicity of UK-1 analogs against MCF-7 and A549 cell
lines
mined and are summarized in Table 2. UK-1, 1, and
all of the other 4-substituted benzoxazole analogs 12–
17 bind copper with high affinity as 1:1 complexes.
The binding curves for these compounds displayed satu-
ration at all concentrations of added Cu2+, so only lower
limits to the association constants could be determined.
In contrast to the 4-substituted benzoxazoles, the 7-
substituted benzoxazole 3 is a relatively poor ligand
for Cu2+. Compound 3 is the only substituted 2-(20-
hydroxyphenyl)benzoxazole that does not appear to
bind Mg2+ by these spectrophotometric titrations;
although, a weak, alternative Mg2+ binding mode for
Compound
IC50 for MCF7a (lM)
IC50 for A549a (lM)
UK-1
1
1.4 0.9
2
2.0 0.5
3
4
12
2
>50
7
>50
35
3
12
1
4
15
10
5
8
40 10
13
14
11
14
1
4
30 10
31
15
16
7
39 17
41 17
>50
13
17
MMCb
2
11 1
0.4 0.1
1
0.20 0.07
this compound has been proposed based on H NMR
and ESI-MS studies.10 The methyl phenyl ether 2 does
not form complexes with either Mg2+ or Cu2+. In con-
trast to the Cu2+ binding ability of the 4-substituted
benzoxazoles UK-1, 1, and 12–17, which is high regard-
less of changes in the 4-substituent, the Mg2+ binding
ability of these compounds is dependent on the nature
of the 4-substituent. The analogs with amide substitu-
ents (e.g., 12–14) do not bind Mg2+ as well as those ana-
logs with ester substituents. As a result, these amides,
particularly 13, are more selective ligands for Cu2+ ver-
sus Mg2+. The critical role played by the benzoxazole 4-
substituent in metal ion recognition is also demonstrated
by a comparison of these substituted benzoxazoles with
2-(20-hydroxyphenyl)benzoxazole (HPBO). Under these
conditions, HPBO does not bind Mg2+ (data not
a Determined using the AlamarBlue assay. See Section 4 for details.
b Mitomycin C.
2.2. Metal ion binding
Because metal binding has been correlated with cytotox-
icity in other benzoxazole compounds,4,6,9,10 the affinity
of the new benzoxazole analogs 12–17 for various metal
ions was studied. Solutions of these compounds in meth-
anol change from colorless to yellow upon the addition
of divalent cations such as Mg2+, Zn2+, Ni2+, and Cu2+
,
indicating complexation between the analogs and the
metal ions. The most pronounced change occurs in the
presence of copper ions. Therefore, the metal complex
formation with both the physiologically abundant mag-
nesium ions as well copper ions for all of the analogs
was monitored by UV/vis spectral changes upon addi-
tion of increasing concentrations of the metal ion salts
in methanol. A marked hyperchromicity in the 350–
450 nm region was observed upon addition of the metal
ions, concomitant with a hypochromicity in the 315–350
region. A clear isosbestic point was present at approxi-
mately 350 nm, with slight variation of the wavelength
of maximum absorbance depending on the analog stud-
ied. Representative data for compounds 1 and 3 for
magnesium and copper binding are shown in Figure 2.
shown). HPBO does complex Cu2+ 22 however; the asso-
,
ciation constant for Cu2+ under these conditions
(4 1 · 105 M ꢀ1) is very similar to that of the 7-substi-
tuted benzoxazole 3 (3.8 · 104
M
ꢀ1) and significantly
lower than any of the 4-substituted benzoxazoles
(Table 2).
2.3. The role of metal ion coordination
The phenol ether 2 does not bind Mg2+ or Cu2+ and is
not cytotoxic to either of the two cancer cell lines exam-
ined here. However, within the series of 2-(20-hydroxy-
phenyl)benzoxazoles, there is no correlation between
either Mg2+ or Cu2+ ion binding affinity and cytotoxic-
ity. Although 3 is the weakest ligand for Mg2+ and Cu2+
ions in the 2-(20-hydroxyphenyl)benzoxazole series, it is
The change in absorbance of the peak in the 410 nm re-
gion was plotted against metal ion concentration to ob-
tain binding isotherms, from which the Mg2+ and Cu2+

1778
M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
Figure 2. Absorbance spectra of compounds 1 (A,B) and 3 (C,D) in the presence of increasing concentrations of Mg2+ (A,C) or Cu2+ (B,D). Arrows
denote the changes in absorbance with increasing metal ion concentrations.
