Synthesis of N-(Methoxycarbonylthienylmethyl)thioureas and evaluation of their interaction with inducible and neuronal nitric oxide synthase

Article abstract of DOI:10.3390/molecules15053121

Two isomeric N-(memoxycarbonylmienylmemyl)mioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethyl- thioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS).

Full text of DOI:10.3390/molecules15053121

Molecules 2010, 15, 3121-3134; doi:10.3390/molecules15053121
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of N-(Methoxycarbonylthienylmethyl)thioureas and
Evaluation of Their Interaction with Inducible and Neuronal
Nitric Oxide Synthase
Ghadeer A.R.Y. Suaifan 1,*, Claire L.M. Goodyer 2 and Michael D. Threadgill 2
1
Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman,
Jordan
2
Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY,
UK; E-Mail: M.D.Threadgill@bath.ac.uk (M.D.T.)
* Author to whom correspondence should be addressed; E-Mail: gh.suaifan@ju.edu.jo;
Tel.: +962-6-5355000; Fax: +962-6-530025.
Received: 21 February 2010; in revised form: 22 April 2010 / Accepted: 26 April 2010 /
Published: 28 April 2010
Abstract: Two isomeric N-(methoxycarbonylthienylmethyl)thioureas were synthesised by
a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with
trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and
addition of ammonia. The interaction of these new thiophene-based thioureas with
inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethyl-
thioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS).
Keywords: nitric oxide synthase; iNOS; stimulation; thienylmethylthioureas
Abbreviations: Nitric Oxide Synthase (NOS); endothelial Nitric Oxide Synthase (eNOS);
neuronal Nitric Oxide Synthase (nNOS); inducible Nitric Oxide Synthase (iNOS); L-
arginine (L-Arg)

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1. Introduction
Nitric oxide (•NO) is biosynthesised from L-arginine in two steps catalysed by nitric oxide synthase
(NOSs) giving L-citrulline as a co-product. There are three isoforms, endothelial NOS (eNOS) and
neuronal NOS (nNOS) (constitutive Ca2+-dependent forms) and an inducible form (iNOS).
Underactivity and overactivity of each of these isoforms can be associated with disease states.
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 [1]. However, if too little NO is produced, this
can lead to conditions such as high blood pressure, angina and impotence [2]. An overexpression of
nNOS in circulating neutrophils has been found in patients with Parkinson’s disease [3] and nNOS
activity is thought to be linked to migraine headaches. NO production by iNOS is essential for the
defence mechanism of an organism; however, overactivity of iNOS has been related to several
pathological conditions, including cancer, arthritis and diabetes [4–5]. Thus Selective inhibition of
each of these three isoforms (eNOS, nNOS and iNOS) has potential applications in medicinal
chemistry. Most potent inhibitors of NOS ligate to the haem iron at the active sites of the enzymes,
through amidines and guanidines and through (iso)thioureas. Early inhibitors included close analogues
of L-Arg, such as Nδ-(iminoethyl)-L-ornithine (L-NIO) [6] and Nω-nitro-L-arginine methyl ester
(L-NAME). [7]
More recent iNOS-selective inhibitors contain cyclic amidines to bind to the haem iron but lack the
amino-acid motif of L-Arg [8,9]. Most interesting are 1400W (1) [10] (Figure 1), an N-benzyl-
acetamidine which is a highly selective inhibitor of rat iNOS, and its lower homologue 2 [11]
(Figure 1), an N-phenylacetamidine which is selective for inhibition of nNOS.
Figure 1. Structures of the iNOS-selective inhibitor 1400W 1, the nNOS-selective lower
homologue 2 and the iNOS stimulators 3 and 4. R = 3-NH2, 4-NH2=, 3-CO2H, 4-CO2H.
S
NH
S
NH
Me
N
NH
NH
Me
NH
NH2
NH
R
R
NH2
NH2
1
2
3
4
We recently reported [12] a series of N-benzyl- and N-phenyl-thioureas and analogous 2-
(substituted-amino)-4,5-dihydrothiazoles; several of these were moderate inhibitors of iNOS but were
without activity against nNOS. More interestingly, N-benzylthioureas 3 (Figure 1) and 2-benzylamino-
4,5-dihydrothiazoles 4 (Figure 1) caused significant stimulation of iNOS activity when the agents were

