Synthesis and metal complexation of chiral 3-mono- or 3,3-bis-allyl-2-hydroxypyrrolopyrazine-1,4-diones

Article abstract of DOI:10.1039/a707341k

A novel synthesis of chiral cyclic hydroxamic acids (4,6,8 and 10) related to cyclodipeptides is described. The crucial reduction of the nitro group of the N-nitroacetyl derivatives of (S)-α-amino acid esters is brought about by zinc-aq. ammonium chloride. The Fe^III and Cu^II complexes of one such cyclic hydroxamic acid 10a have been prepared and their DNAse activity investigated.

Full text of DOI:10.1039/a707341k

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Synthesis and metal complexation of chiral 3-mono- or
3,3-bis-allyl-2-hydroxypyrrolopyrazine-1,4-diones
Pabba Chittari, Vasant R. Jadhav, K. Nagappa Ganesh* and
Srinivasachari Rajappa*
Division of Organic Chemistry (Syn), National Chemical Laboratory, Pune 411008, India
A novel synthesis of chiral cyclic hydroxamic acids (4, 6, 8 and 10) related to cyclodipeptides is described.
The crucial reduction of the nitro group of the N-nitroacetyl derivatives of (S)-á-amino acid esters is
brought about by zinc–aq. ammonium chloride. The FeIII and CuII complexes of one such cyclic
hydroxamic acid 10a have been prepared and their DNAse activity investigated.
restricted. Neither of them can provide easy access to asym-
Introduction
metric molecules with predetermined stereochemistry; nor can
they lead to products incorporating an α,α-disubstituted glycine
unit in the piperazinedione ring.
Hydroxamic acids, both natural and synthetic, linear as well as
cyclic, have been reported to possess interesting biological
activity, especially in iron solubilization and transport.1 Desfer-
rioxamine B (Desferal) is a drug prescribed for reducing the
iron overload in patients suffering from thalassemia. The possi-
bility of using hydroxamic acids in drug delivery systems has
been discussed recently.2 We now report the synthesis of novel
hydroxamic acid siderophores based on the piperazine-2,5-
dione† skeleton.3
Generally, the synthesis of 1-hydroxypiperazine-2,5-diones
has been achieved by cyclisation of dipeptide precursors in
which either of the two amino acid units has an N-hydroxy
group.4 The cyclisation of the dipeptide having the N-hydroxy
amino acid at the N-terminus leads to low yields of 1-hydroxy-
piperazine-2,5-dione because of the weak nucleophilicity of the
hydroxylamine nitrogen.5 The synthesis of the requisite linear
dipeptide with an N-hydroxy amino acid is in itself a tedious
process. A second general method involves the direct oxidation
of amides or amines in the presence of a suitable catalyst.6 It is
obvious that the scope of both these synthetic routes is rather
In this article, we present an extension of our preliminary
report7 on a general synthesis of cyclic 1-hydroxypiperazine-
2,5-diones with a chiral centre at C-3, and with the possibility
of incorporating either one or two alkyl substituents at position
6. The FeIII and CuII metal complexes of one such synthetic
hydroxamic acid, viz. the 6,6-bis(allyl)-1-hydroxy derivative of
cyclo[Gly-(S)-Pro] have been prepared. The DNA cleavage
properties of these two metal complexes are reported.
Results and discussion
Synthesis
The synthesis is an extrapolation of our earlier work on the use
of the nitroacetyl group as a peptide synthon.8 The method-
ology involves the following steps: (i) synthesis of N-nitroacetyl
amino acid esters from 1,1-bis(methylthio)-2-nitroethene, (ii)
mono- or bis-allylation at the active methylene by Pd0-catalysed
allylation, (iii) reduction of the nitro group to a hydroxyl-
amine, (iv) cyclisation (Scheme 1). In a pilot study, the
nitroacetyl derivative 1 of ethyl (S)-prolinate was subjected to
reduction with the following reagents: (a) aluminium amalgam,
(b) SnCl2/PhSH/Et3N,9 (c) Zn/AcOH and (d) Zn/aq. NH4Cl/
† Piperazine-2,5-dione in this context refers to the pyrrolo[1,2-a]-
pyrazine fused ring system (see Experimental section). The locants refer
to the position on the piperazine ring and not to the pyrrolopyrazine
ring as a whole.
R2
R2
R2
O
O
O
OR3
OR3
OR3
O2N
O2N
O2N
N
N
N
R4 R4
O
O
O
R1
R1
R1
R4
R2
R2
R2
O
O
O
OR3
OR3
OR3
HOHN
HOHN
R4
HOHN
N
N
N
R4
O
R1
O
R4
R1
O
R1
R1
N
R1
N
R1
N
R2
R2
R2
O
O
O
R4
N
O
N
R4
O
N
O
R4
OH
OH
OH
Scheme 1
J. Chem. Soc., Perkin Trans. 1, 1998
1319

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led exclusively to the 1-hydroxypiperazine-2,5-dione 4 in 85%
yield (Scheme 2).
O
N
Al–Hg
The N-nitroacetyl derivatives 5 of (S)-valine ethyl ester and
(S)-phenylalanine ethyl ester could similarly be reduced with
Zn/aq. NH4Cl/EtOH and cyclised to the corresponding (3S)-
1-hydroxy-3-alkylpiperazine-2,5-diones 6, but in lower yields
(40–45%) [eqn. (2)].
H
N
H
O
2
O
O
O2N
N
SnCl2/PhSH/Et3N
The next objective was to introduce either one or two alkyl
substituents on the glycine moiety of 4 and 6. The preferred
mode of introducing such substituents at the nitroacetyl stage
was through allylation using Pd0 catalysis. Mono-substitution
of N-nitroacetyl (S)-proline ethyl ester with allyl, methylallyl or
cinnamyl groups leads to a mixture of diastereomers as re-
ported earlier. It has also been shown that the major diastereo-
mer has the (S)-configuration at the newly generated chiral
centre.8b Reduction of this diastereomeric mixture (7; de: 25–
34%) using Zn/aq. NH4Cl/EtOH, and subsequent reflux in
ethanol led to the 6-mono-substituted-1-hydroxypiperazine-
2,5-diones 8 as a mixture of diastereomers with almost the
same de. The chemical yields were good (75–85%) [eqn. (3)].