ESI-MS in the presence of Cu2+ or Ni2+; whereas, the
relatively non-cytotoxic analogs 15 and 16 do not.23
Even the 7-substituted benzoxazole 3 forms ligand–me-
tal ion–DNA complexes, although these complexes are
formed in much lower abundance when compared to
those formed in the presence of 1.10 Thus, despite its
greatly diminished metal ion binding ability, 3 retains
some ability to undergo metal ion-mediated DNA bind-
ing and is also moderately cytotoxic, compared to 1.
Table 2. Association constants for Mg2+ and Cu2+ binding in MeOH
(Mꢀ1
)
Compound
Mg2+
Cu2+
UK-1
1
5
7
1 · 105
>5 · 107
3 · 105
>4 · 107
2
No binding
No binding
1.5 0.4 · 105
No binding
3.8 0.4 · 104
>3 · 107
3
12
13
14
15
16
17
6
2 · 104
>3 · 107
1.1 0.2 · 104
>6 · 107
2.4 0.4 · 104
>2 · 107
These studies cast doubt on the role of magnesium bind-
ing in the cytotoxicity of these 2-(20-hydroxy-
phenyl)benzoxazoles. In methanol, 7-substituted
benzoxazole 3 does not bind Mg2+ ions, yet this com-
pound is moderately cytotoxic. Despite the pronounced
effect of 4-position substituents on Mg2+ ion binding
affinity in the 2-(20-hydroxyphenyl)benzoxazole series,
there are only relatively subtle variations in cytotoxicity.
Our recent ESI-MS investigation of the metal ion bind-
ing of these compounds failed to demonstrate formation
of Mg2+ complexes in aqueous buffers, although com-
plexes were observed in methanol.23 These and previous
ESI-MS studies10 failed to demonstrate formation of li-
gand–Mg2+–DNA complexes with UK-1 or these ana-
logs, although ligand–Cu2+–DNA complexes were
readily observed. ESI-MS also confirmed the formation
3
8
1 · 105
3 · 105
>7 · 107
>7 · 107
cytotoxic, although somewhat less so than 1. The tetra-
ethyleneglycolester 17 binds both Mg2+ and Cu2+ ions
better than UK-1 or 1, yet it is not more cytotoxic than
these compounds.
While metal ion binding affinity per se does not predict
cytotoxicity within this series of 2-(20-hydroxy-
phenyl)benzoxazoles, the ability of these compounds
to bind to DNA in the presence of metal ions is corre-
lated with cytotoxicity. The cytotoxic analogs 1, 12–
14, and 17 all demonstrate enhanced DNA binding by

M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
Table 3. Cytotoxicity of UK-1 alone and in the presence of Cu2+
1779
2-(20-hydroxyphenyl)benzoxazoles, including UK-1,
bind to copper with greater affinity than magnesium,
but the variations in cytotoxicity for these benzoxazoles
do not correlate with either Mg2+ or Cu2+ ion binding
ability. Recent ESI-MS studies have shown that UK-1
does not form Mg2+ ion complexes in water, but that
UK-1 and analogs can form complexes with Cu2+ and
undergo Cu2+-mediated DNA binding. Taken together,
these results indicate that UK-1 and analogs may exert
their cytotoxic effects by interaction with Cu2+ or other
transition metal ions, rather than Mg2+, and that metal
ion-mediated DNA binding, rather than metal ion bind-
ing affinity, is important for the cytotoxic effect of these
compounds. The potential role of Cu2+ ions in the cyto-
toxic action of UK-1 is further supported by the obser-
vation that UK-1 in the presence of CuCl2 displays
enhanced cytotoxicity to MCF-7 and A549 cells when
compared to UK-1 alone.
Compound
IC50 (lM)
MCF-7 cells
IC50 (lM)
A549 cells
24 h
31
72 h
24 h
72 h
UK-1
5
1.7 0.2 17
2
1.0 0.1
UK-1+10 lM CuCl2 4.6 0.6 1.2 0.2 4.5 0.5 0.2 0.04
Mitomycin-C 1.2 0.2 4.5 lM 0.24 0.03
5
2
of 1:1 and in some cases 2:1 complexes between UK-1 or
these benzoxazoles and Cu2+ in aqueous solution.
2.4. Increased cytotoxicity of UK-1 in the presence of
copper
The cytotoxicity of UK-1 was directly compared with
that of UK-1 in the presence of added CuCl2 in both
MCF-7 and A549 cells (Table 3). In both cell lines, the
addition of 10 lM CuCl2 did not affect the growth rate
(data not shown). The combination of CuCl2 and UK-1
displays enhanced cytotoxicity relative to UK-1 alone.
