The Synthesis and Testing of α-(Hydroxymethyl)pyrroles as DNA Binding Agents

Article abstract of DOI:10.1071/CH04183

The α-(hydroxymethyl)pyrroles 16a and 16b were prepared and shown to be cytotoxic against the P388 cancer cell line. Ethyl 5-hydroxymethyl-1H-pyrrole-2-carboxylate 18 was inactive, demonstrating that an α-(hydroxymethyl)pyTrole group alone is not sufficient for activity. Compound 16b has been shown to bind to DNA with reasonable affinity.

Full text of DOI:10.1071/CH04183

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 1073–1077
The Synthesis and Testing of α-(Hydroxymethyl)pyrroles
as DNA Binding Agents
A
A,B
Derek C. Martyn and Andrew D. Abell
A
Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Corresponding author. Email: andrew.abell@canterbury.ac.nz
B
The α-(hydroxymethyl)pyrroles 16a and 16b were prepared and shown to be cytotoxic against the P388
cancer cell line. Ethyl 5-hydroxymethyl-1H-pyrrole-2-carboxylate 18 was inactive, demonstrating that an
α-(hydroxymethyl)pyrrole group alone is not sufficient for activity. Compound 16b has been shown to bind to
DNA with reasonable affinity.
Manuscript received: 30 July 2004.
Final version: 21 September 2004.
Introduction
Nu
N
H
α-(Hydroxymethyl)pyrroles of type 1 are an important class
of π-excessive heterocycle that are particularly susceptible
to nucleophilic substitution due to the intermediacy of the
3
NuϪ
[1–6]
non-aromatic azafulvene 2 (Scheme 1).
This inher-
ϪH O
2
OH
ent reactivity has been utilized by nature in the biosyn-
thesis of (hydroxymethyl)bilane, a key intermediate to
N
H
N
[2]
vitamin B12 and the like, and also by medicinal and
organic chemists. For example, we recently reported some
α-(hydroxymethyl)pyrroles, substituted on nitrogen with an
electron-withdrawing group (EWG), as inhibitors of serine
1
2
OH
A
N
N
ϩ
[3]
proteases, see general structure 4.
EWG
EWG
We postulate that that the introduction of an EWG on
nitrogen, as in 4, would suppress the formation of the cor-
responding azafulvenium species 5, and hence render the
4
5
[
4,5]
system more stable to nucleophilic displacement.
The
O
H
N
reactive pathway (A in Scheme 1) could then be switched on
by removing the EWG in a controlled manner. Our idea was
HO
N
H
N
H
CO2Me
[7]
O
to choose an EWG that resembled the P1 residue of a serine
proteasesubstrate, sothattheenzymewouldacceptitasasub-
Ph
6
[
3]
strateandcatalyzeitsremoval(seestepAinScheme1). The
thus liberated azafulvene could then inactivate the enzyme
by covalent attachment to an active site residue, represented
Scheme 1.
−
by Nu in Scheme 1. We also recently reported a related
however, none of these have the hydroxymethyl substitu-
tent at the optimum α-position for azafulvene formation.
An α-(hydroxymethyl)pyrrole moiety is, however, found in
a series of metabolites, isolated from the Atlantic sponge
Mycale micracanthoxea, that displays moderate activity
[1]
series of α-(hydroxymethyl)pyrrole-based protease inhibitors
[
4]
that contain a C2 peptide-based acyl group, for example 6.
These compounds mimic an extended peptide conformation
[
4,7]
that is found in natural substrates of these proteases.
[
12]
We now report the results of initial work on targeting
DNA by appending a bicyclic aromatic substituent, which is
against five cancer cell lines.
These compounds pos-
sess an acyl side chain, of variable length and unsaturation;
for representative examples see 10a, 10b, Scheme 2. While
these derivatives displayed only moderate activity against the
P388 murine leukaemia cell line, they do suggest that an
α-(hydroxymethyl)pyrrole moiety might provide a suitable
scaffold for the generation of more potent compounds with
[8]
known to bind to DNA, onto an α-(hydroxymethyl)pyrrole.
