DNA-directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles

Article abstract of DOI:10.1021/jm9803119

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9- anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized an

Full text of DOI:10.1021/jm9803119

4744
J . Med. Chem. 1998, 41, 4744-4754
DNA-Dir ected Alk yla tin g Agen ts. 7. Syn th esis, DNA In ter a ction , a n d An titu m or
Activity of Bis(h yd r oxym eth yl)- a n d Bis(ca r ba m a te)-Su bstitu ted P yr r olizin es
a n d Im id a zoles
Graham J . Atwell, J un-Yao Fan, Karin Tan, and William A. Denny*
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland,
Private Bag 92019, Auckland, New Zealand
Received May 5, 1998
A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and
related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinyl-
amino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-
specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were
prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine
dicarboxylates, coupling of these with the desired carrier, and reduction to give the required
bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators,
followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and
some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand
cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the
gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of
G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more
selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5′-NC*C*N,
5′-NGC*N and 5′-NC*GN sequences in the PCR stopping assay (* indicates block sites). The
data suggest that these targeted compounds, like the known thioimidazole bis(carbamate)
carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no
alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated
broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic
(IC50s >1 µM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic
than the corresponding minor-groove-targeted ones. This was true also for the more reactive
thioimidazole bis(carbamates) (IC50s 0.8 and 11 µM, respectively), but both were more active
than the analogous “untargeted” carmethizole (IC50 20 µM). The bis(hydroxymethyl)pyrrolizine
analogues were the most cytotoxic, with IC50s as low as 0.03 µM.
Alkylating agents remain an effective class of anti-
cancer drugs, whose cytotoxic and therapeutic effects
derive primarily from their ability to form DNA inter-
strand cross-links.1 There is renewed interest in this
general class of drugs, following recent demonstrations
that both their sequence and regioselectivity of DNA
alkylation can be altered by attaching them to a variety
of DNA-affinic carriers and that this can result in a
modified spectrum of biological action.2-6
cross-link with the short oligonucleotide 5′-ACGT at the
5′-CG site via the exocyclic amino groups of guanine
residues in the minor groove.12
While nitrogen mustards [N,N-bis(2-chloroethyl)-
amines] have been the most widely used DNA inter-
strand cross-linking agents, other classes of compounds
with similar activity are known. One class is the bis-
(hydroxymethyl)pyrrolizines and corresponding bis(car-
bamates). These “vinylogous carbinolamines” were de-
veloped initially from the pyrrolizine alkaloids and led
to compounds such as IPP (1)7 and the thioimidazole
carmethizole (3), the latter of which has reached clinical
evaluation.8,9 Related bis-alcohols such as 2 are also
DNA alkylators.10 The mechanism of interaction of these
compounds with DNA is considered to be via SN1
reactions that result in electrophilic methides.11 A recent
study of 1 showed that it forms an interstrand
As with the mustard alkylators, an important con-
temporary question is the degree to which the spectra
of both DNA alkylation and biological activity of the bis-
(hydroxymethyl)pyrrolizines and analogues are altered
by linking them to DNA-affinic carriers. A recent study13
of the distamycin analogue 4 showed that it was about
1000-fold more potent than the corresponding “untar-
geted” alkylator 5. In this paper we study the DNA
alkylating ability and cytotoxicities of a series of these
alkylating units of varying reactivity linked to either
the DNA-intercalating carrier 9-anilinoacridine14 (com-
pounds 15-18, 30) or the DNA minor groove binding
carrier 4-(4-quinolinylamino)benzamide15 (compounds
22-25, 32, 37).
10.1021/jm9803119 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/28/1998

DNA-Directed Alkylating Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4745
Sch em e 1a
Ch em istr y
The target bis(N-methylcarbamates) 17, 18, 24, and
25 were synthesized as shown in Schemes 2 or 3, via
the key intermediates 11 or 12 (Scheme 1). Alkylation
of imidazole 7 and the thioimidazole 8 with the alkyl
bromide 6 furnished the diesters 9 and 10. Subsequent
nitro group reduction by Pt/C-catalyzed hydrogenation
gave the amino diesters 11 and 12. Lithium aluminum
hydride reduction of diester 11 gave diol 13 (Scheme
2), which was coupled with 9-chloroacridine to provide
the anilinoacridine 15, and treatment of 15 with methyl
isocyanate employing dibutyltin diacetate as catalyst8
yielded the desired bis(carbamate) 17. The amino di-
ester 12 (Scheme 2) was coupled with 9-chloroacridine
under acid-catalyzed conditions to give the anilinoacri-
dine 13, and subsequent hydride reduction yielded diol
16. This was carbamoylated with methyl isocyanate to
provide the desired product 18.
a
(i) K2CO3/DMF/70-75 °C/2.5 h; (ii) Pt-C/H2/MeOH/20 °C/2 h.
Sch em e 2a
The zwitterionic acid 19 was reacted with amines 11
and 12, employing BOP-Cl as the coupling reagent, to
give the diesters 20 and 21, respectively, in moderate
yields (Scheme 3). Attempts to effect this reaction with
a number of other coupling reagents were unsuccessful.
Reduction of 20 and 21 with lithium aluminum hydride
gave diols 22 and 23, which were subsequently carbam-
oylated to provide the target compounds 24 and 25.
Scheme 4 outlines the preparation of the bis(hy-
droxymethyl)pyrrolizines 30, 32, and 37. The N-acyl-
prolines 26 and 33, which were obtained by reaction of
(S)-proline with the appropriate acid chloride under
Schotten-Baumann conditions,10 were heated with
acetic anhydride/dimethyl acetylenedicarboxylate to
give the 1,3-dipolar cycloaddition products 27 and 34,
respectively.10 Nitro group reduction by Pt/C-catalyzed
hydrogenation yielded amines 28 and 35. Compound 28
was coupled with 9-chloroacridine to furnish the anili-
noacridine 29, and subsequent reduction of this with
lithium aluminum hydride yielded the desired diol 30.
The pyrrolizine diols 32 and 37 were obtained by
hydride reduction of the corresponding diesters 31 and
36, which in turn were prepared by BOP-Cl induced
coupling of acid 19 with amines 28 and 35, respectively.
a
(i) LiAlH4/THF/10-20 °C/3 h; (ii) 9-chloroacridine/MeOH/20
°C/2-4 h; (iii) MeNCO/Bu2Sn(OAc)2/THF/20 °C/1.5-2.5 h.
Sch em e 3a
a
(i) Bis(2-oxo-3-oxazolidinyl)phosphinic chloride/DMF/20 °C/2
h, then 11 and 12/iPr2EtN/20 °C/6 h; (ii) LiAlH4/THF/10-20 °C/4
h; (iii) MeNCO/Bu2Sn(OAc)2/DMF/20 °C/2 h.
Resu lts a n d Discu ssion
diffusion and alkylation processes. Compound 15 pro-
vided a concentration-dependent alteration in the degree
of superhelicity of the DNA, analogous to the effect
seen16 with the classical DNA intercalator ethidium
bromide (results not shown). At a drug concentration
of 1 µM, the presence of the relaxed (type II) form of
the DNA, migrating more slowly than the supercoiled
Mod e of Non cova len t DNA Bin d in g. The com-
pounds studied are listed in Table 1. The ability of the
acridine-linked compound 15 to intercalate was deter-
mined by electrophoresis studies with negatively su-
percoiled pBR322 (type I DNA) on 1% agarose gels
containing varied concentrations of drug (Figure 1).
These studies were carried out at 4 °C to minimize drug

4746 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Atwell et al.
Sch em e 4a
F igu r e 2. DNA interstrand cross-linking by the acridine-
targeted and minor groove-targeted compounds at drug con-
centration of 0.25 mM. Linear pBR322 (EcoRI digested, 2 µg)
was incubated with drugs at 37 °C for 30 min prior to
denaturation in the mixture of SDS and CH3HgOH at room
temperature for 45 min and then was subjected to electro-
phoresis through 1% agarose. Control double-stranded DNA
contains no drug and is nondenatured (DS) or denatured (SS).
Lanes 1-13 represent compounds 15-18, 30, 22-25, 32, 37,
3, and 2, respectively.
pounds toward calf thymus DNA at 37 °C in pH 7.0 SHE
buffer was determined by determining kobs (the rate of
loss of unbound drug) by HPLC. This kinetic rate
constant is the sum of the rate of drug loss by both
hydrolysis and by irreversible alkylation of the DNA.
By this measure, under these conditions the bis(hy-
droxymethyl)imidazoles 15, 16, 22, and 23 and the
related 2-unsubstituted bis(carbamates) 17 and 24 were
very stable, suggesting little DNA interaction. In con-
trast, carmethizole (3) and the related 2-thioimidazole
bis(carbamates) 18 and 25 had half-lives of 86, 72, and
130 min, respectively, under these conditions, and these
were broadly similar to that (128 min) reported previ-
ously for carmethizole at 25 °C in pH 7.4 phosphate
buffer in the absence of DNA.17 The most reactive
compounds were the pyrrolizine analogues 30, 32, and
37, with half-lives of 62, 62, and 73 min, respectively
(see Table 1 for kobs values).
a
(i) Dimethyl acetylenedicarboxylate/Ac2O/130-140 °C/2 h; (ii)
Pt-C/H2/MeOH/THF/20 °C/4 h; (iii) 9-chloroacridine/MeOH/H+/20-
60 °C/30 min; (iv) 19/bis(2-oxo-2-oxazolidinyl)phosphinic chloride/
DMF/iPr2EtN/20 °C/7.5 h; (v) LiAlH4/THF/20 °C/3-19 h.
Ta ble 1. Physicochemical and Biological Data for Analogues of
Carmethizole and Related Agents
alkylating
unit
kobs
P388b
IC50 (µM)
a
no.
(s-1 × 104)
Standard
3
carmethizole
1.34
>20
Intercalating Carriers
bis(OH)imidazole
Efficien cy of DNA In ter str a n d Cr oss-Lin k in g.
Previous studies12,13 using short DNA oligomers have
shown that both untargeted and targeted analogues (1
and 4, respectively) can cross-link double-stranded
DNA. In the present work, the comparative interstrand
cross-linking by the drugs was investigated by incubat-
ing them (0.25 mM) for 5, 30, or 60 min with pBR322
plasmid DNA linearized with EcoRI (2 µg), using
methylmercury hydroxide and SDS as denaturing re-
agents.18 2-Mercaptoethanol was used to renature drug
cross-linked DNA, which was separated from denatured
single-strand DNA on an agarose gel. Maximal cross-
linking was seen after 30 min incubation, and these
comparative results are shown in Figure 2. No cross-
linking was shown at any time point by the bis-
(hydroxymethyl)imidazoles 15 and 22 (lanes 1 and 6 in
Figure 2), the bis(hydroxymethyl)thioimidazoles 16 and
23 (lanes 2 and 7), or the imidazole bis(carbamates) 17
and 24 (lanes 3 and 8). However, the more reactive
thioimidazole bis(carbamates) 18 and 25 did cross-link
at 0.25 mM (lanes 4 and 9), and the pyrrolizine
analogues (30, 32, 37) were even more effective (lanes
5, 10, and 11). High cross-linking efficiencies were also
observed for the “untargeted” alkylators 3 and 2 (lanes
12 and 13).
