Disparate SAR data of griseofulvin analogues for the dermatophytes Trichophyton mentagrophytes, T. rubrum, and MDA-MB-231 cancer cells

Article abstract of DOI:10.1021/jm200835c

Griseofulvin and 53 analogues of this compound have been tested against the pathogenic dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes as well as against the breast cancer cell line MDA-MB-231. The modifications to griseofulvin include the 4, 5, 6, 2′, 3′, and 4′ positions. The SAR of the griseofulvin analogues toward the two fungi followed the same trend with the majority being less active than griseofulvin and none had more than twice the potency of the parent compound. A comparison of the antifungal and the anticancer SAR revealed distinct differences, as the majority of analogues showed increased activity against the cancer cell line MDA-MB-231, highlighted by 2′-benzyloxy-2′-demethoxy-griseofulvin, which showed low activity against both fungi but was among the most potent compounds against MDA-MB-231 cancer cells. Tubulin has been proposed as the target of griseofulvin in both fungal and mammalian cells, but the differences revealed by this SAR study strongly suggest that the mode-of-action of the compound class toward fungi and mammalian cancer cells is different.

Full text of DOI:10.1021/jm200835c

Article
pubs.acs.org/jmc
Disparate SAR Data of Griseofulvin Analogues for the
Dermatophytes Trichophyton mentagrophytes, T. rubrum, and
MDA-MB-231 Cancer Cells
Mads H. Rønnest,†,‡ Marc S. Raab,§,∥ Simon Anderhub,∥ Sven Boesen,‡ Alwin Kramer,∥
̈
Thomas O. Larsen,*,‡ and Mads H. Clausen*,†
†Center for Nanomedicine and Theranostics & Department of Chemistry, Technical University of Denmark, Kemitorvet,
Building 201, DK-2800 Kgs. Lyngby, Denmark
‡Center for Microbial Biotechnology, Department of Systems Biology, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark
§Max-Eder Group “Experimental Therapies for Hematologic Malignancies”, German Cancer Research Center (DKFZ) and
Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 280 (TP4), 69120 Heidelberg, Germany
∥Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ) and Department of Internal
Medicine V, University of Heidelberg, Im Neuenheimer Feld 280 (TP4), 69120 Heidelberg, Germany
S
* Supporting Information
ABSTRACT: Griseofulvin and 53 analogues of this com-
pound have been tested against the pathogenic dermatophytes
Trichophyton rubrum and Trichophyton mentagrophytes as well
as against the breast cancer cell line MDA-MB-231. The
modifications to griseofulvin include the 4, 5, 6, 2′, 3′, and 4′
positions. The SAR of the griseofulvin analogues toward the
two fungi followed the same trend with the majority being less
active than griseofulvin and none had more than twice the potency of the parent compound. A comparison of the antifungal and
the anticancer SAR revealed distinct differences, as the majority of analogues showed increased activity against the cancer cell line
MDA-MB-231, highlighted by 2′-benzyloxy-2′-demethoxy-griseofulvin, which showed low activity against both fungi but was
among the most potent compounds against MDA-MB-231 cancer cells. Tubulin has been proposed as the target of griseofulvin
in both fungal and mammalian cells, but the differences revealed by this SAR study strongly suggest that the mode-of-action of
the compound class toward fungi and mammalian cancer cells is different.
More than 400 griseofulvin analogues have been reported
since its discovery, and the activity of over 300 of these have
been compared with griseofulvin (1) against six dermatophytes
in a study by Crosse et al.11 The curling of hyphae in Botrytis
allii were also tested, but this phenotype did not correlate with
the growth inhibitory effect of the analogues.11 Griseofulvin
analogues with modifications at positions 4, 6, 2′, and 3′ as well
as isogriseofulvin analogues with modifications at the 4, 6, 3′,
and 4′ positions were tested. No 2′ analogues excelled in growth
inhibition of the dermatophytes,11 but elongation did increase
the curling of hyphae with the optimal analogues being
2′-propoxy (46) and 2′-butoxy analogues.12 Most griseofulvin
analogues tested showed increased potency against some
dermatophytes but exhibiting lower activity against others.
Position 5 has been functionalized with nitro (9) and amine
(10) groups, rendering the analogues inactive at relevant con-
centrations against four dermatophytes.13 An ethoxycarbonyl
group has also been introduced in this position and the ana-
logue found to be inactive.14 The 4′ position has also been
INTRODUCTION
■
Griseofulvin (1, see Figure 1) was one of the first antifungal
natural products isolated1 from filamentous fungi and has been
Figure 1. The structure of griseofulvin (1), griseofulvic acid (2), and
isogriseofulvin (3). The rings A, B, and C as well as the positions
modified in this study in 1 are shown.
known as an antifungal agent for decades.2,3 The compound
was, until the approval of Terbinafine4 by the US Food and
Drug Administration5 in 2007, the only drug available for
treatment of tinea capitis,6 a superficial fungal skin infection
caused by dermatophytes, which predominantly affects
children.7 Upon administration to man, griseofulvin accumu-
lates in the skin (stratum corneum), where it presumably binds
to keratin.8 The mode of action is still not determined, but
tubulin binding has been proposed.9,10
Received: June 27, 2011
Published: December 19, 2011
© 2011 American Chemical Society
652
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
examined with the 4′ alcohol analogue (50) being inactive and
the 4′ oxime (36) being 7-fold less potent compared to 1, with
both analogues tested against Microsporum gypseum.15
an improvement on prior methods (Scheme 1).22,27 Alkylation
of 4 to synthesize 5 and 6 has previously been described.22,27
a
Although the initial isolation of 1 was completed in 1939,1
both anticancer16−19 and antiviral20 properties of griseofulvin
have been discovered recently. Three analogues tested for
the former by Oda et al.21 against chinese hamster V79 cells
showed increased cytotoxicity, with 2′-propoxy-2′demethoxy-
griseofulvin being the most potent (46, IC50 0.7 μM; 1, 8 μM),
and it was proposed that additional structural modifications at
the 2′ position could enhance activity further. This was sup-
ported by ourselves in a whole-cell phenotype-based anticancer
assay for spindle multipolarity induction, where increased
activity was seen for analogues with modifications in the 2′
position, with the 2′-benzyloxy-2′-demethoxy-griseofulvin ana-
logue (17) being the most potent compound tested.22 Multiple
papers state that griseofulvin arrests several cancer cell lines in
G2/M phase of the cell cycle.17−19 Several investigators have
proposed tubulin as the main target for griseofulvin, although
for mammalian cells this suggestion is not undis-
puted.9,17,18,20,21,23,24 Recently, Panda et al.25 proposed two
griseofulvin binding sites on tubulin using molecular docking
studies and similar to Oda21 reported that microtubule dyna-
mics were disrupted by 1. Using the hepatitis C virus-1b cell
culture system Huh7/Rep-Feo, Jin et al. reported that G2/M
phase arrest in infected cells was induced by griseofulvin (1).20
It was speculated that the effect was due to interaction with
microtubule polymerization.20
Scheme 1
a
(a) MgI2, toluene, Et2O; (b) RBr, Ag2O, dioxane; (c) HOAc, 2 M
H2SO4; (d) 0.66 M NaOH; (e) MeOH, CSA; (f) MeOH, CSA (13)
or EtBr, Ag2O, dioxane, then MeOH, CSA (14).
Position 4 analogues 7 and 8 were prepared from 4 with Ag2O
and the appropriate alkyl bromide in dioxane as solvent.27 The
syntheses of 9 and 10 have already been described.22,28
Griseofulvin exhibits activity against fungi and mammalian
cancer cells as well as suppressing RNA replication by the hepa-
titis C virus, with tubulin having been proposed to be involved
in all three cases. Tubulins are very conserved within different
eukaryotic cell types,24 and most of the variation among dif-
ferent tubulin isoforms is found in the amino acids near the
C-terminus, which form a ridge on the surface of microtubules.
