Pyrazino[1,2-a]indole-1,4-diones, simple analogues of gliotoxin, as selective inhibitors of geranylgeranyltransferase I

Article abstract of DOI:10.1016/j.bmcl.2003.08.022

Some pyrazino[1,2-a]indole-1,4-diones, structurally simplified analogues of the natural mycotoxin gliotoxin, have been synthesised and investigated as inhibitors of prenyltransferases; one compound, 3-acetylthio-9-methoxy-2-methyl-2,3-dihydropyrazino[1,2-a]indole-1,4-dione 10 shows slightly greater selectivity (8-fold) for geranylgeranyltransferase type I (GGTase I) than gliotoxin tself.

Full text of DOI:10.1016/j.bmcl.2003.08.022

Bioorganic & Medicinal ChemistryLetters 13 (2003) 3661–3663
Pyrazino[1,2-a]indole-1,4-diones, Simple Analogues of Gliotoxin,
as Selective Inhibitors of Geranylgeranyltransferase I
David M. Vigushin,a,* Greg Brooke,a David Willows,b R. Charles Coombesb
and Christopher J. Moodyb,*
aDepartment of Cancer Medicine, 6th Floor MRC Cyclotron Building, Imperial College of Science, Technology and Medicine,
Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK
bDepartment of Chemistry, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
Received 5 August 2003; accepted 12 August 2003
Abstract—Some pyrazino[1,2-a]indole-1,4-diones, structurallysimplified analogues of the natural mycotoxin gliotoxin, have been
synthesised and investigated as inhibitors of prenyltransferases; one compound, 3-acetylthio-9-methoxy-2-methyl-2,3-dihydropyr-
azino[1,2-a]indole-1,4-dione 10 shows slightly greater selectivity (8-fold) for geranylgeranyltransferase type I (GGTase I) than
gliotoxin tself.
# 2003 Elsevier Ltd. All rights reserved.
Activating point mutations in the human ras proto-
oncogenes are associated with 20–30% of all human
cancers, including 90% of pancreatic adenocarcinomas
and 50% of colorectal carcinomas.1,2 Ki-ras is the most
commonlymutated oncogene in human cancers and is
therefore an attractive potential therapeutic target.3 All
four mammalian Ras isoforms, Ha-Ras, N-Ras and the
splice variants of Ki-Ras, Ki4A-Ras and Ki4B-Ras
require prenylation (post-translational modification by
an isoprenoid lipid) for subcellular plasma membrane
attachment and biological activity.4 Protein prenylation
is catalysed by farnesyltransferase (FTase) or ger-
anylgeranyltransferase I (GGTase I), members of the
protein prenyltransferase family that covalently attach a
farnesyl or geranylgeranyl unit, respectively to the C-
terminal tetrapeptide of Ras and other specific proteins
in eukaryotic cells. The related GGTase II specifically
attaches two geranylgeranyl groups to newly synthe-
sised Rab proteins. Both wild type Ras and oncogenic
Ras variants require prenylation for their biological
and/or transforming activities.5À8 These observations
have led to the development of prenyltransferase
enzyme inhibitors for the treatment of cancer.
Of the Ras isoforms, onlyHa-Ras is exclusivelyfarne-
sylated. However, Ki-Ras is more commonly associated
with human cancers and both Ki-Ras and N-Ras are
also substrates for GGTase I.9,10 Whereas Ki-Ras and
N-Ras are normallyfarnesylated in cells, in cells treated
with an FTase inhibitor (FTI) theyare also geranylger-
anylated by GGTase I.11,12 Selective FTase inhibition is
therefore insufficient to inhibit Ki-Ras dependent cell
proliferation. Treatment with a GGTase I inhibitor
(GGTI) can inhibit proliferation of oncogenic Ki-Ras
or N-Ras transformed tumour cells in culture or in nude
mouse xenograft models.13 However, inhibition of Ki-
ras prenylation in human cancer cells and in normal and
tumour tissues in animal models requires a combination
of an FTI and GGTI.14,15
Gliotoxin is a natural epidithiodiketopiperazine myco-
toxin with immunosuppressive and antimicrobial activ-
ity. Gliotoxin and related fungal metabolites gliovirin
and sporedesmin are low molecular weight non-polar
compounds characterised byan intramolecular disulfide
bridge that is the active moiety.16À18 The observation
that gliotoxin inhibits FTase at low micromolar con-
centrations,19À21 and has antiproliferative activityin
lymphosarcoma cells22 stimulated our interest in this
compound as a potential anticancer agent. Our recent
work has shown that gliotoxin is a dual FTI-GGTI with
potent antitumour activityagainst breast cancer and
limited toxicityin vivo in a rodent mammarycarcinoma
*Corresponding author. For biology: fax: +44-208-383-4650; email:
d.vigushin@imperial.ac.uk (D. Vigushin); for chemistry: tel.: +44-
1392-263429; fax: +44-1392-263434 email: c.j.moody@ex.ac.uk (C.J.
Moody).
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.022