15
16
17
18
30
NDc
v.slow
ND
12
1.3
16
0.8
1.3
bis(OH)thioimidazole
imidazole bis(carbamate)
thioimidazole bis(carbamate)
bis(OH)pyrrolizine
1.61
1.87
Minor Groove Carriers
22
23
24
25
32
37
bis(OH)imidazole
ND
>20
>20
>20
11
bis(OH)thioimidazole
imidazole bis(carbamate)
thioimidazole bis(carbamate)
bis(OH)pyrrolizine
ND
ND
0.88
1.86
1.58
0.11
0.03
bis(OH)pyrrolizine
Rate constants (s-1 × 104) for loss of drug when incubated with
calf thymus DNA in SHE buffer (pH 7.0) at 37 C, as determined
by HPLC (see text). IC50; concentration of drug (in µM) to reduce
cell numbers to 50% of control values after a 72 h exposure. Values
are the average of three independent determinations. c ND; not
determined.
a
°
b
F igu r e 1. Unwinding of supercoiled plasmid pBR322 on 1%
agarose gel containing various concentrations of compound 15.
Drug concentrations are labeled on the bottom of the gel.
Sequ en ce Selectivity of Dr u g Alk yla tion Deter -
m in ed by P olym er a se Ch a in Rea ction (P CR) Stop
Assa y. However, despite this observed cross-linking
activity, none of the compounds showed alkylation
patterns in the standard strand cleavage assays (heat
( piperazine)19,20 (data not shown). The major products
of the reaction of 1 and 4 with DNA have been
form, indicates intercalation by the drug. As the drug
concentration is further increased, positive superhelical
turns are generated, resulting in an increase in mobility.
Kin etics of Alk yla tion (Cova len t Bin d in g). An
estimate of the comparative reactivities of the com-

DNA-Directed Alkylating Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4747
shown12,13 (by isolation and structural identification) to
be at the exocyclic 2-amino group of guanine, and it is
known21 that thermal strand cleavage methods do not
readily cleave this type of adduct. Nevertheless, while
these assays give no data on these adducts, the lack of
other cleavage sites in the gels suggests that the present
analogues do not react significantly at N7 and N3 purine
sites.
To further examine the sequence specificity of DNA
alkylation by these compounds, the PCR technique was
used in conjunction with the gpt region of plasmid
gpt2Eco (Figures 3 and 4). In this technique (polymerase
stop assay),22,23 the target DNA is reacted with drug,
denatured, and reannealed with a complementary syn-
thetic oligonucleotide primer which carries a single 32P
radioactive label at its 5′-terminus. This radioactive
primer is then extended by copying the DNA strand in
the nearby region of interest with Taq polymerase. DNA
synthesis stops when the polymerase encounters any
blocking lesion (inter- and intrastrand cross-links and
monoalkylation events, including adducts at the gua-
nine 2-amino group). This creates a truncated strand
which still has the 5′-end of the original primer but a
unique 3′-end corresponding to the blockage site and can
be identified as usual by separation of the collection of
extended primers on high-resolution DNA sequence gels
followed by autoradiography. Only extended primers are
observed upon autoradiography because the 32P end
label on the primer is the only source of radioactivity.24
Figures 3 and 4 show that the primer extension
technique (primers 1 and 2 labeled by 32P, respectively)
can identify certain modified DNA sequences at single
nucleotide resolution, and the patterns of alkylation by
the more reactive drugs (18, 25, 30, 32, 37) are shown.
The PCR blocking sites for the thioimidazole bis-
(carbamates) 18 and 25 (lanes 1 and 2 in both Figures
3 and 4) are quite different from the patterns caused
by the pyrrolizine analogues 30, 32, 37 (lanes 4-6 in
both Figures 3 and 4). The thioimidazole bis(carbam-
ates) 18 and 25 clearly show higher DNA sequence
recognition, with the PCR blocking sites mainly in
C-rich regions (Figures 3 and 4). This suggests that the
alkylation sites of these compounds are at guanines on
the opposite strand of the DNA. The intercalator 18
(lane 1 in both Figures 3 and 4) is more reactive than
the minor-groove-targeted 25 (lane 2 in both Figures 3
and 4), a result consistent with the previous drug
stability studies.
Figure 5 summarizes the sites blocked by compound
18 (the gels are shown in Figures 3 and 4). The
sequences 5′-NC*C*N, 5′-NGC*N, and 5′-NC*GN are
found to be the most favored blocking sites (* indicate
block sites). This suggests that drug alkylation may
occur at guanines at the sequences 5′-NGGN, 5′-NCGN,
and 5′-NGCN on the opposite strand of DNA (underlines
represent alkylation sites). The untargeted thioimida-
zole bis(carbamate) carmethizole (3) shows a blocking
pattern similar to those of 18 and 25, although higher
intensity bands are also observed at sequences 5′-
TTAC*TGG and 5′-AGC*TAC*GAT at the top of the gel
in Figure 3. In contrast, the PCR assay shows little
sequence specificity in blocking sites for the more
reactive pyrrolizine analogues 30, 32, 37 (lanes 4-6 in
both Figures 3 and 4) and for the untargeted pyrrolizine
F igu r e 3. Primer 1 (labeled at 5′-end) extension blocked by
the drug alkylation on the template DNA. Control tracks
labeled “Ct” contained no drug. Tracks labeled A, G, C, and T
represent the DNA sequence reactions upon the primer
extension in the solution containing ddNTP mixtures and Taq
polymerase. Lanes 1-7 represent compounds 18, 25, 3, 30,
32, 37, 2.
2 (lane 7 in both Figures 3 and 4). For these compounds
the blocked sites also occur one base next to the actual
alkylation sites, probably due to the bulky nature of the
pyrrolizine moiety.
Overall, Figures 2-5 provide strong evidence that
these drugs (both targeted and untargeted) cross-link
double-stranded DNA through alkylation at guanine

4748 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Atwell et al.
F igu r e 5. Summary of the PCR blocking sites generated by
compound 18 (from Figures 3 and 4).
These alkylation sites represent sites of drug monoalky-
lation and intrastrand and interstrand cross-linking.
The PCR reaction (Figures 3 and 4) shows clear differ-
ences in alkylation patterns between the thioimidazole
bis(carbamate) and the more reactive pyrrolizines, with
the former alkylating with higher specificity.
Cytotoxicity. The compounds were evaluated for
cytotoxicity against P388 murine leukemia cells in
culture, and the results, as IC50 values, are shown in
Table 1. The cytotoxicities correlated broadly with the
reactivities of the alkylating units as judged by the drug
stability studies. The least reactive bis(hydroxymethyl)-
imidazoles 15, 16, 22, and 23 and the related 2-unsub-
stituted bis(carbamates) 17 and 24 were the least active,
with IC50s >1 µM. A comparison of pairs of intercalator-
linked and minor groove binder linked pairs of com-
pounds bearing indentical alkylating functions (15 &
22, 16 & 23, 17 & 24) showed in each case that the
intercalator-linked compounds were the more active.
Comparison of the more reactive thioimidazole bis-
(carbamates) 3, 18, and 25 again showed the interca-
lator-linked analogue 18 to be the more potent (IC50
)
0.8 compared to 11 µM), but both 18 and 25 were more
effective than the untargeted carmethizole 3 (IC50 > 20
µM). As well as being the most reactive analogues, the
pyrrolizines 30, 32 and 37 were also the most cytotoxic,
with IC50s of 1.3, 0.11, and 0.03 µM, respectively (Table
1). In this series the minor-groove-targeted compounds
were the more cytotoxic.
Con clu sion s
This study was designed to explore the degree to
which the patterns of both DNA alkylation and biologi-
cal activity of the bis(hydroxymethyl)pyrrolizines and
analogues could be altered by attachment to various
DNA-affinic carriers. The unwinding experiments show
that some of the acridine-linked compounds (e.g., 15)
do intercalate prior to alkylation and had slightly faster
rates of alkylation than the corresponding parent com-
pounds, suggesting that initial intercalation by the
acridine moiety somewhat enhances subsequent DNA
alkylation. Overall, the bis(hydroxymethyl)imidazoles,
bis(hydroxymethyl)thioimidazoles, and imidazole bis-
(carbamates) are weak DNA alkylating agents, with the
pyrrolizines being more effective. The cleavage and
polymerase stop assays suggest that alkylation occurs
F igu r e 4. Primer 2 (labeled at 5′-end) extension blocked by
the drug alkylation on the template DNA. Control tracks
labeled “Ct” contained no drug. Tracks labeled A, G, C, and T
represent the DNA sequence reactions upon the primer
extension in the solution containing ddNTP mixtures and Taq
polymerase. Lanes 1-7 represent compounds 18, 25, 3, 30,
32, 37, 2.
residues (via the 2-amino group) and that there are few
alkylations at N3 and N7 guanine and adenine sites.
Although a recent study of the untargeted drug 1
showed that it formed an interstrand cross-link with the
short oligonucleotide at sequence 5′-ACGT, we have
found that the sequences 5′-NGGN, 5′-NCGN, and 5′-
NGCN are the favored alkylation sites by the drugs.

DNA-Directed Alkylating Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4749
°C, gave the unstable 12 (99% yield) which was used im-
mediately: 1H NMR [(CD3)2SO] δ 6.84 (d, J ) 8.3 Hz, 2 H,
H-2′,6′), 6.49 (d, J ) 8.3 Hz, 2 H, H-3′,5′), 4.88 (br s, 2 H, NH2),
4.25 (q, J ) 7.1 Hz, 2 H, CH2CH3), 4.24 (q, J ) 7.1 Hz, 2 H,
CH2CH3), 4.04 (t, J ) 7.8 Hz, 2 H, NCH2), 2.61 (s, 3 H, SCH3),
2.42 (t, J ) 7.6 Hz, 2 H, NCH2CH2CH2), 1.91-1.81 (m, 2 H,
NCH2CH2), 1.26 (t, J ) 7.0 Hz, 3 H, CH2CH3), 1.24 (t, J ) 7.1
Hz, 3 H, CH2CH3). HRMS (DEI) [M+]: found, 391.1561; calcd
for C19H25N3O4S, 391.1566.