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added to the enzyme preparation simultaneously with the substrate. Therefore, in this work, our aim
was to synthesis a new target in which benzene has been deplaced with the approximately isosteric
thiophene. This rigidity introduced into the target is designed to maximise interaction and, more
importantly, to differentiate between various NOS isoforms. Information from potent but non-isoform-
selective NOS inhibitors suggested that 4,5-dihydrothiazole, thiourea and imidazole are good head
groups for haem-iron-binding. So, This work extended our previous work into two exemplary N-
thienylmethylthioureas.
2. Results and Discussion
Retrosynthetic analysis suggested that the commercially available 2-methylthiophene (5) would be
an ideal starting material. Abstraction of the relatively acidic thiophene 2-proton by butyl lithium
would give an appropriate nucleophile for the reaction with a reactive carbonyl electrophile. Therefore,
lithiation of 5 with butyl lithium at 0 °C, followed by quench with ethyl chloroformate (a reactive
carboxy electrophile equivalent) gave the required ester 6 in 68% optimised yield (Scheme 1).
Scheme 1. Synthetic route to target thioureidomethylthiophene 13.
iii
i,ii
Br
CO2Et
CO2Et
Me
Me
S
S
S
6
7
8
5
iii
CF3OCHN
iv, v
CbzHN
CO2Et
CO2R
S
S
9: R = H
10: R = Me
vi
vii
SCN
H3N
CO2Me
CO2Me
S
S
11
12
Br
viii
H
N
H2N
CO2Me
S
13
S
Reagents: i, BuLi, THF, ii, EtO2CCl, THF; iii, NBS, HCIO4, hexane; iv, CF3CONH2, KOBut, THF;
v, NaOH, aq.MeOH, ∆; vi, MeOH, H2SO4; vii, HBr, HOAc; viii, CSCI2, CaCO3, H2O, CHCI3; ix,
NH3, CH2CI2.

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A similar lithiation of 5 with butyl lithium, followed by quench with methyl chloroformate,
afforded mixture of products. Thus the precise nature of the trapping electrophile was important.
Bromination using N-bromosuccinimide (NBS) in the presence of perchloric acid introduced
functionality to the methyl group, although the bromomethyl analogue 7 was obtained in only 7%
isolated yield; ethyl 4-bromo-5-methylthiophene-2-carboxylate and ethyl 4-bromo-5-bromomethyl-
thiophene-2-carboxylate were also identified in the reaction mixture. Thiourea is nucleophilic at sulfur;
thus direct displacement of the bromine of 7 would give the isothiourea, rather than the target thiourea
13, so the thiourea had to be introduced stepwise. Replacement of the bromine with the anion derived
from trifluoroacetamide provided one nitrogen in 8. From here, no conditions were found which would
remove the trifluoroacetyl masking group from the primary amine while leaving the ester unscathed.
Hence vigorous base-hydrolysis cleaved both the amide and the ester to give the highly polar
5-(aminomethyl)thiophene-2-carboxylate which could only be isolated from the reaction mixture after
convertion in situ to the Cbz-protected derivative 9. Now, the methyl ester 10 was formed and the Cbz
was removed quantitatively and selectively, using hydrogen bromide, to afford 11. From here, our
previously developed method [12–14] was employed to convert the primary amine firstly to the iso-
thiocyanate 12 (with thiophosgene) and then to the required thiourea 13.
Scheme 2 shows the synthetic approaches to the 2,4-disubstituted target thiourea 25. Attempted
introduction of a carbonyl substituent to 3-methylthiophene 14, by lithiation and reaction with
dimethylformamide (DMF), gave an inseparable mixture of the regioisomeric aldehydes 15a,b.
Oxidation of this mixture to the carboxylic acids 16a,b was achieved with silver(I) oxide but this also
failed to allow isolation of the pure required regioisomer 16b. Similarly, quench of the lithiated species
derived from 14 with methyl chloroformate gave a 1:4 mixture of the esters 17a and 17b, respectively.
Unfortunately, this mixture was again inseparable. Radical bromination of the mixture was achieved
by treatment with one equivalent of NBS, using dibenzoyl peroxide as radical initiator. However, a
mixture of unreacted starting material, ring-brominated products, and side-chain brominated (and
polybrominated) products were produced. Repeated attempts using two equivalents of NBS gave
higher percentages of ring-brominated products. Separation of the mixture via chromatography
afforded 4.5% of 18. As in the 2,5-disubstituted sequence, reaction with trifluoroacetamide anion gave
a moderate yield of the secondary amide 19 but, despite the presence of a large excess of the nucleo-
phile, a small quantity of the dialkylated product 20 was also isolated. Hydrolysis of the
trifluoroacetamide and the ester functional groups of 19 was carried out simultaneously using sodium
hydroxide. In the same reaction flask, a Schotten-Baumann reaction was carried out using benzyl
chloroformate, giving, after acid work-up, the Cbz compound 21 in 82% yield. This was esterified with
methanol and H2SO4 to give the ester 22 in high yield. As before, the protecting Cbz group of 22 was
cleaved quantitatively upon treatment with hydrogen bromide in acetic acid. Compound 23 was then
converted to the isothiocyanate 24 with thiophosgene in the presence of CaCO3. Reaction of 24 with
ammonia gave the thiourea 25 in excellent yield.

