Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Article abstract of DOI:10.1016/S0968-0896(03)00451-6

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N ^δ-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 °C afforded the corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile substituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation was shown to proceed by an S_N2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihydrothiazoles, with N-(3-aminomethylphenyl)thiourea (IC₅₀=13 μM vs rat neuronal NOS and IC₅₀=23 μM vs rat inducible NOS) and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC₅₀=13 μM vs rat neuronal NOS and IC₅₀=19 μM vs human inducible NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible NOS in a time-dependent manner.

Full text of DOI:10.1016/S0968-0896(03)00451-6

Bioorganic & Medicinal Chemistry 11 (2003) 4189–4206
Synthesis of N-Benzyl- and N-Phenyl-2-amino-4,5-
dihydrothiazoles and Thioureas and Evaluation as Modulators of
the Isoforms of Nitric Oxide Synthase
Claire L. M. Goodyer,a Edwin C. Chinje,b Mohammed Jaffar,b Ian J. Stratfordb
and Michael D. Threadgilla,*
aDepartment of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
bMRC Experimental Oncology Laboratory, School of Pharmacy and Pharmaceutical Sciences, University of Manchester,
Oxford Road, Manchester M13 9PL, UK
Received 20 May 2003; accepted 2 July 2003
Abstract—Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases,
including cancer. Using 1400W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and Nd-(4,5-dihydrothiazol-2-yl)ornithine as
lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS.
Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thio-
phosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles
were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 ꢀC afforded the
corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile sub-
stituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation
was shown to proceed by an SN2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihy-
drothiazoles, with N-(3-aminomethylphenyl)thiourea (IC50=13 mM vs rat neuronal NOS and IC50=23 mM vs rat inducible NOS)
and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC50=13 mM vs rat neuronal NOS and IC50=19 mM vs human inducible
NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible
NOS in a time-dependent manner.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Excessive NO production by eNOS within blood vessel
walls is thought to be the basis for conditions such as
septic- and cytokine-induced circulatory shock. In these
conditions, the sGc–cGMP pathway is excessively acti-
vated, which leads to high levels of NO and so con-
tributes to profound vasodilatation and hypotension.1
Nitric oxide (ꢁ NO) is the smallest known messenger
molecule in biological systems. It is synthesised from
l-arginine 1 by the various isoforms of nitric oxide
synthase (NOS), yielding l-citrulline 3 as a co-product.
The process comprises two separate mono-oxygenation
steps with NG-hydroxyarginine 2 as an intermediate
(Scheme 1). Both steps require molecular oxygen (O2)
and NADPH. There are two main groups of isoforms of
NOS, a constitutive Ca2+/ calmodulin-dependent type
(cNOS) and an inducible Ca2+/ calmodulin-indepen-
dent form (iNOS). cNOS can be further sub-divided
into endothelial and neuronal forms (eNOS and nNOS,
respectively). Underactivity and overactivity of each of
these isoforms can be associated with disease states.
*Corresponding author. Tel.: +44-1225-386840; fax: +44-1225-
386114; e-mail: m.d.threadgill@bath.ac.uk
Scheme 1. Conversion of l-arginine 1 to l-citrulline 3 and nitric oxide,
via l-NG-hydroxyarginine 2, mediated by nitric oxide synthases.
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00451-6

4190
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
However, if too little NO is produced, this can lead to
conditions such as high blood pressure, angina and
impotence. Recently, it has been shown that a reduction
in the activity and synthesis of NO within the endothe-
lium may contribute to the initiation and progression of
atherosclerosis.2 Release of NO from the pelvic nerve
neurons located in the human corpus cavernosum is
known to cause penile erection. NOS inhibitors have
been shown to prevent this action while nitric oxide
sources mimic the effect.3 An overexpression of nNOS
in circulating neutrophils has been found in patients
with Parkinson’s disease4 and nNOS activity is thought
to be linked to migraine headaches; these are believed to
result from abnormal activity in large cerebral blood
vessels and high levels of nNOS occur in the vasodilator
nerves that supply the large cerebral blood vessels.5 NO
production by iNOS is essential for the defence
mechanism of an organism; however, NO produced by
iNOS has been related to several pathological condi-
tions, including cancer,6 arthritis7 and diabetes.8 Thus
selective and potent inhibition of the isoforms of NOS is
an important goal in medicinal chemistry.
esters and dipeptides,14 which are also NOS inhibitors,
the ligand is a nitroguanidine; imidazoles have also been
used as ligands for haem-Fe in our non-isoform-selec-
tive inhibitor 815 and related compounds.16 Substitution
on the imidazole of 8 and replacement with less elec-
tron-rich heterocycles led to weaker inhibition,15 which
is consistent with their weaker potential ligation to iron.
More recent highly iNOS-selective inhibitors17ꢂ22 con-
tain cyclic amidines (e.g. 922) to bind to the iron but lack
the amino-acid motif. Most interesting are the N-(3-
aminomethylbenzyl)acetamidine 1400W 10 (a highly
selective inhibitor of rat iNOS)23 and the lower homo-
logue 11 (a selective inhibitor of nNOS);24 this pair of
compounds illustrates exquisitely the possibility of
switching isoform selectivity radically through subtle
changes in the spatial relationship between the haem-
Fe-binding group and remote functionalities. Sulfur-
containing groups have also been used as the ligands for
iron. The simplest such compound, thiocitrulline 12 is
relatively unselective,25 as is our potent inhibitor 1326
and N-aryl-S-alkylisothioureas such as 14.27 The tetra-
hydrobiopterin binding site has also received some
attention in the development of isoform-selective inhi-
bitors.28,29 7-Nitroindazole 15, which is reported to be
competitive with both the substrate 1 and with tetra-
hydrobiopterin, is claimed30 to be moderately selective
for inhibition of the nNOS isoform.
The structures of several reported inhibitors of NOS are
shown in Figure 1. Many of the early inhibitors, for
example l-NMMA 4,9 are close analogues of the sub-
strate l-arginine 1. Isosteric replacement of the terminal
guanidine of 1 with an acetamidine gives the non-selec-
tive l-NIO 5a.10 The homologue l-NIL 5b,11 however,
shows some selectivity for inhibition of iNOS. This
selectivity (iNOS vs eNOS) increases when the carbox-
ylate of 5b is replaced by the diol motif in 612 but iNOS
vs nNOS selectivity is poor. In each of these inhibitors,
a binding motif can be recognised in which the guani-
dine/amidine ligates to the haem iron at the active site
of the enzyme, while additional binding contacts recog-
nise the amine, the carboxylate and, possibly, the N–H
near the haem ligand. In NG-nitroarginine 713 and its
In the present study, we sought to explore the inhibitory
activity of hybrids between the highly selective 10 and
11 and our potent dihydrothiazole 13. The structures of
the designed target compounds are shown in Figure 2.
In particular, we sought to explore whether the isoform
selectivity shown by 10 and 11 could be translated into
analogous compounds carrying different haem-ligating
head groups. We report here the synthesis of these series
of compounds and their evaluation as inhibitors of nNOS
and iNOS. Although Collins et al.24 note particularly the
Figure 1. Structures of known inhibitors of the isoforms of NOS.

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4191
previously developed method,26 the benzylamines 21
were treated with thiophosgene in the presence of cal-
cium carbonate as an insoluble mild base in a mixed
organic/aqueous solvent system. The corresponding
isothiocyanates 22c–i,k were obtained in yields ranging
from 37 to 83%. Simple treatment of 22c–i,k with
ammonia gave the N-benzylthioureas 16c–i,k in good to
excellent yields. In some cases, deprotection or further
modification of the substituent was necessary. Acid-
olysis removed the Boc protecting groups from 16c,d to
give the analogous N-(aminomethyl)benzylthioureas
16a,b as their bis(trifluoroacetate) salts. Selective reduc-
tion of the nitro groups of 16e,f with tin(II) chloride
provided the aminobenzylthioureas 16l,m, the lower
homologues of 16a,b. Hydrolysis of the protecting esters
in 16i,k with hydrobromic acid afforded the required
carboxylic acids 16n,j, respectively.
Figure 2. Structures of target N-benzyl and N-phenyl thioureas and
2-amino-4,5-dihydrothiazoles.
2-Amino-4,5-dihydrothiazoles can be prepared by at
least three independent routes. Firstly, the dihy-
drothiazole can be introduced as a single unit. Stokker
et al.31 used this approach in synthesising the corre-
sponding 2-benzylamino-4,5-dihydrothiazole by treat-
ment of 5-t-butyl-2-hydroxy-3-iodobenzylamine with
2-methylthio-4,5-dihydrothiazole 26 in boiling ethanol.
Other workers32 have used similar conditions for reac-
tions of primary amines with this electrophilic dihy-
drothiazole synthon. However, Hirashima et al.33 noted
that the reaction of 26 with substituted 2-phenylethyl-
amines gave only low yields of 2-amino-4,5-dihy-
drothiazoles, even under forcing conditions in boiling
pentan-1-ol. In our laboratory, 3-methoxybenzylamine
21g and 4-methoxybenzylamine 21h failed to react with
26 in boiling ethanol and the condensation required
strongly forcing conditions, heating the amines 21g,h
with 26 in the absence of solvent at 180 ꢀC for 4 h, to
achieve even moderate yields of the 2-(methox-
ybenzylamino)-4,5-dihydrothiazoles 17g,h, respectively.
Under these conditions, substituents more sensitive than
methoxy would not be expected to survive; indeed,
treatment of the mono-Boc-protected diamine 21c with
neat 26 at this temperature led only to unidentifiable
degradation products. Secondly, double alkylation of
N-substituted thioureas with 1,2-dibromoethane gives
2-alkylamino-4,5-dihydrothiazoles but the yields
achievable by this method are usually poor to modest.26
Thirdly, Caujolle et al. reported34 introduction of the
CH2CH2 unit in two steps, reaction of the iso-
thiocyanate with 2-aminoethanol to give the N-(2-
hydroxyethyl)thiourea and acid-catalysed cyclisation.
Hence, as shown in Scheme 3, the benzylisothiocyanates
22c–k were treated with 2-aminoethanol in boiling ace-
tone to give the corresponding N-benzylthioureas 24c–k
in satisfactory yields. In the cases of isothiocyanates
carrying strong electron-withdrawing groups on the
benzene ring, low yields of the 3-(benzylaminothio-
carbonyl)-2,2-dimethyltetrahydro-1,3-oxazoles 25e,f,k
were also formed. These products of formal condensa-
tion with the solvent are unlikely to have arisen from
reaction of the hydroxyethylthioureas with acetone,
since this process should not be sensitive to the sub-
stitution on the benzene ring in the manner observed. It
is more likely that 2,2-dimethyltetrahydro-1,3-oxazole is
importance of the 3-aminomethyl group for binding of
10 and 11 to NOS isoforms, we used a range of different
substituents on the benzene ring to test whether this
substitution is still optimal when the ligand for the haem
iron is sulfur, rather than nitrogen.
Chemical Synthesis
The synthesis of the first series of target compounds, the
N-benzylthioureas is shown in Scheme 2. The primary
strategy was to add ammonia to the electrophilic iso-
thiocyanate unit in the benzylisothiocyanates 22. A
common route was developed to prepare these from the
corresponding benzylamines 21. However, certain addi-
tional substituents on the benzyl group required prior
protection. 1,3-Bis(aminomethyl)benzene 21a was trea-
ted carefully with a sub-stoichiometric amount of di-
tert-butyl dicarbonate to give, after a simple aqueous
workup to remove excess 21a, a high yield of the crys-
talline mono-Boc derivative 21c. Similar treatment of
the analogous para diamine 21b furnished the corre-
sponding mono-Boc derivative 21d. To protect the car-
boxylic acid in the 4-aminomethylbenzoic acid 21j, the
methyl ester 21k was formed in the usual way by reac-
tion with acidic methanol. However, the corresponding
3-aminomethylbenzoic acid is not commercially avail-
able and the corresponding methyl ester 21i had to be
synthesised by an alternative route. Using a nitrile as a
synthon for the aminomethyl unit, 3-cyanobenzoic acid
20n was converted to its methyl ester 20i; the relatively
modest yield of 42% may have been due to competing
Pinner reaction of the nitrile with the acidic methanol.
Hydrogenation of 20i gave two products, the required
simple reduction product 21i and the secondary amine
23. The latter is formed by transimination of the inter-
mediate iminomethylbenzene with the primary amine
21i and subsequent hydrogenation of the new imine.
The 3-nitro-, 4-nitro- 3-methoxy- and 4-methox-
ybenzylamines 21e–h, respectively, are commercially
available;
stituted)benzylamines 21 was now in place. Using our
thus
the
set
of
(protected/sub-

4192
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
formed by reversible condensation of 2-aminoethanol
with acetone; this heterocycle is a sterically hindered
nucleophile at nitrogen (owing to the adjacent gem-
dimethyl) and reacts only with the more electrophilic
isothiocyanates.
benzylamino)-4,5-dihydrothiazole 17n. Since these
cyclisation conditions are relatively forcing and cleavage
of sensitive substituents is a risk, a milder cyclisation
was developed. N-(2-Hydroxyethyl)-N0-(3-methoxy-
benzyl)thiourea 24g was treated with neat trifluoroacetic
1
acid at ambient temperature for 2 h; direct H NMR
Treatment of the isomeric N-(2-hydroxyethyl)thioureas
24c,d with boiling hydrochloric acid for prolonged per-
iods efficiently closed the dihydrothiazole rings and
simultaneously removed the Boc protection, giving
17a,b in satisfactory yields as their dihydrochloride
salts. Similar treatment of the para-substituted ester 24k
again closed the dihydrothiazole ring and hydrolysed
the ester protection to afford the target carboxylic acid
17j. However, application of this method to the meta
isomer 24i gave incomplete ester hydrolysis; the product
mixture was re-esterified to give 17i for purification
before acid-catalysed hydrolytic deprotection with aqu-
eous trifluoroacetic acid to give the 2-(3-carboxy-
analysis showed that cyclisation to 17g was complete.
Cyclisation of the nitrobenzyl analogues 24e,f to 17e,f
with trifluoroacetic acid required longer reaction time.
Finally, the nitrobenzylaminodihydrothiazoles were
reduced to the corresponding aminobenzyl com-
pounds 17l,m with tin(II) chloride under neutral con-
ditions.
The preparations of the lower homologues, the N-phe-
nylthioureas 18 and the 2-phenylamino-4,5-dihy-
drothiazoles 19, followed sequences similar to those for
the benzyl compounds above, although a smaller range
of substituents was investigated (Scheme 4). The
Scheme 2. Synthesis of N-benzylthioureas 16. Reagents and conditions: (i) MeOH, SOCl2, 4 d; (ii) MeOH, H2, Pd/C, 16 h; (iii) Boc2O (0.3 equiv),
Et3N, CH2Cl2, 16 h; (iv) CSCl2, CaCO3, H2O, CHCl3, 16 h; (v) NH3, CH2Cl2, 3.5 h, 0 ꢀC; (vi) CF3CO2H, 5 min; (vii) SnCl2, EtOH, reflux, 1 h; (viii)
aq. HBr (50%), 16 h.

