Photoactivated DNA cleavage by compounds structurally related to the bithiazole moiety of bleomycin

Article abstract of DOI:10.1016/S0968-0896(01)00124-9

The syntheses of several novel halogenated bithiazoles structurally related to the bithiazole moiety of bleomycin A₅ are described. Also described is the ability of these compounds to mediate photoactivated NA cleavage. Chlorinated bithiazole analogues were shown to be much more active than an analogous brominated derivative. DNA strand scission activity was strictly light dependent and was accompanied by dechlorination of the bithiazole nucleus, apparently in a stoichiometric fashion. Inhibition of DNA cleavage in the presence of DMSO, as well as photoaddition to 1-octene by both brominated and chlorinated bithiazole derivatives, suggest strongly that the initial step in photoactivated DNA cleavage involves homolysis of the thiazole carbon-haloge bond. The chlorinated bithiazoles were found to mediate sequence selective cleavage of a ³²P-end labeled DNA, although the selectivity observed was not the same as that of bleomycin itself. The implications of this observation are discussed. Copyright

Full text of DOI:10.1016/S0968-0896(01)00124-9

Bioorganic & Medicinal Chemistry 9 (2001) 2303–2314
Photoactivated DNA Cleavage by Compounds Structurally
Related to the Bithiazole Moiety of Bleomycin
James C. Quada, Jr, Didier Boturyn and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22904, USA
Received 16 February 2001; accepted 5 April 2001
Abstract—The syntheses of several novel halogenated bithiazoles structurally related to the bithiazole moiety of bleomycin A5 are
described. Also described is the ability of these compounds to mediate photoactivated DNA cleavage. Chlorinated bithiazole ana-
logues were shown to be much more active than an analogous brominated derivative. DNA strand scission activity was strictly light
dependent and was accompanied by dechlorination of the bithiazole nucleus, apparently in a stoichiometric fashion. Inhibition of
DNA cleavage in the presence of DMSO, as well as photoaddition to 1-octene by both brominated and chlorinated bithiazole
derivatives, suggest strongly that the initial step in photoactivated DNA cleavage involves homolysis of the thiazole carbon-halogen
bond. The chlorinated bithiazoles were found to mediate sequence selective cleavage of a 32P-end labeled DNA, although the
selectivity observed was not the same as that of bleomycin itself. The implications of this observation are discussed. # 2001 Elsevier
Science Ltd. All rights reserved.
The bleomycins (BLMs) are a class of antitumor
agents1 believed to mediate their cytotoxic effects at the
level of DNA,2 and possibly also RNA3 degradation.
Extensive mechanistic studies have focused on the
molecular details of activation of the metal–O2 com-
plexes of bleomycin and subsequent DNA degradation,2
as well as the structural elements in BLM that control
sequence selective DNA interaction.4 The bithiazole
moiety of BLM has been shown to bind to DNA,5 but is
not the primary source of the characteristic sequence
selective cleavage of DNA by BLM,6 which involves the
pyrimidine moieties of many 50-GC-30 and 50-GT-30
sequences.7 To study the nature of bithiazole–DNA
interaction, we sought to equip the bithiazole with a
small reporter group, analogous to those used to study
DNA binding by other ligands.8
of those resulting from UV photolysis of carbon tetra-
chloride11 are formed. These results implicate homolysis
of the carbon–chlorine bond of chlorpromazine as the
initial event in photoactivated DNA cleavage.
We hypothesized that a chlorinated bithiazole irradiated
in the presence of DNA could, like chlorpromazine,
undergo photohomolysis to produce reactive species
capable of DNA cleavage. Reported herein is the
synthesis of halogenated bithiazole molecules (1a–d)
structurally related to bleomycin A5, and their ability to
mediate remarkably efficient photoactivated DNA clea-
vage.12 Also described are mechanistic experiments that
support a cleavage mechanism involving initial photo-
homolysis of the carbon–halogen bond, and conditions
that facilitate extensive DNA cleavage.
Chlorpromazine is a member of the promazine class of
antipsychotic drugs, which are characterized by photo-
toxic and photosensitizing side effects.9 Chlorpromazine
mediates photoactivated DNA cleavage which is unaf-
fected by oxygen radical scavengers such as t-butanol,
sodium benzoate, and sodium formate.10 EPR experi-
ments have shown that chlorpromazine photolysis (330
nm) does not involve active oxygen species, and that
spin adducts characteristic of a phenyl free radical10c or
Results and Discussion
Synthesis of halogenated bithiazole derivatives
It has been shown that thiazoles can be deprotonated
with lithium bases and substituted with a variety of
electrophiles,13 and experimental conditions were opti-
mized for lithium diisopropylamide lithiation and in situ
halogenation using hexachloroethane. N-Boc protected
ethyl (2-aminoethyl)thiazole-4-carboxylate 214 (Scheme
1) was treated with excess lithium diisopropylamide and
hexachloroethane at À78 ꢀC to produce chlorothiazole 3
*Corresponding author. Tel.:+1-804-924-3906; fax: +1-804-924-
7856; e-mail: sidhecht@virginia.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00124-9

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J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
HBr as it evolved during cyclization.16 The crude
hydroxylated intermediate was then dehydrated with
trifluoroacetic anhydride in pyridine17 at À20 ꢀC to
afford monochlorobithiazole ethyl ester 6 in 70% yield.
Although not the most convenient method for pre-
paring the requisite halogenated bithiazoles, this route
established the position of chlorination unambigu-
ously.
More direct access to the halogenated bithiazoles was
sought starting from preformed bithiazole derivatives.
Due to low yields and side reactions in the lithiation–
halogenation method of preparing chlorothiazoles,
other methods of halogenation were also explored. Fol-
lowing published reports18,19 of radical halogenation of
in 39% yield. Ester 3 was converted to amide 4 using
ammonia, and then to thioamide 5 via the agency of
Lawesson’s reagent15 (62% yield for two steps).
Hantzsch cyclization of 5 with ethyl bromopyruvate was
performed in the presence of 1,2-epoxybutane to trap
Scheme 1. Halobithiazole synthesis: (a) iPr2NLi, Cl3CCCl3, THF, 39%; (b) NH3, EtOH, 92%; (c) Lawesson’s reagent, THF, 67%; (d) ethyl
bromopyruvate, 1,2-epoxybutane, to toluene; (e) (CF3CO)2O, 70%; (f) (8a) N-chlorosuccinimide, hn, 74%; (8b) iPr2NLi, Cl3CCCl3, THF, 21%;
(8c) iPr2NLi, Cl3CCCl3, THF, 25%; (8d) N-bromosuccinimide, CH3CN, hn, 42 %.

