Novel route to chaetomellic acid A and analogues: Serendipitous discovery of a more competent FTase inhibitor

Article abstract of DOI:10.1016/j.bmc.2012.10.034

A new practical route to chaetomellic acid A (ACA), based on the copper catalysed radical cyclization (RC) of (Z)-3-(2,2-dichloropropanoyl)-2- pentadecylidene-1,3-thiazinane, is described. Remarkably, the process entailed: (i) a one-pot preparation of the intermediate N-α-perchloroacyl-2-(Z)- alkyliden-1,3-thiazinanes starting from N-(3-hydroxypropyl)palmitamide, (ii) a two step smooth transformation of the RC products into ACA and (iii) only one intermediate chromatographic purification step. The method offers a versatile approach to the preparation of ACA analogues, through the synthesis of an intermediate maleic anhydride with a vinylic group at the end of the aliphatic tail, a function that can be transformed through a thiol-ene coupling. Serendipitously, the disodium salt of 2-(9-(butylthio)nonyl)-3-methylmaleic acid, that we prepared as a representative sulfurated ACA analogue, was a more competent FTase inhibitor than ACA. This behaviour was analysed by a molecular docking study.

Full text of DOI:10.1016/j.bmc.2012.10.034

Bioorganic & Medicinal Chemistry 21 (2013) 348–358
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel route to chaetomellic acid A and analogues: Serendipitous discovery of
a more competent FTase inhibitor
a
b
c
a
a,
⇑
Franco Bellesia , Seoung-ryoung Choi , Fulvia Felluga , Giuliano Fiscaletti , Franco Ghelfi ,
a
d
b
a
e
Maria Cristina Menziani , Andrew F. Parsons , C. Dale Poulter , Fabrizio Roncaglia , Massimo Sabbatini ,
Domenico Spinelli
f
a
Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Modena, Via Campi 183, I-40125 Modena, Italy
Department of Chemistry, University of Utah, 315 South 1400 East RM 2020, Salt Lake City, UT 84112, USA
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, Via L.Giorgeri 1, I-34127 Trieste, Italy
Department of Chemistry, University of York, Heslington, York YO10 5DD, UK
Dipartimento di Patologia Sistematica, Università degli studi di Napoli ‘‘Federico II’’, Via S. Pansini 5, I-80131 Napoli, Italy
Dipartimento di Chimica ‘‘G. Ciamician’’, Università degli Studi di Bologna, Via Selmi 2, I-40126 Bologna, Italy
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2012
Revised 28 September 2012
Accepted 2 October 2012
Available online 29 October 2012
A new practical route to chaetomellic acid A (ACA), based on the copper catalysed radical cyclization (RC)
of (Z)-3-(2,2-dichloropropanoyl)-2-pentadecylidene-1,3-thiazinane, is described. Remarkably, the pro-
cess entailed: (i) a one-pot preparation of the intermediate N-a-perchloroacyl-2-(Z)-alkyliden-1,3-thia-
zinanes starting from N-(3-hydroxypropyl)palmitamide, (ii) a two step smooth transformation of the
RC products into ACA and (iii) only one intermediate chromatographic purification step. The method
offers a versatile approach to the preparation of ACA analogues, through the synthesis of an intermediate
maleic anhydride with a vinylic group at the end of the aliphatic tail, a function that can be transformed
through a thiol–ene coupling. Serendipitously, the disodium salt of 2-(9-(butylthio)nonyl)-3-methylma-
leic acid, that we prepared as a representative sulfurated ACA analogue, was a more competent FTase
inhibitor than ACA. This behaviour was analysed by a molecular docking study.
Keywords:
Chaetomellic acid A
Farnesyl pyrophosphate
FTase inhibitors
Modelling studies
Radical cyclization
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
Farnesylation is required for the subcellular localization and
transformation activity of the oncogenic variants of Ras.1
a,2c,5
Con-
Prenylation of proteins with polyisoprenoids is an important
sequently, the inhibition of farnesylation prevents localization of
these proteins at the cell membrane, thereby prohibiting cell trans-
formation. For this reason, the development of farnesyltransferase
(FTase) inhibitors have emerged as novel class of pharmaceutical
post-translational modification, which plays a major role in cell
1
proliferation of both normal and cancerous cells. Proteins, which
undergo prenylation, are all characterized by a CAAX motif at their
carboxy terminus, where C is a cysteine residue, A is any aliphatic
amino acid, and X could be variable (usually alanine, serine, methi-
1
a,6
agents in noncytotoxic anticancer therapy.
Enzymatic and crys-
tallographic techniques have explained the mechanism of FTase
catalysis, demonstrating that farnesylation proceeds via an ordered
mechanism with farnesyl pyrophosphate (FPP) binding first, fol-
lowed by the CAAX moiety of the Ras-protein and then by the far-
1
,2
onine, or glutamine).
Prenylation can occur via the covalent
attachment of either a 15-carbon farnesyl moiety or a 20-carbon
geranylgeranyl moiety to the cysteine of CAAX motif-containing
proteins, the process being catalysed by farnesyltransferase (FTase)
1
d,6,7
nesyl transfer to the cysteine residue C.
1d
or geranylgeranyltransferase (GGTase) enzymes. Geranylgerany-
lation is found to be responsible for the prenylation of 80–90% of
Inhibitors design is largely based on the CAAX motif, the
farnesyl moiety, or both (in the latter case inhibitors mimic the
3
1b,1c,4
1a,1d,6a,8
prenylated proteins; whereas, lamins A and B,
the fungal
subunit of heterotrimeric
are farnesylated.
transition state).
Also several natural products are able
These substances have been generally
2
a
2e,2f
1a,9
mating factor,
c
-transducin,
G-proteins and Ras-proteins
the
c
to inhibit the FTase.
4
2c,4
⇑
Corresponding author. Tel.: +39 059 2055049; fax: +39 059 373543.
E-mail address: franco.ghelfi@unimore.it (F. Ghelfi).
Figure 1. Chaetomellic acid A (1).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.034

F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
349
identified through screening program of microbial natural prod-
9
ucts. It was just during one of these screening tests that a Merck’s
team isolated, from extracts of the coelomycete Chaetomella
acutiseta, chaetomellic acid A (ACA) 1 (Fig. 1),10 a dicarboxylic acid
that inhibited, in its dianionic form 2, the recombinant human
FTase, with an IC50 value of 55 nM.11 Chaetomellic acid A (1) has
a high tendency to cyclize and, in fact, it was isolated as anhydride
1
0a
3.
The cyclic form, however, is unstable under mild basic
conditions (pH 7.5) and is readily hydrolysed to the dicarboxylate
10a
anion 2, the biologically active component (Scheme 1).
Figure 2. Structural similarity between the dianionic form of chaetomellic acid A 2
FTase activity of the diacid anion of 1 is noncompetitive to-
wards the acceptor peptide Ras, but is highly competitive with re-
spect to farnesyl pyrophosphate (FPP).11 This may be explained by
the structural similarity between the dicarboxylate anion 2 and
FPP, since both possess a hydrophilic head group bound to a hydro-
and farnesyl pyrophosphate (FPP).
1
8a,19
(
i) alkylation of maleic precursors
and (ii) assembly of the
2
0
pivotal 1,4-dicarbonyl group.
1
0a
Singh prepared a number of analogues of 1, characterized by a
modified hydrophobic tail (having one C@C double bond or a
shorter chain length) or by the replacement of the methyl in the
polar head with H, hydroxymethyl or larger groups. No modified
ACA, however, showed more potent inhibition than 2 against the
recombinant human FTase. Also the groups of Vederas and Poulter
phobic tail (Fig. 2).
The maleate unit aligns well with the corre-
sponding diphosphate moiety, since the negatively charged oxygen
atoms can achieve a spacing within 0.1 Å,12 while the difference in
2
0c
the distance between the carboxylic carbons and between the
phosphorus atoms is only 0.4 Å.10a The flexible nature of the ali-
phatic chain, instead, permits it to fill the same space, as the hydro-
phobic end of FPP, upon binding to the enzyme.