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4193
commercially available methoxyphenylisothiocyanates
29g,h reacted with ammonia to give the corresponding
thioureas 18g,h. Protection of the aliphatic amine
nitrogens in the nitrobenzylamines 21e,f with Boc (giv-
ing 27c,d) was followed by reduction of the nitro group
with tin(II) chloride (under neutral conditions to avoid
deprotection) afforded the Boc-amino-anilines 28c,d.
The methyl esters 28q,r were formed by treatment of the
aminophenylacetic acids 28o,p with methanol and thio-
nyl chloride. As in the benzylamine series, amines
28c,d,q,r were converted efficiently to the iso-
thiocyanates 29c,d,q,r with thiophosgene. Again, these
reacted with ammonia to furnish the thioureas 18c,d,q,r.
Acidolytic deprotection removed the Boc groups, giving
the aminomethylphenylthioureas 18a,b as their tri-
fluoroacetate salts, whereas hydrolysis with aqueous
acid yielded the carboxymethylthioureas 18o,p. As
shown in Scheme 5, the same set of phenylisothiocyan-
ates 29c,d,g,h,q,r was used to prepare the N-(2-hydroxy-
ethyl)thioureas 30c,d,g,h, q,r; in contrast to the benzyl
series, there was no evidence of formation of the acetone
adducts (the 2,2-dimethyltetrahydrooxazoles). The con-
ditions for the cyclisations were selected according to
the type of simultaneous deprotection also required.
Cyclisation/deprotection of 30c was effected by both the
boiling hydrochloric acid and the trifluoroacetic acid
methods, giving 19aA and 19aB, respectively. The para-
substituted isomer 19b was prepared by the tri-
fluoroacetic acid method only, as were the two 2-
(methoxyphenylamino)-4,5-dihydrothiazoles
19g,h.
Boiling hydrochloric acid ring-closed and deprotected
the esters 30q,r, giving the 2-(carboxymethyl-
phenylamino)-4,5-dihydrothiazoles 19o,p, respectively,
in good yield.
Scheme 3. Synthesis of N-benzyl-N0-(2-hydroxyethyl)thioureas 24 and 2-(benzylamino)-4,5-dihydrothiazoles 17. Reagents and conditions: (i)
H2N(CH2)2OH, acetone, reflux 4 h; (ii) aq HCl (6 M), reflux, 36 h (17a,b,j); (iii) CF3CO2H, 16 h (17e); iv, CF3CO2H, reflux, 15 h (17f); (v) CF3CO2H,
2 h (17g); (vi) aq HCl (6 M), reflux, 40 h, then MeOH, SOCl2, 4 d (17i); (vii) aq CF3CO2H (50%), reflux, 16 h (17n); (viii) SnCl2, EtOH, reflux, 1 h;
(ix) SnCl2, EtOH, reflux 1.5 h; (x) 26, 180 ꢀC, 4 h.

4194
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
Scheme 4. Synthesis of N-phenylthioureas 18. Reagents and conditions: (i) Boc2O, Et3N, CH2Cl2, 16 h; (ii) SnCl2, EtOH, reflux, 30 min (28c);
(iii) SnCl2, EtOH, reflux, 1 h (28d); (iv) MeOH, SOCl2, 4 d; (v) CSCl2, CaCO3, H2O, CHCl3, 2 h (29c); (vi) CSCl2, CaCO3, H2O, CHCl3, 16 h
(29d,q,r); (vii) NH3, CH2Cl2, 3.5 h; (viii) CF3CO2H, 5 min; (ix) CF3CO2H, 2 h; (x) aq HCl (1 M), 9 d; (xi) aq HCl (6 M), 16 h.
Scheme 5. Synthesis of N-phenyl-N0-(2-hydroxyethyl)thioureas 30 and 2-(phenylamino)-4,5-dihydrothiazoles 19. Reagents and conditions: (i)
H2N(CH2)2OH, acetone, reflux, 2 h (30c,g); (ii) H2N(CH2)2OH, acetone, reflux, 4 h (30d,q,r); (iii) H2N(CH2)2OH, acetone, reflux, 1.5 h (30h); (iv) aq
HCl (6 M), reflux, 43 h (19aA); (v) CF3CO2H, 5 min (19aB); (vi) CF3CO2H, 2 h (19b); (vii) CF3CO2H, 3 h (19g); (viii) CF3CO2H, reflux, 15 h (19h);
(ix) aq HCl (6 M), reflux, 36 h (19o); (x) aq HCl (6 M), 16 h.

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4195
the enzymic reaction by addition of [14C]-arginine. This
pre-incubation has been reported to be optimum for the
inhibitory activity of 1400W 11, which is a slow-binding
selective inhibitor of iNOS;23 this pre-incubation was
investigated since many of the test compounds can be
considered to be analogues of 11 and may also be slow-
binding. We have also noted35 that an amino-acid-type
inhibitor, Ne-homothiocitrulline methyl ester requires at
least 5 min pre-incubation with rat nNOS to exert its
full inhibitory potency. IC50 values were determined
(with 15 min pre-incubation) for compounds 18a,b and
19a, which showed consistent activity in the general
screen. Compounds 16a,h, 17a, 18a and 19a were also
evaluated at 100 mM for their inhibition of rat iNOS,
without pre-incubation.
The results of the biological evaluation of the test com-
pounds are shown in Table 1. Pre-incubation of the
compounds with the rat nNOS preparation has little or
no effect on the inhibition of this isoform by most of the
compounds. Many of the compounds (16b,g,h,j,l–n,
17g,h, j,l–n, 18g,h,o,p, 19g,h,o,p, 24g,h and 30g,h) are
inactive or have only weak activity against this isoform.
Significant activity was shown by all the new com-
pounds carrying the aminomethyl group on the benzene
ring, consistent with the view of Collins et al.24 that this
group is optimal in the amidine series of inhibitors, for
example 10 and 11. Thus inhibitory activity was shown
by 16a and by 17a, the N-benzylthiourea and the 2-
benzylamino-4,5-dihydrothiazole most closely related to
1400W 11 with an aminomethyl group located meta on
the benzene ring. The corresponding para-substituted
analogues 16b and 17b, respectively, were less potent.
The N-(aminomethylphenyl)thioureas 18a,b and the 2-
(aminomethylphenylamino)-4,5-dihydrothiazoles 19a,b
were also active. Again, the meta substituted isomers
were more potent than the corresponding para com-
pounds, in that 18a had IC50=13 mM, whereas 18b had
IC50=41 mM, when assayed with 15-min pre-incu-
bation. Interestingly, 7-nitroindazole 15, which is
claimed to be selective for nNOS inhibition,30 showed
IC50=40 mM in this system, making compounds 18a
and 19a more potent than this lead compound in our
nNOS system and similar in potency to l-NMMA 4,
1400W 10 and thiocitrulline 12.
Scheme 6. Stereochemical outcome of acid-catalysed cyclisation to
form the 4,5-dihydrothiazole. Reagents and conditions: (i) R-1-amino-
propan-2-ol, acetone, reflux, 2 h; (ii) aq HCl (6 M), reflux, 24 h.
To investigate briefly the mechanism of the acid-cata-
lysed ring-closure of the 2-hydroxyethylthioureas, the
homochiral R-N-(2-hydroxypropyl)thiourea 31 was syn-
thesised by treatment of 3-methoxyphenylisothiocyanate
29g with R-1-aminopropan-2-ol (Scheme 6). Cyclisation
with boiling hydrochloric acid gave the corresponding 2-
(3-methoxyphenylamino)-5-methyl-4,5-dihydrothiazole
20
D
32, with specific rotation ½aŠ ¼ ꢂ32:4ꢀ. Since the race-
mate has not been formed, the cyclisation proceeds, at
least in part, by an SN2-like process.
Biological Evaluation
The N-benzylthioureas 16, the 2-benzylamino-4,5-dihy-
drothiazoles 17, the N-phenylthioureas 18, the 2-
phenylamino-4,5-dihydrothiazoles 19 and selected N-(2-
hydroxyethyl)thioureas 24 and 30 were evaluated for
their inhibition of the activities of the isoforms of NOS,
generally according to the method published pre-
viously.15 The known inhibitors l-NMMA 4, 1400W
10, thiocitrulline 12 and 7-nitroindazole 15 were also
subjected to the test system, for comparison. Inhibition
of the activity of nNOS was measured using an enzyme
preparation from rat brain (in which the large majority
of the NOS activity is nNOS), whereas most of the
assays of activity against iNOS were performed using a
preparation of recombinant human iNOS overexpressed
in an HT1080 cell line.35 Selected compounds were also
evaluated for inhibition of iNOS as a crude preparation
from the lungs of rats previously treated with lipopoly-
saccharide (LPS). The assays were based on the con-
version of [14C]-arginine to [14C]-citrulline. As a screen
for inhibitory activity, all compounds were tested at
100 mM concentration against human iNOS and rat
nNOS. Assays were performed in two modes, simulta-
neous addition of the test compound to the enzyme
preparation and of [14C]-arginine (to start the enzymic
reaction) and pre-incubation of the test compound with
the enzyme preparation for 10 min before initiation of
In contrast with the results for nNOS, pre-incubation of
the test compounds with the human iNOS enzyme pre-
paration had a profound effect on the inhibition. The
inhibition caused by 16g,h, 17a,g,h, 18g and 19g
appeared to decrease to a greater or lesser extent with
pre-incubation, although these compounds had only
moderate potency. In contrast, the inhibition of human
iNOS by the most potent compound, 19a, increased
with pre-incubation, suggesting that slow binding may
be involved. This effect was also observed with the
weaker inhibitor 19b. The most potent inhibitor of
human iNOS was the meta-aminomethylphenylamino-
4,5-dihydrothiazole 19a, with IC50=19 mM. Interest-
ingly, the corresponding thiourea 18a was much less
potent, with IC50=260 mM. Again, 19a, was more
potent in this system than 7-nitroindazole 15 but less
potent than the other ‘standard’ inhibitors 4, 10 and 12.

4196
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
Most striking, however, was the strong stimulation of
the activity of human iNOS by 16j,l,m,n, 17j,m, 18o, 19p
and 24g. In each case, pre-incubation abolished this sti-
mulation and, in some cases, led to weak inhibition.
Compounds 17l,n and 24h were weaker stimulators
(with no pre-incubation) but switched to become sig-
nificant inhibitors when pre-incubated with the enzyme
preparation for 10 min before initiation of the enzyme-
catalysed generation of nitric oxide. We have previously
noted15 the phenomenon of stimulation of rat iNOS
activity by S-2-amino-5-(3-nitrotriazol-1-yl)pentanoic
acid and by S-2-amino-5-(3-aminotriazol-1-yl)pentanoic
acid, although the effects were much weaker. A struc-
ture–activity tendency is evident for this stimulatory
effect. The most effective stimulators in the benzyl series
16, 17 carry either amines or carboxylic acids attached
directly to the benzene ring. In the phenyl series, only
18o and 19p are stimulatory; these carry CH2CO2H as
substituents on the benzene ring, giving the same dis-
tance between the carboxylate and the sulfur as in their
stimulatory isomers 16j and 16n, respectively. The
molecular origin of this stimulatory effect remains
unclear, although it is consistent with a model in which
there are two binding sites for these compounds, the
substrate (arginine)-binding site at the catalytic centre
and a (possibly remote) allosteric site. To rationalise the
data in terms of this model, the binding of the com-
pounds to the arginine site would be inhibitory (and
competitive with arginine) and binding to the allosteric
site would be stimulatory, possibly through inducing a
conformational change in the enzyme protein. To fit the
observed dependence on pre-incubation, binding to
the allosteric site would be fast (leading to stimula-
tion of nitric oxide synthesis without pre-incubation),
whereas inhibitory binding to the substrate-binding
site would be slow. Then the overall effect observed
after pre-incubation would be the sum of the stimu-
latory and inhibitory effects (leading to apparent
diminution of stimulation or switch to apparent inhi-
bition).
Table 1. Inhibition of human iNOS, rat iNOS and rat nNOS by the thioureas 16,18, the N-(2-hydroxyethyl)thioureas 24, 30, the 4,5-dihy-
drothiazoles 17, 19 and, for comparison, by the known inhibitors l-NMMA 4, 1400W 10, thiocitrulline 12 and 7-nitroindazole 15
Compd
% inhibition (human iNOS)a
10 min
% inhibition (rat iNOS)a
% inhibition (rat nNOS)a,b
No
IC50
(15 min
No
pre-incubationb
No
10 min
IC50 (15 min
pre-incubationb pre-incubationb
pre-incubationb pre-incubationb pre-incubation)b
pre-incubation)b
(mM)
(mM)
4
10
12
15
<4
<4
<5
24
94ꢃ1
96ꢃ1
9
12
17
40
79ꢃ1
82ꢃ1
77ꢃ2
34ꢃ1
68ꢃ2
44ꢃ2
32ꢃ4
7ꢃ3
59ꢃ1
39ꢃ5
74ꢃ3
47ꢃ1
20ꢃ2
6ꢃ4
16a
16b
16g
16h
16j
16l
16m
16n
17a
17b
17g
17h
17j
17l
17m
17n
18a
23ꢃ2
5ꢃ3
29ꢃ5
11ꢃ6
35ꢃ2
ꢂ3ꢃ1
8ꢃ1
26ꢃ6
2ꢃ5
14ꢃ1
6ꢃ1
ꢂ56ꢃ12
ꢂ58ꢃ1
ꢂ55ꢃ11
ꢂ46ꢃ5
48ꢃ1
3ꢃ1
ꢂ0.4ꢃ0.4
ꢂ1ꢃ6
ꢂ0.3ꢃ1
14ꢃ1
9ꢃ1
9ꢃ6
12ꢃ1
7ꢃ2
12ꢃ2
ꢂ0.4ꢃ2
59ꢃ1
65ꢃ2
14ꢃ1
13ꢃ1
ꢂ11ꢃ1
17ꢃ5
12ꢃ1
13ꢃ3
67ꢃ1
26ꢃ3
51ꢃ2
8ꢃ4
5ꢃ3
55ꢃ6
ꢂ30ꢃ2
34ꢃ4
33ꢃ3
8ꢃ1
26ꢃ6
3ꢃ13
5ꢃ1
10ꢃ2
ꢂ45ꢃ1
ꢂ36ꢃ1
ꢂ47ꢃ10
ꢂ39ꢃ1
35ꢃ4
0.4ꢃ4
5ꢃ2
12ꢃ2
19ꢃ2
34ꢃ3
40ꢃ5
8ꢃ2
5ꢃ3
260
89
98ꢃ1
98ꢃ3
13
41
(IC50=23 mM)
(IC50=10 mM)
48ꢃ4
18b
18g
18h
18o
18p
19a
57ꢃ1
17ꢃ6
52ꢃ1
2ꢃ1
44ꢃ1
5ꢃ1
ꢂ4ꢃ1
ꢂ1ꢃ3
9ꢃ6
26ꢃ7
28ꢃ1
ꢂ3ꢃ14
13ꢃ3
86ꢃ1
4ꢃ2
ꢂ65ꢃ5
15ꢃ2
5ꢃ2
ꢂ7ꢃ8
66ꢃ1
15ꢃ6
89ꢃ1
62ꢃ2
19
34ꢃ1
13
(IC50=190 mM)
(IC50=21 mM)
56ꢃ1
19b
19g
19h
19o
19p
24g
24h
30g
30h
16ꢃ4
26ꢃ2
40ꢃ1
ꢂ1ꢃ12
7ꢃ3
56ꢃ1
20ꢃ2
17ꢃ1
9ꢃ3
7ꢃ1
1ꢃ1
ꢂ2ꢃ1
4ꢃ2
ꢂ1ꢃ4
ꢂ58ꢃ4
ꢂ58ꢃ7
ꢂ25ꢃ1
ꢂ10ꢃ1
6ꢃ3
ꢂ8ꢃ14
9ꢃ2
9ꢃ2
14ꢃ5
5ꢃ1
8ꢃ1
ꢂ1ꢃ4
ꢂ1ꢃ1
0ꢃ0.6
2ꢃ1
44ꢃ5
ꢂ1ꢃ1
6ꢃ2
5ꢃ1
1ꢃ1
2ꢃ3
aConcentration of test compound 100 mM.
bPre-incubation refers to the time between addition of the test compound and addition of [14C]-arginine to initiate the enzymic reaction.