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Scheme 2. Synthetics route to target thioureidomethylthiophene 25.
3125
iv, v
Me
Me
Me
Me
i, ii
iii
iv
CHO
CO2H
CO2Me
S
S
S
15a
16a
17a
Me
Me
Me
+
+
+
S
14
CHO
CO2H
CO2Me
S
S
S
15b
16b
17b
Br
vi
MeO2C
CO2Me
CO2Me
CO2R
S
18
19
S
N
vii
CF3OCHN
+
CF3
O
CO2Me
S
S
20
viii
SCN
CbzHN
NH3
x
ix
Br
CO2Me
CO2Me
S
S
S
xi
23
21: R = H
24
S
22: R = Me
NH2
N
H
CO2Me
S
25
Reagents: i, BuLi, THF, ii, DMF, Et2O; iii, Ag2O, NaOH, H2O; iv, MeOH, H2SO4; v, EtO2CCI,
THF; vi, NBS, (PhCO2)2, CCI4; vii, CF3CONH2, KOBut, THF; viii, NaOH, aq.MeOH, ∆; ix,
MeOH, H2SO4; x, HBr, HOAc; xi, CSCI2, CaCO3, H2O, CHCI3; xii, NH3, CH2CI2
Biological Evaluation
Compounds 13 and 25 were evaluated for their effects on the activity of rat brain nNOS and of
recombinant human iNOS (hiNOS) at a test concentration of 100 µM using the L-[U-14C]-arginine to
L-[U-14C]-citrulline conversion assay. 1400W 1 [10] and N-(3-aminobenzyl)thiourea 3 (R = 3-NH2)
[12] were used for comparison. Compounds were evaluated either by adding the radiolabelled
substrate simultaneously with the inhibitor or with a pre-incubation of 10 min before the substrate was
added. This pre-incubation has been reported to be optimum for the inhibitory activity of

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1400W 1[10]. This pre-incubation was investigated since test compounds can be considered to be
analogues of the slow inhibitor 1400W 1.
As expected, 1400W 1 was a potent inhibitor of iNOS activity, both with and without pre-
incubation with the enzyme. N-(3-Aminobenzyl)thiourea 3 was inactive towards the rat nNOS isoform
but, as noted previously [12], stimulated formation of L-[U-14C]-citrulline from L-[U-14C]-arginine
when the agent was added simultaneously with the substrate. The N-(thienylmethyl)thioureas 13 and
25 showed similar activity to that of 3, stimulating human iNOS activity but not modulating the
activity of this isoform at all when the compounds were pre-incubated with the enzyme before addition
of the substrate. Neither 13 nor 25 affected the activity of rat nNOS, with or without pre-incubation.
Table 1. Stimulation of human iNOS and rat nNOS activity by 1400W 1, N-(3-amino-
benzyl)thiourea 3 and the N-thienylmethylthioureas 13 and 25.
% Stimulation of hiNOS
activity a
IC50 (µM)
hiNOS
% Stimulation of nNOS
activitya
Compound
t = 0 min b
-79 ± 1
t = 10 min b t = 15 min b
t = 0 min b
ND
t = 10 min b
1
3 (R = 3-NH2)
13
-82 ± 1
+9 ± 1
-4 ± 3
<4
ND
+58 ± 1
+62 ± 2
+1 ± 6
-1 ± 4
+6 ± 1
-9 ± 6
+3 ± 3
+6 ± 7
25
+37 ± 27
-8 ± 11
a
b
Concentration of test compound 100 μM; t refers to the time between addition of the test
compound and addition of L-[U-14C]-arginine to initiate the enzymic reaction.
3. Experimental
3.1. General
NMR data were recorded on either JEOL/Varian GX 270 or EX 400 spectrometers, using solutions
in deuteriochlorform (CDCl3), unless otherwise stated. IR spectra of samples were recorded as
potassium bromide (KBr) discs, unless otherwise stated. Mass Spectra were recorded using a VG
Analytical Mass Spectrometer in the FAB positive ion mode, unless otherwise stated. Elemental
analyses was determined using Exeter Analytical Inc. CE-440 Elemental Analyzer. Solutions in
organic solvents were dried with magnesium sulfate (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.
3.2. Chemistry
Ethyl 5-methylthiophene-2-carboxylate (6). 2-Methylthiophene (5, 20.0 g, 204 mmol) was added to
butyl lithium (220 mmol) in dry tetrahydrofuran (400 mL) at 0 °C. The mixture was stirred at 0 °C for
3 h before being added to ethyl chloroformate (28.2 g, 260 mmol) in tetrahydrofuran (200 mL) at 0 °C.
The mixture was stirred at 20 °C for 16 h, and then poured onto ice. The organic layer was washed
with water, hydrochloric acid (1 M), aq. sodium hydrogen carbonate and water. Drying, evaporation