Introduction of a second identical allyl group on the α-carbon
of the N-nitroacetyl moiety of ester 7 helped to remove the com-
plexity generated by the presence of two asymmetric centres in
the molecule. At the same time, it created a quaternary carbon
adjacent to the nitrogen atom destined to become part of the
hydroxamic acid functionality; this would indeed be a welcome
feature of the product. Reduction of the tertiary nitro group in
these compounds (9a–d) with Zn/aq. NH4Cl/EtOH proceeded
well; the in situ generated hydroxylamines were cyclised by
refluxing in ethanol to yield the 6,6-bis(allyl)-1-hydroxypiper-
azine-2,5-diones (10a–d) in 70–85% yields [eqn. (4)].
In contrast to the proline-containing peptides discussed
above, the α-mono-substituted‡ N-nitroacetyl (S)-phenyl-
alanine 11 or α,α-bis-substituted derivatives 13 of N-nitro-
acetyl-(S)-valine ester or N-nitroacetyl-(S)-phenylalanine ester
did not lead to the desired cyclic hydroxamic acids on con-
trolled reduction with Zn/aq. NH4Cl/EtOH. In the case of the
monoallyl derivative 11, the product obtained was the oxime
12 (70% yield). The α,α-bis(allyl) derivatives 13‡ apparently
gave the corresponding hydroxylamines 14a–b, which how-
ever refused to undergo cyclisation to the hydroxamic acids
(Scheme 3).
HO-N
(1)
N
CO2Et
1
3
CO2Et
Zn/aq. NH4Cl/EtOH
O
N
H
O
N
OH
4
R2
H
R2
O
O
O
N
OR3
Zn/aq. NH4Cl/EtOH
O2N
(2)
N
H
N
O
OH
5a–b
a R2= -CH(CH3)2
b R2 -CH2Ph
6a–b
O
O2N
O
N
N
N
H
Zn/aq. NH4Cl/EtOH
H
O
(3)
H
CO2Et
R2
OH
R2
R1
7a–c
R1
8a–c
a R1 and R2 = H
b R1 = H, R2 = CH3
c R1 = Ph, R2 = H
O
O
N
N
O2N
N
Zn/aq. NH4Cl/EtOH
R2
H
(4)
R1
R1
O
R1
R1
CO2Et
Ph
Ph
OH
O
O
R2
R2
9a–d
HON
Ph
OEt
O2N
OEt
Zn/aq. NH4Cl/EtOH
N
N
R
H
H
10a–d
H
O
O
a R1 and R2 = H
b R1 = CH3, R2 = H
c R1 = H, R2 = Ph
12
11
Ph
d R1 = H, R2 = -CH2OAc
Scheme 2
R2
R2
EtOH [Scheme 2, eqn. (1)]. The Al–Hg reagent reduced the
primary nitro group directly to the amine; this was followed by
cyclisation to give exclusively cyclo[Gly-(S)-Pro] 2. Obviously
the use of this reagent had resulted in ‘over reduction’. In con-
trast, the SnCl2/PhSH/Et3N system led to incomplete reduction;
the product obtained was the oxime 3. A base catalysed proto-
tropic shift at the nitroso stage had intervened to prevent fur-
ther reduction to the hydroxylamine. The zinc-mediated reduc-
tions were more successful. Treatment of 1 with Zn/AcOH gave
a mixture of the corresponding amine and the hydroxylamine,
both of which then underwent cyclisation to the piperazine-2,5-
diones 2 and 4 respectively. It is known that controlling the pH
of the reaction mixture is crucial for the partial reduction of the
nitro group to the level of hydroxylamine.10 It seemed appropri-
ate therefore to use Zn/aq. NH4Cl/EtOH under neutral pH to
achieve the desired transformation. In the event, reduction of 1
under these conditions, followed by reflux to induce cyclisation
O
O
OEt
O2N
OEt
HOHN
N
N
Zn/aq. NH4Cl/EtOH
H
H
O
O
a R2= -CH(CH3)2
b R2 = -CH2Ph
14a–b
13a–b
Scheme 3
In the 13C NMR spectra, there was a characteristic downfield
shift of about 10 ppm for C-6 in the case of the N-hydroxy
compounds compared to the parent piperazine-2,5-dione. All
‡ In compounds of this type the α-position of substituents is with
respect to the carbonyl group attached to the nitrogen atom of the
amino acid.
1320
J. Chem. Soc., Perkin Trans. 1, 1998

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Optical rotations were measured on a JASCO-181 digital
polarimeter with a Na light (5893 Å) source. IR spectra
were recorded on a Perkin-Elmer Infracord spectrometer. UV
spectra were recorded on a Perkin-Elmer UV–Visible (λ) spec-
trometer. 1H and 13C NMR spectra were recorded on a Bruker
WH-90, Bruker AC-200 or Bruker MSL-300 using tetramethyl-
silane as internal standard, δ values are reported in ppm. Mass
spectra were recorded on a Finnigan-MAT-1020B spectrometer.
Elemental analyses were performed in our analytical group.
ESR and CV were obtained from the Physical Chemistry group
of the National Chemical Laboratory. The pBR322 (form I)
and Agarose gel were obtained from Bangalore, Genie, India.
Synthesis of nitro acetamides. Nitro acetamides 1, 5a and 5b
were prepared by the reaction of the respective amino acid
esters with 1,1-bis(methylthio)-2-nitroethylene in the presence
of a catalytic amount of PTSA in acetonitrile at room temper-
ature according to our earlier reports.8a,b
Fig. 1 Cleavage of pBR322 plasmid DNA by FeIII and CuII complex
of 10a. Forms I, II and III represent closed circular supercoiled, open
circular and linear DNA respectively. All lanes contain pBR322 plas-
mid DNA with the following additions. Lane 1: only DNA; Lane 2:
H2O2; Lane 3: ME; Lane 4: FeIII complex 15 ϩ H2O2 ϩ ME; Lane 5:
CuII complex 16 ϩ H2O2; Lane 6: Free ligand 10a ϩ H2O2 ϩ ME.
the cyclic hydroxamic acids exhibited a characteristic deep
violet colour with a trace of FeCl3.
Metal complexes
Several recent reports in the literature have dealt with the chem-
istry of the metal complexes of hydroxamic acids11 and their
ability to act as artificial nucleases.12 The DNAse activity of the
FeIII, CoII, NiII and CuII complexes of desferrioxamine have
been investigated in our laboratory.13
The 6,6-bis(allyl)-1-hydroxypiperazine-2,5-dione 10a in
which C-3† has the (S)-configuration, was used for our initial
studies on the possible application of the metal complexes of
such peptide-related hydroxamic acids for DNA cleavage. The
ferric trishydroxamate complex 15 and the CuII dihydroxamate
16 were prepared and characterised by IR, UV–VIS and ESR
spectroscopy and CV (Scheme 4). Fig. 1 shows the agarose gel
First and second allylation of N-nitroacetyl amino acid esters:
general procedure A
To a solution of the N-nitroacetyl derivative (1 mmol) in
degassed acetonitrile (15 ml), DBU (1 mmol) was added and
stirred under argon for 5 min. Pd(dba)2 (3 mol%) and PPh3 (12
mol%) were added to the mixture and stirred for another 5 min.