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4197
Examination of the activity of the most potent inhibi-
tors of the activities of the isoforms of NOS reveals
moderate selectivity. Interestingly, the claimed30 nNOS-
selective inhibitor 15 showed no selectivity for rat NOS
over human iNOS, although this may have been a spe-
cies effect. Whereas 2-(3-aminomethylphenylamino)-4,5-
dihydrothiazole 19a was the most potent inhibitor of
human iNOS (IC50=19 mM after 15 min pre-incu-
bation), it was similarly active against rat nNOS
(IC50=13 mM after 15 min pre-incubation); interest-
ingly, it was much less potent against rat iNOS
(IC50=190 mM with no pre-incubation) than against rat
nNOS (IC50=21 mM with no pre-incubation). A differ-
ent species effect was seen with the corresponding N-(3-
aminomethylphenyl)thiourea 18a. Here, little selectivity
was seen between inhibition of rat iNOS (IC50=23 mM
with no pre-incubation) and inhibition of rat nNOS
(IC50=10 mM with no pre-incubation) but the com-
pound was 20-fold selective for inhibition of rat nNOS
(IC50=13 mM with 15-min pre-incubation) over inhibi-
tion of human iNOS (IC50=260 mM with 15-min pre-
incubation). The para isomer 18b, however, showed
only 2-fold selectivity in the same (rat nNOS vs human
iNOS) comparison.
mechanistic origin of this stimulation, which may have
experimental or therapeutic applications, will be the
subject of intense further study.
Experimental
NMR data were recorded on either JEOL/Varian GX
270 or EX 400 spectrometers, using solutions in CDCl3,
unless otherwise stated. IR spectra were recorded on
samples as KBr discs, unless otherwise stated. Mass
Spectra were recorded using a VG Analytical Mass
Spectrometer in the FAB positive ion mode, unless
otherwise stated. Solutions in organic solvents were
dried with MgSO4 and solvents were evaporated under
reduced pressure. Experiments were conducted at
ambient temperature, unless otherwise stated. Melting
points were determined using a Reichert-Jung Thermo
Galen Kofler block.
N-(3-(Aminomethyl)phenylmethyl)thiourea bis(trifluoro-
acetate) salt (16a). Compound 16c (100 mg, 300 mmol)
was stirred in CF3CO2H (3 mL) for 5 min. Evaporation
gave 16a (140 mg, 99%) as a colourless hygroscopic
gum: IR (film) nmax 1172, 1780, 3200 cmꢂ1; NMR
((CD3)2SO) dH 4.00 (2H, m, CH2N+H3), 4.65 (2H, m,
CH2NH), 7.15 (3H, br, N+H3), 7.32 (4H, m, Ar-H4),
8.10 (4H, br, NH+N+H3); MS m/z 196.0905 (M+H)
(C9H14N3S requires 196.0908), 179 (MꢂNH3), 162
(Mꢂ2 ꢄ NH3).
Conclusions
In this paper, we have described our design of novel
inhibitors of the isoforms of NOS, replacing the ami-
dines in the highly isoform-selective inhibitors 1400W
11 and its lower homologue 12 with thiourea and 4,5-
dihydrothiazole units for ligation to the haem iron; this
replacement was based on the potent activity of thioci-
trulline 13 and of the analogous dihydrothiazole 14. The
effect of varying the nature and position of the sub-
stituents on the aryl ring were also examined. The
thioureas were synthesised in good yields by reaction of
the corresponding isothiocyanates with ammonia,
whereas the 2-(substituted amino)-4,5-dihydrothiazoles
were prepared by two routes, reaction of benzylamines
with 2-methylthio-4,5-dihydrothiazole under forcing
conditions or acid-catalysed cyclisation of N-(2-hydroxy-
ethyl)thioureas.
N-(4-(Aminomethyl)phenylmethyl)thiourea bis(trifluoro-
acetate) salt (16b). Compound 16d was treated with
CF3CO2H, as for the synthesis of 16a (reaction time
2 h), to give 16b (99%) as white crystals: mp 197–199 ꢀC;
IR nmax 1188, 3293 cmꢂ1; NMR ((CD3)2SO) dH 3.99
(4H, s, 2 ꢄ CH2), 4.64 (3H, br, NH3), 7.29 (1H, br,
NH), 7.30 (2H, d, J=8.0 Hz, Ar 3,5-H2), 7.37 (2H, d,
J=8.0 Hz, Ar 2,6-H2), 8.16 (3H, br, N+H3); MS m/z
196.0908 (M+H) (C9H14N3S requires 196.0920), 179
(MꢂNH3).
1,1-Dimethylethyl N-(3-(thioureidomethyl)phenylmethyl)-
carbamate (16c). NH3 was passed through 22c (400 mg,
1.5 mmol) in CH2Cl2 (40 mL) for 30 min. The mixture
was stirred for 3 h at 0 ꢀC. Evaporation and chromato-
graphy (EtOAc/hexane 4:1) gave 16c (300 mg, 72%) as
white crystals: mp 70–72 ꢀC; IR nmax 1164, 1608,
3308 cmꢂ1; NMR dH 1.40 (9H, s, But), 4.18 (2H, br,
CH2), 4.62 (2H, br, CH2), 5.30 (1H, br, NH), 6.01 (2H,
br, NH2), 7.21 (5H, m, Ar-H4+NH); MS m/z 296.1426
(M+H) (C14H22N3O2S requires 296.1433), 240
(MꢂMe2C¼CH2); Found C, 56.50; H, 7.11;
C14H21N3O2S requires C, 56.50; H, 7.09%.
The most potent inhibitory activity was seen where the
substituent was aminomethyl, the same substituent as in
11 and 12, although the position (meta or para) of this
substituent was of limited importance. The isoform-
selectivity for inhibition of iNOS and nNOS, where
observed, was generally limited. However, 18a showed
useful selectivity for inhibition of rat nNOS over human
iNOS. Most compounds showed modest inhibitory
potency but the activities of 18a and 19a were compar-
able to those of the widely used experimental inhibitor
thiocitrulline 13,15 of 815 and of our previous o-iso-
thiourea ornithine-based inhibitors, Nd-(imino(isopro-
pylthio))methyl)ornithine,26 Nd-(4,5-dihydro-1,3-thiazin-
2-yl)ornithine26 and Nd-(4,5-dihydro-1,3-thiazol-2-yl)or-
nithine 14.26 Most striking, however, is the stimulation of
the activity of human iNOS by the N-(aminobenzyl) and
N-(carboxybenzyl) thioureas and 2-(aminobenzylamino)
and 2-(carboxybenzyl) 4,5-dihydrothiazoles. The
1,1-Dimethylethyl N-(4-(thioureidomethyl)phenylmethyl)-
carbamate (16d). Compound 22d was treated with
NH3, as for the synthesis of 16c (chromatographic
eluant EtOAc), to give 16d (71%) as pale yellow crystals:
mp 104–106 ꢀC; IR nmax 1171, 1686, 3355 cmꢂ1; NMR
((CD3)2SO) dH 1.38 (9H, s, But), 4.08 (2H, d, J=6.0 Hz,
CH2), 4.57 (2H, br, CH2), 7.20 (6H, m, Ar-H4+NH2),
7.37 (1H, br, NH), 7.98 (1H, br, NH); MS m/z 591

4198
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
(2 M+H), 296.1423 (M+H) (C14H22N3O3S requires
296.1421), 249 (MꢂMe2C¼CH2); Found C, 56.6: H,
7.09; N, 13.90; C14H21N3O2S requires C, 56.72; H, 7.16;
N, 14.23%.
Methyl 4-(thioureidomethyl)benzoate (16k). Compound
22k was treated with NH3, as for the synthesis of 16h, to
give 16k (99%) as white crystals: mp 131–133 ꢀC; IR
nmax 1179, 1711, 3409 cmꢂ1; NMR ((CD3)2SO) dH 3.82
(3H, s, Me), 4.70 (2H, s, CH2), 7.18 (2H, br, NH2), 7.38
(2H, d, J=8.2 Hz, Ar 3,5-H2), 7.90 (2H, d, J=8.2 Hz,
Ar 2,6-H2), 8.06 (1H, br NH); MS m/z 225.0690
(M+H) (C10H13N2O2S requires 225.0698).
N-(3-Nitrophenylmethyl)thiourea (16e). Compound 22e
was treated with NH3, as for the synthesis of 16d, to
give 16e (68%) as yellow crystals: mp 143–145 ꢀC; IR nmax
1159, 1347, 1529, 3292 cmꢂ1; NMR dH 4.77 (2H, s, CH2),
7.20 (2H, br, NH2), 7.64 (1H, dd, J=8.2, 7.8 Hz, Ar 5-H),
7.74 (1H, d, J=7.8 Hz, Ar 4-H), 8.10 (1H, s, Ar 2-H), 8.11
(1H, d, J=8.2 Hz, Ar 6-H); MS m/z 212.0498 (M+H)
(C8H10N3O2S requires 212.0494), 196 (MꢂNH2).
N-(3-Aminophenylmethyl)thiourea (16l). Compound 16e
(90 mg, 0.4 mmol) was boiled under reflux with
.
SnCl2 2H2O (200 mg, 1.2 mmol) in EtOH (5 mL) for 1 h.
The solution was cooled to 0 ꢀC, basified with 5% aq
NaHCO3, extracted with EtOAc and washed with satu-
rated brine. Drying, evaporation and chromatography
(EtOAc/hexane 5:1) gave 16l (40 mg, 55%) as pale buff
N-(4-Nitrophenylmethyl)thiourea (16f). Compound 22f
was treated with NH3, as for the synthesis of 16d, to
give 16f (69%) as a colourless oil: (lit.36 mp 113.5–
crystals: mp 141–143 ꢀC; IR nmax 1166, 3289 cmꢂ1
;
115 ꢀC); IR (film) nmax 1159, 1344, 1563, 3213 cmꢂ1
;
NMR (CD3OD) dH 3.34 (2H, s, CH2), 4.25 (1H, br,
NH), 4.58 (2H, br, NH2), 6.62 (3H, m, Ar 2,4,6-H3),
6.68 (1H, br, NH), 7.05 (1H, t, J=7.8 Hz, Ar 5-H); MS
m/z 182.0757 (M+H) (C8H12N3S requires 182.0752).
NMR dH 4.82 (2H, br, CH2), 5.82 (2H, br, NH2), 7.26
(1H, s, NH), 7.50 (2H, d, J=8.6 Hz, Ar 2,6-H2), 8.21
(2H, d, J=8.6 Hz, Ar 3,5-H2); MS m/z 422 (2 M+H),
212.0490 (M+H) (C8H10N3O2S requires 212.0494), 196
(MꢂNH2).
N-(4-Aminophenylmethyl)thiourea (16m). Compound
16f was treated with SnCl2, as for the synthesis of 16l
(reaction time 3 h; chromatographic eluant EtOAc/
AcOH/ hexane (10:1:1), to give 16m (95%) as pale
N-(3-Methoxyphenylmethyl)thiourea (16g). Compound
22g was treated with NH3, as for the synthesis of 16d, to
give 16g (99%) as a colourless oil: (lit.37 mp 102 ꢀC); IR
(film) nmax 1046, 2835, 3273 cmꢂ1; NMR dH 3.80 (3H, s,
Me), 4.20 (1H, br, NH), 4.77 (2H, m, CH2), 5.83 (2H, s,
NH2), 6.86 (3H, m, Ar 2,4,6-H3), 7.27 (1H, m, Ar 5-H);
MS m/z 197.0751 (M+H) (C9H13N2OS requires
197.0749), 121 (MꢂNHCSNH2).
orange crystals: mp >270 ꢀC; IR nmax 1179, 3422 cmꢂ1
;
NMR (CD3OD) dH 4.54 (2H, s, CH2), 6.69 (2H, d,
J=8.2 Hz, Ar 3,5-H2), 7.06 (2H, d, J=8.2 Hz, Ar 2,6-
H2); MS m/z 182.0746 (M+H) (C8H12N3S requires
182.0752), 164 (MꢂNH3).
3-(Thioureidomethyl)benzoic acid hydrobromide (16n).
Compound 16i (80 mg, 360 mmol) was stirred in aq HBr
(50%, 5 mL) for 16 h. Evaporation gave 16n (70 mg,
98%) as a colourless hygroscopic gum: IR (film) nmax
1704, 3298 cmꢂ1; NMR (CD3OD) dH 3.89 (2H, s, CH2),
7.45 (1H, t, J=7.8 Hz, Ar 5-H), 7.57 (1H, d, J=7.8 Hz,
Ar 4-H), 7.91 (1H, d, J=7.8 Hz, Ar 6-H), 8.08 (1H, s,
Ar 2-H); MS m/z 225.0708 (M+H) (C10H13N2O2S
requires 225.0698).
N-(4-Methoxyphenylmethyl)thiourea (16h). Compound
22h was treated with NH3, as for the synthesis of 16c
[chromatographic eluant EtOAc/hexane (1:1)], to give
16h (99%) as white crystals: mp 124–126 ꢀC (lit.38 mp
135 ꢀC); IR nmax 1177, 2800, 3165 cmꢂ1; NMR dH
3.87 (3H, s, Me), 4.38 (2H, br, CH2), 5.75 (2H, br,
NH2), 6.88 (1H, br, NH), 6.90 (2H, d, J=8.4 Hz, Ar
3,5-H2), 7.23 (2H, d, J=8.4 Hz, Ar 2,6-H2); MS m/z
393 (2 M+H), 197.0749 (M+H) (C9H13N2OS
requires 197.0751); Found C, 54.80: H, 6.13; N,
14.14; C9H12N2OS requires C, 54.30; H, 6.11; N,
14.10%.
2-(3-(Aminomethyl)phenylmethylamino)-4,5-dihydrothi-
azole dihydrochloride (17a). Compound 24c (83 mg,
240 mmol) was boiled under reflux for 36 h in aq HCl
(6 M, 4 mL). Evaporation gave 17a (59 mg, 47%) as a
colourless hygroscopic gum: IR (film) nmax 1632,
3429 cmꢂ1; NMR dH 3.63 (2H, t, J=7.4 Hz, thiazole 5-
H2), 3.99 (2H, t, J=7.4 Hz, thiazole 4-H2), 4.06 (2H,
brq, J=5.9 Hz, CH2N+H3), 4.77 (2H, d, J=6.2 Hz,
ArCH2Nthiazole) 7.50 (4H, m, Ar-H4), 8.70 (3H, br,
N+H3), 10.89 (2H, s, 2 ꢄ NH); MS m/z 222.1067
(M+H) (C11H16N3S requires 222.1065).
Methyl 3-(thioureidomethyl)benzoate (16i). Compound
22i was treated with NH3, as for the synthesis of 16d, to
give 16i (99%) as pale yellow crystals: mp 123–125 ꢀC;
IR nmax 1202, 1710, 3418 cmꢂ1; NMR (CD3OD) dH 3.89
(3H, s, Me), 4.88 (2H, s, CH2), 7.44 (1H, t, J=7.8 Hz,
Ar 5-H), 7.56 (1H, d, J=7.8 Hz, Ar 4-H), 7.90 (1H, d,
J=7.8 Hz, Ar 6-H), 8.03 (1H, s, Ar 2-H); MS m/z 449
(2 M+H), 225.0708 (M+H) (C10H13N2O2S requires
225.0698), 211 (MꢂMe).
2-(4-(Aminomethyl)phenylmethylamino)-4,5-dihydrothi-
azole dihydrochloride (17b). Compound 24d (100 mg,
0.29 mmol) was boiled under reflux in aq HCl (6 M,
6 mL) for 36 h. Evaporation and recrystallisation
(PriOH) gave 17b (40 mg, 47%) as pale yellow crystals:
mp 198–200 ꢀC; IR nmax 1654, 3425 cmꢂ1; NMR
((CD3)2SO) dH 3.64 (2H, t, J=7.4 Hz, thiazole 5-H2),
4.04 (2H, m, thiazole 4-H2), 4.14 (2H, m, CH2NHthia-
zole), 4.61 (2H, s, CH2N+H3), 7.44 (2H, m, Ar 3,5-H2),
4-(Thioureidomethyl)benzoic acid hydrobromide (16j).
Compound 16k was treated with HBr, as for the synth-
esis of 16n, to give 16j (99%) as a colourless hygroscopic
gum: IR (film) nmax 1176, 1705, 3382 cmꢂ1; NMR
(CD3OD) dH 3.88 (2H, s, CH2), 7.41 (2H, d, J=8.3 Hz,
Ar 3,5-H2), 7.97 (2H, d, J=8.3 Hz, Ar 2,6-H2); MS m/z
211.0541 (M+H) (C9H11N2O2S requires 211.0547).

