Benzofuranyl 3,5-bis-polyamine derivatives as time-dependent inhibitors of trypanothione reductase

Article abstract of DOI:10.1016/S0968-0896(03)00344-4

The synthesis and evaluation of 3,5-disubstituted benzofuran derivatives as time-dependent inhibitors of the protozoan oxidoreductase trypanothione reductase are reported. These molecules were designed as simplified mimetics of the naturally occurring spermidine-bridged macrocyclic alkaloid lunarine 1, a known time-dependent inhibitor of trypanothione reductase. In this series of compounds the bis-polyaminoacrylamide derivatives 2-4 were all shown to be competitive inhibitors, but only the bis-4-methyl-piperazin-1-yl-propylacrylamide derivative 4 displayed time-dependent activity. The kinetics of time dependent inactivation of trypanothione reductase by 1 and 4 have been determined and are compared and discussed herein.

Full text of DOI:10.1016/S0968-0896(03)00344-4

Bioorganic & Medicinal Chemistry 11 (2003) 3683–3693
Benzofuranyl 3,5-bis-Polyamine Derivatives as Time-Dependent
Inhibitors of Trypanothione Reductase
Chris J. Hamilton,a,y Ahilan Saravanamuthu,b
Alan H. Fairlambb and Ian M. Egglestona,*
aDivision of Biological Chemistry & Molecular Microbiology, Faculty of Life Sciences,
Carnelley Building, University of Dundee, Dundee DD1 4HN, UK
bDivision of Biological Chemistry & Molecular Microbiology, Faculty of Life Sciences,
Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK
Received 10 July 2002; accepted 16 May 2003
Abstract—The synthesis and evaluation of 3,5-disubstituted benzofuran derivatives as time-dependent inhibitors of the protozoan
oxidoreductase trypanothione reductase are reported. These molecules were designed as simplified mimetics of the naturally
occurring spermidine-bridged macrocyclic alkaloid lunarine 1, a known time-dependent inhibitor of trypanothione reductase. In
this series of compounds the bis-polyaminoacrylamide derivatives 2–4 were all shown to be competitive inhibitors, but only the bis-
4-methyl-piperazin-1-yl-propylacrylamide derivative 4 displayed time-dependent activity. The kinetics of time dependent inactiva-
tion of trypanothione reductase by 1 and 4 have been determined and are compared and discussed herein.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
reductase (TryR; EC 1.6.4.8). The structures of the
disulfide substrates for TryR and GR are illustrated in
Figure 1.
Parasitic protozoa belonging to the genera Trypano-
soma and Leishmania cause a number of severe infec-
tious diseases in humans and their domestic livestock in
tropical countries. In humans, these include Chagas’
disease (caused by Trypanosoma cruzi), African sleeping
sickness (Trypanosoma brucei rhodesiense and T.b. gam-
biense), and visceral, mucocutaneous and cutaneous
leishmaniasis (caused by various species of Leishmania).
TryR is an NADPH-dependent flavoprotein oxido-
reductase which maintains an intracellular reducing
environment by the recycling of trypanothione disulfide
(T[S])2) to its dithiol form, T[SH]2. Trypanothione is
oxidised back to T[S]2 following reaction with poten-
tially damaging radicals and oxidants generated by
aerobic metabolism and by host macrophages. By
maintaining a high intracellular ratio of T[SH]2 to T[S]2,
the TryR redox cycle is a primary line of defence for
these parasites against respiratory burst responses from
the mammalian host. Disabling the function of TryR in
Leishmania2 and T. brucei3 has been shown to markedly
increase the parasites’ sensitivity to oxidative stress.
T[S]2 differs from GSSG by the presence of a spermi-
dine cross-link between the two glycyl carboxyl groups
(compare GSSG and T[S]2 in Fig. 1). Due to structural
and charge differences between T[S]2 and GSSG, TryR
and GR are mutually exclusive with respect to sub-
strate specificity.4 Thus the essential requirement for
TryR, its absence in host metabolism and the potential
to design specific inhibitors make it an attractive
therapeutic target.
A potential drug target in trypanosomes and leishma-
nia has been identified through the discovery of a
fundamental difference between the redox defence
system of the trypanosomal/leishmanial parasite and
the infected host.1 The mammalian redox defence sys-
tem, based on glutathione (l-g-glutamyl-l-cysteinyl-
glycine) and glutathione disulfide reductase (GR; EC
1.6.4.2), is replaced in trypanosomatids by an analo-
gous system based on trypanothione [N1,N8-bis(glu-
tathionyl)spermidine] and trypanothione disulfide
*Corresponding author. E-mail: i.m.eggleston@dundee.ac.uk
yCurrent address: Centre for Carbohydrate Chemistry, School of
Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00344-4

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C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
Figure 1. Native disulfide substrates for TryR (T[S]2) and GR (GSSG) and the time dependent inhibitor of TryR lunarine 1.
Various molecules have been identified as classical inhi-
bitors of TryR such as hydrophobic linear polyamine
derivatives5 and the naturally occurring bis(tetrahy-
drocinnamoyl)spermine, kukoamine A.6 Recently, the
spermidine-based macrocyclic alkaloid lunarine 1 (Fig.
1) was identified as a competitive, time-dependent inhi-
bitor of TryR in the reduced state (TryRred).7 This
alkaloid is composed of a spermidine chain with the
terminal nitrogen atoms forming amide linkages with
two a,b-unsaturated carboxylic acid functions disposed
upon an unusual 3-oxohexahydrodibenzofuranyl tri-
cyclic scaffold.8 A possible mechanism for this time-
dependent inhibition involves the covalent modification
of a redox-active cysteine residue in the active site of
TryRred (C53) by conjugate addition to one of these
unsaturated amide moieties in the lunarine macrocycle
(Fig. 2). This is supported by both the requirement for
the enzyme in its reduced form7 and the presence of a
potential Michael acceptor unit in the inhibitor.
pared some benzofuranyl-based acyclic bis-polyamine
analogues 2–4 of lunarine 1 where the skew boat cyclo-
hexanone moiety9 has been removed leaving a planar
bicyclic benzofuranyl scaffold (Fig. 3). Acyclic bis-poly-
amine derivatives were chosen since bis-polyamine
functionalised disulfides such as the naturally occurring
N1-glutathionylspermidine disulfide4b and the synthetic
bis-dimethylaminopropyl- and bis-N4-methylpiperazinyl
amides of Ellman’s reagent (DTNB)10 are known TryR
substrates. These three polyamine chains were chosen
for functionalisation of a 3,5-disubstituted benzofuranyl
template to give potential inhibitors 2–4. Presented
herein are the syntheses and inhibitory properties of
these compounds.