Compounds of this type have the potential to alkylate
[
9]
DNA and hence act as anticancer agents.
of hydroxymethylpyrrole-based DNA alkylating agents are
Examples
[
10] [11]
[11]
and 9 (Scheme 2);
known and these include 7,
8,
©
CSIRO 2004
10.1071/CH04183
0004-9425/04/111073

1
074
D. C. Martyn and A. D. Abell
HO
HO
NH2
O
O
X
N
HO
N
X
(a), (b)
HO
Ph
N
(
c)
1
1
1a X ϭ CH
1b X ϭ N
12a X ϭ CH
12b X ϭ N
7
O
O
N
H
HO
NH
HN
N
OHC
CO2H
N
H
X
8
HO
(d )
14
OH
1
1
3a X ϭ CH
3b X ϭ N
O
N
O
R1
NH
HN
N
H
O
X
1
1
5a X ϭ CH, R1 ϭ CHO
9
N
5b X ϭ N, R1 ϭ CHO
(
e)
1
1
6a X ϭ CH, R ϭ CH2OH
R1
1
16b
X ϭ N, R ϭ CH2OH
2
R1
(CH2)7
HO
10a
Scheme 3. Reagents and conditions: (a) Succinic anhydride, PhMe,
N
H
◦
5
reflux, 15 min; (b) CH3CO2Na, Ac2O, 70–80 C, 4.5 h (12a, 86% over
O
i
two steps), (12b, 79% over two steps); (c) NaBH4, 6 : 1 Pr OH/H2O,
10b
(CH2)17Me
room temp., 17 h (13a, 71%), 13b; (d ) BOP-Cl, Et3N, CH2Cl2, room
◦
temp., 64 h, 15a, 15b. (e) LiBH4,THF, −78 C, 1 h, then room temp., 1 h
Scheme 2. Hydroxymethylpyrrole-based antitumour agents.
(
16a, 18% over two steps from 13a), (16b, 11% over three steps from
1
2b).
the judicious choice of functionality at C2. We now report
the synthesis and in vitro testing, against P388 cancer cells,
of compounds in which the aliphatic chain of 10 is replaced
by a bicyclic aryl group (naphthalene or quinoline) which is
attached to the α-(hydroxymethyl)pyrrole via an ester linker.
Aryl groups of this type are known to facilitate non-covalent
(
a)
HO
OHC
N
H
CO2Et
N
H
CO2Et
17
18
◦
Scheme 4. Reagents and conditions: (a) LiBH4,THF, −78 C, 1 h, then
[8]
room temp., 1 h (76%).
binding to DNA.
Table 1. P388 cancer cell line cytotoxicity assay
results
Results and Discussion
The synthesis of the substituted α-(hydroxymethyl)pyrrole
Compound
ID50 [µM]
1
6a is outlined in Scheme 3. The amine 11a was treated
1
1
18
6a
6b
468
314
>739
9.4
with succinic anhydride in refluxing toluene, followed by
the addition of sodium acetate in acetic anhydride
the succinimide 12a in 86% yield over two steps. The suc-
cinimide 12a was then reduced with sodium borohydride in
6
[13]
to give
1
9
[
14]
: 1 propan-2-ol/water
to give 13a in 71% yield. The
[15]
alcohol 13a was coupled with the pyrrole acid 14,
the presence of BOP-Cl,
in
to give aldehyde 15a, which
the assays, was prepared by reduction of the formylpyrrole
[
16]
[15]
ester 17 with lithium borohydride as outlined in Scheme 4.