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4199
7.53 (2H, m, Ar 2,6-H2); MS m/z 443 (2 M+H),
222.1061 (M+H) (C11H16N3S requires 222.1065), 205
(MꢂNH3).
Methyl 3-((4,5-dihydrothiazol-2-ylamino)methyl)benzoate
hydrochloride (17i). Compound 24i (70 mg, 260 mmol)
was boiled under reflux for 40 h in aq HCl (6 M, 5 mL).
The evaporation residue was stirred with MeOH
(70 mL) and SOCl2 (0.5 mL) for 4 d. Evaporation gave
17i (50 mg, 67%) as a pale buff gum: IR (film) nmax
1640, 1718, 3398 cmꢂ1; NMR dH 3.51 (2H, br, CH2),
3.92 (3H, s, Me), 4.02 (2H, br, CH2), 4.54 (2H, s,
CH2Ar), 7.47 (1H, t, J=7.4 Hz, Ar-H5), 7.57 (1H, d,
J=7.4 Hz, Ar-H4), 7.95 (1H, s, Ar-H2), 7.99 (1H, d,
J=7.4 Hz, Ar-H4), 10.40 (1H, br, NH), 10.93 (1H, br,
NH): MS m/z 251.0859 (M+H) (C12H14N2O2S requires
251.0854).
2-(3-Nitrophenylmethylamino)-4,5-dihydrothiazole
tri-
fluoroacetate salt (17e). Compound 24e (120 mg,
470 mmol) was stirred for 16 h with CF3CO2H (5 mL).
Evaporation gave 17e (170 mg, 99%) as a colourless
hygroscopic gum: IR (film) nmax 1352, 1532, 1679,
3170 cmꢂ1; NMR dH 3.55 (2H, t, J=7.8 Hz, thiazole
5-H2), 4.05 (2H, t, J=7.8 Hz, thiazole 4-H2), 4.59
(2H, d, J=4.7 Hz, ArCH2), 7.61 (1H, dd, J=8.2,
7.8 Hz, Ar 5-H), 7.69 (1H, d, J=7.8 Hz, Ar 6-H),
8.17 (1H, s, Ar 2-H), 8.21 (1H, d, J=8.2 Hz, Ar 4-
H), 11.94 (1H, br, NH), 12.32 (1H, br NH); MS m/z
475 (2 M+H), 238.0640 (M+H) (C10H12N3O2S
requires 238.0650).
4-(4,5-Dihydrothiazol-2-ylaminomethyl)benzoic
acid
hydrochloride (17j). Compound 24k was treated with aq
HCl, as for the synthesis of 17a, to give 17j (99%) as
pale yellow crystals: mp 138–140 ꢀC; IR (film) nmax
1656, 1697, 3430 cmꢂ1; NMR ((CD3)2SO) dH 3.57 (2H,
t, J=7.4 Hz, thiazole 5-H2), 3.92 (2H, t, J=7.4 Hz,
thiazole 4-H2), 4.73 (2H, s, CH2Ar), 7.46 (2H, d,
J=8.2 Hz, Ar 3,5-H2), 7.95 (2H, d, J=8.2 Hz, Ar 2,6-
H2), 10.66 (1H, br, NH), 13.05 (1H, br, NH); MS m/z
237.0688 (M+H) (C11H13N2O2S requires 237.0698).
2-(4-Nitrophenylmethylamino)-4,5-dihydrothiazole
tri-
fluoroacetate salt (17f). Compound 24f (200 mg,
780 mmol) was boiled under reflux in CF3CO2H (5 mL)
for 15 h. Evaporation gave 17f (240 mg, 99%) as a col-
ourless hygroscopic gum: IR (film) nmax 1347, 1524,
1678, 3173 cmꢂ1; NMR dH 3.54 (2H, t, J=7.8 Hz, thia-
zole 5-H2), 4.04 (2H, t, J=7.8 Hz, thiazole 4-H2), 4.59
(2H, d, J=5.1 Hz, CH2Ar), 7.49 (2H, d, J=8.6 Hz, Ar
2,6-H2), 8.23 (2H, d, J=8.6 Hz, Ar 3,5-H2), MS m/z
238.0639 (M+H) (C10H12N3O2S requires 238.0650),
222 (MꢂNH2).
2-(3-Aminophenylmethylamino)-4,5-dihydrothiazole (17l).
Compound 17e was treated with SnCl2, as for the
synthesis of 16l (chromatography omitted), to give 17l
(150 mg, 99%) as a colourless oil: IR (film) nmax 1618;
3391 cmꢂ1; NMR (CD3OD) dH 3.30 (2H, m, thiazole 5-
H2), 3.90 (2H, m, thiazole 4-H2), 4.30 (2H, br,
CH2Ar), 6.62 (2H, m, Ar 4,6-H2), 6.67 (1H, s, Ar 2-
H), 7.04 (1H, t, J=7.4 Hz, Ar 5-H); MS m/z 208.0905
2 - (3 - Methoxyphenylmethylamino) - 4,5 - dihydrothiazole
(17g). Method A. Compound 24g (290 mg, 1.2 mmol)
was stirred in CF3CO2H (5 mL) for 2 h. Evaporation
and chromatography (EtOAc/MeOH 5:1) gave 17g
(200 mg, 75%) as white crystals: mp 94–96 ꢀC; IR (film)
nmax 1681, 3200 cmꢂ1; NMR dH 3.46 (2H, t, J=7.4 Hz,
thiazole 5-H2), 3.82 (3H, s, Me), 3.95 (2H, t, J=7.8 Hz,
thiazole 4-H2), 4.43 (2H, d, J=5.6 Hz, CH2Ar), 6.87
(3H, m, Ar 2,4,6-H3), 7.28 (1H, m, Ar 5-H), 12.25 (1H,
s, NH), 12.36 (1H, s, NH); NMR dC 31.1, 48.7, 51.3,
55.2, 112.9, 113.9, 119.6, 130.0, 136.5, 160.0, 174.6; MS
m/z 223.0890 (M+H) (C11H15N2OS requires 223.0905).
(M+H)
(C10H14N3S
requires
208.0908),
196
(Mꢂaminodihydrothiazole).
2-(4-Aminophenylmethylamino)-4,5-dihydrothiazole (17m).
Compound 17f was treated with SnCl2, as for the
synthesis of 17l (reaction time 1.5 h), to give 17m (38%)
as a colourless oil: IR (film) nmax 1609, 3324 cmꢂ1
;
NMR dH 3.33 (2H, t, J=7.4 Hz, thiazole 5-H2), 3.64
(2H, br, NH2), 4.02 (2H, t, J=7.4 Hz, thiazole 4-H2),
6.64 (2H, d, J=8.6 Hz, Ar 2,6-H2), 7.11 (2H, d,
J=8.6 Hz, Ar 3,5-H2); NMR 35.7, 49.2), 60.4, 115.4,
129.0, 129.3, 146.0, 161.9; MS m/z 208.0911 (M+H)
(C10H14N3S requires 208.0908).
2-(3-Methoxyphenylmethyl)-4,5-dihydrothiazole
(17g).
Method B. 3-Methoxybenzylamine 21g was heated
with 2-methylthio-4,5-dihydrothiazole 26 (320 mg,
2.4 mmol) at 180 ꢀC for 4 h. Evaporation and chroma-
tography (EtOAc/ MeOH 5:1) gave 17g (163 mg, 30%)
with properties as above.
3-(4,5-Dihydrothiazol-2-ylaminomethyl)benzoic acid tri-
fluoroacetate salt (17n). Compound 17i (70 mg,
280 mmol) was stirred under reflux in aq CF3CO2H
(50%, 5 mL) for 16 h. Evaporation gave 17n (70 mg,
99%) as a colourless hygroscopic gum: IR nmax 1645,
1696, 3433 cmꢂ1; NMR (CD3OD) dH 3.65 (2H, t,
J=7.8 Hz, thiazole 5-H2), 4.04 (2H, t, J=7.8 Hz, thia-
zole 4-H2), 4.60 (2H, m, CH2Ar), 7.54 (3H, m, Ar-H3),
8.02 (1H, m, Ar-H); MS m/z 237.0698 (M+H)
(C11H13N2O2S requires 237.0698).
2 - (4 - Methoxyphenylmethylamino) - 4,5 - dihydrothiazole
(17h). 4-Methoxybenzylamine 21h was treated with 26,
as for the synthesis of 17g (Method B) [chromato-
graphic eluant CH2Cl2/MeOH (10:1)] to give 17h (30%)
as white crystals: mp 77–79 ꢀC (lit.33 mp 84–85 ꢀC);
NMR dH 3.38 (2H, t, J=7.4 Hz, thiazole 5-H2), 3.80
(3H, s, Me), 4.04 (2H, t, J=7.4 Hz, thiazole 4-H2), 4.40
(2H, s, ArCH2), 6.87 (2H, d, J=8.8 Hz, Ar 3,5-H2), 7.25
(2H, d, J=8.8 Hz, Ar 2,6-H2); IR nmax 1610, 2780,
3207 cmꢂ1; MS m/z 223.0912 (M+H) (C11H15N2OS
requires 223.0905); Found C, 58.25: H, 6.44; N, 12.35:
C11H14N2OS. 0.25H2O requires C, 58.29; H, 6.40; N,
12.36%.
N-(3-Aminomethylphenyl)thiourea bis(trifluoroacetate)
salt (18a). Compound 18c was treated with CF3CO2H,
as for the synthesis of 16a, to give 18a (99%) as a col-
ourless hygroscopic gum: IR (film) nmax 1170,
3407 cmꢂ1; NMR ((CD3)2SO) dH 4.02 (2H, brq,

