New prodrugs derived from 6-aminodopamine and 4-aminophenol as candidates for melanocyte-directed enzyme prodrug therapy (MDEPT)

Article abstract of DOI:10.1039/b506404j

Two novel tyrosinase mediated drug delivery pathways have been investigated for the selective delivery of cytotoxic units to melanocytes from urea and thiourea prodrugs. The synthesis of these prodrugs is reported, as well as oximetry data that illustrate that the targets are substrates for tyrosinase. The stability of each of the prodrugs in (i) phosphate buffer and (ii) bovine serum is discussed, and the urea prodrugs are identified as lead candidates for further studies. Finally, HPLC studies and preliminary cytotoxicity studies in a melanotic and an amelanotic cell line, that illustrate the feasibility of the approach, are presented. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b506404j

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A R T I C L E
New prodrugs derived from 6-aminodopamine and 4-aminophenol
as candidates for melanocyte-directed enzyme prodrug therapy
(MDEPT)†
Sarah Knaggs,a Hugh Malkin,a Helen M. I. Osborn,*b Nana Aba O. Williamsa and
Parveen Yaqoobc
a School of Chemistry, University of Reading, Whiteknights, Reading, UK RG6 6AD
b School of Pharmacy, University of Reading, Whiteknights, Reading, UK RG6 6AD.
E-mail: h.m.i.Osborn@rdg.ac.uk; Fax: +44 (0) 118 378 6331; Tel: +44 (0) 118 378 7338
c School of Food Biosciences, University of Reading, Whiteknights, Reading, UK RG6 6AP
Received 9th May 2005, Accepted 8th September 2005
First published as an Advance Article on the web 5th October 2005
Two novel tyrosinase mediated drug delivery pathways have been investigated for the selective delivery of
cytotoxic units to melanocytes from urea and thiourea prodrugs. The synthesis of these prodrugs is reported, as well
as oximetry data that illustrate that the targets are substrates for tyrosinase. The stability of each of the prodrugs
in (i) phosphate buffer and (ii) bovine serum is discussed, and the urea prodrugs are identified as lead candidates for
further studies. Finally, HPLC studies and preliminary cytotoxicity studies in a melanotic and an amelanotic cell line,
that illustrate the feasibility of the approach, are presented.
In this paper we wish to describe our recent work in this
Introduction
area that has involved the synthesis of further classes of urea
Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT) is
and thiourea prodrugs that rely on complementary tyrosinase
an acronym coined for a prodrug strategy directed towards
mediated pathways for drug release. An analysis of the ability
the treatment of melanoma.1 MDEPT differs from some other
of the prodrugs to act as tyrosinase substrates and release cyto-
toxic units upon exposure to tyrosinase, in vitro, is also presented.
A nitrogen mustard (aniline N-mustard) has been selected
enzyme prodrug therapies such as Antibody-Directed Enzyme
Prodrug Therapy (ADEPT)2 in that the activating enzyme,
tyrosinase, necessary for drug release is naturally expressed
within the host and does not need to be artificially introduced.
as the cytotoxic agent for selective delivery since N-mustards
have been approved for the clinical treatment of melanoma,5
This therefore overcomes a major drawback of some current
and have also been incorporated within ADEPT strategies.6
prodrug delivery systems that require the delivery of sufficient
Moreover, it has been demonstrated that alkylating agents can
non-mammalian enzyme to the tumour site, as well as prodrug
be administered repeatedly with less induced resistance than
activation in a selective manner, for effective therapy. It has
other classes of anticancer drugs.7 The tyrosinase substrates and
been reported that when melanocytes become malignant, the
genes expressing tyrosinase become up-regulated, resulting in
a marked increase in the tyrosinase levels within the cancer-
the cytotoxic unit are connected within the prodrugs via urea or
thiourea linkages, to probe the stabilities of the different linkages
in vitro.
ous cells.3 Thus, since tyrosinase is naturally present in the
The general structure of the first class of prodrugs is illustrated
tumour and virtually absent from other cells it provides an
in Fig. 1.
in-built drug delivery mechanism that will be selective for
The trigger units, 4-aminophenol or 4-amino-2-hydroxy-
melanoma tumours over both healthy cells and normal, healthy
phenol, were found to be substrates for tyrosinase by oximetry,
melanocytes. A number of tyrosinase dependent prodrug strate-
oxidising at 70% of the rate of the oxidation of L-tyrosine, the
gies have been investigated for the treatment of melanoma.3,4
For example, non-toxic phenol and catechol prodrugs have
natural substrate for tyrosinase. It was hypothesised that the
prodrugs could be activated via the mechanism described below
in Scheme 1. Since the onset of drug release will be dependent
been oxidised by tyrosinase to afford toxic quinones within
the vicinity of melanoma tumours.4 Initial studies within the
upon tyrosinase activation, this approach should allow drug
MDEPT strategy have also illustrated that tyrosinase can
release solely within the melanoma tumour.
be utilised to mediate the release of a cytotoxic agent from
Preliminary results suggesting the feasibility of this approach
carbamate and urea prodrugs via a cyclisation–drug release
have recently been reported from our laboratory where we have
illustrated that amines can be liberated from ureas derived
mechanism.1
from 4-aminophenol, upon exposure to tyrosinase—this has
culminated in the development of novel enzyme labile protecting
† This paper is dedicated to Professor S. V. Ley on the occasion of his
60th birthday.
group methodology.8
Fig. 1 Structures of novel prodrugs for use within MDEPT: Series 1.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0 T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
4 0 0 2
©

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Scheme 1 Proposed drug release pathway.
The general structure of the second group of prodrugs is
displayed in Fig. 2.
Results and discussion
Synthesis of the prodrugs
It was envisaged that syntheses of the urea and thiourea prodrugs
from both Series 1 and Series 2 could be achieved via reaction of
aniline N-mustard with isocyanates and isothiocyanates derived
from the trigger units (Scheme 3). The N-mustard (7) was
prepared according to multi-step literature procedures.10 Entry
to the urea prodrug (1) was achieved from isocyanate (9), which
was itself prepared in 96% yield by reaction of benzyl protected
phenol amine (8) with four equivalents of triphosgene for 2 h.
A sharp peak at 2267 cm−1 in the IR spectrum, characteristic
of the isocyanate stretch was indicative of the formation of (9).
With isocyanate (9) available, coupling to the N-mustard (7)
Fig. 2 Structures of novel prodrugs for use within MDEPT: Series 2.
The release mechanism for this series of prodrugs elaborates
a report that 6-aminodopamine is a good substrate for ty-
rosinase, with oxidation occurring to afford the corresponding
orthoquinone.9 This orthoquinone then undergoes a rapid in-
tramolecular cyclization to initiate excision of the 6-substituent.
This study therefore sought to investigate whether the tyrosinase
mediated manipulation could be expanded to allow delivery of
a cytotoxic moiety to malignant melanocytes as illustrated in
Scheme 2.
was achieved by reaction in DCM under N2
to yield (10),
(g)
Scheme 3, in 50% yield. This was converted to prodrug (1)
in 81% yield by deprotection of the benzyl group using 10%
Pd/C and H2 (g). In a similar fashion, amine (8) was converted
to the isothiocyanate derivative (11) using thiophosgene in
an excellent yield of 90%. Subsequent coupling to the N-
mustard (7) afforded thiourea (12) in 48% yield. However,
removal of the benzyl ether protecting group from (12) using
Scheme 2 Proposed drug release pathway.
Scheme 3 Synthesis of prodrug (1) and attempted synthesis of prodrug (2) via benzyl protected 4-aminophenol. (i) X = O, triphosgene, EtOAc,
reflux, 2 h, 96%; X = S, thiophosgene, EtOAc, rt, 2 h, 90%; (ii) X = O, N-mustard (7), DCM, rt, 12 h, 50%; X = S, N-mustard (7), DCM, rt, 24 h,
48%; (iii) Pd/C, H2, EtOAc, rt, 2 h, 81%.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0
4 0 0 3

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Scheme 4 Synthesis of prodrug (2) via silyl protected 4-aminophenol. (i) TBDMSCl, imidazole, THF, rt, 90%; (ii) thiophosgene, DCM, reflux, 87%;
(iii) N-mustard (7), DCM, rt, 55%; (iv) TBAF, rt, 62%.