1-[3-[4-(9-Acr idin ylam in o)ph en yl]pr opyl]-4,5-bis(N-m e-
th ylca r ba m oyloxym eth yl)im id a zole (17) (Sch em e 2). A
solution of 11 (900 mg, 2.84 mmol) in anhydrous THF (30 mL)
was added dropwise over a 1 h period to a stirred suspension
of LiAlH4 (755 mg, 19.89 mmol) in anhydrous THF (60 mL) at
20 °C. The mixture was stirred for a further 2 h at 20 °C and
then treated dropwise and sequentially with water (0.76 mL),
15% aqueous NaOH (0.76 mL), and water (2.3 mL). The
mixture was warmed, filtered through a Celite pad, and the
solids were washed with hot THF. The filtrate was concen-
trated to dryness under reduced pressure below 30 °C, and
the residue was extracted with hot CH2Cl2, filtered, and then
reevaporated. The residue was crystallized twice from EtOAc
to give 1-[3-(4-aminophenyl)propyl]-4,5-bis(hydroxymethyl)-
imidazole (13) (420 mg, 57%): mp 121-122 °C (hygroscopic);
1H NMR [(CD3)2SO] δ 7.51 (s, 1 H, H-2), 6.84 (d, J ) 8.3 Hz,
2 H, H-2′,6′), 6.48 (d, J ) 8.3 Hz, 2 H, H-3′,5′), 4.86 (t, J ) 5.3
Hz, 1 H, OH), 4.83 (s, 2 H, NH2), 4.59 (t, J ) 5.5 Hz, 1 H, OH),
4.44 (d, J ) 5.1 Hz, 2 H, CH2OH), 4.32 (d, J ) 5.5 Hz, 2 H,
CH2OH), 3.92 (t, J ) 7.3 Hz, 2 H, NCH2), 2.40 (t, J ) 7.8 Hz,
2 H, NCH2CH2CH2), 2.00-1.90 (m, 2 H, NCH2CH2). Anal.
(C14H19N3O2‚0.5H2O) C, H, N.
9-Chloroacridine (0.32 g, 1.50 mmol) was added to a solution
of 13 (0.44 g, 1.68 mmol) in anhydrous MeOH (25 mL), and
the mixture was stirred at 20 °C for 4 h and then heated under
reflux for 15 min. Addition of EtOAc to the cooled solution
provided a semisolid which was dissolved in warm water.
Filtration and basification with aqueous Na2CO3 yielded a
crude product which was crystallized twice from MeOH/EtOAc
to give 1-[3-[4-(9-acridinylamino)phenyl]propyl]-4,5-bis(hy-
droxymethyl)imidazole (15) (0.58 g, 88%): mp 201 °C; 1H NMR
[(CD3)2SO] δ 10.83 (s), 9.17 (s), 8.30 (br s), 8.14 (d, J ) 8.6
Hz), 8.08 (d, J ) 8.7 Hz), 7.76 (t, J ) 7.4 Hz), 7.55 (d, J ) 9.3
Hz), 7.42 (t, J ) 7.5 Hz), 7.28 (d, J ) 8.0 Hz), 7.16 (d, J ) 8.2
Hz), 7.05 (d, J ) 4.2 Hz), 6.78 (d, J ) 8.3 Hz), 6.68 (d, J ) 8.2
Hz) [identifiable anilinoacridine protons, mixture of imino and
amino forms], 7.56 (s, H-2), 4.92 (t, J ) 5.2 Hz, 1 H, OH), 4.63
(t, J ) 5.5 Hz, 1 H, OH), 4.48 (d, J ) 5.1 Hz, 2 H, CH2OH),
4.34 (d, J ) 5.4 Hz, 2 H, CH2OH), 3.99 (t, J ) 7.3 Hz, 2 H,
NCH2), 2.59 (t, J ) 7.6 Hz, 2 H, NCH2CH2CH2), 2.15-2.02
(m, 2 H, NCH2CH2). HRMS (DEI) [M+]: found, 438.2047; calcd
for C27H26N4O2, 438.2056. Anal. (C27H26N4O2) C, H, N.
only at the 2-amino group of guanine residues, with the
nature of the carrier group not significantly modulating
the pattern. This is at variance with other work, which
shows that the sites of alkylation of both nitrogen
mustards19,25 (N7 of guanine) and cyclopropylindolines26
(N3 of adenine) can be altered by appropriate targeting,
and suggests that this particular covalent reaction can
only take place at exocyclic primary amines. The cyto-
toxicities of the compounds correlated broadly with their
reactivities, and for compounds bearing identical alky-
lating units, the intercalator-linked analogues were in
most cases the more potent. Both of the targeted
thioimidazole bis(carbamates) were more cytotoxic than
carmethizole, demonstrating the utility of DNA-target-
ing. The pyrrolizine analogues were the most reactive
and the most cytotoxic, with the longer chain compound
37 having an IC50 of 0.03 µM.
Exp er im en ta l Section
Analyses were carried out in the Microchemical Laboratory,
University of Otago, Dunedin, New Zealand. Melting points
were determined on an Electrothermal 2300 digital melting
point apparatus and are as read. NMR spectra were obtained
on a Bruker AC-200 or AM-400 spectrometer and are refer-
enced to Me4Si. Mass spectra were determined on a VG 707
mass spectrometer at nominal 5000 resolution. Thin-layer
chromatography was carried out on aluminum-backed silica
gel plates (Merck 60 F254). Column chromatography was
carried out on silica gel, (Merck 230-400 mesh). Compounds
for biological evaluation were of >99% purity, as determined
by reverse-phase HPLC.
Dim eth yl 1-[3-(4-Am in op h en yl)p r op yl]im id a zole-4,5-
d ica r boxyla te (11) a n d Dieth yl 2-(Meth ylth io)-1-[3-(4-
a m in op h en yl)p r op yl]im id a zole-4,5-d ica r b oxyla t e (12)
(Sch em e 1). A mixture of 3-(4-nitrophenyl)propyl bromide (6)
(3.15 g, 12.9 mmol), dimethyl imidazole-4,5-dicarboxylate (7)
(2.48 g, 13.5 mmol), powdered K2CO3 (2.68 g, 19.4 mmol), and
DMF (30 mL) was stirred at 70 °C for 2.5 h. The hot mixture
was filtered, the solids were washed with DMF, and the filtrate
was concentrated under reduced pressure below 60 °C. Addi-
tion of water provided a solid which was chromatographed on
silica gel. Elution with EtOAc/MeOH (20:1) gave dimethyl 1-[3-
(4-nitrophenyl)propyl]imidazole-4,5-dicarboxylate (9) (3.95 g,
1
88%): mp (EtOAc/i-Pr2O) 78-79 °C; H NMR (CDCl3) δ 8.16
(d, J ) 8.6 Hz, 2 H, H-3′,5′), 7.54 (s, 1 H, H-2), 7.33 (d, J ) 8.6
Hz, 2 H, H-2′,6′), 4.27 (t, J ) 7.2 Hz, 2 H, NCH2), 3.93 (s, 3 H,
CH3), 3.92 (s, 3 H, CH3), 2.76 (t, J ) 7.9 Hz, 2 H, NCH2-
CH2CH2), 2.23-2.12 (m, 2 H, NCH2CH2). Anal. (C16H17N3O6)
C, H, N.
A suspension of 15 (0.48 mg, 1.09 mmol) in anhydrous THF
(50 mL) was treated with methyl isocyanate (0.25 g, 4.39
mmol) followed by dibutyltin diacetate (1 drop) and stirred at
20 °C for 2.5 h. The resulting homogeneous solution was
concentrated under reduced pressure below 30 °C, and the
residue was triturated with EtOAc and cooled for a prolonged
period to give 17 (0.55 g, 91%): mp MeOH/EtOAc) 198-199
Similar reaction of 6 and diethyl 2-(methylthio)imidazole-
4,5-dicarboxylate8 (8), followed by chromatography on silica
gel, eluting with EtOAc, gave diethyl 2-(methylthio)-1-[3-(4-
nitrophenyl)propyl]imidazole-4,5-dicarboxylate (10) (90% yield)
as an oil: 1H NMR (CDCl3) δ 8.16 (d, J ) 8.7 Hz, 2 H, H-3′,5′),
7.34 (d, J ) 8.7 Hz, 2 H, H-2′,6′), 4.38 (q, J ) 7.0 Hz, 2 H,
CH2CH3), 4.32 (q, J ) 7.0 Hz, 2 H, CH2CH3), 4.20 (t, J ) 7.6
Hz, 2 H, NCH2), 2.79 (t, J ) 7.9 Hz, 2 H, NCH2CH2CH2), 2.70
(s, 3 H, SCH3), 2.16-2.06 (m, 2 H, NHCH2CH2), 1.38 (t, J )
7.1 Hz, 3 H, CH2CH3), 1.34 (t, J ) 7.1 Hz, 3 H, CH2CH3). Anal.
(C19H23N3O6S‚0.5H2O) C, H, N, S.
A solution of 9 (4.00 g, 11.5 mmol) in MeOH (40 mL) was
hydrogenated over Pt/C at 50 psi for 2 h. The crude product
was chromatographed on silica gel, eluting with EtOAc/MeOH
(19:1) to give 11 (3.47 g, 95%) as a waxy solid: mp 59-60 °C;
1H NMR [(CD3)2SO] δ 7.97 (s, 1 H, H-2), 6.82 (d, J ) 8.2 Hz,
2 H, H-2′,6′), 6.49 (d, J ) 8.3 Hz, 2 H, H-3′,5′), 4.86 (br s, 2 H,
NH2), 4.14 (t, J ) 7.2 Hz, 2 H, NCH2), 3.80 (s, 3 H, CH3), 3.78
(s, 3 H, CH3), 2.36 (t, J ) 7.7 Hz, 2 H, NCH2CH2CH2), 2.01-
1.82 (m, 2 H, NCH2CH2). Anal. (C16H19N3O4) C, H, N.
Similar reduction of 10, followed by removal of the catalyst
and evaporation under reduced pressure (0.1 Torr) below 30
1
°C; H NMR [(CD3)2SO] δ 10.83 (s), 9.16 (s), 8.13 (br s), 7.76
(t, J ) 7.6 Hz), 7.69 (d, J ) 8.9 Hz), 7.42 (t, J ) 7.6 Hz), 7.28
(d, J ) 8.1 Hz), 7.16 (d, J ) 8.1 Hz), 6.77 (d, J ) 8.2 Hz), 6.68
(d, J ) 8.2 Hz) [identifiable anilinoacridine protons, mixture
of imino and amino forms], 7.70 (s, H-2), 7.06 (q, J ) 6.8 Hz,
1 H, NHCH3), 6.94 (br d, J ) 4.4 Hz, 1 H, NHCH3), 5.10 (s, 2
H, CH2O), 4.92 (s, 2 H, CH2O), 3.99 (t, J ) 7.3 Hz, 2 H, NCH2),
2.64-2.48 (m, 8 H, 2 × CH3, NCH2CH2CH2), 2.10-1.99 (m, 2
H, NCH2CH2). HRMS (DAB) [M + H]+: found, 553.2552;
C31H33N6O4 requires 553.2563. Anal. (C31H32N6O4) C, H, N.