J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
2305
Scheme 2. Synthesis of halobithiazoles 1a–1e; (a) 3-aminopropanol; (b) p-toluenesulfonyl chloride, pyridine; (c) (Boc)1,4-diaminobutane; (d)
CF3COOH or HCl.
heterocyclic compounds, photohalogenation of N-Boc
protected methyl 2-(20-aminoethyl)-2,40-bithiazole 714
was attempted. A degassed acetonitrile solution of N-
chlorosuccinimide and 7 was irradiated in Pyrex glass-
ware using a 100 watt low pressure mercury lamp at
25 ꢀC. This procedure provided chlorobithiazole methyl
ester 8a in 74% yield. Treatment of 8a with excess 3-
aminopropanol in methanol effected conversion into
hydroxypropyl amide 9a in 80% yield; the product was
identical in all respects to 9a produced by treatment of 6
with 3-aminopropanol. Hydroxypropyl amide 9a was
converted to the respective tosylate by treatment with 5
equiv of p-toluenesulfonyl chloride in dry pyridine at 0–
4 ꢀC for 22 h, then 50 equiv of 1,4-diaminobutane was
added; this procedure afforded Boc-protected chloro-
bithiazole 10a in 58% yield from 9a. Boc deprotection
was accomplished with trifluoroacetic acid in dichloro-
methane, providing chlorobithiazole 1a as a light yellow
solid in 49% yield.
25% yield, as well as monochlorobithiazole (11%) and
starting material (20% recovery).
Aminolysis of the mono- and bischlorobithiazole
methyl esters (8b and 8c, respectively) was accomplished
using 50 equiv of 3-aminopropanol to provide hy-
droxypropylamides 9b (68% yield) and 9c (75% yield)
(Scheme 2). That 9b was chlorinated on ring B (cf.
Scheme 1) was confirmed by comparison of the chemi-
cal shift of the thiazole proton in 9b (7.78 ppm) with the
thiazole proton in authentic 9a (8.17 ppm); the latter
was chlorinated on ring A. This chemical shift difference
was consistent for each intermediate in both mono-
chlorinated series. Tosylation of 9b was accomplished
using 5 equiv of p-toluenesulfonyl chloride in dry pyri-
dine (0–4 ꢀC) and provided the tosylate in 72% yield.
Treatment with 50 equiv of 1,4-diaminobutane in dry
pyridine afforded Boc-protected monochlorobithiazole
10b in 63% yield. The tosylate derived from 9c was not
isolated, but was treated directly with 50 equiv of 1,4-
diaminobutane, to give bischlorobithiazole 10c in 60%
yield. Deprotection of both 10b and 10c (HCl, CH2Cl2,
25 ꢀC)20 and purification by reversed-phase chromato-
graphy provided monochlorobithiazole 1b in 67% yield
and bischlorobithiazole 1c in 78% yield.
While photohalogenation of the bithiazole moiety pro-
vided the N-terminal chlorobithiazole in good yield, no
C-terminal chlorinated product was formed under any
condition attempted. Although the lithiation-halogena-
tion procedure used for the conversion 2!3 had pro-
ceeded in rather low yield, we nonetheless attempted this
procedure on bithiazole 7. It was found that lithiation and
in situ halogenation of 7 with optimal amounts of base and
halogenating agent could provide predominantly the C-
terminal monochloroinated bithiazole 8b or bischlor-
obithiazole 8c, which could be separated chromato-
graphically. Using 2.5 equiv of lithium diisopropylamide
and 4 equiv of hexachlorethane, monochlorobithiazole 8b
was obtained in 21% yield, along with bischlorobithiazole
8c (6%) and starting material (62% recovery).
The success of the photochlorination procedure in pro-
viding monochlorobithiazole derivatized on ring A led
Figure 1. Relaxation of supercoiled pBR322 DNA by chlorobithiazole
1b. The incubation mixtures including 200 ng of DNA were irradiated
for 5 min with 1000 watt high pressure (lanes 3–7) or 100 watt low
pressure (lanes 8–12) mercury lamps. Lanes 1 and 2 were not irra-
diated. Lane 1, DNA alone; lane 2, 10 mM Fe2++55 mM H2O2; lane
3, DNA alone; lane 4, 100 nM 1b; lane 5, 10 nM 1b; lane 6, 1 nM 1b;
lane 7, 100 pM 1b; lane 8, DNA alone; lane 9, 100 nM 1b; lane 10,
10 nM 1b; lane 11, 1 nM 1b; lane 12, 100 pM 1b.
Attempts to improve the yield of monochlorobithiazole
with more lithium diisopropylamide and hexa-
chloroethane resulted instead in increased yields of
bischlorobithiazole 8c. The best yield of bischloro-
bithiazole was achieved with 3.2equiv each of lithium
diisopropylamide and hexachlorethane, providing 8c in

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J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
to the attempted photobromination of 7 using N-bro-
mosuccinimide. Equimolar amounts of N-bromosucci-
nimide and bithiazole 7 were irradiated in deoxygenated
acetonitrile; monobromobithiazole 8d was obtained in
42% yield. The thiazole proton of monobromobithiazole
resonated at 8.26 ppm in the 1H NMR spectrum, almost
identical to the chemical shift of the thiazole proton of
monochlorobithiazole 8a (8.24 ppm). Ten equiv of ami-
nopropanol in methanol was employed to convert
monobromobithiazole ester 8d to the respective hydro-
xypropylamide 9d in 82% yield. The tosylate was pre-
pared from 9d in 58% yield using 5 equiv of p-
toluenesulfonyl chloride in dry pyridine. In order to
reduce the amount of diaminobutane for conversion of
the tosylate to the requisite spermidine derivative 10d,
and thereby facilitate purification, tosylate 9d was treated
with 4.3 equiv of mono Boc protected 1,4-diaminobutane
to afford N,N 0-bis tert-Boc-protected bromobithiazole
10d in 16% yield. Deprotection was accomplished with
trifluoroacetic acid in dichloromethane to afford mono-
bromobithiazole 1d in 50% yield.
chlorobithiazole 1b with a 1000 watt high pressure mer-
cury lamp or a 100 watt low pressure mercury lamp. Lanes
4 and 5 show greater extents of DNA plasmid relaxation
(1000 watt lamp) than can be seen in lanes 9 and 10 (100
watt lamp). Other experiments (data not shown) have also
demonstrated the requirement for light in chlor-
obithiazole-mediated DNA scission, and that increased
irradiation time results in increased DNA cleavage.
It may be noted that greater than 50% conversion of
Form I ! Form II DNA was achieved using 10 nM 1b.
Since the concentration of DNA plasmid employed was
ꢁ3–4 nM, this represents an exceptionally efficient pro-
cess for DNA nicking, and suggests that the bithiazole
must be activated essentially stoichiometrically and also
likely binds to its DNA target to facilitate cleavage. The
putative association of the bithiazoles with DNA prior
to cleavage is also supported by the observation that
cleavage by 1b was more efficient at 0 ꢀC than at 25 ꢀC
(data not shown).
In order to define the structural nature of the apparent
requirement for DNA binding, protected mono-
chlorobithiazole 8b was tested in a DNA plasmid
relaxation assay in direct comparison with mono-
chlorobithiazole 1b. As shown in Figure 2, concentra-
tion dependent conversion of Form I to Form II DNA
resulted when 1b was employed (lanes 4, 6, and 8), but
irradiation of protected analogue 8 (which should bind
DNA weakly if at all since it lacks a spermidine sub-
stituent) in the presence of Form I DNA failed to pro-
duce DNA strand scission at identical concentrations
(lanes 5, 7, and 9). These experiments begin to demon-
strate the remarkable chemistry of these agents: very high
efficiency transduction of light into DNA cleavage, which
is proportional to irradiation time, extent of bithiazole
halogenation and intensity of irradiation, suggesting that
halogen may be intimately involved in the cleavage pro-
cess. That DNA binding is required for cleavage to occur
suggests that the active species may be formed in close
proximity to the DNA target, assuming that both 8b and
1b are activated to the same extent as seems reasonable.
DNA cleavage activity
A preliminary report12 illustrated a comparison of the
DNA cleavage activity of photoactivated mono-
chlorobithiazole 1b and bischlorobithiazole 1c, showing
that the extent of chlorine substitution was directly
proportional to the extent of DNA cleavage observed at
a constant level of irradiation. Another DNA plasmid
relaxation experiment is shown in Figure 1, in which a
single chlorobithiazole was irradiated with lamps having
different intensities. The figure illustrates DNA cleavage
resulting from irradiation of a solution containing
100 nM to 100 pM concentrations of mono-
Figure 2. Relaxation of supercoiled pBR322 DNA by chloro-
bithiazoles 1b and 8b. The incubation mixtures including 200 ng of
DNA were irradiated (lanes 3–9) with a 100 watt low pressure mercury
lamp for 10 min. Lane 1, DNA alone; lane 2, 10 mM Fe2++55 mM
H2O2; lane 3, DNA alone; lane 4, 200 nM 1b; lane 5, 200 nM 8b; lane
6, 100 nM 1b; lane 7, 100 nm 8b; lane 8, 50 nM 1b; lane 9, 50 nM 8b.
Mechanism of chlorobithiazole activation for DNA
cleavage
Having defined the conditions required to obtain effec-
tive photoactivated DNA cleavage, experiments were
Figure 3. Relaxation of supercoiled pBR322 DNA by chlor-
obithiazoles 1a–c. The incubation mixtures including 200 ng of DNA
were irradiated with a 100 watt low pressure mercury lamp for 20 min.
The reaction mixtures in lanes 2and 4–10 were irradiated. Lanes 1 and
3 were not irradiated. Lanes 1 and 2, DNA alone; lanes 3 and 4,
DNA+10% DMSO; lane 5, 100 nM 1a; lane 6, 100 nM 1a+10%
DMSO; lane 7, 100 nM 1b; lane 8, 100 nM 1b+10% DMSO; lane 9,
100 nM 1c; lane 10, 100 nM 1c+10% DMSO.
Figure 4. Relaxation of supercoiled pGEM3Zf(+) DNA by chlor-
obithiazole 1a in the presence and absence of O2. Lane 1 was not
irradiated. Reaction mixtures including 200 ng of DNA in lanes 2–6
were irradiated under argon (lanes 4 and 6) or an ambient atmosphere.
Lanes 1 and 2, DNA alone; lanes 3 and 4, 100 mM Fe(II)ꢂ BLM A2;
lanes 5 and 6, 50 nM 1a.