Finally, a solution of the allyl acetate (1 mmol) in acetonitrile
(5 ml) was added and the mixture stirred for 8–10 h. After
cooling to Ϫ20 ЊC, the mixture was made acidic by addition of
5% aq. HCl and extracted with ethyl acetate, the organic extract
washed well with brine, dried (Na2SO4), and evaporated; the
orange gum was further purified by column chromatography
(silica gel, 60–120 mesh, light petroleum–AcOEt)§ to give the
pure monoallyl-substituted N-nitroacetyl derivative. The above
pure monoallyl product was further subjected to a second
allylation repeating the above general procedure.
O
N
H
N
O
The monoallyl N-nitroacetyl derivatives 7a–c, 11 and the bis-
(allyl) N-nitroacetyl derivatives 9a–c, were prepared according
to the above general procedure. Their spectral data have already
been reported in our earlier publication.8c
OH
10a
(S)-Ethyl N-[2-nitro-2,2-bis(4-acetyloxybut-2-en-1-yl)acetyl]-
prolinate 9d
Compound 9d was synthesised from N-nitroacetyl-(S)-proline
ethyl ester 1. The procedure involved two successive allylation
sequences using cis-but-2-ene-1,4-diyl diacetate as described in
the above general procedure A.
The first allylation. The mono-substituted N-nitroacetyl-(S)-
proline ethyl ester was obtained in 50% yield as a diastereo-
meric mixture (de 32%)16 from N-nitroacetyl-(S)-proline ethyl
ester 1, cis-butene-1,4-diyl diacetate, Pd(dba)2 (3 mol%) and
PPh3 (12 mol%) as described in general procedure A.
νmax(CHCl3)/cmϪ1 1740, 1675, 1580, 1450, 1380, 1220, 1100 and
1040; δH(CDCl3) 1.2–1.4 (m, 3H, CH3), 2.1 (2s, 3H, COCH3),
2.0–2.4 (m, 4H, 2CH2), 2.8–3.25 (m, 2H, allylic CCH2), 3.6–3.9
(m, 2H, NCH2), 4.1–4.3 (m, 2H, OCH2), 4.5–4.7 (m, 3H, NCH,
CuCl2, EtOH–H2O, pH ~ 8
FeCl3, EtOH–H2O , pH ~ 6
O
C
N
O
Fe+3
C
N
Cu+2
O
O
3
2
Fe(III) complex 15
Cu(II) complex 16
Scheme 4
electrophoresis pattern of DNA cleavage by these two metal
complexes. Both the complexes were able to convert supercoiled
DNA (form I) into either open circular (form II) or linear (form
III) DNA, thus acting as effective Fenton-type reagents.14 Inter-
estingly, while the ferric complex 15 required both an oxidising
agent (H2O2) and a reducing agent [mercaptoethanol (ME)] for
its DNAse activity, the CuII complex 16 was able to cleave DNA
only in the presence of H2O2, indicating that the CuII–DNA
complex may be directly reduced by the peroxide to produce the
corresponding DNA Cu-hydroperoxo species15 thereby leading
to DNA damage. Neither the free ligand nor H2O2 (or ME)
were found to have any DNAse activity under identical reaction
conditions.
᎐CH C᎐), 5.2–5.4 (m, 1H, O NCH), 5.6–5.9 (m, 2H, olefinic);
᎐
2
2
δC(CDCl3) 14.19 (14.08) (CH3), 20.89 (COCH3), 21.50, 24.73,
29.12 (29.03), 30.98 (CH2), 33.03 (32.93) (allylic CH2), 47.65
(47.56) (NCH2), 59.63 (59.87) (NCH), 61.40 (OCH2), 64.11
(allylic OCH2), 85.57 (85.79) (O2NCH), 126.58 (126.82), 129.88
(C᎐C), 161.9 (161.0), 170.64, 170.90, 179.18 (CO) (Found: C,
᎐
52.45; H, 6.58; N, 8.40. C15H22N2O7 requires C, 52.62; H, 6.47;
N, 8.18%).
Second allylation. The diastereomeric mixture of monoallyl
product obtained above was subjected to a second allylation
using the same general procedure A to give the bis(allyl) prod-
uct 9d as gum. [α]D Ϫ44.5 (c 0.6, MeOH); νmax(CHCl3)/cmϪ1
1740, 1660, 1550, 1430, 1380, 1220, 1030; δH(CDCl3) 1.26 (t,
3H, CH3), 1.8–2.3 (m, 4H, 2CH2), 2.05 (2s, 6H, COCH3), 2.75–
Experimental
General
Mps were recorded on a Campbell-Electronic-Thermonic
instrument in open capillary tubes and are quoted uncorrected.
§ Light petroleum refers to the fraction with bp 60–80 ЊC.
J. Chem. Soc., Perkin Trans. 1, 1998
1321

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3.0 (m, 4H, allylic CH2), 3.2–3.5 (2m, 2H, NCH2), 4.2 (q, 2H,
OCH2), 4.4–4.7 (m, 5H, NCH, allylic CH2), 5.4–5.9 (m, 4H,
olefinic); δC(CDCl3) 14.39 (CH3), 21.03 (COCH3), 25.05 (CH2),
28.22 (CH2), 36.50 and 38.71 (allylic CH2), 47.12 (NCH2),
61.14 and 61.43 (NCH, OCH2), 64.34 and 64.29 (allylic
203–205 ЊC (lit.,17 209–210 ЊC); νmax(CHCl3)/cmϪ1 3400, 1670,
1450, 1300; δH([2H6]DMSO) 1.5–2.55 (m, 4H, 2CH2), 3.2–4.2
(m, 5H, 2NCH2 and NCH), 6.8 (br s, 1H, NH); δC(CDCl3)
22.14 (CH2), 28.16 (CH2), 45.02 (CH2), 46.31 (CH2), 58.33
(CH), 163.54 (CO), 169.93 (CO).