This enhanced cytotoxicity is most pronounced after
24-h incubation in the case of MCF-7 cells. In contrast,
in the case of A549 cells, the enhanced cytotoxicity of
UK-1 plus CuCl2 relative to UK-1 alone is more pro-
nounced after 72-h exposure than 24-h exposure.
4. Experimental
4.1. General
Compounds 1, 2, 3, 4, 5, and UK-1 were synthesized as
described previously.5,6,10 AlamarBlue reagent was pur-
chased from Biosource and mitomycin-C (MMC) was
purchased from MP Biomedicals. Cell viability assays
were carried out in sterile black polystyrene 96-well plates
from Nunc. Spectroscopic metal ion binding studies were
carried out in HPLC grade MeOH. The MCF-7 and
A549 cell lines were kind gifts from Profs. Shawn Bratton
and Bob Krug (University of Texas at Austin), respec-
tively, from ATCC Biology Products. The medium for
the MCF7 cell line was the improved MEM (Richter’s
Modification) with L-glutamine, supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin
(from MediaTech). The A549 cells were cultured in
Ham’s F-12 media (Kaighn’s Modification) supple-
mented with 10% fetal bovine serum and 1% penicillin/
streptomycin. All other reagents were purchased from
The enhanced cytotoxicity of the UK-1 in the presence
of copper supports a role for the copper complex in
the mode of action of UK-1. The formation of the cop-
per complex of UK-1 in cells may be limited by the rel-
atively low availability of free Cu2+ ions. Addition of
Cu2+ ions increases the effectiveness of UK-1 by increas-
ing the amount of the complex available to the cellular
target.
3. Conclusions
This study has shown that it is possible to modify the
minimalized UK-1 analog 4-carbomethoxy-2-(20-
hydroxyphenyl)benzoxazole (1) at the C4 position with
certain groups and still retain activity against cancer cell
lines. In particular, changing the methyl ester of 1 to a
small amide group, as in 13, retains anticancer activity,
but as the length of the amide N-substituent increases,
cytotoxicity decreases. This trend of decreasing cytotox-
icity with increasing chain length is also seen with the
ethyl ester 12; however, the incorporation of additional
metal ion coordination sites in the side chain, as in the
tetraethyleneglycol ester 17, can recapture this lost activ-
ity while also increasing water solubility.
1
major commercial sources. Unless otherwise noted, H
and 13C NMR spectra were determined in CDCl3 on a
spectrometer operating at 300 and 75.5 MHz, respec-
tively. All mass spectra were obtained by chemical ioniza-
tion using methane as the ionizing gas. Chromatography
refers to flash chromatography on silica gel, and Rf values
were determined using silica gel–GF TLC plates (Merck)
using the solvent system indicated.
4.1.1. 2-(20-Benzyloxyphenyl)-benzoxazole-4-carboxylic
acid ethyl ester (6). 2-(20-Benzyloxyphenyl)-benzoxaz-
ole-4-carboxylic acid (4, 142 mg, 0.41 mmol) was dis-
solved in absolute EtOH (1.25 mL) in a flask fitted
with a drying tube. Thionyl chloride (45 ll, 0.62 mmol)
was then added dropwise while stirring, and the reaction
mixture was left stirring overnight at room temperature.
The reaction mixture was poured over ice, extracted
with CH2Cl2, washed with sodium bicarbonate, 1%
HCl, and water, dried over anhydrous Na2SO4, and fil-
tered. The product was then concentrated by rotory-
evaporation and purified by column chromatography
(20% EtOAc in hexanes) to give a white solid (103 mg,
The Mg2+ and Cu2+ ion binding ability of these UK-1
analogs were determined spectrophotometrically in
methanol. While the Cu2+ ion binding affinity of these
4-substituted benzoxazole analogs is relatively unaf-
fected by the nature of the 4-position substituents exam-
ined in this study, the Mg2+ ion binding affinities vary
with the nature of this substituent. Both Mg2+ and
Cu2+ ion binding ability are greatly diminished in the
regioisomeric 7-substituted benzoxazole 3. All of these

1780
M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
69% yield): mp 112–114 ꢁC; Rf 0.30 (20% EtOAc in hex-
4.1.4. 2-(20-Benzyloxyphenyl)-benzoxazole-4-carboxylic
acid (6-hydroxyphenyl)amide (9). Following the general
procedure described above, compound 3 (232 mg,
0.67 mmol) was coupled with 262 mg of aminohexanol
(2.24 mmol). Compound 9 was obtained after column
chromatography as white crystals (135 mg, 45% yield):
1
anes); H NMR d 1.44 (t, 3H, J = 7.2 Hz), 4.49 (q, 2H,
J = 7.2 Hz), 5.27 (s, 2H), 7.07–7.12 (m, 2H), 7.27–7.49
(m, 5H), 7.63 (d, 2H, J = 7.8 Hz), 7.71 (dd, 1H,
J = 8.4, 1.0 Hz), 8.00 (dd, 1H, J = 7.5, 1.2 Hz), 8.28
(dd, 1H, J = 8.1, 1.8 Hz); 13C NMR d 14.30, 61.07,
70.47, 113.62, 114.38, 116.28, 120.94, 122.31, 123.95,
126.40, 126.69, 127.57, 128.37, 131.91, 133.05, 136.60,
141.49, 151.22, 157.59, 163.61, 165.12; CIMS m/z 374
(MH+); HRMS m/z calcd for C23H20NO4: 374.1392,
found: 374.1389.