Therefore, variations among different isoforms are expected to
affect primarily the association of accessory proteins with the
surface of microtubules rather than microtubule polymerization
per se. In case tubulin is the sole target of griseofulvin in both
fungi and mammalian cells, the activity profile of an array of
analogues against these cell types should be similar. To test this
hypothesis, we decided to carry out an SAR study of griseo-
fulvin analogues. This is, to the best of our knowledge, the first
study of griseofulvin (1) and analogues thereof which compares
antifungal and anticancer SAR from growth inhibition assays.
Griseofulvin (1) and 54 griseofulvin analogues (11 reported
for the first time here) have been tested in an antifungal assay
against Trichophyton mentagrophytes and Trichophyton rubrum,
two dermatophytes causing tinea capitis.7 All compounds were
also tested in an anticancer assay against the human cell line
MDA-MB-231 (breast adenocarcinoma), which was chosen
because this cell line represents a common cancer type and is
known to harbor supernumerary centrosomes which are regu-
larly clustered into a bipolar mitotic spindle array in a high
percentage of cells.26 All analogues, having alterations at the 4,
5, 6, 2′, 3′, and 4′ positions, have been synthesized from com-
mercially available griseofulvin in one to five synthetic steps, as
described here or following published procedures.
For the preparation of 11, 12, and 13, see Rønnest et al.29
and Arkley et al.27 Compound 12 was alkylated using Ag2O and
EtBr followed by repeated solvolysis in MeOH with camphor
sulfonic acid (CSA) to afford 14. The dichloro analogues 15
and 16 were synthesized using 2,30 POCl3, LiCl, and dioxane,
a modification of a known method (Scheme 2).31 The
a
Scheme 2
a
(a) LiCl, POCl3, dioxane.
compounds 17−33 were synthesized by 1,4-addition of the
corresponding alcohol to 16 using either NaH or 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) as base (Scheme 3).31 The iso-
griseofulvin analogues 3 and 35 were synthesized in the same
manner but from 15. Compound 3 has previously been
prepared by treating 2 with excess diazomethane, yielding both
1 and 3.32 Compounds 26 and 38 have been reported in a
patent,33 which is also the case for 19, 21, and 22.34 The dimer
34, which was conveniently synthesized together with 22 by a
second 1,4-addition of 22 to 16, has previously been published
in a Japanese patent.35
The analogues with an oxime functionality 36, 37, and 38
were synthesized from the corresponding ketones (1, 17, and 26)
using hydroxylamine hydrochloride in ethanol and DMSO.13,22
Isogriseofulvin (39−44) and griseofulvin analogues (45−49
and 17) were synthesized simultaneously in pairs by solvolysis
RESULTS AND DISCUSSION
■
Chemistry. The 4-phenol (4) was synthesized from 1 by
treatment with freshly prepared MgI2, procured by sonica-
tion of Mg and I2 in Et2O/toluene, affording 4 in 99% yield,
653
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
a
Scheme 3
Figure 2. The analogues 50−55 were prepared according to literature
procedures.22 Geodin (56) was isolated as previously described.36
that all compounds were soluble in the assay media, which for
some compounds were not the case at higher concentrations.
Antifungal SAR for T. rubrum and T. mentagrophytes.
All compounds in the position 4 series of 4−8 were less active
than griseofulvin (1) against the two fungi and 8 was
completely inactive. For T. rubrum, 4 and 6 were inactive, 7
had low activity, and 5 was, with an IC50 of 2.0 μM, appro-
ximately five times less potent than griseofulvin. For
T. mentagrophytes, analogue 4 showed low activity and the
rest were less potent than 1: 5 (0.29 μM), 6 (0.25 μM), and 7
(0.17 μM). The activity of 6 has previously been reported against
a number of dermatophytes (Epidermophyton floccosum, Micro-
sporum canis, Trichophyton interdigitale, Trichophyton persicolor,
T. mentagrophytes, and T. rubrum) and found to be less active
than 1 against all of them.11
a
(a) RH, DBU, THF, or dioxane; (b) hydroxylamine, NaOAc, EtOH,
DMSO.
with the respective alcohols and catalytic CSA and subsequently
separated by chromatography (Scheme 4).
a
Scheme 4
The nitro (9) and amine (10) position 5 analogues were
inactive against both fungi. Compound 10 has also been
reported as inactive, but 9 has been reported as weakly active
with inhibition for both fungi starting at around 25 μM.13 Other
position 5 analogues (ethoxycarbonyl,14 methoxy, methyl, or
chloro37) have been reported to have lower activity than 1
against dermatophytes.
a
(a) ROH, CSA, dioxane (17, 42−44, 48, and 49).
Whereas the position 6 phenols 13 and 11 were inactive
against both fungi, the 6-ethyl analogue (14) had an IC50 of
1.3 μM against T. rubrum and was as active as griseofulvin (1)
against T. mentagrophytes with an IC50 of 0.062 μM, which is in
accordance with the literature.11
Analogues 50−55 were synthesized according to published
procedures (Figure 2).22 The fungal secondary metabolite
geodin (56) was recently isolated from Aspergillus terreus.36
Please see the Supporting Information for experimental details
and spectral data.
The 2′ series is the most extensively studied due to the
increased activity observed in the anticancer phenotype-based
assay19 by these analogues. For T. rubrum, elongation to 2′-
ethyloxy (45, 0.69 μM) and 2′-propyloxy (46, 0.62 μM)
lowered the activity 2-fold, confirming the findings of Crosse
et al.11 The bulkier 2′-isopropyloxy (47, 3.1 μM) was 10 times less
potent. Increasing bulkiness through 2′-cyclopropylmethoxy
(48, 1.5 μM), cyclopentyloxy (49, 2.5 μM), and benzyloxy (17,
1.1 μM) enol ethers did not reveal a trend, but all three were
less active toward T. rubrum than 1. No correlation between
the size of the 2′ substituent and the activity was seen for
T. mentagrophytes with 46 (0.050 μM) retaining the activity
of 1 and IC50 values of 0.25 μM and 0.11 μM for 45 and
47, respectively. Both 45 and 46 have been reported to retain
the activity of 1 against T. mentagrophytes by Crosse et al.11
Assays. Griseofulvin (1), geodin (56), and 53 griseofulvin
analogues covering variations on six positions (see Figure 1)
were tested against two dermatophytes (T. rubrum and T.
mentagrophytes) and against the breast cancer cell line MDA-
MB-231 in a cytotoxicity assay. For all the test results the
following definitions were used: if no activity was observed at
50 μM, a given compound was deemed inactive; if activity was
observed but 50% inhibition was not reached at 50 μM, a given
compound is described as having low activity and no IC50 value
is calculated (see Supporting Information for examples). The
IC50 of griseofulvin (1) was determined to be 0.38 0.048 μM
against the T. rubrum isolate and 0.058
0.018 μM against
the T. mentagrophytes isolate. All IC50 values and 95% confidence
intervals are given in Table 1. All dilution series were made
starting with 50 μM of the tested compound in order to ensure
654
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
a
Table 1. All Available IC50 Values from the Screen
STD
IC50
(μM)
T. menta-
grophytes
(μM)
STD
IC50
(μM)
STD
IC50
(μM)
STD
IC50
(μM)
T. menta-
grophytes
(μM)
STD
IC50
(μM)
STD
Cmp. T. rubrum
cytotox
(μM)
Cmp. T. rubrum
no. (μM)
cytotox
(μM)
IC50
no.