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1
and distillation gave 6 (23.0 g, 68%) bp~1 120°C (lit. [15] bp5 87-89°C); H-NMR δ 1.33 (3 H, t,
J = 7.0 Hz, CH2CH3), 2.48 (3 H, brs, 5-Me), 4.30 (2 H, q, J = 7.0 Hz, CH2), 6.72 (1 H, dq, J = 3.7, 1.2
Hz, 4-H), 7.57 (1 H, d, J = 3.7Hz, 3-H).
Ethyl 5-bromomethylthiophene-2-carboxylate (7). Compound 6 (1.00 g, 5.9 mmol) was stirred with N-
bromosuccinimide (1.08 g, 6.1 mmol) and perchloric acid (60%, 30 μL) in hexane (3.0 mL) for 24 h.
The evaporation residue, in EtOAc, was washed with saturated aq. sodium sulfite solution. Drying,
1
evaporation and chromatography (hexane / EtOAc 9:1) gave 7 (160 mg, 11%) as a colourless oil: H-
NMR δ 1.37 (3 H, t, J = 7.0 Hz, Me), 4.35 (2 H, q, J = 7.0 Hz, OCH2), 4.68 (2 H, s, CH2Br), 7.08 (1 H,
dt, J = 3.9, 0.8 Hz, 4-H), 7.63 (1 H, d, J = 3.9 Hz, 3-H); MS m/z 251/249 (M + H), 170 (M + H – Br);
Found C, 38.60; H, 3.77; C8H9BrO2S requires C, 38.55; H, 3.64%.
Ethyl 5-(trifluoroacetamidomethyl)thiophene-2-carboxylate (8). Trifluoroacetamide (410 mg,
3.6 mmol) was stirred with potassium t-butoxide (410 mg, 3.6 mmol) in dry tetrahydrofuran (4 mL) for
1 h. Compound 7 (130 mg, 520 μmol) in dry tetrahydrofuran (3 mL) was added and the mixture was
stirred for 15 h. The evaporation residue, in dichloromethane, was washed with water, hydrochloric
acid (1 M) and water. Drying and evaporation gave 8 (90 mg, 62%) as a pale yellow solid: mp 65-66
°C: IR νmax 3333, 1719, 1683 cm-1; 1H-NMR δ 1.37 (3 H, t, J = 7.0 Hz, Me), 4.34 (2 H, q, J = 7.0 Hz,
OCH2), 4.72 (2 H, d, J = 5.9 Hz, CH2N), 7.03 (1 H, dt, J = 3.9, 0.8 Hz, 4-H), 7.67 (1 H, d, J = 3.5 Hz,
3-H); NMR δF -76.2 (3 F, s, CF3); MS m/z 282.0413 (M + H) (C10H11F3NO3S requires 282.0412);
Found C, 42.90; H, 3.49; N, 4.98; C10H10F3NO3S requires C, 42.68; H, 3.59; N, 4.98%.
5-(Benzyloxycarbonylaminomethyl)thiophene-2-carboxylic acid (9). Compound 8 (3.50 g, 12.0 mmol)
was boiled under reflux with sodium hydroxide (3.93 g, 98 mmol) in water (50 mL) and methanol
(50 mL) for 16 h. The methanol was evaporated. Benzyl chloroformate (5.1 mL, 36 mmol) was added
at 0 °C and the mixture was stirred vigorously for 4 h. The mixture was washed with diethyl ether. The
aqueous layer was acidified (hydrochloric acid) and extracted with ethyl acetate. Drying and
evaporation gave 9 (3.33 g, 92%) as a yellow solid: mp 142-145 °C; IR νmax 3344, 2700, 1694, 1547,
1528 cm-1; 1H-NMR [(CD3)2SO] δ 4.36 (2 H, d, J = 6.2 Hz, CH2N), 5.04 (2 H, s, CH2O), 6.98 (1 H, d,
J = 3.5 Hz, 4-H), 7.3 (5 H, m, Ph-H5), 7.55 (1 H, d, J = 3.5 Hz, 3-H), 8.02 (1 H, t, J = 6 Hz, NH); MS
m/z 292.0644 (M + H) (C14H14NO4S requires 292.0640); Found: C, 57.50; H, 4.41; N, 4.63;
C14H13NO4S requires C, 57.70; H, 4.50; N, 4.81%.
Methyl 5-(benzyloxycarbonylaminomethyl)thiophene-2-carboxylate (10). Compound 9 (500 mg, 1.7
mmol) was boiled under reflux with sulfuric acid (1.0 mL) in methanol (50 mL) for 2 d. Aq. sodium
hydrogen carbonate was added until no further bubbling occurred. The suspension was filtered. The
evaporation residue was washed with water and dried to afford 10 (460 mg, 88%) as a white solid: mp
80-83 °C; 1H-NMR δ 3.87 (3 H, s, Me), 4.55 (2 H, d, J = 5.9 Hz, CH2N), 5.14 (2 H, s, CH2O), 5.23 (1
H, br, NH), 6.95 (1 H, d, J = 3.5 Hz, 4-H), 7.31-7.36 (5 H, m, Ph-H5), 7.64 (1 H, d, J = 3.5 Hz, 3-H);