Results
Synthesis
As part of our ongoing investigations into the mechan-
istic details of TryR inhibition by the Lunaria alkaloids
and the design of simplified analogues, we have pre-
The common benzofuranyl derivative 11 that was
required in this work was prepared as outlined in
Schemes 1 and 2.
The tribromo derivative 6 was easily prepared from
5-bromobenzofuran 511 by treatment with bromine in
dichloromethane (Scheme 1)12 and, due to its instability,
was immediately treated with KOH13 to give the
3,5-dibromobenzofuran 7 in excellent yield (98%).
Compound 7 was then treated with copper(I) cyanide in
DMF under reflux14 to obtain the dinitrile 8 in 52%
yield.15 The relatively low yield of this reaction was due
to significant mechanical loss during the treatment of
the reaction products with acidic FeCl3, which was a
necessary step as residual copper(I) salts complex with
the aromatic nitriles making separation difficult [FeCl3
Figure 2. Proposed mechanism for time-dependent inactivation of
TryR by the reversible formation of a covalent adduct between an
active site thiol (C53) and one of the a,b-unsaturated amide groups of
lunarine 1.

C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
3685
Figure 3. Structure of target compounds 2–4.
Scheme 1. Reagents and conditions: (i) Br2, CH2Cl2, 40min; (ii) KOH, EtOH, reflux, 15 min; (iii) CuCN, DMF, reflux, 16 h, then FeCl 3, 1.5 M HCl,
70 ꢀC, 30min; (iv) DIBAL, CH 2Cl2, À15 ꢀC, 3 min, then 1 M HCl(aq), 0 ꢀC.
Three polyamines were required for preparation of the
acyclic benzofuranyl bis-polyamine derivatives 2–4.
3-Dimethylaminopropylamine was commercially avail-
able, while the selectively masked spermidine 15 was
prepared as previously described.8 N-Methylpiper-
azinylpropylamine 14 was prepared as shown in Scheme
3, nitrile derivative 13 being prepared from N-methyl-
piperazine 12 using a modification of a previously
reported procedure,16 then hydrogenated over a rho-
dium catalyst to give the free amine 14,17 albeit in low
yield (19%).
The bis-polyamine derivatives were prepared from the
diacid 11 using two different amide bond-forming pro-
Scheme 2. Reagents and conditions: (i) Ph3P¼CHCO2Et, DMF,
cedures (Scheme 4). Compounds 3 and 4 were prepared
via the in situ formation of the mixed anhydride of 11
and diphenylphosphinic chloride,18 which was then
reflux, 4 h; (ii) LiOH, THF/H2O/MeOH, 4 h.
treatment oxidises them to the non-complexing cop-
per(II) species].15 Compound 8 was then reduced to the
dialdehyde 9 by brief treatment with DIBAL at À15 ꢀC.
Lower reaction temperatures could not be employed in
this case due to precipitation of the starting material.
treated with 3-dimethylaminopropylamine or 14 to give
3 and 4 in yields of 35 and 42%, respectively. Activation
of 11 as the bis-pentafluorophenyl ester followed by
treatment with the selectively protected spermidine
derivative 158 gave the tert-butoxycarbonyl-protected
bis-spermidine analogue 16 in 41% yield. Cleavage of
the tert-butoxycarbonyl protecting groups with 4 M
HCl in dioxane then gave 2 in essentially quantitative
yield as the hydrochloride salt.
To complete the preparation of diacid 11, dialdehyde 9
was first converted to the bis-a,b-unsaturated ester 10
by reaction with (ethoxycarbonylmethylene)-triphenyl-
phosphorane in 64% yield (Scheme 2). Saponification
of 10 by treatment with aqueous LiOH then gave the
di-carboxylate 11 in 88% isolated yield after ion
exchange chromatography. Although the dilithium salt
of 11 was readily soluble in water, the free acid was
only sparingly soluble in both water and most organic
solvents, and some product was therefore lost through
precipitation on the cation exchange column that was
used to isolate 11.
Inhibition assays
Compounds 2–4 and 10, 11 and 16 were initially
screened for time-dependent inhibition of TryR using a
microplate assay which we have developed for this pur-
pose.19 The inhibition assays were carried out using 100
mM inhibitor concentrations in the presence of 1 mM
trypanothione
disulfide
and
excess
NADPH.

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C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
Scheme 3. Reagents and conditions: (i) CH2¼CHCN, MeOH, 2.5 h; (ii) 5% Rh/C, H2 (2–3 atm), EtOH saturated with NH3, 20 h.
Scheme 4. Reagents and conditions: (i) Ph2P(O)Cl, Et3N, DMF, À10 ꢀC 1 h then add RNH2, 6 h; (ii) C6F5OH, EDCI, DMAP, DMF, 3 h then
RNH2, CH2Cl2, 2 h; (iii) 4 M HCl/dioxane, 1 h.
5,50-Dithiobis-(2-nitrobenzoic acid) (DTNB) was inclu-
ded in the assay mixture to mediate the rapid re-oxida-
tion of the product T[SH]2 back to T[S]2. In this
manner, the continuous recycling of T[SH]2 ensured
that the concentration of substrate remained constant
for the duration of these prolonged assays such that any
decrease in enzyme activity over time could be assigned
to time-dependent inhibition rather than substrate
depletion. In order to screen for time-dependent inacti-
vation of TryR, these continuous assays were monitored
for 60min following the addition of inhibitor.
analysis of these compounds showed them to be simple
competitive inhibitors of TryR, with respect to T[S]2,
with respective Ki values of 114 (ꢁ14) and 196 (ꢁ19)
mM (compared to a substrate Km value of 28 (ꢁ4) mM
(Fig. 4). The methylpiperazine functionalised bis-poly-
amine 4 displayed time-dependent activity against TryR
over a 60-min incubation period in the microplate assay.
A further detailed analysis of this compound was there-
fore carried out alongside lunarine 1 to deduce both the
kinetic parameters and likely mechanism of time-
dependent inhibition of TryR with 4 as outlined below.