was finally reduced with lithium borohydride to give 16a
P388 Cancer Cell Line Cytotoxicity Assay
in 18% yield over two steps. A heterocyclic ring system
[8]
known to interact with DNA was also appended to the
α-(hydroxymethyl)pyrrole moiety in an analogous fashion,
using quinolin-8-ylamine 11b as shown in Scheme 3. In par-
ticular, 11b was treated with succinic anhydride, followed by
sodium acetate/acetic anhydride, to give the succinimide 12b
in 79% yield over two steps. Reduction of 12b with sodium
borohydride gave the alcohol 13b, which was subsequently
coupled to 14 to give 15b.This was then reduced with lithium
borohydride to give the desired product 16b. The simple
α-(hydroxymethyl)pyrrole ester 18, required as a control in
Compounds 16a, 16b, 18, and 19 were assayed against
[
17]
the P388 cancer cell line,
and the resulting ID50 val-
ues are given in Table 1. The hydroxymethylpyrrole 18 was
assayed to determine if an α-(hydroxymethyl)pyrrole moiety
alone is sufficient for activity. The tricyclic heterocycle, 3,6-
diaminoacridine (Scheme 5, compound 19), was assayed as a
representative intercalator to compare its cytotoxicity to that
of 16a and 16b.
From the results in Table 1 it is apparent that both 16a
and 16b are cytotoxic, with ID50 values of 468 and 314 µM,

α-(Hydroxymethyl)pyrroles as DNA Binding Agents
1075
NH2
BrϪ
where this ring system is known to have a very high affinity
for DNA. Importantly, an α-(hydroxymethyl)pyrrole moiety
alone is not sufficient for activity, where 18 was effectively
inactive. Pyrrole 16b was shown, by a DNA binding assay,
to have moderate affinity for oligonucleotides, but at a lower
level than the classical intercalator, ethidium bromide—this
result demonstrates that compounds of type 16 are able to
bind to DNA.
Nϩ
H N
2
N
NH2
H2N
Et
Ph
20
19
Scheme 5. Assay compounds.
Experimental
respectively. However, 18 displayed minimal activity, indicat-
ing that a hydroxymethylpyrrole moiety alone is not sufficient
for cytotoxicity and that an aromatic substituent enhances
activity. As might be expected, all compounds proved to be
less active than the known and potent intercalator 19.
All reactions carried out under an argon atmosphere using oven-dried
glassware. Analytical TLC was performed on plastic-backed Merck
Kieselgel KG60F254 silica plates, and visualized using short-wave UV
light. Flash chromatography was performed using 230–400 mesh Merck
Silica Gel 60 under positive pressure. Petroleum ether refers to the frac-
◦
tion collected between 60–70 C. All solvents were purified and dried
according to standard procedures and compounds 14 and 17 were syn-
Deoxyribonucleic Acid Binding Assay
[
15]
thesized by literature procedures.
All melting points were obtained
We next chose to test the ability of the most active com-
pound 16b to bind to DNA since this represents an import-
ant component of the proposed mechanism of action of
this case of compound. The method reported by Baguley
on an Electrothermal apparatus and are uncorrected. Infrared spectra
were obtained using a Shimadzu 8201PC series FTIR interfaced with
an Intel 486 PC operating Shimadzu’s HyperIR software. Spectra were
1
obtained in KBr (diffuse reflectance method). H NMR spectra were
obtained on a Varian Inova spectrometer, operating at 500 MHz. Cou-
pling constants (J ) are quoted in Hz. 13C NMR spectra were obtained
on a Varian Unity 300 spectrometer, operating at 75 MHz, with a delay
[18]
et al.
of 16b relative to ethidium bromide (Scheme 5, compound
0), by measuring the competition for DNA binding sites.
was used to determine the DNA binding activity
(
D1) of 1 s. Chemical shifts are reported in parts per million (ppm) on the
2
δ scale. Two-dimensional NMR experiments, including COSY, HSQC,
HMBC, and CIGAR, were obtained on the Varian Inova spectrometer
operating at 500 MHz. The HSQC, HMBC, and CIGAR experiments
The assay used relies on measurement of the fluorescence
emitted by 20 upon binding to DNA. The addition of a
second DNA binding agent (16b in this case) results in
a reduction in fluorescence, due to displacement of 20
from the genetic material. Using this method, compound
were all obtained with a delay (D ) of 1 s. Electron-impact (EI) mass
1
spectra were detected on a Kratos MS80 RFA mass spectrometer oper-
ating at 4000 V (accelerating potential) and 70 eV (ionization energy)
◦
using a source temperature of 200–250 C. Electrospray ionization (ES)
1
6b was determined to have a 3000-fold lower affinity for
mass spectra were detected on a micromass LCT TOF mass spectrome-
3
−1
poly[dA–dT] (K 4.6 × 10 M ) and poly[dG–dC] (K 3.0 ×
◦
ter, with a probe voltage of 3200 V, temperature of 150 C, and a source
temperature of 80 C.
