5- and 6-Membered (thio)lactones are prodrug type carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.018

The inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) with (thio)coumarins has been recently reported (Maresca et al., J. Am. Chem. Soc. 2009, 131, 3057). Here we demonstrate that a series of γ- and δ-(thio)lactones also act as mechanism based, prodrug type CA inhibitors, similar to the (thio)coumarins. Through the esterase activity of CA, these compounds are hydrolyzed in situ to the corresponding hydroxy/keto/mercapto acids which thereafter act as inhibitors. CA isoforms I and IX were efficiently inhibited by simple such compounds, with K_Is in the range of 0.92-19.1 μM, whereas CA II was not inhibited at all. Isoform-selective CA inhibitors which spare the ubiquitous off-target CA II may have interesting applications for example for selectively inhibiting the tumor-associated CA IX, a validated anticancer target.

Full text of DOI:10.1016/j.bmcl.2011.11.018

Bioorganic & Medicinal Chemistry Letters 22 (2012) 267–270
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5- and 6-Membered (thio)lactones are prodrug type carbonic
anhydrase inhibitors
⇑
Fabrizio Carta, Alfonso Maresca, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) with (thio)coumarins has been
Received 10 October 2011
Revised 4 November 2011
Accepted 6 November 2011
Available online 11 November 2011
recently reported (Maresca et al., J. Am. Chem. Soc. 2009, 131, 3057). Here we demonstrate that a series
of
thio)coumarins. Through the esterase activity of CA, these compounds are hydrolyzed in situ to the
corresponding hydroxy/keto/mercapto acids which thereafter act as inhibitors. CA isoforms I and IX were
efficiently inhibited by simple such compounds, with K s in the range of 0.92–19.1 M, whereas CA II was
c- and d-(thio)lactones also act as mechanism based, prodrug type CA inhibitors, similar to the
(
I
l
Keywords:
Carbonic anhydrase
Lactone
Thiolactone
Coumarin
not inhibited at all. Isoform-selective CA inhibitors which spare the ubiquitous off-target CA II may have
interesting applications for example for selectively inhibiting the tumor-associated CA IX, a validated
anticancer target.
Ó 2011 Elsevier Ltd. All rights reserved.
Prodrug
Esterase activity
Isoform-selective inhibitor
The classical inhibitors of the metalloenzyme carbonic anhy-
drase, CA (EC 4.2.1.1) are the primary sulfonamides, RSO NH
and their isosteres, such as the sulfamates/sulfamides) which
the Australian plant Leionema ellipticum)3a as well as simple cou-
marin and many of their diversely substituted congeners in differ-
ent positions of the heterocyclic ring, are hydrolyzed within the CA
2
2
(
are in clinical use for more than 50 years as diuretics and system-
ically acting antiglaucoma drugs.1 In fact there are around 30 clin-
ically used drugs (or agents in clinical development) belonging to
active site with formation of the 2-hydroxy-cinnamic acids, which
,2
3,4
represent the de facto enzyme inhibitors.
The 2-hydroxy-cin-
namic acids formed in this way occlude the entrance to the enzyme
active site, a mechanism never evidenced before for any other CA
1
,2
these classes, which show significant CA inhibitory activity.
4
Apart their use as diuretics and antiglaucoma agents, recently it
has emerged that they have potential as anticonvulsant, antiobe-
sity, anticancer, and antiinfective drugs.1 However barriers to
the design of CA inhibitors (CAIs) as therapeutic agents are related
to the high number of isoforms present in mammals (i.e., 16 CAs, of
which 13 have catalytic activity), their rather diffuse localization in
many tissues/organs, and the lack of isozyme selectivity of most of
the presently available inhibitors of the sulfonamide/sulfamate
type (although sulfonamides which selectively inhibit CA IX over
CA II, CA VA/B over CA II or CA VII over CA II have been recently
inhibitor. Thiocoumarins were then showed to behave similarly.
Several interesting facts emerged during these studies3 : (i) this
new class of CAIs binds in hydrolyzed form at the entrance of the
CA active site (which differs significantly between the various iso-
forms) and does not interact with the metal ion, constituting thus a
new category of mechanism-based inhibitors; (ii) the formed
substituted-cinnamic acids were observed bound within the CA
active site either as the cis- or trans-2-hydroxycinnamic acids
(although these derivatives are stable in solution only as trans iso-
mers); (iii) a very high level of isoform selectivity has been
observed for many coumarins/thiocoumarins assayed so far as
,4
,2
3
–7
reported).