1
mp 139–141ꢁC; Rf 0.54 (10% MeOH in CH2Cl2); H
NMR d 1.35 (m, 4H), 1.52 (m, 4H), 2.47 (s, 1H), 3.38,
(q, 2H, J = 6.6 Hz), 3.57, (t, 2H, J = 6.6 Hz), 5.22 (s,
2H), 7.09 (m, 2H), 7.26–7.50 (m, 7H), 7.60 (d, 1H,
J = 7.5 Hz), 8.14 (t, 2H, J = 7.4 Hz), 9.16 (m, 1H); 13C
NMR d 25.19, 26.58, 29.37, 32.47, 39.34, 62.37, 70.45,
113.17, 113.70, 115.56, 120.99, 123.82, 124.62, 125.29,
126.64, 127.80, 128.44, 131.29, 133.35, 136.33, 139.28,
150.14, 157.60, 162.12, 164.27; CIMS m/z 445 (MH+);
HRMS m/z calcd for C27H28N2O4: 445.2127, found:
445.2126.
4.1.2. 2-(20-Benzyloxy-phenyl)-benzoxazole-4-carboxylic
acid methylamide (7). Methylaluminumchloride-methyl-
amide24 was prepared from MeNH2HCl (675 mg,
10 mmol) and Me3Al (2 M solution in hexanes, 5 mL)
in 10 mL dry benzene. Compound
5
(100 mg,
0.28 mmol) was dissolved in 2.8 mL dry benzene. To this
solution was added 830 lL of the aluminum amide solu-
tion (0.67 M, 0.56 mmol), and the mixture heated under
reflux overnight. The reaction mixture was then cooled
to room temperature and quenched with 5% HCl. The
organic layer was separated and the aqueous layer was
extracted three times with EtOAc. The organic layers
were combined, dried over MgSO4, and concentrated
by rotory-evaporation. The product was re-crystallized
from EtOAc/hexanes to give light pink needles (49 mg,
49% yield): mp 158.5–160 ꢁC; Rf 0.74 (10% MeOH in
4.1.5. 2-(20-Benzyloxy-phenyl)-benzoxazole-4-carboxylic
acid [2-(20-hydroxy-ethoxy)-ethyl]-amide (10). Following
the general procedure described above, compound 3
(112 mg, 0.32 mmol) was coupled with 113 mg of 2-(2-
aminoethoxy)ethanol (1.08 mmol). Compound 10 was
obtained after chromatography as
a white solid
(120 mg, 86% yield): mp 91–94 ꢁC; Rf 0.68 (10% MeOH
1
in CH2Cl2); H NMR d 2.2 (s(br), 1H), 3.54–3.67 (m,
8H), 5.28 (s, 2H), 7.12–7.15 (m, 2H), 7.29–7.53 (m,
7H), 7.65–7.68 (m, 1H, J = 8.0, 0.8 Hz), 8.16–8.20 (m,
2H), 9.42 (m, 1H); 13C NMR d 39.22, 61.69, 69.86,
70.77, 72.08, 113.47, 114.01, 115.80, 121.21, 123.80,
124.76, 125.53, 126.92, 127.96, 128.55, 131.56, 133.54,
136.45, 139.42, 150.38, 157.72, 162.49, 164.56; CIMS
m/z 433 (MH+); HRMS m/z calcd for C25H24N2O5:
433.1763, found: 433.1747.