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D. M. Vigushin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3661–3663
model.23 In contrast, other investigators have found
that concurrent in vivo administration of an FTI and
GGTI is verypoorlytolerated and often lethal in nude
mouse tumour xenograft models.24 The antitumour
efficacyand remarkable lack of toxicityof gliotoxin
therefore led us to design, synthesise and evaluate a
series of simple structural analogues.
and the 4-methoxyderivative 2.25 Formation of the acid
chloride bytreatment with thionyl chloride in toluene
followed byreaction with sarcosine ethyl ester gave the
amides 326 and 427 in 74 and 84% yield, respectively
(Scheme 1). Treatment with magnesium in methanol
resulted in reduction of the indole ring,28 and cyclisation
of the resulting indoline to give the tetrahydro pyr-
azino[1,2-a]indoles 5 and 6.27
Alternatively, the indole-2-carboxylic acids could be
converted directly into the dihydropyrazino[1,2-a]-
indoles 726 and 827 bycarbodiimide mediated coupling
to sarcosine ethyl ester followed by cyclisation. Sulfur
substituents were introduced byinitial radical bromina-
tion at C-3,29 followed byreaction with excess potas-
sium thioacetate to give the 3-acetylthio derivatives 9
and 10. Hydrolysis of the thioacetate gave the corre-
sponding thiols which were converted into the S-benzyl
Chemistry
The analogues chosen for study share the tricyclic pyr-
azino[1,2-a]indole-1,4-dione ring structure of gliotoxin,
and are either unsubstituted in the diketopiperazine ring
or contain a sulfur substituent. The starting materials
were commerciallyavailable indole-2-carboxylic acid
1
disulfides 11 and 12 byreaction with
zylthiophthalimide30 (Scheme 1).
S-ben-
Table 1. In vitro inhibition of recombinant protein prenyl trans-
ferases
Compd
FTase
IC50 (mM)
GGTase I
IC50 (mM)
5
6
7
>100
>100
>100
>100
>100
>100
8
>100
748Æ263
>100
90.3Æ8.0
10
11
12
>100
>100
>100
>100
*
Figure 1. Inhibition of FTase ( ) and GGTase I (ꢁ) enzyme activities
bythe pyrazino[1,2- a]indole-1,4-dione 10. Recombinant human FTase
and GGTase I were incubated in the presence of various concentra-
tions of 10. Transfer of farnesyl from [3H]FPP and geranylgeranyl
from [3H]GGPP into recombinant human Ha-Ras-CVLS and Ha-Ras-
CVLL, respectively, was determined. Each value represents a single
incubation, except for control values taken as 100%, which are the
mean of triplicate determinations. A blank value, determined in par-
allel reactions without protein substrate, was subtracted from each
value. IC50 values were determined graphicallyusing non-linear
regression analysis to fit the inhibition data to the appropriate dose–
response curves.
Scheme 1. Reagents and conditions: (i) SOCl2, toluene, 50 ꢀC; (ii)
sarcosine ethyl ester (2 equiv), ether; (iii) Mg, MeOH; (iv) sarcosine
ethyl ester hydrochloride, Et3N, CH2Cl2, EDCI, C6F5OH; (v) NBS,
(PhCOO)2, CCl4; (vi) KSAc (4 equiv), CH2Cl2; (vii) HCl, MeOH; (viii)
PhthNSBn, toluene, 80 ꢀC.