DDQ11 was unsuccessful and the starting material was instead
recovered.
thus affording the partially deprotected target. Removal of the
methyl ether protecting group to afford the target prodrug (3)
was accomplished in 91% yield using BBr3 at low temperature,
Scheme 5.
Synthesis of prodrug (4) required reaction of the commercially
available isothiocyanate (20) with N-mustard (7) to afford
thiourea (21) in 93% yield. Subsequent deprotection of the
methyl ether protecting groups, again with BBr3, afforded
prodrug (4) in 92% yield, Scheme 6.
Preparation of prodrugs (5) and (6) from Series 2 again
involved the coupling of protected isocyanate and isothiocyanate
derivatives (27) and (28) with aniline N-mustard (7) as illustrated
in Scheme 7. The isocyanate (27) and isothiocyanate (28) were
prepared by reaction of protected amine (26) with phosgene and
thiophosgene respectively.
Synthesis of amine (26) commenced with nitration of
dopamine (22) using sodium nitrite and a 20% solution of
sulfuric acid in water at 0 ◦C, according to the procedure of
Napolitano et al.9 Precipitation of the hydrogen sulfate salt
(23) occurred in the reaction vessel, and after filtration, washing
and drying, it could be isolated as a mustard coloured solid in
93% yield. Introduction of a Boc protecting group to amine
(23) using Boc2O and NaOH in DMF proved low yielding
(typically 22%). However, use of Boc–ON12 and NEt3 in a
THF–H2O mixture allowed access to the desired Boc-protected
amine (24) in 59%. Subsequent acetyl protection of the catechol
moiety using Ac2O and pyridine proceeded well to afford nitro
derivative (25) in quantitative yield. Nitro reduction, to afford
An alternative protecting group for 4-aminophenol was
therefore employed—thus, 4-aminophenol was converted to
amine (13) by treatment with tert-butyldimethylsilylchloride in
the presence of a weak base, imidazole, in 90% yield, Scheme 4.
Amine (13) was next converted to the thioisocyanate (14) by
reaction with thiophosgene, in 87% yield. Subsequent reaction
of (14) with the N-mustard (7) then afforded the protected
prodrug (15), in 55% yield. The deprotection of the silyl group
was achieved by using TBAF as a source of fluoride ions and
in this way the target prodrug (2) was obtained in 62% yield,
Scheme 4.
For comparative purposes, prodrug (1) was also synthesised
using the silyl protected 4-aminophenol starting material (13),
in an overall yield of 49%. This compared favourably with
the overall yield of 34% obtained when benzyl protected 4-
aminophenol (8) was utilised as the starting material.
Synthesis of the catechol analogue of (1), namely prodrug
(3), was achieved from commercially available amine (16). Thus
alcohol (16) was converted to silyl ether (17) in 65% yield using
TBDMSCl and a catalytic amount of DMAP. Silyl ether (17)
was then converted to the isocyanate (18), by treatment with
triphosgene, in 93% yield, and this was then coupled to the
N-mustard (7) to afford urea (19) in 71% yield. It was hoped
that (19) could then be deprotected using conc. HCl to afford
prodrug (3). These conditions, however, failed to deprotect
the methyl ether group even after prolonged reaction times,
Scheme 5 Synthesis of prodrug (3). (i) TBDMSCl, DMAP, NEt3, THF, rt, 65%; (ii) triphosgene, EtOAc, reflux, 93%; (iii) N-mustard (7), DCM, rt,
71%; (iv) conc. HCl, reflux, 77% then BBr3, DCM, −78 ◦C, 91%.
Scheme 6 Synthesis of prodrug (4). (i) N-Mustard (7), DCM, rt, 93%; (ii) BBr3, DCM, 92%.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0
4 0 0 4

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Scheme 7 Synthesis of prodrugs (5) and (6). (i) NaNO2, H2SO4, H2O, 93%; (ii) Boc–ON, NEt3, THF, H2O, 59%; (iii) Ac2O, pyridine, 100%; (iv) H2,
Pd/C, 70%; (v) thiophosgene or triphosgene (Y = S and O respectively); (vi) pyridine, rt, 16 h, Y = O, 63%, Y = S, 71% (all yields quoted over 2
steps); (vii) 2 M HCl, acetone, room temperature, 2–4 h, Y = O, 100%, Y = S, 67%.
amine (26) was first attempted using Raney nickel and hydrazine
but this resulted in a poor yield of formation of amine (26)
(25%) with some O-acetyl deprotection also occurring under
these reaction conditions. Hydrogenation of (25) over Pd/C
proved more effective, affording amine (26) in 70% yield after
purification by column chromatography. Amine (26) was next
converted to either the isocyanate (27) or isothiocyanate (28)
using triphosgene or thiophosgene respectively. The isocyanate
(27) and isothiocyanate (28) were then reacted with the N-
mustard (7) in pyridine at room temperature for 16 h to allow
entry to the protected urea (29) and thiourea prodrugs (30)
in synthetically useful yields. Removal of the acetate and Boc
protecting groups from (29) and (30) was easily achieved in good
to excellent yields for both targets upon treatment with 2 M HCl,
in acetone, without any decomposition of the resulting urea (5)
and thiourea (6) prodrugs.
(6) contrasted with that obtained for 6-hydroxydopamine which
proved to be a better substrate for tyrosinase than L-tyrosine
(Rmax = 30 nanomole/min).
In addition to oximetry studies, HPLC studies were also
performed to assess the viability of the tyrosinase mediated
drug release protocol. Each prodrug was therefore solubilised
in phosphate buffer, treated with tyrosinase and the solution
analysed by HPLC for evidence of drug release. Table 1 displays
the results obtained and illustrates that whilst urea linked
prodrugs showed successful drug release, the thiourea linked
prodrugs proved less effective. This is in contrast with the
oximetry results that had suggested that both urea and thiourea
prodrugs (1)–(4) were good substrates for tyrosinase. It is
possible that drug release from prodrugs (2) and (4) was less
effective than expected due to the generation of an inhibitor
of tyrosinase—this hypothesis is supported by the observation
that phenylthiourea is an inhibitor of tyrosinase.14 It should be
noted that for the urea linked prodrugs, the efficiency of drug
release, as evidenced by the quantitative HPLC assays, reflected
the ability of the prodrugs to act as substrates for tyrosinase.
Thus drug release from the urea linked prodrugs (1) and (3) was
more effective than from prodrug (5).
Tyrosinase mediated processing of the prodrugs
Attention next focused on probing the ability of prodrugs (1)–(6)
to be processed by tyrosinase, to effect drug release. Although
human tyrosinase is not commercially available, mushroom-
derived tyrosinase (E.C. 1.14.18.1) can be readily obtained, and
so was utilised in this programme. Studies have shown that
mushroom tyrosinase is a good model for human tyrosinase
with the active sites of both mushroom and human tyrosinase
containing a common binuclear copper centre.13
When molecules act as substrates for tyrosinase, oxygen
is removed from the surrounding solution, and this can be
measured via oximetry. The rate of oxygen depletion corre-
lates with the tyrosinase-mediated oxidation of the substrates.
Oxidation of each prodrug (1)–(6) was therefore monitored
via oximetry and the rates of oxidation were compared with
that for a natural substrate of tyrosinase, namely L-tyrosine
(Rmax = 17 nanomoles/min). Prodrugs (1)–(4) showed very
similar oxidation profiles, and were oxidised at 70–78% the rate
of oxidation of L-tyrosine. In contrast, prodrugs (5) and (6)
demonstrated slower oxidation profiles, and were oxidised at
50% and 25% of the rate of oxidation of L-tyrosine, respectively.
The relatively slow oxidation rates afforded by prodrugs (5) and
Table 1 Tyrosinase mediated drug release studies
Prodrug
(1)
t1/2
Comment
85 min
—
Complete consumption of prodrug, and
generation of drug, was observed
(2)
No evidence of drug release, even after 4 h.