J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
2307
designed to elucidate the mechanism of photoactivation.
Dimethyl sulfoxide has been used with spin trapping
reagents to probe the photoreactivity of chlorpromazi-
ne.10a The authors interpreted their results in terms of
the reaction of promazine and chlorine radicals with
DMSO. Therefore, an experiment was performed (Fig.
3) to determine the ability of DMSO to inhibit DNA
cleavage by the chlorobithiazoles. Photoactivated DNA
cleavage by all compounds except bischlorobithiazole 1c
was diminished in the presence of 10% (v/v) DMSO
(lanes 6, 8, and 10).
Dimethyl sulfoxide has also been shown to trap active
oxygen species,21 so the requirement for oxygen in DNA
cleavage by the chlorobithiazoles was studied. As shown
in Figure 4, under conditions that were sufficiently
anaerobic to substantially suppress the relaxation of
supercoiled plasmid DNA by 100 mM Fe(II)ꢂ bleomycin
A2, DNA cleavage by 1a was essentially unaffected by
the presence or absence of dioxygen. In this particular
experiment, a slight enhancement of cleavage was actu-
ally observed when oxygen was absent. An analogous
effect has been documented in a study of chlorproma-
zine.10a The inhibition seen in the above experiment
utilizing DMSO must, therefore, have arisen from the
interaction of DMSO with some other species formed
upon irradiation of the chlorobithiazoles.
In order to determine the fate of the C–Cl bond of the
chlorobithiazole during irradiation with DNA, mono-
chlorobithiazole 1b (100 mM concentration) was irra-
diated in the presence of 400 mg of calf thymus DNA for
5, 10, or 20 min (Fig. 5). A control reaction was also
performed in which the same amounts of DNA and 1b
were incubated together in the absence of light for 20
min. The DNA was precipitated and the supernatant
was assayed by C18 reversed-phase HPLC. The control
reaction resulted in trace A and the peak at 13.4 min
corresponded to 1b as judged by co-injection with
authentic 1b. With increasing irradiation time the peak
corresponding to 1b at 13.4 min diminished and a new
peak (at 12.3 min) increased concurrently; few other
peaks were found in the HPLC traces. Co-injection of
the trace C sample (10-min irradiation) and a roughly
equal amount of authentic nonchlorinated analogue
1e22 caused the peak at 12.3 min to approximately
double in size. In addition, the UV spectra associated
with the peaks at 12.3 and 13.4 min matched the UV
spectra of nonchlorinated bithiazole 1e and mono-
chlorobithiazole 1b, respectively. These results showed
that in the presence of DNA, chlorobithiazole 1b was
dechlorinated with very few side products, under con-
ditions utilized for DNA cleavage. Photohomolysis of
the C–Cl bond in chlorobithiazole would produce
chlorine atoms and carbon-centered thiazole radicals,
which could be quenched by abstraction of deoxyribose
hydrogen atoms from DNA in much the same manner
as has been demonstrated for the enediyne antibiotics.23
Figure 5. Dechlorination of chlorobithiazole 1b during irradiation in
the presence of calf thymus DNA. Aliquots of the irradiated solution
were analyzed by C18 reversed-phase HPLC. A, nonirradiated control;
B, 5 min irradiation; C, 10 min irradiation; D, 20 min irradiation. An
authentic sample of 1e was found to co-elute with the peak at 12.3
min.
Scheme 3. Photo-addition of 8b and 8d to 1-octene.

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J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
In fact, it has been shown that Clꢂ can mediate DNA
cleavage.24
products. These were isolated and structures assigned as
2-bithiazolyl-1-chlorooctane 11 (31% yield) and 1-
bithiazolyl-2-chlorooctane 12 (39% yield) on the basis
of mass spectral and NMR data. As a control reaction
monochlorobithiazole 8b was heated at reflux in 1-
octene for 1 h in the absence of light. There was no
reaction and the starting material was recovered quan-
titatively, which provides support for a radical process.
Monobromobithiazole 8d was also shown to undergo
photoinduced addition to the double bond of 1-octene,
but only 1-bithiazolyl-2-bromooctane 13 was formed
(34% yield). This is the same type of product that is
formed by bromotrichloromethane26 in olefin photo-
additions. A mechanism that could account for the dif-
ferent product distribution in these photoadditions
might involve initial attack on the olefin at C-1, to form
the more stable stable 2ꢀ free radical intermediate. In
the case of 8b, either the thiazole radical or chlorine
atom may initiate attack on the double bond, then the
remaining two radicals could combine to form the
observed products 11 and 12. The formation of only one
photoadduct from 1-octene and bromobithiazole 9 may
indicate that only the thiazole radical was responsible
for initial attack on the double bond, and the less reac-
tive bromine atom quenched the intermediate 2ꢀ octenyl
radical to complete the addition. Taken together with
the observed difference between chlorobithiazole 1a and
bromobithiazole 1d as DNA cleaving agents, the photo-
addition data support the interpretation that both C–Cl
and C–Br bonds are efficiently photohomolyzed by
irradiation, but that the bromine atom so liberated is
less reactive than the chlorine atom in the event that
initiates DNA cleavage, presumably abstraction of a
sugar hydrogen atom. While the enediyne antibiotics
utilize an aryl radical to abstract H atoms from DNA, the
present data argue that DNA cleavage by the chlor-
obithiazoles involves Clꢂ, a conclusion that is consistent
with the observation of Armitage and Schuster.24,27
Having demonstrated C–Cl bond cleavage under the
conditions of the photoactivated DNA cleavage reac-
tion, analogue 1d was prepared in which chlorine was
replaced with bromine. The comparative photoactivated
DNA cleavage of chlorobithiazole 1a and bromobithia-
zole 1d has been reported;12 the bromobithiazole analo-
gue induced no DNA cleavage at concentrations up to
200 nM, whereas chlorobithiazole analogue 1a was able
to completely convert supercoiled DNA into products
under the same conditions. If photocleavage is initiated
via homolysis of the C–X bond, diminished DNA clea-
vage by the bromobithiazole could mean that the bro-
mobithiazole homolyzes to a lesser extent, or that the
bromine atom, once formed, is less efficient as a DNA
cleavage agent than the chlorine atom. These possibi-
lities were investigated by studying the relative photo-
chemical reactivies of the halobithiazoles. Photoinduced
radical addition to olefins is known for organic
halides.25 Irradiation of monochlorobithiazole 8b in
deoxygenated 1-octene for 1 h (Scheme 3) produced two
Sequence selectivity of DNA cleavage
Also investigated has been the sequence selectivity of
DNA cleavage by the chlorobithiazoles. The sequence
selectivities of 50-32P end labeled DNA cleavage by
chlorobithiazoles 1a–1c are compared in Figure 6. DNA
cleavage by 2 mM Fe(II)ꢂ bleomycin A2+O2 is shown for
comparison (lane 3), and lanes 5–7, respectively,
demonstrate the patterns of DNA cleavage resulting
from treatment with 1 mM 1a–1c. At the 50-GTGTAT-30
site (nucleotides 82–87),28 Fe(II)ꢂ BLM A2 cleaves at T83
and T85, monochlorobithiazole 1a has a cleavage band
at A86, and monochlorobithiazole 1b exhibits cleavage
bands at T87. At this position, the orientations of clea-
vage sites lend themselves to the interpretation that 1a–
1c bind in the minor groove of DNA in a single orienta-
tion, consistent with findings for chlorobleomycin deri-
vatives prepared using these abbreviated bithiazoles.29
That these bands are close to sites of Feꢂ BLM-mediated
cleavage of this substrate (cf. lane 3) supports the idea29
that the bithiazole moiety may in some cases reinforce
the sequence selectivity of DNA binding defined by the
metal binding domain of bleomycin, and thereby create
strongly preferred sites for DNA cleavage.6 However,
Figure 6. Cleavage of a 50-32P end labeled 158 base pair DNA duplex
by Fe(II)ꢂ BLM A2 or by nonchlorinated bithiazole 1e and chlor-
obithiazoles 1a–c during irradiation with a 100 watt low pressure
mercury lamp apparatus for 15 min. The reaction mixtures in lanes 2
and 4–7 were irradiated. Lanes 1 and 2, DNA alone; lane 3, 2 mM
Fe(II)ꢂ BLM A2; lane 4, 1 mM nonchlorinated bithiazole 1e; lane 5,
1 mM chlorobithiazole 1a; lane 6, 1 mM chlorobithiazole 1b; lane 7,
1 mM bischlorobithiazole 1c; lanes 8–11, Maxam-Gilbert G, G+A, C,
and C+T reactions, respectively.