Crucial advances have been ultimately made in this
field, with many novel chemotypes reported to behave as potent
CAIs, with CA II-selective, CA IX-selective, CA XII-selective, CA
CAIs in addition to the sulfonamides,3
,4,7–10
among which the
XIII-selective inhibitors detected so far.
3,4,9,10
This has been ex-
coumarins/thiocoumarins are undoubtedly the most interesting
plained by the fact that these inhibitors bind in the most variable
region of the CA active site, at its entrance, unlike the sulfonamides
and their isosteres which bind deep within the active site cavity
which is very much conserved between all the catalytically active
3
,4
ones. Coumarin and thiocoumarins were in fact recently discov-
ered to act as CAIs, and their inhibition mechanism deciphered in
3
,4
detail. We demonstrated that the natural product 6-(1S-hydro-
xy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (isolated from
7
–10
isoforms.
Recently, this group also showed that a substituted
coumarin CA IX-selective potent inhibitor also shows strong
anticancer activity in vitro, inhibiting the growth of both primary
tumors and metastases in an animal model of breast cancer.11
⇑
Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.018

2
68
F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 267–270
Considering that coumarins are benzopyranones, that is, benzo-
annealed d-lactones, we decided to investigate the aliphatic - and
and they are also drug targets for some pathologies, but quite
1
,2
c
often, also off-targets. CA IX is on the other hand an anticancer
drug target useful for the treatment and imaging of hypoxic can-
cers, with several agents targeting CA IX in Phase I–III clinical
d-lactones (and their thioanalogues) for possible CA inhibitory
activity. Here we report that these compounds constitute indeed
a new class of CAIs which like the coumarins/thiocoumarins, seem
to be mechanism-based inhibitors, that is, to be hydrolyzed within
the enzyme active site and the inhibition to be achieved by the
1
2,13
trials.
As for the coumarins, we have incubated compounds 1–9 with
the three CA isoforms for periods of 15 min–24 h, and then asay
(
thio)lactone hydrolysis product.
the CA inhibition (for the physiologic reaction, i.e., CO
2
hydration
1
4
to bicarbonate) by a stopped flow method. As for the coumarins
O
3
,4,10,11
KOH
investigated earlier,
we have observed very low inhibitory
K
activity after 15 min incubation time (which is the normal one
HO
O
O
O
1
4
for assaying sulfonamide CAIs, data not shown) but the inhibi-
tory power of compounds 1–9 started to be enhanced by prolong-
ing the incubation time. A progressive increase of the inhibition
has been observed up to 6 h incubation time, after which the inhib-
itory activity was stable (data not shown), exactly as for the cou-
1
1a
The structures of the nine (thio)lactones 1–9 investigated here
as possible CAIs are shown above. Both 5- and 6-membered ring,
saturated compounds (4 and 5), as well as derivatives incorporat-
ing one (1, 6 and 7) or two double bonds (2, 3, 8 and 9) have been
included in our study. Only the thiolactones with exocyclic sulfur
were investigated so far (derivatives 8 and 9) for their interaction
with CAs.
3
,4
marins. Data shown in Table 1 are those after 6 h incubation of
enzyme with the (thio)lactone (or coumarin). Indeed, we also in-
cluded the inhibition data of the three CAs with the coumarin 10,
determined in exactly the same conditions as for the (thio)lactones
1
–9. It may be observed that human (h) isoforms hCA I and hCA IX
are inhibited by (thio)lactones 1–9 generally in the low micromo-
lar range, whereas hCA II is not inhibited at all (K s >100 M). Thus,
best activity has been observed against hCA IX, for which com-
pounds 1–9 showed K s in the range of 0.92–9.51 M. The most ac-
tive inhibitor was -valerolactone 5 and 5,6-dihydro-2H-pyran-2-
I
l
R
I
l
c
O
1
O
R
O
O
O
4
O
one 1 against the tumor-associated isoform. Against hCA I the
activity range was wider, with several compounds being low
2: R = H
: R = Me
micromolar inhibitors (e.g., 5 and 8, K
effective inhibitors, with K s <10 M (1 , 2, 4, 9) whereas the remain-
ing ones (3, 6 and 7) were less effective hCA I inhibitors (K s of
3.6–68.7 M)—see Table 1. It is interesting to note that the cou-
marin 10 was a rather effective, low micromolar hCA I and II inhib-
itor (K s of 3.1–9.2 M) but was ineffective as a hCA IX inhibitor (K
100 M). It is clear that both saturated as well as unsaturated 5-
I
s <5 lM), other being still
3
R
I
l
I
R
1
l
O
O
R
O
S
O
O
I
l
I
>
l
6: R =H
: R = OMe
8
: R = H
9: R = Me
5
7
and 6-ring membered lactones/thiolactones may lead to effective
CAIs (at least against isoforms hCA I and IX) and that the presence
of different substituents on these rings strongly influences enzyme
inhibitory activity. For example, the presence of the methoxy
group in 7 leads to a strong loss of activity against hCA I of this
Three CA isoforms were included in this study, as shown in
Table 1: the cytosolic CA I and II and the transmembrane, tumor-
associated CA IX. The reasons for investigating them are rather
straightforward: CA I and II are the most abundant CAs in the body
compound compared to the parent, unsubstituted
c-lactone 6,
whereas against hCA IX the activity of the methoxy-derivative 7
is slightly improved compared to the unsubstituted compound 6.