With the absence of any polyamine side chains, neither
the diester 10 nor the diacid 11 showed any inhibitory
activity towards TryR at 100-mM concentrations. 11
was also inactive against human GR at 100-mM con-
centrations in the presence of 80 mM GSSG substrate
(Km=64 mM).
Time-dependent inhibition can arise from three different
mechanisms (Fig. 5). In classical inhibition mechanisms,
the steady state equilibrium between enzyme and inhi-
bitor is rapidly established (usually within milliseconds).
Simple slow binding (mechanism B) occurs when the
inhibitor binds to the enzyme like a classical inhibitor
but with a slower binding rate such that the steady state
is attained over a much longer timescale of several sec-
onds or even min. With an enzyme isomerisation
mechanism (C), the Michaelis–Menten complex (EI) is
rapidly formed followed by slower conformational
Inhibition of TryR by the tert-butoxycarbonyl pro-
tected bis-spermidine derivative 16 was also found to be
negligible, but in the initial microplate assay both 2 and
3 inhibited TryR in a linear manner. A more detailed

C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
3687
Figure 4. (a) Double reciprocal plots of initial rate against substrate concentration (T[S]2) in the presence of compound 2 at 0 mM (*); 100 mM (*);
and 200 mM (&) concentrations. (b) Double reciprocal plots of initial rate against substrate concentration (T[S]2) in the presence of compound 3 at 0
mM (*); 293 mM (*); and 596 mM (&) concentrations.
Figure 5. The basic mechanisms of time-dependent inhibition.
changes in either the enzyme or inhibitor resulting in a
more tightly bound complex (EI*). Irreversible inhibi-
tion proceeds by the same rapid binding step followed
by a rate-limiting covalent modification of the enzyme
(D).
respectively). The reaction progress curves for TryR in
the presence of a range of concentrations of 4 (Fig. 6a)
indicate a slow establishment of a final steady state
velocity as a result of either mechanism (B) or (C). The
concentration-dependent values for the initial rate (vi),
steady-state rate (vs), the apparent rate constant for
establishing the steady state equilibrium between EI and
EI* (k0) and the displacement of the curve from the
vertical ordinate (d) were determined by non-linear fit-
ting of these data to eq 1.20
The time-dependent inhibition mechanism for 4 and 1
was investigated using an extended continuous assay19
to monitor the time-dependent effect of different inhi-
bitor concentrations on enzyme activity (Figs 6 and 7,

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C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
Figure 6. Inhibition of TryR by 4. (a) Reduction in enzyme activity over time as a function of inhibitor concentration: no inhibitor (*); plus inhi-
bitor: 12.5 mM (*); 25 mM (&); 50 mM (&); 100 mM (~); 200 mM (~). (b) Re-plot of variation in vi (*) and vs (*) with respect to concentration of
4. These data were fitted to equations (2) and (3) to derive values for Ki and Ki*, respectively (Table 1). (c) Variation of the reciprocal of vi (*) and vs
(*) as a function of concentration of 4. (d) Double reciprocal plot of vi against substrate concentration in the presence of no inhibitor (*); plus
inhibitor: 100 mM (*); 200 mM (&) to demonstrate the competitive nature of inhibition by 4.
0
k5=k6 ¼ Ki=Kꢀi À 1
ð4Þ
P ¼ vst þ ðvi À vsÞð1 À eÀk tÞ=k0 þ d
ð1Þ
For each inhibitor concentration, the value for k6 was
determined from the relationship in eq 5.
The fact that both vi and vs vary as a function of inhi-
bitor concentration for 4 (Fig. 6a and b) clearly indi-
cates that a two-step isomerisation mechanism is
operating [i.e., mechanism (C)] where a Michaelis-type
inhibitor complex (EI) forms as an intermediate and
two inhibition constants must therefore be considered.
The first (Ki) is the dissociation constant for the initial
EI complex and the second (Ki*) is the dissociation
constant for the EI* complex that is subsequently
formed. Non-linear curve fitting of these data using eqs
2 and 3 (Fig. 6b) was used to derive values for Ki and
Ki*, respectively for 4.
k6 ¼ vsk0=vi
ð5Þ
Finally, the mean value for k6 was substituted into eq 4
to calculate k5.
An identical analysis was performed for 121 using the
above procedures (see Fig 7) in order to extract the
same kinetic parameters for time-dependent inhibition
of TryR. This data is summarised in Table 1.
vi ¼ vi¼0=ð1 þ I=Kið1 þ S=KmÞÞ
vs ¼ vi¼0=ð1 þ I=Kꢀi ð1 þ S=KmÞÞ
ð2Þ
ð3Þ
Discussion
Comparing 4 with 1, it is apparent that the quite sub-
stantial structural modifications made actually have
relatively little effect on the initial dissociation constant
for this inhibitor with TryR. On the other hand, a 3-fold
reduction in Ki* does result; a difference which is also
reflected in the isomerisation constants k5/k6 (see Table
1). It should be noted that the differences in Ki* are a
consequence of differences in the forward isomerisation
constants k5 as the reverse isomerisation constants for 4
and 1 are effectively the same. As the absolute k5 values
are relatively low it takes a long time (many min) to
establish the steady state equilibrium between EI and
where S is the substrate concentration and Km the
apparent Michaelis–Menten constant for TryR. The
variation in initial and steady state rates as a function of
inhibitor concentration is shown in Fig. 6c where the
slopes of the two lines illustrate the difference in the
magnitudes of Ki and Ki*. The ratio between the for-
ward and reverse isomerisation steps (k5/k6) for the
interconversion between EI and EI* was calculated
using eq 4.

C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
3689
Figure 7. Inhibition of TryR by 1. (a) Reduction in enzyme activity over time as a function of inhibitor concentration: no inhibitor (*); plus inhi-
bitor: 31.25 mM (*); 62.5 mM (&); 125 mM (&); 250 mM (~); 500 mM. (b) Re-plot of variation in vi (*) and vs (*) with respect to concentration of
1. These data were fitted to eqs 2 and 3 to derive values for Ki and Ki*, respectively (Table 1). (c) Variation of the reciprocal of vi (*) and vs (*) as a
function of concentration of 1. (d) Double reciprocal plot of vi against substrate concentration in the presence of no inhibitor (*); plus inhibitor: 100
mM (*); 200 mM (&) to demonstrate the competitive nature of inhibition by 1.
potency. Whilst the structural differences between the
polyamine units of 2–4 appear to have little effect on
their potency as competitive inhibitors of TryR, their
efficacy as time-dependent inhibitors is much more sen-
sitive to such alterations. Neither the bis-spermidine 2
nor the bis-dimethylamino-propylamine derivative 3
exhibit time-dependent inhibition kinetics with TryR
during the timescale of the continuous enzyme inhibi-
tion experiments (60min) in contrast to bis-methylpi-
perazinyl derivative 4.