1-[3-[4-(9-Acr id in yla m in o)p h en yl]p r op yl]-2-(m et h yl-
th io)-4,5-bis(N-m eth ylcar bam oyloxym eth yl)im idazole (18)
(Sch em e 2). A mixture of powdered 9-chloroacridine (1.07 g,
5.01 mmol) and freshly prepared 12 (2.07 g, 5.29 mmol) in
MeOH (25 mL) was stirred at 20 °C until homogeneous and
then treated with 12 N HCl (0.05 mL). After the mixture was

4750 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Atwell et al.
stirred at 20 °C for a further 1 h, it was heated under reflux
for 30 min and then concentrated to 5 mL and diluted with
EtOAc. The resulting solid was crystallized from MeOH/
aqueous KHCO3, followed by purification by alumina chroma-
tography (elution with MeOH). Appropriate fractions were
pooled, triturated with i-Pr2O, and crystallized from MeOH/
i-Pr2O to give diethyl 1-[3-[4-(9-acridinylamino)phenyl]propyl]-
2-(methylthio)imidazole-4,5-dicarboxylate (14) (1.71 g, 60%):
mp 143-145 °C; 1H NMR [(CD3)2SO] δ 10.83 (s), 9.18 (s), 8.31
(br s), 8.14 (d, J ) 8.7 Hz), 8.08 (d, J ) 8.6 Hz), 7.76 (t, J )
7.5 Hz), 7.42 (t, J ) 7.5 Hz), 7.29 (d, J ) 8.1 Hz), 7.16 (d, J )
8.1 Hz), 7.04 (d, J ) 8.0 Hz), 6.78 (d, J ) 7.9 Hz), 6.69 (d, J )
8.1 Hz) [identifiable anilinoacridine protons, mixture of imino
and amino forms], 4.28 (q, J ) 7.0 Hz, 2 H, CH2CH3), 4.26 (q,
J ) 7.0 Hz, 2 H, CH2CH3), 4.10 (t, J ) 7.7 Hz, 2 H, NCH2),
2.65-2.58 (m, 5 H, SCH3, NCH2CH2CH2), 2.04-1.90 (m, 2 H,
NCH2CH2), 1.27 (t, J ) 7.1 Hz, 6 H, 2 × CH2CH3). HRMS (EI)
[M+]: found, 568.2158. calcd for C32H32N4O4S, 568.2144. Anal.
(C32H32N4O4S) C, H, N, S.
crystallized twice from MeOH/H2O to give dimethyl 1-[3-[4-
[4-(4-quinolinylamino)benzamido]phenyl]propyl]imidazole-4,5-
dicarboxylate (20) (1.20 g, 70%): mp 206-207 °C; 1H NMR
[(CD3)2SO] δ 10.11 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.58 (d, J
) 4.9 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 8.5 Hz, 1 H,
quinoline H-8), 8.02 (s, 1 H, H-2), 8.01 (d, J ) 7.5 Hz, 2 H,
PhH), 7.93 (d, J ) 8.4 Hz, 1 H, quinoline H-5), 7.74 (t, J ) 8.2
Hz, 1 H, quinoline H-7), 7.72 (d, J ) 7.9 Hz, 2 H, PhH), 7.58
(t, J ) 7.6 Hz, 1 H, quinoline H-6), 7.48 (d, J ) 8.2 Hz, 2 H,
PhH), 7.21 (d, J ) 4.8 Hz, 1 H, quinoline H-3), 7.18 (d, J ) 8.2
Hz, 2 H, PhH), 4.20 (t, J ) 7.2 Hz, 2 H, NCH2), 3.82 (s, 3 H,
CH3), 3.79 (s, 3 H, CH3), 2.54 (t, J ) 7.7 Hz, 2 H, NCH2-
CH2CH2), 2.08-1.96 (m, 2 H, NCH2CH2). Anal. (C32H29N5O5)
C, H, N.
A hot solution of 20 (1.36 g, 2.41 mmol) in anhydrous THF
(300 mL) was cooled to 20 °C and added dropwise over a 1 h
period to a stirred suspension of LiAlH4 (1.10 g, 29.0 mmol)
in anhydrous THF (130 mL) at 20 °C. The mixture was stirred
for
a further 2 h at 20 °C, then treated dropwise and
A solution of 14 (300 mg, 0.53 mmol) in anhydrous THF
(25 mL) was added dropwise over a 1 h period to a stirred
suspension of LiAlH4 (200 mg, 5.27 mmol) in anhydrous THF
(60 mL) at 5-10 °C. The mixture was stirred for a further 2 h
at 10 °C, treated dropwise and sequentially with water (0.2
mL), 15% aqueous NaOH (0.2 mL), and water (0.6 mL). The
mixture was filtered through a Celite pad, and the solids were
washed with hot THF. The filtrate was concentrated under
reduced pressure below 30 °C to 30 mL and then diluted with
aqueous KHCO3. The precipitated solid was extracted with hot
EtOAc, impurities were removed by filtration, and the solution
was then concentrated and diluted with i-Pr2O to provide a
crude product. Crystallization from THF-iPr2O gave 1-[3-[4-
(9-acridinylamino)phenyl]propyl]-2-(methylthio)-4,5-bis(2-hy-
droxymethyl)imidazole (16) (210 mg, 82%): mp 186-187 °C;
1H NMR [(CD3)2SO] δ 10.82 (s), 9.18 (s), 8.31 (br s), 8.14 (d, J
) 8.6 Hz), 8.07 (d, J ) 8.6 Hz), 7.75 (t, J ) 7.6 Hz), 7.41 (t, J
) 7.3 Hz), 7.28 (d, J ) 7.7 Hz), 7.17 (d, J ) 8.3 Hz), 7.06 (d, J
) 8.4 Hz), 6.79 (d, J ) 8.4 Hz), 6.69 (d, J ) 8.3 Hz) [identifiable
anilinoacridine protons, mixture of imino and amino forms],
4.94 (t, J ) 5.3 Hz, 1 H, OH), 4.68 (t, J ) 5.6 Hz, 1 H, OH),
4.46 (d, J ) 5.3 Hz, 2 H, CH2OH), 4.32 (d, J ) 5.6 Hz, 2 H,
CH2OH), 3.93 (t, J ) 7.8 Hz, 2 H, NCH2), 2.64 (t, J ) 7.4 Hz,
2 H, NCH2CH2CH2), 2.57 (s, 3 H, SCH3), 2.05-1.96 (m, 2 H,
NCH2CH2). HRMS (FAB) [M + H]+: Found, 485.1998; calcd
for C28H29N4O2S, 485.2011. Anal. (C28H28N4O2S) C, H, N, Cl.
sequentially with water (1.1 mL), 15% aqueous NaOH (1.1
mL), and water (3.3 mL). The mixture was diluted with MeOH
(250 mL), warmed, and filtered through a Celite pad, and the
solids were washed with MeOH/THF (1:1). The filtrate was
concentrated under reduced pressure to 50 mL, and aqueous
Na2CO3 was then added. The resulting solid was crystallized
successively from MeOH/EtOAc and MeOH to give 1-[3-[4-[4-
(4-quinolinylamino)benzamido]phenyl]propyl]-4,5-bis(hydroxym-
1
ethyl)imidazole (22) (0.76 g, 63%): mp 149-153 °C; H NMR
[(CD3)2SO] δ 10.11 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.59 (d, J
) 5.2 Hz, 1 H, quinoline H-2), 8.39 (d, J ) 7.9 Hz, 1 H,
quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.94 (d, J ) 8.0
Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.5 Hz, 1 H, quinoline
H-7), 7.72 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.5 Hz, 1 H,
quinoline H-6), 7.56 (s, 1 H, H-2), 7.48 (d, J ) 8.7 Hz, 2 H,
PhH), 7.21 (d, J ) 5.1 Hz, 1 H, quinoline H-3), 7.20 (d, J ) 8.5
Hz, 2 H, PhH), 4.91 (t, J ) 5.3 Hz, 1 H, OH), 4.62 (t, J ) 5.5
Hz, 1 H, OH), 4.48 (d, J ) 5.2 Hz, 2 H, CH2OH), 4.34 (d, J )
5.4 Hz, 2 H, CH2OH), 3.98 (t, J ) 7.3 Hz, 2 H, NCH2), 2.57 (t,
J ) 7.8 Hz, 2 H, NCH2CH2CH2), 2.12-2.01 (m, 2 H, NCH2CH2).
HRMS (FAB) [M + H]+: found, 508.2343; calcd for C30H30N5O3,
508.2349. Anal. (C30H29N5O3‚H2O) C, N. H: found, 6.4; requires
5.9%.
A mixture of 22 (140 mg, 0.28 mmol), methyl isocyanate (96
mg, 1.68 mmol), anhydrous DMF (1 mL), and anhydrous THF
(2 mL) was treated with dibutyltin diacetate (1 drop) and
stirred at 20 °C for 2 h. The mixture was diluted with aqueous
Na2CO3, and the resulting solid was dried and dissolved in
MeOH at 20 °C. The solution was clarified by filtration, diluted
with EtOAc, and then concentrated under reduced pressure
below 30 °C. The product which separated was crystallized
from MeOH/EtOAc to give 24 (143 mg, 83%): mp 198-199
°C; 1H NMR [(CD3)2SO] δ 10.11 (s, 1 H, NH), 9.26 (s, 1 H, NH),
8.58 (d, J ) 5.2 Hz, 1 H, quinoline H-2), 8.39 (d, J ) 8.2 Hz,
1 H, quinoline H-8), 8.00 (d, J ) 8.6 Hz, 2 H, PhH), 7.94 (d, J
) 8.3 Hz, 1 H, quinoline H-5), 7.77-7.67 (m, 4 H, H-2,
quinoline H-7, PhH), 7.58 (t, J ) 7.5 Hz, 1 H, quinoline H-6),
7.48 (d, J ) 8.5 Hz, 2 H, PhH), 7.24-7.15 (m, 3 H, quinoline
H-3, PhH), 7.06 (q, J ) 4.6 Hz, 1 H, NHCH3), 6.94 (q, J ) 4.5
Hz, 1 H, NHCH3), 5.10 (s, 2 H, CH2O), 4.92 (s, 2 H, CH2O),
3.97 (t, J ) 7.3 Hz, 2 H, NCH2), 2.62-2.50 (m, 8 H, 2 × CH3,
NCH2CH2CH2), 2.10-1.96 (m, 2 H, NCH2CH2). HRMS (FAB)
[M + H]+: found, 622.2780; calcd for C34H36N7O5, 622.2778.