Formation of a metabolite was observed, RT =
2.0 min. t1/2 = 110 min
(3)
(4)
58 min
—
Complete consumption of prodrug, and
generation of drug, was observed
Some drug release observed, but the major
components were two metabolites, RT = 1.8,
RT = 4.0 min
(5)
(6)
100 min
—
Slow release of drug is evident, with complete
drug release eventually being observed
No evidence for drug release, but instead
formation of a single metabolite is evident
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0
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Table 2 Stability profiles for prodrugs (1)–(6)
C18 reverse phase column (250 mm × 4.5 mm). Compounds
were detected by UV at 254 nm. Method 1: 0 min H2O (100%),
5 min H2O (100%), 25 min MeCN (100%), 30 min MeCN
(100%), 35 min H2O (100%), flow rate 1 mL min−1. Method
2: 0 min H2O (100%), 5 min H2O (100%), 25 min 1,4-dioxane
(100%), 30 min 1,4-dioxane (100%), 35 min H2O (100%), flow
rate 1 mL min−1. Method 3: H2O–MeCN (3 : 7, v/v), flow
rate 1 mL min−1. Oximetry was performed using a YSI 5300
biological oxygen monitor, utilising a KCl electrolyte on a
membrane bound oxygen probe. Readings were recorded on an
ABB SE120 chart recorder operating at 1 cm min−1 chart speed
and 10 mV sensitivity. Unless stated otherwise, all chemicals
and materials were obtained from the Sigma-Aldrich Chemical
Company, the B. D. H. Merck Chemical Company or Lancaster
Chemicals and were used as received. Silica gel for column
chromatography was obtained from Merck, with a pore diameter
of 6 nm.
Prodrug
Phosphate buffera
Bovine seruma
(1)
(2)
(3)
(4)
(5)
(6)
100
98
99
65
45
98
0, t1/2 = 20 min
17, t1/2 = 110 min
98
96
80
0, t1/2 = 10 min
a % of the prodrug remaining after 5 h.
In order to ensure that drug release was truly dependent on
tyrosinase, the stability of each prodrug in phosphate buffer, and
in bovine serum, was examined. Thus the prodrugs were exposed
to phosphate buffer or bovine serum, and the rate of undesirable
drug release was determined by HPLC analysis. The results of
these studies are illustrated in Table 2.
In all cases it was evident that the urea linked prodrugs (1), (3)
and (5) were of greater stability in aqueous and biological media
than the thiourea linked prodrugs (2), (4) and (6). This, together
with the tyrosinase mediated drug release data, suggested that
the urea prodrugs were better candidates for MDEPT than their
thiourea counterparts
4-Benzyloxyphenyl isocyanate (9)
Et3N (0.3 mL, 2.12 mmol) was added to a solution of 4-
benzyloxyaniline hydrochloride salt (0.5 g, 2.12 mmol) in EtOAc
(40 mL). The mixture was kept at 0–5 ◦C and triphosgene (2.5 g,
8.48 mmol, 4 eq.) was added. The reaction mixture was gradually
brought to reflux at 77 ◦C for 2 h. The progress of the reaction
was monitored by IR spectroscopy and TLC analysis. Excess
solvent was removed in vacuo and the crude product was flashed
through a short pad of SiO2 to yield the pure produ◦ct (9) as
a pale pi◦nk crystalline solid (0.48 g, 96%). Mp 59–60 C (lit.17
The final stage of the study involved an analysis of the
cytotoxicity of the lead urea prodrugs (1), (3) and (5) in a
tyrosinase rich (FF1, 0.55 nM/min/mg protein) and tyrosinase
absent (A375) cell line.15,16 Pleasingly greater cytotoxicity was
evident in the tyrosinase rich cell line (IC50 = 10.2, 15.2 and
9.7 lM for (1), (3) and (5) respectively) than in the tyrosinase
absent cell line (IC50 = 20.2, 25.8 and 30.2 lM for (1), (3)
and (5) respectively). The N-mustard (7) exhibited an IC50
of 1 lM in each cell line. These results therefore support
the hypothesis that tyrosinase can be utilised for the selective
release of a cytotoxic drug from the two new series of prodrugs
described herein. Whilst it is hypothesised that orthoquinones
are generated as intermediates in the proposed drug release
pathways, and orthoquinones are known to be cytotoxic, the
biological effect for the protocol described herein is likely to
be dominated by the DNA cross-linking properties of the N-
mustard thus released. This is due to the rapid reaction of
orthoquinones with cellular glutathione which, in the absence
of ancillary treatment to depress glutathione synthesis, makes it
difficult for the requisite quinone levels to be attained.3c
1
59–61.5 C); Rf 0.54 (hexane–EtOAc, 7 : 3, v/v); H NMR: d
7.27–7.35 (5H, m, ArH), 7.03 (2H, d, J 9.0 ArH ‘o’ to NCO),
6.92 (2H, d, J 9.0 ArH ‘m’ to NCO), 5.07 (2H, s, OCH2); 13C
NMR: d 155.6 (ArCOBn), 137.4 (ArCCH2), 127.8–129.0 (8 ×
ArCH), 121.1 (NCO), 115.7 (2 × ArCH), 70.7 (CH2); IR (thin
film) m cm−1 2267 s (NCO), 1243 s; m/z (CI) 91 (100%), 225 (M +
H, 30%); Found 225.0785. [C14H11NO2 + H]+ requires 225.0790.
1(4-Benzyloxy-3-{4-[bis-(2-chloroethyl)-amino]-phenyl} urea (10)
Benzylisocyanate (9) (0.4 g, 1.91 mmol) was dissolved in DCM
(40 mL) and freshly prepared N-mustard (7) (0.4 g, 1.91 mmol)
was added. The reaction mixture was left to stir for 12 h,
concentrated in vacuo and purified by column chromatography
(DCM–MeOH, 95 : 5, v/v) to yield the desired product (10) as
a white solid (0.43 g, 50%). Mp 114–116 ◦C; Rf 0.38 (DCM–
1
MeOH, 95 : 5, v/v); H NMR: d 7.36–7.39 (5H, m, ArHBn),
7.19–7.26 (4H, m, ArH, ‘o’ to urea), 6.95 (2H, d, J 9.0 ArH ‘o’
to OBn), 6.67 (2H, d, J 9.0 ArH, ‘o’ to NCH2CH2Cl), 6.39 (1H,
br, s, NH), 6.31 (1H, br, s, NH), 5.02 (2H, s, CH2), 3.73–3.78
Conclusions
In conclusion, the synthesis and analysis of a range of pro-
drugs derived from 6-aminodopamine and 4-aminophenol, of
potential use within MDEPT, has been described. Promising
results have been obtained that indicate that the urea prodrugs
(1), (3) and (5) are good leads for further studies. Future work
will seek to further increase the differential cytotoxicity of the
prodrugs within tyrosinase rich and tyrosinase absent cell lines
to maximise the impact of this targeted approach.
(4H, m, 2 × CH2CH2Cl), 3.59–3.66 (4H, m, 2 × CH2CH2Cl);
13
=
C NMR: d 155.5 (ArCOBn), 152.4 (C O), 137.9 (2 × ArCN
and ArCOBn), 128.1–129.3 (5 × ArCH), 126.0 (2 × ArCNH),
124.5 (4 × ArCH ‘o’ to urea), 116.2 (2 × ArCH ‘o’ to OBn),
113.5 (2 × ArCH ‘o’ to NCH2CH2Cl), 71.0 (CH2), 54.3 (2 ×
CH2CH2Cl), 41.1 (2 × CH2Cl); IR (thin film) m cm−1 3321 s
NH, 3035, 2686 s, 2531 s, 1707 s, 1503 s, 1240 s and 908; m/z
(CI) 91 (100%), 200 (45%), 458 (M + H, 35Cl, 35Cl, 20%), 460
(M + H, 35Cl, 37Cl, 13%), 462 (M + H 37Cl, 37Cl, 2%); Found
458.1412. C24H2535Cl2N3O2 + H+ requires 458.1403; Found C,
62.39; H, 5.52; N, 9.61. C24H25Cl2N3O2 requires C, 62.89; H,
5.50; N, 9.16%.