Table 1. Kinetic parameters for time-dependent inhibition of TryR
by 1 and 4
Compd Ki (mM) Ki* (mM) k5/k6 k5 (minÀ1
)
k6 (minÀ1
)
4
1
213 (ꢁ9) 34 (ꢁ6) 5.2
304 (ꢁ2) 114 (ꢁ2) 1.7
0.0329
0.0128
0.0063 (ꢁ0.0006)
0.0077 (ꢁ0.0024)
EI*. This made it possible to determine the type of
slow binding inhibition (e.g., competitive, non-compe-
titive, etc.) by taking initial rate measurements (the first
60s) over a range of substrate and 4 concentrations.
The subsequent double reciprocal plot shows 4 to be a
competitive inhibitor of TryR with respect to T[S]2
(Fig. 6d).
One possible explanation for the observed time-depen-
dence of 4 is as follows: first an equilibrium is rapidly
established between 4 and TryR where a Michaelis-type
complex (EI) is formed. This may be followed by a
much slower conformational change in order to position
one of the a,b-unsaturated amide units in line for
nucleophilic attack by an active site thiol to give a
reversible covalent adduct (EI*). Relating this to
mechanism (C), k5 would reflect the conformational
reorientation of the inhibitor and if the conjugate addi-
tion step were effectively instantaneous, k6 would reflect
the reverse reaction. For 4, k5 is 3 times greater than
that determined for lunarine 1, and so this might be
attributed to the greater flexibility of the open chain
structure of 4 that enables it to undergo a more rapid
conformational change during the EI to EI* transition.
In contrast, the conformational flexibility of 1 is more
restricted by the spermidine bridge. The off rates (k6) for
4 and 1 are effectively the same which is to be expected
The initial dissociation constants (Ki) for 2 and 4 are
comparable to that of lunarine 1, despite the simulta-
neous replacement of both the fused tricyclic scaffold of
1 with a planar bicyclic benzofuranyl ring system and
also the bridging spermidine macrocycle with two linear
alkylamines. The bis-polyamine derivatives 2–4 are all
competitive inhibitors of TryR (with respect to T[S]2)
which may be explained by the affinity of their poly-
amine ‘side chains’ for the negatively charged spermi-
dine binding pocket in the enzyme active site. Neither
the neutral ethyl diester 10 nor the anionic diacid 11 are
inhibitors of TryR, while the extremely bulky side
chains on the tert-butoxycarbonyl protected bis-spermi-
dine derivative 16 are also detrimental to inhibitor

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C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
if k6 relates to a retro-Michael addition process since its
rate will be governed by the leaving group ability of the
b-carbon substituent (the same C53 residue in both
cases).
at the EPSRC National Mass Spectrometry Service
(University of Wales Swansea). Flash chromatography
was performed on columns of silica gel (Fluorochem,
Silica Gel 60; 40–63 m). Reverse phase chromatography
was performed using Supelco DiscoveryTM DSC-18
solid phase extraction tubes. Petroleum ether bp
40–60 ꢀC (referred to as petrol) was distilled through a
vigreux column prior to use. All other solvents were
dried using standard procedures.
Alternatively, if 4 or 1 were to undergo conjugate addi-
tion without any further change to the conformation of
the initial EI complex, then the rate of nucleophilic
attack by C53 alone should be what dictates the rate of
EI* formation (reflected by k5). That is, if the inhibitor
were to dock in the active site such that a Michael
acceptor unit and C53 are well aligned for conjugate
addition, then the rate of conversion from EI to EI* will
be faster than for the case where inhibitor binding leads
to a less favourable mutual alignment. The 3-fold
reduction in rate of time-dependent inactivation with 1
might therefore be simply because it is docked in the
active site in a comparatively less favourable orientation
for Michael addition relative to 4. In these terms, the
absence of time-dependent inhibition with 2 and 3 may
then again be ascribed to their particular polyamine
moieties that do not allow them to adopt an active-site-
bound orientation that is favourable for reaction with
C53.
Recombinant T. cruzi TryR was expressed in Escher-
ichia coli and purified, as previously described.22 The
enzyme was stored as a suspension in 70% ammonium
sulfate solution at 4 ꢀC and extensively dialysed against
the assay buffer before use. Glutathione reductase was
purified to homogeneity from human erythrocytes.23
Trypanothione disulfide was purchased from Bachem
and other biochemical reagents from Sigma. Spectro-
photometric assays of TryR and GR were carried out
on a temperature controlled Shimadzu UV–vis record-
ing spectrophotometer. Microtitre plate assays were
carried out in 96-well plates on a Molecular Devices
ThermoMax microplate reader.
Enzyme assays
For TryR, the standard assay mixture (1 mL) contained
TryR (1 mU), 40mM 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES) (pH 7.5), 1 mM
EDTA, 0.14 mM NADPH and varying concentrations
of inhibitor/substrate. Inhibitor stock solutions were
made up using co-solvent mixtures of assay buffer and
DMSO; final assay mixtures contained 2% DMSO.
Enzyme mixtures were pre-incubated with NADPH for
5 min at 27 ꢀC prior to initiating the reaction by the
addition of substrate. Enzyme activity was monitored
by the decrease in absorbance at 340nm due to
NADPH oxidation. For Ki determinations, inhibition
assays were carried out at three different inhibitor con-
centrations and five substrate concentrations (100, 60,
40, 30 and 20 mM). Initial rates (v) were measured from
the linear region of product formation and Ki values
were determined by weighted non-linear regression
analysis of the hyperbola plot of v against substrate
concentration (S) using the equation for linear compe-
titive inhibition in GraFit (Erithacus Software Ltd). GR
assays were carried out in a similar manner using a
buffer system composed of 100 mM KH2PO4 (pH 7.0),
200 mM KCl and 1 mM EDTA.24
Conclusion
The results presented herein demonstrate that a greatly
simplified achiral analogue of the natural product
lunarine 1 may exhibit comparable properties as a time-
dependent inhibitor of TryR. Such results are encoura-
ging as the synthetic complexity of 1 is an obstacle
towards its further development as a lead chemothera-
peutic against the range of orphan diseases associated
with trypanosomal parasites. Further detailed investi-
gations of the mechanism and minimal structural and
functional requirements of time dependent-inhibitors
for 1 and related compounds are ongoing and will be
reported in due course.