25
synthesized some ACA analogues, namely those with R = n-C12H ,
1
9a
farnesyl, or geranylgeranyl. These substances were evaluated for
inhibition of the yeast FTase and, unlike Singh’s observations, the
compounds with the shorter tail showed greater activity than
ACA (1).
Unfortunately 1 did not inhibit Ras processing in ras-trans-
formed NIH3T3, perhaps because of poor penetration in these cells
or its untimely enzymatic transformation.11 However, Bach, on
studying the capacity of branch-specific inhibitors of the cytosolic
isoprenoid pathway (downstream from mevalonic acid) to block
cell cycle progression in tobacco BY-2 cells, observed that ACA
In spite of the variety of known methods, all of the reported
synthetic procedures to 3 suffer from one or more of the following
disadvantages: (i) low yields, (ii) costly reagents, (iii) unstable pre-
cursors and/or reactants, (iv) harmful solvents, (v) unwieldy proto-
cols, or (vi) lack of versatility.
(
1) behaved like a true cell cycle inhibitor, in that it led to a specific
1
3
arrest in the cell cycle. Vederas, instead, in an effort to character-
ize the FPP binding site of rubber transferase showed that 1 is able
We tried to overpass these disadvantages and a few years ago
devised an interesting way to prepare 3, where the anhydride moi-
ety was constructed through the atom-transfer radical cyclization
(ATRC) of N-(benzyl)- or better N-(2-pyridyl)-N-(3-chloro-2-prope-
nylamino)-2,2-dichlorohexadecanamides 4, followed by the func-
to inhibit, in vitro, rubber biosynthesis promoted by rubber trans-
ferase from Hevea Brasiliensis.14 Besides, the same author found
that 1 was also able to reduce the activity of the PBP1b (penicil-
lin-binding protein 1b), albeit with modest potency.15 Recently,
Sabbatini has shown that the inhibition of the Ras/ERK1/2 pathway
tional rearrangement (FR) of the intermediate trichloro
c
-lactams
by 1 resulted in a beneficial effect on acute ischemia-reperfusion
2
1a,21b
1
6
5 (Scheme 2).
The same strategy was then applied to
injury in rats, preserving either renal function and histology.
N-(2-pyridyl)-N-allyl-2,2-dichlorohexadecanamide 6 (Scheme 3),
the preparation of which uses a more accessible secondary allyla-
mine. The lack of the Cl atom in the allyl moiety, in this case, called
for the introduction of an oxidative extra step in the synthetic
These results were the consequence of the reduced apoptosis/
necrosis of renal cells observed after oxidative stress in rats, as also
shown both in human tubular and endothelial cells in culture. ACA
(1) selectively inhibited the membrane-bound Ha-Ras (a pro-
2
1c
route.
apoptotic pathway), since it did not alter the membrane-bound
Ki-Ras (an antiapoptotic pathway), nor different prenylated
16
intracellular proteins like Rab. Similar results have been also
described in an experimental murine model of ischemic stroke
(
exocytoxic lesion), in which ACA (1) administration increased
the intracellular concentration of inactive Ha-Ras, significantly
reducing the production of superoxide anion and the volume of
cerebral necrotic tissue, following the improved survival of hypoxic
1
7
neuronal cells.
Because of its biological activity1
0a,11,13–17
and potential employ-
ment as therapeutic agents,18 chaetomellic acid A (1) has been the
subject of considerable synthetic efforts. In general, the synthesis
of anhydride 3 has been targeted since this compound is easier
to manipulate/isolate than diacid 1. The synthetic strategies
investigated can be roughly grouped into two general categories:
Scheme 2. Reagents and conditions: (a) (1) (COCl)
benzyl-3-Cl-2-propenylamine (PG = Bn) or 2-(3-chloro-2-propenylamino)pyridine
PG = 2-Py), Py, 23 °C, 1 h. (b) CuCl-TMEDA, CH
CH
(2) H /H
2 2 2
, CH Cl , DMF, 23 °C, 2 h; (2) N-
1
0
(
2
3
CN, argon, 60 °C, 20 h. (c) (1) Na ,
O. (d) (1) KOH, CH OH/THF, reflux, 2 h;
OH/diethyl ether, 25 °C, 20 h; (2) H SO 4 N, 110 °C, 2 h.
+
Scheme 1. pH dependent equilibrium between the active dianionic open form 2
and the anhydride chaetomellic A (3).
3
OH/diethyl ether, 25 °C, 20 h; (2) H /H
2
3
+
0
2
O. (e) (1) Na , CH
3
2
4

3
50
F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
Scheme 3. Reagents and conditions: (a) CuCl–TMEDA, toluene, argon, 60 °C, 24 h.
0
(
b) Na , toluene/MeOH, 25 °C, 24 h. (c)
H
2
SO
procedure: (1) Na , toluene/MeOH, 25 °C, 24 h; (2) H
MnO , CH Cl , 25 °C, 20 h. (f) H SO /H O, 110 °C, 4 h.
4
/H
2
O, 110 °C, 3 h. (d) One-pot
0
2
SO /H O, 110 °C, 3 h. (e)
4
2
2
2
2
2
4
2
Both of these procedures have the same two important draw-
backs. The former is the use of long chain -perchlorocarboxylic
a
acids (these starting materials are not very easy to prepare), while
the latter is the lack of a perfect control of the ATRC stereoselectiv-
ity. This is a critical event for the economy of the process since the
FR reaction is viable only in the case of cis-lactams,2
1b,22
and the
cis/trans ratio of the
c
-lactams, delivered by the ATRC step, is
Scheme 4. Possible mechanism for the RC of N-
cyclic ketene-N,S-acetals A.
a-perchloroacyl six membered
acceptable only when these lactams carry a bulky substituent at
C-3 and a chloro- or a dichloro-methyl group at C-4. In fact, when
we tested the viability of the complementary strategy, in which the
methyl substituent is introduced through the acid reagent while
the long aliphatic chain is added through the allylamino moiety,
the stereoselectivity of the ATRC lactam was disappointing (the di-
the expected anhydrides F in a more than acceptable overall yield
from the starting enamides A (Scheme 5).
2
3b
The new process bypasses the stereoselectivity problem of the
ATRC step in the ATRC-FR method. Moreover its versatility is no
longer reliant on the somewhat exotic functionalized 2,2-dichloro-
acyl chlorides, but can rely on the more accessible functionalized
carboxylic acids. In addition we have recently developed an easy,
chloro-c-lactam from the cyclization of N-benzyl-N-[(E)-2-hexe-
nyl)]-2,2-dichloropropanamide was recovered as a 66/34 cis/trans
mixture of four diastereomers).21c Since a practical route to func-
tionalized 2,2-dichloro acids appeared not viable, the ATRC-FR ap-
proach to chaetomellic acid A analogues lost any appeal.
cheap and efficient preparation of short-chain
chlorides, from -perhalogenation of the corresponding unfunc-
tionalized acyl halides with Cl , using tetraalkylammonium chlo-
a-perchloroacyl
a
Now as part of a project about the acute kidney injury (AKI)
from ischaemia-reperfusion in rat, we were asked to develop a ver-
satile way to chaetomellic acid A (1), and analogues, for the pre-
vention of the ischemic damage, through the inhibition of the
pathway Ras/ERK1/2. Here we describe the new synthetic method
and the serendipitous discovery of an ACA analogue having a high-
er affinity for the FTase than the natural product.
2
2
4
ride as a catalyst.