3
−1
7
−1
◦
1
0 M ) than 20 (K 1.10 M for both oligonucleotides).
While the binding constant for 16b was considerably lower
than for 20, it must be noted that 20 has an extremely high
General Procedure A: Succinimide Formation
from Aromatic Amines
[11]
affinity for DNA. A separate assay,
to determine if 16b
binds to the genetic material by intercalation, could not be
performed owing to the insufficient solubility of 16b in the
assay conditions. However, an intercalative binding mode for
Astirredsolutionofthearomaticamine(1equiv.)andsuccinicanhydride
(
1 equiv.) in dry toluene (approx. 0.25 M), under an argon atmosphere,
was heated at reflux for 15 min. After cooling to room temperature, the
solid was collected by filtration and washed with cold petroleum ether.
The material was dissolved in a mixture of ethyl acetate (60 mL) and
5% aqueous sodium hydroxide (60 mL), the layers were separated, and
the organic phase was extracted with 5% aqueous sodium hydroxide
1
6b to DNA is considered unlikely, as generally a tricyclic
ring system is considered the ‘minimum’ for intercalation,
[8,19]
although exceptions do exist.
The inherentreactivity ofhydroxymethylpyrroles has been
(
20 mL). The combined aqueous fractions were acidified to pH ∼1 with
concentrated hydrochloric acid, and the resultant solid was allowed to
stand for 3 h, after which it was collected by filtration, washed with water
[
2]
put to good use by nature in the biosynthesis of vitamin B12
and also medicinal chemists to generate inhibitors of serine
(
10 mL), and dried under reduced pressure. A mixture of the result-
[3–6]
proteases.
This moiety is also found in several natural
ing N-arylsuccinamic acid (1 equiv.) and anhydrous sodium acetate
product-based DNA alkylating agents (see earlier for a dis-
cussion), but none of these have the optimum combination
of an intercalating substituent and the hydroxymethylpyr-
(0.33 equiv.) in dry acetic anhydride (approx. 0.25 M) was heated to
◦
7
0–80 C for 4.5 h, under an argon atmosphere. The mixture was cooled
to room temperature, poured onto water (60 mL), and extracted with
dichloromethane (3 × 20 mL). The combined organic fractions were
washed with aqueous saturated ammonium chloride (20 mL), dried
[1]
role group at the α-position (see Scheme 2). In this paper
we have presented initial studies towards this goal with the
development of a general method for the preparation of
α-hydroxymethylpyrroles to which is appended an aromatic
(MgSO ), and the solvent was removed by evaporation under reduced
4
pressure. Residual solvent was removed from the desired product by
◦
Kugelrohr distillation (up to 140 C). See individual experiments for
details.
[8]
group that is known to interact with DNA, see 16a and
6b. Scope exists to develop the potency of these com-
1
General Procedure B: Succinimide Reduction/Ring Opening
with Sodium Borohydride
pounds with optimization of intercalator and the linker chain.
Compounds 16a and 16b displayed micromolar cytotoxic-
ity towards cancer P388 cell line, but as expected both were
less active than the representative and potent intercalator 19,
To a stirred suspension of the succinimide (1 equiv.) in 6 : 1 propan-2-
ol/water (approx. 0.1 M) was added sodium borohydride (2.5 equiv.),
and the resulting suspension was stirred at room temperature for 17 h.

1
076
D. C. Martyn and A. D. Abell
The mixture was filtered through Dowex-50W resin and the filtrate evap-
orated to dryness under reduced pressure. See individual experiments
for details.