(μM)
(μM)
1
0.38
inactive
inactive
inactive
2.0
0.05
0.058
0.02
18
4
30 low activity
31 low activity
low activity
0.70
19
11
2
3
2
inactive low activity
inactive inactive
inactive low activity
inactive
inactive
inactive
inactive
20
inactive
inactive
inactive
inactive
4
0.6
3
inactive
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
0.96
inactive
inactive
inactive
1.3
0.4
0.14
0.07
8.1
2
4
inactive
inactive
inactive
inactive
inactive low activity
-
5
0.7
0.29
0.25
0.1
inactive
8.5
2
6
inactive
low activity
inactive
inactive
inactive
inactive
inactive
1.3
inactive
0.009
inactive low activity
25
5
7
0.17
0.02
low activity
17
0.5
0.9
0.19
0.30
0.1
0.2
12
2
8
inactive
inactive
inactive
inactive
inactive
0.1
inactive
inactive
inactive
inactive
inactive
0.062
inactive
low activity
0.26
inactive
inactive
inactive
inactive
2
7.9
1.4
0.3
0.8
9
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
0.69
inactive
inactive
inactive
8.3
10
11
13
14
15
inactive low activity
inactive low activity
inactive low activity
inactive low activity
inactive low activity
inactive low activity
low activity
43
48
6
10
10
4
inactive low activity
0.02
36
1.0
3.2
2.1
4.7
6.5
1.8
7.0
2.8
7.2
17
3
0.1
0.3
0.2
0.7
1
52
inactive
inactive
inactive
27
16 low activity
low activity
13
17
18
19
20
21
1.1
1.3
0.2
0.4
0.1
0.02
0.01
0.1
0.2
0.09
1
0.25
0.050
0.08
0.02
0.05
0.02
0.09
3
0.058
0.041
0.24
0.62
16
5
0.83
1.0
very wide
0.2
3.1
0.11
11
3
2
0.3
0.9
0.6
1
1.5
0.3
0.8
0.060
9.4
1.5
0.2
0.15
0.1
2.5
0.24
3.2
0.5
22 low activity
23 6.1
24 low activity
1.7
1
50 low activity
51 low activity
low activity
3.0
inactive
inactive
inactive
22
inactive
inactive
inactive
1
7
1.6
3
1
0.28
0.2
2
52
53
54
55
56
inactive
inactive
1.6
inactive
inactive
inactive
25
26
27
inactive
2.6
inactive
0.3
inactive
0.25
inactive
0.09
inactive
3
5.8
13
1
inactive low activity
2
0.4
0.23
1.1
0.08
0.5
8.0
1
inactive
inactive
inactive
1.3
4.7
32
0.5
6
inactive
inactive
inactive
inactive
low activity
9.9
28 low activity
29 low activity
inactive
inactive
1
0.35
0.2
10
2
Table 1. continued
a
If no activity was observed at 50 μM, a given compound is deemed inactive. If activity was observed, but 50% inhibition was not reached at 50 μM, a
given compound is described as having low activity and the IC50 value is not calculated.
Analogue 48 (0.060 μM) had the same activity as 1, but 17
(0.26 μM) and 49 (0.24 μM) were about four times less potent.
The activities of 17, 45, and 46 have been reported against
T. mentagrophytes and T. rubrum, and all showed lower activity
when tested against the latter.11 Lower activity than 1 toward
T. mentagrophytes was also reported for 17, but 45 and 46 had
similar potency.11 The three compounds were also tested
against E. floccosum, M. canis, T. interdigitale, and T. persicolor
displaying the same trends, with 17 having lower activity and
45 and 46 retaining the activity of 1.11
The rest of the 2′ series includes bulkier analogues (25−27),
para-substituted variations of 17 (19−22), two sets of ortho-,
meta-, and para-analogues (28−30 and 31−33), modification
of the linker part (23 and 24), and a vinyl sulfide analogue 18.
Of the bulkier 2′ analogues, the naphthalen-1-ylmethoxy 26
was the only active analogue and 25 and 27 were inactive
against both fungi. With IC50 values of 2.6 μM (T. rubrum) and
0.25 μM (T. mentagrophytes), compound 26 is three and four
times less potent than 1, respectively.
The three 2′ pyridine analogues (28−30) had low activity
toward T. rubrum, and 30 also showed low activity toward
T. mentagrophytes. Compounds 28 and 29 had IC50 values of
1.3 and 0.35 μM, meaning they were approximately 22 and 6
times less potent toward T. rubrum compared to 1, respectively.
The series with ortho-, meta-, and para-methoxy groups revealed
33 as inactive against both fungi, 31 with low activity toward
T. rubrum and an IC50 of 0.70 μM against T. mentagrophytes.
The meta-substituted analogue 32 (0.96 μM) was 3-fold less
active against T. rubrum compared to 1 and displayed a 2-fold
decrease in activity against T. mentagrophytes with an IC50 of
0.14 μM.
The 2′-phenoxy analogue (23) had IC50 values of 6.1 μM
against T. rubrum and 1.6 μM against T. mentagrophytes, while
the phenylethoxy analogue (24) had low activity against
T. rubrum and an IC50 of 0.28 μM against T. mentagrophytes.
Lower activity for 23 has previously been reported.11 The vinyl
sulfide analogue 18 (1.3 μM) was as active as 17 against
T. rubrum, but against T. mentagrophytes the IC50 (0.058 μM)
was equal to that of griseofulvin (1).
The 3′-dimethyl analogue 52 was inactive against both fungi.
The series of 3′ analogues include three 3′-iodo analogues with
2′ modifications, 2′-methoxy (53), 2′-propoxy (54), and 2′-
benzyloxy (55). The 3′-iodo griseofulvin (53) was inactive
against T. rubrum and had low activity toward T. mentagrophytes
in accordance with Crosse et al.11 The 2′-benzyloxy (55) ana-
logue was inactive against T. rubrum and close to 20 times less
potent (1.1 μM) than 1 against T. mentagrophytes. Analogue 55
was also four times less potent than 17, which has no 3′ iodo
group. The propoxy (54) analogue showed activity against both
fungi (T. rubrum 1.6 μM and T. mentagrophytes 0.23 μM) but
was less potent than both 1 and the analogue 46, which does
not contain iodide. Analogue 54 has been reported to be the
most potent at inducing curling of hyphae but less active
against all dermatophytes spare one.11
655
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
The introduction of an oxime functionality on the parent
compounds 1, 17, and 26 afforded the analogues 36, 37, and 38
and lowered the activity toward both fungi compared to the
parent ketones (38 was inactive against both fungi altogether).
The IC50 values for 36 were 1.3 μM against T. rubrum and
0.19 μM against T. mentagrophytes, which is in accordance with
previously published data.13 Analogue 36 has also been
reported active against M. gypseum.15 Compound 37 had IC50
values of 7.9 μM against T. rubrum and of 0.30 μM against
T. mentagrophytes, 20 and 3 times less potent than 1,
respectively. The 4′ alcohol 50 exhibited low activity toward
both fungi, which was not in agreement with prior work, as it
has been reported that 50 causes 100% growth inhibition of
T. rubrum and visible inhibition against T. mentagrophytes at
28 μM.13 Compound 50 has however been tested inactive
against M. gypseum.15
The series including isogriseofulvin (3) and isogriseofulvin
analogues 35 and 39−44 was inactive against T. rubrum and
showed low activity against T. mentagrophytes. This is in accor-
dance with the literature, where 3 and 39 have been reported
to be less active than griseofulvin against a number of derma-
tophytes (E. floccosum, M. canis, T. interdigitale, T. persicolor,
T. mentagrophytes, and T. rubrum).11
Griseofulvic acid (2) was inactive against T. rubrum and had
low activity against T. mentagrophytes contrary to prior work,
which reported 2 to be inactive toward T. mentagrophytes but
having visible growth inhibition at 30 μM against T. rubrum.13
The griseofulvin dimer 34 was inactive against both fungi. The
dichloro analogue 16 showed low activity toward both fungi,
while 15 was inactive against both. The reduced analogue 51
had low activity against T. rubrum and an IC50 of 3.0 μM
against T. mentagrophytes, which is 50 times less potent com-
pared to 1. Geodin (56) was inactive against both fungi.