The same phenomenon has been observed for coumarins, where
the substitution pattern on the coumarin ring strongly influenced
3
,4,9,10
Table 1
enzyme inhibitory activity and isoform-selectivity.
CA inhibition data with (thio)lactones 1–9 and 5-hydroxy-2-pentenoic acid 1a by a
We wanted to understand whether the inhibition mechanism of
CAs with (thio)lactones resembles the coumarin inhibition of these
enzymes. We have demonstrated earlier that coumarin 10, is being
stopped-flow CO
2
hydrase assay. Compouds were preincubated with enzyme for
5 min–24 h. Data for 1–9 are at 6 h preincubation time whereas the ones for 1a at
5 min incubation time. The inhibition data with the standard coumarin inhibitor 10
1
1
are also included
a
Compound
K
I
(l
M)
hCA I
hCA II
hCA IX
1.35
1
2
3
4
5
6
7
8
9
8.03
7.97
19.1
9.60
4.69
13.6
68.7
2.48
7.52
8.46
3.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.2
COOH
1.60
2.47
3.66
0.92
4.42
3.38
7.45
9.51
1.15
O
O
OH
10
10a
COOH
1
1
1
a
0
0a
b
>100
>100
O
1
O
HO
b
3.4
9.3
1a
a
Errors were in the range of 5–10% of the reported data (from 3 different assays,
data not shown).
Scheme 1. CA-mediated-hydrolysis of coumarin 10 to 2-hydroxy-trans-cinnamic
acid 10a and of lactone 1 to 5-hydroxy-2-pentenoic acid 1a.
b
From ref.3

F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 267–270
269
hydrolyzed to the trans-2-hydroxycinnamic acid 10a,3 by the CA
esterase activity. Thus, lactone 1 has been hydrolyzed to the corre-
sponding hydroxyl acid 1a (Scheme 1) which has been isolated,
purified and assayed as a CA I (Table 1). Data of Table 1 show that
lactone 1 and hydroxyl acid 1a have basically the same CA inhibi-
tory activity against the three investigated isoforms (within the
limits of the experimental errors of the assay method), which is ex-
actly the situation observed for the coumarin 10 and its opened
form, trans-2-hydroxycinnamic acid 10a. We are thus confident
that (thio)lactones possess a CA inhibition mechanism similar to
that of coumarins, that is, they act as prodrugs, being hydrolyzed
by the esterase activity of these enzymes, with formation of hydro-
xy/keto/mercapto acids which thereafter act as inhibitors. We are
however not sure whether these compounds bind in the same ac-
tive site region as the hydrolyzed coumarins, or whether they coor-
dinate to the Zn(II) ion of the enzyme. In fact both situations are
plausible, since the antiepileptic lacosamide 11 (an aliphatic com-
pound) was found bound at the entrance of the hCA II active site, in
the coumarin binding region,15 but some carboxylates, such as ace-
tate, were found coordinated to the Zn(II) ion in the X-ray crystal
24, 1; (b) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.;
Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol. 2009, 92, 423; (c)
Ahlskog, J. K. J.; Dumelin, C. E.; Trüssel, S.; Marlind, J.; Neri, D. Bioorg. Med.
Chem. Lett. 2009, 19, 4851; (d) Ahlskog, J. K.; Schliemann, C.; Mårlind, J.;
Qureshi, U.; Ammar, A.; Pedleym, R. B.; Neri, D. Br. J. Cancer 2009, 101, 645.
Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med.Chem. 2010, 53, 5511.
6
.
.
7
Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. X-Ray
Crystallography of CA Inhibitors and its Importance in Drug Design. In Drug
Design of Zinc-Enzyme Inhibitors: Functional, Structural and Disease Applications;
Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009; pp 73–138.
(a) Švastová, E.; Hulıkova, A.; Rafajová, M.; Zat’ovi cˇ ová, M.; Gibadulinova, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.; Pastorek, J. FEBS Lett. 2004,
´
8
.
5
77, 439; (b) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.;
Vaughan-Jones, R. D. J. Biol. Chem. 2008, 283, 20473; (c) Pacchiano, F.; Carta, F.;
McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.; Dedhar, S.; Supuran, C. T. J.
Med. Chem. 2011, 54, 1896.
Maresca, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 4511.
0. Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
9.
1
7255.
11. Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle,
A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.;
Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.;
Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.;
Dedhar, S. Cancer Res. 2011, 71, 3364.
12. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De
Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233.
16
structures of some isoforms, such as CA XIII. Further studies are
necessary thus to decipher the detailed inhibition mechanism of
CAs with (thio)lactones.1
1
3. Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767.
14. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. An applied photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO hydration
7,18
2
activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
R
O
R
O
as buffer, and 20 mM Na
following the initial rates of the CA-catalyzed CO
period of 10–100 s. The CO concentrations ranged from 1.7 to 17 mM for the
2
SO
4
(for maintaining constant the ionic strength),
2
hydration reaction for a
Lawesson's Reagent
Toluene
R=R1= H; 8
2
R=R = Me; 9
determination of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled-deionized water with 1–2%
DMSO, and dilutions up to 1 nM were done thereafter with distilled-deionized
water. Inhibitor and enzyme solutions were preincubated together for 15 min–
1
R1
S
R1
O
OMe
O
7
2 h at room temperature (15 min) or 4 °C (all other incubation times) prior to
H
N
assay, in order to allow for the formation of the E-I complex or for the eventual
active site mediated hydrolysis of the inhibitor. Data reported in Table 1 show
the inhibition after 6 h incubation, which led to the completion of the in situ
hydrolysis of the (thio)lactone/coumarin and formation of the hydrolysis
products.3 The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3, as reported earlier,3 and represent the mean from at
least three different determinations. CA isoforms were recombinant ones
obtained in house as reported earlier.3
N
H
O
1
1: lacosamide
b,4
b,4
1
1
1
5. Temperini, C.; Innocenti, A.; Scozzafava, A.; Parkkila, S.; Supuran, C. T. J. Med.
In conclusion, we report here a new class of mechanism-based
Chem. 2010, 53, 850.
CA I, the (thio)lactones, which similar to the coumarins are hydro-
lyzed by the CA esterase activity, and the formed products than act
as low micromolar inhibitors of some isoforms, but not of CA II, an
off-target isozyme. Considering that the tumor-associated CA IX
was the mostly inhibited isoform by this new class of CAIs, and that
this is a recently validated antitumor target, this discovery brings
interesting prospects for designing CA IX-selective agents belong-
ing to novel chemotypes.
6. Di Fiore, A.; Monti, S. M.; Hilvo, M.; Parkkila, S.; Romano, V.; Scaloni, A.;
Pedone, C.; Scozzafava, A.; Supuran, C. T.; De Simone, G. Proteins 2009, 74, 164.
7. Lactones 1–7 were commercially available (Sigma–Aldrich, Milan, Italy)
whereas the thiolactones 8 and 9 were prepared from the corresponding
lactones 2 and 3 by thionylation with Lawesson’s reagent. Nuclear magnetic
1
13
( H, C) spectra were determined in D O or DMSO and were recorded using a
2
Bruker Advance III 400 MHz instrument. The chemical shifts (d scale) are
reported in parts per million (ppm) and the coupling constants (J) are
expressed in hertz (Hz). Splitting patterns are designated as follows: m,
multiplet. Thin layer chromatography (TLC) was carried out on Merck Silica Gel
6
0 F254 aluminum backed plates. Elution of the plates was carried out using
ethyl acetate/n-hexane systems. Visualization was achieved with UV light at
54 nm, by dipping into a 0.5% aqueous potassium permanganate solution, by
Acknowledgment
2
Hanessian’s stain solution and heating with a hot air gun or by exposure to
iodine. 5,6-Dihydropyran-2-one for synthesis is commercially available from
Sigma–Aldrich (Milan, Italy), and was used without further purification. All
other solvents and chemicals were used as supplied from Aldrich Chemical Co.,
Acros, Fisher, Alfa Aesar or Lancaster Synthesis. All CA isozymes were
recombinant ones produced and purified in our laboratory as described
This research was financed by an FP7 EU project (Metoxia).