Experimental
General
Melting points were recorded on an Electrothermal
IA9000 series digital melting point apparatus, using
open capillaries and are quoted uncorrected. IR spectra
were recorded on a Nicolet 205 FT spectrophotometer.
UV spectra were recorded on a Perkin-Elmer Lambda
16 spectrophotometer.
For the microtitre plate assays, the final assay mixtures
(0.25 mL) contained TryR (1 mU) in the presence of 40
mM HEPES (pH 7.5), 1 mM EDTA, 0.15 mM
NADPH, 25 mM DTNB, 1 mM T[S]2 and inhibitor (100
mM). Enzyme mixtures were pre-incubated with
NADPH for 5 min at 27 ꢀC prior to initiating the
enzyme reaction by the addition of substrate followed
by inhibitor. Following inhibitor addition, enzyme
activity was followed by the increase in absorbance at
410nm, due to formation of the 5-thio-2-nitrobenzoic
acid di-anion. Substrate depletion did not contribute to
any deviation from linearity in these experiments as
T[S]2 concentrations were kept at a constant level by
1
NMR Spectra: H NMR spectra were recorded on a
Bruker Avance DPX 300 FT-spectrometer at 300 MHz,
13C NMR spectra were recorded on the same spectro-
meter at 75 MHz. Chemical shifts (d) are expressed in
ppm and coupling constants (J) are given in Hz. Low
resolution mass spectra were recorded on a VC 70-S
double focusing mass spectrometer in Electron Impact
(EI) mode, or on a VG Quattro triple quadrupole elec-
trospray mass spectrometer (ESI). Accurate mass mea-
surements were obtained on the VC 70-S instrument, or

C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
3691
the rapid DTNB-mediated re-oxidation of the T[SH]2
product.
THF (0.5 mL) was then added dropwise and a pre-
cipitate slowly began to form whilst stirring at room
temperature for 14 h. The reaction was filtered and the
solids washed with EtOAc. The combined filtrates were
washed with saturated aqueous Na2CO3 (10mL) and
the organics were dried (MgSO4). The solvent was
evaporated to give a residue that was purified by reverse
phase chromatography [MeOH/MeCN (9:1) as eluent]
to give 3 (6 mg, 25%).
N-[3-(4-Amino-butylamino)-propyl]-3-(3{2-[3-(4-amino-
butylamino)-propylcarbamoyl]-vinyl}-benzofuran-5-yl)-
acrylamide (tetra-hydrochloride salt) (2). A solution of
16 (26 mg, 0.028 mmol) dissolved in 4 M HCl in dioxane
(10mL) was stirred at room temperature for 1 h. The
solvent was then evaporated and the resulting residue
was coevaporated several times with ether to give 2 as a
white solid (18.5 mg, 100%). dH (300 MHz, D2O);
1.52–1.74 (8H, m, 4ꢂCH2), 1.77–1.93 (4H, m, 2ꢂCH2),
2.83–3.08 (12H, m, 6ꢂCH2), 3.23–3.36 (4H, m,
2ꢂCH2), 6.29 (1H, d, J=16.0) and 6.34 (1H, d, J=15.6,
H-9 and H-12), 7.22 (2H, d, J=15.8, H-8 and H-11),
7.30(1H, d, J=9.0, H-7), 7.35 (1H, d, J=9.3, H-6), 7.46
(1H, s, H-2), 7.82 (1H, s, H-4); dC (75 MHz, D2O);
24.09, 25.21, 27.07, 37.58, 40.07, 46.50, 48.25 (7ꢂCH2),
113.51 (C-7), 118.63 (C-3), 120.10, 121.04, 122.38 (C-4,
C-9, C-12), 126.17 (C-6 overlapping C-3a), 131.35
(C-11), 131.81 (C-5), 142.54 (C-8), 150.49 (C-2), 157.76
(C-7a), 170.11 (C¼O), 170.20 (C¼O); [Found: (ESI+)
513.3563 [M+H]+, C28H45N6O3 requires 513.3547];
m/z (ESI+) 513 (60%, [M+H]+), 484 (100).
N-[3-(4-Methyl-piperazine-1-yl)-propyl]-3-(3-{2-[3-(4-
methyl-piperazin-1-yl)-propylcarbamoyl]-vinyl}-benzo-
furan-5-yl)-acrylamide (4). A solution of the bis-penta-
fluorophenol ester of 11 (38 mg, 0.064 mmol) and
DIPEA (60mg, 0.464 mmol) in CH 2Cl2 (0.5 mL), was
treated with a solution of 14 (25 mg, 0.159 mmol) in
CH2Cl2 (0.5 mL) added dropwise and was left to stir at
room temperature for 3 h. The reaction mixture was
diluted with CH2Cl2 then washed with saturated aqu-
eous NaHCO3 (10mL), dried (MgSO ) and the solvent
4
was evaporated to give a solid. This was purified by
chromatography eluting with a 4:1 mixture of CHCl3
and EtOH (saturated with ammonia) to give the desired
product as a white solid (14 mg, 42%). dH (300 MHz,
CDCl3) 1.77–1.83 (4H, m, 2ꢂCH2CH2CH2) 2.28 (3H, s,
CH3), 2.29 (3H, s, CH3), 2.38–2.83 (20H, m, 10ꢂCH2),
3.48 (2H, q, J=6.2, NHCH2), 3.50(2H, q, J=6.4,
NHCH2), 6.45 (1H, d, J=15.6, ¼CH-9), 6.63 (1H, d,
J=15.8, ¼CH-12), 7.43 (1H, d, J=8.6) and 7.48 (1H, d,
J=8.6, H-6 and H-7), 7.67 (1H, d, J=15.8, ¼CH-11),
7.69, d, J=15.6, ¼CH-10), 7.62–7.69 (1H, br, NH over-
lapping H-10), 7.80 (1H, s, H-2), 7.87 (1H, s, H-4), 7.84–
7.92 (1H, br, NH overlapping H-4); dC (75 MHz, CDCl3)
25.70, 25.82 (NHCH2CH2), 39.81, 39.89 (NHCH2),
46.36, 46.45 (CH3), 53.38, 53.43 (NHCH2CH2CH2),
55.70(N CH2CH2NMe), 57.47, 57.53 (NCH2CH2NMe),
112.69 (C-7), 118.40(C-3), 121.27, 121.55 (C-9, C-4),
122.58 (C-12), 124.50(C-6), 126.17 (C-3a), 129.92 (C-11),
131.14 (C-5), 140.73 (C-8), 147.88 (C-2), 156.96 (C-7a),
166.33 (C¼O); [Found: (ESI+) 537.3547 [M+H]+,
C30H44N6O3 requires 537.3547]; m/z 537 (100%,
[M+H]+).