Experimental
All NMR spectra were recorded on a Bruker WM250, Bruker
AC250, Bruker Avance DPX 250, Bruker AMX400 or Jeol
AX400 spectrometer, using CHCl3 as an internal standard
unless otherwise stated (7.26 ppm for 1H NMR, 77.0 ppm
for 13C NMR). 13C spectra were recorded using Distortionless
Enhancement by Polarisation Transfer. Mass spectra were
recorded on a Fisons VG Autospec. Infra red spectra were
recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer.
Melting points were determined using an Electrothermal digital
melting point apparatus, and are uncorrected.
1-{4-[Bis-(2-chloroethyl)-amino]-phenyl}-3-(4-hydroxyphenyl)
urea (1)
Benzyl ether (10) (0.34 g, 0.742 mmol) was dissolved in EtOAc
(40 mL). Pd/C (10%) (0.15 g) was added and the suspension
degassed using a water aspirator. H2
was then added via
(g)
balloons and the reaction was left to stir for 2 h. The reaction
R
HPLC was performed on a Perkin-Elmer 410 series LC pump
fitted with a Gilson 231 auto-sampler injector, using a LUNA
mixture was filtered through a pad of Celiteꢀ and the filtrate
was concentrated in vacuo to dryness. The residue was purified
4 0 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0

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by column chromatography (DCM–MeOH, 96 : 4, v/v) to give
(1) as a colourless powder (0.22 g, 81%). Mp 160–162 ◦C; Rf
0.38, (DCM–MeOH, 95 : 5, v/v); 1H NMR (MeOH-d4): d 7.16–
7.09 (4H, m, ArH ‘o’ to urea), 6.71 (2H, d, J 9.0, ArH ‘o’ to
OH), 6.59 (2H, d, J 9.0, ArH ‘o’ to NCH2CH2Cl), 6.20 (1H, s, br,
NH), 6.13 (1H, br s, NH), 5.15 (1H, br s, OH), 3.60–365 (4H, m,
2 × CH2CH2Cl), 3.53–3.57 (4H, m, 2 × CH2CH2Cl); 13C NMR
and extracted with Et2O (2 × 50 mL). The organic extracts were
combined, dried over MgSO4, and concentrated in vacuo to give
a dark yellow oil. This was purified by column chromatography
(EtOAc–hexane, 1 : 1, v/v) to give the pure product (13) as a
brown oil (3.7 g, 90%). Rf 0.38 (hexane–EtOAc, 1 : 1, v/v); 1H
NMR: d 6.51 (2H, d, J 9.0, ArH ‘o’ to NH2), 6.42 (2H, d, J 9.0,
ArH ‘m’ to NH2), 3.21 (2H, br s, NH2), 0.81 (9H, s, tBu), 0.10
(6H, s, 2 × CH3); 13C NMR: d 148.6 (ArCOTBDMS), 140.7
(ArCNH2), 121.1 (2 × ArCH ‘o’ to OTBDMS), 116.7 (2 ×
ArCH ‘m’ to OTBDMS), 26.1 (3 × CH3), 18.5 (CCH3), −4.0
(2 × CH3); IR (thin film) m cm−1 3300 (NH2), 2268 (NCO); m/z
(CI) 224 (M + H, 100%); Found 224.1467. [C12H21NOSi + H]+
requires 224.1471.
=
(MeOH-d4): d 157.2 (ArCOH), 155.0 (C O), 144.6 (ArCN),
132.6 (ArCNH), 131.1 (ArCNH), 124.1 (2 × ArCH ‘o’ to urea),
123.8 (2 × ArCH ‘o’ to urea), 116.8 (2 × ArCH ‘o’ to ArCOH),
114.5 (2 × ArCH ‘o’ to NCH2CH2Cl), 55.1 (2 × CH2CH2Cl),
42.2 (2 × CH2CH2Cl); IR (thin film) m cm−1 3321 br (OH), 1700 s,
1575 s; m/z (CI) 260 (100%), 368 (M + H, 35Cl, 35Cl, 30%), 370
(M + H, 35Cl, 37Cl, 18%), 372 (M + H 37Cl, 37Cl 3%); Found
368.0924. [C17H1935Cl2N3O2 + H]+ requires 368.0933; Found C,
54.45; H, 5.19; N, 10.91. C17H19Cl2N3O2 requires C, 55.45; H,
5.20; N, 11.40%; HPLC: tR 3.8 min (method 3).
4-(tert-Butyldimethylsilanyloxy)-phenyl isothiocyanate (14)
4-(tert-Butyldimethylsilanyloxy)-phenyl aniline (13) (0.5 g,
2.24 mmol) was dissolved in DCM (20 mL) and cooled in ice.
Thiophosgene (0.7 mL, 8.96 mmol) was added dropwise whilst
stirring then the solution was gradually brought to reflux at
37 ◦C under an inert atmosphere for 2 h. The reaction mixture
was cooled to room temperature and concentrated in vacuo,
to give a crude yellow product. Flash column chromatography
(hexane–EtOAc, 7 : 3, v/v) yielded the pure product (14) as a
pale yellow oil (0.52 g, 87%). Rf 0.39 (hexane–EtOAc, 7 : 3, v/v);
1H NMR: d 6.91 (2H, d, J 9.0, ArH ‘o’ to NCS), 6.60 (2H, d,
4-Benzyloxyphenyl isothiocyanate (11)
Et3N (0.3 mL, 2.12 mmol) was added to a solution of 4-
benzyloxyaniline hydrochloride salt (0.5 g, 2.12 mmol) in EtOAc
(40 mL). The mixture was kept at 0–5 ◦C, and thiophosgene
(0.65 mL, 8.48 mmol, 4 eq.) was added. The reaction was stirred
for 24 h at room temperature, filtered and concentrated in vacuo
to obtain a peach oil. Flash chromatography (hexane–EtOAc,
1 : 1, v/v) afforded 4-benzyloxyphenyl isothiocyanate (11) as a
t
J 9.0 ArH ‘m’ to NCS), 0.79 (9H, s, Bu), 0.01 (6H, s, 2 ×
◦
pal◦e peach crystalline solid (0.49 g, 96%). Mp 54–56 C (lit.18
CH3); 13C NMR: d 155.3 (NCS), 127.3 (2 × ArCH ‘o’ to NCS),
126.9 (2 × ArCOTBDMS and ArCNCS), 121.4 (2 × ArCH ‘m’
to NCS), 26.0 (3 × CH3), 18.6 (CCH3), −4.0 (2 × CH3); IR
(thin film) m cm−1 2106 s, 1501; m/z (CI) 265 (M+ 100%); Found
265.0960. [C13H19NOSSi]+ requires 265.0957.
1
60 C); Rf 0.75 (hexane–EtOAc, 1 : 1, v/v); H NMR: d 7.26–
7.28 (5H, m, ArH), 7.10 (2H, d, J 9.0 ArH, ‘o’ to NCS), 6.84
(2H, d, J 9.0, ArH, ‘m’ to NCS), 4.98 (2H, m, CH2); 13C NMR:
d 158.1 (ArCOBn), 136.7 (ArCBn), 127.4–129.1 (8 × ArCH),
124.2 (NCS), 116.2 (2 × ArCH ‘o’ to OBn), 70.7 (CH2); IR (thin
film) m cm−1 2176 (NCS); m/z (CI) 91 (100%), 241 (M+, 40%);
Found 241.0569. [C14H11NOS]+ requires 241.0561.