1
CH2Cl2); H NMR d 2.82 (d, 3H, J = 4.8 Hz), 5.24 (s,
2H), 7.11–7.17 (m, 2H), 7.32–7.44 (m, 4 H), 7.50–7.55
(m, 3H), 7.65 (m, 1H, J = 8.0, 0.8 Hz), 8.16–8.19 (m,
2H), 8.90 (s, 1H); 13C NMR d 26.20, 70.64, 113.15,
113.56, 115.50, 121.03, 123.89, 124.69, 125.29, 127.03,
128.00, 128.56, 131.29, 133.40, 136.29, 139.47, 150.01,
157.77, 161.84, 164.92; CIMS m/z 359 (MH+); HRMS
m/z calcd for C22H19N2O3: 359.1396, found 359.1398.
4.1.6. 2-(20-Benzyloxyphenyl)-benzoxazole-4-carboxylic
acid 2-{2-[2-(20-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl
ester (11). Following the general procedure described
above, compound 3 (112 mg, 0.32 mmol) was coupled
with 210 mg of tetraethyleneglycol (1.08 mmol). Com-
pound 11 was obtained after column chromatography
as a transparent oil (86 mg, 51% yield): Rf 0.76 (10%
4.1.3. General procedure for CDI coupling: 2-(20-benzyl-
oxy-phenyl)-benzoxazole-4-carboxylic acid butylamide
(8). The benzoxazole acid imidazolide was prepared by
dissolving carboxylic acid 4 (306 mg, 0.89 mmol) in
dry CH2Cl2, approximately 6 mL/g, and adding portion-
wise 1.3 equivalents of carbonyldiimidazole (CDI) with
stirring for 1 h at room temperature, until the evolution
of CO2 ceased. Butylamine (215 mg, 3 mmol) was added
to the reaction mixture, which was heated under reflux
for 2 days. The reaction mixture was diluted with
CH2Cl2 and washed with H2O (2·), 0.1 N HCl (2·),
H2O (2·), 0.1 N NaOH, and H2O (2·). The organic
layer was dried over anhydrous Na2SO4, and the solvent
was evaporated. Compound 8 was obtained after col-
umn chromatography as white needle-like crystals
(157 mg, 45% yield): mp 122–123 ꢁC; Rf 0.22 (20%
1
MeOH in CH2Cl2); H NMR d 2.6 (s(br), 1H), 3.52–
3.54 (m, 2H), 3.58–3.70 (m, 8H), 3.72–3.74 (m, 2H),
3.87 (t, 2H, J = 4.8 Hz), 4.58 (t, 2H, J = 4.8 Hz), 5.27
(s, 2H), 7.06–7.11 (m, 2H), 7.28–7.48 (m, 5H), 7.61 (d,
2H, J = 7.2 Hz), 7.71 (dd, 1H, J = 8.0, 0.8 Hz), 8.01
(dd, 1H, J = 7.6, 0.8 Hz), 8.27 (dd, 1H, J = 8.2,
1.8 Hz); 13C NMR d 61.54, 64.18, 69.08, 70.16, 70.44,
70.48, 70.58, 72.37, 113.60, 114.61, 116.23, 120.95,
121.80, 123.96, 126.57, 126.68, 127.59, 128.40, 131.97,
133.12, 136.57, 141.57, 151.27, 157.59, 163.78, 164.86;
CIMS m/z 522 (MH+); HRMS m/z calcd for
C29H31NO8: 521.2050, found: 521.2036.
EtOAc in hexanes); 1H NMR
d 0.89 (t, 3H,
J = 7.2 Hz), 1.34–1.42 (m, 2H), 1.49–1.57 (m, 2H),
3.42, (q, 2H, J = 6.4 Hz), 5.29 (s, 2H), 7.11–7.15 (m,
2H), 7.29–7.52 (m, 7H), 7.66 (dd, 1H, J = 8.0, 0.8 Hz),
8.16–8.20 (m, 2H), 9.16 (m, 1H); 13C NMR d 13.68,
20.16, 31.53, 39.31, 70.50, 113.11, 113.76, 115.72,
121.03, 124.08, 124.64, 125.35, 126.67, 127.86, 128.48,
131.36, 133.33, 136.38, 139.35, 150.21, 157.67, 162.11,
164.16; CIMS m/z 401 (MH+); HRMS m/z calcd for
C25H24N2O3: 401.1865, found: 401.1870.