Anal. (C34H35N7O5‚2H2O). C, N. H: found, 5.5; requires 6.0%.
2-(Meth ylth io)-1-[3-[4-[4-(4-qu in olin yla m in o)ben za m i-
d o]p h en yl]p r op yl]-4,5-bis(N-m eth ylca r ba m oyloxym eth -
yl)im id a zole (25) (Sch em e 3). A suspension of 19 (1.60 g,
6.05 mmol) in DMF (25 mL) was treated with bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (97%, 1.91 g, 7.26 mmol) and
stirred at 20 °C for 2 h. The mixture was cooled to -5 °C and
treated sequentially with N,N-diisopropylethylamine (1.73 g,
13.38 mmol) and freshly prepared 12 (2.49 g, 6.36 mmol). After
stirring at 20 °C for a further 6 h, the mixture was diluted
with aqueous KHCO3 to provide a semisolid. This was dried,
triturated with EtOAc, and crystallized successively from
A mixture of 16 (402 mg, 0.83 mmol) and methyl isocyanate
(284 mg, 4.98 mmol) in DMF (4 mL) and anhydrous THF (4
mL) was treated with dibutyltin diacetate (2 drops) and stirred
at 20 °C for 1.5 h. The mixture was concentrated under
reduced pressure below 30 °C to remove THF and then diluted
with aqueous Na2CO3. The resulting solid was crystallized
from MeOH/i-P2O and then from MeOH to give 18 (446 mg,
1
90%): mp 159-162 °C; H NMR [(CD3)2SO] δ 10.82 (s), 9.17
(s), 8.31 (br s), 8.14 (d, J ) 8.6 Hz), 8.08 (d, J ) 8.7 Hz), 7.76
(t, J ) 7.1 Hz), 7.41 (t, J ) 7.5 Hz), 7.28 (d, J ) 7.8 Hz), 7.18
(d, J ) 8.2 Hz), 6.78 (d, J ) 8.4 Hz), 6.69 (d, J ) 8.2 Hz)
[identifiable anilinoacridine protons, mixture of imino and
amino forms], 7.11-7.00 (m, 1 H, NHCH3), 6.96 (q, J ) 4.5
Hz, 1 H, NHCH3), 5.09 (s, 2 H, CH2O), 4.90 (s, 2 H, CH2O),
3.89 (t, J ) 7.9 Hz, 2 H, NCH2), 2.63 (t, J ) 7.2 Hz, 2 H, NCH2-
CH2CH2), 2.59-2.50 (m, 9 H, SCH3, 2 × NHCH3), 2.01-1.91
(m, 2 H, NCH2CH2). HRMS (FAB) [M + H]+: found, 599.2427;
calcd for C32H35N6O4S, 599.2441. Anal. (C32H34N6O4S) C, H,
N, S.
1-[3-[4-[4-(4-Qu in olin yla m in o)b e n za m id o]p h e n yl]-
p r op yl]-4,5-b is(N -m e t h ylca r b a m oyloxym e t h yl)im id a -
zole (24) (Sch em e 3). A suspension of 4-(4-quinolinylamino)-
benzoic acid27 (19) (0.80 g, 3.03 mmol) in DMF (12 mL) was
treated with bis(2-oxo-3-oxazolidinyl)phosphinic chloride (97%,
0.95 g, 3.62 mmol) and stirred at 20 °C for 2 h. The mixture
was cooled to -5 °C and treated with diisopropylethylamine
(0.86 g, 6.65 mmol) immediately followed by 11 (1.01 g, 3.18
mmol). After stirring at 20 °C for a further 6 h, the mixture
was diluted with aqueous KHCO3, and the resulting solid was

DNA-Directed Alkylating Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4751
MeOH/EtOAc/i-Pr2O and then EtOAc to give diethyl 2-(me-
thylthio)-1-[3-[4-[4-(4-quinolinylamino)benzamido]-phenyl]-
propyl]imidazole-4,5-dicarboxylate (21) (2.38 g, 62%): mp 148-
150 °C; 1H NMR [(CD3)2SO] δ 10.11 (s, 1 H, NH), 9.24 (s, 1 H,
NH), 8.59 (d, J ) 5.2 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 7.9
Hz, 1 H, quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.94
(d, J ) 8.1 Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.6 Hz, 1 H,
quinoline H-7), 7.72 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.7
Hz, 1 H, quinoline H-6), 7.48 (d, J ) 8.7 Hz, 2 H, PhH), 7.24-
7.15 (m, 3 H, quinoline H-3, PhH), 4.27 (q, J ) 6.9 Hz, 2 H,
CH2CH3), 4.25 (q, J ) 6.9 Hz, 2 H, CH2CH3, 4.10 (t, J ) 7.6
Hz, 2 H, NCH2), 2.63 (s, 3 H, SCH3), 2.60 (t, J ) 7.6 Hz, 2 H,
NCH2CH2CH2), 2.03-1.90 (m, 2 H, NCH2CH2), 1.27 (t, J )
7.1 Hz, 3 H, CH2CH3), 1.26 (t, J ) 7.1 Hz, 3 H, CH2CH3). Anal.
(C35H35N5O5S) C, H, N, S.
gel, eluting with EtOAc, to give crude (S)-1-[4-(4-nitrophenyl)-
butyryl]pyrrolidine-2-carboxylic acid (26) (8.03 g, 78%) as a
gum: 1H NMR (CDCl3) δ 8.14 (d, J ) 8.7 Hz, 2 H, H-3′,5′), 7.8
(br s, 1 H, CO2H), 7.36 (d, J ) 8.7 Hz, 2 H, H-2′,6′), 4.61-4.54
(m, 1 H, CHCO2H), 3.59-3.49 (m, 1 H, CH), 3.48-3.39 (m, 1
H, CH), 2.81 (t, J ) 7.7 Hz, 2 H, CH2), 2.45-2.30 (m, 3 H,
CH2, CH), 2.13-1.97 (m, 5 H, 2 × CH2, CH). HRMS (DEI) [M+]:
found, 306.1217; calcd for C15H18N2O5, 306.1216.
A mixture of 26 (7.00 g, 23 mmol), dimethyl acetylenedi-
carboxylate (5.7 mL, 46 mmol), and Ac2O (57 mL) was stirred
and heated at 130-140 °C until CO2 evolution had ceased (ca.
2 h). The mixture was concentrated under reduced pressure,
and the residue was shaken with water to give an oil which
was chromatographed on silica gel. Elution with EtOAc gave
a solid that was crystallized twice from EtOAc/petroleum ether
(decolorizing charcoal) to give dimethyl 2,3-dihydro-5-[3-(4-
nitrophenyl)propyl]-1H-pyrrolizine-6,7-dicarboxylate (27) (6.48
A solution of 21 (1.15 g, 1.80 mmol) in anhydrous THF (125
mL) was added dropwise over a 1 h period to a stirred
suspension of LiAlH4 (0.68 g, 17.92 mmol) in anhydrous THF
(70 mL) at 10 °C. After the mixture was stirred at 10 °C for a
further 3 h, it was treated dropwise and sequentially with
water (0.7 mL), 15% aqueous NaOH (0.7 mL), and water (2.1
mL). This mixture was diluted with MeOH (200 mL), heated,
and filtered through a Celite pad, and the solids were washed
with MeOH/THF (1:1). The filtrate was concentrated under
reduced pressure below 30 °C to 50 mL and then diluted with
aqueous Na2CO3 to precipitate the crude product. Two crystal-
lizations from MeOH/EtOAc gave 2-(methylthio)-1-[3-[4-[4-(4-
quinolinylamino)benzamido]phenyl]propyl]-4,5-bis(2-hydroxym-
1
g, 73%): mp 94 °C; H NMR (CDCl3) δ 8.14 (d, J ) 8.8 Hz, 2
H, H-3′,5′), 7.32 (d, J ) 8.8 Hz, 2 H, H-2′,6′), 3.83 (t, J ) 7.1
Hz, 2 H, NCH2), 3.80 (s, 3 H, CH3), 3.78 (s, 3 H, CH3), 3.03 (t,
J ) 7.5 Hz, 2 H, CH2), 2.86-2.72 (m, 4 H, 2 × CH2), 2.58-
2.40 (m, 2 H, NCH2CH2), 2.03-1.84 (m, 2 H, PhCH2CH2). Anal.
(C20H22N2O6) C, H, N.
A solution of 27 (3.50 g, 9.1 mmol) in 1:1 MeOH/THF (40
mL) was hydrogenated over Pt/C at 60 psi for 2 h. The crude
product was chromatographed on silica gel, eluting with CH2-
Cl2/EtOAc (3:2), to give dimethyl 2,3-dihydro-5-[3-(4-ami-
nophenyl)propyl]-1H-pyrrolizine-6,7-dicarboxylate (28) (3.07 g,
1
1
ethyl)imidazole (23) (0.76 g, 76%): mp 196-197 °C; H NMR
95%): mp (EtOAc/petroleum ether) 121 °C; H NMR (CDCl3)
[(CD3)2SO] δ 10.11 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.58 (d, J
) 5.2 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 8.0 Hz, 1 H,
quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.94 (d, J ) 8.2
Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.6 Hz, 1 H, quinoline
H-7), 7.72 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.3 Hz, 1 H,
quinoline H-6), 7.48 (d, J ) 8.6 Hz, 2 H, PhH), 7.24-7.18 (m,
3 H, quinoline H-3, PhH), 4.94 (t, J ) 5.3 Hz, 1 H, OH), 4.68
(t, J ) 5.5 Hz, 1 H, OH), 4.45 (d, J ) 5.1 Hz, 2 H, CH2OH),
4.32 (d, J ) 5.3 Hz, 2 H, CH2OH), 3.92 (t, J ) 7.7 Hz, 2 H,
NCH2), 2.62 (t, J ) 7.6 Hz, 2 H, NCH2CH2CH2), 2.52 (s, 3 H,
SCH3), 2.05-1.93 (m, 2 H, NCH2CH2). HRMS (FAB) [M + H]+:
found, 554.2216; calcd for C31H32N5O3S, 554.2226. Anal.
(C31H31N5O3S) C, H, N, S.