OCH ), 94.97 (C), 125.76 and 126.37 (C᎐C), 131.00–131.06
(C᎐C), 163.68, 170.78 and 171.71 (CO) (Found: C, 55.75; H,
᎐
᎐
2
Ethyl N-hydroxyiminoacetyl-(S)-prolinate 3
To a solution of anhydrous SnCl2 (285 mg, 1.5 mmol) in
benzene (10 ml), at 30 ЊC thiophenol (0.62 ml, 6 mmol) and
triethylamine (0.62 ml, 4.5 mmol) were added, followed by
the addition of N-nitroacetamide derivative 1 (0.75 mmol).
The mixture was stirred for 6 h. After completion of the
reaction, the organic layer was loaded directly on a silica gel
column for chromatography. Elution with CH2Cl2–MeOH
gave the product 3 (60%). νmax(Nujol)/cmϪ1 3200–2700 (br),
1750, 1650, 1600, 1470, 1380, 1200, 1020; δH(CDCl3) 1.2 (m,
3H, CH3), 2.0 (m, 4H, 2CH2), 3.8 (m, 2H, CH2), 4.57 (m, 1H,
NCH), 4.9 (m, 1H, CH), 10.3 (br s, 1H, OH) (Found: C,
50.33; H, 6.74; N, 13.35. C9H14N2O4 requires C, 50.46; H,
6.58; N, 13.07%).
6.6; N, 6.28. C21H30N2O9 requires C, 55.50; H, 6.64; N, 6.16%).
Ethyl N-[2-nitro-2,2-bis(2-methylprop-2-en-1-yl)acetyl]-(S)-
valinate 13a
Compound 13a was synthesised from N-nitroacetyl-(S)-valine
ethyl ester 5a. The procedure involved two successive allylations
using methylallyl chloride as described in the above general
procedure A.
The first allylation. The mono-substituted N-nitroacetyl-(S)-
valine ethyl ester was obtained as a diastereomeric mixture
(60%); νmax(neat)/cmϪ1 3340, 1755, 1680, 1580, 1380; δH(CDCl3)
0.85–0.9 (m, 6H, 2CH3), 1.25 (t, 3H, CH3), 1.53–1.62 (2s, 3H,
CH3), 2.1 (m, 1H, CH), 2.85 (m, 2H, allylic CH2), 4.15 (m, 2H,
OCH ), 4.4 (m, 1H, CHα), 4.75–4.85 (2s, 2H, ᎐CH ), 5.2 (m,
1H, NO2CH), 6.85 (br s, 1H, NH).
᎐
2
2
Preparation of 1-hydroxypiperazine-2,5-diones
General procedure B. To a solution of the N-nitroacetyl-(S)-
proline ethyl ester (1 mmol) in EtOH (5 ml) was added aq.
NH4Cl (50%; 5 ml) followed by zinc dust (390 mg, 6 mmol) in
small portions at 30 ЊC. The mixture was allowed to stir at
30 ЊC for 12–24 h, during which the reaction was monitored by
TLC. After the completion of the reaction, the zinc dust was
filtered off and the filtrate was concentrated under reduced
pressure. The residue was taken up in anhydrous EtOH, the
insoluble NH4Cl filtered off and the filtrate refluxed for 3–6 h
to ensure completion of the cyclisation. Finally the solution
was concentrated and the residue was purified by rapid chro-
matography on silica (light petroleum–EtOAc). The isolated
product gave as expected a deep violet colour with ethanolic
FeCl3 solution indicating clearly the formation of a hydroxamic
acid.
Second allylation. The diastereomeric mixture of monoallyl
products obtained above was subjected to a second allylation
using the same general procedure to give 13a as a gum (73%);
νmax(CHCl3)/cmϪ1 3340, 1740, 1670, 1540, 1520, 1450, 1380,
1220; δH(CDCl3) 0.85 (d, 6H, 2CH3), 1.2 (t, 3H, CH3), 1.6 (s,
6H, 2CH3), 2.15 (m, 1H, CH), 2.9 (m, 4H, allylic CH2), 4.1 (q,
2H, OCH2), 4.35 (m, 1H, CHα), 4.65–4.85 (m, 4H, allylic CH2),
7.8 (br d, 1H, NH); δC(CDCl3) 14.07 (CH3), 17.83, 18.74
(2CH3), 22.86, 23.13 (2CH3), 31.04 (CH), 45.24, 45.74 (allylic
CH2), 58.13 [NCH(α)], 61.19 (OCH2), 96.97 (C), 116.19, 116.56
(᎐CH ), 138.29, 138.48 (᎐C), 165.70 (CO), 170.74 (CO)
᎐
᎐
2
(Found: C, 60.11; H, 8.13; N, 8.46. C17H28N2O5 requires C,
59.98; H, 8.29; N, 8.22%).
Ethyl N-[2-nitro-2,2-bis(2-methylprop-2-en-1-yl)acetyl]-(S)-
phenylalaninate 13b
Compound 13b was synthesised from N-nitroacetyl-(S)-
phenylalanine ethyl ester 5b. The procedure involved two suc-
cessive Pd0-catalysed allylations using methylallyl chloride as
described in the above general procedure A.
The first allylation. The mono-substituted N-nitroacetyl-(S)-
phenylalanine ethyl ester was obtained as a diastereomeric mix-
ture (65%); νmax(neat)/cmϪ1 3350, 1760, 1660, 1550, 1440, 1380;
δH(CDCl3) 1.26 (t, 3H, CH3), 1.75 (s, 3H, CH3), 2.84 (m, 2H,
allylic CH2), 3.15 (m, 2H, CH2), 4.2 (q, 2H, OCH2), 4.8 (2s, 2H,
(8aS)-2-Hydroxyperhydropyrrolo[1,2-a]pyrazine-1,4-dione 4
The product 4 was obtained as a white solid from N-nitroacetyl-
(S)-proline ethyl ester 1 (85%), mp 115 ЊC (lit.,4a 114 ЊC); [α]D
Ϫ132.9 (c 0.4, MeOH);4a δH(D2O) 1.8–2.5 (m, 4H, 2CH2), 3.55
(m, 2H, CH2), 4.22 (d, 1H, CH), 4.37 (m, 1H, NCH), 4.6 (d, 1H,
CH); δC(D2O) 23.3 (γCH2), 29.7 (βCH2), 47.12 (δCH2), 55.73
(αЈCH2), 60.12 (αCH), 166.6 (CO), 165.1 (CO); m/z 170 (Mϩ),
153 (M Ϫ 17), 142, 128, 111, 98, 83, 70 (base peak) (Found: C,
48.32; H, 5.58; N, 15.84. C7H10N2O3ؒ0.25H2O requires C, 48.12;
H, 5.76; N, 15.92%).