References and notes
4,8–10
1.
2.
3.
(a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3467.
(a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (b)
Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3233.
(a) Vu, H.; Pham, N. B.; Quinn, R. J. J. Biomol. Screen. 2008, 13, 265; (b) Maresca,
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Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057.
earlier.
Synthesis of potassium (E)-5-hydroxypent-2-enoate 1a.18
5,6-Dihydropyran-2-one 1 (0.2 g, 2.1 mmol) was dissolved in 2 mL of water.
Potassium hydroxide (0.056 g, 2.6 mmol) was added and the reaction mixture
was left stirring at room temperature until complete (TLC monitoring). The
solvent was then removed in vacuo to obtain a light brown hygroscopic solid in
80% yield. Silica gel TLC R
O) 2.40 (3H, m, 4-H ), 3.71 (2H, t J 6.4, 5-H
6.58 (1H, dt J 15.6, 6.8, 3-H); d (100 MHz, D O) 176.2, 141.9, 129.1, 60.9, 34.9.
Synthesis of 2H-pyran-2-thione 8 and 4,6-dimethyl-2H-pyran-2-thione 9.
2H-Pyran-2-one or 4,6-dimethyl- -pyrone (0.1 g, 1.0 eq) was dissolved in dry
f
0.01 (Ethyl acetate /n-hex 50%, v/v); d
H
(400 MHz,
4
.
.
Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C.
T. J. Med. Chem. 2010, 53, 335.
(a) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.; Kienninger,
J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin, P.; Wouters, B. G.;
Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.; Gasmann,
M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme Inhib. Med. Chem. 2009,
D
2
2
2
), 5.89 (1H, dt J 15.6, 1.6, 2-H),
c
2
19
5
a
toluene (3.0 ml) and Lawesson’s reagent (1.5 eq) was added. The mixture was
stirred at 80 °C under a nitrogen atmosphere until starting material was

2
70
F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 267–270
1
9
consumed (TLC monitoring) and then solvent was removed under vacuo. The
residue was partitioned between H O and ethyl acetate, the aqueous layer was
extracted with ethyl acetate (3 Â 15 ml). The organic layer was dried over
Na SO , filtered and concentrated under vacuo to afford a solid that was
4,6-Dimethyl-2H-pyran-2-thione 9: 75% yield; m.p. 72–74 °C (Lit. 70.5 °C
sublimation); silica gel TLC R
À1
2
f
0.59 (Ethyl acetate/n-hexane 50% v/v); m cm
) 2.13 (3H, s), 2.37 (3H, s), 6.60 (1H, s,
6
(100 MHz, DMSO-d ) 197.5 (C@S), 168.2, 154.2,
(KBr) 1645, 1540; d (400 MHz, DMSO-d
H
6
2
4
5-H), 7.06 (1H, s, 3-H); d
C
purified by silica gel column chromatography eluting 50% ethyl acetate/n-
hexane v/v.
127.4, 112.1, 21.3, 20.4.
18. Manning, P.T.; Misko, T.P. Preparation of amino acid derivatives as inhibitors of
inducible nitric oxide synthase for use in combination therapy with alkylating
agents. WO Pat. 2005, 025620 A2.
19. Defoin, A.; Augelmann, G.; Fritz, H.; Geffroy, G.; Schmidlin, C.; Streith, J. Helv.
Chim. Acta 1985, 1998.
1
9
2
H-Pyran-2-thione 8: 27% yield; m.p. 53–55 °C (Lit. 50.5–51.0 °C benzene/
À1
cyclohexane); silica gel TLC R
KBr) 1630, 1535; d (400 MHz, DMSO-d
, 5-H), 7.30 (1H, d, J 8.0, 6-H), 6.85 (1H, m, 4, 5-H); d
98.4 (C@S), 160.2, 133.4, 133.0, 102.0.
f
0.18 (Ethyl acetate/n-hexane 20% v/v); m cm
(
4
1
H
6
) 8.29 (1H, d, J 8.0, 3-H), 7.46 (1H, m,
(100 MHz, DMSO-d
C
6
)