J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
2309
the gel in Figure 6 also reflects chlorobithiazole cleavage
sites not close to any site of cleavage mediated by
Fe(II)ꢂ BLM (cf. lane 3 and lanes 5–7), and vice versa,
indicating that the sites preferred for DNA binding by
the metal binding and bithiazole domains of bleomycin
are not always the same.
stirring to allow excess ammonia to evaporate. The
solution was then concentrated under diminished pres-
sure, and the residue was applied to a flash silica gel
column, which was washed with 2:1 ethyl acetate–hex-
anes, affording Boc-protected 5-chlorothiazole amide 4
as a light yellow gum: yield 96 mg (92%); 1H NMR
(CDCl3) d 1.43 (s, 9H), 3.09 (t, 2H), 3.52 (t, 2H), 6.13 (br
s, 1H) and 7.15 (br s, 1H); mass spectrum m/z 308 and
306 (M+H)+, 252 and 250, and 208 and 206; HRMS m/
z 307.0547 (M+), C11H1637ClN3O3S requires 307.0570).
The accumulated data support a mechanism for DNA
cleavage initiated via carbon–halogen bond photo-
homolysis. Chlorobithiazole-derived radical species are
among the most efficient DNA damaging agents known,
and their utility in study of DNA–bleomycin interac-
tions has the wherewithal to provide further insight into
the nature of bleomycin–DNA interaction.
2-[2-(t-Butoxycarbonyl)amino]ethyl 5-chlorothiazole-4-
thiocarboxamide (5)
A solution containing 96 mg (0.32mmol) of Boc-pro-
tected 5-chlorothiazole amide and 78 mg (0.19 mmol) of
Lawesson’s reagent in 10 mL of dry THF under argon
was heated at reflux with stirring for 75 min. The solu-
tion was then concentrated under diminished pressure
and the residue was applied to a column of flash silica
gel and washed with 1:1 ethyl acetate–hexanes, afford-
ing N-Boc 5-chlorothiazole thioamide 5 as a yellow
Experimental
All irradiations were performed in Pyrex glassware in a
Rayonet preparative photochemical reactor, type RS,
from Southern New England Ultraviolet Company that
was equipped with four RUL 2537A lamps, or with a
high pressure mercury capillary lamp, Model BH61B,
from TJ Sales. 1H NMR spectra were recorded at
300 MHz; chemical ionization mass spectra were
obtained using methane as reagent gas. Melting points
are uncorrected. Blenoxane was obtained from Bristol-
Myers Squibb Pharmaceuticals and was fractionated to
provide bleomycin A2 as described.30 Supercoiled plas-
mid DNAs were obtained from Promega (Madison,
WI). Reagent grade water for bioassays was produced
from a Millipore Milli-Q system.
1
solid: yield 67 mg (67%); H NMR (CDCl3) d 1.44 (s,
9H), 3.22 (t, 2H) and 3.60 (q, 2H); mass spectrum m/z
324 and 322 (M+H)+, 268 and 266, 232, and 224 and
222; HR-MS m/z 321.0394 (M+) (C11H1635ClN3O2S2
requires 321.0372).
Ethyl 20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-chloro-
2,40-bithiazole-4-carboxylate (6). A solution containing
105 mg (0.33 mmol) of N-Boc 5-chlorothiazole thioa-
mide 5, 83 mg (0.42mmol) of ethyl bromopyruvate, and
26 mg (0.36 mmol) of 1,2-epoxybutane in 10 mL of dry
toluene under argon was heated at reflux for 30 min and
then concentrated under diminished pressure. The resi-
due was dissolved in 25 mL of ethyl acetate and the
solution was washed with three 25-mL portions of
water, three 25-mL portions of saturated brine, and
then dried over anhydrous MgSO4. Concentration
under diminished pressure afforded a brown gum that
was dissolved in 10 mL of dry CH2Cl2. Dry pyridine
(129 mg, 1.6 mmol) was added to the mixture, which was
then cooled to À20 ꢀC. To this cooled solution was
added dropwise 205 mg (0.98 mmol) of trifluoroacetic
anhydride. The reaction mixture was stirred at À20 ꢀC
for 10 min and then concentrated under diminished
pressure. The residue was applied to a flash silica gel
column, that was washed with 2:1 hexanes–ethyl acetate
to provide N-Boc protected 50-chloro-2,40-bithiazole
ester 6 as colorless microcrystals from acetone–hexanes:
Ethyl
2-[2-(tert-Butoxycarbonyl)amino]ethyl-5-chloro-
thiazole-4-carboxylate (3). A solution containing 1.6 g
(5.4 mmol) of N-Boc-thiazole ethyl ester 2 and 5.71 g
(24.1 mmol) of Cl3CCCl3 in 120 mL of dry THF was
cooled to À78 ꢀC with stirring under argon. Lithium
diisopropylamide (33.8 mmol, 22.5 mL of a 1.5 M solu-
tion) was added dropwise over a period of 45 min. The
reaction mixture was stirred for 30 min then diluted
with 10 mL of water. The reaction mixture was parti-
tioned between 100 mL of water and 250 mL of hexanes,
and the organic layer was concentrated under dimin-
ished pressure. The residue was applied to a flash silica
gel column,31 and was washed with 1:2ethyl acetate–
hexanes to provide N-Boc-aminoethyl 5-chlorothiazole
ester 3 as colorless needles from acetone–hexanes: yield
0.7 g (39%); mp 88 ꢀC; H NMR (CDCl3) d 1.38–1.47
1
1
(m, 12H), 3.15 (t, 2H), 3.52 (q, 2H), 4.43 (q, 2H), and
4.88 (br s, 1H); mass spectrum, m/z 335, 337 and 279.
Anal. calcd for C13H19ClN2O4S: C, 46.63; H, 5.72; N,
8.37. Found: C, 46.69; H, 5.72; N, 8.37.
yield 94 mg (70%); mp 92–93 ꢀC; H NMR (CDCl3) d
1.42 (m, 12H), 3.16 (t, 2H), 3.56 (q, 2H), 4.42 (q, 2H),
5.00 (br s, 1H) and 8.21 (s, 1H); mass spectrum m/z 420
and 418 (M+H)+, 364 and 362, and 320 and 318. Anal.
calcd for C16H20ClN3O4S2: C, 45.98; H, 4.82; N, 10.05.
Found: C, 46.07; H, 4.79; N, 10.04.
2-[2-(tert-Butoxycarbonyl)amino]ethyl 5-chlorothiazole-
4-carboxamide (4). To a solution of 116 mg (0.35 mmol)
of Boc-chlorothiazole ethyl ester 3 in 7 mL of ethanol in
a heavy walled Pyrex tube cooled in dry ice was added
ammonia gas for 5 min (until the volume increased by
ꢁ1 mL). The tube was then sealed and the solution was
stirred at 25 ꢀC for 3 days. The tube was again cooled in
dry ice and opened, then warmed to 25 ꢀC slowly with
Methyl
20-[2-(t-Butoxycarbonyl)amino]ethyl-50-chloro-
2,40-bithiazole-4-carboxylate (8a). A solution containing
25 mg (69 mmol) of N-Boc protected bithiazole ester 7
and 12mg (92 mmol) of N-chlorosuccinimide in 3 mL of
acetonitrile in a test tube was stirred and purged with a
stream of argon gas for 20 min, then irradiated at 25 ꢀC