᎐CH ), 5.17 [m, 2H, NO CH, CH(α)], 6.74 (br d, 1H, NH), 7.1–
᎐
2
2
7.25 (m, 5H, ArH).
(3S)-1-Hydroxy-3-isopropylpiperazine-2,5-dione 6a
Second allylation. The diastereomeric mixture of mono-
allylated product obtained above was subjected to a second
allylation using the same general procedure to give the bis-
(allyl) product 13b as a gum (70%); νmax(CHCl3)/cmϪ1 3320,
1750, 1660, 1540, 1450, 1380; δH(CDCl3) 1.22 (t, 3H, CH3),
1.4, 1.53 (2s, 6H, 2CH3), 2.7–3.2 (m, 4H, 2CH2), 4.15 (q, 2H,
Reduction of N-nitroacetyl-(S)-valine ethyl ester 5a according
to general procedure
B gave (3S)-1-hydroxy-3-isopropyl-
piperazine-2,5-dione 6a (40%), mp 115 ЊC (lit.,4a 115 ЊC); [α]D
Ϫ33.5 (c 1, MeOH);4a δH([2H6]DMSO) 0.83–0.92 (2d, 6H,
2CH3), 2.15 (m, 1H, CH), 3.7 (m, 1H, CH), 4.0–4.15 (2d, 2H,
CH2), 8.26 (s, 1H, NH); δC([2H6]DMSO) 17.04 and 18.87
(2CH3), 33.24 (CH), 53.54 (NCH2), 59.58 (NCH), 162.22 and
165.07 (CO); m/z 172 (Mϩ), 155 (M Ϫ 17), 144, 130, 113, 101
(base peak), 85, 72 (Found: C, 48.05; H, 7.0; N, 16.03.
C7H12N2O3 requires C, 48.33; H, 7.02; N, 16.27%).
OCH ), 4.6–5.0 [m, 5H, 2C᎐CH , CH(α)], 7.25 (m, 5H, ArH),
᎐
2
2
7.8 (br d, 1H, NH) (Found: C, 65.13; H, 7.38; N, 7.12.
C21H28N2O5 requires C, 64.92; H, 7.26; N, 7.21%).
Reduction of N-nitroacetyl-(S)-proline ethyl ester
(8aS)-Perhydropyrrolo[1,2-a]pyrazine-1,4-dione 2. To the N-
nitroacetyl-(S)-proline ethyl ester 1 (230 mg, 1 mmol) in eth-
anol was added freshly prepared Al–Hg (300 mg) and the
mixture was stirred at 30 ЊC until the complete disappearance
of the starting material (ca. 8 h). After completion of the reac-
tion the inorganic material was filtered off and the filtrate was
concentrated under reduced pressure to afford a crude product.
Purification of the crude product on a silica gel column with
light petroleum and EtOAc as eluents, initially 7:3 to remove
impurities, finally 1:1 to elute the product, yielded 2 (70%), mp
(3S)-1-Hydroxy-3-benzylpiperazine-2,5-dione 6b
Reduction of N-nitroacetyl-(S)-phenylalanine ethyl ester 5b
according to the general procedure B gave (3S)-1-hydroxy-3-
benzylpiperazine-2,5-dione 6b (45%), mp 232 ЊC (lit.,4a 232 ЊC);
[α]D 18.2 (c 1, DMF);4a νmax(CHCl3)/cmϪ1 1650; δH([2H6]-
DMSO) 2.88 (dd, 1H, CH), 3.05 (d, 1H, CH), 3.15 (dd, 1H,
CH), 3.7 (d, 1H, CH), 4.22 (m, 1H, CH), 7.2 (m, 5H, PhH), 8.2
(br d, 1H, NH); m/z 220 (Mϩ), 192, 162, 120, 91 (base peak)
(Found: C, 58.71; H, 5.31; N, 12.64. C11H12N2O3ؒ0.3H2O
requires C, 58.55; H, 5.35; N, 12.42%).
1322
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
(8aS)-Perhydro-2-hydroxy-3-(prop-2-en-1-yl)pyrrolo[1,2-a]-
pyrazine-1,4-dione 8a
Reduction of the monoallyl derivative 7a (de = 29%) according
to the general procedure B gave the 2-hydroxypyrrolopyrazine-
1,4-dione 8a (80%) as a gum (de = 30%),16 νmax(KBr)/cmϪ1 3471,
3100, 2920, 1648; δH(D2O) 1.5–2.25 (m, 4H, 2CH2), 2.4–2.9 (m,
2H, allylic CH2), 3.2–3.6 (m, 2H, NCH2), 4.1 [m, 1H, NCH(α)],
allylic CH2), 3.4–3.8 (m, 2H, NCH2), 4.1 [dd, 1H, NCH(α)],
5.1–5.9 (m, 6H, CH᎐CH ), 8.3 (br s, 1H, OH); δ (CDCl ) 21.38,
29.99 (β- and γ-C), 39.27, 39.87 (allylic CH2), 44.73 (NCH2),
᎐
2
C
3
57.46 [NCH(α)], 72.56 (C), 119.77, 120.56 (᎐CH ), 131.0,
᎐
2
131.41 (᎐CH᎐), 162.28 (CO), 164.16 (CO); m/z 250 (Mϩ), 233
᎐
(M Ϫ 17), 209, 181 (base peak), 112, 70 (Found: C, 62.41;
H, 7.01; N, 11.28. C13H18N2O3 requires C, 62.38; H, 7.24; N,
11.19%).
4.2–4.5 (m, 1H, CHNOH), 5.0–5.8 (m, 3H, CH᎐CH ); δ (D O)
᎐
2
C
2
22.81 (22.41) (γC), 29.87 (30.37) (βC), 35.20 (33.66) (allylic
CH2), 46.85 (46.27) (NCH2), 59.58 [NCH(α)], 66.62 (64.8)
(8aS)-Perhydro-2-hydroxy-3,3-bis(2-methylprop-2-en-1-yl)-
pyrrolo[1,2-a]pyrazine-1,4-dione 10b
(αЈC), 121.83, 122.51 (᎐CH ), 132.03, 132.2 (᎐CH), 165.94
᎐
᎐
2
(CO), 166.77 (CO) (Found: C, 57.27; H, 6.48; N, 13.05.
C10H14N2O3 requires C, 57.14; H, 6.70; N, 13.32%).