Of the 55 compounds tested, 27 analogues were inactive or
exhibited low activity for both fungi (see Table 1). Nine of the
compounds were active against both fungi within the same
order of magnitude compared to griseofulvin (17, 20, 21, 23,
26, 32, 36, 49, and 54).
5 analogues 9 and 10 were inactive in the cytotoxicity assay as
they were in the assay for multipolarity induction.
The 6-phenol griseofulvic acid analogue 11 was inactive, and
the 6-phenol griseofulvin analogue (13) showed low activity.
The 6-ethyl griseofulvin (14) analogue was approximately 50%
less active than griseofulvin. The two dichloro analogues 15 and
16 were some of the most cytotoxic among the tested
analogues, with IC50 values of 1.0 and 3.2 μM, respectively. 2′-
Chloro analogue 16 has previously been observed by us to have
a different phenotype from all other analogues in the cell-based
assay for multipolarity induction.22 The highly electrophilic
nature of 16 prompted an investigation of the ability of this
analogue, together with 1 and 17, to act as Michael acceptors.
Briefly, the three compounds were incubated in buffer with
two different potential nucleophiles, 2-aminoethanol and
2-mercaptoethanol, for up to seven days (see Supporting
Information for details). The experiments showed that neither
1 nor 17 underwent addition of the nucleophiles, whereas 16
did react with 2-mercaptoethanol to form the Michael
addition/elimination product, which was the major constituent
after 72 h of incubation. This confirms that the highly electron
withdrawing properties of the chlorine atom enable 16 to react
as a general alkylating agent and one could imagine the for-
mation of a covalent bond to reactive cysteine residues in
proteins. The experiment also shows that in the absence of the
chlorine atom, this type of reactivity is not present for the
griseofulvin analogues. Furthermore, the three representa-
tive compounds were found to be stable in the absence of
nucleophiles for 1 week of incubation, showing only a slight
decrease in purity.
When elongating the 2′ position from the parent methoxy
(1) to ethoxy (45) and propoxy (46) and then further in-
creasing the bulkiness with isopropoxy (47), cyclopentoxy
(49), and benzyloxy (17), the activity increased through the
series (see Figure 3), plateauing with 17 and 49 at 2.1 and
There were however some compounds that fared differently
against the two fungi. The compounds 6, 7, 22, 24, 28, 29,
31, 51, and 55 were all inactive or had low activity toward
T. rubrum but had IC50 values in the range of 0.25−3.0 μM
against T. mentagrophytes. For other analogues (14, 18, 19, 37,
47, and 48) the activity against T. mentagrophytes was similar to
that of 1 but against T. rubrum it was significantly lower. This is
in accordance with earlier observations by Crosse et al., demon-
strating that a given analogue was more active against some
fungi but less active against others.11
Anticancer SAR. Looking at the IC50 values for the 16
analogues (1, 17, 18, 20, 23−25, 27, 36, 37, 45−49, and 54)
tested active in both the multipolarity assay19 and the cytotoxicity-
based assay there was good correlation between the data with an
R2 of 0.70 (see Supporting Information).
Figure 3. The activity increases with the number of carbon atoms at
the 2′ position from griseofulvin itself to the 2′ ethoxy (45), propoxy
(46), isopropoxy (47), and cyclopropylmethoxy (48) before leveling
out with the cyclopentoxy (49) and benzyloxy (17) analogues. The
bulkier analogues 25, 27, and 26 were less active than the benzyloxy
analogue (17).
The IC50 of griseofulvin (1) was determined to be 18 4 μM
3.2 μM, respectively. The same trend was seen when these
compounds were tested in the phenotype-based multipolarity
assay. Moving from the benzyloxy derived analogues to even
bulkier groups like the naphthalen-1-ylmethoxy (26, 13 μM),
biphenylmethoxy (25, 5.8 μM), and 1-adamantylmethoxy (27,
4.7 μM) analogues, the activity did not increase further. The
three compounds were still more potent than griseofulvin
though, which was also observed for 25 and 27 in the phenotype-
based assay. The trend seen for substituents of increasing size
(20
1 μM in the phenotype-based spindle multipolarity
assay22), while an IC50 of 25 4 μM against HeLa cells was
found by Panda et al.18 In the position 4 series, 4, 5, and 7 were
all inactive but 6 and 8 harboring aromatic moieties had
activities similar to griseofulvin (1), with IC50 values of 20 (6)
and 17 μM (8). This indicates a mode of action for cytotoxicity
that does not involve induction of multipolar mitosis, as 6
was inactive in the multipolarity assay. Both the position
656
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
does not indicate that increasing lipophilicity also leads to
increasing potency, which would be the case if transport over
the cell membrane was a determining parameter for activity.
This is further illustrated by the analogues 17, 23, and 24,
where the former is the most active (Table 1), indicating that a
binding event is responsible for the activity.
increased potency against the cancer cell line but against the
two fungal strains the activity was lower than for griseofulvin
(1). Looking at the 4 position, there were inconsistencies
throughout all three cell types. The ethyl analogue 5 was active
against both fungi but inactive toward the cancer cells, while
the bulkier naphtyl analogue 8 was inactive against both fungi
but more potent than griseofulvin against the MDA-MB-231
cells. Analogues 6 and 7 were more potent than 5 against
T. mentagrophytes, but both compounds were inactive or showed
low activity toward T. rubrum. The most potent analogue
toward the cancer cells (15) was inactive against both fungi and
the isomer (16), which was among the most active compounds,
only had low activity toward the two fungi. The two bulky
analogues 25 and 27 as well as the dimer (34) were inactive in
the antifungal assays but were all more potent than griseofulvin
against the cancer cell line. It is also worth noticing that geodin
(56) was inactive against both fungi but twice as potent as 1
against the cancer cell line.
The introduction of an oxime at the 4′ position (36, 37, and
38) decreased the potency compared to the parent compounds
(1, 17, and 26) against both fungi. The opposite effect was seen
for the cancer cell line, where the introduction of this moiety
increased the activity for all three compounds.
There were however some similar trends for all three cell
types. A number of analogues were inactive or had low activity
toward both fungi and cancer cells. Among these were the
position five analogues (9, 10), isogriseofulvin (3), and the series
of isogriseofulvin analogues (39−44), with 35 as the sole
exception. The two 6 phenols (11, 13) were virtually inactive
against all cell types, which was the same for 50−52.
A number of variations of 17 have been tested (18−24, 28−
30, and 31−33), and although all except 33 (low activity), 28
(32 μM), and (24, 17 μM; 30, 19 μM) were more active than
1, only 20 was as active as 17 with an IC50 of 1.8 μM. The
difference in activity between the phenoxy (23, 7.2 μM) and
phenylethoxy (24, 17 μM) analogues was less pronounced in
the cytotoxicity assay than in the multipolarity assay with two
and a half orders of magnitude compared to the approximately
7-fold difference in the phenotype-based assay. The dimer (34)
was about twice as active as 1 with an IC50 value of 8.5 μM.