4-(Naphthalen-1-ylamino)-4-oxobutyl-5-hydroxymethyl-1H-
pyrrole-2-carboxylate 16a
[15]
Pyrrole acid 14
(100 mg, 0.72 mmol) was coupled to alcohol 13a
(
247 mg, 1.08 mmol) using general procedure C. The crude product
General Procedure C: Couplings Using Bis(2-oxo-3-
oxazolidinyl)phosphinic Chloride
was purified by flash chromatography (ethyl acetate/petroleum ether,
2 : 1) to give 15a (95 mg) as an orange solid, which was not purified
further. This material was reduced with lithium borohydride (12 mg,
[
15]
(1 equiv.), alco-
To a stirred suspension of the formylpyrrole acid 14
hol 13 (1.5 equiv.), and bis(2-oxo-3-oxazolidinyl)phosphinic chloride
2 equiv.) in dry dichloromethane (approx. 0.07 M), at room tempera-
0
.54 mmol) by general procedure D and the crude product purified by
flash chromatography (ethyl acetate/petroleum ether, 5 : 1, then ethyl
(
acetate), followed by recrystallization from ethyl acetate, to give 16a
ture under an argon atmosphere, was added dry triethylamine (2 equiv.).
The resulting suspension was stirred for 64 h, after which it was diluted
with ethyl acetate (20 mL), washed with 10% aqueous sodium bicarbon-
ate (10 mL), saturated aqueous brine (10 mL), dried (MgSO4), and the
solvent removed by evaporation under reduced pressure. See individual
experiments for details.
◦
(
46 mg, 18% over two steps) as a white solid, mp 154–156 C (Found:
C 68.2, H 5.8, N 7.8. C20H20N2O4 requires C 68.2, H 5.7, N 8.0%).
−
1
νmax (KBr/cm ) 1500.5, 1531.4, 1654.8, 1685.7, 3282.6, 3433.1. δH
500 MHz, (CD3)2SO, Me4Si] 2.15 (2 H, m, CH2CH2CH2), 2.74 (2 H,
[
t, J 7.3, CH2CO), 4.37 (2 H, t, J 6.3, CO2CH2), 4.51 (2 H, d, J 5.4,
HOCH2), 5.15 (1 H, t, J 5.9, HOCH2), 6.17 (1 H, m, pyrrole H4), 6.86
(
1 H, s, pyrrole H3), 7.60 (3 H, m, ArH), 7.76 (1 H, d, J 7.3, ArH), 7.85
General Procedure D: Lithium Borohydride Reductions
(1 H, d, J 8.3, ArH), 8.03 (1 H, m, ArH), 8.14 (1 H, m, ArH), 10.05 (1 H,
br s, CONH), 11.73 (1 H, br s, pyrrole NH). δC [75 MHz, (CD3)2SO,
Me4Si] 24.9 (CH2CH2CH2), 32.5 (CH2CO), 56.1 (HOCH2), 63.1
A stirred solution of the formylpyrrole 15 (1 equiv.), in dry THF
◦
(
(
approx. 0.03 M) under an argon atmosphere, was cooled to −78 C
(
CO2CH2), 108.2 (pyrrole C4), 115.7 (pyrrole C3), 121.2 (pyrrole C2)
dry ice/acetone). Lithium borohydride (2 equiv.) was added, and the
◦
122.0, 122.9, 125.4, 125.8, 126.0, 126.2, 128.0, 128.3, 133.7, 133.9
resulting solution was stirred at −78 C for 1 h, then warmed to room
temperature and stirred for an additional 1 h. Water (10 mL) was care-
fully added to quench the reaction. The solution was extracted with
ethyl acetate (3 × 10 mL), the combined organic phases were washed
with saturated aqueous brine (10 mL), dried (MgSO4), and the solvent
was removed by evaporation under reduced pressure. See individual
experiments for details.
(
ArC), 139.4 (pyrrole C5), 160.7 (CO2), 171.6 (CONH). m/z (ES)
+
3
75.1321 (M + Na, C20H20N2NaO4 requires 375.1321).