Reduction of the bis(methylallyl) derivative 9b8c by the general
procedure B gave the 3,3-bis(methylallyl)-2-hydroxypyrrolo-
pyrazine-1,4-dione 10b as a gum (80%); [α]D Ϫ55.4 (c 1,
MeOH); νmax(CHCl3)/cmϪ1 3423, 2926, 1660, 1446, 1218;
δH(CDCl3) 1.6–1.8 (2s, 6H, CH3), 1.5–2.5 (m, 4H, 2CH2), 2.5–
2.9 (m, 4H, allylic CH2), 3.4–3.75 (2m, 2H, CH2), 4.7–4.9 (m,
4H, ᎐CH ); m/z 278 (Mϩ), 261 (M Ϫ 17) (Found: C, 64.51;
(8aS)-Perhydro-2-hydroxy-3-(2-methylprop-2-en-1-yl)pyrrolo-
[1,2-a]pyrazine-1,4-dione 8b
Reduction of the diastereomeric mixture of the monoallyl
derivative 7b8c (de = 25%) according to the general procedure B
gave the 2-hydroxypyrrolopyrazine-1,4-dione 8b (77%) as a
gum (de = 23%); νmax(CHCl3)/cmϪ1 3300, 2900, 1660, 1450,
1220, 1070; δH(CDCl3) 1.5–2.4 (m, 4H, β and γCH2), 1.7 and
1.85 (2s, 3H, CH3), 2.6–3.0 (m, 2H, allylic CH2), 3.3–3.8 (m, 2H,
NCH2), 4.1 [m, 1H, NCH(α)], 4.5–4.6 (m, 1H, CHNOH), 4.9
᎐
2
H, 8.01; N, 10.01. C15H22N2O3 requires C, 64.73; H, 7.95; N,
10.06%).
(8aS)-Perhydro-2-hydroxy-3,3-bis(3-phenylprop-2-en-1-yl)-
pyrrolo[1,2-a]pyrazine-1,4-dione 10c
(2s, 2H, ᎐CH ); δ (CDCl ) 22.0 (21.60) (γC), 23.0 (allylic CH ),
Reduction of the bis(cinnamyl) derivative 9c8c by the general
᎐
2
C
3
3
29.29 (29.75) (βC), 35.83 (37.63) (allylic CH2), 45.15 (NCH2),
57.74 [NCH(α)], 63.94 (αЈC), 116.26 (olefinic CH2), 139.81
᎐
206, 196, 166, 151, 141 (base peak), 123, 110, 97, 70 (Found: C,
59.13; H, 7.04; N, 12.58. C11H16N2O3 requires C, 58.90; H, 7.19;
N, 12.49%).
procedure B gave the 3,3-bis(cinnamyl)-2-hydroxypyrrolo-
pyrazine-1,4-dione 10c as a gum (70%); νmax(CHCl3)/cmϪ1
3200–3400 (br), 3100, 2900, 1660, 1500, 1460, 1230; δH(CDCl3)
1.5–2.7 (m, 4H, 2CH2), 2.8–3.0 (m, 4H, allylic CH2), 3.2–4.1 (m,
(᎐C), 162.27 (CO), 163.53 (CO); m/z 224 (Mϩ), 207 (M Ϫ 17),
3H, CHα, CH ), 6.0–6.6 (m, 4H, HC᎐CH), 7.4 (m, 10H, Ar-H);
᎐
2
δC(CDCl3) 21.3, 29.8 (β-, γ-C), 38.5, 39.0 (allylic CH2), 44.9
(NCH ), 57.3 [NCH(α)], 73.4 (C), 122.3, 122.9 (᎐CH), 126.05,
᎐
2
(8aS)-Perhydro-2-hydroxy-3-(3-phenylprop-2-en-1-yl)pyrrolo-
[1,2-a]pyrazine-1,4-dione 8c
126.16, 127.3, 127.5, 128.3 and 128.5 (aromatic), 135.0, 135.4
(᎐CH᎐), 136.95, 137.2 (aromatic), 162 (CO), 164.4 (CO); m/z
᎐
Reduction of the diastereomeric mixture of the monocinnamyl
derivative 7c8c (de = 34%) according to general procedure B
gave the 2-hydroxy-3-cinnamylpyrrolopyrazine-1,4-dione 8c
(70%) as a gum (de = 38%); νmax(CHCl3)/cmϪ1 3340, 3100, 2920,
1660, 1450, 1220, 1070; δH(CDCl3) 1.5–2.25 (m, 4H, β- and γ-
CH2), 2.7–3.0 (m, 2H, allylic CH2), 3.2–3.7 (m, 2H, NCH2), 3.9–
4.1 [m, 1H, NCH(α)], 4.4 (m, 1H, CHNOH), 5.9–6.15 (m, 1H,
402 (Mϩ), 385 (M Ϫ 17), 357, 285, 257 (100%), 117, 91 (A small
sample of the product was further purified for microanalysis
by preparative TLC. Found: C, 71.55; H, 6.04; N, 6.38.
C25H26N2O3ؒH2O requires C, 71.40; H, 6.23; N, 6.66%).
(8aS)-Perhydro-2-hydroxy-3,3-bis(4-acetoxybut-2-en-1-yl)-
pyrrolo[1,2-a]pyrazine-1,4-dione 10d
᎐CH), 6.4 (m, 1H, ᎐CH), 7.0–7.2 (m, 5H, ArH); δ (CDCl )
Reduction of the bis(acetoxyallyl) derivative 9d by the general
procedure B gave the 3,3-bis(acetoxyallyl)-2-hydroxypyrrolo-
pyrazine-1,4-dione 10d as a gum (70%); [α]D Ϫ27.58 (c 2.4,
MeOH); νmax(CHCl3)/cmϪ1 3331, 2929, 1737, 1659, 1650, 1453,
1383; δH(CDCl3) 1.7–2.5 (m, 4H, 2CH2), 2.0 (2s, 6H, COCH3),
2.5–3.0 (m, 4H, allylic CH2), 3.4–3.9 (2m, 2H, NCH2), 4.1 (dd,
1H, NCH), 4.45 (m, 4H, allylic OCH2), 5.4–5.8 (m, 4H,
᎐
᎐
C
3
22.12 (21.91) (γC), 29.57 (30.21) (βC), 33.6 and 34.0 (allylic
CH2), 44.75 (45.31) (NCH2), 57.95, 60.24, 65.41, 122.25, 126.39,
128.0, 128.78, 135.70, 137.04, 161.5 and 162 (CO), 163.5, 164.0
(CO); m/z 286 (Mϩ), 269 (M Ϫ 17), 241, 152, 117, 77, 70.