The introduction of an oxime functionality at the 4′ position
(36) increased potency 2-fold to 12 μM, an increase in activity
that was also seen in the phenotype-based assay. Introducing
the oxime to analogues 17 and 26 affording 37 and 38 im-
proved the activity further for both compounds. Apart from 15,
37 is the most active analogue in the cytotoxicity assay with
an IC50 of 1.4 μM. The stability of the oximes in PBS buffer
(pH 7.4) was tested, and less than 5% hydrolysis to the parent
ketones could be detected after 48 h (data not shown).
The isogriseofulvin analogues tested in the multipolarity
assay (3 and 39−44) were all inactive. In the cytotoxicity assay,
35 retained the activity of 1, while the rest were either inactive
(3), showed low activity (39, 44), or were less potent (40,
43 μM; 41, 48 μM; 42, 52 μM; 43, 27 μM).
Geodin (56) did not induce multipolar mitoses and was in
that respect deemed inactive.36 Geodin (56) is however twice
as cytotoxic as griseofulvin when tested against the MDA-MB-
231 cell line, with an IC50 of 9.9 μM opposed to 18 μM for 1.
The reduced analogues 50 and 51 as well as the 3′-dimethyl
analogue 52 were inactive in both anticancer assays. In the 3′
iodo series, 53 (22 μM) retained activity, the 2′ propoxy
analogue (54, 8.0 μM) had increased activity, and 55 exhibited
low activity. 53 and 55 were inactive in the phenotype-based
assay, but 54 was more potent than 1.
CONCLUSION
■
The first comparison of antifungal and anticancer SAR for
griseofulvin analogues is presented in this work covering 53
analogues of griseofulvin as well as the natural product geodin
(56). All compounds have been tested against T. rubrum,
T. mentagrophytes, and in a cytotoxicity assay against MDA-MB-
231 breast cancer cells.
Even though there were similarities between the SAR of the
two fungi, with some compounds showing no activity against
both fungi and some active against both, there were also
some differences. Analogues 46 and 48 retain the activity of
griseofulvin against T. mentagrophytes but were 2- and 4-fold
less active against T. rubrum than 1, respectively. This is in
accordance with reported observations by Crosse et al. that
analogues differ in activity against different dermatophytes.11
We show that there is a good correlation between the IC50
values from analogues that were active in both the phenotype-
based assay for spindle multipolarity22 and the cytotoxicity
assay used in this work with an R2 of 0.70. Comparing the two
fungal SARs with the anticancer cytotoxicity SAR afforded
distinct differences. The analogues 8, 25, 27, and 38 were inac-
tive or had low potency against both fungi but either retained
the activity (8) or were at least 2-fold more active against the
cancer cells compared to 1. The 2′-benzyloxy analogue (17)
was less active against both fungi in this study and against six
dermatophytes in the study by Crosse et al.11 This analogue is
however nine times more potent than 1 against the cancer cell
line MDA-MB-231. It is noteworthy that the two dichloro
compounds 15 and 16 were inactive against both fungi but
featured as some of the most cytotoxic agents against the cancer
cell line, possibly due to their electrophilic nature, which is
unique among the tested analogues.
Anticancer SAR versus Antifungal SAR. The difference
between the antifungal and anticancer data is illustrated in
Figure 4, demonstrating that most of the analogues had
Figure 4. The IC50 values for each cell line have been normalized
defining the activity of griseofulvin as having a value of 1. It is seen that
of the 53 analogues the majority was less active against the two fungi
opposed to the anticancer activity where most analogues were more
active than griseofulvin.
657
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
water from a Milli-Q gradient fitted with a Millipore 0.22 μm filter
(cat. no. MPGL04001), both from Millipore (Billerica, MA, USA).
Each well contained 99 μL of RPMI media with inoculum, 99 μL of
Milli-Q water, and 2 μL of DMSO. Microplates were incubated for
7 days at 37 °C in a WTB binder incubator from VWR (Radnor, PA,
US), at which point the OD of the wells were measured at 530 μM on
a BioTek Synergy 2 (Brøndby, Denmark) using Gen5TM 1.10.8
software. The half-maximal inhibitory concentration (IC50) values
were obtained with GraphPad Prism5 (ver. 5.04) from GraphPad
Software (La Jolla, CA, USA) using the “nonlinear regression, sig-
moidal dose−response (variable slope)” function. The fitting method
used was “automatic outlier elimination”,39 which eliminated less than
1.1% of data points.
Cytotoxicity Assay. MDA-MBA-231 human adenocarcinoma cells
were cultivated in Leibovitz’s L-15 medium in the presence of 10%
fetal calf serum at 37 °C. Cell viability was examined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma
Chemical, St. Louis, MO) colorimetric assay, as previously described.40
Briefly, cells were plated in 96-well microtiter plates at a density of
(2−3) × 104 cells per well, and each plate was incubated for 24 and/or
48 h, with MTT added to each well for at least 4 h. The absorbance of
each well was measured at 570/630 nm using a spectrophotometer
(Molecular Devices, Sunnyvale, CA). Each condition was analyzed in
at least three replicates, and the results are presented as the mean
standard deviation of replicates of a representative experiment that was
repeated at least three times.
The differences in activity observed for the two fungi and the
MDA-MB-231 cell line could rise from a number of factors,
such as transport over the cell membrane, efflux pumps, or
different modes of action. We find it unlikely that transport or
efflux pumps could explain that the majority of analogues were
more active against MDA-MB-231 cells and less active against
both fungi. Our conclusion therefore is that the mode-of-action
of griseofulvin(s) toward fungal and mammalian cells is
different, making it unlikely that tubulin itself constitutes the
main cellular target in both fungi and mammalian cells. Because
it has been shown that griseofulvin leads to mitotic arrest in
both fungal and mammalian cells, an alternative explanation is
that griseofulvin disrupts microtubule dynamics without
directly interacting with tubulin, e.g., through interaction with
microtubule-associated proteins (MAPs).
EXPERIMENTAL SECTION
General. Starting materials, reagents, and solvents were purchased
from Sigma-Aldrich and used without further purification. H NMR
■
1
spectra were recorded using either a Varian Unity Inova 500 MHz
spectrometer or a Varian Mercury 300 MHz spectrometer both from
Agilent (Santa Clara, CA, US). 13C NMR spectra were recorded using
either a Varian Mercury 300 MHz or a Bruker AC 200 MHz from
Bruker Optics (Ettlingen, Germany). Chemical shifts were measured
in ppm and coupling constants in Hz. When CDCl3 was used as
solvent, the residual peak was used as internal reference at δ 7.27 for
1H NMR and δ 77.00 for 13C NMR spectra. IR spectra were recorded
using a Bruker Alpha ATR and measured in cm−1. All melting points
are uncorrected. TLC was performed on aluminum sheets precoated
with silica gel 60 F254 (Merck 1.05554.0001). Compounds were
visualized by charring after dipping in a solution of 1% KMnO4, 6.7%
K2CO3, and 0.08% NaOH in water. UV visualization was done using a
model UVGL-25 Mineralight lamp. High-resolution LC-DAD-MS was
performed on an Agilent 1100 system equipped with a photodiode
array detector (DAD) and coupled to a LCT orthogonal time-of-flight
mass spectrometer (Waters-Micromass, Manchester, UK) with a
Z-spray electrospray ionization (ESI) source and a LockSpray probe
(M + H 556.2771) and controlled by MassLynx 4.0 software. LC-MS
calibration from m/z 100−900 was done with a PEG mixture.