D. M. Vigushin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3661–3663
3663
Biological Results and Discussion
10. James, G. L.; Goldstein, J. L.; Brown, M. S. J. Biol.
Chem. 1995, 270, 6221.
11. Whyte, D. B.; Kirschmeier, P.; Hockenberry, T. N.;
Nunez-Oliva, I.; James, L.; Catino, J. J.; Bishop, W. R.; Pai,
J. K. J. Biol. Chem. 1997, 272, 14459.
12. Rowell, C. A.; Kowalczyk, J. J.; Lewis, M. D.; Garcia,
A. M. J. Biol. Chem. 1997, 272, 14093.
FTase and GGTase I enzyme inhibition assays measur-
ing the amount of [3H]FPP and [3H]GGPP incorporated
into recombinant human Ha-Ras-CVLS and Ha-Ras-
CVLL, respectivelywere performed as previously
described,31 and the results are shown in Table 1.
13. Sebti, S. M.; Hamilton, A. D. Expert Opin. Investig. Drugs
2000, 9, 2767.
The preliminarydata show that the compounds are not
particularlypotent inhibitors of FTase or GGTase I,
with all except one compound having IC50 values >100
mM.32 Compound 10, however, is an inhibitor of
GGTase I with an IC50 of 90.3 mM and interestingly
14. Sun, J.; Qian, Y.; Hamilton, A. D.; Sebti, S. M. Oncogene
1998, 16, 1467.
15. Lerner, E. C.; Zhang, T. T.; Knowles, D. B.; Qian, Y.;
Hamilton, A. D.; Sebti, S. M. Oncogene 1997, 15, 1283.
16. Johnson, J. R.; Bruce, W. F.; Dutcher, J. D. J. Am. Chem.
Soc. 1943, 65, 2005.
exhibits ca. 8-fold selectivityover FTase (IC 748 mM)
50
17. Bruce, W. F.; Dutcher, J. D.; Johnson, J. R.; Miller, L. L.
J. Am. Chem. Soc. 1944, 66, 614.
(Fig. 1).
18. Fridrichsons, J.; Mathieson, A. M. Acta Crystallogr. 1967,
23, 439.
19. Hara, M.; Han, M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
3333.
20. Nagase, T.; Kawata, S.; Tamura, S.; Matsuda, Y.; Inui,
Y.; Yamasaki, E.; Ishiguro, H.; Ito, T.; Miyagawa, J.; Mitsui,
H.; Yamamoto, K.; Kinoshita, M.; Matsuzawa, Y. Br. J.
Cancer 1997, 76, 1001.
Although the simple analogues are not as potent as
gliotoxin itself (IC50 80 and 17 mM vs FTase as GGTase I,
respectively23), compound 10 shares the same selectivity
for GGTase I (ca. 8-fold, cf. 5-fold for gliotoxin), and
therefore forms the basis for the design of further
analogues.
21. Van der Pyl, D.; Inokoshi, J.; Shiomi, K.; Yang, H.;
Takeshima, H.; Omura, S. J. Antibiot. 1992, 45, 1802.
22. Kidd, J. G. Science 1947, 105, 511.
Acknowledgements
23. Vigushin, D. M. Unpublished data.
We thank the EPSRC for a studentship to D.W., and
Charitable Funds for Hammersmith Hospital.
24. Lobell, R. B.; Omer, C. A.; Abrams, M. T.; Bhim-
nathwala, H. G.; Brucker, M. J.; Buser, C. A.; Davide, J. P.;
deSolms, S. J.; Dinsmore, C. J.; Ellis-Hutchings, M. S.; Kral,
A. M.; Liu, D.; Lumma, W. C.; Machotka, S. V.; Rands, E.;
Williams, T. M.; Graham, S. L.; Hartman, G. D.; Oliff, A. I.;
Heimbrook, D. C.; Kohl, N. E. Cancer Res. 2001, 61, 8758.
25. Blaike, K. G.; Perkin, W. H. J. Chem. Soc. 1924, 296.
26. Johnson, J. R.; Andreen, J. H.; Holley, A. D. J. Am.
Chem. Soc. 1947, 69, 2370.
27. Compound 4, mp 153–154 ꢀC; compound 5, mp 154–
155 ꢀC; compound 6, mp 196–197 ꢀC.
28. Youn, I. K.; Yon, G. H.; Pak, C. S. Tetrahedron Lett.
1986, 27, 2409.
29. Trown, P. W. Biochem. Biophys. Res. Commun. 1968, 33,
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31. Marson, C. M.; Rioja, A. S.; Brooke, G.; Coombes, R. C.;
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32. Manyof the compounds were insoluble in 5% DMSO at
concentrations exceeding 500 mM. For the assay, a test com-
pound must be soluble in 5% DMSO at 5 times the desired
final concentration. We therefore had to report the IC50 results
as >100 mM.
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