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MS m/z 398 (M + glycerol + H), 306.0813 (M + H) (C15H16NO4S requires 306.0800), 259 (M -
CO2Me); Found C, 58.99; H, 5.00; N, 4.52; C15H15NO4S requires C, 58.98; H, 4.95; N, 4.59%.
Methyl 5-(aminomethyl)thiophene-2-carboxylate hydrobromide (11). Compound 10 (100 mg,
330 μmol) was stirred with hydrogen bromide in AcOH (15%, 2.0 mL) for 20 min. The evaporation
residue was washed (10 ×) with diethyl ether and dried to afford 11 (83 mg, quant.) as a white solid:
mp 212-215 °C; 1H-NMR [(CD3)2SO] δ 3.83 (3 H, s, Me), 4.31 (2 H, s, CH2), 7.30 (1 H, d, J = 3.9 Hz,
4-H), 7.76 (1 H, d, J = 3.9 Hz, 3-H), 8.26 (3 H, br, N+H3); MS m/z 325 (M + mNBA + H), 172.0437
(M + H) (C7H10NO2S requires 172.0432); Found H, 4.01; N, 5.56; C7H9NO2S requires H, 4.00; N,
5.56%.
Methyl 5-(isothiocyanatomethyl)thiophene-2-carboxylate (12). Compound 11 (210 mg, 830 μmol) was
stirred with calcium carbonate (90 mg, 870 μmol) and thiophosgene (200 mg, 1.7 mmol) in water
(1.5 mL) and chloroform (10 mL) for 20 h. The solvent was evaporated from the organic layer.
Chromatography (hexane / ethyl acetate 7:3) gave 12 (110 mg, 62%) as a colourless oil: IR (film) νmax
2074, 1711 cm-1; 1H-NMR δ 3.87 (3 H, s, Me), 4.85 (2 H, s, CH2), 7.03 (1 H, dt, J = 3.9, 0.8 Hz, 4-H),
7.76 (1 H, d, J = 3.9 Hz, 3-H); MS m/z 367 (M + mNBA + H), 214.0003 (M + H) (C8H8NO2S2 requires
213.9997), 182 (M - S); Found C, 44.90; H, 3.32; N, 6.55. C8H7NO2S2 requires C, 45.05; H, 3.31; N,
6.57%.
Methyl 5-(thioureidomethyl)thiophene-2-carboxylate (13). Ammonia was passed through 12 (60 mg,
280 μmol) in dichloromethane at 0°C for 20 min. The mixture was stirred for 3 h at 0 °C. Evaporation
gave 13 (65 mg, quant.) as a white solid: mp 131-132 °C: IR νmax 3398, 1703, 1277 cm-1; 1H-NMR
(CD3OD) δ 3.84 (3 H, s, Me), 4.93 (2 H, d, J = 5.9 Hz, CH2N), 7.06 (1 H, dt, J = 3.7, 0.7 Hz, 4-H),
7.65 (1 H, d, J = 4.0 Hz, 3-H); MS m/z 231.0255 (M + H) (C8H11N2OS2 requires 231.0262); Found C,
41.50; H, 4.32; N, 12.06; C15H15NO4S requires C, 41.72; H, 4.38; N, 12.16%.
3-Methylthiophene-2-carboxaldehyde (15a) and 4-methylthiophene-2-carboxaldehyde (15b). 3-
Methylthiophene 18 (10.0 g, 102 mmol) was added slowly to butyl lithium (2.0 M in hexane, 55 mL,
110 mmol) in dry diethyl ether (175 mL) at 0 °C and the mixture was stirred for 3 h at 0 °C, before
being added slowly to dimethylformamide (10.2 g, 140 mmol) in dry diethyl ether (35 mL) at 0 °C.
The mixture was stirred for 16 h and poured onto ice. The organic layer was washed with water,
hydrochloric acid (1 M), aq. sodium hydrogen carbonate and water. Drying, evaporation and
distillation (ca. 1 torr) gave a mixture of 15a and 15b (12.6 g, 61%) as a colourless liquid: 1H-NMR δ
2.32 [2.3 H, s, Me (15b)], 2.58 [0.7 H, s, Me (15a)], 6.97 [0.22 H, d, J = 5.2 Hz, 4-H (15a)], 7.36 [0.78
H, d, J = 1.3 Hz, 5-H (15b)], 7.58 [0.78 H, d, J = 1.3 Hz, 3-H (15b)], 7.63 [0.22 H, d, J = 5.2 Hz, 5-H
(15a)], 9.86 [0.78 H, d, J = 1.3 Hz, CHO (15b)], 10.04 [0.22 H, d, J = 1.3 Hz, CHO (15a)].
3-Methylthiophene-2-carboxylic acid (16a) and 4-methylthiophene-2-carboxylic acid (16b). Silver(I)
oxide (4.6 g, 20 mmol) was stirred with mixture 19a,b (2.5 g, 20 mmol) and sodium hydroxide (3.2 g,