Standard separation involved a LUNA 2 column with an acetonitrile
(50 ppm TFA) in water gradient starting from 15% to 100% over
25 min with a flow rate of 0.3 mL/min. Purity of all compounds was
found to be greater than 95% by LC-DAD. Microanalyses were ob-
General Procedure for the Synthesis of Position 4 Ethers (7
and 8). The appropriate alkyl bromide (1.2 mmol, 3 equiv) was added
to a solution of 4 (0.4 mmol, 1 equiv), Ag2O (1.2 mmol, 3 equiv), and
1,4-dioxane (5 mL). The mixture was stirred at 50 °C for 18 h and
then cooled to 20 °C. EtOAc (10 mL) was added to the solution, and
the mixture was washed with brine (15 mL). The aqueous phase was
extracted with EtOAc (2 × 10 mL), and the combined organic phases
were dried (MgSO4) and concentrated. The residue was purified by
column chromatography (EtOAc:heptane 1:3) to afford the desired
product.
(2S,6′R)-(7-Chloro-6-ethoxy-4-methoxy-benzofuran-3-one)-
2-spiro-1′-(2′-methoxy-6′-methyl-cyclohex-2′-ene-4′-one) 14.
Ethyl bromide (0.9 mmol, 3 equiv) was added to a solution of 12
(0.3 mmol, 1 equiv), Ag2O (0.9 mmol, 3 equiv), and 1,4-dioxane
(3 mL). The mixture was stirred at 50 °C for 18 h and then cooled to
20 °C. EtOAc (10 mL) was added to the solution, and the mixture was
washed with brine (15 mL). The aqueous phase was extracted with
EtOAc (2 × 10 mL), and the combined organic phases were dried
(MgSO4) and concentrated. The following process was repeated three
times: The residue was dissolved in MeOH (10 mL), CSA (0.1 mmol)
was added, and the mixture was heated to 65 °C for 18 h. EtOAc
(10 mL) was added to the solution, and the mixture was washed with
satd aq NaHCO3 (15 mL). The aqueous phase was extracted with
EtOAc (2 × 10 mL), and the combined organic phases were dried
(MgSO4) and concentrated. The residue was purified by column
chromatography (EtOAc:heptane 1:3) to afford the desired product
14. Yield, 5 mg (5%) (yellow oil); Rf value (EtOAc/heptane, 5:1),
0.43. IR (neat, cm−1): 1709, 1666, 1613, 1587 (lit.41). 1H NMR
(CDCl3, 300 MHz): δ 6.13 (1H, s), 5.56 (1H, s), 4.25 (2H, q, J =
7.0 Hz), 3.98 (3H, s), 3.63 (3H, s), 3.05 (1H, dd, J = 16.7, 13.4 Hz),
2.86 (1H,), 2.45 (1H, dd, J = 16.7, 4.7 Hz), 1.56 (3H, t, J = 7.0 Hz),
0.98 (3H, d, J = 6.7 Hz). 13C NMR (CDCl3, 50 MHz): δ 197.0, 192.4,
170.8, 169.6, 164.1, 157.7, 104.9 (2C), 93.4, 90.7, 90.2, 65.8, 56.6, 56.3,
40.1, 36.4, 14.6, 14.2; [α]20D = +252° (c = 0.5 in CCl3) (Lit.27). HRMS
(ESI+) calcd for [M + H]+ [C18H20ClNO6]+ 367.0948, found
367.0949.
tained from H. Kolbe Mikroanalytisches Laboratorium in Mulheim an
̈
der Ruhr, Germany. The purity of all tested compounds was found to
be >95% by HPLC.
Antifungal Assay. The fungal micro broth dilution assay was
performed in sterile flat bottomed microplates (cat. no. 655101) and
lids (cat. no. 656161) from Greiner Bio-One GmbH (Frickenhausen,
Germany). Each microplate accommodated five dilution series starting
at 50 μM of a given analogue. Each plate was also fitted with four
negative and four positive control wells. The fungi T. rubrum
(IBT29284) and T. mentagrophytes (IBT2724) were kept in the IBT
fungal collection at the Department of Systems Biology at the
Technical University of Denmark. The identity of the fungal cultures
was verified by microscopy after the last test round in the antifungal
assay. For testing against T. rubrum, a 4-fold dilution series was used,
resulting in 10 concentrations with the lowest at 0.2 μM. For
T. mentagrophytes, an 8-fold dilution series was used, resulting in 10
concentrations with the last one at 0.4 pM.
Inoculum preparation was performed in accordance with the
document M38-A238 from the “Committee for Clinical Laboratory
Standards”. Inoculum was adjusted to 0.90−1.5 OD at 530 nM on a
Genesys 10uv from Thermo Electron Corporation (Waltham, MA,
US). The inoculums were diluted 50-fold with RPMI media (with
L-glutamine, without NaHCO3, cat. no. R6504 in MOPS buffer (cat.
no.69947)), both from Sigma-Aldrich (St Louis, MO, USA). The pH
was adjusted to 7.00 with 1 M NaOH, and the volume with Milli-Q
General Procedure for the Synthesis of 2′ Enol Ethers (19,
21, 22, 26, 28−33). To a solution of 16 (0.65 mmol, 1 equiv) in 1,4-
dioxane (3 mL, 0.2 M) was added the desired alcohol (1.30 mmol,
2 equiv) and DBU (1.63 mmol, 2.5 equiv). The mixture was heated to
100 °C and stirred for 12 h. The mixture was then cooled to 20 °C,
and excess reagent was quenched with satd aq NH4Cl (30 mL). The
aqueous phase was extracted with EtOAc (3 × 30 mL), and the
combined organic phases were dried (MgSO4) and concentrated. The
658
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
residue was purified by column chromatography (EtOAc:heptane: 1:3)
affording the product. When possible, the product was recrystallized
from EtOAc/heptane.
compounds 3, 7, 8, 15, 19, 21, 22, 26, 28−35, and 38−44. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(2S,6′R)-(7-Chloro-4,6-dimethoxy-benzofuran-3-one)-2-
spiro-1′-(2′-(naphthalen-1-ylmethoxy)-6′-methylcyclohex-2′-
ene-4′-one-4′-oxime) 38. To a solution of 26 (0.08 mmol, 1.0
equiv) in EtOH (2 mL) and DMSO (1 mL) was added hydroxylamine
hydrochloride (0.30 mmol, 3.5 equiv) and sodium acetate (0.37 mmol,
4.3 equiv). The mixture was stirred at 75 °C for 18 h, allowed to reach
20 °C, and diluted with EtOAc (5 mL). The mixture was washed with
brine (3 × 4 mL). The organic phase was dried (MgSO4) and
concentrated. The crude mixture was purified by column chromatog-
raphy (EtOAc:heptane 1:1) to afford 38 (40 mg, 96%) as a yellow oil.
Rf values (EtOAc/heptane, 5:1), 0.60 and 0.51. IR (neat, cm−1): 1704,
AUTHOR INFORMATION
Corresponding Author
*For T.O.L.: phone, +45 4525 2632; E-mail, tol@bio.dtu.dk.
For M.H.C.: phone, +45 4525 2131; E-mail, mhc@kemi.dtu.dk.
■
ACKNOWLEDGMENTS
■
We thank Anja Irsigler, Christine Immel, and Sigrid Heil for
cytotoxicity assay testing. We thank Brian Dideriksen for
assistance with stability studies. We thank the Danish Research
Council (reference no. 274-07-0561), the Deutsche Krebshilfe
(grant no. 107739), and the Karen Krieger Foundation for
financial support.