4200
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
J=5.9 Hz, CH2), 7.09 (3H, br, N+H3), 7.20 (1H, m, Ar
4-H), 7.40 (2H, m, Ar 5,6-H2), 7.50 (1H, s, Ar 2-H), 8.16
(3H, br, N+H3), 9.87 (1H, s, NH); NMR ((CD3)2SO) dC
42.3, 123.4, 123.5, 124.8, 129.1, 134.5, 139.5, 181.1; MS
m/z 182.0768 (M+H) (C8H12N3S requires 182.0752),
164 (MꢂNH3).
3-Thioureidophenylacetic acid hydrochloride (18o). Com-
pound 18q (50 mg, 220 mmol) was stirred in aq HCl
(1 M, 3 mL) for 9 d. Evaporation gave 18o (40 mg, 86%)
as white crystals: mp 159–161 ꢀC (lit.41 mp 174–176 ꢀC);
IR nmax 1157, 1730, 2500, 3337 cmꢂ1
;
NMR
(CD3OD)&#32;dH 3.67 (2H, s, CH2), 7.15 (1H, d,
J=7.4 Hz, Ar 4-H), 7.23 (1H, d, J=8.6 Hz, Ar 6-H),
7.25 (1H, s, Ar 2-H), 7.35 (1H, dd, J=8.6, 7.4 Hz, Ar 5-
H); MS m/z 211.0541 (M+H) (C9H11N2O2S requires
211.0531).
N-(4-Aminomethylphenyl)thiourea bis(trifluoroacetate)
salt (18b). Compound 18d was treated with CF3CO2H,
as for the synthesis of 16a (reaction time 2 h), to give
18b (99%) as a colourless hygroscopic gum: IR (film)
n
max 1173, 3369 cmꢂ1; NMR ((CD3)2SO) dH 3.96 (2H, q,
4-Thioureidophenylacetic acid hydrochloride (18p). Com-
pound 18r (50 mg, 220 mmol) was stirred for 16 h in aq
HCl (6 M, 5 mL). Evaporation gave 18p (60 mg, 99%) as
white crystals: mp 198–200 ꢀC (lit.40 mp 200–203 ꢀC); IR
nmax 1181, 1699, 3313 cmꢂ1; NMR (CD3OD) dH 3.65
(2H, s, CH2), 7.26–7.32 (4H, m, Ar-H4); MS m/z
211.0541 (M+H) (C9H11N2O2S requires 211.0531).
J=5.6 Hz, CH2), 7.38 (2H, d, J=8.2 Hz, Ar 2,6-H2),
7.51 (2H, d, J=8.2 Hz, Ar 3,5-H2), 7.51 (3H, br,
N+H3), 8.11 (3H, br, N+H3), 9.83 (1H, s, NH); MS m/z
182.0748 (M+H) (C8H12N3S requires 182.0752), 164
(MꢂNH3).
1,1-Dimethylethyl N-(3-thioureidophenylmethyl)carbamate
(18c). Compound 29c was treated with NH3, as for the
synthesis of 16c, to give 18c (38%) as pale yellow crys-
tals: mp 198–200 ꢀC; IR nmax 1173, 3291 cmꢂ1, NMR dH
1.37 (9H, s, But), 4.01 (2H, d, J=7.4 Hz, CH2), 6.96
(1H, d, J=7.4 Hz, NH), 7.23 (1H, d, J=7.8 Hz, Ar-H),
7.16–7.29 (5H, m, Ar-H3+NH2), 9.78 (1H, s, NH); MS
m/z 282.1295 (M+H) (C13H20N3O2S requires
282.1276), 226 (MꢂMe2C¼CH2).
Methyl 3-thioureidophenylacetate (18q). Compound 29q
was treated with NH3, as for the synthesis of 16h, to
give 18q (99%) as pale yellow crystals: mp 131–133 ꢀC;
IR nmax 1160, 1730, 3409 cmꢂ1; NMR ((CD3)2SO) dH
3.61 (3H, s, Me), 3.65 (2H, s, CH2), 7.00 (1H, d,
J=7.4 Hz, Ar 4-H), 7.23 (1H, dd, J=8.6, 7.4 Hz, Ar 5-
H), 7.25 (1H, s, Ar 2-H), 7.33 (1H, d, J=8.6 Hz, Ar 6-
H), 7.35 (1H, br, NH), 9.71 (1H, s, NH); NMR
((CD3)2SO)C 40.1, 51.8, 121.6, 123.8, 125.4, 128.7,
134.9, 139.2, 171.5, 181.1; MS m/z 225.0694 (M+H)
(C10H13N2O2S) requires 225.0698).
1,1-Dimethylethyl N-(4-thioureidophenylmethyl)carbamate
(18d). Compound 29d was treated with NH3, as for the
synthesis of 16c, to give 18d (66%) as white crystals: mp
89–91 ꢀC; IR nmax 1187, 1690, 3298 cmꢂ1; NMR dH 1.46
(9H, s, But), 4.30 (2H, d, J=6.0 Hz, CH2), 5.03 (1H, br,
NH), 6.25 (2H, br, NH2), 7.19 (2H, d, J=8.2 Hz, Ar
3,5-H2), 7.33 (2H, d, J=8.2 Hz, Ar 2,6-H2), 8.39 (1H,
br, NH); MS m/z 282.1276 (M+H) (C13H20N3O2S
requires 282.1276), 226 (MꢂMe2C¼CH2); Found C,
54.40: H, 6.73; N, 14.20: C13H19N3O2S 0.5H2O requires
C, 53.87; H, 6.78; N, 14.49%.
Methyl 4-thioureidophenylacetate (18r). Compound 29r
was treated with NH3, as for the synthesis of 16d, to
give 18r (93%) as a white powder: mp 121–123 ꢀC; IR
nmax 1718, 3168, 3341 cmꢂ1; NMR ((CD3)2SO) dH 3.59
(3H, s, Me), 3.62 (2H, s, CH2), 7.18 (2H, d, J=8.2 Hz,
Ar 3,5-H2), 7.31 (2H, d, J=8.2 Hz, Ar 2,6-H2), 7.43
(2H, br, NH2), 9.65 (1H, s, NH); MS m/z 225.0687
(M+H) (C10H13N2O2S requires 225.0698).
N-(3-Methoxyphenyl)thiourea (18g). 3-Methoxyphenyl-
isothiocyanate 29g was treated with NH3, as for the
synthesis of 16c (chromatography omitted), to give 18g
(99%) as white crystals: mp 160–162 ꢀC (lit.39 mp
160 ꢀC); IR nmax 1166, 3149 cmꢂ1; NMR dH 3.73 (3H, s,
Me), 6.67 (1H, d, J=7.6 Hz, Ar 4-H), 6.90 (1H, d,
J=7.6 Hz, Ar 6-H), 7.10 (1H, s, Ar 2-H), 7.23 (1H, t,
J=7.6 Hz, Ar 5-H), 7.53 (2H, br, NH2) 9.62 (1H, s,
NH); NMR ((CD3)2SO) dC 55.1, 108.6, 110.0, 115.0,
129.6, 140.3, 159.3, 180.9; MS m/z 365 (2 M+H),
183.0598 (M+H) (C8H11N2OS requires 183.0592);
Found C, 52.50: H, 5.53; N, 15.40; C8H10N2OS requires
C, 52.72; H, 5.53; N, 15.37%.
2-(3-(Aminomethyl)phenylamino)-4,5-dihydrothiazole di-
hydrochloride (19aA). Compound 30c was treated with
HCl, as for the synthesis of 17a (reaction time 43 h), to
give 19aA (99%) as pale buff crystals: mp 208–210 ꢀC;
IR nmax 1640, 3432 cmꢂ1; NMR ((CD3)2SO) dH 3.65
(2H, t, J=7.7 Hz, thiazole 5-H2), 4.01 (2H, t, J=7.7 Hz,
thiazole 4-H2), 4.11 (2H, m, ArCH2), 7.39 (1H, d,
J=7.5 Hz, Ar 4-H), 7.56 (2H, m, Ar 5,6-H2), 7.62 (1H, s,
Ar 2-H), 8.66 (3H, s, N+H3), 10.5 (1H, br, NH), 12.7 (1H,
br, NH); MS m/z 208.0918 (M+H) (C10H14N3S requires
208.0908); Found C, 37.91: H, 6.06; N, 13.29; C10H13N3S
2H2O 2HCl requires C, 38.50; H, 5.67; N, 13.00%.
2-(3-(Aminomethylphenylamino)-4,5-dihydrothiazole bis
(trifluoroacetate) salt (19aB). Compound 30c was trea-
ted with CF3CO2H, as for the synthesis of 16a, to give
19aB (99%) as a colourless hygroscopic gum: IR (film)
N-(4-Methoxyphenyl)thiourea (18h). 4-Methoxyphenyl-
isothiocyanate 29h was treated with NH3, as for the
synthesis of 18g, to give 18h (99%) as white crystals:
mp 198–200 ꢀC (lit.40 mp 210 ꢀC); IR nmax 1171, 2838,
3154 cmꢂ1; NMR dH 3.71 (3H, s, Me), 6.89 (2H, d,
J=8.8 Hz, Ar 3,5-H2), 7.21 (2H, d, J=8.8 Hz, Ar
2,6-H2), 7.23 (2H, br, NH2), 9.47 (1H, s, NH); MS
n
max 1674, 3398 cmꢂ1; NMR ((CD3)2SO) dH 3.62 (2H, t,
J=7.7 Hz, thiazole 5-H2), 4.0 (4H, m, thiazole 4-
H2+ArCH2), 7.30 (4H, m, Ar-H4), 7.86 (1H, br, NH),
8.18 (3H, m, N+H3), 9.73 (1H, br, NH); MS m/z
208.0915 (M+H) (C10H14N3S requires 208.0908), 191
(MꢂNH3).
m/z
183.0592).
183.0592
(M+H)
(C8H10N2OS
requires

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4201
2 - (4 - (Aminomethyl)phenylamino) - 4,5 - dihydrothiazole
bis(trifluoroacetate) salt (19b). Compound 30d was trea-
ted with CF3CO2H, as for the synthesis of 16a (reaction
time 2 h), to give 19b (99%) as a colourless hygroscopic
gum: IR (film) nmax 1643, 3400 cmꢂ1; NMR ((CD3)2SO)
dH 3.60 (2H, t, J=7.4 Hz, thiazole 5-H2), 4.00 (2H, t,
J=7.4 Hz, thiazole 4-H2), 4.03 (2H, q, J=6.0 Hz
CH2N+H3), 4.59 (1H, br, NH), 7.37 (2H, d, J=8.2 Hz,
Ar 2,6-H2), 7.54 (2H, d, J=8.2 Hz, Ar 5,6-H2), 8.26
(3H, br, NH3); MS m/z 208.0914 (M+H) (C10H14N3S
requires 208.0908), 191 (MꢂNH3).
1,1-Dimethylethyl N-(3-(aminomethyl)phenylmethyl)car-
bamate (21c). Di(t-butyl) dicarbonate (1.0 g, 4.9 mmol)
was added slowly to 1,3-bis(aminomethyl)benzene 21a
(2.0 g, 15 mmol) and Et3N (2.9 g, 29 mmol) in CH2Cl2
(15 mL) at 0 ꢀC and the mixture was stirred for 16 h. The
evaporation residue, in CH2Cl2, was washed with aq
NaHCO3 and dried. Evaporation gave 21c (900 mg,
78%) as white crystals: mp 61–64 ꢀC (lit.43 oil); NMR
dH 1.51 (9H, s, But), 1.67 (2H, s, NH2), 3.90 (2H, d,
J=4.3 Hz, CH2NH2), 4.34 (2H, s, CH2NHBoc), 5.10
(1H, br, NH), 7.25 (4H, m, Ar-H4); MS m/z 237
(M+H), 181 (MꢂMe2C¼CH2), 164 (MꢂButO).
2-(3-Methoxyphenylamino)-4,5-dihydrothiazole
(19g).
Compound 30g (200 mg, 890 mmol) was stirred in
CF3CO2H (5 mL) for 3 h. Evaporation and chromato-
graphy (EtOAc) gave 19g (130 mg, 70%) as pale buff
1,1-Dimethylethyl N-(4-(aminomethyl)phenylmethyl)car-
bamate (21d). 1,4-Bis(aminomethyl)benzene 21b was
treated with Boc2O, as for the synthesis of 21c, to give
21d (930 mg, 80%) as a colourless oil: (lit.44 solid) NMR
dH 1.46 (9H, s, But), 1.52 (2H, br, NH2), 3.85 (2H, s,
CH2NH2), 4.29 (2H, d, J=6.0 Hz, CH2NHBoc), 5.89
(1H, br, NH), 7.24–7.28 (4H, m, Ar-H4); MS m/z 237
(M+H), 181 (MꢂMe2C¼CH2), 164 (MꢂButO).
crystals: mp 80–82 ꢀC; IR nmax 1674, 2850, 3238 cmꢂ1
;
NMR ((CD3)2SO) dH 4.17 (3H, s, Me), 3.92 (2H, t,
J=7.6 Hz, thiazole 5-H2), 4.35 (2H, t, J=7.6 Hz, thia-
zole 4-H2) 7.24 (1H, d, J=8.2 Hz, Ar 4-H), 7.34 (1H, d,
J=8.2 Hz, Ar 6-H), 7.41 (1H, s, NH), 7.73 (1H, t,
J=8.2 Hz, Ar 5-H), 7.74 (1H, s, Ar 2-H); MS m/z
209.0749 (M+H) (C10H13N2OS requires 209.0743).
Methyl 3-(aminomethyl)benzoate (21i) and di(3-methoxy-
carbonylphenylmethyl)amine (23). Compound 20i
(900 mg, 5.6 mmol) in MeOH (30 mL) was treated with
Pd/C (10%) and H2 for 16 h. Filtration (Celite#), eva-
poration and chromatography (EtOAc) gave 23
(150 mg, 9%) as a colourless oil: (lit.45 oil); IR (film)
nmax 1721, 3336 cmꢂ1; NMR dH 3.84 (4H, s, 2 ꢄ CH2),
3.91 (6H, s, 2 ꢄ Me), 7.40 (2H, t, J=7.8 Hz, 2 ꢄ Ar 5-
H), 7.52 (2H, d, J=7.8 Hz, 2 ꢄ Ar 4-H), 7.93 (2H, d,
J=7.8 Hz, 2 ꢄ Ar 6-H), 8.02 (2H, s, 2 ꢄ Ar 2-H); MS
m/z 314 (M+H). Further elution gave 21i (330 mg,
36%) as white crystals: mp 37–39 ꢀC (lit.45 oil); IR (film)
nmax 1719, 3453 cmꢂ1; NMR dH 3.92 (3H, s, Me), 3.95
(2H, s, CH2), 7.40 (1H, t, J=7.8 Hz, Ar 5-H), 7.53 (1H,
d, J=7.8 Hz, Ar 4-H), 7.92 (1H, d, J=7.8 Hz, Ar 6-H),
8.01 (1H, s, Ar 2-H); MS m/z 166 (M+H), 121
(MꢂNH2).
2-(4-Methoxyphenylamino)-4,5-dihydrothiazole trifluoro-
acetate salt (19h). Compound 30h (200 mg, 880 mmol)
was boiled under reflux in CF3CO2H (5 mL) for 15 h.
Evaporation gave 19h (220 mg, 99%) as white crystals:
mp 101–103 ꢀC: IR nmax 1674, 2750, 3409 cmꢂ1; NMR
((CD3)2SO) dH 2.29 (3H, s, Me), 3.60 (2H, m, CH2) 3.75
(2H, m, CH2), 7.02 (2H, d, J=6.9 Hz, Ar 3,5-H2), 7.25
(2H, d, J=6.9 Hz, Ar 2,6-H2), 7.97 (1H, br, NH); MS
m/z 209.0758 (M+H) (C10H13N2OS requires 209.0749).
3-(4,5-Dihydrothiazol-2-ylamino)phenylacetic acid hydro-
chloride (19o). Compound 30q was treated with aq HCl,
as for the synthesis of 17a, to give 19o (99%) as a col-
ourless hygroscopic gum: IR (film) nmax 1633, 1714,
3450 cmꢂ1; NMR ((CD3)2SO) dH 3.55 (2H, t, J=7.6 Hz,
thiazole 5-H2), 3.93 (2H, t, J=7.6 Hz, thiazole 4-H2),
5.75 (2H, s, CH2Ar), 7.25 (3H, m, Ar-H3), 7.44 (2H, m,
Ar-H+NH); NMR ((CD3)2SO) dC 31.0, 40.5, 48.7,
122.0, 124.3, 129.1, 129.8, 135.8 136.8, 171.2, 173.6; MS
m/z 237.0698 (M+H) (C11H13N2O2S requires
237.0698).
Methyl 4-(aminomethyl)benzoate hydrochloride (21k). 4-
Aminomethylbenzoic acid 21j was treated with MeOH
and SOCl2, as for the synthesis of 28q, to give 21k (2.2 g,
99%) as white crystals: mp 153–155 ꢀC (lit.46 mp 235–
238 ꢀC); NMR ((CD3)2SO) dH 3.84 (3H, s, Me), 4.08
(2H, s, CH2), 7.64 (2H, d, J=8.2 Hz, Ar 3,5-H2), 7.96
(2H, d, J=8.2 Hz, Ar 2,6-H2), 8.62 (3H, br, NH3); MS
m/z 166 (M+H), 150 (MꢂNH2).
4-(4,5-Dihydrothiazol-2-ylamino)phenylacetic acid hydro-
chloride (19p). Compound 30r was treated with aq HCl,
as for the synthesis of 18p, to give 19p (99%) as a col-
ourless hygroscopic gum: IR (film) nmax 1633, 1736,
3423 cmꢂ1: NMR (CD3OD) dH 3.67 (4H, s, thiazole 5-
CH2, ArCH2), 4.04 (2H, s, thiazole 4-CH2), 7.30 (2H, d,
J=8.2 Hz, Ar 3,5-H2), 7.43 (2H, d, J=8.2 Hz, Ar 2,6-
H2); MS m/z 237.0697 (M+H) (C11H13N2O2S requires
237.0698).
1,1-Dimethylethyl N-(3-(isothiocyanatomethyl)phenylme-
thyl)carbamate (22c). Compound 21c (900 mg,
3.9 mmol), CaCO3 (400 mg, 4.0 mmol), water (3 mL),
thiophosgene (900 mg, 7.8 mmol) and CHCl3 (25 mL)
were stirred vigorously for 16 h. The mixture was
extracted with CHCl3. Drying, evaporation and chro-
matography (EtOAc/hexane 1:3) gave 22c (400 mg,
37%) as a colourless oil (lit.44 mp 43 ꢀC); IR (film) nmax
1670, 2060, 3353 cmꢂ1; NMR dH 1.46 (9H, s, But), 4.33
(2H, d, J=5.6 Hz, CH2NHBoc), 4.70 (2H, s, CH2NCS),
4.91 (1H, br, NH), 7.22–7.27 (3H, m, Ar 2,4,6-H3), 7.34
(1H, t, J=7.8 Hz, Ar 6-H); MS m/z 279.1163 (M+H)
Methyl 3-cyanobenzoate (20i). 3-Cyanobenzoic acid 20n
was treated with MeOH and SOCl2, as for the synthesis
of 28q, to give 20i (42%) as white crystals: mp 38–40 ꢀC
(lit.42 mp 64–65 ꢀC); IR nmax 1720, 2228 cmꢂ1; NMR dH
3.96 (3H, s, Me), 7.95 (1H, t, J=7.8 Hz, Ar 5-H), 7.84 (1H,
d, J=7.8 Hz, Ar 4-H), 8.27 (1H, d, J=7.8 Hz, Ar 6-H),
8.34 (1H, s, Ar 2-H); MS m/z 162 (M+H), 147 (MꢂMe).
(C14H19N2O2S
requires
279.1167),
223
(MꢂMe2C¼CH2), 179 (MꢂBoc), 164 (MꢂBocNH).