2310
J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
through a water filter for 40 min in a Rayonet appara-
tus. The solution was concentrated under diminished
pressure and the residue was purified by preparative
silica gel TLC (development was with 1:1 ethyl acetate–
hexanes), to afford N-Boc protected 50-chlorobithiazole
ester 8a as colorless microcrystals from ethyl acetate:
yield 20 mg (74%); mp 132 ꢀC; 1H NMR (CDCl3) d 1.42
(s, 9H), 3.16 (t, 2H), 3.57 (q, 2H), 3.97 (s, 3H), 4.96 (br
s, 1H) and 8.24 (s, 1H); mass spectrum m/z 406 and 404
(M+H)+, 350 and 348, and 306 and 304. Anal. calcd
for C15H18ClN3O4S2: C, 44.61; H, 4.49; N, 10.40.
Found: C, 44.84; H, 4.50; N, 10.37.
residue was applied to an Amberlite XAD-2column
(31ꢃ1.3 cm), which was washed successively with 5% aq
NaCl, water and methanol. The water and methanol
fractions containing the desired product were pooled and
concentrated under diminished pressure. The residue was
purified by preparative silica gel TLC, development with
4:1 methanol–NH4OH to afford chlorobithiazole 1a as a
1
pale yellow solid: yield 96 mg (49%); H NMR (D2O) d
1.51 (s, 4H), 1.80 (m, 2H), 2.58–2.78 (m, 6H), 2.98 (s,
4H), 3.30–3.42(m, 2H) and 7.94 (s, 1H); mass spectrum
m/z 419 and 417 (M+H)+; HR-MS m/z 417.1291
(M+H)+ (C16H2635ClN6OS2 requires 417.1299).
20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-chloro-2,40-bi-
thiazole-4-(3-hydroxypropyl)carboxamide (9a). A solu-
tion containing 600 mg (1.64 mmol) of N-Boc protected
50-chlorobithiazole ester 8a and 6.14 g (81.7 mmol) of 3-
aminopropanol in 12mL of methanol was stirred at
25 ꢀC for 11 h, and was then concentrated under dimin-
ished pressure. The residue was applied to a column of
flash silica gel, which was washed with ethyl acetate and
acetone to afford 1.24 g of 50-chlorobithiazole 3-hydro-
xypropylamide 9a as a yellow solid that crystallized
from acetone as colorless prisms: yield 586 mg (80%);
Methyl 20-[2-(tert-Butoxycarbonyl)amino]ethyl-5-chloro-
2,40-bithiazole-4-carboxylate (8b). To 2.0 g (5.4 mmol)
of bithiazole 7 and 5.14 g (21.7 mmol) of Cl3CCCl3 in
100 mL of dry THF was added 13.6 mmol (9.03 mL of a
1.5 M solution) of lithium diisopropylamide using the
same procedure as described above for lithiation-halo-
genation of thiazole 2, which gave after purification
Boc-protected 5-chlorobithiazole (8b) as colorless nee-
dles from hexanes–acetone: yield 466 mg (21%), plus
recovered starting material, 1.24 g (62%); mp 128.5–
129 ꢀC; 1H NMR (CDCl3) d 1.44 (s, 9H), 3.2 2 (t, 2 H), 3.59
(q, 2H), 3.98 (s, 3H), 4.99 (br s, 1H) and 7.97 (s, 1H); mass
spectrum m/z 406 and 404 (M+H)+, 350 and 348, and 306
and 304. Anal. calcd for C15H18ClN3O4S2: C, 44.61; H,
4.49; N, 10.40. Found: C, 44.72 ; H, 4.49; N, 10.41.
mp 142 ꢀC; H NMR (CDCl3) d 1.44 (s, 9H), 1.82(m,
1
2H), 3.18 (q, 2H), 3.55–3.74 (m, 6H), 4.95 (br s, 1H),
7.73 (br s, 1H) and 8.17 (s, 1H); mass spectrum m/z 449
and 447 (M+H)+, 393 and 391, and 349 and 347. Anal.
calcd for C17H23ClN4O4S2: C, 45.68; H, 5.19; N, 12.53.
Found: C, 45.74; H, 5.17; N, 12.57.
20-[2-(t-Butoxycarbonyl)amino]ethyl-5-chloro-2,40-bithia-
zole-4-(3-hydroxypropyl)carboxamide (9b). N-Boc pro-
tected 5-chlorobithiazole (8b) (455 mg, 1.1 mmol) and
4.24 g (56.5 mmol) of 3-aminopropanol in 25 mL of
THF were allowed to react for 44 h and the reaction
mixture was worked up as described for 50-chloro-
bithiazole 9a to provide N-Boc protected 5-chloro-
bithiazole hydroxypropylamide 9b as colorless
microcrystals from CH2Cl2–acetone: yield 345 mg
(68%); mp 118–119 ꢀC; 1H NMR (CDCl3) d 1.40 (s,
9H), 1.78 (m, 2H), 3.17 (t, 2H), 3.5–3.6 (m, 4H), 3.67 (t,
2H), 5.18 (br s, 1H), 7.73 (t, 1H) and 7.78 (s, 1H); mass
spectrum m/z 449 and 447 (M+H)+, and 349 and 347.
Anal. calcd for C17H23ClN4O4S2: C, 45.68; H, 5.19; N,
12.53. Found: C, 45.72; H, 5.15; N, 12.57.
20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-chloro-2,40-bi-
thiazole - 4 - [3 - (4 - aminobutyl)aminopropyl]carboxamide
(10a). A solution containing 455 mg (1.02mmol) of 5 0-
chlorobithiazole 3-hydroxypropylamide 9a and 975 mg
(5.1 mmol) of p-toluenesulfonyl chloride in 40 mL of dry
pyridine was stirred at 0–4 ꢀC for 23 h. The reaction
mixture was then treated with 4.4 g (51 mmol) of 1,4-
diaminobutane and stirred at 25 ꢀC for 16 h. The solu-
tion was concentrated under diminished pressure and
the residue was applied to a flash silica gel column,
which was washed successively with CHCl3, 1:1
methanol–CHCl3, 1:1:0.01 methanol–CHCl3–NH4OH,
99:1 MeOH–NH4OH, and finally 9:1 MeOH–NH4OH.
The product, N-Boc 50-chlorobithiazole 10a, eluted
from the column with the 9:1 methanol–NH4OH wash;
concentration afforded the product as a tan solid: yield
304 mg (58%); 1H NMR (CDCl3) d 1.30–1.55 (m, 13H),
1.88 (m, 2H), 2.65–2.83 (m, 6H), 3.14 (t, 2H), 3.47–3.57
(m, 4H), 5.37 (br s, 1H), 7.97 (br s, 1H) and 8.14 (s, 1H);
mass spectrum m/z 519 and 517 (M+H)+, and 419
and 417; FAB HR-MS m/z 517.1851(M+H)+
(C21H3435ClN6O3S2 requires 517.1824).
20 -[2-(tert-Butoxycarbonyl)amino]ethyl-5-chloro-2,40 -bi-
thiazole-4-[3-(O-p-toluenesulfonyl)propyl]carboxamide. A
solution containing 173 mg (0.39 mmol) of N-Boc pro-
tected 5-chlorobithiazole hydroxypropylamide 9b and
372mg (1.95 mmol) of p-toluenesulfonyl chloride in 10 mL
of dry pyridine under argon at 0–4 ꢀC was stirred for
17 h, and was then diluted with 10 mL of dry toluene and
concentrated under diminished pressure. The residue was
applied to a flash silica gel column and washed with 2:1
ethyl acetate–hexanes to afford the tosylate as a color-
less foam: yield 167 mg (72%); 1H NMR (CDCl3) d 1.41
(s, 9H), 1.97 (m, 2H), 2.39 (s, 3H), 3.18 (t, 2H), 3.45–3.6 (m,
4H), 4.13 (t, 2H), 5.01 (br s, 1H), 7.28 (d, J=8.1 Hz,
2H), 7.69 (br s, 1H), 7.76 (d, J=8.1 Hz, 2H) and 7.97 (s, 1H).
20-Aminoethyl-50-chloro-2,40-bithiazole-4-[3-(4-aminobu-
tyl)aminopropyl]carboxamide (1a). To a solution of
240 mg (0.46 mmol) of N-Boc 50-chlorobithiazole 10a in
20 mL of dry CH2Cl2 at 25 ꢀC was added 6 mL of tri-
fluoroacetic acid with stirring. The reaction mixture was
stirred at 25 ꢀC for 3 h and then concentrated under
diminished pressure. The residue was coevaporated
successively with portions of CH2Cl2, methanol, and
then water under diminished pressure, and then the
20 -[2-(tert-Butoxycarbonyl)amino]ethyl-5-chloro-2,40 -bi-
thiazole - 4 - [3 - (4 - aminobutyl)aminopropyl]carboxamide
(10b). A solution containing 100 mg (0.17 mmol) of 5-