4
2
-(Quinolin-8-ylamino)-4-oxobutyl-5-hydroxymethyl-1H-pyrrole-
-carboxylate 16b
The succinimide 12b (496 mg, 2.19 mmol) was reduced with sodium
borohydride (207 mg, 5.48 mmol) using general procedure B. The crude
material was purified by flash chromatography (ethyl acetate, then ethyl
acetate/methanol, 10 : 1) to give 13b (335 mg) as a sticky white solid.
δH (500 MHz, CD3OD, Me4Si) 1.99 (2 H, m, CH2CH2CH2), 2.68 (2 H,
t, J 7.6, CH2CO), 3.67 (2 H, t, J 6.3, HOCH2), 7.53 (2 H, m, ArH), 7.61
N-(Naphthalen-1-yl)pyrrolidine-2,5-dione 12a
Naphthalen-1-ylamine 11a (1.00 g, 6.98 mmol) was treated with suc-
cinic anhydride (699 mg, 6.98 mmol) followed by anhydrous sodium
acetate (191 mg, 2.33 mmol) according to general procedure A to give
(
1 H, m, ArH), 8.29 (1 H, dd, J 2.0, 8.3, ArH), 8.62 (1 H, d, J 7.8, ArH),
◦
1
2a (1.36 g, 86% over two steps) as a light pink solid, mp 153–154 C
[15]
8
.86 (1 H, d, J 1.5, 4.4, ArH). Pyrrole acid 14
(135 mg, 0.97 mmol)
[
13] ◦
(
lit.
CH2CH2), 7.33 (1 H, dd, J 1.0, 7.3, ArH), 7.53 (4 H, m, ArH), 7.93
2 H, m, ArH).
147–149 C). δH (500 MHz, CDCl3, Me4Si) 2.96–3.09 (4 H, m,
was coupled to 13b (335 mg, 1.45 mmol) according to general pro-
cedure C. The crude material was purified by flash chromatography
(
(
ethyl acetate/petroleum ether, 2 : 1) to give 15b as an orange solid. This
material was reduced with lithium borohydride (19 mg, 0.85 mmol) by
general procedure D and the crude material purified by flash chromato-
graphy (ethyl acetate/petroleum ether, 4 : 1, then ethyl acetate), followed
N-(Quinolin-8-yl)pyrrolidine-2,5-dione 12b
Quinolin-8-ylamine 11b (500 mg, 3.47 mmol) was treated with succinic
anhydride (347 mg, 3.47 mmol) followed by sodium acetate (95 mg,
by recrystallization from ethyl acetate/petroleum ether, to give 16b
◦
(
86 mg, 11% over three steps) as a tan solid, mp 118–119 C (Found:
1
.16 mmol) according to general procedureA to give 12b (620 mg, 79%
C 64.5, H 5.6, N 11.6. C19H19N3O4 requires C 64.6, H 5.4, N 11.9%).
◦
−1
)
over two steps) as a light brown solid, mp 134–136 C. νmax (KBr/cm
−1
νmax (KBr/cm ) 1539.1, 1652.9, 1708.8, 2949.0, 3261.4, 3348.2. δH
1
2
502.4, 1701.1, 1778.2, 2947.0, 3051.2. δH (500 MHz, CDCl3, Me4Si)
.97, 3.13 (4 H, 2 m, CH2CH2), 7.44 (1 H, dd, J 3.9, 8.3, ArH), 7.62
(
500 MHz, CDCl3, Me4Si) 2.25 (2 H, m, CH2CH2CH2), 2.57 (1 H,
br s, OH), 2.71 (2 H, t, J 7.1, CH2CO), 4.39 (2 H, t, J 6.3, CO2CH2),
.63 (2 H, s, HOCH2), 6.17 (1 H, t, J 3.2, pyrrole 4H), 6.83 (1 H, dd,
(
2 H, m, ArH), 7.93 (1 H, dd, J 2.0, 7.8, ArH), 8.20 (1 H, dd, J 1.5,
4
9
.8, ArH), 8.87 (1 H, dd, J 1.7, 4.2, ArH). δC (75 MHz, CDCl3, Me4Si)
J 2.4, 3.4, pyrrole H3), 7.43 (1 H, dd, J 4.4, 8.3, ArH), 7.51 (2 H, m,
ArH), 8.14 (1 H, dd, J 1.7, 8.1, ArH), 8.74 (2 H, m, ArH), 9.77, 9.85
2
9.0 (2 C, CH2CH2), 122.0, 126.2, 129.3, 129.7, 129.8, 136.6, 150.8
+
(
ArC), 176.9 (2 CO2). m/z (EI) 226.0744 (M , C13H10N2O2 requires
(2 H, 2 br s, CONH and pyrrole NH). δC (75 MHz, CDCl3, Me4Si) 24.2
(CH2CH2CH2), 33.7 (CH2CO), 56.7 (HOCH2), 62.5 (CO2CH2), 107.2
(pyrrole C4), 114.9 (pyrrole C3), 116.3, 121.2, 126.8, 127.4, 133.6,
2
26.0742).