δ 6.95 (d, J ) 8.4 Hz, 2 H, H-2′,6′), 6.62 (d, J ) 8.4 Hz, 2 H,
H-3′,5′), 3.79 (s, 3 H, CH3), 3.77 (s, 3 H, CH3), 3.77 (t, J ) 7.2
Hz, 2 H, NCH2; partially obscured), 3.56 (br s, 2 H, NH2), 3.01
(t, J ) 7.5 Hz, 2 H, CH2), 2.73 (t, J ) 7.8 Hz, 2 H, CH2), 2.54
(t, J ) 7.6 Hz, 2 H, CH2), 2.54-2.36 (m, 2 H, NCH2CH2), 1.91-
1.72 (m, 2 H, PhCH2CH2). Anal. (C20H24N2O4) C, H, N.
A solution of 28 (0.40 g, 1.12 mmol) in MeOH (25 mL) was
treated in one portion with powdered 9-chloroacridine (0.22
g, 1.03 mmol). The mixture was stirred at 20 °C until
homogeneous, 12 N HCl (half drop) was added, and the
solution was heated under reflux for 15 min. The solution was
diluted with EtOAc, concentrated, and cooled. The resulting
orange crystals were treated with MeOH/aqueous KHCO3 to
provide a solid which was crystallized from EtOAc/CH2Cl2/i-
Pr2O to give dimethyl 2,3-dihydro-5-[3-[4-(9-acridinylamino)-
phenyl]propyl]-1H-pyrrolizine-6,7-dicarboxylate (29) (0.48 g,
87%): mp 73-77 °C; 1H NMR [(CD3)2SO] δ 10.82 (s), 9.15 (s),
8.30 (br s), 8.13 (d, J ) 8.6 Hz), 8.07 (d, J ) 8.6 Hz), 7.75 (t,
J ) 7.5 Hz), 7.41 (t, J ) 7.5 Hz), 7.28 (d, J ) 8.1 Hz), 7.14 (d,
J ) 8.2 Hz), 7.03 (d, J ) 8.1 Hz), 6.77 (d, J ) 8.1 Hz), 6.67 (d,
J ) 8.2 Hz) [identifiable anilinoacridine protons, mixture of
imino and amino forms], 3.92 (t, J ) 7.1 Hz, 2 H, NCH2), 3.66
(s, 3 H, CH3), 3.65 (s, 3 H, CH3), 2.91 (t, J ) 7.4 Hz, 2 H, CH2),
2.70 (t, J ) 7.7 Hz, 2 H, CH2), 2.59 (t, J ) 7.4 Hz, 2 H, CH2),
2.49-2.36 (m, 2 H, NCH2CH2), 1.88-1.74 (m, PhCH2CH2).
HRMS (EI) [M+]: found, 533.2293; calcd for C33H31N3O4,
533.2314. Anal. (C33H31N3O4) C, H, N.
A solution of 29 (0.60 g, 1.12 mmol) in anhydrous THF (80
mL) was added dropwise over a 1 h period to a stirred
suspension of LiAlH4 (0.45 g, 1.19 mmol) in anhydrous THF
(80 mL) at 20 °C. The mixture was stirred for a further 2 h at
20 °C and then treated dropwise and sequentially with water
(0.45 mL), 15% aqueous NaOH (0.45 mL), and water (1.4 mL).
The mixture was warmed and filtered through a Celite pad,
and the solids were washed with hot THF. The filtrate was
concentrated to dryness under reduced pressure below 30 °C.
The residue was dissolved in hot anhydrous THF, diluted with
i-Pr2O to precipitate impurities (which were removed by
filtration), and then further diluted with i-Pr2O to provide the
crude product. Crystallization from THF/EtOAc/i-Pr2O gave
30 (0.38 g, 71%): mp 265-270 °C (hygroscopic); 1H NMR
[(CD3)2SO] δ 10.81 (s), 9.16 (s), 8.31 (br s), 8.14 (d, J ) 8.7
Hz), 8.07 (d, J ) 8.7 Hz), 7.75 (t, J ) 7.4 Hz), 7.41 (t, J ) 7.5
Hz), 7.28 (d, J ) 8.0 Hz), 7.15 (d, J ) 8.2 Hz), 7.04 (d, J ) 8.3
Hz), 6.78 (d, J ) 8.2 Hz), 6.67 (d, J ) 8.2 Hz) [identifiable
A mixture of 23 (360 mg. 0.65 mmol) in DMF (4 mL) and
THF (4 mL) was treated with methyl isocyanate (222 mg, 3.90
mmol), followed by dibutyltin diacetate (2 drops), and stirred
at 20 °C for 2 h. The mixture was concentrated under reduced
pressure below 30 °C to remove THF and then diluted with
aqueous Na2CO3. The resulting solid was dissolved in warm
MeOH and clarified by filtration, and then EtOAc was added.
The precipitated solid was crystallized from MeOH/EtOAc to
1
give 25 (370 mg, 85%): mp 181-182 °C; H NMR [(CD3)2SO]
δ 10.11 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.59 (d, J ) 5.2 Hz, 1
H, quinoline H-2), 8.38 (d, J ) 7.9 Hz, 1 H, quinoline H-8),
8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.93 (d, J ) 7.9 Hz, 1 H,
quinoline H-5), 7.74 (t, J ) 7.6 Hz, 1 H, quinoline H-7), 7.72
(d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.6 Hz, 1 H, quinoline
H-6), 7.48 (d, J ) 8.7 Hz, 2 H, PhH), 7.24-7.17 (m, 3 H,
quinoline H-3, PhH), 7.08 (q, J ) 4.6 Hz, 1 H, NHCH3), 6.97
(q, J ) 4.6 Hz, 1 H, NHCH3), 5.08 (s, 2 H, CH2O), 4.90 (s, 2 H,
CH2O), 3.87 (t, J ) 7.8 Hz, 2 H, NCH2), 2.61 (t, J ) 7.3 Hz, 2
H, NCH2CH2CH2), 2.59 (d, J ) 4.5 Hz, 3 H, NHCH3), 2.55 (d,
J ) 4.7 Hz, 3 H, NHCH3), 2.54 (s, 3 H, SCH3), 2.03-1.88 (m,
2 H, NCH2CH2). HRMS (FAB) [M + H]+: found, 668.2679.
calcd for C35H38N7O5S, 668.2655. Anal. (C35H37N7O5S) C, H,
N, S.
2,3-Dih yd r o-5-[3-[4-(9-a cr id in yla m in o)p h en yl]p r op yl]-
6,7-bis(h yd r oxym eth yl)-1H-p yr r olizin e (30) (Sch em e 4).
A solution of 4-(4-nitrophenyl)butyryl chloride (7.62 g, 33
mmol) in Et2O (40 mL) was added dropwise to a vigorously
stirred solution of (S)-proline (4.62 g, 40 mmol) and NaOH
(2.91 g, 73 mmol) in H2O (28 mL) at 5 °C. The mixture was
stirred at 5 °C for 1 h, then at 20 °C for 1 h, followed by
concentration to remove Et2O. Acidification with 12 N HCl
gave a precipitated oil which was chromatographed on silica

4752 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Atwell et al.
anilinoacridine protons, mixture of imino and amino forms],
4.43 (t, J ) 5.1 Hz, 1 H, OH), 4.33-4.23 (1 H, OH; obscured),
4.30 (d, J ) 4.9 Hz, 2 H, CH2OH), 4.27 (d, J ) 4.7 Hz, 2 H,
CH2OH), 3.76 (t, J ) 6.9 Hz, 2 H, NCH2), 2.71 (t, J ) 7.2 Hz,
2 H, CH2), 2.60 (t, J ) 7.4 Hz, 2 H, CH2), 2.53 (t, J ) 7.4 Hz,
2 H, CH2), 2.41-2.30 (m, 2 H, NCH2CH2), 1.83-1.68 (m, 2 H,
PhCH2CH2). HRMS (FAB) [M + H]+: found, 478.2486; calcd
for C31H32N3O2, 478.2495. Anal. (C31H31N3O2) H, N. C: found,
77.4; requires 77.9%.
) 8.7 Hz, 2 H, H-3′,5′), 7.26 (d, J ) 8.7 Hz, 2 H, H-2′,6′), 3.82
(s, 3 H, CH3), 3.79 (s, 3 H, CH3), 3.52 (t, J ) 7.2 Hz, 2 H, NCH2),
3.10-2.92 (m, 6 H, 3 × CH2), 2.46-2.27 (m, 2 H, NCH2CH2).
Anal. (C19H20N2O6) C, H, N.
Hydrogenation of 34 in MeOH/THF (1:1) over Pt/C at 60
psi gave dimethyl 2,3-dihydro-5-[2-(4-aminophenyl)ethyl]-1H-
pyrrolizine-6,7-dicarboxylate (35) (92% yield): mp (EtOAc/
petroleum ether) 153-154 °C; 1H NMR (CDCl3) δ 6.82 (d, J )
7.9 Hz, 2 H, H-2′,6′), 6.56 (d, J ) 7.9 Hz, 2 H, H-3′,5′), 3.83 (s,
3 H, CH3), 3.78 (s, 3 H, CH3), 3.59 (br s, 2 H, NH2), 3.35 (t, J
) 7.2 Hz, 2 H, NCH2), 2.98-2.87 (m, 4 H, 2 × CH2), 2.76 (t, J
) 7.2 Hz, 2 H, CH2), 2.33-2.23 (m, 2 H, NCH2CH2). Anal.
(C19H22N2O4) C, H, N.
Reaction of 35 with 19 by the method described for 31 gave
a crude product that was triturated with MeOH/EtOAc and
crystallized from THF/MeOH/H2O to give dimethyl 2,3-dihy-
dro-5-[2-[4-[4-(4-quinolinylamino)benzamido]phenyl]ethyl]-1H-
pyrrolizine-6,7-dicarboxylate (36) (75% yield): mp 208-209 °C;
1H NMR [(CD3)2SO] δ 10.12 (s, 1 H, NH), 9.26 (s, 1 H, NH),
8.59 (d, J ) 5.2 Hz, 1 H, quinoline H-2), 8.39 (d, J ) 8.4 Hz,
1 H, quinoline H-8), 8.02 (d, J ) 8.6 Hz, 2 H, PhH), 7.94 (d, J
) 8.4 Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.6 Hz, 1 H,
quinoline H-7), 7.69 (d, J ) 8.3 Hz, 2 H, PhH), 7.59 (t, J ) 7.6
Hz, 1 H, quinoline H-6), 7.49 (d, J ) 8.6 Hz, 2 H, PhH), 7.22
(d, J ) 5.2 Hz, 1 H, quinoline H-3), 7.10 (d, J ) 8.3 Hz, 2 H,
PhH), 3.74 (t, J ) 7.1 Hz, 2 H, NCH2), 3.69 (s, 3 H, CH3), 3.67
(s, 3 H, CH3), 2.94 (t, J ) 7.5 Hz, 2 H, CH2), 2.89 (t, J ) 7.4
Hz, 2 H, CH2), 2.78 (t, J ) 7.5 Hz, 2 H, CH2), 2.39-2.28 (m, 2
H, NCH2CH2). Anal. (C35H32N4O5) C, H, N.