However, the new method is still unpractical and unsuited for
large-scale preparation. The starting enamides A are secured
through acylation of 2-alkyl-5,6-dihydro-4H-1,3-thiazines, sub-
strates that can be obtained following a literature procedure,25
involving thionation of N-(3-hydroxypropyl)-carboxyamides with
the Lawesson’s reagent (LR). In our hands the preparation of 5,6-
dihydro-4H-1,3-thiazines was too expensive and characterized by
a susceptible and too lengthy work-up; furthermore LR has also a
repulsive high equivalent weight. In addition, the deprotection of
the thioacetal group in E (following the method of Hajipour)26
was exceedingly long (around 2 d) and used a large amount of solid
2
2
. Results and discussion
.1. Synthesis of ACA
To solve the intrinsic problems of the ATRC-FR paths to maleic
anhydrides, we have recently studied the copper catalyzed radical
cyclization (RC) of N- -perchloroacyl cyclic ketene-N,X(X = O, NR,
S)-acetals. The RC of N- -perchloroacyl-2-(Z)-alkyliden-1,3-thiaz-
inane A was shown to be, by far, the most efficient and selective
2
3
reagents (Scheme 5, step c).
a
After a search of literature, looking for a more appropriate
method for thionation of amides, our attention was attracted to
the use of cheap Berzelius reagent (P S10). This reagent is
4
a
2
3
reaction (Scheme 4). The catalytic cycle begins with the forma-
tion of a carbamoyl methyl radical, which leads to a cascade of
reactions, including a radical polar crossover step, culminating in
the formation of the maleimide nucleus (D and E), or of a direct
precursor of it (E) (Scheme 4). The typical large prevalence of E
over D likely means that the quenching of the acyliminium cation
B by C is kinetically favoured in comparison with the oxidative
homocoupling of C.
The disulfide D and thioacetal E, without being isolated, were
subjected to an oxidative treatment to reveal the C@O group,
masked as an S,S-acetal in E. The crude products from this step
were then hydrolyzed according to Argade’s method,1 affording
Scheme 5. Preparation of the 3,4-disubstituted-2,5-furandiones. Reagents and
conditions: (a) CuCl (5 mol %), TMEDA (10 mol %), CH CN, Na CO , argon, 17 h,
0 °C; (b) silica–sulfuric acid, NaNO , SiO /H O 3:2, CH Cl , 45 °C, 40 h; (c) KOH,
CH OH–THF, reflux 2 h; (d) HCl (10%), rt.
3
2
3
3
3
2
2
2
2
9c
3

F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
351
characterized by an attractive low equivalent weight,27 which,
when combined with hexamethyldisiloxane (HMDO), issues better
or comparable) results than LR.2
7,28
(
4 10
The combination of P S -
HMDO is known as the Curphey reagent (CR). HMDO has the
function to scavenge the reactive electrophilic polythiophosphates
(
thionation by-products), before they cause any side reaction.
Thionation of 7 was carried out with the CR in CH Cl at reflux
Scheme 6). For an effective transformation, however, the amount
2
2
(
of water present in the reaction mixture must be kept low. As 7 is
recovered by filtration from water suspensions, it is the major
source of humidity. Thus it is recommended to use a dry starting
hydroxyamide (e.g. re-crystallized material from diethyl ether).
Otherwise the H
the [N,O]-migration of the thioacyl or acyl moieties. The prob-
lems associated with the presence of H O can be also downsized
by working in open vessels, since the volatile H S has the chance
2
S, which comes from hydrolytic processes, causes
29
Scheme 7. Reactions of the N-2,2-dichloropropanoyl ketene-N,S-acetal 10.
Reagents and conditions: (a) CuCl (10 mol %), TMEDA (20 mol %), CH
3:2), Na CO , argon, 30 °C, 19–24 h; (b) KI, H O, rt, 24 h; (c) NaOH, THF/H
12 h, acidic work-up.
3
CN/toluene
2
(
2
O, rt,
2
3
2
2
to escape from the reaction mixture through the condenser.
Astonishingly the MS-spectra of the unique peak, observed dur-
ing the GC-MS analysis of the final reaction mixture, was peculiar
of the cyclic thioimidate 9 and not of the expected hydroxythioa-
mide 8. Soon we realized that this was indeed an instrumental
fake. We speculated that the thermal stress, to which 8 was sub-
jected in the injection chamber, resulted in cyclization to 9. In
fact on heating at 150 °C (2–3 h), under vacuum and a slight flow
of argon, the crude reaction mixture from the thionation step, after
evaporation of the volatiles, gave 9, also free of the silylated side-
products. The intermediate thioimidate is, however, somewhat
sensitive to hydrolysis, and cannot be chromatographed on silica-
gel. But it was indeed quite clean and was acylated, as such, using
carbonyl group from S,S-acetals,32 could be helpful. Particularly
+
attractive is the possibility to generate iodine (or I reagents)
3
3
in situ, through the oxidation of the iodide anion. After a number
of tests, we found it convenient to add KI (100 mg/5 mmol of 10)
and a few drops of water (5–10/5 mmol of 10) to the open Schlenk
tube, once the RC reaction was completed (Scheme 7, path ii). A
possible mechanism for the conversion of 12 into 11 is outlined
in Scheme 8. The process is intramolecular, as shown in the
scheme, when the ‘endo’ sulfur is attacked, otherwise it could be
intermolecular.
3
0
Finally, the hydrolysis of chromatographed 11, adapting Ar-
gade’s protocol, gave the targeted 3a in good yield (94%).1 More
appropriately, it was possible to carry out the hydrolytic step di-
rectly on the crude extract of 11 (Scheme 7, path iii). In this way,
since a purification step is spared, the process gives a higher global
yield (71% against 66%).
9c
2
,2-dichloropropanoyl chloride, affording the N-acyl-ketene-N,S-
acetal 10 (a relatively robust molecule, that was purified by chro-
matography) in high yield (80–90%) (Scheme 6). Because analogues
of N-acyl-ketene-N,S-acetals typically have a Z configured C@C
bond,2 the same geometry was assigned to 10, and to the other
enamides we prepared in this work.
3b
2
.2. A route for the synthesis of tailor made ACA analogues
Using a reasonably pure sample of 10 (a condition that has to be
maintained also with the other enamides we prepared), the radical
cyclization proceeded smoothly giving, as expected, the disulfide
Aiming to develop an intrinsically versatile process, capable of
delivering tailor-made ACA analogues, we targeted the construc-
tion of 18 (Scheme 9), a chaetomellic anhydride with a vinyl group
at the end of a shorter aliphatic chain. An intermediate of this type
1
1 and the thioacetal 12 (Scheme 7, path i).
The delatentization of the C@O function, hidden as an S,S-acetal
can be generally achieved by heavy metals coordination (such as
2
+
2+
31
2
is particularly valuable, since on the terminal CH@CH one can add
Hg or Cu ), by alkylation or by oxidative methods. While we
were studying a more appropriate protocol than that of Hajipour,
we incidentally observed that a crude extract from the RC of 10,
left on the laboratory bench for 3 weeks, contained only the sym-
metric dimer 11. Evidently, residual copper and air promoted the
oxidative deprotection of 12 and its transformation into 11. We
were unable to repeat this excellent result, but a large conversion
of 12 into 11 was always experienced.
thiols through a radical addition. This reaction, also known as
3
4
thiol–ene coupling, is a useful ‘click’ methodology.
The starting N-(3-hydroxypropyl)undec-10-enamide 13 can be
easily obtained by manipulation of undecylenic acid, an interesting
3
5
renewable resource, industrially produced on heating ricinoleic
To make the deprotection of 12 reproducible and achievable in
reasonable times, we considered that an oxidative method based
on I , a reagent effectively employed for the regeneration of the
2
Scheme 6. Preparation of the N-2,2-dichloropropanoyl ketene-N,S-acetal 10.
Scheme 8. Possible mechanism for deprotection of the thioacetal moiety in 12.

3
52
F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
Scheme 11. Preparation of anhydride 19. Reagents and conditions: (a) P
HMDO, CH Cl , reflux, 5 h; (b) 150 °C, 2 h, under vacuum; (c) CH CCl COCl, Py,
CH Cl , 0–25 °C, 20 h; (d) (i) CuCl (10 mol %), TMEDA (20 mol %), CH CN/toluene
3:2), Na CO , argon, 30 °C, 19–24 h, (ii) KI, H O, rt, 24 h, (iii) NaOH, THF/H O, rt,
2 h, acidic work-up.