1
1613, 1590. H NMR (500 MHz, CDCl3): δ 7.82−7.73 (3H, m),
7.50−7.42 (2H, m), 7.37−7.32 (2H, m), 6.54 (0.4H, s), 5.99 (0.6H, s),
5.96 (0.4H, s), 5.84 (0.6H, s), 5.31 (0.4H, d, J = 12.0 Hz), 5.29 (0.6H,
d, J = 12.0 Hz), 5.26 (0.4H, d, J = 11.9 Hz), 5.23 (0.6H, d, J = 11.9
Hz), 3.95 (1.8H, s), 3.93 (1.2H, s), 3.91 (1.8H, s), 3.90 (1.2H, s), 3.15
(0.6 H, dd, J = 16.8, 4.9 Hz), 3.06 (0.4 H, dd, J = 15.0, 13.3 Hz), 2.75
(0.6 H, dd, J = 16.8, 13.1 Hz), 2.70−2.57 (1 H, m), 2.44 (0.4 H, dd,
J = 15.0, 4.1 Hz), 0.98 (1.8H, d, J = 6.7 Hz), 0.98 (1.2H, d, 6.8 Hz).
13C NMR (75 MHz, CDCl3): δ 194.0 (0.5C, s), 193.9 (0.5C, s), 169.3
ABBREVIATIONS USED
■
CSA, camphor sulfonic acid; DBU, 1,8-diazabicyclo[5.4.0]-
undec-7-ene; MAP, microtubule-associated protein; PBS,
phosphate-buffered saline; SAR, structure−activity relationship
(0.5C, s), 164.0 (0.5C, s), 160.0 (0.5C, s), 157.3 (0.5C, s), 157.3
(0.5C, s), 157.3 (0.5C, s), 155.0 (0.5C, s), 151.8 (0.5C, s), 133.3
(0.5C, s), 131.2 (0.5C, s), 131.1 (0.5C, s), 130.8 (0.5C, s), 130.6
(0.5C, s), 128.9 (0.5C, s), 128.9 (0.5C, s), 128.8 (0.5C, s), 128.3
(0.5C, s), 128.2 (0.5C, s), 128.1 (0.5C, s), 126.3 (0.5C, s), 126.3
(0.5C, s), 126.2 (0.5C, s), 126.0 (0.5C, s), 125.7 (0.5C, s), 125.6
(0.5C, s), 125.2 (0.5C, s), 125.0 (0.5C, s), 124.9 (0.5C, s), 123.6
(0.5C, s), 123.5 (0.5C, s), 105.6 (0.5C, s), 105.5 (0.5C, s), 100.5
(0.5C, s), 96.9 (0.5C, s), 96.8 (0.5C, s), 93.9 (0.5C, s), 91.5 (0.5C, s),
91.4 (0.5C, s), 89.0 (0.5C, s), 89.0 (0.5C, s), 69.4 (0.5C, s), 69.0
(0.5C, s), 56.7 (1 C, s), 56.1 (0.5C, s), 56.1 (0.5C, s), 36.4 (0.5C, s),
35.2 (0.5C, s), 30.9 (0.5C, s), 25.5 (0.5C, s), 14.4 (0.5 C, s), 14.3
(0.5 C, s); [α]20D = +112° (c = 1.0 in CDCl3). HRMS (ESI+) calcd for
M + H [C27H25ClNO6]+ 494.1370, found 494.1371.
REFERENCES
■
(1) Oxford, A. E.; Raistrick, H.; Simonart, P. Studies in the
biochemistry of micro-organisms. LX. Griseofulvin, C17H17O6Cl, a
metabolic product of Penicillium griseo-fulvum Dierckx. Biochem. J.
1939, 33, 240−248.
(2) Gentles, J. C. Experimental Ringworm in Guinea PigsOral
Treatment with Griseofulvin. Nature 1958, 182, 476−477.
(3) Williams, D. I.; Marten, R. H.; Sarkany, I. Oral Treatment of
Ringworm with Griseofulvin. Lancet 1958, 2, 1212−1213.
(4) Alles, R.; Bekker, D.; Bonni, Z.; Khirsh, S.; Kal, B. O.; Kelle, E. U.
L. R.; Majer, F. K.; Shtjutts, A.; Uill, J. A. Terbinafine Solid Medicinal
Formulation for Oral Administration. Russian Patent RU2298402,
2007.
(5) Terbinafine. FDA: Washington, DC, 11/11/2011; http://www.
fda.gov/ForConsumers/ConsumerUpdates/ucm048710.htm.
(6) Gupta, A. K.; Summerbell, R. C. Tinea capitis. Med. Mycol. 2000,
38, 255−287.
(7) Seebacher, C.; Abeck, D.; Brasch, J.; Cornely, O.; Daeschlein, G.;
Effendy, I.; Ginter-Hanselmayer, G.; Haake, N.; Hamm, G.; Hipler, C.;
Hof, H.; Korting, H. C.; Kramer, A.; Mayser, P.; Ruhnke, M.; Schlacke,
K. H.; Tietz, H. J. Tinea capitis: ringworm of the scalp. Mycoses 2007,
50, 218−226.
The analogue 35 was synthesized according to the general pro-
cedure for 2′ enol ethers with the starting material being 15 instead
of 16.
(2S,6′R)-(7-Chloro-4,6-dimethoxy-benzofuran-3-on)-2-spiro-
1′-(4′-(3-methoxybenzyl)oxy-6′-methyl-cyclohex-3′-en-2′-
one] 35. Yield: 83 mg (32%) (white crystals); Rf value (EtOAc/
heptane, 5:1): 0.65; mp 173−175 °C. IR (neat, cm−1): 1691, 1653,
1
1605, 1584. H NMR (CDCl3, 300 MHz): δ 7.37−7.27 (1H, m),
7.02−6.84 (3H, m), 6.09 (1H, s), 5.54 (1H, d, J = 1.0 Hz), 5.01−4.85
(2H, m), 4.01 (3H, s), 3.93 (3H, s), 3.84 (3H, s), 3.26 (1H, ddd, J =
17.5, 11.9, 1.0 Hz), 2.96−2.80 (1H, m), 2.54 (1H, dd, J = 17.5,
5.6 Hz), 1.03 (3H, d, J = 6.6 Hz). 13C NMR (CDCl3, 50 MHz): δ
191.7, 188.8, 177.7, 169.7, 164.5, 159.9, 157.7, 136.0, 129.7, 120.0,
114.2, 113.2, 105.1, 100.7, 97.3, 95.1, 89.5, 71.0, 57.0, 56.2, 55.3, 35.2,
(8) Sobue, S.; Sekiguchi, K.; Nabeshima, T. Intracutaneous
distributions of fluconazole, itraconazole, and griseofulvin in guinea
pigs and binding to human stratum corneum. Antimicrob. Agents
Chemother. 2004, 48, 216−223.
33.2 14.5; [α]20 = +179° (c = 0.6 in CCl3). HRMS (ESI+) calcd for
D
[M + H]+ [C24H24ClO7]+ 459,1211, found 459.1216.
(9) Chaudhuri, A. R.; Luduena, R. F. Griseofulvin: Interaction With
̃
General Procedure for the Synthesis of Enol Ethers by
Solvolysis (42 and 43). CSA (0.1 mmol, 0.1 equiv) was added to a
solution of griseofulvic acid (0.6 mmol, 1 equiv) in the appropriate
alcohol (6 mL, 0.1 M). The mixture was stirred at 100 °C for 6 h and
then cooled to 20 °C. EtOAc (20 mL) was added to the solution, and
the mixture was washed with satd aq NaH2PO4 (20 mL) and water
(20 mL). The combined aqueous phases were extracted with EtOAc
(3 × 20 mL), and the combined organic phases were dried (MgSO4)
and concentrated. The residue was purified by column chromatog-
raphy (toluene:CH2Cl2:EtOAc 7:7:1) to afford the desired products.
When possible, the products were recrystallized from EtOAc/heptane.