4202
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
1,1-Dimethylethyl N-(4-isothiocyanatomethyl)phenylme-
thyl)carbamate (22d). Compound 21d was treated with
thiophosgene, as for the synthesis of 22c, to give 22d
(860 mg, 81%) as pale yellow crystals: mp 80–82 ꢀC
(lit.44 mp 74 ꢀC); IR nmax 1682, 2091, 3358 cmꢂ1; NMR
dH 1.46 (9H, s, But), 4.33 (2H, m, CH2NHBoc), 4.70 (2H,
s, CH2), 4.88 (1H, br, NH), 7.24–7.35 (4H, m, Ar-H4);
MS m/z 279 (M+H), 164 (MꢂButO).
(210 mg, 1.0 mmol) in acetone (1 mL) was added drop-
wise during 30 min to 2-aminoethanol (60 mg, 1.0 mmol)
in acetone (1 mL). The mixture was boiled under reflux
for 4 h. Evaporation and chromatography (EtOAc/hex-
ane 1:1) gave 24c (90 mg, 30%) as a colourless oil: IR
(film) nmax 1170, 1642, 3413 cmꢂ1; NMR ((CD3)2SO) dH
1.45 (9H, s, But), 1.75 (1H, s, NHBoc), 3.75 (2H, m,
CH2CH2O), 4.01 (2H, t, J=6.2 Hz, CH2O), 4.26 (2H,
m, ArCH2thiourea), 4.85 (2H, d, J=4.7 Hz, ArCH2-
NBoc), 5.4 (1H, br, NH or OH), 7.16–7.33 (4H, m, Ar-
H4); MS m/z 340.1695 (M+H) (C16H26N3O3S requires
340.1697), 284 (MꢂMe2C¼CH2), 179, 164.
3-Nitrophenylmethylisothiocyanate (22e). 3-Nitrobenzyl-
amine 21e was treated with thiophosgene, as for the
synthesis of 22c (chromatographic eluant ꢀEtOAc), to
give 22e (61%) as yellow crystals: mp 65–67 C (lit.47 mp
65–67 ꢀC); IR nmax 1347, 1526, 2135 cmꢂ1; NMR dH
4.82 (2H, s, CH2), 7.62 (1H, t, J=7.8 Hz, Ar 5-H), 7.69
(1H, d, J=7.8, Hz, Ar 4-H), 8.20 (1H, s, Ar 2-H), 8.21
(1H, d, J=7.8 Hz, Ar 6-H); MS m/z 193 (MꢂH).
1,1-Dimethylethyl N-(4-(N-(2-hydroxyethyl)thioureido-
methyl)phenylmethyl)carbamate (24d). Compound 22d
was treated with 2-aminoethanol, as for the synthesis of
24c (chromatography omitted), to give 24d (41%) as an
oil: NMR dH 1.44 (9H, s, But), 3.69 (4H, m, 2 ꢄ CH2),
4.24 (2H, d, J=5.4 Hz, ArCH2), 4.67 (2H, s, ArCH2),
5.04 (1H, s, OH), 6.88 (1H, br, NH), 7.18 (1H, br, NH),
7.20 (2H, d, J=7.0 Hz, Ar 3,5-H2), 7.26 (3H, m, Ar 2,6-
H2+NH); IR (film) nmax 1171, 1682, 3297 cmꢂ1; MS m/
z 340.1695 (M+H) (C16H26N3O3S requires 340.1684),
679 (2M+H), 284 (MꢂMe2C¼CH2).
4-Nitrophenylmethylisothiocyanate (22f). 4-Nitrobenzy-
lamine 21f was treated with thiophosgene, as for the
synthesis of 22c (chromatography omitted), to give 22f
(56%) as a colourless oil: (lit.36 mp 37–38 ꢀC); IR (film)
n
CH2), 7.52 (2H, d, J=8.9 Hz, Ar 2,6-H2), 8.25 (2H, d,
J=8.9 Hz, Ar 3,5-H2); MS m/z 195 (M+H).
max 1347, 1531, 2070, 3079 cmꢂ1; NMR dH 4.88 (2H, s,
N-(2-Hydroxyethyl)-N0 -(3-nitrophenylmethyl)thiourea
(24e) and 2,2-dimethyl-3-(N-(3-nitrophenylmethyl)ami-
nothiocarbonyl)tetrahydrooxazole (25e). Compound 22e
was treated with 2-aminoethanol, as for the synthesis of
24c (chromatographic eluant EtOAc), to give 25e
(50 mg, 19%) as a colourless oil: IR (film) nmax 1144,
1346, 1534, 3413 cmꢂ1; NMR dH 1.80 (6H, s, 2 ꢄ Me),
3.82 (2H, br, oxazole 4-H2), 4.05 (4H, t, J=6.6 Hz,
oxazole 5-H2), 5.02 (2H, d, J=5.4 Hz, CH2Ar), 5.70
(1H, br, NH), 7.55 (1H, dd, J=8.2, 7.8 Hz, Ar 5-H),
7.72 (1H, d, J=7.8 Hz, Ar 4-H), 8.15 (1H, d, J=8.2 Hz,
Ar 6-H), 8.16 (1H, s, Ar 2-H); MS m/z 296.1066
(M+H) (C13H18N3O3S requires 296.1069). Further elu-
tion gave 24e (180 mg, 70%) as pale buff crystals: mp
79–81 ꢀC; IR nmax 1144, 1348, 1531, 3402 cmꢂ1; NMR
3-Methoxyphenylmethylisothiocyanate (22g). 3-Methoxy-
benzylamine 21g was treated with thiophosgene, as for
the synthesis of 22c (chromatographic eluant EtOAc/
hexane (1:1)), to give 22g (40%) as a colourless oil:
(lit.37 oil): IR (film) nmax 2094 cmꢂ1; NMR dH 3.82
(3H, s, Me), 4.68 (2H, s, CH2), 6.87 (3H, m, Ar 2,4,6-
H3), 7.30 (1H, t, J=7.8 Hz, Ar 5-H); MS m/z 180.0460
(M+H)
(MꢂNCS).
(C9H10NOS
requires
180.0483),
121
4-Methoxyphenylmethylisothiocyanate (22h). 4-Methoxy-
benzylamine 18h was treated with thiophosgene, as for
the synthesis of 22g, to give 22h (80%) as a pale yellow
oil: (lit.48 oil); IR (film) nmax 2087 cmꢂ1; NMR dH 3.81
(3H, s, Me), 4.63 (2H, s, CH2), 6.93 (2H, d, J=8.2 Hz,
Ar 3,5-H2), 7.22 (2H, d, J=8.2 Hz, Ar 2,6-H2); MS m/z
179 (M), 121 (MꢂNCS).
d
H 3.61 (2H, br, NCH2), 3.82 (2H, t, J=4.7 Hz, OCH2),
4.89 (2H, d, J=5.4 Hz, ArCH2), 6.78 (1H, br, OH), 7.28
(1H, br, NH), 7.50 (1H, t, J=7.4 Hz, Ar 5-H), 7.77 (1H,
d, J=7.4 Hz, Ar 4-H), 8.11 (1H, d, J=7.4 Hz, Ar 6-H),
8.16 (1H, s, Ar 2-H); MS m/z 256.0753 (M+H)
(C10H14N3O3S requires 256.0756).
Methyl 4-(isothiocyanatomethyl)benzoate (22k). Com-
pound 21k was treated with thiophosgene, as for the
synthesis of 22f, to give 22k (83%) as a pale yellow oil:
(lit.49 oil); IR (film) nmax 1715, 2089 cmꢂ1; NMR
((CD3)2SO) dH 3.88 (3H, s, Me), 5.05 (2H, s, CH2), 7.49
(2H, d, J=8.4 Hz, Ar 3,5-H2), 7.98 (2H, d, J=8.4 Hz,
Ar 2,6-H2); MS m/z 208 (M+H), 192 (MꢂMe).
N-(2-Hydroxyethyl)-N0 -(4-nitrophenylmethyl)thiourea
(24f) and 2,2-dimethyl-3-(N-(4-nitrophenylmethyl)ami-
nothiocarbonyl)tetrahydrooxazole (25f). Compound
22f was treated with 2-aminoethanol, as for the synth-
esis of 24e and 25e, to give 25f (13%) as a colourless oil:
IR (film) nmax 1143, 1346, 1541, 3380 cmꢂ1; NMR dH
1.79 (6H, s, 2 ꢄ Me), 3.82 (2H, m, CH2N), 4.05 (2H, t,
J=6.2 Hz, OCH2), 5.03 (2H, d, J=5.4 Hz, ArCH2), 5.67
(1H, br, NH), 7.49 (2H, d, J=8.8 Hz, Ar 2,6-H2) 8.17
(2H, d, J=8.8 Hz, Ar 3,5-H2); MS m/z 296.1063
(M+H) (C13H18N3O3S requires 296.1069). Further elu-
tion gave 24f (34%) as a colourless oil: IR (film) nmax
1160, 1346, 1562, 3368 cmꢂ1; NMR dH 3.62 (2H, br,
CH2Ar), 3.82 (2H, t, J=5.0 Hz, NCH2), 4.90 (2H, d,
J=5.8 Hz, OCH2), 6.54 (1H, br, OH), 7.10 (1H, br,
NH), 7.26 (1H, br NH), 7.50 (2H, d, J=8.9 Hz, Ar
Methyl 3-(isothiocyanatomethyl)benzoate (22i). Com-
pound 21i was treated with thiophosgene, as for the
synthesis of 22e, to give 22i (51%) as a colourless oil:
(lit.50 oil); IR (film) nmax 1722, 2101 cmꢂ1; NMR dH 3.94
(3H, s, Me), 4.79 (2H, s, CH2), 7.50 (1H, t, J=7.8Hz, Ar
5-H), 7.54 (1H, d, J=7.8 Hz Ar 4-H), 8.0 (1H, d,
J=7.8 Hz, Ar 6-H), 8.03 (1H, s, Ar 2-H); MS m/z 208.0434
(C10H10NO2S requires 208.0432), 149 (MꢂNCS).
1,1-Dimethylethyl N-(3-(N0-(2-hydroxyethyl)thioureidome-
thyl)phenylmethyl)carbamate (24c). Compound 22c