1-{4-[Bis-(2-chloroethyl)-amino]-phenyl}-3-(4-hydroxyphenyl)
thiourea (2)
To the silyl ether thiourea (15) (210 mg, 0.422 mmol) in
anhydrous THF (10 mL) was added Bu4NF (1 M in THF,
0.42 mL). After 45 min, the solution was concentrated and
purified by flash chromatography (hexane–EtOAc, 5 : 2, v/v)
to afford the desired product (2) as a pale peach crystalline solid
(100 mg, 62%). Mp 104–105 ◦C; Rf 0.25 (hexane–EtOAc, 5 : 2,
1-(4-Benzyloxyphenyl)-3-{4-[bis-(2-chloroethylamino]-phenyl}
thiourea (12)
The N-mustard salt (7) (0.5 g, 1.63 mmol) was dissolved in
DCM (15 mL), and NEt3 (0.46 mL, 3.28 mmol) and benzyl
isothiocynate (11) (0.4 g, 1.63 mmol) were added. The reaction
1
mixture was left to stir under N2
for 24 h. The reaction
v/v); H NMR (MeOH-d4): d 7.75 (2H, br s, 2 × NH), 7.12–
(g)
was partitioned between DCM–H2O (1 : 1, 2 × 20 mL) and
the aqueous layer was extracted with DCM (2 × 20 mL).
The organic phases were collected and dried over MgSO4 and
concentrated in vacuo to give a pale brown solid which was
purified by chromatography (hexane–EtOAc, 3 : 7, v/v) to afford
(12) (0.37 g, 48%) as a pale pink powder. Mp 114–116 ◦C; Rf
6.92 (4H, m, ArH ‘o’ to thiourea), 6.69 (2H, d, J 9.0, ArH
‘o’ to OH), 6.51 (2H, d, J 9.0, ArH ‘o’ to NCH2CH2Cl), 5.04
(1H, br s, OH), 3.60–3.69 (4H, m, 2 × CH2CH2Cl), 3.49–3.54
(4H, m, 2 × CH2CH2Cl); 13C NMR (MeOH-d4): d 182.6 (C S),
=
157.6 (ArCOH), 146.8 (ArCN), 132.5 (2 × ArCNH), 129.0 (4 ×
ArCH ‘o’ to thiourea), 116.9 (2 × ArCH ‘o’ to OH), 113.9 (2 ×
ArCH ‘o’ to ArCNCH2CH2Cl), 54.9 (2 × CH2CH2Cl), 42.0
(2 × CH2CH2Cl); IR (thin film) m cm−1 3351 br (OH), 1697,
1576; m/z (CI) 384 (M + H, 35Cl, 35Cl, 100%), 386 (M + H,
35Cl, 37Cl, 65%), 388 (M + H, 37Cl, 37Cl, 10%); Found 384.3229.
[C17H1935Cl2N3OS + H]+ requires 384.3238; Found C, 52.98; H,
5.35; N, 10.46. C17H19Cl2N3OS requires C, 53.13; H, 4.98; N,
10.93%; HPLC: tR 4.0 min (method 3).
1
0.67 (hexane–EtOAc, 3 : 7, v/v); H NMR: d 7.30–7.35 (7H,
m, ArHBn, and ‘o’ to OBn), 7.22–7.14 (4H, m, ArH ‘o’ to
thiourea), 6.90 (2H, d, J 9.0 ArH ‘o’ to NCH2CH2Cl), 6.63 (1H,
br s, NH), 6.60 (1H, br s, NH), 4.99 (2H, s, CH2), 3.71–3.79
(4H, m, 2 × CH2CH2Cl), 3.58–3.62 (4H, m, 2 × CH2CH2Cl);
13
=
C NMR: d 181.6 (C S), 158.5 (ArCO), 137.5 (2 × ArCN and
ArC), 136.9 (2 × ArCNH), 128.4–128.5 (5 × ArCHBn), 127.8–
127.9 (4 × ArCH ‘o’ to thiourea), 116.0 (2 × ArCH ‘o’ to OBn),
112.7 (2 × ArCH ‘o’ to NCH2CH2Cl), 70.7 (CH2), 53.9 (2 ×
CH2CH2Cl), 40.6 (2 × CH2CH2Cl); IR (thin film) m cm−1 3351 s
(NH), 2060, 1697, 1576, 1237, 1142, 967 and 919; m/z (CI) 475
(M + H, 35Cl, 35Cl 25%), 477 (M + H, 35Cl, 37Cl, 15%), 479
(M + H 37Cl, 37Cl 2%); Found 475.1168. [C24H2535Cl2N3OS +
H]+ requires 475.1208.
3-(tert-Butyldimethylsilanyloxy)-4-methoxyphenyl amine (17)
5-Amino-2-methoxyphenol (16) (2.0 g, 14.37 mmol) was dis-
solved in DCM (50 mL) and added to a solution of tert-
butyldimethyl silyl chloride (2.2 g, 14.37 mmol) in DCM
(10 mL). NEt3 (2 mL, 14.37 mmol) was added, followed by
a catalytic amount of DMAP and the solution was stirred
rapidly. A white precipitate formed immediately. After 30 min
the reaction mixture was poured onto H2O (150 mL) and
extracted with Et2O (2 × 50 mL). The organic extracts were
combined, dried over MgSO4, and concentrated in vacuo to give
a dark yellow oil. This was purified by column chromatography
(EtOAc–hexane, 1.5 : 1, v/v) to give the pure product (17) as
a brown oil (2.38 g, 65%). Rf 0.67 (hexane–EtOAc, 2 : 8, v/v);
4-(tert-Butyldimethylsilanyloxy)-phenyl aniline (13)
To a solution of imidazole (2.0 g, 29 mmol) and 4-aminophenol
(2.0 g, 18.3 mmol) in THF (50 mL) was added tert-butyldimethyl
silyl chloride (3.6 g, 24 mmol) with rapid stirring at room
temperature. A white precipitate formed immediately. After
30 min the reaction mixture was poured onto H2O (150 mL)
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0
4 0 0 7

View Article Online
1H NMR: d 6.52 (1H, d, J 8.0 ArH ‘o’ to OCH3), 6.10–6.13
(2H, m, ArH ‘m’ to OCH3), 3.57 (3H, s, OCH3), 3.08 (2H, br, s,
J 9.0, ArH), 7.58 (2H, d, J 9.0, ArH), 7.01 (1H, t, J 2.0 ArH),
6.74 (2H, d, J 2.0, ArH), 4.04 (4H, t, J 13.0, 2 × CH2CH2Cl),
3.32 (4H, t, J 13.0, CH2CH2Cl); 13C NMR (MeOH-d4): d 155.3
t
NH2), 0.84 (9H, s, Bu), 0.01 (6H, s, CH3); 13C NMR: d 146.4
=
(COMe), 144.7 (COTBDMS), 141.0 (CNH2), 114.7 (ArCH ‘o’
to OMe), 109.9 (CH ‘m’ to OMe), 108.6 (ArCH ‘m’ to OMe),
56.9 (OCH3), 26.1 (3 × CH3), 18.8 (CCH3), −4.3 (2 × CH3); IR
(thin film) m cm−1 3367 (NH), 2955, 2858 m (OMe), 1711; m/z
(CI) 254 (M + H, 100%); Found 254.1576. [C13H23NO2Si + H]+
requires 254.1577.
(C O), 146.2 (ArCOH), 142.5 (ArCOH), 142.2 (ArCN), 132.0
(ArCNH), 131.1 (ArCNH), 123.1 (2 × ArCH), 121.3 (ArCH),
116.3 (ArCH), 113.1 (2 × ArCH), 109.9 (ArCH), 59.7 (2 ×
CH2CH2Cl), 38.3 (2 × CH2CH2Cl); IR (thin film) m cm−1 3422
br, 1642; m/z (CI) 384 (M + H, 35Cl, 35Cl, 55%), 386 (M + H,
35Cl, 37Cl, 33%), 388 (M + H, 37Cl, 37Cl, 5%); Found 384.0881.