Reduction of diester 36 using the method described for 32,
followed by crystallization of the crude product from MeOH/
EtOAc (twice) then THF/EtOAc, gave 37 (61% yield): mp 222-
227 °C; 1H NMR [(CD3)2SO] δ 10.09 (s, 1 H, NH), 9.24 (s, 1 H,
NH), 8.59 (d, J ) 5.2 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 8.2
Hz, 1 H, quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.94
(d, J ) 8.3 Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.6 Hz, 1 H,
quinoline H-7), 7.69 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.3
Hz, 1 H, quinoline H-6), 7.48 (d, J ) 8.6 Hz, 2 H, PhH), 7.21
(d, J ) 5.2 Hz, 1 H, quinoline H-3), 7.16 (d, J ) 8.5 Hz, 2 H,
PhH), 4.40 (t, J ) 4.6 Hz, 1 H, OH), 4.33-4.25 (1 H, OH,
obscured), 4.30 (d, J ) 4.8 Hz, 2 H, CH2OH), 4.27 (d, J ) 4.6
Hz, 2 H, CH2OH), 3.67 (t, J ) 6.9 Hz, 2 H, NCH2), 2.80-2.65
(m, 6 H, 3 × CH2), 2.37-2.25 (m, 2 H, NCH2CH2). Anal.
(C33H32N4O3‚H2O) C, H, N.
2,3-Dih yd r o-5-[3-[4-[4-(4-qu in olin yla m in o)ben za m id o]-
p h en yl]p r op yl]-6,7-b is(h yd r oxym et h yl)-1H -p yr r olizin e
(32) (Sch em e 4). A suspension of 19 (1.11 g, 4.20 mmol) in
DMF (15 mL) was treated with bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (1.32 g of 97%, 5.03 mmol). The mixture
was stirred at 20 °C for 1.5 h, then cooled to -5 °C, and treated
with diisopropylethylamine (1.19 g, 9.24 mmol) immediately
followed by amine 28 (1.57 g, 4.41 mmol). After being stirred
at 20 °C for a further 6 h, the mixture was diluted with
aqueous KHCO3. Trituration of the resulting solid with cold
MeOH provided a crude product which was crystallized from
DMF/MeOH/H2O to give dimethyl 2,3-dihydro-5-[3-[4-[4-(4-
quinolinylamino)benzamido]phenyl]propyl]-1H-pyrrolizine-6,7-
dicarboxylate (31) (1.79 g, 71%): mp 257 °C; 1H NMR
[(CD3)2SO] δ 10.09 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.58 (d, J
) 5.2 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 7.9 Hz, 1 H,
quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.94 (d, J ) 8.1
Hz, 1 H, quinoline H-5), 7.74 (td, J ) 7.1, 1.2 Hz, 1 H, quinoline
H-7), 7.70 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (td, J ) 7.6, 1.1 Hz,
1 H, quinoline H-6), 7.48 (d, J ) 8.7 Hz, 2 H, PhH), 7.21 (d, J
) 5.2 Hz, 1 H, quinoline H-3), 7.18 (d, J ) 8.6 Hz, 2 H, PhH),
3.91 (t, J ) 7.2 Hz, 2 H, NCH2), 3.65 (s, 6 H, 2 × CH3), 2.91 (t,
J ) 7.5 Hz, 2 H, CH2), 2.69 (t, J ) 7.7 Hz, 2 H, CH2), 2.57 (t,
J ) 7.6 Hz, 2 H, CH2), 2.48-2.36 (m, 2 H, NCH2CH2), 1.87-
1.73 (m, J ) 7.6 Hz, 2 H, PhCH2CH2). Anal. (C36H34N4O5) C,
H, N.
Powdered diester 31 (750 mg, 1.24 mmol) was added
portionwise over a 1.5 h period to a stirred suspension of
LiAlH4 (470 mg, 12.38 mmol) in THF (210 mL) at 20 °C under
N2. Stirring was continued for 18 h at 20 °C and then the
mixture was treated dropwise and sequentially with water (0.5
mL), 15% aqueous NaOH (0.5 mL), and water (1.5 mL). The
warmed mixture was filtered and the solids were washed with
hot THF. The filtrate was concentrated under reduced pressure
below 30 °C to 15 mL and diluted with EtOAc to provide the
crude product. Repeated crystallization from dry THF/i-Pr2O
gave 32 (428 mg, 63%): mp 225-230 °C (hygroscopic); 1H NMR
[(CD3)2SO] δ 10.09 (s, 1 H, NH), 9.24 (s, 1 H, NH), 8.58 (d, J
) 5.2 Hz, 1 H, quinoline H-2), 8.38 (d, J ) 8.0 Hz, 1 H,
quinoline H-8), 8.01 (d, J ) 8.7 Hz, 2 H, PhH), 7.93 (d, J ) 7.8
Hz, 1 H, quinoline H-5), 7.74 (t, J ) 7.7 Hz, 1 H, quinoline
H-7), 7.70 (d, J ) 8.5 Hz, 2 H, PhH), 7.58 (t, J ) 7.7 Hz, 1 H,
quinoline H-6), 7.48 (d, J ) 8.7 Hz, 2 H, PhH), 7.23-7.15 (m,
3 H, H-3, quinoline H-3, PhH), 4.42 (t, J ) 5.2 Hz, 1 H, OH),
4.30 (d, J ) 5.3 Hz, 3 H, CH2OH; OH obscured), 4.26 (d, J )
5.0 Hz, 2 H, CH2OH), 3.76 (t, J ) 6.9 Hz, 2 H, NCH2), 2.70 (t,
J ) 7.3 Hz, 2 H, CH2), 2.58 (t, J ) 7.6 Hz, 2 H, CH2), 2.52 (t,
J ) 7.6 Hz, 2 H, CH2), 2.40-2.30 (m, 2 H, NCH2CH2), 1.82-
1.70 (m, 2 H, PhCH2CH2). HRMS (FAB) [M + H]+: found,
547.2735; calcd for C34H35N4O3, 547.2709. Anal. (C34H34N4O3‚
0.5H2O) C, H, N.
Rea ctivity of Com p ou n d s w ith Ca lf Th ym u s DNA.
Stock solutions (10 mM) of the compounds in DMSO were
prepared freshly. Calf thymus DNA was dissolved in SHE
buffer (2 mM Hepes/9 mM NaCl/10 µM EDTA, pH 7.0), and
the solution was adjusted to an accurately determined con-
centration of between 7.5 and 8.5 µM (in base pairs). Solutions
of drug (100 µL) and DNA (900 µL) were mixed and im-
mediately placed at 37 °C. At various times (up to 8 h), 45 µL
aliquots of the mixture were removed and mixed with 20 µL
of 3 N aqueous NaOAc solution and 65 µL of 1-butanol. The
mixture was vortexed vigorously and then centrifuged briefly.
The top butanol layer contained the unreacted drug, including
drug that may have bound noncovalently with DNA. An
aliquot (25 µL) of the 1-butanol layer was injected directly into
the HPLC, using a 300 × 3.9 mm Bondclone C-18 column and
isocratic elution on
a Phillips PU-4100 instrument with
2,3-Dih yd r o-5-[2-[4-[4-(4-qu in olin yla m in o)ben za m id o]-
ph en yl]eth yl]-6,7-bis(h ydr oxym eth yl)-1H-pyr r olizin e (37).
Reaction of 3-(4-nitrophenyl)propionyl chloride with (S)-proline
as described for 26 gave crude (S)-1-[3-(-4-nitrophenyl)prop-
ionyl]pyrrolidine-2-carboxylic acid (33) (80%) as a gum: 1H
NMR (CDCl3) δ 8.1 (br s, 1 H, CO2H), 8.13 (d, J ) 8.8 Hz, 2 H,
H-3′,5′), 7.40 (d, J ) 8.8 Hz, 2 H, H-2′,6′), 4.63-4.50 (m, 1 H,
CHCO2H), 3.70-3.35 (m, 2 H, CH2), 3.20-3.02 (m, 2 H, CH2),
2.80-2.65 (m, 2 H, CH2), 2.33-1.85 (m, 4 H, 2 × CH2). HRMS
(EI) [M+]: found, 292.1053; calcd for C14H16N2O5, 292.1059.
Unicam software. The mobile phase was a mixture of A,
acetonitrile/water (80:20), and B, 0.05 M sodium formate buffer
(pH 4.5). The ratio of these two solvents varied (for different
compounds) from 70% to 50% of the organic phase. Detection
was at 254 nm using a Phillips PU 4120 diode array detector.
The data were fitted to the equation ln(A)t ) -kobst + ln(A)o,
where (A)t is the integrated peak area of the unreacted drug
at time t and kobs is kinetic rate constant observed for the sum
of the rate of drug loss by both by hydrolysis and by alkylation
of the DNA. A plot of ln(A)t versus time yielded straight-line
plots of slope -kobs
.
Reaction of 33 with dimethyl acetylenedicarboxylate by the
method described for 27 gave dimethyl 2,3-dihydro-5-[2-(4-
nitrophenyl)ethyl]-1H-pyrrolizine-6,7-dicarboxylate (34) (73%
yield): mp (EtOAc) 104-105 °C; 1H NMR (CDCl3) δ 8.12 (d, J
DNA La belin g. The primers 1 and 2 (10 pmol; complemen-
tary to the gpt2Eco template DNA) were 5′-labeled using
[γ-32P]-ATP and T4 polynucleotide kinase. The sample was

DNA-Directed Alkylating Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4753
(5) Hartley, J . A.; Wyatt, M. D.; Garbiras, B. J .; Richter, C.; Lee,
M. Probing the importance of the second chloroethyl arm of a
benzoic acid mustard derivative of an imidazole-containing
analogue of distamycin. Biorg. Med. Chem. Lett. 1994, 4, 2412-
2416.
(6) Broggini, M.; Coley, H. M.; Mongelli, N.; Pesenti, E.; Wyatt, M.
D.; Hartley, J . A.; D’Incalci, M. DNA sequence-specific adenine
alkylation by the novel antitumor drug tallimustine (FCE
24517), a benzoyl nitrogen mustard derivative of distamycin.
Nucleic Acids Res. 1995, 23, 81-87.