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4203
2,6-H2), 8.19 (2H, d, J=8.9 Hz, Ar 3,5-H2); MS m/z
256.0755 (M+H) (C8H10N3O2S requires 256.0756).
evaporation residue, in CH2Cl2, was washed with aq
NaHCO3 and dried. Evaporation and chromatography
(CH2Cl2) gave 27c (900 mg, 56%) as white crystals: mp
124–126 ꢀC (lit.51 mp 75–76 ꢀC); NMR dH 1.39 (9H, s,
But), 4.35 (2H, d, J=6.1 Hz, CH2), 5.23 (1H, br, NH),
7.40 (1H, dd, J=8.9, 7.8 Hz, Ar 5-H), 7.54 (1H, d,
J=7.8 Hz, Ar 6-H), 8.03 (1H, d, J=8.9 Hz, Ar 4-H),
8.04 (1H, s, Ar 2-H); MS m/z 253.1184 (M+H)
(C12H17N2O4 requires 253.1188), 197 (MꢂMe2C¼CH2);
Found C, 57.30: H, 6.35; N, 11.20; C12H15N2O4
requires C, 57.14; H, 6.35; N, 11.11%.
N-(2-Hydroxyethyl)-N0 -(3-methoxyphenylmethyl)thio-
urea (24g). Compound 22g was treated with 2-ami-
noethanol, as for the synthesis of 24e and 25e, to give
24g (85%) as a colourless oil: NMR ((CD3)2SO) dH 3.36
(4H, m, 2 ꢄ CH2), 3.67 (3H, s, Me), 4.55 (2H, m,
CH2Ar), 4.78 (1H, s, OH), 6.80 (1H, dd, J=7.4, 2.1 Hz,
Ar 6-H), 6.86 (1H, dd, J=7.4, 2.1 Hz, Ar 4-H), 6.85
(1H, s, Ar 2-H), 7.23 (1H, dd, J=7.4 Hz, Ar 5-H), 7.52
(1H, br, NH), 7.92 (1H, br, NH); NMR ((CD3)2SO) dC
46.7, 48.5, 55.3, 112.9, 113.2, 119.7, 129.7, 159.6; MS m/z
241.1009 (M+H) (C11H17N2O2S requires 241.1011).
1,1-Dimethylethyl
N-(4-nitrophenylmethyl)carbamate
(27d). 4-Nitrobenzylamine 21f was treated with Boc2O,
as for the synthesis of 27c, to give 27d (91%) as white
crystals: mp 111–114 ꢀC (lit.52 mp 109–110 ꢀC); NMR
dH 1.53 (9H, s, But), 4.42 (2H, d, J=5.9 Hz, CH2), 5.05
(1H, br, NH), 7.44 (2H, d, J=8.6 Hz, Ar 2,6-H2), 8.19
(2H, d, J=8.6 Hz, Ar 3,5-H2); MS m/z 505 (2 M+H),
406 (2 M+HꢂBoc), 275 (M+Na), 253 (M+H), 197
(MꢂMe2C¼CH2), 180 ( MꢂButO).
N-(2-Hydroxyethyl)-N0 -(4-methoxyphenylmethyl)thio-
urea (24h). 3-Methoxyphenylisothiocyanate 22h was
treated with 2-aminoethanol, as for the synthesis of 24e
and 25e (reaction time 2 h), to give 24h (47%) as a col-
ourless oil: IR (film) nmax 1171, 3336 cmꢂ1; NMR dH
3.49 (3H, s, Me), 3.66 (2H, br, CH2N), 3.81 (2H, d,
J=4.8 Hz, CH2O), 4.58 (2H, s, ArCH2), 6.48 (1H, br,
NH), 6.74 (1H, br, NH), 6.87 (2H, d, J=8.6 Hz, Ar 3,5-
H2), 7.25 (2H, d, J=8.6 Hz, Ar 2,6-H2); MS m/z
241.1011 (M+H) (C11H17N2O2S requires 241.1018).
1,1-Dimethylethyl N-(3-aminophenylmethyl)carbamate
(28c). Compound 27c was treated with SnCl2, as for
the synthesis of 28d (reaction time 30 min), to give 28c
(31%) as a pale buff oil (lit.53 oil); NMR dH 1.40
(9H, s, But), 3.59 (2H, br, NH2), 4.15 (2H, d,
J=5.7 Hz, CH2), 4.70 (1H, br, NH), 6.55 (3H, m, Ar
2,4,6-H3), 7.06 (1H t, J=7.7 Hz, Ar 5-H); MS m/z
222 (M), 167 (MꢂMe2C¼CH2), 121 (MꢂBoc), 106
(MꢂBocNH).
Methyl 3-(N0-(2-hydroxyethyl)thioureidomethyl)benzoate
(24i). Compound 22i was treated with 2-aminoethanol,
as for the synthesis of 24e and 25e (reaction time 2.5 h),
to give 24i (77%) as a colourless oil: IR (film) nmax 1197,
1715, 3355 cmꢂ1; NMR (CD3OD) dH 3.60 (2H, m,
CH2), 3.66 (2H, m, CH2), 3.89 (3H, s, Me), 4.80 (2H, s,
CH2Ar), 7.44 (1H, t, J=7.8 Hz, Ar-H5), 7.56 (1H, d,
J=7.8 Hz, Ar-H4), 7.88 (1H, d, J=7.8 Hz, Ar-H4), 7.96
(1H, s, Ar-H2); MS m/z 537 (2 M+H), 269.0955
(M+H) (C12H17N2O3S requires 269.0960).
1,1-Dimethylethyl N-(4-aminophenylmethyl)carbamate
(28d). Compound 27d was treated with SnCl2, as for
the synthesis of 16l (chromatography omitted), to give
28d (31%) as a colourless oil: (lit.54 mp 75–76 ꢀC);
NMR dH 1.45 (9H s But), 3.8 (2H, br, NH2), 4.18 (2H,
d, J=5.1 Hz, CH2), 4.73 (1H, br, NH), 6.64 (2H, d,
J=8.2 Hz, Ar 2,6-H2), 7.07 (2H, d, J=8.2 Hz, Ar 3,5-
H2); MS m/z 445 (2 M+H), 222 (M+H), 165
(MꢂMe2C¼CH2).
Methyl 4-(N0-(2-hydroxyethyl)thioureidomethyl)benzoate
(24k) and 2,2-dimethyl-3-(N-(4-methoxycarbonylphenyl-
methyl)aminothiocarbonyl)tetrahydrooxazole
(25k).
Compound 22k was treated with 2-aminoethanol, as
for the synthesis of 24e and 25e, to give 25k (280 mg,
43%) as white crystals: mp 105–107 ꢀC; IR nmax 1199,
1701, 3359 cmꢂ1; NMR ((CD3)2SO) ndH 1.69 (6H, s,
Me2C), 3.82 (3H, s, OMe), 3.64 (2H, t, J=6.3 Hz, oxa-
zole 4-CH2), 3.96 (2H, t, J=6.3 Hz, oxazole 5-H2), 4.82
(2H, d, J=5.5 Hz, ArCH2), 7.39 (2H, d, J=8.2 Hz, Ar
3,5-H2), 7.88 (2H, d, J=8.2 Hz, Ar 2,6-H2), 7.84 (1H,
br, NH); MS m/z 309.1263 (M+H) (C15H21N2O3S
requires 309.1273). Further elution gave 24k (64%) as
pale buff crystals: mp 75–77 ꢀC; IR (film) nmax 1194,
1714, 3351 cmꢂ1; NMR ((CD3)2SO) dH 3.47 (4H, m, 2 ꢄ
CH2), 3.82 (3H, s, Me), 4.74 (3H, m, CH2Ar+OH),
7.38 (2H, d, J=8.2 Hz, Ar 3,5-H2), 7.89 (2H, d,
J=8.2 Hz, Ar 2,6-H2), 7.58 (1H, br, NH), 8.01 (1H, br,
NH); MS m/z 269.0961 (M+H) (C12H16N2O3S requires
269.0960).
Methyl 3-aminophenylacetate hydrochloride (28q). 3-
Aminophenylacetic acid 28o (2.0 g, 13.2 mmol) was stir-
red with MeOH (350 mL) and SOCl2 (20 mL) for 4 d.
Evaporation gave 28q (2.6 g, 99%) as a colourless
hygroscopic gum: (lit.55 mp 167–170 ꢀC); NMR
((CD3)2SO) dH 3.60 (2H, s, CH2), 3.41 (3H, br, N+H3),
3.74 (3H, s, Me), 7.15 (3H, m, Ar-H3), 7.41 (1H, m, Ar
5-H); MS m/z 166 (M+H).
Methyl 4-aminophenylacetate hydrochloride (28r). 4-
Aminophenylacetic acid 28p was treated with MeOH
and SOCl2, as for the synthesis of 28q, to give 28r (99%)
as off-white crystals: mp 118–120 ꢀC (lit.56 mp 197–
199 ꢀC); NMR ((CD3)2SO) dH 3.47 (3H, br, NH3), 3.59
(3H, s, Me), 3.68 (2H, s, CH2), 7.19 (2H, d, J=8.6 Hz,
Ar 3,5-H2), 7.30 (2H, d, J=8.6 Hz, Ar 2,6-H2); MS m/z
166 (M+H), 121 (MꢂCO2H).
1,1-Dimethylethyl
N-(3-nitrophenylmethyl)carbamate
(27c). Di-tert-butyl dicarbonate (1.7 g, 7.8 mmol) was
added slowly to 3-nitrobenzylamine 21e (1.0 g,
6.6 mmol) and Et3N (1.1 g, 11 mmol) in CH2Cl2 (25 mL)
at 0 ꢀC and the mixture was stirred for 16 h. The
1,1-Dimethylethyl N-(3-isothiocyanatophenylmethyl)car-
bamate (29c). Compound 28c was treated with thio-
phosgene, as for the synthesis of 22c (reaction time 2 d),

4204
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
to give 29c (70%) as white crystals: mp 70–72 ꢀC; IR
nmax 1675, 2100, 3180 cmꢂ1; NMR ((CD3)2SO) dH 1.39
(9H, s, But), 4.12 (2H, d, J=5.9 Hz, CH2), 7.23 (3H, m,
Ar 2,4,6-H3), 7.43 (1H, t, J=7.7 Hz, Ar 5-H), 7.46 (1H,
br, NH); MS m/z 265.1012 (M+H) (C13H17N2O2S
requires 265.1011), 209 (MꢂMe2C¼CH2); Found C,
58.50; H, 6.02; N, 10.40; C13H16N2O2S requires C,
59.00; H, 6.06; N, 10.60%.
N-(2-Hydroxyethyl)-N0-(3-methoxyphenyl)thiourea (30g).
3-Methoxyphenylisothiocyanate 29g was treated with
2-aminoethanol, as for the synthesis of 24d (reaction
time 2 h), to give 30g (84%) as white crystals: mp 129–
131 ꢀC; IR nmax 1149, 2900, 3186 cmꢂ1
;
NMR
((CD3)2SO) dH 3.52 (4H, m, 2 ꢄ CH2), 3.70 (3H, s, Me),
4.80 (1H, br, OH), 6.65 (1H, d, J=8.2 Hz, Ar 6-H), 6.90
(1H, d, J=7.4 Hz, Ar 4-H), 7.20 (2H, m, Ar 2,5-H2),
7.71 (1H, br, NH), 9.41 (1H, s, NH); MS m/z 452
(2 M+H), 227.0846 (M+H) (C10H15N2O2S requires
227.0854); Found C, 53.4: H, 6.28; N, 12.36;
C10H14N2O2S requires C, 53.08; H, 6.24; N, 12.38%.
1,1-Dimethylethyl N-(4-isothiocyanatophenylmethyl)car-
bamate (29d). Compound 28d was treated with thio-
phosgene, as for the synthesis of 22c, to give 29d (26%)
as a pale yellow powder: mp 113–115 ꢀC; IR nmax 1683,
2123, 3366 cmꢂ1; NMR dH 1.46 (9H, s, But), 4.29 (2H,
d, J=5.6 Hz, CH2), 4.91 (1H, br, NH), 7.18 (2H, d,
J=8.6 Hz, Ar 2,6-H2), 7.27 (2H, d, J=8.6 Hz, Ar 3,5-
H2); MS m/z 529 (2 M+H), 265.1006 (M+H)
N-(2-Hydroxyethyl)-N0-(4-methoxyphenyl)thiourea (30h).
4-Methoxyphenylisothiocyanate 29h was treated with
2-aminoethanol, as for the synthesis of 24e and 25e
(reaction time 1.5 h), to give 30h (88%) as pale buff
crystals: mp 147 ꢀC (lit.58 mp 146–147 ꢀC); IR nmax 1165,
2835, 3189, 3646 cmꢂ1; NMR ((CD3)2SO) dH 3.35 (3H,
s, Me), 3.73 (4H, s, 2 ꢄ CH2), 4.79 (1H, br, OH), 6.88
(2H, d, J=8.6 Hz, Ar 3,5-H2), 7.24 (2H, d, J=8.6 Hz,
Ar 2,6-H2), 7.46 (1H, br, NH), 9.41 (1H, s, NH); MS
m/z 227.0850 (M+H) (C10H15N2O2S requires
227.0854); Found C, 53.0; H, 6.17; N, 12.2;
C11H14N2O2S requires C, 53.08; H, 6.24; N, 12.38%.
(C13H17N2O2S
requires
265.1011),
209
(MꢂMe2C¼CH2); Found C, 58.10: H, 5.92; N, 10.30;
C13H16N2O2S 0.25H2O requires C, 58.08; H, 6.19; N,
10.42%.
Methyl 3-isothiocyanatophenylacetate (29q). Compound
28q was treated with thiophosgene, as for the synthesis
of 22g, to give 29q (77%) as a pale yellow liquid: IR
(film) nmax 1738, 2119 cmꢂ1; NMR ((CD3)2SO) dH 3.61
(2H, s, CH2), 3.71 (3H, s, Me), 7.26 (3H, m, Ar-H3),
7.30 (1H, t, J=7.8 Hz, Ar 5-H); MS m/z 208.0432
(M+H) (C10H10NO2S requires 208.0432), 192
(M+HꢂMe), 148 (M+HꢂNCS).
Methyl 3-(N0-(2-hydroxyethyl)thioureido)phenylacetate
(30q). Compound 29q was treated with 2-aminoetha-
nol, as for the synthesis of 24e and 25e, to give 30q
(64%) as a colourless oil: IR (film) nmax 1061, 1732,
3293 cmꢂ1; NMR ((CD3)2SO) dH 3.34 (2H, m, CH2N),
3.52 (2H, br, CH2O), 3.59 (3H, s, Me), 3.63 (2H, m,
CH2Ar), 4.70 (1H, s, OH), 6.97 (1H, d, J=7.4 Hz, Ar 4-
H) 7.24 (1H, dd, J=7.8, 6.3 Hz, Ar 5-H), 7.28 (1H, s, Ar
2-H), 7.33 (1H, d, J=6.3 Hz, Ar 6-H), 7.69 (1H, s, NH),
9.60 (1H, br, NH); MS m/z 537 (2 M+H), 269.0953
(M+H) (C12H17N2O3S requires 269.0960).
Methyl 4-isothiocyanatophenylacetate (29r). Compound
28r was treated with thiophosgene, as for the synthesis
of 22f, to give 29r (83%) as pale buff oil: (lit.57 mp 168–
170 ꢀC); IR nmax 1738, 2120 cmꢂ1; NMR ((CD3)2SO) dH
3.61 (3H, s, Me), 3.73 (2H, s, CH2), 7.34 (2H, d,
J=8.6 Hz, Ar 2,6-H2), 7.39 (2H, d, J=8.6 Hz, Ar 3,5-
H2); MS m/z 208 (M+H), 192 (MꢂMe).
1,1-Dimethylethyl N-(3-(N0-(2-hydroxyethyl)thioureido)-
phenylmethyl)carbamate (30c). Compound 28c was
treated with 2-aminoethanol, as for the synthesis of 24e
and 25e (reaction time 2 h), to give 30c (43%) as a col-
ourless oil; IR nmax (film) 1164, 1693, 3380 cmꢂ1; NMR
((CD3)2SO) dH 1.39 (9H, s, But), 3.50 (2H, br, CH2NH),
3.52 (2H, m, CH2O), 4.04 (2H, d, J=6.6 Hz,
CH2NHBoc), 4.80 (1H, s, OH), 6.96 (1H, d, J=7.4 Hz,
Ar 4-H), 7.23 (2H, m, NH+Ar 2-H), 7.36 (2H, m, Ar
5,6-H2), 7.66 (1H, br, NH), 9.60 (1H, br, NH); MS m/z
651 (2 M+H), 326.1541 (M+H) (C15H24N3O3S
requires 326.1538), 270 (MꢂMe2C¼CH2).
1,1-Dimethylethyl N-4-(N0-(2-hydroxyethyl)thioureido-
phenylmethyl)carbamate (30d). Compound 29d was
treated with 2-aminoethanol, as for the synthesis of 24e
and 25e, to give 30d (45%) as a colourless oil; IR (film)
nmax 1166, 1689, 3323 cmꢂ1; NMR ((CD3)2SO) dH 1.39
(9H, s, But), 3.32 (2H, m, CH2), 3.52 (2H, m, CH2), 4.07
(2H, d, J=6.2 Hz, CH2NHBoc), 4.94 (1H, br, OH), 7.15
(2H, d, J=8.2 Hz, Ar 3,5-H2), 7.33 (2H, d, J=8.2 Hz,
Ar 2,6-H2), 7.38 (1H, br, NH), 7.65 (1H, br, NH), 9.57
(1H, br, NH); MS m/z 326.1552 (M+H) (C15H24N3O3S
requires 326.1538).
Methyl 4-(N0-(2-hydroxyethyl)thioureido)phenylacetate
(30r). Compound 29r was treated with 2-aminoethanol,
as for the synthesis of 24e and 25e, to give 30r (27%) as
pale yellow crystals: mp 53–55 ꢀC; IR nmax 1169, 1730,
3325, 3480 cmꢂ1; NMR dH 2.35 (1H, br, OH), 3.64 (2H,
s, CH2), 3.72 (3H, s, Me), 3.80 (4H, m, 2 ꢄ CH2), 6.56
(1H, br, NH), 7.20 (2H, d, J=7.8 Hz, Ar 3,5-H2), 7.33
(2H, d, J=7.8 Hz, Ar 2,6-H2), 7.94 (1H, br, NH); MS
m/z 537 (2 M+H), 269.0933 (M+H) (C12H16N2O3S
requires 269.0960).
(R)-N-(2-Hydroxypropyl)-N0-(3-methoxyphenyl)thiourea
(31). Compound 29g (500 mg, 3.0 mmol) in acetone
(2.1 mL) was added dropwise during 30 min to (R)-1-
aminopropan-2-ol (420 mg, 4.0 mmol) in acetone
(2.1 mL). The mixture was boiled under reflux for 2 h.
Evaporation and chromatography (EtOAc/hexane 1:1)
gave 31 (270 mg, 38%) as a colourless oil: NMR dH 1.22
(3H, d, J=6.3 Hz, Me), 3.46 (1H, m, CHNH), 3.81 (3H,
s, OMe), 3.94 (1H, m, CHNH), 4.02 (1H, CHOH),
6.64 (1H, br, NH), 6.78 (3H, m, Ar 2,4,6-H3), 7.33
(1H, t, J=8.2 Hz, Ar 5-H), 7.76 (1H, br, NH); IR
20
D
, MeOH); MS m/z 241.1007 (C11H17N2O2S
(film) nmax 1180, 3369 cmꢂ1; ½aŠ ¼ ꢂ7:2ꢀ (c 1.4 mg
mLꢂ1
requires 241.1011).