4 10
S /
2
2
3
2
2
2
3
(
2
3
2
2
1
Scheme 9. Preparation of anhydride 18. Reagents and conditions: (a) P
CH Cl , reflux, 5 h; (b) 150 °C, 2 h, under vacuum; (c) CH CCl COCl, Py, CH
5 °C, 20 h; (d) CuCl (10 mol %), TMEDA (20 mol %), CH CN/toluene (3:2), Na
O, 24 h, (ii) NaOH, THF/H O, rt, 12 h, acidic work-
4
S
10/HMDO,
Cl , 0–
3
CO ,
2
2
3
2
2
2
2.3. FTase assays
2
3
2
argon, 30 °C, 19–24 h; (e) (i) KI, H
2
2
Taking in mind the previously pointed out pharmacological
activity of ACA, we then examined the efficacy of the new chaeto-
mellic analogue against the FTase, to see if the replacement of the
up.
acid, the main component of castor oil.36 Hydroxyamide 13, trea-
tenth CH in the aliphatic tail with sulfur hadn’t impaired the inhi-
2
bition power. This required the preliminary conversion of the
ted as usual, gave the enamide 16 in high yield (Scheme 9). Ena-
mide 16 was next subjected to the RC, which, after the addition
of KI during the work-up procedure, afforded the symmetric disul-
fide 17 or, once it was hydrolyzed, the targeted anhydride 18
anhydride 19 into the corresponding disodium salt 27 (Fig. 3).
The conversion was efficiently achieved in four easy steps: (i) reac-
tion of the anhydride with a stoichiometric amount of NaOH
2
in THF/H O, (ii) evaporation of the solution under vacuum, (iii)
(Scheme 9, path i and ii, respectively).
re-dissolution of the remaining material in water, and finally (iv)
With the anhydride 18 in our hand, we were ready to test the
1
9a
freeze-drying.
Using this procedure we also prepared the
thio-click reaction. We focused on the preparation of 19, an isoster-
ic ACA analogue. Thus, the radical addition of butanethiol to 18 had
to be realized. Since anhydride 18 carried two olefinic functions a
problem of chemoselectivity could raise. The two C@C bonds are,
however, quite different: one is electron-poor and tetrasubstituted,
whereas the other is electron rich and monosubstituted. As the thi-
sodium maleates 2 and 26, from the anhydrides 3 and 18 secured
in this work, and 28, from 3-hexadecyl-4-methylfuran-2,5-dione2
that we had in stock (Fig. 3).
These substances were evaluated for inhibition against yeast
and rat FTases, using the continuous fluorescence assay.40 In both
assays we used the same dansylpeptide (dansyl-Gly-Cys-Val-Ile-Ala).
As far as we know, there is no reported IC50 value with dansyl-
GCVIA for inhibition of rat FTase. For example, peptidomimetic
inhibitors were tested with dansyl-Gly-Cys-Val-Leu-Ser for
inhibition of rat FTase, showing submicromolar activities. We
believe that there is no major difference between dansyl-GCVIA
and dansyl-GCVLS as a peptide substrate for inhibition of rat
FTase. Since FTases are promiscuous with regard to the CAAX box,
the exact sequence should not be a problem. Indeed Singh tried
the same Ras-CVLS with both FTases, and he did not raise any
1c
37
yl radical is electrophilic and the rate of radical attack controlled
by steric and polar factors,38 we anticipated that attack at the api-
cal methylene carbon should be favored.3
4d
At the beginning we tried the initiation of the radical chain at
room temperature, using organoboranes (such as triethylborane
41
or 2-ethylbenzo[d][1,3,2]dioxaborole),39 but procedure and results
were unappealing. We then resorted to AIBN, and gratifyingly an
almost quantitative addition of butanethiol to the apical C@C
was smoothly attained. No involvement of the maleic end, as fore-
seen, was noted (Scheme 10).
2
0c
doubt about the results of his tests.
his study on substrate recognition by wild FTase, observed that
dansyl-GCVIA and dansyl-GCVLS were similarly recognized,
Interestingly Fierke, during
Alternatively 19 was also prepared, always in good yields, from
4
2
the
11-(butylthio)-N-(3-hydroxypropyl)-undecanamide
20
(Scheme 11), by bringing forward the thiol–ene coupling on the
peptide
ꢀ1 ꢀ1
respectively: kcat/K
M
120 and 170 (mM
s
), and kcat > 0.2
starting hydroxyamide 13. It is noted that the thioether function
in the aliphatic chain survived the oxidative deprotection of the
thioacetal intermediate (not shown), and was not converted into
a sulfoxide group.
ꢀ1
and 0.4 (s ). In our IC50 measurements, the concentration of rat
FTase was 10 times increased (15 nM against 1.5 nM) to compen-
sate a possible lower affinity of dansyl-GCVIA for the enzyme and
Scheme 10. Radical addition of butanethiol to the terminal C@C group of 18.
Figure 3. Sodium maleates used in the FTase assays.

F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
353
Table 1
a
Inhibition of yeast and rat FTases by ACA and analogues
No.
Inhibitor
Yeast FTase IC50
(l
M ± SD)
Rat FTase IC50 (lM ± SD)
1
2
3
4
2
16.7 ± 0.9 (17 ± 3)18a
13.4 ± 1.1
3.5 ± 0.3
0.91 ± 0.08
0.49 ± 0.05
0.19 ± 0.01
2.6 ± 0.3
26
27
28
65.6 ± 16.7
a
For conditions see ‘FTase assays’ in the Section 4.
to increase the rate at which the bound substrate is converted to
product. The IC50 values are summarized in Table 1.
The value we determined for the sodium salt of chaetomellic
acid A (2) against yeast FTase, compared very favorably to that
1
9a
previously measured (Table 1, no. 1).
analogue 27 was approximately 5 times more potent than ACA
Table 1 no. 1 and 3), showing an IC50 value (3.5 M) near that of
chaetomellic acid C 29 (4 M) and of the farnesylated ACA
analogue 30 (2.4 M) (Fig. 4).
Astonishingly the thia-
(
l
l
19a
l
This result confirms that it is
unnecessary to retain the hydrophobic farnesyl group for having
potent FPP analogues inhibitors, contrary to what was previously
4
3
20c
claimed. Since FPP has more affinity for mammalian FTases,
20c
inhibition, as expected, was better against the rat Ftase (Table 1);
above all, this second round of assays stressed the same order of
activity between the four sodium maleates tested.
The size of the tail appears critical,20c in fact with chains P16
the inhibition power collapses (Table 1, no. 4). Unexpectedly the
intermediate 26, notwithstanding it shorter C-9 chain (which,
however, implement a terminal vinyl group), gave an IC50 at least
analogous to that of the natural inhibitor 2.
Figure 5. (a) Alignment of FPP, in the conformation assumed in the 1FT2 pdb
structure (blue), FPT-II FPP, in the conformation assumed in the 1TN8 pdb structure
(
yellow), and of the thia-analogue 27 in the quasi-extended conformation chosen
(atom colors: carbon atoms are in green, oxygen atoms in red, and sulfur atom in
orange). (b) Superposition of the molecular volume of 27 (green) and the volume of
the supermolecule (white) formed by FPP and FPT-II FPP. In the figure the hydrogen
atoms are omitted for clarity.
2
.4. Molecular modelling
A posteriori computational analysis has been carried out on the
interaction of thia-analogue 27 with FTase in order to get insights
into its moderate increased inhibition potency with respect to the
parent compound 2.