J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
2311
chlorobithiazole tosylate and 736 mg (8.4 mmol) of 1,4-
diaminobutane in 6 mL of dry pyridine under argon was
stirred at 25 ꢀC for 17 h, after which 6 mL of dry toluene
was added and the mixture was concentrated under
diminished pressure. The residue was applied to a flash
silica gel column, which was washed successively with
2:1:0.01 CHCl3–MeOH–NH4OH, 1:1:0.01 CHCl3–
MeOH–NH4OH, and finally 9:1 methanol–NH4OH,
affording N-Boc protected 5-chlorobithiazole 10b as a
calcd for C15H17Cl2N3O4S2: C, 41.10; H, 3.91 ; N, 9.59.
Found: C, 41.71; H, 3.96; N, 9.49.
20-[2-(tert-Butoxycarbonyl)amino]ethyl-5,50-dichloro-2,40-
bithiazole-4-(3-hydroxypropyl)carboxamide (9c). N-Boc
protected 5,50-dichlorobithiazole ester 8c (139 mg,
0.32mmol) and 1.18 g (15.75 mmol) of 3-aminopropanol
in 1 mL of methanol were allowed to react over a period
of 24 h; purification was carried out as described for 50-
chlorobithiazole 9a to afford N-Boc protected 5,50-
dichlorobithiazole 9c, as colorless prisms from acetone–
CH2Cl2: yield 114 mg (75%); mp 169 ꢀC; 1H NMR
(CDCl3) d 1.43 (s, 9H), 1.82 (m, 2H), 3.14 (t, 2H), 3.50–
3.64 (m, 4H), 3.72(br s, 2H), 5.02(br s, 1H) and 7.75
(br s, 1H); mass spectrum m/z 483 and 481 (M+H)+
and 381.
1
colorless solid: yield 54 mg (63%); H NMR (CDCl3) d
1.44 (s, 9H), 1.52 (m, 2H), 1.62 (m, 2H), 1.88 (m, 2H),
2.64–2.74 (m, 4H), 2.81 (t, 2H), 3.22 (t, 2H), 3.50–3.63
(m, 4H), 5.14 (br s, 1H), 7.91 (s, 1H) and 8.05 (br s, 1H);
mass spectrum m/z 519 and 517 (M+H)+, and 419
and 417; FAB HR-MS m/z 517.1846 (M+H)+
(C21H3435ClN6O3S2 requires 517.1824).
20-Aminoethyl-5-chloro-2,40-bithiazole-4-[3-(4-aminobut-
yl)aminopropyl]carboxamide (1b). To a solution of
54 mg (0.1 mmol) of N-Boc 5-chlorobithiazole 10b in
5 mL of dry CH2Cl2 under argon at 0–4 ꢀC was added
2.1 mmol (2.1 mL of a 1.0 M solution in diethyl ether) of
hydrogen chloride in a dropwise fashion. The reaction
mixture was stirred at 25 ꢀC for 50 min, and was then
concentrated under diminished pressure. The residue
was dissolved in dry CH2Cl2, and again concentrated.
The residue was then applied to a column of C2 bonded
silica gel (Analytichem International) and washed with
water, affording 42mg of impure product and 9 mg of
unreacted starting material. The impure product was
purified further on an MPLC system (Rainin) using a
C8 reversed-phase column with water as the eluant; this
afforded free chlorobithiazole 1b as a cream-colored
20-[2-(tert-Butoxycarbonyl)amino]ethyl-5,50-dichloro-2,40-
bithiazole-4-[3-(4-aminobutyl)aminopropyl]carboxamide
(10c). To a solution of 114 mg (0.24 mmol) of N-Boc
protected 5,50-dichlorobithiazole hydroxypropylamide
9c in 5 mL of dry pyridine at 0–4 ꢀC was added 181 mg
(0.95 mmol) of p-toluenesulfonyl chloride and the solu-
tion was stirred at 0–4 ꢀC for 6.5 h. 1,4-Diaminobutane
(1.04 g, 11.9 mmol) was then added and the resulting
solution was stirred at 25 ꢀC for 12h. The reaction mix-
ture was concentrated under diminished pressure and
the residue was applied to a flash silica gel column and
washed with 1:2:0.02 methanol–CHCl3–NH4OH to
afford a product contaminated with diaminobutane.
The product was again applied to a column of flash
silica gel and washed with the same eluant, affording
Boc-dichlorobithiazole 10c as a yellow gum: yield 81 mg
1
1
powder: yield 29 mg (67%); H NMR (D2O) d 1.70 (s,
(60%); H NMR (CDCl3) d 1.38–1.60 (m, 13H), 1.80
4H), 1.95 (br s, 2H), 2.90–3.10 (m, 6H), 3.32–3.46 (m,
6H) and 7.99 (s, 1H); mass spectrum m/z 419 and 417
(M+H)+; FAB HR-MS m/z 419.1304 (M+H)+
(C16H2637ClN6OS2 requires 419.1269).
(m, 2H), 2.63 (q, 4H), 2.73 (t, 2H), 3.15 (t, 2H), 3.47–
3.59 (m, 4H), 5.15 (br s, 1H) and 7.88 (br s, 1H); mass
spectrum m/z 555, 553,and 551 (M+H)+; FAB HR-MS
m/z 551.1456 (M+H)+ (C21H3335Cl2N6O3S2 requires
551.1434).
Methyl 20-[2-(tert-Butoxycarbonyl)amino]ethyl-5,50-di-
chloro-2,40-bithiazole-4-carboxylate (8c). To a solution
containing 200 mg (0.54 mmol) N-Boc protected bithia-
zole ester 7 in 7 mL of dry THF under argon at À78 ꢀC
was added 186 mg (1.73 mmol) of lithium diisopropyl-
amide dropwise as a 1.5 M solution in THF-cyclohex-
ane over a period of 10 min. The reaction mixture was
stirred for 10 min at À78 ꢀC, then 410 mg (1.73 mmol) of
Cl3CCCl3 was added as a solution in 1.5 mL of dry THF
over a 5-min period. The reaction mixture was stirred at
À78 ꢀC for 10 min, quenched by the addition of 3 mL of
water, and concentrated to 3 mL under diminished
pressure. Twenty milliliters of ethyl acetate was added
and the organic solution was washed with three 20-mL
portions of water and two 20-mL portions of saturated
brine, then dried over MgSO4. The reaction mixture was
concentrated under diminished pressure and the residue
was applied to a flash silica gel column and washed with
1:1 ethyl acetate–hexanes to afford N-Boc protected
5,50-dichlorobithiazole ester (8c) as colorless needles
20-Aminoethyl-5,50-dichloro-2,40-bithiazole-4-[3-(4-amino-
butyl)aminopropyl]carboxamide (1c). A solution con-
taining 78 mg (0.14 mmol) of N-Boc protected
dichlorobithiazole 10c in 2mL of dry CH 2Cl2 under
argon at 0–4 ꢀC was treated with 2.76 mmol of hydrogen
chloride (30 min reaction time) and purified as for Boc-
5-chlorobithiazole 1b, affording dichlorobithiazole 1c as
an off-white powder: yield 50 mg (79%); 1NMR (D2O) d
1.70–1.82 (m, 4H), 2.03 (m, 2H), 2.98–3.17 (m, 6H) and
3.37–3.54 (m, 6H); mass spectrum m/z 453 and 451
(M+H)+; FAB HR-MS m/z 451.0910 (M+H)+
(C16H2535Cl2N6OS2 requires 451.0909).
Methyl 20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-bromo-
2,40-bithiazole-4-carboxylate (8d). A solution containing
1.00 g (2.71 mmol) of N-Boc protected bithiazole ester 7
and 496 mg (2.79 mmol) of N-bromosuccinimide in
100 mL of dry acetonitrile was stirred and purged with a
stream of argon gas for 12min, then irradiated at 0–
4 ꢀC with stirring through a water filter in a Rayonet
apparatus for 30 min. The solution was concentrated
under diminished pressure and the residue was applied
to a flash silica gel column, which was washed with 1:1
from acetone: yield 60 mg (25%); mp 136–137 ꢀC; H
1
NMR (CDCl3) d 1.43 (s, 9H), 3.15 (t, 2H), 3.56 (q, 2H),
3.97 (s, 3H) and 4.92(br s, 1H); mass spectrum m/z 440
and 438 (M+H)+ 384 and 382, and 340 and 338. Anal.