N-(3-Dimethylaminopropyl)-3-{3-[2-(3-dimethylamino-
propylcarbamoyl)-vinyl]-benzofuran-5-yl}-acrylamide (3).
Method A. A solution of the bis-pentafluorophenyl ester
of 11 (38 mg, 0.064 mmol) and DIPEA (60 mg, 0.464
mmol) in CH2Cl2 (0.5 mL), was treated with a solution
of dimethylaminopropylamine (19 mg, 0.186 mmol) in
CH2Cl2 (0.5 mL), added dropwise, and the mixture was
left to stir at room temperature for 3 h. The reaction
mixture was then diluted with CH2Cl2 (10mL) washed
with saturated NaHCO3 (10mL) and the organics were
dried (MgSO4). The solvent was evaporated to give a
solid. This was purified by chromatography eluting with
a 4:1 mixture of CHCl3 and EtOH (saturated with
ammonia) to give 3 as a white solid (4 mg, 15%). dH
(300 MHz, CDCl3) 1.72–1.83 (4H, m, 2ꢂCH2), 2.28
(12H, s, 4ꢂCH3), 2.45 (4H, t, J=6.4, 2ꢂCH2),
3.47–3.55 (4H, m, 2ꢂNHCH2), 6.45 (1H, d, J=15.6,
H-9), 6.60(1H, d, J=15.8, H-12), 7.42–7.58 (4H, m,
H-6, H-7, 2ꢂNH), 7.68 (1H, d, J=15.8, H-11), 7.71
(1H, d, J=15.6, H-8), 7.81 (1H, s, H-2), 7.89 (1H, br,
H-4); dC (75 MHz, CDCl3) 26.66, 26.96 (NHCH2CH2),
39.54, 39.82 (NHCH2), 45.84 (CH3), 58.60, 58.88
(CH2NMe2), 112.59 (C-7), 118.43 (C-3), 121.16, 121.29
(C-9, C-4), 122.51 (C-12), 124.88 (C-6), 126.23 (C-3a),
129.92 (C-11), 131.18 (C-5), 140.83 (C-8), 147.65 (C-2),
156.95 (C-7a), 166.37 (C¼O); [Found: (ESI+) 427.2707
[M+H]+, C24H35N4O3 requires 427.2703]; m/z 427
(100%, [M+H]+).
2,3,5-Tribromo-2, 3-dihydrobenzofuran (6). A solution of
bromine (0.87 g, 16.92 mmol) in CH2Cl2 (20mL) was
added, dropwise over 5 min, to a solution of 5-bromo-
benzofuran 511 (3.03 g, 15.38 mmol) in CH2Cl2 (20mL)
and the mixture was stirred at room temperature for 40
min. The reaction mixture was washed with 0.05 M
sodium thiosulfate (20mL) followed by water (20mL)
and the organics were then dried (MgSO4) and the sol-
vent was evaporated to give 6 as a white solid (5.95 g).
This was immediately carried through the next step
without further purification. dH (300 MHz, CDCl3) 5.69
(1H, s, 3-H), 6.89 (1H, s, 2-H), 6.96 (1H, d, J=8.6,
7-H), 7.49 (1H, dd, J=8.6, 2.1, 6-H), 7.64 (1H, d,
J=2.1, 4-H).
Method B. A solution of 11 (36 mg, 0.062 mmol) and
triethylamine (14 mg, 0.139 mmol) in a 1:1 mixture of
THF/pyridine (2 mL), cooled to À12 ꢀC, was treated
with a solution of diphenylphosphinic chloride (31 mg,
0.131 mmol) in THF (0.5 mL), added dropwise. This
was stirred at À12 ꢀC for 60min giving a cloudy mix-
ture. A solution of dimethylaminopropylamine (13 mg,
0.127 mmol), and triethylamine (14 mg, 0.139 mmol) in
3,5-Dibromobenzofuran (7). Potassium hydroxide pow-
der (1.75 g, 31.19 mmol) was added in one portion to a
suspension of 6 (5.95 g, 15.38 mmol) in absolute EtOH
(25 mL). The solids immediately dissolved, and a fine
white precipitate (KBr) began to form. The reaction

3692
C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
mixture was heated under reflux for 15 min then
allowed to cool before the solids were filtered off and
washed with EtOH. The combined filtrates were evapo-
rated and the residue obtained was then partitioned
between petrol/EtOAc (1:1, 60mL) and water (15 mL).
The organics were washed with water (2ꢂ15 mL) and
the aqueous layers back-extracted with petrol (3ꢂ10
mL). The combined organics were dried (MgSO4) and
the solvent was evaporated to give the crude product.
This was then purified by chromatography (petrol elu-
ent) to give 7 as a white solid (4.16 g, 98% over two
steps). dH (300 MHz, CDCl3) 7.38 (1H, d, J=8.8, 7-H),
7.46 (1H, dd, J=8.7, 1.9, 6-H), 7.66 (1H, s, 2-H), 7.70
(1H, d, J=1.9, 4-H); dC (75 MHz, CDCl3) 97.50(C-3),
113.69 (C-7), 117.05 (C-5), 123.05 (C-4), 128.90 (C-6),
129.44 (C-3a), 144.24 (C-2), 153.60(C-7a); [Found: (EI)
273.86516, C8H4Br2O requires 273.86289]; m/z 274, 276,
278 (100%, 1:2:1, M+), 197 (10, MÀBr).