After an extensive analysis of the X-ray structures of FTase
available in the PDB data bank, the X-ray structure of rat FTase
ences; (Fig. 5a and b) the best fit of the molecular volume of 27 and
the volume of the supermolecule formed by FPP and FPT-II FPP,
which can be considered to reflect the overall shape and the con-
formational flexibility of the enzyme binding site (Fig. 5b).
complexed with farnesyl pyrophosphate (FPP) (PDB ref code
1
The structural motif of hydrophilic head group of 27 is well
accommodated into the highly positively charged pocket, located
near the subunit interface and adjacent to the catalytic zinc ion,
which constitutes the site of the diphosphate moiety of farnesyl
diphosphate (FPP) in the crystal structures of the binary and ter-
FT2)4 and of the ternary complex in which the rat FTase interacts
4
with the FPT-II FPP analog and the substrate peptide CVLS (PDB ref
4
5
code 1TN8) were selected. Superposition of the two 3D struc-
tures by alignment of all enzyme C atoms shows that the struc-
a
7
c,44,46
tures of the enzyme in these complexes are essentially identical,
and the location and conformation of the isoprenoid and nonreac-
tive isoprenoid analogs are very similar.
In fact, only a few minor side chain rearrangements are ob-
served in the proximity of the anionic head binding sites of the iso-
prenoid analogs, and of the C-terminal carboxylate residues of the
CVLS peptide.
The choice of the conformation of the thia-analogue 27 (among
the many low-energy quasi-extended conformations it can as-
sume) to be considered for docking experiments was based on:
nary complexes.
dues K164, Y200, and H201 from the
This pocket is formed by amino acid resi-
-subunit of the enzyme
a
and Y300, K294, R291, H248 from the b-subunit (Fig. 6, top).
The hydrophobic tail of inhibitor 27 perfectly fits the length
(14 Å) of the hydrophobic funnel-shaped groove of the enzyme, ex-
tended along one side of the cavity and it interacts with a number
of conserved aromatic residues (Fig. 6, bottom and inset). These
are: Y154, W102, and Y205 of the b-subunit; they are located at
the bottom of the hydrophobic cavity and are considered to consti-
tute the discriminants for the maximum length of the ligands,44
C254, G250, Y251, W303, Y361 and the aliphatic portion of the
side-chain of R202 of the b-subunit; Y166, H201, and aliphatic por-
(
a) the best alignment with the isoprenoid analogs, taken as refer-
tion of the side-chain of K164 of the a-subunit.
The CVLS peptide sandwiches compound 27 against the wall of
the hydrophobic cavity, sequestering the 20% of the solvent acces-
sible surface, still available after the establishment of the binary
complex, from the solvent permeating the cavity (Fig. 6). A direct
van der Waals contact between the Leu residue of the CVLS peptide
and the aliphatic tail of 27 is observed. It is worth noting that the
same interaction is achieved by Ile, uponmutation of the CAAX
peptide used in the docking experiments into the one used in the
experimental measurements (CVIA).
Figure 4. Structure of the ACA analogues 29 and 30, and their IC50 values against
yeast FTase.1
9a

3
54
F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
Figure 6. The interaction of inhibitor 27 and the FTase binding site. The enzyme a-subunit is represented in grey, the b-subunit is represented in yellow. Aminoacid residues
involved in the interations are colored by element type (grey: carbon, blue: nitrogen, red: oxygen). Compound 27 is represented according to the color code of Figure 5. The
hydrogen atoms are not displayed for clarity. Top: focus on the interactions of the anionic head of 27 with the FTase binding site; bottom: focus on the interactions
established by the hydrophobic tail of compound 27; inset: an overall view of the ternary complex FTase-27-peptide (CVLS).
Interestingly, the sulfur atom in the aliphatic chain is found sur-
rounded by three aromatic residues (W303, Y251, and Y156), R202,
C254, and in direct contact with the oxygen atom of the backbone
chain of G250. Thus, the high versatility of the divalent sulfur atom
towards both electron-rich and electron-poor atoms is perfectly
satisfied in this environment.47 In particular, the clear orientation
preference for sulfur relative to oxygen atom of G250 could be a
reason of the improved inhibitory power observed with respect
to the parent ligand (compound 2) and it is worth of further
investigations.
and efficient one-pot procedure, thanks to the use of the Curphey
reagent in the thionation step, and (ii) the required deprotection
of the intermediate thioacetal was easily achieved through
the addition of KI, during the work-up procedure of the radical
cyclization reaction. Remarkably, conversion of the starting
N-(3-hydroxypropyl)palmitamide into ACA (1) entailed only one
intermediate chromatographic purification step.
Since the process uses carboxylic acid as starting materials, this
allowed us to develop a versatile approach to the preparation of
ACA analogues, through the synthesis of an intermediate maleic
anhydride with a vinylic group at the end of the aliphatic tail, a
function that can be transformed through a thiol–ene coupling.
Alternatively, the same thia-analogue can be also efficiently se-
cured, by bringing forward the thio-click reaction on the starting
N-(3-hydroxypropyl)-unsaturated amide. This more rigid alterna-
tive well matches with the large-scale preparation of ACA ana-
logues, as it avoids the use of a valuable maleic anhydride
intermediate.
3
. Conclusion
In this article we have described a new route to chaetomellic
acid A (1). The process implements our recent method for the
preparation of maleic anhydrides, which is based on the copper
catalyzed radical cyclization of N-a-perchloroacyl-2-(Z)-alkyl-
iden-1,3-tiazinanes. To make the process more appealing two
critical adjustments had to be introduced: (i) the preparation of
the intermediate enamide was realized with a practical, cheap
Finally, we serendipitously observed that the sodium salt 27,
prepared from the representative sulfurated anhydride 19, was a

F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
355
more competent FTase inhibitor than ACA (1). On the ground of a
dissolved in dry CH
2
Cl
2
(15 mL) and pyridine (50 mmol, 4.03 mL)
molecular modeling study we attributed the improved inhibitory
power of 27 to the direct contact of the sulfur atom with the oxy-
gen atom of the backbone chain of G250. This result and the capa-
bility to assemble tailor made thia-analogues of ACA favorably
meet and will be further investigated to identify competent mole-
cules for the prevention of the ischemic damage.
added. The stirred solution was then cooled at 0 °C (ice bath) and
2,2-dichloropropanoyl chloride (30 mmol, 4,84 g), diluted in
CH Cl (5 mL), was slowly dropped (10 min), through a dropping
2 2
funnel. Subsequently the ice-bath was removed. The mixture was
thermostatted at 25 °C (20 h), afterwards it was diluted with water
(40 mL). When all the solid, which developed during the acylation,
was dissolved in the added water, the mixture was poured in a sep-
aration funnel, further diluted with other water (60 mL) and ex-
4
4
. Experimental part
tracted with CH
2
Cl
2
(4 ꢁ 20 mL) (the separation of the phases is
rather slow). The organic extracts were collected and evaporated.
The crude mixture was purified by flash-chromatography on silica
gel, eluting with a petroleum ether (PE, bp 40–70 °C)/diethyl ether
.1. General
Reagents and solvents were standard grade commercial prod-
(
2
Et O) gradient (from 100/0 to 90/10). The enamide 10 was recov-
ucts, purchased from Aldrich or Acros, and generally used without
further purification. CH Cl was dried over 3 Å sieves (5% w/v).
Hexamethyldisiloxane was commercial or obtained following a
ered as a yellow oil (7.77 g, yield 89%); [HRMS found 436.2198.