[C17H1935Cl2N3O3S + H]+ requires 384.0882; HPLC: tR 4.0 min
(method 3).
tert-Butyl-(5-isocyanato-2-methoxyphenoxy)-dimethylsilane (18)
Amine (17) (0.5 g, 1.98 mmol) was dissolved in EtOAc (20 mL)
and triphosgene (2.3 g, 7.90 mmol, 4 eq.) was added. The
reaction was gradually brought to reflux at 77 ◦C and was
monitored at this temperature for 2 h. The resultant mixture was
concentrated in vacuo and purified by column chromatography
(EtOAc–hexane, 7 : 3, v/v) to obtain the pure isocyanate (18)
(0.51 g, 93%) as a dark brown oil. Rf 0.77 (EtOAc–hexane, 7 : 3,
v/v); 1H NMR: d 6.61–6.78 (3H, m, ArH), 3.63 (3H, s, OCH3),
1-{4-[Bis-(2-Chloroethyl)-amino]-phenyl-3-(3,4-
dimethoxyphenyl)-thiourea (21)
To a stirred solution of N-mustard salt (7) (0.5 g, 1.63 mmol)
and NEt3 (0.5 mL, 3.26 mmol) in DCM was added 3, 4-
dihydroxyphenyl isocyanate (20) (0.22 g, 1.63 mmol). The
reaction was left to stir under an inert atmosphere until complete
disappearance of the NCO stretch in the IR spectrum was
observed, and total consumption of the N-mustard (7) was
evident by TLC analysis. After 1.5 days, the reaction was washed
with water, and extracted with DCM (30 mL). The organic
extracts were combined and dried over MgSO4, filtered and
concentrated in vacuo. The crude mixture was purified by column
chromatography (DCM–acetone, 7 : 1, v/v) to yield a colourless
powder which was crystallised from hot toluene to give (21)
as pale yellow crystals (0.63 g, 93%). Mp 104–107 ◦C; Rf 0.4
(EtOAc–hexane, 6 : 4, v/v); 1H NMR (DMSO-d6): d 7.62 (2H,
br s, 2 × NH), 7.25 (2H, d, J 9.0, ArH), 6.98 (1H, br s, ArH),
6.86 (2H, br s, ArH), 6.69 (2H, d, J 9.0, ArH), 3.88 (6H, s, 2 ×
t
0.84 (9H, s, Bu), 0.02 (6H, s, 2 × CH3); 13C NMR: d 149.7
(COMe), 145.9 (COTBDMS), 131.0 (ArCN), 126.3 (NCO),
118.2–118.0 (3 × ArCH), 56.1 (OCH3), 26.0 (3 × CH3), 18.8
(CCH3), −4.3 (2 × CH3); IR (thin film) m cm−1 3325, 2859
(OCH3), 2273 (NCO), 1520, and 841.
1-{4-[Bis-(2-chloroethyl)-amino]-phenyl}-3-[3-tert-
butyldimethylsilanyloxy)-4-methoxyphenyl] urea (19)
N-Mustard (7) (0.5 g, 1.63 mmol) was dissolved in DCM (15 mL)
and NEt3 (0.34 mL, 2.45 mmol) was added. The reaction
mixture was stirred for 5 min, then the protected isocyanate
(18) (0.45 g, 1.63 mmol) was added and the mixture stirred
for 24 h. The resulting mixture was partitioned between H2O–
DCM and then purified by chromatography (EtOAc–hexane, 7 :
3, v/v) to obtain (19) (0.6 g, 71%) as a very pale yellow liquid.
OCH3), 3.72–3.80 (4H, m, 2 × CH2CH2Cl), 3.44–3.52 (4H, m,
13
=
2 × CH2CH2Cl); C NMR (DMSO-d6): d 180.9 (C S), 148.2
(ArCOMe), 145.4 (ArCOMe), 127.9 (ArCN and 2 × ArCNH),
118.2 (ArCH), 112.3 (ArCH), 109.9 (4 × ArCH), 56.1 (2 ×
CH2CH2Cl), 53.5 (2 × OCH3), 40.3 (2 × CH2CH2Cl); IR (thin
film) m cm−1 1513; m/z (CI) 428 (M + H, 35Cl, 35Cl, 60%), 430
(M + H, 35Cl, 37Cl, 35%), 432 (M + H, 37Cl, 37Cl, 6%); Found
428.0971. [C19H2335Cl2N3O2S + H]+ requires 428.0967; Found C,
53.16; H, 5.40; N, 9.75. C19H23Cl2N3O2S requires C, 53.27; H,
5.41; N, 9.80%.
1
Rf 0.65 (hexane–EtOAc, 3 : 7, v/v); H NMR: d 7.03 (2H, d,
J 9, ArH ‘m’ to NCH2CH2Cl), 6.61–6.71 (3H, m, 1 × ArH
‘o’ to OCH3 and 2 × ArH ‘o’ to NCH2CH2Cl), 6.50 (2H, d,
J 9, ArH ‘m’ to OCH3), 6.05 (1H, br s, NH), 6.04 (1H, br s
NH), 3.62 (3H, s, OCH3), 3.59–3.40 (8H, m, 2 × CH2CH2Cl),
t
0.83 (9H, s, Bu), 0.01 (6H, s, 2 × CH3); 13C NMR: d 150.1
=
(C O), 149.5 (COMe), 141.1 (ArCOTBDMS), 140.0 (ArCN),
131.1 (ArCNH), 126.5 (ArCNH), 120.5 (2 × ArCH), 113.2 (2 ×
ArCH), 112.9 (3 × ArCH), 56.2 (2 × CH2CH2Cl), 54.1 (OCH3),
40.8 (2 × CH2CH2Cl) 26.1 (3 × CH3), 18.8 (CCH3), −4.2 (2 ×
CH3); IR (thin film) m cm−1 3327 br, 1500 s, 1223 s, 898; m/z
(CI) 254 (100%), 512 (M + H, 35Cl, 35Cl, 25%), 514 (M + H,
35Cl, 37Cl, 15%), 516 (M + H, 37Cl, 37Cl, 3%); Found 512.1911.
[C24H3535Cl2N3O3Si + H]+ requires 512.1904.
1-{4-[Bis-(2-chloroethyl)-amino]-phenyl-3-(3,4-
dimethoxyphenyl)-thiourea (4)
The dimethoxy urea (21) (200 mg, 0.147 mmol) was dissolved in
dry DCM (6 mL) and cooled to −78 ◦C (dry ice–acetone) under
a stream of nitrogen. Boron tribromide (1.2 mL, 1 M solution
in dichloromethane) was added slowly. After the addition of
boron tribromide the pale yellow/green solution was allowed to
warm to room temperature over 4 h. The reaction mixture was
quenched with brine (10%), extracted into dichloromethane (3 ×
30 mL), dried over MgSO4 and the solvents removed in vacuo to
produce a light brown oil (184 mg, 98%). This was purified by
column chromatography (DCM–MeOH, 9 : 1, v/v) to afford (4)
as a pale mustard solid (174 mg, 92%). Mp 212–220 ◦C; Rf 0.33
1-{4-[Bis-(2-chloroethyl)-amino]-phenyl}-3-(3,4-
dihydroxyphenyl) urea (3)
Urea (19) (0.6 g, 1.17 mmol) was dissolved in the minimum
amount of conc. HCl and heated at reflux at 130 ◦C for 2 h.
The product was washed with water and extracted with DCM
(4 × 15 mL). The organic extracts were combined and dried over
MgSO4, filtered and concentrated in vacuo. The residue (0.35 g,
0.486 mmol) was dissolved in dry DCM and cooled to −78 ◦C
(dry ice–acetone) under a stream of nitrogen. Boron tribromide
(1.3 mL, 1 M solution in dichloromethane) was added slowly.
After the addition of boron tribromide, the pale yellow/green
solution was allowed to warm to room temperature over 4 h.