4.1.7. General procedure for hydrogenolysis: 2-(20-
hydroxyphenyl)benzoxazole-4-carboxylic acid ethyl ester
(12). The benzyl ether 6 (103 mg, 0.28 mmol) was dis-
solved in absolute EtOH (4 mL/mmol), and an equal
weight of 10% palladium on activated carbon was
added. The reaction mixture was placed under a balloon
of hydrogen for approximately 2 h. After verifying by
TLC that the reaction had gone to completion, the solu-

M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
1781
tion was filtered through Celite and washed with EtOAc
and CH2Cl2. The solution was then evaporated under
reduced pressure conditions, and the compound purified
by column chromatography (10% EtOAc in hexanes) to
obtain compound 12 as white crystals (75 mg, 96%
yield): mp 116–118 ꢁC; Rf 0.56 (20% EtOAc in hexanes);
1H NMR (400 MHz) d 1.51 (t, 3H, J = 7.2 Hz), 4.46 (q,
2H, J = 7.2 Hz), 7.00 (ddd, 1H, J = 8.0, 7.4, 1.2 Hz),
7.12 (dd, 1H, J = 8.4, 0.8 Hz), 7.41–7.46 (m, 2H), 7.70
(dd, 1H, J = 8.4, 1.0 Hz), 8.01 (dd, 1H, J = 7.8,
1.4 Hz), 8.06 (dd, 1H, J = 7.8, 2.0 Hz), 11.80 (s(br),
1H); 13C NMR d 14.23, 61.34, 109.82, 114.66, 117.51,
119.43, 121.39, 124.59, 127.02, 127.24, 134.05, 139.20,
149.57, 159.30, 164.06, 165.03; CIMS m/z 284 (MH+);
HRMS m/z calcd for C16H14NO4: 284.0923, found:
284.0926; EA calcd for C16H13NO4: C, 67.84; H, 4.63;
N, 4.94; found: C, 67.63; H, 4.52; N, 4.91.
MeOH in CH2Cl2) to afford an off-white solid (87 mg,
90% yield): mp 145.5–147 ꢁC; Rf 0.54 (10% MeOH in
CH2Cl2); 1H NMR (400 MHz) d 1.47–1.49 (m, 4 H),
1.59–1.62 (m, 2H), 1.70–1.74 (m, 2H), 1.82 (s(br), 1H),
3.58 (dt, 2H, J = 6.8, 6.0 Hz), 3.64 (t, 2H, J = 6.4 Hz),
7.03 (t, 1H, J = 7.4 Hz), 7.10 (d, 1H, J = 8.4 Hz), 7.44–
7.48 (m, 2H), 7.70 (d, 1H, J = 8.0 Hz), 7.83 (m, 1H),
8.03 (dd, 1H, J = 8.0, 1.2 Hz), 8.16 (d, 1H, J = 7.6 Hz),
10.44 (s, 1H); 13C NMR d 25.30, 26.72, 29.55, 32.54,
39.71, 62.62, 109.89, 113.53, 117.46, 120.27, 124.04,
125.51, 126.48, 127.74, 134.60, 137.20, 148.95, 158.30,
163.40, 163.84; CIMS m/z 355 (MH+); HRMS m/z calcd
for C20H23N2O4: 355.1658, found: 355.1660; EA calcd
for C20H22N2O4: C, 67.78; H, 6.26; N, 7.90; found: C,
67.64; H, 6.33; N, 7.48.
4.1.11. 2-(20-Hydroxyphenyl)benzoxazole-4-carboxylic
acid [2-(20-hydroxy-ethoxy)-ethyl]-amide (16). Following
the general procedure for hydrogenolysis, compound
10 (111 mg, 0.27 mmol) was de-protected to give com-
pound 16, which was purified by column chromatogra-
phy (10% MeOH in CH2Cl2), affording white crystals
(56 mg, 63% yield): mp 157–159 ꢁC; Rf 0.68 (10% MeOH
4.1.8.
2-(20-Hydroxyphenyl)benzoxazole-4-carboxylic
acid methylamide (13). Following the general procedure
for hydrogenolysis, compound 7 (44 mg, 0.12 mmol)
was de-protected to give compound 13, which was puri-
fied by column chromatography (10% MeOH in
CH2Cl2) and re-crystallized from MeOH in CH2Cl2 to
afford white crystals (27.87 mg, 85% yield): mp 225.5–
1
in CH2Cl2); H NMR d 2.65 (s(br), 1H), 3.67 (m, 2H),
3.74 (m, 2H, J), 3.80–3.84 (m, 4 H), 7.05 (ddd, 1H,
J = 8.1, 6.9, 1.2 Hz), 7.12 (dd, 1H, J = 8.4, 0.8 Hz),
7.45–7.50 (m, 2H), 7.72 (dd, 1H, J = 8.0, 0.8 Hz), 8.04
(dd, 1H, J = 8.2, 1.4 Hz), 8.19 (dd, 1H, J = 7.4,
1.0 Hz), 8.38 (m, 1H), 10.80 (s, 1H); 13C NMR d 39.41,
61.93, 70.08, 73.24, 109.93, 113.70, 117.59, 120.62,
123.87, 125.56, 126.54, 127.71, 134.69, 137.29, 149.01,
157.72, 163.35, 163.64; CIMS m/z 343 (MH+); HRMS
m/z calcd for C18H19N2O5: 343.1294, found: 343.1285;
EA calcd for C18H18N2O5+(H2O)1/3: C, 62.06; H, 5.40;
N, 8.04; found: C, 62.24; H, 5.19; N, 7.69.