2312
J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
ethyl acetate–hexanes to afford N-Boc protected 50-bro-
mobithiazole ester 8d as colorless microcrystalꢀs from
563 and 561, and 463 and 461; FAB HR-MS m/z
661.1843 (M+H)+ (C26H4279BrN6O5S2 requires
661.1843).
1
ethyl acetate: yield 510 mg (42%); mp 118–120 C; H
NMR (CDCl3) d 1.44 (s, 9H), 3.18 (t, 2H), 3.58 (q, 2H),
3.97 (s, 3H), 4.96 (br s, 1H) and 8.26 (s, 1H); mass
spectrum m/z 450 and 448 (M+H)+. Anal. calcd for
C15H18BrN3O4S2: C, 40.18; H, 4.05; N, 9.37. Found: C,
40.28; H, 4.02; N, 9.35.
20 -Aminoethyl-50 -bromo-2,40 -bithiazole-4-[3-(4-amino-
butyl)aminopropyl]carboxamide (1d).
A solution of
42mg (63.5 mmol) of bis N-Boc protected 50-bromo-
bithiazole 10d in 15 mL of dry CH2Cl2 and 6 mL of tri-
fluoroacetic acid was stirred at 25 ꢀC for 1 h and was
then concentrated under diminished pressure. The resi-
due was coevaporated successively with portions of
CH2Cl2 and water under diminished pressure, and the
residue was then purified by preparative silica gel TLC
using 4:1 methanol–NH4OH for development, to pro-
vide mono bromobithiazole 1d as a cream-colored
20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-bromo-2,40-bi-
thiazole-4-(3-hydroxypropyl)carboxamide (9d). A solu-
tion containing 386 mg (0.86 mmol) of N-Boc protected
50-bromobithiazole and 646 mg (8.61 mmol) of 3-ami-
nopropanol in 20 mL of methanol was stirred at 25 ꢀC
for 41 h and worked up as described above for 50-
chlorobithiazole 9a to afford N-Boc protected 50-bro-
mobithiazole hydroxypropylamide 9d as colorless
microcrystals from acetone: yield 348 mg (82%); mp
1
powder: yield 15 mg (50%); H NMR (D2O) d 1.61 (s,
4H), 1.75–1.98 (m, 2H), 2.68–3.08 (m, 8H), 3.26–3.46
(m, 4H) and 7.93 (s, 1H); mass spectrum m/z 463 and
461 (M+H)+, and 383; FAB HR-MS m/z 463.0742
(M+H)+ (C16H2681BrN6OS2 requires 463.0772).
ꢀ
1
142 C; H NMR (CDCl3) d 1.44 (s, 9H), 1.83 (m, 2H),
3.20 (q, 2H), 3.55–3.74 (m, 6H), 4.96 (br s, 1H), 7.73 (br
s, 1H) and 8.18 (s, 1H); mass spectrum m/z 493 and 491
(M+H)+, and 393 and 391. Anal. calcd for
C17H23BrN4O4S2: C, 41.55; H, 4.72; N, 11.40. Found:
C, 41.99; H, 4.72; N, 11.36.
Agarose gel bioassay
DNA cleavage reactions were performed in 20 mL (total
volume) of 0.5 mM Na cacodylate, pH 7.2, containing
200 ng of supercoiled pBR322 DNA, and the other
components described in the figure legends. Reactions
were performed with or without irradiation, using a
Pyrex beaker as sample holder, a cooling bath, and a
UV filter. All irradiations were performed with the
reaction tubes packed in ice. Reaction mixtures were
diluted with 5 mL of a loading buffer containing
0.27 M Tris–acetate, pH 7.8, 13 mM EDTA, 40%
glycerol, 0.4% sodium dodecylsulfate, and 3.2% bro-
mophenol blue, and the entire reaction mixture was
then loaded onto a 1.2% (w/v) agarose gel containing
40 mM Tris–acetate, pH 7.8, 1 mM EDTA, and 1 mg/
mL of ethidium bromide.28 Horizontal gel electro-
phoresis was carried out in the same buffer and
cleavage products were visualized by UV transillumi-
nation.
20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-bromo-2,40-bi-
thiazole-4-[3-(O-p-toluenesulfonyl)propyl]carboxamide. A
solution containing 340 mg (0.69 mol) of N-Boc pro-
tected 50-bromobithiazole hydroxypropylamide 9d and
658 mg (3.45 mmol) of p-toluenesulfonyl chloride in
20 mL of dry pyridine was stirred at 0–4 ꢀC for 8 h and
was then concentrated under diminished pressure. The
residue was applied to a flash silica gel column and
washed with 2:1 ethyl acetate–hexanes to provide 50-
bromobithiazole tosylate as a colorless foam: yield
1
261 mg (58%); H NMR (CDCl3) d 1.44 (s, 9H), 2.02
(m, 2H), 2.42 (s, 3H), 3.18 (t, 2H), 3.52–3.61 (m,
4H), 4.16 (t, 2H), 5.00 (br s, 1H), 7.32 (d, 2H,
J=8.1 Hz), 7.66 (br s, 1H), 7.79 (d, 2H, J=8.1 Hz)
and 8.13 (s, 1H); mass spectrum m/z 475 and 473
(M-OTs)+.
20-[2-(tert-Butoxycarbonyl)amino]ethyl-50-bromo-2,40-bi-
thiazole-4-[3-(4-[tert-butoxycarbonyl]aminobutyl)amino-
HPLC assay
propyl] carboxamide (10d).
A
solution containing
A solution containing 400 mg of calf thymus DNA and
100 mM monochlorobithiazole 1b in 200 mL of 4.2mM
Na cacodylate, pH 7.2, was irradiated at 0–4 ꢀC for 5–
20 min or incubated in the absence of light at 0–4 ꢀC for
20 min. The DNA was removed by precipitation and
the supernatant was concentrated to dryness under
diminished pressure. C18 Reversed-phase HPLC ana-
lysis (100ꢃ4.6 mm column) utilized a flow rate of
1 mL/min and a linear gradient program of 0!40%
CH3CN in 0.1% aqueous trifluoroacetic acid over a
period of 20 min with detection at 295 nm. A diode
array detector recorded UV spectra at the apex of each
peak.
260 mg (0.40 mmol) of bromobithiazole tosylate deriva-
tive and 325 mg (1.73 mmol) of mono-N-Boc-1,4-dia-
minobutane in 10 mL of dry pyridine was stirred at
25 ꢀC for 26 h, after which 10 mL of water was added
and the reaction mixture was concentrated under
diminished pressure. The residue was applied to a flash
silica gel column and washed with 8:1:0.01 CHCl3–
methanol–NH4OH. The fractions containing the pro-
duct were pooled and concentrated and the residue was
purified by preparative silica gel TLC using 8:1:0.01
CHCl3-methanol-NH4OH for development. Bis N-Boc
protected 50-bromobithiazole 10d was obtained as a
colorless glass: yield 42mg (16%); 1H NMR (CDCl3)
d 1.42(s, 9H) 1.44 (s, 9H), 1.54 (m, H2 ), 1.67 (m,
2H), 2.00 (m, 2H), 2.79 (m, 2H) and 2.87 (m, 2H),
3.09 (m, 2H), 3.17 (t, 2H), 3.53–3.63 (m, 4H), 4.94
(br s, 1H), 5.05 (br s, 1H), 7.82(br t, 1H) and 8.17
(s, 1H); mass spectrum m/z 663 and 661 (M+H)+,
Sequence gel bioassay
A 10-mL reaction mixture contained 5 mM Na cacody-
late, pH 7.5, 5 mM (final nucleotide concentration)
sonicated calf thymus DNA, and 50-[32P] labeled duplex

J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
2313
DNA 158 bp in length (derived from plasmid pBR322
by HindIII cleavage, dephosphorylation with alkaline
phosphatase, 32P labeling with T4 polynucleotide
kinase, EcoRV cleavage, and native PAGE isolation).32
Other conditions are described in the figure legends.
Solutions of Fe(NH4)2(SO4)2 were prepared immedi-
ately before use. Reactions were performed as described
above for the agarose gel bioassay. Then, 5 mL of load-
ing solution (10 M urea, 1.5 mM EDTA, 0.05% (w/v)
each of bromophenol blue and xylene cyanol) was
added and one-half of each reaction mixture was
applied to the gel. DNA sequencing reactions were per-
formed according to the method of Maxam and Gil-
bert.33 The denaturing 10% polyacrylamide gel (7.5 M
urea) contained 89 mM Tris–borate, pH 8.0, and 2mM
EDTA.28 Gels were run in the same buffer and analyzed
by autoradiography.
of Health Research Grants CA76297 and CA77284,
awarded by the National Cancer Institute.
References and Notes
1. (a) Umezawa, H. In Bleomycin: Current Status and New
Developments; Carter, S. K., Crooke, S. T., Umezawa, H.
Eds.; Academic: New York, 1978; pp 15–20. (b) Sikic, B. I.;
Rozencweig, M., Carter, S. K., Eds. Bleomycin Chemotherapy;
Academic: Orlando, FL, 1985.
2. (a) Hecht, S. M. Acc.Chem.Res. 1986, 19, 383. (b) Stubbe,
J.; Kozarich, J. W. Chem.Rev. 1987, 87, 1107. (c) Natrajan,
A.; Hecht, S. M. In Molecular Aspects of Anticancer Drug–
DNA Interactions; Neidle, S., Waring, M., Eds.; Macmillan:
London, 1993; pp 197–242. (d) Kane, S. A.; Hecht, S. M.
Prog.Nucleic Acid Res.Mol.Biol.
R. M. Chem.Rev. 1998, 98, 1153.
1994, 49, 313. (e) Burger,
3. (a) Magliozzo, R. S.; Peisach, J.; Ciriolo, M. R. Mol.
Pharmacol. 1989, 35, 428. (b) Carter, B. J.; de Vroom, E.;
Long, E. C.; van der Marel, G. A.; van Boom, J. H.; Hecht,
Photolysis of chlorobithiazole in 1-octene
A solution of 1.0 mg (2.5 mmol) of chlorobithiazole
methyl ester 8b in 3 mL of 1-octene was purged with
argon gas for 20 min. The solution was irradiated in a
Rayonet apparatus for 1 h at 30 ꢀC in a water bath with
a Pyrex filter. The reaction mixture was concentrated
under diminished pressure and the residue was pur-
ified by preparative silica gel TLC, development
with 2:1 hexanes–ethyl acetate, to afford two pro-
ducts. The faster moving product was found to be
1-bithiazolyl-2-chlorooctane 12, isolated as a color-
less glass: yield 0.5 mg (39%); 1H NMR (CDCl3) d
0.83 (t, 3H), 1.21 (s, 10H), 1.44 (s, 9H), 2.73 (dd, 1H,
J=15, 6 Hz), 3.02(dd, 1H, J=15, 10 Hz), 3.23 (t, 2H),
3.55–3.65 (m, 3H), 3.88 (s, 3H), 4.97 (br s, 1H) and 8.08
(s, 1H); mass spectrum m/z 581 and 516 (M+H)+, and
462and 460. The slower moving product was found to
be the regioisomer, 2-bithiazolyl-1-chlorooctane 11, also
isolated as a colorless glass: yield 0.4 mg (31%); 1H
NMR (CDCl3) d 0.86 (t, 3H), 1.25 (s, 10H), 1.45 (s, 9H),
2.48 (m, 1H), 2.78 (dd, 1H, J=12, 12 Hz), 3.12–3.25 (m,
3H), 3.57 (q, 2H), 3.92 (s, 3H), 4.97 (br s, 1H) and 8.08
(s, 1H); mass spectrum m/z 518 and 516 (M+H)+, and
462and 460.
S. M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9373. (c) Hut-
¨
tenhofer, A.; Hudson, S.; Noller, H. F.; Mascharak, P. K. J.
Biol.Chem. 1992, 267, 24471. (d) Holmes, C. E.; Carter, B. J.;
Hecht, S. M. Biochemistry 1993, 32, 4293. (e) Dix, D. J.; Lin,
P.-N.; McKenzie, A. R.; Walden, W. E.; Theil, E. C. J.Mol.
Biol. 1993, 231, 230. (f) Hecht, S. M. Bioconjugate Chem. 1994,
5, 513. (g) Hecht, S. M. In The Many Faces of RNA; Eggle-
ston, D. S., Prescott, C. D., Pearson, N. D., Eds.; Academic:
London, 1998, pp 3–17 (h) Hecht, S. M. J.Nat.Prod. 2000,
63, 158.
4. (a) Takeshita, M.; Kappen, L. S.; Grollman, A. P.; Eisen-
berg, M.; Goldberg, I. H. Biochemistry 1981, 20, 7599. (b)
Kross, J.; Henner, W. D.; Hecht, S. M.; Haseltine, W. A.
Biochemistry 1982, 21, 4310. (c) Shipley, J. B.; Hecht, S. M.
Chem.Res.Toxicol. 1988, 1, 2 5.
5. (a) Chien, M.; Grollman, A. P.; Horwitz, S. B. Biochemistry
1977, 16, 3641. (b) Lin, S. Y.; Grollman, A. P. Biochemistry
1981, 20, 7589. (c) Kross, J.; Henner, W. D.; Haseltine, W. A.;
Rodriguez, L.; Levin, M. D.; Hecht, S. M. Biochemistry
1982, 21, 3711. (d) Kilkuskie, R. E.; Suguna, H.; Yellin, B.;
Murugesan, N.; Hecht, S. M. J.Am.Chem.Soc.
1985, 107,
260. (e) Hamamichi, N.; Natrajan, A.; Hecht, S. M. J.Am.
Chem.Soc. 1992, 114, 6278.
6. (a) Sugiyama, H.; Kilkuskie, R. E.; Chang, L.-H.; Ma, L.-
T.; Hecht, S. M.; van der Marel, G. A.; van Boom, J. H. J.
Am.Chem.Soc. 1986, 108, 3852. (b) Carter, B. J.; Murty, V. S.;
Reddy, K. S.; Wang, S.-N. J.Biol.Chem. 1990, 265, 4193. (c)
Guajardo, R. J.; Hudson, S. E.; Brown, S. J.; Mascharak, P. K.
J.Am.Chem.Soc. 1993, 115, 7971.
Photolysis of bromobithiazole in 1-octene
A solution of 10 mg (23 mmol) of bromobithiazole
methyl ester 8d in 30 mL of 1-octene was treated and
worked up as described above for the photolysis of
chlorobithiazole 8d, to afford 1-bithiazolyl-2-bro-
mooctane 13 as a yellow oil: yield 4.5 mg (34%); 1H
NMR (CDCl3) d 0.88 (t, 3H), 1.29 (s, 10H), 1.45 (s,
9H), 3.12–3.20 (m, 2H), 3.55-3.6 (m, 3H), 3.95 (s,
3H), 5.09 (br s, 1H) and 8.15 (s, 1H); mass spectrum
m/z 562and 560 (M+H) +, 506 and 504, and 462
and 460.
7. (a) D’Andrea, A. D.; Haseltine, W. A. Proc.Natl.Acad.
Sci. U.S.A. 1978, 75, 3608. (b) Takeshita, M.; Grollman, A. P.;
Ohtsubo, E.; Ohtsubo, H. Proc. Natl. Acad. Sci. U.S.A. 1978,
75, 5983. (c) Mirabelli, C. K.; Ting, A.; Huang, C.-H.; Mong,
S.; Crooke, S. T. Cancer Res. 1982, 42, 2779.
8. (a) Youngquist, R. S.; Dervan, P. B. J.Am.Chem.Soc.
1985, 107, 5528. (b) Baker, B. F.; Dervan, P. B. J.Am.Chem.
Soc. 1985, 107, 8266.
9. (a) Epstein, J. H.; Brunsting, L. A.; Peterson, M. C.;
Schwarz, B. E. J.Invest.Dermatol. 1957, 28, 329. (b) Kirsch-
baum, B. A.; Beerman, H. Am.J.Med.Sci. 1964, 248, 445. (c)
Satanove, M.; McIntosh, J. S. J.Am.Med.Assoc. 1967, 200,
209.
10. (a) Decuyper, J.; Piette, J.; Lopez, M.; Merville, M.-P.;
van de Vorst, A. Biochem.Pharmacol. 1984, 33, 4025. (b)
Chignell, C. F.; Motten, A. G.; Buettner, G. R. Environ.
Health Perspect. 1985, 64, 103. (c) Motten, A. G.; Buettner,
G. R.; Chignell, C. F. Photochem.Photobiol. 1985, 42, 9.
Acknowledgements
This paper is dedicated to Professor Peter Dervan on
the occasion of his receipt of the 2000 Tetrahedron
Prize. This work was supported by National Institutes