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80 mmol) in water (50 mL) for 16 h. The mixture was filtered, acidified (hydrochloric acid) and
extracted with dichloromethane. Drying, evaporation and recrystallisation (hexane) gave a mixture of
1
16a and 16b (2.30 g, 82%) as white crystals: mp 118-120 °C. H-NMR δ 2.30 [2.7 H, s, Me (16b)],
2.57 [0.3 H, s, Me (16a)], 6.95 [0.1 H, d, J = 4.7 Hz, 4-H (16a)], 7.13 [0.9 H, m, 5-H (16b)], 7.48 [1 H,
d, J = 5.1 Hz, 5-H (16a)], 7.69 [0.9 H, d, J = 1.6 Hz, 3-H (16b)]; MS m/z 143.0169 (M + H) (C6H7O2S
requires 143.0167).
Methyl 3-methylthiophene-2-carboxylate (17a) and methyl 4-methylthiophene-2-carboxylate (17b). 3-
Methylthiophene 14 (5.0 g, 51 mmol) was added slowly to butyl lithium (2.0 M in tetrahydrofuran,
28 mL, 56 mmol) in dry tetrahydrofuran (100 mL) at 0 °C. The mixture was stirred for 3 h at 0 °C,
before being added to methyl chloroformate (6.33 g, 67 mmol) in dry tetrahydrofuran (20 mL) at 0 °C.
The mixture was stirred for 16 h and poured onto ice. The organic layer was washed with water,
hydrochloric acid (1 M), aq. sodium hydrogen carbonate and water. Drying, evaporation and
1
distillation (ca. 1 torr) gave a mixture of 17a and 17b (5.00 g, 62%) as a colourless oil: H-NMR δ
2.28 [2.4 H, s, Me (17b)], 2.56 [0.6 H, s, Me (17a)], 3.86 (3 H, s, OMe), 6.91 [0.2 H, d, J = 5.1 Hz, 4-
H (17a)], 7.13 [0.8 H, m, 5-H (17b)], 7.38 [0.2 H, d, J = 5.1 Hz, 5-H (17a)], 7.60 [1 H, d, J = 1.6 Hz,
3-H (17b)].
Methyl 4-bromomethylthiophene-2-carboxylate (18). Mixture 17a,b (2.48 g, 16 mmol) was boiled
under reflux with dibenzoyl peroxide (80 mg, 330 μmol) and N-bromosuccinimide (2.83 g, 16 mmol)
in tetrachloromethane (40 mL) for 17 h. Additional dibenzoyl peroxide (50 mg, 210 μmol) was added
every 10 min for 1 h and then every 30 min (twice). Evaporation and chromatography (hexane/ethyl
1
acetate 9:1) gave 18 (170 mg, 5%) as a pale buff oil; H-NMR δ 3.89 (3 H, Me), 4.46 (2 H, s, CH2),
7.49 (1 H, d, J = 1.6 Hz, 5-H), 7.80 (1 H, d, J = 1.6 Hz, 3-H).
Methyl 4-(trifluoroacetamidomethyl)thiophene-2-carboxylate (19) and N,N-bis((2-methoxycarbonyl-
thien-4-yl)methyl)trifluoroacetamide (20). Trifluoroacetamide (570 mg, 5.1 mmol) in dry THF (2 mL)
was stirred with KOBut (570 mg, 5.1 mmol) in dry THF (2 mL) for 1 h. Compound 18 (170 mg,
0.72 mmol) in dry THF (2 mL) was added slowly and the mixture was stirred for 15 h. The
evaporation residue, in CH2Cl2, was washed with H2O, aq. HCl (1 M) and H2O. Drying, evaporation
and chromatography (CHCl3 / EtOAc 19:1) afforded 20 (10 g, 5%) as a yellow solid: IR (film) νmax
1
(NH), 1711 (CF3C=O), 1603 (amide I), 1541 (amide II) cm-1; H-NMR δ 3.89 (3 H, s, MeCO), 3.90
(3 H, s, MeCO), 4.50 (2 H, s, CH2), 4.54 (2 H, s, CH2), 7.34 (1 H, brs, 5-H), 7.36 (1 H, brs, 5-H), 7.57
19
(1 H, d, J = 1.6 Hz, 3-H), 7.60 (1 H, d, J = 1.6 Hz, 3-H); F-NMR δ -68.67 (3 F, s, CF3); MS m/z
422.0340 (M + H) (C16 H15F3NO5S2 requires 422.0344). Further elution gave 19 (70 mg, 35%) as a
yellow solid: mp 80-83°C; IR νmax 3305 (NH), 1703 (CF3C=O), 1562 (amide) cm-1; 1H-NMR δ 3.89 (3
H, s, Me), 4.53 (2 H, d, J = 5.8 Hz, CH2), 6.61 (1 H, br, NH), 7.45 (1 H, m, 5-H), 7.71 (1 H, d,
19
J = 1.7 Hz, 3-H); F-NMR δ -76.2 (3 F, s, CF3); MS m/z 268.0255 (M + H) (C9H9O3F3NS requires
268.0246).