2
2
+
C
m
22
H
40Cl
2
NOS (M+H) requires 436.2202]; R
f
(95% PE/Et
(500 MHz, CDCl ) 0.89 (t, J
), 1.27 (br s, 20H, (CH 10), 1.35 (m, 2H, CH ),
CH@C), 2.11 (br, 2H, SCH CH CH N), 2.29 (m,
C), 2.87 (br s, 2H, CH S), 4.17 (br,
N), 6.10 (br, 1H, CH@C); d (125 MHz, CDCl ) 14.1, 22.6,
6.3 (broad), 28.6 (broad), 28.9, 29.3, 29.35, 29.4, 29.6, 29.65 (4
), 29.7, 31.9, 36.9, 50.1, 80.2, 131.6 (broad and
weak, C@), 136.2 (broad and weak, CH@), 163.4; ESI-MS: 436.6
2
O) 0.55;
ꢀ1
4
8
max (neat) 2924, 2853, 1663 cm ; d
H
3
literature procedure. 2,2-Dicloropropanoyl chloride was prepared
by chlorination with Cl of propionyl chloride in the presence of
tetrabutylammonium chloride. The starting N-(3-hydroxypro-
7.1 Hz, 3H, CH
1.44 (m, 2H, CH
2H, CH
2H, CH
2
3
CH
2
2
)
2
2
24
2
CH
2
2
2
2
4
9
50
2
CH@C), 2.34 (s, 3H, CH
3
2
pyl)palmitamide 7 and N-(3-hydroxypropyl)undec-10-enamide
3 were secured in high yields (on the scale of 100–200 g) from the
1
2
C
3
corresponding acids, after methylation and ensuing amino-
de-methoxylation with 3-aminopropanol. The substitution is
overlapped CH
2
helped by the addition of K
2 3
CO 10 mol % and of THF to solubilize
+
[
M+H] .
the mixture of reagents (heating at reflux was required with methyl
2
9,51
palmitate).
The 11-(butylthio)-N-(3-hydroxypropyl)undecan-
4
.2.2. Radical cyclization of (Z)-3-(2,2-dichloropropanoyl)-2-
pentadecylidene-1,3-thiazinane (10)
CuCl (0.42 mmol, 0.042 g), Na CO
substrate 10 (4.2 mmol, 1.84 g) were weighed into an oven dried
Schlenk tube, then CH CN/toluene 1:1 (6.3 mL) and TMEDA
0.2 mmol, 126 L) were added under argon. The mixture was stir-
red at 30 °C and after 24 h diluted with water (30 mL) and ex-
tracted with CH Cl
(3 ꢁ 15 mL). The combined organic layers
were concentrated under vacuum. Flash chromatography of the
recovered material on silica gel, eluting with a PE/Et O gradient
from 100/0 to 20/80) gave the disulfide 11 (0.45 g, 28%) as a white
wax, [HRMS found 761.5324. C44
(M+H)+ requires
O) 0.42, and the thioacetal 12 (0.92 g,
8%) as a white solid [HRMS found 745.5380. C44
amide 20 was prepared in quantitative yield by standard radical
3
4d,52
addition of butanethiol to 13, using AIBN as initiator.
silica gels used for flash chromatography was Silica Gel 60 Merck
.040–0.063 mm. TLC were performed on silica coated plates
The
2
3
(4.6 mmol, 0.488 g) and the
0
Merck 60 F254, using UV light (254 nM) or cerium molybdate
solutions to visualize the spots.
3
(
l
1
13
H NMR and
C NMR spectra were recorded on ‘Varian
13
5
00 MHz’ or Jeol GSX 400. C NMR were obtained with full proton
decoupling. H NMR and C NMR signals attribution was based on
2
2
1
13
1
1
1
13
gradient-enhanced H, H-DQF-COSY, H, C-Edited-HSQC, and
2
1
13
(
H, C-HMBC experiments, run with standard pulses programmes.
77 2 4 2
H N O S
IR spectra were recorded on a ‘FT-IR Perkin Elmer 1600 Series’
while MS spectra on a ‘HP G1800C GCD System Series II’. High
resolution mass spectra (HRMS) were obtained on an ‘Agilent
7
5
f 2
61,5319], R (60% PE/Et
77 2 3 2
H N O S
+
(
f 2
M+H) requires 745,5370], R (60% PE/Et O) 0.20, mp 63–64 °C.
6
520 Accurate-Mass Q-TOF LC/MS’ and ESI-MS were run on a
Bruker Esquire 4000 instrument.
0
0
4
.2.2.1.
1,1 -[3,3 -Disulfanediylbis(propane-3,1-diyl)]bis(3-
max (Nu-
): 0.88 (t, J 7.0 Hz,
), 1.29 (8H, (CH ), 1.52
C@C), 1.96 (s, 6H, 2 ꢁ CH ), 1.97 (quint, J
CH CH
N), 2.36 (t, J 7.3 Hz, 4H, 2 ꢁ CH
S), 3.57 (t, J 6.9 Hz, 4H,
2 C 3
N); d (125 MHz, CDCl ): 8.5, 13.9, 22.5, 23.5, 28.0, 28.1,
Recombinant yeast FTase was produced in Escherichia coli and
purified over HisTrap affinity column (HisTrap™ HP, GE Health-
care) as previously described.53 Rat FTase was purchased from Jena
methyl-4-tetradecyl-1H-pyrrole-2,5-dione) (11).
m
ꢀ
1
jol) 1768 and 1708 cm ; d
H, CH CH
), 1.26 (br s, 36H, 2 ꢁ (CH
bm, 4H, 2 ꢁ CH CH
.9 Hz, 4H, 2 ꢁ SCH
H 3
(500 MHz, CDCl
6
(
6
3
2
2
)
9
2 4
)
Bioscience (Germany). n-Dodecyl-b-D-maltoside was purchased
from ACROS (Geel, Belgium) and dansyl-Gly-Cys-Val-Ile-Ala was
synthesized from HSC Core Research Facilities at University of
Utah. Fluorescent signals for yeast FTase assays were recorded on
a SpexFluoroMax spectrofluorimeter (Jobin Yuan Spex, Edison, NJ).
2
2
3
2
2
2
2
-
C@C), 2.68 (t, J 6.9 Hz, 4H, 2 ꢁ CH
2
2
2
1
ꢁ CH
9.1, 29.2, 29.35, 29.40, 29.5, 31.8, 35.4, 36.5, 136.9, 141.2, 171.9,
72.2; ESI-MS: 783.8 [M+Na] .
+
4
4
.2. Preparation of ACA and analogues
4
.2.2.2. 3-Methyl-1-{3-(8-methyl-6-oxo-7-tetradecyl-3,4,6,8a-
.2.1. Preparation of (Z)-3-(2,2-dichloropropanoyl)-2-
tetrahydro-2H-pyrrolo[2,1-b][1,3]thiazin-8a-ylthio)propyl}-4-
tetradecyl-1H-pyrrole-2,5-dione (12). max (neat) 1706,
) 0.89 (2t, J 7.0 Hz each, 6H,
pentadecylidene-1,3-thiazinane (10)
m
ꢀ1
In a one necked 100 mL round bottom flask, fitted with a con-
1
2
1
1
1
1
1
3
681 cm ; d
ꢁ CH CH ), 1.26 (br, 43H), 1.38 (m, 2H, CH
.60 (m, 2H, CHaxH+CH), 1.65 (quint, J 7.0 Hz, 2H, CH
.83 (t, J 7.0 Hz, 2H, CH S), 1.91 (s, 3H, CH ), 1.96 (s, 3H, CH
.99 (bdt, 1H, CHeqH), 2.37 (m, 3H, CH
H 3
(500 MHz, CDCl
denser, dry N-(3-hydroxypropyl)palmitamide
.27 g) and P 10 (3.68 mmol, 1.63 g) were weighed. Then dry
CH Cl (20 mL) and hexamethyldisiloxane (33.4 mmol, 7.16 mL)
were added. The mixture, under stirring, was than heated at reflux
5 h). Afterwards the solvent was evaporated, and the remaining
7
(20.0 mmol,
3
2
2
), 1.51 (m, 2H, CH
CH CH
2
),
2
),
3
),
6
4
S
2
2
2
2
2
3
2
-C@C+CHH-C@C), 2.49 (m,
(
H, CHH-C@C), 2.67 (dt, J 13.2, 1.3 Hz, 1H, CHeqHS), 3.08 (dt, J
3.2, 3.0 Hz, 1H, CHaxHN,), 3.34 (dt, J 13.2, 1.3 Hz, 1H, CHaxHS),
oil was stirred at 150 °C (2–3 h) under vacuum (8–9 mm Hg), while
argon, delivered through a capillary, bubbled inside the liquid
.45 (m, 2H, CH
125 MHz, CDCl
6.80, 27.80, 27.82, 28.20, 28.83, 29.26, 29.33, 29.34, 29.35,
2
N), 4.30 (br dt, J 13.2, 1.3 Hz, 1H, CHeqHN); d
C
(
this helps the removal of silylphosphate, issued as side-product).