The reaction mixture was quenched by the addition of NaHCO3
(10%), extracted into dichloromethane (3 × 30 mL), dried over
MgSO4 and the solvents removed in vacuo to produce a light
brown oil. This was purified by column chromatography (DCM–
MeOH, 9 : 1, v/v) to afford prodrug (3)◦ as a pale mustard
solid (170 mg, 70% over 2 steps). Mp 112 C; Rf 0.37 (DCM–
MeOH, 9 : 1, v/v); 1H NMR (MeOH-d4): d 7.69 (2H, d,
1
(DCM–MeOH, 9 : 1, v/v); H NMR (MeOH-d4): d 9.23 (1H,
br s, OH), 9.16 (1H, br s, OH), 9.06 (1H, br s, NH), 8.80 (1H, br s,
NH), 7.32 (2H, d, J 9.0, ArH), 6.84 (1H, d, J 2.0, ArH), 6.56–
6.74 (4H, m, ArH), 3.79 (8H, br s, 2 × CH2CH2Cl); 13C NMR
=
(MeOH-d4): d 179.9 (C S), 155.3 (ArCOH), 144.1 (ArCOH),
143.2 (ArCN), 131.0 (ArCNH), 129.5 (ArCNH), 126.7 (2 ×
ArCH), 115.9 (ArCH), 115 (ArCH), 113.1 (ArCH), 111.9 (2 ×
ArCH), 55.3 (2 × CH2CH2Cl), 41.5 (2 × CH2CH2Cl); IR (thin
film) m cm−1 3449 br, 1620; m/z (CI) 400 (M + H, 35Cl, 35Cl, 40%),
402 (M + H, 35Cl, 37Cl, 27%), 404 (M + H, 37Cl, 37Cl, 4%); Found
400.0661. [C17H1935Cl2N3O2S +H]+ requires 400.0654; Found C,
49.53; H, 4.90; N, 9.75. C17H19Cl2N3O2S·H2O requires C, 48.81;
H, 5.06; N, 10.04%; HPLC: tR 3.5 min (method 3).
4 0 0 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0

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N-tert-Butoxycarbonyl-6-nitrodopamine (24)
2-Acetoxy-4(3-{4-[bis-(2-chloroethyl) amino]phenyl}ureido)-5-
(2-tert-butoxycarbonylaminoethyl)phenyl acetate (29)
6-Nitrodopamine hydrogen sulfate salt (23)9 (5.4 g, 18.2 mmol)
was suspended in THF–H2O (5 : 1 v/v, 60 mL) with triethy-
lamine (6.4 mL, 45.6 mmol). Boc–ON (5.39 g, 22.0 mmol)
was added and the reaction mixture was stirred at room
temperature for 24 h. The solvent was then removed under
reduced pressure and to the residue was added EtOAc (200 mL)
and MeOH (50 mL). This was then dried (MgSO4), filtered
and reduced to dryness. The crude product was purified by
column chromatography (DCM–MeOH, 96 : 4, v/v) to yield
amine (24) as a yellow solid (3.19 g, 59%). Mp 169–171 ◦C;
Amine (26) (150 mg, 0.43 mmol) was solubilised in DCM
◦
(4 mL) and cooled to 0 C. Triphosgene (0.27 mL, 0.57 mmol
of a 20% solution in toluene) was added dropwise, followed
by the addition of triethylamine (0.13 mL, 0.94 mmol). The
reaction mixture was stirred at 0 ◦C for 1 hour before the
addition of aniline mustard dihydrochloride salt (7) (143 mg,
0.47 mmol) and pyridine (2 mL). The reaction was stirred at
room temperature for 16 h, diluted with DCM (10 mL), dried
over MgSO4, filtered and the filtrate reduced to dryness. The
crude residue was purified by column chromatography (hexane–
EtOAc, 1 : 1, v/v) to yield the pure product (29) as a brown foam
1
Rf 0.45 (hexane–EtOAc, 1 : 1, v/v); H NMR (MeOH-d4): d
7.54 (1H, s, H5), 6.72 (1H, s, H2), 3.32 (2H, t, J 6.8, CH2),
2.98 (2H, t, J 6.9, CH2), 1.41 (9H, s, 3 × CH3); 13C NMR:
d 158.9 (NHCOO), 152.6 (ArC), 145.6 (ArC), 142.1 (ArC),
130.0 (ArC), 119.9 (ArCH), 113.8 (ArCH), 80.4 (C[CH3]3),
42.2 (CH2), 35.4 (CH2), 29.2 (C[CH3]3); IR (thin film) m cm−1
3401, 1674, 1455, 1394, 1368, 1329, 1286, 1161, 1018; Found C,
52.13; H, 6.03; N, 9.03. C13H18N2O6 requires C, 52.35; H, 6.08;
N, 9.39%.
1
(163 mg, 63%). Rf 0.56 (Hexane–EtOAc, 1 : 1, v/v); H NMR
(MeOH-d4): d 8.76 (1H, bs, NH-urea), 8.30 (1H, s, H6), 7.73
(1H, bs, NH-urea), 7.40 (2H, d, J 9.0, 2ArH), 6.92 (1H, s, H3),
6.75 (2H, d, J 9.0, 2ArH), 5.35 (1H, t, J 6.0, NHBoc), 3.74–
3.60 (8H, m, 4 × CH2), 3.20–3.11 (2H, m, CH2), 2.78 (2H, t,
J 8.0, CH2), 2.28 (3H, s, OCOCH3), 2.27 (3H, s, OCOCH3), 1.56
(9H, s, 3 × CH3); 13C NMR (MeOH-d4): d 169.1 (OCOCH3),
168.9 (OCOCH3), 158.4 (NHCOO), 153.4 (NHCOO), 141.4
(ArC), 137.1 (ArC), 136.3 (ArC), 132.4 (ArC), 124.3 (ArCH),
123.8 (ArCH), 121.6 (ArCH), 114.6 (ArCH), 114.1 (ArCH),
81.5 (C[CH3]3), 54.6 (2 × CH2), 40.7 (2 × CH2 + ArCH2), 33.1
(CH2N), 29.0 (C[CH3]3), 21.1 (2 × OCOCH3); IR (thin film)
m cm−1 3380, 2977, 1769, 1705, 1661, 1597, 1516, 1421, 1367,
1201, 1104, 1013; m/z (FAB) 633 (15%, M + Na), 610 (21),
511 (7), 154 (100); Found 610.1979. C28H36N4O7Cl2 requires
610.1961; Found C, 54.01; H, 6.07; N, 9.11. C28H36N4O7Cl2
requires C, 54.98; H, 5.95; N, 9.16%.
2-Acetoxy-5-(2-tert-butoxycarbonylaminoethyl)-4-nitrophenyl
acetate (25)
N-tert-Butoxycarbonyl-6-nitrodopamine
(24)
(0.75
g,
2.51 mmol) was solubilised in pyridine (15 mL) and acetic
anhydride (0.52 mL, 5.53 mmol) was added. The reaction
mixture was stirred at room temperature for 16 h. DCM
(20 mL) and water (20 mL) were added to the reaction mixture
and the two phases were partitioned. The organic phase was
washed with water (20 mL), dried (MgSO4), filtered and reduced
to dryness. The product (25) was isolated as a waxy yellow solid
(834 mg, 88%). Mp 89–92 ◦C; Rf 0.57 (hexane–EtOAc, 1 : 1,
v/v); 1H NMR: d 7.84 (1H, s, H5), 7.17 (1H, s, H2), 4.71 (1H,
bs, NHBoc), 3.38 (2H, q, J 6.8, CH2), 3.04 (2H, t, J 6.9, CH2),
2.49 (6H, s, 2 × OCOCH3), 1.36 (9H, s, 3 × CH3); 13C NMR:
d 169.0 (OCOCH3), 167.7 (OCOCH3), 156.3 (NHCOO), 146.6
(ArC), 146.1 (ArC), 141.1 (ArC), 133.9 (ArC), 127.7 (ArCH),
121.3 (ArCH), 79.9 (C[CH3]3), 41.1 (CH2), 33.8 (CH2), 28.7
(C[CH3]3), 21.0 (OCOCH3), 20.9 (OCOCH3); IR (thin film)
m cm−1 1778, 1649, 1530, 1436, 1371, 1273, 1199, 1145; m/z
(FAB) 405 (45%, M + Na), 327 (90), 283 (100); Found 405.1262.
C17H22N2O8Na requires 405.1274.
2-Acetoxy-4-(3-{4-[bis-(2-chloroethyl) amino]phenyl}thioureido)-
5-(2-tert-butoxycarbonylaminoethyl)phenyl acetate (30)
Amine (26) (200 mg, 0.57 mmol) was solubilised in DCM (4 mL)
and cooled to 0 ◦C. Thiophosgene (43 lL, 0.57 mmol) was added
dropwise, followed by the addition of triethylamine (0.17 mL,
1.25 mmol). The reaction mixture was stirred at 0 ◦C for 1 hour
before the addition of aniline mustard dihydrochloride salt (7)
(191 mg, 0.62 mmol) and pyridine (2 mL). The reaction was
stirred at room temperature for 16 h, diluted with DCM (10 mL),
dried over MgSO4, filtered and the filtrate reduced to dryness.