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4-(Benzyloxycarbonylaminomethyl)thiophene-2-carboxylic acid (21). Compound 19 (200 mg,
0.75 mmol) was boiled under reflux with NaOH (300 mg, 7.5 mmol) in H2O (3 mL) and MeOH
(3 mL) for 16 h. The MeOH was evaporated. Benzyl chloroformate (380 mg, 2.2 mmol) was added at
0 °C and the mixture was stirred vigorously for 4 h, before being washed with EtOAc. The aqueous
layer was acidified (aq. HCl) and extracted with EtOAc. Drying, evaporation and chromatography
1
(EtOAc) afforded 21 (180 mg, 82%) as a white solid: mp 149-151 °C; H-NMR [(CD3)2SO] δ 4.08
(2 H, d, J = 5.5 Hz, CH2N), 5.02 (2 H, s, CH2O), 7.33-7.36 (5 H, m, Ph-H5), 7.57 (1 H, m, 5-H), 7.60
(1 H, d, J = 1.2 Hz, 3-H), 7.82 (1 H, t, J = 5.9 Hz, NH); MS m/z 315.0503 (M + Na)
12
(13C1 C13H13NNaO4S requires 315.0496), 314.0462 (M + Na) (12C14H13NNaO4S requires 3140463).
Methyl 4-(benzyloxycarbonylaminomethyl)thiophene-2-carboxylate (22). Compound 21 (500 mg,
1.7 mmol) was boiled under reflux with H2SO4 (1 mL) and MeOH (50 mL) for 2 d. Aq. NaHCO3 was
added until no further bubbling occurred. The evaporation residue, in CHCl3 was washed with H2O.
Drying and evaporation afforded 22 (430 mg, 82%) as a solid: 64-65ºC; 1H-NMR δ 3.88 (3 H, s, Me),
4.35 (2 H, d, J = 5.9 Hz, CH2N), 5.13 (3 H, br, CH2O + NH), 7.34-7.38 (6 H, m, Ph-H5 +5-H), 7.71
(1 H, br, 3-H); MS m/z 306.0809 (M + H) (C15H16NO4S requires 306.0800).
Methyl 4-(aminomethyl)thiophene-2-carboxylate hydrobromide (23). Compound 22 (280 mg,
0.92 mmol) in AcOH (2.8 mL) was stirred with 30% HBr in AcOH (2.8 mL) for 20 min. The
evaporation residue was washed with Et2O (10 ×) and dried to afford 23 (230 mg, quant.) as a white
solid: mp 203-205 °C; NMR ((CD3)2SO) δΗ 3.85 (3 H, s, Me), 4.08 (2 H, s, CH2), 7.95 (1 H, d, J = 1.6 Hz,
5-H), 7.97 (1 H, m, 3-H), 8.13 (3 H, br, N+H3); MS m/z 172.0439 (M + H) C7H10NO2S requires
172.0432); Found C, 33.20; H, 3.88; N, 5.44; C7H9NO2S·HBr requires C, 33.33; H, 4.00; N, 5.56%.
Methyl 4-(isothiocyanatomethyl)thiophene-2-carboxylate (24). Compound 23 (200 mg, 0.8 mmol) was
stirred with CaCO3 (90 mg, 0.93 mmol) and thiophosgene (180 mg, 1.6 mmol) in H2O (2.0 mL) and
CHCl3 (10 mL) for 20 h. The solvent was evaporated from the organic layer. Chromatography
(CH2Cl2) afford 24 (104 mg, 62%) as a white solid: mp 69-70 °C: IR νmax 2115 (NCS), 1711 (C=O)
cm-1; 1H-NMR δ 3.90 (3 H, s, Me), 4.71 (2 H, s, CH2), 7.48 (1 H, dt, J = 2.0, 0.8 Hz, 5-H), 7.75 (1 H,
d, J = 2.0 Hz, 3-H); MS m/z 214 (M + H); Found C, 45.30; H, 3.37; N, 6.66. C8H7NO2S2 requires C,
45.03; H, 3.31; N, 6.57%.
Methyl 4-(thioureidomethyl)thiophene-2-carboxylate (25). NH3 was bubbled through 24 (60 mg,
0.28 mmol) in CH2Cl2 (3 mL) at 0°C for 20 min. The mixture was stirred for 3 hr at 0 °C. Evaporation
afforded 25 (61 mg, 95%.) as a white solid: mp 131-132 °C: 1H-NMR δ 3.87 (3 H, s, Me), 4.64 (2 H,
br, CH2), 5.98 (2 H, br, NH2), 6.77 (1 H, br, NH), 7.46 (1 H, br, 5-H), 7.72 (1 H, d, J = 1.6 Hz, 3-H);
MS m/z 231.0272 (M + H) C8H11N2O2S2 requires 231.0262); Found C, 41.39; H, 4.29; N, 11.96;
C15H15NO4S requires C, 41.72; H, 4.38; N, 12.16%.