Ethyl N-[(2-hydroxyimino)-5-phenylpent-4-enoyl]-(S)-
phenylalaninate 12
HC᎐CH); δ (CDCl ) 20.61 (COCH ), 21.53 (CH ), 29.93
᎐
C
3
3
2
Compound 12 was obtained as a solid by reduction of the
mono-substituted N-nitroacetyl-(S)-phenylalanine ethyl ester
11 according to the general procedure B, mp 110 ЊC; [α]D ϩ6.8
(c 1, MeOH); νmax(CHCl3)/cmϪ1 3400, 3300, 1740, 1670, 1520,
1230, 1020; δH(CDCl3) 1.3 (t, 3H, CH3), 3.0 (d, 2H, CH2), 3.48
(d, 2H, CH2), 4.1 (q, 2H, OCH2), 4.85 [m, 1H, CH(α)], 6.6 (m,
(CH2), 37.60, 38.27 (allylic CH2), 44.80 (NCH2), 57.07 (NCH),
64.04, 64.30 (allylic OCH2), 72.81 (C), 127.05, 127.72, 128.15,
129.47, 129.60, 130.09 and 130.30 (olefinic), 162.22, 163.83,
170.33 and 170.44 (CO) (Found: C, 57.94; H, 6.46; N, 7.32.
C19H26N2O7 requires C, 57.86; H, 6.63; N, 7.10%).
2H, CH᎐CH), 7.0–7.5 (m, 11H, ArH, NH), 9.62 (br s, 1H,
Ethyl N-[N-hydroxy-2,2-bis(2-methylprop-2-en-1-yl)glycyl]-(S)-
valinate 14a
᎐
OH); δC(CDCl3) 13.72 (CH3), 26.82 (CH2), 34.84 (CH2), 53.4
[NCH(α)], 61.47 (OCH2), 122.65, 126.02, 126.84, 126.96,
128.18, 128.27, 129.02, 132.63, 135.63 and 137.21 (aromatic and
Reduction of the bis(methylallyl) derivative 13a of N-
nitroacetyl-(S)-valine ethyl ester by the general procedure B
gave the hydroxylamine derivative 14a (74%) as a solid, mp
116 ЊC; [α]D 6.6 (c 0.5, MeOH); νmax(CHCl3)/cmϪ1 3400, 1740,
1650, 1540, 1470, 1450, 1390, 1310, 1270, 1220, 1040;
δH(CDCl3) 0.85 (m, 6H, 2CH3), 1.2 (t, 3H, CH3), 1.62 (s, 6H,
2CH3), 2.1 (m, 1H, CH), 2.2–2.7 (m, 4H, allylic CH2), 4.1 (q,
olefinic), 151.97 (C᎐N), 162.96 (CO), 171.74 (CO); m/z 380
᎐
(Mϩ), 363 (M Ϫ 17), 335, 307, 192, 170, 144, 115, 91 (Found: C,
69.64; H, 6.12; N, 7.53. C22H24N2O4 requires C, 69.46; H, 6.35;
N, 7.36%).
(8aS)-Perhydro-2-hydroxy-3,3-bis(prop-2-en-1-yl)pyrrolo[1,2-a]-
pyrazine-1,4-dione 10a
2H, OCH ), 4.8–5.2 (m, 5H, NH, ᎐CH ), 7.15 (d, 1H, NH);
᎐
2 2
δC(CDCl3) 14.08 (CH3), 17.78, 18.89 (2CH3), 24.45, 24.53
(2CH3), 30.93 (CH), 40.72, 41.97 (allylic CH2), 57.18 [NCH(α)],
Reduction of the bisallyl derivative 9a8c by the general pro-
cedure B gave the 3,3-bis(allyl)-2-hydroxypyrrolopyrazine-1,4-
dione 10a as a solid, mp 102 ЊC (85%); [α]D Ϫ80.3 (c 1, MeOH);
νmax(CHCl3)/cmϪ1 3300–3100 (br), 2900, 1660, 1440, 1300, 1220;
δH(CDCl3) 1.5–2.4 (m, 4H, β- and γ-CH2), 2.6–2.9 (m, 4H,
69.16 (C), 115.56 (᎐CH ), 141.15, 141.51 (᎐C), 172.39, 172.98
᎐
᎐
2
(CO); m/z 326 (Mϩ), 309 (M Ϫ 17), 281, 271 (base peak), 253,
197, 154, 144 (Found: C, 62.27; H, 9.43; N, 8.78. C17H30N2O4
requires C, 62.55; H, 9.25; N, 8.58%).
J. Chem. Soc., Perkin Trans. 1, 1998
1323

View Article Online
Ethyl N-[N-hydroxy-2,2-bis(2-methylprop-2-en-1-yl)glycyl]-(S)-
phenylalaninate 14b
Acknowledgements
We thank the CSIR for financial support (to S. R.) under the
Emeritus Scientist scheme, and for the award of Senior
Research Fellowships (to P. C. and V. R. J.).
Reduction of the bis(methylallyl) derivative 13b of N-nitro-
acetyl-(S)-phenylalanine ethyl ester by the general procedure B
gave the hydroxylamine derivative 14b (81%); νmax(CHCl3)/cmϪ1
3400, 1740, 1680, 1530, 1280, 1030; δH(CDCl3) 1.25 (t, 3H,
CH3), 1.65, 1.75 (2s, 6H, CH3), 2.25–2.65 (m, 4H, allylic CH2),
3.1 (d, 2H, CH2), 4.15 (q, 2H, OCH2), 4.5–5.0 [m, 6H, NH,
References
1 (a) M. J. Miller, Chem. Rev., 1989, 89, 1563; (b) H. C. J. Ottenheijm
and J. D. M. Herscheid, Chem. Rev., 1986, 86, 697; (c) J. B. Neilands,
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2 M. J. Miller and F. Malouin, Acc. Chem. Res., 1993, 26, 241.
3 S. Rajappa and M. V. Natekar, Adv. Heterocycl. Chem., 1993,
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4 (a) M. Akiyama, A. Katoh and Y. Tsuchiya, J. Chem. Soc., Perkin
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48, 2584.
CH(α), ᎐CH ], 7.0–7.3 (m, 6H, NH, ArH); δ (CDCl ) 13.89
᎐
2
C
3
(CH3), 24.31 (CH3), 30.06 (CH2), 40.96, 41.45 (allylic CH2),
52.88 [NCH(α)], 61.28 (OCH2), 68.52 (C), 115.41, 115.46
(᎐CH ), 126.93, 128.38, 129.14 and 136.06 (aromatic), 141.06,
᎐
2
141.30 (᎐C), 171.77 (CO), 172.65 (CO); m/z 374 (Mϩ), 357
᎐
(M Ϫ 17), 319 (base peak), 283, 245, 192, 154, 146, 138, 120, 91
(Found: C, 67.31; H, 8.14; N, 7.33. C21H30N2O4 requires C,
67.35; H, 8.06; N, 7.48%).