(EI) 174.03058, C10H6O3 requires 174.03170]; m/z 174
(100%, M+), 173 (100, MÀH), 145 (37, MÀCHO).
3-[3-(2-Ethoxycarbonyl-vinyl)-benzofuran-5-yl]-acrylic
acid ethyl ester (10). A solution of 9 (107 mg, 0.614
mmol) and (carbethoxymethylene)triphenyl-phosphor-
ane (540mg, 1.55 mmol) in DMF (6 mL) was heated
under reflux for 4 h. The reaction mixture was then
diluted with EtOAc (20mL) and washed with water (10
mL). The organics were dried (MgSO4) and the solvent
was evaporated to give a brown solid. The crude pro-
duct was adsorbed onto silica and purified by chroma-
tography (CH2Cl2 eluent) to give 10 as a white solid
(124 mg, 64%). Mp (EtOAc) 98–99 ꢀC; lmax (CHCl3)/
nm 258 (e 37,722), 270(34,897), 294 (28,652); dH
(300 MHz, CDCl3) 1.37 (3H, t, J=7.1, CH3), 1.38 (3H,
t, J=7.1, CH3), 4.29 (2H, q, J=7.1, CH2), 4.31 (2H, q,
J=7.1, CH2), 6.48 (1H, d, J=16.0, H-9), 6.56 (1H, d,
J=16.1, H-12), 7.52 (1H, d, J=8.6, H-7), 7.57 (1H, dd,
J=8.7, 0.9, H-6), 7.77 (1H, d, J=16.1, H-11), 7.81 (1H,
d, J=16.0, H-8), 7.90 (1H, s, H-2), 7.97 (1H, s, H-4); dC
(75 MHz, CDCl3) 14.55, 14.75 (2ꢂCH3), 60.95, 61.07
(2ꢂCH2), 112.91 (C-7), 118.36 (C-3), 118.40(C-9),
119.44 (C-12), 121.58 (C-4), 125.64 (C-6), 125.92 (C-3a),
131.02 (C-5), 134.17 (C-11), 144.79 (C-8), 148.89 (C-2),
157.42 (C-7a), 167.26 (C¼O); [Found: (EI) 314.11726,
C18H18O5 requires 314.11543]; m/z 314 (100%, M+),
269 (42, MÀOEt).
Benzofuran-3,5-dicarbonitrile (8). A mixture of 7 (221
mg, 0.80 mmol) and copper(I) cyanide (184 mg, 2.05
mmol) in anhydrous DMF (2 mL) was heated under
reflux overnight (the mixture changed from a green
slurry to a deep red/brown solution during this time).
The reaction mixture was allowed to cool before pour-
ing into a solution of 1 M FeCl3 in 1.5 M aqueous HCl
(5 mL). After heating at 70 ꢀC for 30min, the slurry was
extracted with dichloromethane (4ꢂ8 mL) and the
combined organics were washed with water, 2 M HCl,
water and then 2 M NaOH (10mL each) The organics
were dried (MgSO4) and the solvent was evaporated to
give a brown solid. The crude product was adsorbed
onto silica and then purified by chromatography
(CH2Cl2/petrol eluent, 8:2) to give 8 as a white solid (67
mg, 50%) which was sufficiently pure for use in the next
step. nmax (CDCl3)/cmÀ1; 2238 (CꢃN); dH (300 MHz,
CDCl3) 7.73 (1H, d, J=8.8, 7-H), 7.77 (1H, dd, J=8.7,
1.5, 6-H), 8.12 (1H, s, 4-H), 8.30(1H, s, 2-H); dC
(75 MHz, CDCl3) 95.41 (C-3), 109.26, 110.73 (CN, C-5),
113.65 (C-7), 117.89 (CN), 125.28 (C-4), 125.42 (C-3a),
3-[3-(2-Carboxy-vinyl)-benzofuran-5-yl]-acrylic acid (11).
A solution of 10 (122 mg, 0.388 mmol), in THF (4 mL)
was treated with an aqueous solution of 3.5 M lithium
hydroxide (2 mL) to give a biphasic mixture. MeOH
was then added dropwise (approx. 2 mL) until a homo-
geneous mixture was obtained which was stirred at
room temperature for 4 h. The solvents were evaporated
to give a white solid, then lithium salts were removed by
passing the crude product through a column of Amber-
lite IR120(plus) cation exchange resin (50% aqueous
THF as eluent). Acidic fractions were collected and the
solvent was evaporated to give 11 as a white solid (88
mg, 88%). dH (300 MHz, DMSO-d6) 6.70(1H, d,
J=16.2, H-9), 6.76 (1H, d, J=16.5, H-12), 7.70–7.85
(4H, m, H-6, H-7, H-8, H-11), 8.42 (1H, s, H-2), 8.57
(1H, s, H-4), 12.25–12.50(2H, br, 2 ꢂCO2H); dC
(75 MHz, DMSO-d6) 112.40(C-7), 117.61 (C-3), 118.86
(C-9), 119.77 (C-12), 121.9 (C-4), 125.07 (C-3a), 125.50
(C-6), 130.70 (C-5), 133.45 (C-11), 144.19, 150.12 (C-2,
C-8), 156.32 (C-7a), 167.74 (C¼O); m/z (ESI+) 282.1
(100%, [M+Na]+).
13.020(C-6), 153.70(C-2), 155.69 (C-7a);
(100%, M+).