(
2
3
) 8.66, 8.98, 14.10, 22.67, 23.69, 25.75, 25.88,
Next the crude 2-pentadecyl-5,6-dihydro-4H-1,3-thiazine 9 was

3
56
F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
2
3
1
9.50, 29.54, 29.56, 29.62, 29.63, 29.65, 29.67, 29.68, 30.14, 31.90,
C@) 135.0 (s, broad and weak, CH@C), 139.1, 163.5; ESI-MS:
364.5 [M+H] , 386.4 [M+Na] .
+
+
1.91, 35.71, 37.04, 74.37, 129.69, 136.93, 141.20, 154.38, 168.12,
+
71.81, 172.10; ESI-MS: 767.8 [M+Na] .
0
0
4
.2.7. Preparation of 1,1 -(3,3 -disulfanediylbis(propane-3,1-
diyl))bis(3-methyl-4-(non-8-enyl)-1H-pyrrole-2,5-dione) (17)
Following the same procedure used to prepare 11, (Z)-3-(2,2-
dichloropropanoyl)-2-(dec-9-enylidene)-1,3-thiazinane 16
0
0
4
.2.3. Preparation of 1,1 -[3,3 -disulfanediylbis(propane-3,1-
diyl)]bis(3-methyl-4-tetradecyl-1H-pyrrole-2,5-dione) (11)
CuCl (0.50 mmol, 0.050 g), Na CO (5.5 mmol, 0.590 g) and the
substrate 10 (5.0 mmol, 2.19 g) were weighed into an oven dried
Schlenk tube, then CH CN/toluene 3:2 (5 mL) and TMEDA
0.2 mmol, 151 L) were added under argon. The mixture was stir-
red at 30 °C and after 19 h the tube was open and KI (100 mg) and a
few drops of water were added. The mixture was vigorously stirred
in the open air for 24 h, after which it was diluted with water
30 mL) and extracted with CH
ganic layers were concentrated under vacuum. Flash chromatogra-
phy of the recovered material on silica gel, eluting with a PE/Et
gradient (from 100/0 to 40/60) gave the disulfide 11 (1.35 g, 71%).
2
3
(
5.0 mmol, 1.83 g) gave 17 as a pale yellow oil (0.99 g, 64%); [HRMS
+
found 617.3450. C34
PE/Et O) 0.52; max (neat) 3455, 3074, 1768, 1703, 1640 cm ; d
400 MHz, CDCl ), 1.35 (4H), 1.50
) 1.28 (br s, 12H, 2 ꢁ (CH
bm, 4H, 2 ꢁ CH CH C@C), 1.94 (s, 6H, 2 ꢁ CH ), 1.94 (m, over-
CH CH N), 2.02 (br q, 7.9 Hz, 4H,
CH@C), 2.35 (t, J 7.7, 4H, 2 ꢁ CH C@C), 2.62 (t, J 7.3 Hz,
H, 2 ꢁ CH S), 3.55 (t, J 7.3 Hz, 4H, 2 ꢁ CH N), 4.91 (part A of an
J 1.2 Hz, 2H, CHH@), 4.97
53 2 4 2 f
H N O S (M+H) requires 617.3441]; R (70%
3
-1
2
m
H
(
l
(
(
3
2 3
)
2
2
3
lapped, 4H, 2 ꢁ SCH
2
2
2
J
2
4
ꢁ CH
2
2
(
2
Cl
2
(3 ꢁ 15 mL). The combined or-
2
2
1.4
AMXY2 system, Jcis 10.2, Jgem 2.1,
ddq, part M of an AMXY2 system, Jtrans 16.9, Jgem 2.1, J 1.2 Hz,
2
O
1,4
(
2
6
H, 2 ꢁ CHH@), 5.78 (part X of an AMXY2, Jtrans 16.9, Jcis 10.3, JXY
.8 Hz, 2H, 2 ꢁ CH@C); d
C
(100.1 MHz, CDCl ) 8.7, 23.6, 28.1,
3
0
0
4
.2.4. Hydrolysis of 1,1 -(3,3 -disulfanediylbis(propane-3,1-
28.2, 28.8, 28.9, 29.1, 29.4, 33.7, 36.0, 36.6, 114.2, 136.9, 139.0,
141.1, 171.9, 172.2; ESI-MS: 639.1 [M+Na] .
+
diyl))bis(3-methyl-4-tetradecyl-1H-pyrrole-2,5-dione) (11)
In a one-necked 25 mL Erlenmeyer flask 11 (2.5 mmol, 1.903 g),
THF (2.5 mL) and a solution of NaOH 5 M (2.5 mL) were added. The
reaction mixture was stirred for 12 h, after which it was treated
with HCl 36% (2 mL), diluted with brine/water 1:1 and extracted
4.2.8. Preparation of 3-methyl-4-(non-8-enyl)furan-2,5-dione
(18)
Following the same procedure used to prepare 3, (Z)-3-(2,2-
dichloropropanoyl)-2-(dec-9-enylidene)-1,3-thiazinane 16
(20 mmol, 7.29 g) gave 18, as a pale yellow oil (3.26 g, overall yield
with CH
silica gel, eluting with a PE/Et
gave the chaetomellic anhydride A 3 (1.45 g, 94%); spectroscopic
2
Cl
2
Flash chromatography of the recovered material on
2
O gradient (from 100/0 to 20/80)
69%); [HRMS found 237.1465.
237.1485]; R (90% PE/Et O) 0.30.
w), 1767 (str), 1673 (w) cm ; d
H, (CH ), 1.35 (2H, CH ), 1.56 (bm, 2H, CH
CH CH@C), 2.05 (s, 3H, CH ), 2.43 (t, J 7.9, 2H, CH
A of an AMXY2 system, Jcis 10.2, Jgem 2.1, J 1.2 Hz, 1H, CH
C
14
H
21
O
3
(M+H)+ requires
max (neat) 3076, 1853, 1821
(400 MHz, CDCl ) 1.28 (br s,
), 2.01 (m, 2H,
C@C), 4.91 (part
@), 4.97
ddq, part M of an AMXY2 system, Jtrans 17.8, Jgem 2.1, J 1.2 Hz, 1H,
2
1b
data are in agreement with those reported in the literature.
f
2
m
H
ꢀ
1
(
6
3
2
)
3
2
2
4
.2.5. Preparation of chaetomellic anhydride A (3)
CuCl (2.00 mmol, 0.200 g), Na CO (22.0 mmol, 2.332 g) and the
substrate 10 (20.0 mmol, 8.73 g) were weighed into an oven dried
Schlenk tube, then CH CN/toluene 3:2 (20 mL) and TMEDA
4.0 mmol, 604 L) were added under argon. The mixture was stir-
2
3
2
2
3
1,4
2
1,4
(
3
CH
CH@C); d
2
@), 5.78 (part X of an AMXY2, Jtrans 17.8, Jcis 10.3, JXY 6.8 Hz, 1H,
(100.1 MHz, CDCl ) 9.5, 24.4, 27.5, 28.7, 28.8, 29.0, 29.3,
3.6, 114.2, 138.9, 140.4, 144.7, 165.8, 166.2; m/z (EI, 70 eV) 236 (2,
(
l
C
3
red at 30 °C and after 19 h the tube was open and KI (400 mg) and a
few drops of water were added. The mixture was vigorously stirred
in the open air for 24 h, after which it was diluted with water
3
+
M ), 191 (100), 163 (21), 126 (97%).