4
-Hydroxy-N-(naphthalen-1-yl)butanamide 13a
136.4, 137.2, 147.4 (ArC), 121.4 (pyrrole C2), 137.8 (pyrrole C5), 160.5
+
(
CO2), 170.3 (CONH). m/z (EI) 353.1362 (M , C19H19N3O4 requires
The succinimide 12a (1.36 g, 6.04 mmol) was treated with sodium boro-
hydride (571 mg, 15.1 mmol) by general procedure B.The crude product
was purified by flash chromatography (ethyl acetate/methanol, 10 : 1),
followed by recrystallization from ethyl acetate, to give 13a (983 mg,
7
3
53.1376).
Ethyl 5-hydroxymethyl-1H-pyrrole-2-carboxylate 18
◦
[15]
1%) as a white solid, mp 102–104 C (Found: C 73.2, H 6.7, N 6.1.
Formylpyrrole ester 17
(150 mg, 0.90 mmol) was reduced with
−
1
C14H15NO2 requires C 73.3, H 6.6, N 6.1%). νmax (KBr/cm ) 1508.2,
541.0, 1652.9, 2950.9, 3274.9. δH (500 MHz, CD3OD, Me4Si) 2.00
2 H, m, CH2CH2CH2), 2.63 (2 H, t, J 7.3, CH2CO), 3.70 (2 H, t, J 6.3,
lithium borohydride (39 mg, 1.79 mmol) using general procedure D.The
crude product was purified by flash chromatography on silica (ethyl
acetate/petroleum ether, 1 : 1) to give 18 (115 mg, 76%) as a white
1
(
◦
HOCH2), 7.50 (3 H, m, ArH), 7.57 (1 H, d, J 7.3, ArH), 7.77 (1 H, d,
J 8.3, ArH), 7.89 (1 H, m, ArH), 7.98 (1 H, d, J 7.8, ArH). δC (75 MHz,
CD3OD, Me4Si) 30.0 (CH2CH2CH2), 34.2 (CH2CO), 62.6 (HOCH2),
solid; mp 76–78 C (Found: C 57.0, H 6.6, N 8.2. C8H11NO3 requires C
−
1
56.8, H 6.6, N 8.3%). νmax (KBr/cm ) 1502.4, 1683.7, 2993.3, 3286.5.
δH (500 MHz, CDCl3, Me4Si) 1.35 (3 H, t, J 7.1, CH2CH3), 4.31 (2 H,
q, J 7.2, CH2CH3), 4.69 (2 H, s, HOCH2), 6.11 (1 H, t, J 3.2, pyrrole
H4), 6.84 (1 H, dd, J 2.4, 3.9, pyrrole H3), 9.51 (1 H, br s, pyrrole NH).
δC [75 MHz, (CD3)2CO, Me4Si] 14.1 (CH2CH3), 57.0 (HOCH2), 59.6
1
23.7, 124.3, 126.7, 127.4, 127.5, 127.8, 129.6, 130.4, 134.4, 135.9
+
(
ArC), 175.7 (CONH). m/z (EI) 229.1102 (M , C14H15NO2 requires
2
29.1103).