The crude residue was purified by column chromatography
(DCM–MeOH, 96 : 4, v/v) to yield the pure product (30) as a
red/brown foam (252 mg, 71%). Mp 89–94 ◦C; Rf 0.78 (DCM–
MeOH, 9 : 1, v/v); 1H NMR (MeOH-d4): d 8.20 (1H, bs, NH-
urea), 8.30 (1H, s, H6), 7.49 (1H, bs, NH-urea), 7.25 (2H, d,
J 9.0, 2ArH), 7.20 (1H, s, H3), 6.60 (2H, d, J 9.0, 2ArH), 4.84
(1H, bs, NHBoc), 3.64 (4H, 2 × t, J 6.5, 2 × CH2), 3.54 (4H,
2 × t, J 7.0, 2 × CH2), 3.24 (2H, q, J 7.0, CH2), 2.70 (2H,
t, J 7.0, CH2), 2.20 (6H, 2 × s, 2 × OCOCH3), 1.32 (9H, s,
3 × CH3); 13C NMR: d 181.9 (NHCOO), 168.6 (OCOCH3),
168.5 (OCOCH3), 158.3 (ArC), 146.2 (ArC), 134.9 (ArC +
NHCSNH), 129.0 (ArC), 125.3 (ArCH), 124.0 (ArC), 112.6
(ArCH), 80.4 (C[CH3]3), 53.9 (2 × CH2), 40.7 (2 × CH2 +
ArCH2), 34.0 (CH2N), 28.7 (C[CH3]3), 21.1 (2 × OCOCH3); IR
(thin film) m cm−1 3344, 2977, 1772, 1695, 1613, 1517, 1455, 1367,
1207, 1013; m/z (FAB) 649 (30%, M + Na), 625 (95), 571 (55);
Found 626.1742. C28H36N4O6SCl2 requires 626.1733; Found C,
52.92; H, 5.73; N, 9.03. C28H36N4O6SCl2 requires C, 53.79; H,
5.78; N, 8.92%.
2-Acetoxy-5-(2-tert-butoxycarbonylaminoethyl)-4-aminophenyl
acetate (26)
2-Acetoxy-5-(2-tert-butoxycarbonylaminoethyl)-4-nitrophenyl
acetate (25) (1.40 g, 3.66 mmol) was solubilised in MeOH
containing 10% palladium on carbon (90 mg). The mixture
was stirred under an atmosphere of hydrogen for 2 h and
then filtered through a short pad of Celite. The filtrate was
reduced to dryness and the crude product was purified by
column chromatography (hexane–EtOAc, 1 : 1). The product
(26) was obtained as a yellow oil that crystallised on standing
(0.9 g, 70%). Mp 140–142 ◦C; Rf 0.37 (hexane–EtOAc, v/v);
1H NMR (MeOH-d4): d 6.80 (1H, s, H5), 6.53 (1H, s, H2),
3.21 (2H, t, J 7.5 CH2), 2.66 (2H, t, J 7.5, CH2), 2.23 (3H, s,
OCOCH3), 2.22 (3H, s, OCOCH3), 1.45 (9H, s, 3 × CH3);
13C NMR (MeOH-d4): d 173.9 (OCOCH3), 173.2 (OCOCH3),
161.6 (NHCOO), 148.6 (ArC), 145.4 (ArC), 137.3 (ArC), 128.2
(ArC), 125.6 (ArCH), 113.6 (ArCH), 83.0 (C[CH3]3), 43.5
(CH2), 35.3 (CH2), 31.7 (C[CH3]3), 23.4 (2 × OCOCH3); IR
(thin film) m cm−1 2526, 1762, 1646, 1508, 1448, 1376, 1216,
1098; m/z (FAB) 375 (25%, M + Na), 352 (100), 310 (42), 297
(27); Found 352.1634. C17H24N2O6 requires 352.1634; Found C,
57.91; H, 6.84; N, 7.87. C17H24N2O6 requires C, 57.94; H, 6.86;
N, 7.95%.
1-[2-(2-Aminoethyl)-4,5-dihydroxyphenyl]-3-{4-[bis-(2-
chloroethyl) amino]-phenyl}urea (5)
Urea (29) (100 mg, 0.16 mmol) was solubilised in acetone (4 mL)
and heated to reflux with 6 M HCl (2 mL) for 4 h. After
removal of the solvent by lyophilisation, the pure product (5)
was obtained as a red/brown solid (104 mg, 100%). Mp 98 ◦C;
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0
4 0 0 9

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Rf 0.19 (MeCN–H2O, 94 : 6, v/v); 1H NMR (MeOH-d4): d 7.62
(2H, d, J 9.0, 2ArH), 7.33 (2H, d, J 9.0, 2ArH), 6.84 (1H, s, H6),
3.99 (4H, t, J 6.5, 2 × CH2), 3.74 (1H, s, H3), 3.66 (4H, 2 × t,
J 6.5, 2 × CH2), 3.15 (2H, t, J 7.5, CH2), 2.89 (2H, t, J 7.5, CH2);
13C NMR (MeOH-d4): d 157.4 (NHCONH), 146.4 (ArC), 146.3
(ArC), 145.8 (ArC), 129.1 (ArC), 125.1 (ArC), 122.6 (ArCH),
122.1 (ArC), 117.8 (ArC), 116.1 (ArCH), 59.6 (2 × CH2), 41.5
(2 × CH2), 39.2 (ArCH2), 30.4 (CH2NH); IR (thin film) m cm−1
2531, 2361, 1645, 1558, 1515, 1456, 1319, 1221; m/z (FAB) 467
(54%, M + K), 427 (100), 233 (35), 209 (33); Found 427.1302.
C19H25N4O3Cl2 requires 427.1304; Found C, 42.76; H, 5.33; N,
9.92. C19H25N4O3Cl2·3HCl requires C, 42.52; H, 5.08; N, 10.44%;
HPLC: tR 21.1 min (method 1), 23.6 min (method 2).
References
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Thiourea (30) (200 mg, 0.36 mmol) was solubilised in acetone
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removal of the solvent by lyophilisation, the pure product (6)
was purified by column chromatography (MeCN–H2O, 94 : 6,
v/v) to yield the pure product as a yellow solid (55 mg, 67%). Mp
226 ◦C; Rf 0.19 (MeCN–H2O, 94 : 6, v/v); 1H NMR (MeOH-d4):
d 7.25 (2H, d, J 9.0, 2ArH), 6.79–6.77 (3H, m, H6 + 2ArH),
6.71 (1H, s, H3), 3.81–3.68 (8H, m, 4 × CH2), 3.18 (2H, t, J 7.5,
CH2), 2.86 (2H, t, J 7.5, CH2); 13C NMR (MeOH-d4): d 183.7
=
(C S), 147.0 (ArC), 146.4 (ArC), 141.8 (ArC), 129.1 (ArC),
127.3 (ArC), 118.1 (2 × ArCH), 117.9 (ArC), 117.6 (ArC), 113.9
(2 × ArCH), 54.9 (2 × CH2), 42.1 (2 × CH2), 41.6 (ArCH2),
30.6 (CH2NH); IR (thin film) m cm−1 2531, 2361, 1645, 1558,
1515, 1456, 1319, 1221; m/z (FAB) 523 (30%, M + 2H + 2K),
443 (75), 409 (20); Found 443.1070. C19H24N3O2SCl2 requires
443.0997; HPLC: tR 20.7 min (method 1), 22.9 min (method 2).
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was incubated in phosphate buffer (900 lL, pH 7.2) at
37 ◦C. Aliquots (100 lL) were removed at various time intervals
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Acknowledgements
We gratefully acknowledge the AICR, the University of Read-
ing’s Research Endowment Trust Fund and the School of
Chemistry, University of Reading, for financial support of this
work.
4 0 1 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 0 2 – 4 0 1 0