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
4205
(S)-2-(3-Methoxyphenylamino)-5-methyl-4,5-dihydrothi-
Acknowledgements
azole hydrochloride (32). Compound 31 (80 mg,
0.3 mmol) was boiled under reflux for 24 h in aq HCl
(6 M, 4 mL). Evaporation gave 32 (70 mg, 99%) as a
colourless hygroscopic gum; NMR dH 1.51 (3H, d,
J=5.9 Hz, thiazole-Me), 3.72 (1H, m, 4-H), 3.81
(3H, s, OMe), 4.11 (2H, m, 4-H+5-H), 6.81 (1H, m,
Ar 6-H), 6.88 (1H, d, J=8.2 Hz, Ar 4-H), 7.30 (1H,
s, Ar 2-H), 7.32 (1H, t, J=8.2 Hz, Ar 5-H), 10.67
We are very grateful to the Association for Interna-
tional Cancer Research for a Project Grant which gen-
erously supported this work. We also thank Dr. S. J.
Black (University of Bath) for the NMR spectra and
Mr. C. Cryer (University of Bath) for the mass spectra.
(1H, br, NH), 12.29 (1H, br, NH); IR (film) nmax
20
½ꢀŠ ¼ ꢂ32:4ꢀ (c 2.6 mg mLꢂ1
1629, 3434 cmꢂ1
MeOH); MS m/z 223.08963 (C11H16N2OS requires
223.0905).
;
,
References and Notes
D
1. Gross, S. S.; Wolin, M. S. Ann. Rev. Physiol. 1995, 57, 737.
2. Napoli, C.; Ignarro, L. J. Nitric Oxide 2001, 5, 88.
3. Kerwin, J. F.; Lancaster, J. R.; Feldman, P. L. J. Med.
Chem. 1995, 32, 4343.
NOS inhibition studies
4. Gatto, E. M.; Riobo, N. A.; Carreras, M. C.; Chernavsky,
A.; Rubio, A.; Satz, M. L.; Poderosos, J. J. Nitric Oxide 2000,
4, 534.
5. Christopherson, K. S.; Bredt, D. S. J. Clin. Invest. 1997,
100, 2424.
Measurements of the inhibitory activity of the test
compounds against rat nNOS and against rat iNOS
were made essentially as described previously by us.15
For the studies with human iNOS, the enzyme was pre-
pared as follows. An optimised mammalian expression
vector (pEFIRES-P, courtesy of Dr. S. Hobbs, Cancer
Research UK Centre for Cancer Therapeutics, ICR,
London) was designed to express human iNOS cDNA
(courtesy of Prof. Ian Charles, University of London).
Expression was linked with the selectable marker gene
(pac) at the level of mRNA and antibiotic selection
(puromycin) directly enforced expression of the cDNA.
This vector was been used to transfect the human
fibrosarcoma cell line, HT1080 and a series of iNOS-
expressing clones were produced. To avoid loss of via-
bility through the cytotoxic consequences of excessive
NO production, clones were grown in the presence of a
non-toxic dose of 7 (100 mM). Routinely, clones were
grown for 48 h in the absence of puromycin and 7 prior
to extracting the iNOS enzyme. Cells were grown to
near confluence and were harvested by trypsinisation.
Cells were then washed twice in cold phosphate-buffered
saline and homogenised in five volumes of ice-cold buf-
fer containing HEPES (10 mM, pH 7.4), sucrose
(320 mM), EDTA (100 mM), dithiothreitol (50 mM), leu-
peptin (10 mg mLꢂ1), soybean trypsin inhibitor (10 mg
mLꢂ1) and aprotinin (2 mg mLꢂ1). The preparations
were then sonicated using an MSE Soniprep 150 for 3 ꢄ
5 s at a nominal frequency of 23 KHz and oscillation
amplitude between 5 and 10 mm. Samples were placed in
ice between each sonication. These suspensions were
allowed to stand in ice for a further 10 min, then cen-
trifuged at 9000g for 15 min at 4 ꢀC. The post-mito-
chondrial supernatant was treated with Dowex-50W
[(200–400), 8% cross-linked, Na+ form] to remove
endogenous arginine. The supernatant was incubated
with the resin for 5 min and centrifuged at 10,000 rpm
for 5 min to pellet the resin. This process was repe-
ated twice, after which the cytosol was treated as free
of endogenous arginine and was used for assays of
inhibition, using the usual protocol with and without
pre-incubation of the test compounds with the pre-
paration.
6. Chinje, E. C.; Stratford, I. J. Essays Biochem. 1997, 32, 61.
7. Evans, C. H.; Stefanovic-Racic, M. Nitric Oxide in Arthri-
tis: It’s Probably There but What Is it Doing? In Nitric Oxide,
Cytochromes P450 and Sexual Steroid Hormones; Lancaster,
J. R., Parkinson, S. F., Eds.; Springer: Berlin, 1997; p 181.
8. McDaniel, M. L.; Kwon, G.; Hill, J. R.; Marshall, C. A.;
Corbett, J. A. Proc. Soc. Exp. Biol. Med. 1996, 211, 24.
9. Gross, S. S.; Steuhr, D. J.; Aisaka, K.; Jaffe, E. A.; Levi,
R.; Griffith, O. W. Biochem. Biophys. Res. Commun. 1990, 270,
96.
10. McCall, T. B.; Feelisch, M.; Palmer, R. M. J.; Moncada,
S. Br. J. Pharmacol. 1991, 102, 234.
11. Moore, W. M.; Webber, R. K.; Jerome, G. M.; Tjeong,
F. S.; Misko, T. P.; Currie, M. G. J. Med. Chem. 1994, 37,
3886.
12. Hallinan, E. A.; Tsymbalov, S.; Finnegan, P. M.; Moore,
W. M.; Currie, M. G.; Jerome, G. M.; Pitzele, B. S. J. Med.
Chem. 1998, 41, 775.
13. Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P.
Biochemistry 1993, 32, 8512.
14. Huang, H.; Martasek, P.; Roman, L. J.; Siler Masters,
B. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147.
15. Ulhaq, S.; Chinje, E. C.; Naylor, M. A.; Jaffar, M.; Strat-
ford, I. J.; Threadgill, M. D. Bioorg. Med. Chem. 1998, 6,
2139.
16. Lee, Y.; Martasek, P.; Roman, L. J.; Siler Masters, B. S.;
Silverman, R. B. Bioorg. Med. Chem. 1999, 7, 1941.
17. Kawanaka, Y.; Kobayashi, K.; Kusuda, S.; Tatsumi, T.;
Murota, M.; Nishiyama, T.; Hisaichi, K.; Fujii, K.; Hirai, K.;
Nishizaki, M.; Naka, M.; Komeno, M.; Nakai, H.; Toda, M.
Bioorg. Med. Chem. 2003, 11, 689.
18. Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerome, G. M.;
Connor, J. R.; Manning, P. T.; Wyatt, P. S.; Misko, T. P.;
Tjoeng, F. S.; Currie, M. G. J. Med. Chem. 1996, 39, 669.
19. Hansen, D. W.; Peterson, K. B.; Trivedi, M.; Kramer,
S. W.; Webber, R. K.; Tjoeng, F. S.; Moore, W. M.; Jerome,
G. M.; Kornmeier, C. M.; Manning, P. T.; Connor, J. R.;
Misko, T. P.; Currie, M. G.; Pitzele, B. S. J. Med. Chem. 1998,
41, 1361.
20. Hagen, T. J.; Bergmanis, A. A.; Kramer, S. W.; Fok,
K. F.; Schmelzer, A. E.; Pitzele, B. S.; Swenton, L.; Jerome,
G. M.; Kornmeier, C. M.; Moore, W. M.; Branson, L. F.;
Connor, J. R.; Manning, P. T.; Currie, M. G.; Hallinan, E. A.
J. Med. Chem. 1998, 41, 3675.
21. Webber, R. K.; Metz, S.; Moore, W. M.; Connor, J. R.;
Currie, M. G.; Fok, K. F.; Hagen, T. J.; Hansen, D. W.; Jer-
ome, G. M.; Manning, P. T.; Pitzele, B. S.; Toth, M. V.; Tri-
The results are shown in Table 1 as the mean of tripli-
cate experimentsꢃSEM.

4206
C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206
vedi, M.; Zupec, M. E.; Tjoeng, F. S. J. Med. Chem. 1998, 41,
96.
37. Ettlinger, M. G.; Lundeen, A. J. J. Am. Chem. Soc. 1956,
78, 1952.
22. Tinker, A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook,
T. R.; Cooper, S. L.; Fraser-Rae, L.; Hallam, K.; Hamley, P.;
McInally, T.; Nicholls, D. J.; Pimm, A. D.; Wallace, A. V. J.
Med. Chem. 2003, 46, 913.
38. Huter, F. Chem. Ber. 1947, 80, 273.
¨
39. Dyson, G. M.; George, H. J.; Hunter, R. F. J. Chem. Soc.
1927, 440.
ˇ
40. Tisler, M.; Vrbaski, Z. J. Org. Chem. 1960, 25, 770.
ˇ
ˇ
23. Garvey, E. P.; Oplinger, J. A.; Furfine, E. S.; Kiff, R. J.;
Laszlo, F.; Whittle, B. J. R.; Knowles, R. G. J. Biol. Chem.
1997, 272, 4959.
41. Richter, P.; Wagner, G.; Michalke, B.; Schwab, M. Phar-
mazie 1974, 29, 307.
42. Friedman, L.; Shechter, H. J. Org. Chem. 1961, 26, 2522.
24. Collins, J. L.; Shearer, B. G.; Oplinger, J. A.; Lee, S.;
Garvey, E. P.; Salter, M.; Duffy, C.; Burnette, T. C.; Furfine,
E. S. J. Med. Chem. 1998, 41, 2858.
25. Narayanan, K.; Griffith, O. W. J. Med. Chem. 1994, 37,
885.
26. Ulhaq, S.; Chinje, E. C.; Naylor, M. A.; Jaffar, M.; Strat-
ford, I. J.; Threadgill, M. D. Bioorg. Med. Chem. 1999, 7,
1787.
43. Lowik, D. W. P. M.; Lowe, C. R. Eur. J. Org. Chem.
¨
2001, 2825.
44. Smith, J.; Liras, J. L.; Schneider, S. E.; Anslyn, E. V. J.
Org. Chem. 1996, 61, 8811.
45. Ashton, P. R.; Balzani, V.; Credi, A.; Kocian, O.; Pasini,
D.; Prodi, L.; Spencer, N.; Stoddart, J. F.; Tolley, M. S.;
Venturi, M.; White, A. J. P.; Williams, D. J. Chem. Eur. J.
1998, 4, 577.
27. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.;
Garvey, E. P.; Furfine, E. S. J. Med. Chem. 1997, 40, 1901.
28. Frolich, L. G.; Kotsonis, P.; Traub, H.; Taghavi-
¨
46. Tamura, N.; Matsushita, Y.; Iwama, T.; Harada, S.;
Kishimoto, S.; Itoh, K. Chem. Pharm. Bull. 1991, 39, 1199.
47. US Patent 3539612, 1966.
Moghaddam, S.; Al-Masoudi, N.; Hofmann, H.; Strobel, H.;
Matter, H.; Pfleiderer, W.; Schmidt, H. H. H. W. J. Med.
Chem. 1999, 42, 4108.
48. Parks, T. E.; Spurlock, L. A. J. Org. Chem. 1973, 38, 3922.
49. Emerson, W. S.; Heimsch, R. A. J. Am. Chem. Soc. 1950,
72, 5152.
29. Matter, H.; Kotsonis, P.; Klingler, O.; Strobel, H.; Fro-
¨
lich, L. G.; Frey, A.; Pfleiderer, W.; Schmidt, H. H. H. W. J.
Med. Chem. 2002, 45, 2923.
50. Kyne, G. M.; Light, M. E.; Hursthouse, M. B.; de Men-
doza, J.; Kilburn, J. D. J. Chem. Soc., Perkin Trans. 1 2001,
1258.
30. Wolff, D. J.; Gribbin, B. J. Arch. Biochem. Biophys. 1994,
311, 300.
51. Woodroofe, C. C.; Zhong, B.; Lu, X.; Silverman, R. B. J.
Chem. Soc., Perkin Trans. 2 2000, 55.
31. Stokker, G. E.; Deanna, A. A.; deSolms, S. J.; Schultz,
E. A.; Smith, R. L.; Cragoe, E. J.; Baer, J. E.; Russo, H. F.;
Watson, L. S. J. Med. Chem. 1982, 25, 735.
32. Clapp, R. C.; Long, L.; Hasselstrom, T. J. Am. Chem.
Soc. 1961, 1666.
33. Hirashima, A.; Yoshii, Y.; Eto, M. Biosci. Biotechnol.
Biochem. 1992, 56, 1062.
34. Caujolle, R.; Amarouch, H.; Payard, M.; Loiseau, P. R.;
Bories, C.; Loiseau, P. M.; Gayral, P. Eur. J. Med. Chem.
1989, 24, 287.
52. Tanaka, K.-I.; Yoshifuji, S.; Nitta, Y. Chem. Pharm. Bull.
1988, 36, 3125.
53. Moloney, G. P.; Martin, G. R.; Mathews, N.; Milne, A.;
Hobbs, H.; Dodsworth, S.; Sang, P. Y.; Knight, C.; Williams,
M.; Maxwell, M.; Glen, R. C. J. Med. Chem. 1999, 42, 2504.
54. Tsuda, Y.; Tada, M.; Wanaka, K.; Okamoto, U.; Hiji-
kata-Okunomiya, A.; Okamoto, S.; Okada, Y. Chem. Pharm.
Bull. 2001, 49, 1457.
55. Jacobs, W. A.; Heidelberger, M. J. Am. Chem. Soc. 1917,
39, 2418.
35. Goodyer, C. L. M.; Chinje, E. C.; Jaffar, M.; Stratford, I.
J.; Threadgill, M. D. Bioorg. Med. Chem. Lett. Submitted for
publication.
36. McKay, A. F.; Garmaise, D. L.; Gaudry, R.; Baker, H. A.;
Paris, G. Y.; Kay, R. W.; Just, G. E.; Schwartz, R. J. Am.
Chem. Soc. 1959, 81, 4328.
56. Jacobson, K. A.; Kirk, K. L.; Padgett, W. L.; Daly, J. W.
J. Med. Chem. 1985, 28, 1341.
57. Shridhar, D. R.; Sastry, C. V. R.; Lal, B.; Gurumurthy,
R.; Parihar, P.; Singh, P. P.; Mur, N.; Rao, C. S.; Junnakar,
A. Y. J. Indian Chem. Soc. 1985, 62, 687.
58. Adcock, B.; Lawson, A. J. Chem. Soc. 1965, 474.