α-(Hydroxymethyl)pyrroles as DNA Binding Agents
1077
(
1
CH2CH3), 108.0 (pyrrole C4), 115.2 (pyrrole C3), 122.4 (pyrrole C2),
38.7 (pyrrole C5), 160.7 (CO2). m/z (EI) 169.0739 (M , C8H11NO3
[5] A. D. Abell, B. K. Nabbs, A. R. Battersby, J. Org. Chem. 1998,
63, 8163. doi:10.1021/JO980573I
+
requires 169.0738).
[6] A. D. Abell, B. K. Nabbs, A. R. Battersby, J. Am. Chem. Soc.
1
998, 120, 1741. doi:10.1021/JA973656+
[7] (a) I. Schechter, A. Berger, Biochem. Biophys. Res. Commun.
967, 27, 157.
P388 Cancer Cell Line Cytotoxicity Assay
1
These assays were carried out according to the literature method of
[17]
−1
in 3 : 1
(b) Note the use of Schechter–Berger nomenclature—the
residues on the N-terminal side of the peptide bond that
is cleaved are denoted (in order) P1–Pn, and those on the
Alley et al.,
using stock solutions of 16a (20 mg mL
−
1
methanol/dimethylformamide), 16b and 18 (10 mg mL in methanol),
−1
and 19 (1 mg mL in 3 : 1 methanol/dimethylformamide).
ꢀ
ꢀ
C-terminus are denoted P1 –Pn . In turn, the corresponding
ꢀ
subsites on the enzyme are denoted Sn–Sn . L. Hedstrom,
Determination of the Binding Constant of 16b to DNA
Chem. Rev. 2002, 102, 4501. doi:10.1021/CR000033X
8] (a) H. Zang, K. S. Gates, Biochemistry 2000, 39, 14968.
doi:10.1021/BI001998D
3
−1
Binding constants of 4.6 × 10 M
for poly[dA–dT] and 3.0 ×
7
[
[
3
−
1
−
1
1
0 M for poly[dG–dC] were calculated for 16b and 1.10 M for
[
18,19]
ethidium bromide 20 according to the literature method.
point of the assay was based on 20% displacement of 20 by 16b due to
the low solubility of 16b in the assay medium.
The end
(
b) M. S. Searle, J. G. Hall, W. A. Denny, L. P. G. Wakelin,
Biochem. J. 1989, 259, 433.
[
20]
9] J. A. Hartley, in Molecular Aspects of Anticancer Drug-DNA
Interactions (Eds S. Neidle, M. Waring) 1993, Vol. 1, p. 1 (CRC
Press: Boca Raton, FL).
Acknowledgments
[
[
10] W. K. Anderson, P. F. Corey, J. Med. Chem. 1977, 20, 812.
11] G. J. Atwell, J.-Y. Fan, K. Tan, W. A. Denny, J. Med. Chem.
1998, 41, 4744. doi:10.1021/JM9803119
The authors thank Professor Bruce Baguley (Auckland Can-
cer Society Research Centre) for performing the DNA bind-
ing assays, and Gill Ellis (Marine Natural Products Group,
Department of Chemistry, University of Canterbury) for car-
ryingouttheP388cytotoxicityassays. Financialsupportfrom
Bright FuturesTopAchievers Doctoral Scholarship (D.C.M.)
and the Royal Society of New Zealand Marsden Fund is also
gratefully acknowledged.
[12] M. J. Ortega, E. Zubia, J. L. Carballo, J. Salva, Tetrahedron
997, 53, 331. doi:10.1016/S0040-4020(96)00989-1
[
1
13] J. L. Hubbard, J. M. Carl III, G. D. Anderson, J. Heterocycl.
Chem. 1992, 29, 720.
[
14] N. Defacqz, R. Touillaux, A. Cordi, J. Marchand-Brynaert,
J. Chem. Soc., Perkin Trans. 1 2001, 2632. doi:10.1039/
B105111N
[
15] (a) D. M. Bailey, R. E. Johnson, N. F. Albertson, Org. Synth.
1
971, 51, 100.
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b) D. M. Wallace, S. H. Leung, M. O. Senge, K. M. Smith,
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[
4] D. C. Martyn, A. J. Vernall, B. M. Clark, A. D. Abell, Org.
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