Metal complexes of 3,3-bis(allyl)-2-hydroxypyrrolopyrazine-
1,4-dione
FeIII trihydroxamate complex 15. The hydroxamic acid 10a
(750 mg, 3 mmol) was dissolved in an ethanol and water mix-
ture (1:4, 10 ml) and allowed to warm on a steam bath. To
this was added hydrated FeCl3 (150 mg, 0.9 mmol) as an
aqueous solution dropwise. An intense violet colour
developed immediately. The pH was adjusted to 6 using
sodium acetate solution. A violet coloured solid soon separ-
ated from the hot solution. After complete removal of ethanol
from the mixture, the solution was allowed to cool to 30 ЊC.
The solid was collected by filtration, thoroughly washed with
cold water and dried over P2O5. The yield of the ferric com-
plex 15 was about 50%; νmax(CHCl3)/cmϪ1 1650, 1600;
5 (a) J. D. M. Herscheid, J. H. Colstee and H. C. J. Ottenheijm, J. Org.
Chem., 1981, 46, 3346; (b) J. D. M. Herscheid, R. J. F. Nivard, M. W.
Tijhuis, H. P. H. Scholten and H. C. J. Ottenheijm, J. Org. Chem.,
1980, 45, 1880.
6 (a) A. Ohta, Chem. Pharm. Bull., 1964, 12, 125; (b) S. I. Murahashi,
T. Oda, T. Sugihara and Y. Masui, J. Org. Chem., 1990, 55, 1744;
(c) S. M. Neset, T. Benneche and K. Undheim, Acta Chem. Scand.,
1993, 47, 1141.
7 P. Chittari, A. Thomas and S. Rajappa, Tetrahedron Lett., 1994, 35,
3793.
8 (a) S. G. Manjunatha, K. V. Reddy and S. Rajappa, Tetrahedron
Lett., 1990, 31, 1327; (b) S. G. Manjunatha and S. Rajappa, J. Chem.
Soc., Chem. Commun., 1991, 372; (c) S. G. Manjunatha, P. Chittari
and S. Rajappa, Helv. Chim. Acta, 1991, 74, 1071; (d) A. Thomas
and S. Rajappa, Helv. Chim. Acta, 1992, 75, 715.
9 M. Bartra, P. Romea, F. Urpi and J. Vilarrasa, Tetrahedron, 1990,
46, 587.
10 M. Hudlicky, Reductions in Organic Chemistry, Wiley, New
York, 1984, 69; (b) H. O. House, Modern Synthetic Reactions,
W. A. Benjamin Inc., CA, 1972; (c) A. S. Kende and J. S. Mendoza,
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11 (a) B. Chatterjee, Coord. Chem. Rev., 1978, 26, 281; (b) G. D.
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12 (a) S. Hashimoto, R. Yamashita and Y. Nakamura, Chem. Lett.,
1992, 1639; (b) S. Hashimoto and Y. Nakamura, J. Chem. Soc.,
Chem. Commun., 1995, 1413.
λmax(MeOH)/nm 420 (ε/dm3 molϪ1 cmϪ1 2340); E Ϫ980 mV
₁
₂
(∆E 100, dropping mercury electrode);18 EPR g 4.2 (Found:
C, 58.12; H, 6.53; N, 10.66. C39H51N6O9Fe requires C, 58.29;
H, 6.35; N, 10.46%).
CuII dihydroxamate complex 16. The hydroxamic acid 10a
(250 mg, 1 mmol) was dissolved in an ethanol and water mix-
ture (1:4, 10 ml) and allowed to warm on a hot water bath. To
this solution was added CuCl2 (0.43 mmol) and the mixture was
stirred for 30 min while a light green colour developed slowly.
The pH of the solution was adjusted to 8 using sodium acetate
solution. A light green coloured solid precipitated immediately.
This was collected, washed thoroughly with cold water and
dried, to yield the complex 16 (56%); νmax(CHCl3)/cmϪ1 1650,
1600; λmax(MeOH)/nm 650 (ε/dm3 molϪ1 cmϪ1 30), 350 (150);
EPR g 2.136 (Found: C, 55.73; H, 6.68; N, 10.11. C26H34-
N4O6Cu requires C, 55.56; H, 6.05; N, 9.97%).
13 (a) R. R. Joshi and K. N. Ganesh, Biochem. Biophys. Res. Commun.,
1992, 182, 588; (b) R. R. Joshi and K. N. Ganesh, FEBS Lett., 1992,
313, 303.
14 J. Cadet and M. Weinfeld, Anal. Chem., 1993, 65, 675.
15 (a) K. Yamamoto and S. Kawanishi, J. Biol. Chem., 1989, 264,
15 435; (b) M. Chikira and Y. Mizukami, Chem. Lett., 1991, 189.
16 De was estimated from the relative peak heights in the 13C NMR
spectra. The chemical shift values of the minor diastereomer are
given in parentheses.
DNA cleavage assay
The standard DNA cleavage reactions were performed in a
total volume of 20 µl consisting of 20 m tris-HCl (pH 7.8),
sodium acetate (2.5 µ), plasmid pBR322 DNA (70 µ), H2O2
(5 µ), mercaptoethanol (0.5 µ) and metal complex 15 or 16
(130 µ). The reactions were done at 37 ЊC for 30 min followed
by addition of gel loading buffer containing bromophenol blue
as dye and then directly loaded on 1% agarose gel for analysis
by electrophoresis at 100 V.19 The gels were stained with ethid-
ium bromide (0.1 µg mlϪ1) and visualised under UV light. The
DNA cleavage reactions in the presence of sodium azide (0.1
), mannitol (0.1 ) and glycerol (2 ) were carried out under
similar conditions as described above.
17 J. Vicar, J. Smolikova and K. Blaha, Collect. Czech. Chem.
Commun., 1972, 37, 4060.
18 We thank Dr P. Ghosh for determining the oxidation potential of
FeIII complex 15 by cyclic voltammetry.
19 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular cloning: a
laboratory manual, Cold Spring Harbor, New York, 1989.
Paper 7/07341K
Received 10th October 1997
Accepted 19th January 1998
1324
J. Chem. Soc., Perkin Trans. 1, 1998