(
50 mL) and extracted with CH
2
Cl
2
(3 ꢁ 15 mL). The combined or-
4
.2.9. Addition of butane-1-thiol to 3-methyl-4-(non-8-
ganic layers were concentrated under vacuum. The recovered
material was diluted with THF (10 mL). Next a solution of NaOH
5
1
enyl)furan-2,5-dione (18)
AIBN (0.2 mmol, 0.033 g) and anhydride 18 (10.0 mmol, 2.36 g)
were weighed into an oven dried Schlenk tube, then 1-butanethiol
M (10 mL) was added. The reaction mixture was stirred for
2 h, after which it was treated with HCl 36% (8 mL), diluted with
(
20.0 mmol, 2.1 mL) were added under argon. The mixture was
brine/water 1:1 and extracted with CH
of the recovered material on silica gel, eluting with a PE/Et
dient (from 100/0 to 20/80) gave the chaetomellic anhydride A 3
4.40 g, 71%).
2
Cl
2
. Flash chromatography
stirred at 80 °C (5 h). Next the unreacted thiol was evaporated un-
der vacuum. Flash chromatography of the recovered material on
silica gel, eluting with a PE/Et
gave 19, as a pale yellow oil (3.07 g, 94%); [HRMS found
2
O gra-
2
O gradient (from 100/0 to 20/80)
(
+
3
27.1980. C18
H
31
O
3
S (M+H) requires 327.1989]; R
f
(90% PE/Et
2
O)
dH
ꢀ
1
4
.2.6. Preparation of (Z)-3-(2,2-dichloropropanoyl)-2-(dec-9-
0.26); mmax (neat) 1855, 1821, 1766 (str), 1673 cm
;
enylidene)-1,3-thiazinane (16)
3 3 2
(400 MHz, CDCl ) 0.89 (t, J 7.5 Hz, 3H, CH CH ), 1.28 (bm, 8H,
Following the same procedure used to prepare 10, N-(3-
hydroxypropyl)undec-10-enamide 13 (40 mmol, 9.66 g) gave 16
CH ), 1.37 (m, 4H, 2 ꢁ CH ) 1.54 (m, 6H, 3 ꢁ CH ), 2.04 (s, 3H,
2
2
2
CH ), 2.34 (t, J 7.8 Hz, 2H, CH C@), 2.47 (t, J 7.4 Hz, 2H, CH S),
3
2
2
as a yellow oil (12.69 g, yield 87%); [HRMS found 364.1240.
2 C 3
2.48 (t, J 7.6 Hz, 2H, CH S); d (100.1 MHz, CDCl ) 9.5, 13.7, 220,
+
C
17
H
28Cl
2
NOS (M+H) requires 364.1263]; R
f
(95% PE/Et
2
O) 0.50;
(500 MHz, CDCl
), 2.05 (dq, 2H, J
CH N), 2.29 (q,
), 2.87 (br s, 2H, CH S),
N), 4.94 (part A of an AMXY2 system, Jcis 10.2,
.1, 1.2 Hz, 1H, CHH@), 5.00 (part M of an AMXY2 system, Jtrans
24.4, 27.5, 28.8, 29.1, 29.12, 29.2, 29.4, 29.6, 31.8, 31.82, 32.1,
ꢀ1
+
m
max (neat) 3075, 2925, 2854 and 1664 cm ; d
.32, 1.38, 1.44 (overlapped multiplets, 10H, (CH
.1, 1.2 Hz, CH CH@CH ), 2.11 (br s, 2H, SCH CH
H, J 7.5 Hz, CH CH@CNS), 2.34 (s, 3H, CH
.17 (br s, 2H, CH
H
3
)
140.4, 144.7, 165.8, 166.2; m/z (EI, 70 eV) 326 (33, M ), 283 (8),
1
7
2
4
2
1
1
2
)
5
269 (52), 201 (74), 191 (32), 126 (38), 61 (100%).
2
2
2
2
2
2
3
2
4.2.10. Preparation of (Z)-3-(2,2-dichloropropanoyl)-2-[10-
(butylthio)decylidene] -1,3-thiazinane (23)
2
Following the same procedure used to prepare 10, 11-(butyl-
thio)-N-(3-hydroxypropyl)undecanamide 20 (20 mmol, 6.64 g)
gave 23 as a pale yellow oil (7.83 g, overall yield 86%); [HRMS
1
,4
6.9, Jgem 2.1, J 1.2 Hz, 1H, CHH@), 5.82 (part X of an AMXY2, Jtrans
3
6.9, Jcis 10.2, J 6.8 Hz, 1H, CH@CH
125 MHz, CDCl ) 26.1, 28.3, 28.7, 28.8 (2 overlapped CH
9.0, 29.6, 33.5, 36.7, 50.0, 80.1, 114.1, 131.6 (broad and weak,
2
), 6.21 (broad, 1H, CH@CNS); d
C
+
(
3
2
), 28.9,
found 454.1780. C21H38Cl NOS (M+H) requires 454.1766]; R
2
2
f
ꢀ1
2
2
(90% PE/Et O) 0.85; mmax (neat) 2926, 2850, 1700, 1667 cm ; dH

F. Bellesia et al. / Bioorg. Med. Chem. 21 (2013) 348–358
357
(
1
400 MHz, CDCl
.39 (m, 2H, CH
SCH CH CH N), 2.26 (m, 2H, CH
J 7.9 Hz, 2H, CH S), 2.49 (t, J 7.9 Hz, 2H, CH
CH S), 4.21 (broad, 2H, CH N), 6.23 (broad, 1H, CH@C); d
100.1 MHz, CDCl ) 13.7, 22.0, 26.2 (broad), 28.6 (broad), 28.9,
9.2, 29.22, 29.3, 29.4, 29.7, 31.8, 31.9, 32.2, 36.8, 50.2 (broad),
0.2 (CCl ), 131.5 (broad and weak, C@), 136.4 (broad and weak,
3
) 0.89 (t, J 7.1 Hz, 3H, CH
CH ), 1.55 (m, 4H), 2.09 (broad, 2H,
CH@C), 2.31 (s, 3H, CH ), 2.48 (t,
S), 2.84 (broad, 2H,
3
CH
2
), 1.26 (broad, 12H),
Standard protonation states, corresponding to pH 7, were assigned
to the amino acid residues.
2
3
2
2
2
2
3
2
2
Acknowledgments
2
2
C
(
2
8
3
We thank the Ministero dell’Istruzione, dell’Università e della
Ricerca (MIUR, PRIN 20085E2LXC-004, 20085E2LXC-003), the
National Institute of Health (NIH Grant GM 21328) and the EU
(under the ERASMUS scheme) for financial support.
2
+
CH@), 163.4 (CO); ESI-MS: 454.6 [M+H] .
4
2
.2.11. Preparation of 3-(9-(butylthio)nonyl)-4-methylfuran-
,5-dione (19) from 23
Supplementary data
Following the same procedure used to prepare 3, (Z)-3-(2,2-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.034.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
dichloropropanoyl)-2-[10-(butylthio)decylidene]-1,3-thiazinane
3 (10 mmol, 4.55 g) gave 19, as a pale yellow oil (2.49 g, overall
yield 76%).
2
4
4
.3. FTase assays
References and notes
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1
(
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(
8
(
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2
O (3–4 mL).
(
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.3.2. Yeast and Rat Farnesyl Transferase assays40
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2
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4
1
(
990, 87, 7673;
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1
0
2
lM ZnCl , 5 mM DTT, 0.04% (w/v) n-dodecyl-b-D-maltoside
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3
4
.
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4
.4. Molecular modelling methods
8
9
.
.
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0. (a) Singh, S. B.; Zink, D. L.; Liesch, J. M.; Goetz, M. A.; Jenkins, R. G.; Nallin-
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4
4
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4
5
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1
5
5
verged to 1eꢀ05 kJ/mol using the CHARMM force-field.

3
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2
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