(7) Anderson, W. K.; New, J . S.; Corey, P. F. Tumor inhibitory
agents: bis(N-alkylcarbamate) derivatives of 2,3-dihydro-5-(3′,4′-
dichlorophenyl)-6,7-bis(hydroxymethyl)-1H-pyrrolizine. Arzneim.-
Forsch. 1980, 30 (I), 765-768.
(8) Anderson, W. K.; Bhattacharjee, D.; Houston, D. M. Design,
synthesis, antineoplastic activity, and chemical properties of bis-
(carbamate) derivatives of 4,5-bis(hydroxymethyl)imidazole. J .
Med. Chem. 1989, 32, 119-127.
(9) Elliot, W. L.; Fry, D. W.; Anderson, W. K., Nelson, J . M.; Hook,
K. E.; Hawkins, P. A.; Leopold, W. R. In vivo and in vitro
evaluation of the alkylating agent carmethizole. Cancer Res.
1991, 51, 4581-4587.
(10) Anderson, W. K.; Corey, P. F. Synthesis and antileukemic
activity of 5-substituted 2,3-dihydro-6,7-bis(hydroxymethyl)-1H-
pyrrolizine diesters. J . Med. Chem. 1977, 20, 812-818.
(11) J arosinski, M. A.; Reddy, P. S.; Anderson, W. K. Synthesis,
chemical reactivity and antitumor evaluation of congeners of
carmethizole hydrochloride; an experimental “acylated vinylo-
gous carbinolamine” tumor inhibitor. J . Med. Chem. 1993, 36,
3618-3627.
then heated at 80 °C for 3 min to inactive the kinase. No
further purification is required for the labeled primer. The
sequences for primers 1 and 2 are shown in Figure 5.
Cr oss-Lin k in g Assa y. A linear pBR322 plasmid DNA (4.36
kb digested at EcoRI, 2 µg) was incubated with drug at
concentration of 0.25 mM in TE buffer (10 mM Tris/1 mM
EDTA, pH 7.4) at 37 °C for 30 min. The reaction adducts were
denatured by adding sodium dodecyl sulfate (SDS) and me-
thylmercury hydroxide at room temperature for 1 h (final
concentrations of 0.1% SDS and 25 mM methylmercury
hydroxide). The denatured samples were mixed with 0.1 vol
of 2-mercaptoethanol and loading buffer, and electrophoresis
was carried out on a 1% agarose gel in TAE buffer containing
10% DMSO at 50 V for 2 h. The gel was stained for 30 min in
TAE buffer containing 0.5 µg/mL ethidium bromide and
photographed using a Polaroid MP 4 Land camera.
Un w in d in g Assa y. Drugs were dissolved in melted 1%
agarose gel solution, and six mini-gels containing various drug
concentrations (0-12 µM) were made at the same size. The
pBR322 DNA (1 µg) was mixed with loading buffer and added
to the mini-gels, then electrophoresed on 1% agarose gel in
TAE buffer at 50 V for 2.5 h in a cool room (4 °C). The gel was
stained for 30 min in TAE buffer containing 0.5 µg/mL
ethidium bromide and photographed using a Polaroid MP 4
Land camera.
P CR Rea ction (P olym er a se Stop Assa y). The template
was the gpt region (503 bp) of plasmid gpt2Eco amplified using
primers 1 and 2. A typical reaction containing 50 ng DNA and
1 µL of 5 mM drug stock in DMSO was carried out in SHE
buffer, pH 7.4, for 3 h at 37 °C (total volume 20 µL). Following
the reactions, excess drugs were moved by DNA precipitating
in ethanol twice. To the dried alkylated DNA was added 5′-
end labeled primers 1 and 2 (0.7 µL, 1 pmol/µL), 3 units of
Taq DNA polymerase, and 1.25 µL of 10× reaction buffer. The
samples were mixed well and chilled on ice before adding 4
µL of 600 mM dNTP solution and one drop of oil. The tubes
were returned to ice before placing in the PCR machine
preheated to 94 °C. The PCR reaction was performed as
follows: cycles 1, 2.5 min at 94 °C; cycles 2-20, 30 s at 94 °C,
30 s at 62 °C, 30 s at 72 °C. The extension products were
denatured at 90 °C for 2 min in sequencing gel loading buffer
before sequencing gel electrophoresis on a 8% denaturing
polyacrylamide gel containing 8.3 M urea. The gels were dried
and autoradiographed at -70 °C with an intensifying screen.
Cytotoxicity Assa ys. These were carried out as previously
described.28 IC50 values were determined with log-phase
cultures in 96-well microtiter plates, with drug present
throughout the growth period (72 h). The IC50 values are the
average of at least two consistent and independent determina-
tions, calculated as the nominal drug concentration needed to
reduce the cell density to 50% of control values. There were
eight control wells on each plate.
(12) Woo, J .; Sigurdsson, S. T.; Hopkins, P. B. DNA interstrand cross-
linking reactions of pyrrole-derived, bifunctional heterocycles:
evidence for a common target site in DNA. J . Am. Chem. Soc.
1993, 115, 3407-3415.
(13) (a) Sigurdsson, S. T.; Rink, S. M.; Hopkins, P. B. Affinity cross-
linking of duplex DNA by a pyrrole-oligopeptide conjugate. J .
Am. Chem. Soc. 1993, 115, 12633-12634. (b) Sigurdsson, S. T.;
Hopkins, P. B. Synthesis and reactions with DNA of a family of
DNA-DNA affinity cross-linking agents. Tetrahedron 1994, 42,
12065-12084.
(14) Waring, M. J . DNA-binding characteristics of acridinylmethane-
sulphonanilide drugs: comparison with antitumour properties.
Eur. J . Cancer 1976, 12, 995-1001.
(15) Braithwaite, A. W.; Baguley, B. C. Existence of an extended
series of antitumor compounds which bind to deoxyribonucleic
acid by nonintercalative means. Biochemistry 1980, 19, 1101-
1106.
(16) Espejo, R. T.; Lebowitz, J . A simple electrophoretic method for
the determination of superhelical density of closed circular DNAs
and for observation of their superhelix density heterogeneity.
Anal. Biochem. 1976, 72, 95-103.
(17) Stella, V. J .; Anderson, W. K.; Bendetti, A.; Waugh, W. A.;
Killion, R. B. Stability of carmethizole hydrochloride (NSC
602668), an experimental cytotoxic agent. Int. J . Pharm. 1991,
71, 157-165.
(18) Maniatis, M. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Press: Plainview, NY 1981.
(19) Prakash, A. S.; Denny, W. A.; Gourdie, T. A.; Valu, K. K.;
Woodgate, P. D.; Wakelin, L. P. G. DNA-directed alkylating
ligands as potential antitumor agents: sequence specificity of
alkylation by intercalating aniline mustards. Biochemistry 1990,
29, 9799-9807.
(20) Kohn, K. W.; Hartley, J . A.; Mattes, W. B. Mechanisms of DNA
sequence selective alkylation of guanine-N7 positions by nitrogen
mustards. Nucleic Acids Res. 1987, 15, 1987, 10531-10549.
(21) Petrusek, R. L.; Anderson, G. L.; Garner, T. F.; Fannin, Q. L.;
Kaplan, D. J .; Zimmer, S. G.; Hurley, L. H. Pyrrolo[1,4]-
benzodiazepine antibiotics. Proposed structures and character-
istics of the in vitro deoxyribonucleic acid adducts of anthramy-
Ack n ow led gm en t. This work was supported by the
Auckland Division of the Cancer Society of New Zealand
and the Health Research Council of New Zealand. J .-
Y.F. is a Cancer Society/Air NZ Repatriation Fellow.
Refer en ces
(1) Hansson, J .; Lewensohn, R.; Ringborg, U.; Nilsson, B. Formation
and removal of DNA cross-links induced by melphalan and
nitrogen mustard in relation to drug-induced cytotoxicity in
human melanoma cells. Cancer Res. 1987, 47, 2631-2637.
(2) Krowicki, K.; Balzarini, J .; De Clercq, E.; Newman, R. A.; Lown,
J . W. Novel DNA groove binding alkylators: design, synthesis
and biological evaluation. J . Med. Chem. 1988, 31, 341-345.
(3) Gourdie, T. A.; Valu, K. K.; Gravatt, G. L.; Boritzki, T. J .;
Baguley, B. C.; Wilson, W. R.; Woodgate, P. D.; Denny, W. A.
DNA-directed alkylating agents. 1. Structure-activity relation-
ships for acridine-linked aniline mustards: consequences of
varying the reactivity of the mustard. J . Med. Chem. 1990, 33,
1177-1186.
cin, tomamycin, sibiromycin and neothramycins
Biochemistry 1981, 20, 1111-1119.
(22) Lane, M. J .; Dabrowiak, J . C.; Vournakis, J . N. Sequence
specificity of actinomycin D and netropsin binding to pBR322
DNA analyzed by protection from DNase I. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 3260-3264.
(23) Fox, K. R.; Howarth, N. R. Investigations into the sequence-
selective binding of mithramycin and related ligands to DNA.
Nucleic Acids Res. 1985, 13, 8695-8714.
(24) Gralla, J . D.; Sasse-Dwight, S.; Poljak, L. G. Formation of
blocking lesions at identical DNA sequences by the nitrosourea
and platinum classes of anticancer drugs. Cancer Res. 1987, 47,
5092-5096.
(25) Boritzki, T. J .; Palmer, B. D.; Coddington, J . M.; Denny, W. A.
Identification of the major lesion from the reaction of an acridine-
targeted aniline mustard with DNA as an adenine N1 adduct.
Chem. Res. Toxicol. 1994, 7, 41-46.
A and B.
(4) Gravatt, G. L.; Baguley, B. C.; Wilson, W. R.; Denny, W. A. DNA-
directed alkylating agents. 6. Synthesis and antitumor activity
of DNA minor groove-targeted aniline mustard analogues of
pibenzimol (Hoechst 33258). J . Med. Chem. 1994, 37, 4338-
4345.

4754 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Atwell et al.
(26) Fan, J .-Y., Tercel, M.; Denny, W. A. Synthesis, DNA binding
and cytotoxicity of 1-[[(-(9-acridiny)lamino]alkyl]carbonyl-3-
(chloromethyl)-6-hydroxyindolines, a new class of DNA-targeted
alkylating agents. Anti-Cancer Drug Design 1997, 12, 277-293.
(27) Fuller, A. T.; Harington, C. R. Rivers, R. P.; Stephens, J . M. L.
Some derivatives of p-aminobenzoic acid. J . Chem. Soc. 1948,
241.
(28) Baguley, B. C.; Wilson, W. R. Comparison of in vivo and in vitro
drug sensitivities of Lewis lung carcinoma and P388 leukemia
to analogues of amsacrine. Eur. J . Clin. Oncol. 1987, 23,
607-613.
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