1
226.5 ꢁC; Rf 0.74 (10% MeOH in hexanes); H NMR d
3.14 (d, 3H, J = 7.2 Hz), 7.06 (ddd, 1H, J = 8.0, 7.2,
1.0 Hz), 7.13 (dd, 1H, J = 8.7, 0.9 Hz), 7.46–7.53 (m,
2H), 7.73 (dd, 1H, J = 7.8, 0.9 Hz), 7.79 (s(br), 1H),
8.06 (dd, 1H, J = 8.1, 1.5 Hz), 8.20 (dd, 1H, J = 7.5,
0.9 Hz), 10.45 (s(br), 1H); 13C NMR d 26.69, 109.89,
113.55, 117.45, 120.25,123.96, 125.48, 126.48, 127.75,
134.57, 137.18, 148.95, 158.27, 163.39, 164.54 CIMS
m/z 269 (MH+); HRMS m/z calcd for C15H13N2O3:
269.0926, found: 269.0926; EA calcd for C15H12N2O3+(-
H2O)1/2: C, 66.52; H, 4.57; N, 10.34; found: C, 66.88, H,
4.42, N, 9.96.
4.1.12. 2-(20-Hydroxyphenyl)benzoxazole-4-carboxylic
acid 2-{2-[2-(20-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl
ester (17). Following the general procedure for hydrog-
enolysis, compound 11 (65 mg, 0.12 mmol) was de-pro-
tected to give compound 17, which was purified by
column chromatography (10% MeOH in CH2Cl2),
affording a yellow oil (46 mg, 85% yield): Rf 0.76 (10%
4.1.9.
2-(20-Hydroxyphenyl)benzoxazole-4-carboxylic
acid butylamide (14). Following the general procedure
for hydrogenolysis, compound 8 (150 mg, 0.37 mmol)
was de-protected to give compound 14, which was
recrystallized from EtOAc/hexanes to give white-flake
like crystals (105.9 mg, 91% yield): mp 181–183 ꢁC; Rf
1
MeOH in CH2Cl2); H NMR (400 MHz) d 2.60 (s(br),
1
0.22 (20% EtOAc in hexanes); H NMR d 0.99 (t, 3H,
1H), 3.52–3.54 (m, 2H), 3.59–3.71 (m, 8H), 3.74–3.77
(m, 2H), 3.91 (t, 2H, J = 4.8 Hz), 4.58, (t, 2H,
J = 4.8 Hz), 6.98 (t, 1H, J = 7.6 Hz), 7.09 (d, 1H,
J = 8 Hz), 7.39–7.46 (m, 2H), 7.76 (dd, 1H, J = 8.0,
0.8 Hz), 7.98 (dd, 1H, J = 8.0, 1.6 Hz), 8.05 (dd, 1H,
J = 7.8, 0.6 Hz), 11.82 (s, 1H); 13C NMR d 61.64, 64.50,
69.10, 70.24, 70.53, 70.61, 72.44, 109.88, 114.90, 117.59,
119.49, 121.16, 124.68, 127.13, 127.46, 134.12, 139.42,
149.67, 159.34, 164.19, 165.04; CIMS m/z 432 (MH+);
HRMS m/z calcd for C22H26NO8: 432.1675, found:
432.1658; EA calcd for C22H25NO8+(H2O)1/3: C, 60.40;
H, 5.91; N, 3.26; found: C, 60.53; H, 5.92; N, 3.10.