m/z 168
Benzofuran-3, 5-dicarbaldehyde (9). A solution of 8 (598
mg, 3.56 mmol) in anhydrous CH2Cl2 (40mL) at
À15 ꢀC was treated with a solution of 1.5 M DIBAL
(5.8 mL, 8.70mmol) in toluene. The reaction was com-
plete within 3 min (by TLC). The reaction mixture was
warmed to 0 ꢀC and 2 M HCl (20mL) was added drop-
wise. The reaction mixture was then allowed to warm to
room temperature, the organic layer was separated and
the aqueous layer was then extracted with CH2Cl2 (10
mL). The combined organics were washed with water,
dried (MgSO4) and the solvent was evaporated to give a
pale yellow solid. This was purified by chromatography
(CH2Cl2 eluent) to give 9 as a pale yellow solid (576 mg,
93%). nmax (CDCl3)/cmÀ1; 1698 (C¼O); dH (300 MHz,
CDCl3) 7.69 (1H, d, J=8.6, 7-H), 8.02 (1H, dd, J=8.6,
1.2, 6-H), 8.39 (1H, s, 4-H), 8.71 (1H, s, 2-H), 10.11
(1H, s, CHO), 10.22 (1H, s, CHO); dC (75 MHz,
DMSO-d6) 113.34 (C-7), 123.05, 123.66 (C-3, C-3a),
125.05 (C-4), 127.38 (C-6), 133.91 (C-5), 158.44 (C-7a),
159.81 (C-2), 186.75 (CHO), 192.94 (CHO); [Found:
Preparation of the bis-pentafluorophenyl ester of 11. A
solution of 11 (32 mg, 0.128 mmol) in DMF (4 mL) at
0 ꢀC was treated with EDC (58 mg, 0.302 mmol),
DMAP (1 mg, 0.008 mmol) and a solution of penta-
fluoro-phenol (58 mg, 0.318 mmol) in DMF (0.5 mL) to
give a yellow mixture that was stirred at room tempera-
ture for 3 h. The reaction mixture was diluted with
EtOAc (20mL), washed with saturated aqueous
NaHCO3 (10mL) and then brine (10mL). The organics
were dried (MgSO4) and the solvent evaporated to give
the bis-pentafluorophenyl ester as a pale brown solid.
This material was then used without further purification.

C. J. Hamilton et al. / Bioorg. Med. Chem. 11 (2003) 3683–3693
3693
3-(4-Methyl-piperazin-1-yl)-propionitrile (13).16 Acrylo-
Acknowledgements
nitrile (0.67 mL, 10.18 mmol) was added dropwise to a
solution of N-methyl-piperazine 12 (1.02 g, 10.18 mmol)
in MeOH (6 mL) and was left to stir at room temperature
for 2.5 h. The solvent was evaporated to give an oily
residue that was purified by elution through a thin bed of
silica (EtOAc/MeOH, 9:1 as eluent) to give 13 as a pale
yellow oil (1.26 g, 81%). dH (300 MHz, CDCl3); 2.26
(3H, s, CH3), 2.34–2.60(8H, m, 4 ꢂNCH2), 2.48 (2H, t,
J=6.8, NCH2CH2), 2.67 (2H, t, J=6.8, CH2CN).
We thank the UNDP/WORLD BANK/WHO (Special
Programme for Research and Training in Tropical Dis-
eases) for financial support (CJH) and the Wellcome Trust
(AHF). We are also grateful to Dr. C. Poupat (Institute de
Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette,
France) for the generous gift of an authentic sample of
(+)-lunarine, and the EPSRC National Mass Spectro-
metry Service, University of Wales Swansea, for accurate
mass measurements on compounds 2–4.
3-(4-Methyl-piperazin-1-yl)-propylamine (14).17 A mix-
ture of 3-(4-methyl-piperazin-1-yl)-propionitrile 13 (583
mg, 3.80mmol) and 5% Rh/C (98 mg, 0.19 mmol) in
2.77 M ethanolic ammonia (8 mL) was stirred under a
hydrogen atmosphere (5 atm pressure) for 20h. The
reaction mixture was then filtered through Celite and the
solvent was evaporated to give an oily residue which was
purified by short-path distillation to give 14 as a colour-
less oil (110mg, 19%). bp 34 ꢀC (0.05 mmHg) (lit.,16
52 ꢀC, 0.3 mmHg); dH (300 MHz, CDCl3); 1.63–1.73 (2H,
References and Notes
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{4-[tert-Butoxycarbonyl-(3-{3-[3-(2-{3-[tert-butoxycarbo-
nyl-(4-tert-butoxycarbonylamino-butyl)-amino]-propyl-
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A solution of 11 (21 mg, 0.081 mmol) and triethylamine
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treated with a solution of diphenylphosphinic chloride
(31 mL, 0.163 mmol) in DMF (0.5 mL) added dropwise.
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cloudy green mixture. A solution of 15 (62 mg, 0.179
mmol), and triethylamine (25 mL, 0.180 mmol) in DMF
(0.5 mL) was then added dropwise and the reaction was
stirred at room temperature for 6 h. The solvent was
evaporated and the residue was then partitioned
between water (5 mL) and EtOAc (5 mL). The aqueous
layer was extracted with EtOAc (3ꢂ5 mL), and the
combined organics were then dried (MgSO4) and the
solvent evaporated to give an oily residue that was
purified by chromatography (EtOAc eluent) to give 16
as a colourless oil (30mg, 41%).
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dH (300 MHz, 323 K, CDCl3); 1.43 (9H, s, 3ꢂCH3), 1.44
(9H, s, 3ꢂCH3), 1.47 (18H, s, 6ꢂCH3), 1.46–2.05 (12H,
m, 6ꢂCH2), 3.08–3.51 (16H, m, 8ꢂCH2), 4.60–4.90
(2H, br, 2ꢂNH), 6.49 (1H, d, J=15.6, H-9), 6.68 (1H,
d, J=15.8, H-12), 6.80–7.22 (2H, br, 2ꢂNH), 7.44 (1H,
d, J=8.5, H-7), 7.49 (1H, d, J=8.3, H-6), 7.69 (1H, d,
J=15.8, H-11), 7.72 (1H, d, J=15.6, H-8), 7.81 (1H, s,
H-2), 7.92 (1H, s, H-4); dC (75 MHz, CDCl3); 26.12
(CH2), 27.76 (CH2), 27.85 (CH2), 28.11 (CH2), 28.84
(CH3), 36.31 (CH2), 36.71 (CH2), 40.47 (CH2), 43.86
(CH2), 44.32 (CH2), 45.36 (CH2), 47.06 (CH2), 79.62,
79.66, 80.08, 80.27 (4ꢂC), 112.55 (C-7), 118.54 (C-3),
121.04 (C-9, C-4, overlapping [as determined by HET-
COR)], 122.46 (C-12), 125.44 (C-6), 126.13 (C-3a),
129.93 (C-11), 131.23 (C-5), 141.05 (C-8), 147.93 (C-2),
156.49 (C-7a), 157.01 (carbamate–C¼O), 166.61
(amide–C¼O); m/z (ESI+) 913 (100%, [M+H]+).
19. Hamilton, C. J.; Sarvanamuthu, A.; Eggleston, I. M.;
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