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3.3. Enzyme study
Measurements of the inhibitory activity of the test compounds against nNOS was perfomed using
an enzyme preparation from rat brain (in which the large majority of the NOS activity is nNOS),
whereas the assay of activity against iNOS was performed using preparation of recombinant human
iNOS (hiNOS) overexpressed in an HT1080 cell line as described previously by us [12].
The assay used the conversion of L-[U-14C]-arginine to L-[U-14C]-citrulline as described previously
by us [16]. Compounds were evaluated at 100 μM and assays were performed in two modes,
simultaneous addition of the test compound to the enzyme preparation and of L-[U-14C]-arginine (to
start the enzymic reaction) and pre-incubation of the test compound with the enzyme preparation for
10 min before initiation of the enzymic reaction by addition of L-[U-14C]-arginine.
Briefly, for the studies with human iNOS, the enzyme was prepared as follows: an optimised
mammalian expression vector (pEFIRES-P, courtesy of Dr. S. Hobbs, Cancer Research UK Centre for
Cancer Therapeutics, ICR, London, U.K.) was designed to express human iNOS cDNA (courtesy of
Prof. Ian Charles, University of London, London, U.K.). 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 iNOSexpressing clones were produced. To avoid loss of viability through the
cytotoxic consequences of excessive NO production, clones were grown in the presence of a non-toxic
dose of NG-nitroarginine (100 mM). Routinely, clones were grown for 48 h in the absence of
puromycin and NG-nitroarginine 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 buffer containing HEPES (10 mM, pH
7.4), sucrose (320 mM), EDTA (100 mM), dithiothreitol (50 mM), leupeptin (10 mg mL-1), soybean
trypsin inhibitor (10 mg mL-1) and aprotinin (2 mg mL-1). The preparations were then sonicated using
an MSE Soniprep 150 for 35 s at a nominal frequency of 23KHz 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 centrifuged at 9,000g for 15 min at 4 °C. The post-mitochondrial
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 repeated 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 preparation. The results are shown in Table 1 as
the mean of triplicate experiments SEM.
4. Conclusions
In this work, we have extended the range of compounds that cause this apparent stimulation of
iNOS activity from benzylthioureas [12] into thienylmethylthioureas. These results are significant and
will be interesting for many scientists.

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Actually, the biochemical origin and mechanism of this stimulation is unclear, although a similar
but weaker stimulation was reported by Ulhaq et al. for S-2-amino-5-(3-nitro-1,2,4-triazol-1-
yl)pentanoic acid, S-2-amino-5-(3-amino-1,2,4-triazol-1-yl)pentanoic acid and S-2-amino-5-(2-
cyanoimidazol-1-yl)pentanoate on iNOS and nNOS [16]. On the other hand, it has been reported that
3-phenyl-3,4-dihydro-1-isoquinolinamine is a weak inhibitor of iNOS and nNOS [17]. However,
analogous 6-phenyl-4-amino-6,7-dihydrothienopyridines, in which the benzo ring has been replaced by
a thieno ring fusion, are potent iNOS and nNOS inhibitors [18].
Several synthetic guanidines and N-hydroxyguanidines can act as artificial substrates for nitric
oxide synthases [19–20]. However, since the assay used by us does not measure nitric oxide
production but rather measures conversion of radiolabelled L-Arg to radiolabelled L-Cit, it is clear that
the increased activity of the iNOS is a genuine stimulation. Therefore, one might speculate that these
compounds interact with the enzyme through more than one binding site under different conditions;
binding to one site would have a stimulatory effect while binding to the other would have inhibitory
effect. The binding to the allosteric stimulatory site may be fast, whereas inhibitory binding of some
known inhibitors (e.g. 1400W) to the catalytic haem site is known to be slow [10].
Recently, it was demonstrated that 1400W is not a slow-binding inhibitor but a time-dependent
inactivator of iNOS [22]. Therefore our result could be also explained as follows: Binding is rapid, but
initially reversible; the rate-determining step is inactivation of the enzyme by heme
modification. Therefore, the "loss" of stimulation after 10 minutes is because the enzyme is becoming
inactivated. This would then explain the stimulation effects reported without preincubation.
Although most effort in this area has gone into inhibiting biosynthesis of •NO, increasing
biosynthesis may have some therapeutic value. Nitric oxide blocks the expression of NADP oxidase,
an enzyme important in the etiology of cardiovascular disease [23]. Also, sildenafil augments •NO
concentrations by inhibiting phosphodiesterase-5 (PDE-5) and is useful in treating erectile disfunction
[24] and, possibly, pulmonary hypertension and acute respiratory distress syndrome (ARDS) [25].
Direct stimulation of NOS isoforms may provide an alternative approach to these beneficial effects.
Also, the thienylmethylthioureas and benzylthioureas reported here and previously [12] will be used
in a future work to provide a pharmacophore from which more potent and more site-selective ligands
may be designed by modeling and docking studies.
Acknowledgements
We are grateful to Ian J. Stratford and Edwin J. Chinje (University of Manchester, U.K.) for helpful
discussions and for allowing access to facilities for the biochemical assays. Also, the authors wish to
thank the Deanship of the Scientific Research at the University of Jordan for providing funds for the
ongoing research on the discovery of new selective and potent Nitric Oxide Synthase Ligands.
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Sample Availability: Samples of the compounds are available from the authors
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