J = 7.2 Hz), 1.45–1.52 (m, 2H), 1.66–1.72 (m, 2H), 3.58
(q, 2H, J = 7.2 Hz), 7.04 (t, 1H, J = 8.4 Hz), 7.12 (d,
1H, J = 8 Hz), 7.42–7.46 (m, 2H), 7.72 (d, 1H,
J = 8.4 Hz), 7.84 (s(br), 1H), 8.04 (dd, 1H, J = 8.2,
1.4 Hz), 8.15 (d, 1H, J = 8.1 Hz), 10.37 (s, 1H); 13C
NMR d 13.72, 20.23, 31.53, 39.60, 109.81, 113.41,
117.43, 120.14, 124.08, 125.42, 126.40, 127.65, 134.50,
137.15, 148.88, 158.33, 163.31, 163,70; CIMS m/z 311
(MH+); HRMS m/z calcd for C18H19N2O3: 311.1396,
found: 311.1397; EA calcd for C18H18N2O3+(H2O)1/4
:
C, 68.69; H, 5.92; N, 8.90; found: C, 68.68; H, 5.73;
N, 8.69.
4.2. Metal ion binding studies
4.1.10. 2-(20-Hydroxyphenyl)benzoxazole-4-carboxylic
acid (6-hydroxyhexyl)amide (15). Following the general
procedure for hydrogenolysis, compound 9 (120 mg,
0.27 mmol) was de-protected to give compound 15,
which was purified by column chromatography (10%
Stock solutions of UK-1 and analogs, Mg(NO3)2, and
Cu(NO3)2 were prepared fresh on the day of the spectro-
photometric titrations. The fraction of complex formed
was measured by using solutions of increasing mole

1782
M. L. McKee, S. M. Kerwin / Bioorg. Med. Chem. 16 (2008) 1775–1783
fraction of metal ion, keeping the concentration of li-
gand constant, at 10 lM, using as a reference the absor-
bance of a solution containing the same concentration
of metal ion without the ligand. The change in absor-
bance was monitored from 600 to 200 nm for each sam-
ple, as a function of the concentration of metal ion. The
maximum absorbance change for the ligand–metal com-
plex was recorded at each metal ion concentration.
Mean Ftest = the averaged measured fluorescence inten-
sities of AlamarBlue reagent after 72-h exposure of the
cells to the test substance at a particular concentration.
Mean Fcrtl = the averaged measured fluorescence
intensities of AlamarBlue reagent after 72-h exposure
of the cells to the vehicle without the test substance.
The IC50, the compound concentration for which the
growth of treated cells from time0 was only 50% as much
as the vehicle-control, was determined by nonlinear
regression fitting of the data to Eq. (4):
Spectrophotometric data were analyzed by Eqs. (1) and
(2). Binding constants were calculated by nonlinear
regression of the experimental points.
Kd ¼ ½C0 ꢀ ðDA=DAmaxÞC0ꢁ½Cm ꢀ ðDA=DAmaxÞC0ꢁ
y ¼ Min þ ðMax ꢀ MinÞ=ð1 þ 10^ððx ꢀ log IC50Þ ꢃ nÞÞ
where:
=½ðDA=DAmaxÞC0ꢁ
ð1Þ
ð4Þ
2
Min, the minimum response plateau (0% Growth); Max,
the maximum response plateau (100% Growth); y, %
Growth at each test compound concentration; n, a fitted
parameter (the Hill slope coefficient).
C0ðDA=DAmaxÞ ꢀ ðC0 þ Cm þ KdÞðDA=DAmaxÞ þ Cm ¼ 0
ð2Þ
where:
Kd is the dissociation constant, C0 is the initial concen-
tration of the UK-1 analog, Cm is the concentration of
the metal ion, DA is the increase in absorption at the
wavelength of maximum absorbance for the ligand–me-
tal ion complex (ꢂ410 nm) upon addition of each metal
ion, and DAmax is the same parameter when the ligand is
totally bound to the metal ion.
Acknowledgments
We thank Chad M. McKee and Dr. John Richburg (The
University of Texas at Austin) for their assistance in the
cell culture work, and financial support from the Na-
tional Institutes of Health (R01 GM65956), the US–
Egypt Science and Technology Joint Fund, administered
through the USDA (BIO9-002-015), and the Robert
Welch Foundation (F-1298).
4.3. Cytotoxicity assays
Cell culture cytotoxicity assays were carried out as de-
scribed previously.6 Briefly, aliquots of 100 ll cell sus-
pension (1–3 · 103 cells) were placed in microtiter
plates in an atmosphere of 5% CO2 at 37 ꢁC. After
24 h, 100 lL of culture media and 2 ll of the compound
in DMSO were added to each well in duplicate, and the
plates incubated for an additional 72 h at 37 ꢁC. There
was no effect on the growth of cells compared to that
of cells in culture media alone at this DMSO concentra-
tion. Compounds, along with mitomycin-C as a positive
control, were evaluated at final concentrations ranging
from 0.001 to 50 lM.
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