2314
J.C.Quada, Jr.et al./ Bioorg.Med.Chem.9 (2001) 2303–2314
(d) Ciulla, T. A.; Epling, G. A.; Kochevar, I. E. Photochem.
Photobiol. 1986, 43, 607.
11. Janzen, E. G.; Stronks, H. J.; Nutter, D. E.; Davis, E. R.;
Blount, H. N.; Lee Poyer, J.; McCay, P. B. Can.J.Chem.
1980, 58, 1596.
23. (a) Kappen, L. S.; Napier, M. A.; Goldberg, I. H. Proc.
Natl. Acad. Sci. U.S.A. 1980, 77, 1970. (b) Zein, N.; Sinha,
A. M.; McGahren, W. J.; Ellestad, G. A. Science 1988, 240,
1198. (c) Sugiura, Y.; Uesawa, Y.; Takahashi, Y.; Kuwahara,
J.; Golik, J.; Doyle, T. W. Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 7672. (d) Sugiura, Y.; Shiraki, T.; Konishi, M.; Oki, T.
Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3831.
12. Quada, J. C., Jr.; Levy, M. J.; Hecht, S. M. J.Am.Chem.
Soc. 1993, 115, 12171.
13. (a) Knaus, G.; Meyers, A. I. J.Org.Chem. 1974, 39, 1192.
(b) Dondoni, A.; Mastellari, A.R.; Medici, A.; Negrini, E.;
Pedrini, P. Synthesis 1986, 757 (c) Sawhney, I.; Wilson, J. R.
24. Armitage, B.; Schuster, G. B. Photochem.Photobiol. 1997,
66, 164.
25. (a) Poutsma, M.L. In Methods in Free-Radical Chemistry;
Huyser, E. S., Ed.; Academic: New York, 1969; Vol. 1, pp
163–186 (b) Thaler, W. A. In Methods in Free-Radical Chem-
istry; Huyser, E. S., Ed.; Academic: New York, 1969; Vol. 2,
pp 214–218.
H. J.Chem.Soc,. Perkin Trans.1
1990, 329.
14. (a) Zee-Cheng, K. Y.; Cheng, C. C. J.Heterocycl.Chem.
1970, 7, 1439. (b) McGowan, D. A.; Jordis, U.; Minster, D. K.;
Hecht, S. M. J.Am.Chem.Soc.
1977, 99, 8078. (c) Sakai,
T. T.; Riordan, J. R.; Booth, T. E.; Glickson, J. D. J.Med.
Chem. 1981, 24, 279.
15. Cava, M. P.; Levinson, M. I. Tetrahedron 1985, 41, 5061.
16. Schmidt, U.; Gleich, P.; Greisser, H.; Utz, R. Synthesis
1986, 992.
26. Kharasch, M. S.; Sage, M. J.Org.Chem. 1949, 14, 537.
27. It may be noted, however, that it is possible that the dif-
ferences observed for bromobithiazole 1d reflect its reaction
via an ionic mechanism. See: Kropp, P. J. Acc.Chem.Res.
1984, 17, 131.
17. Campagna, F.; Carotti, A.; Casini, G. Tetrahedron Lett.
1977, 21, 1813.
18. Anderson, A. G.; Tober, T. Y. J.Org.Chem. 1980, 45, 1695.
19. Phillips, R. S.; Cohen, L. A. Tetrahedron Lett. 1983, 24,
5555.
28. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular
Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, NY, 1989.
29. Zuber, G.; Quada, J. C., Jr.; Hecht, S. M. J.Am.Chem.
Soc. 1998, 120, 9368.
20. Stahl, G. L.; Walter, R.; Smith, C. W. J.Org.Chem. 1978,
43, 2285.
30. Chien, M.; Grollman, A. P.; Horwitz, S. B. Biochemistry
1977, 16, 3641.
21. Repine, J. E.; Pfenninger, O. W.; Talmage, D. W.; Berger,
E. M.; Pettijohn, D. E. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,
1001.
22. Bithiazole 1e was prepared from 714 in analogy with the
syntheses of 1a–1d. See: Kane, S. A.; Sasaki, H.; Hecht, S. M.
J.Am.Chem.Soc. 1995, 117, 9107.
31. Still, W. C.; Khan, J.; Mitra, A. J.Org.Chem. 1978, 43,
2923.
32. Sugiyama, H.; Ehrenfeld, G. M.; Shipley, J. B.; Kilkuskie,
R. E.; Chang, L.-H.; Hecht, S. M. J.Nat.Prod. 1985, 48, 869.
33. Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65,
499.