Normal and Subtilisin-Treated Tubulin. Drug Dev. Res. 2001, 53,
44−49.
(10) Wehland, J.; Herzog, W.; Weber, K. Interaction of Griseofulvin
with Microtubules, Microtubule Protein and Tubulin. J. Mol. Biol.
1977, 111, 329−342.
(11) Crosse, R.; Rhodes, A.; Mcwillia, R. Some Relations between
Chemical Structure and Antifungal Effects of Griseofulvin Analogues.
J. Gen. Microbiol. 1964, 34, 51−65.
(12) Crowdy, S. H.; Grove, J. F.; McCloskey, P. Translocation of
Antibiotics in Higher Plants 0.4. Systemic Fungicidal Activity and
Chemical Structure in Griseofulvin Relatives. Biochem. J. 1959, 72,
241−249.
(13) Delgado, L.; De Croos, P. Z.; Lu, M. C. H.; Currie, B. L.
Structure Modification and Biological Activity of Some Griseofulvin
Derivatives. Kaohsiung J. Med. Sci. 1992, 8, 632−639.
(14) Yamato, M.; Yoshida, H.; Ikezawa, K.; Kohashi, Y. Synthesis of a
Griseofulvin Analogue. Chem. Pharm. Bull. 1986, 34, 71−76.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental data for compounds 3, 7, 8, 19, 21, 22, 26,
28−34, and 39−44, as well as copies of NMR spectra for
659
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660

Journal of Medicinal Chemistry
Article
(15) Fischer, L. J.; Riegelman, S. Absorption and Activity of Some
Derivatives of Griseofulvin. J. Pharm. Sci. 1967, 56, 469−476.
(16) Use of Griseofulvin for Inhibiting the Growth of Cancers. (The
Proctor & Gamble Company) . Patent WO9705870A2, 1997.
(17) Ho, Y. S.; Duh, J. S.; Jeng, J. H.; Wang, Y. J.; Liang, Y. C.; Lin,
C. H.; Tseng, C. J.; Yu, C. F.; Chen, R. J.; Lin, J. K. Griseofulvin
potentiates antitumorigenesis effects of nocodazole through induction
of apoptosis and G2/M cell cycle arrest in human colorectal cancer
cells. Int. J. Cancer 2001, 91, 393−401.
(37) Takeuchi, Y.; Watanabe, I.; Misumi, K.; Irie, M.; Hirose, Y.;
Hirata, K.; Yamato, M.; Harayama, T. Syntheses and Antifungal
Activity of DL-Griseofulvin and Its Congeners. III. Chem. Pharm. Bull.
1997, 45, 2011−2015.
(38) Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Filamentous Fungi; Approved Standard M38-A2; National
Committee for Clinical Laboratory Standards: Wayne, PA, 2002.
(39) Motulsky, H. J.; Brown, R. E. Detecting outliers when fitting
data with nonlinear regressiona new method based on robust
nonlinear regression and the false discovery rate. BMC Bioinform.
2006, 7, 123.
(18) Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Kinetic
suppression of microtubule dynamic instability by griseofulvin:
implications for its possible use in the treatment of cancer. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 9878−9883.
(40) Hideshima, T.; Chauhan, D.; Hayashi, T.; Podar, K.; Akiyama,
M.; Mitsiades, C.; Mitsiades, N.; Gong, B.; Bonham, L.; de Vries, P.;
Munshi, N.; Richardson, P. G.; Singer, J. W.; Anderson, K. C.
Antitumor activity of lysophosphatidic acid acyltransferase-beta
inhibitors, a novel class of agents, in multiple myeloma. Cancer Res.
2003, 63, 8428−8436.
(19) Rebacz, B.; Larsen, T. O.; Clausen, M. H.; Rønnest, M. H.;
Loffler, H.; Ho, A. D.; Kramer, A. Identification of griseofulvin as an
̈
inhibitor of centrosomal clustering in a phenotype-based screen.
Cancer Res. 2007, 67, 6342−6350.
(41) Page, J. E.; Staniforth, S. E. Griseofulvin Analogues. Part V.
Infrared Absorption. J. Chem. Soc. 1962, 1292−1303.
(20) Jin, H.; Yamashita, A.; Maekawa, S.; Yang, P. T.; He, L. M.;
Takayanagi, S.; Wakita, T.; Sakamoto, N.; Enomoto, N.; Ito, M.
Griseofulvin, an oral antifungal agent, suppresses hepatitis C virus
replication in vitro. Hepatol. Res. 2008, 38, 909−918.
(21) Oda, T. Effects of 2′-Demethoxy-2′-propoxygriseofulvin on
Microtubule Distribution in Chinese Hamster V79 cells. J. Antibiot.
2006, 59, 114−116.
(22) Rønnest, M. H.; Rebacz, B.; Markworth, L.; Terp, A. H.; Larsen,
T. O.; Kramer, A.; Clausen, M. H. Synthesis and Structure−Activity
̈
Relationship of Griseofulvin Analogues as Inhibitors of Centrosomal
Clustering in Cancer Cells. J. Med. Chem. 2009, 52, 3342−3347.
(23) Grisham, L. M.; Wilson, L.; Bensch, K. G. Antimitotic Action of
Griseofulvin does not Involve Distruption of Microtubules. Nature
1973, 244, 294−296.
(24) Zomorodian, K.; Uthman, U.; Tarazooie, B.; Rezaie, S. The
effect of griseofulvin on the gene regulation of alpha-tubulin in the
dermatophyte pathogen Trichophyton rubrum. J. Infect. Chemother
2007, 13, 373−379.
(25) Rathinasamy, K.; Jindal, B.; Asthana, J.; Singh, P.; Balaji, P. V.;
Panda, D. Griseofulvin stabilizes microtubule dynamics, activates p53
and inhibits the proliferation of MCF-7 cells synergistically with
vinblastine. BMC Cancer 2010, 10, 213.
(26) Ganem, N. J.; Godinho, S. A.; Pellman, D. A mechanism linking
extra centrosomes to chromosomal instability. Nature 2009, 460, 278−
283.
(27) Arkley, V.; Gregory, G. I.; Attenbur, J.; Walker, T. Griseofulvin
Analogues. Part I. Modification of the Aromatic Ring. J. Chem. Soc.
1962, 1260−1268.
(28) Arkley, V.; Walker, T.; Gregory, G. I. Griseofulvin Analogues.
Part VI. Dechlorogriseofulvin and Some of Its Derivatives. J. Chem.
Soc. 1963, 1603−1610.
(29) Rønnest, M. H.; Harris, P.; Gotfredsen, C. H.; Larsen, T. O.;
Clausen, M. H. Synthesis and single crystal X-ray analysis of two
griseofulvin metabolites. Tetrahedron Lett. 2010, 51, 5881−5882.
(30) Process of Preparing Griseofulvic Acid. (Glaxo Group Limited)
U.S. Patent US3102122, 1959.
(31) Stephenson, L.; Walker, T.; Warburton, W. K. A.; Webb, G. B.
Griseofulvin Analogues. Part IV. The Preparation and Properties of
Some Chlorides. J. Chem. Soc. 1962, 1282−1292.
(32) Grove, J. F.; MacMillan, J.; Mulholland, T. P. C; Rogers,
M. A. T. Griseofulvin. Part 1. J. Chem. Soc. 1952, 3949−3958.
(33) Clausen M. H.; Kramer A.; Laresen T. O.; Rebacz B.
̈
Griseofulvin Analogues for the Treatment of Cancer by Inhibition of
Centrosomal Clustering. Patent WO 2009000937, 2008.
(34) Preparation of griseofulvin analogs for the treatment of cancer
by inhibition of centrosomal clustering. Patent WO2010072770, 2010.
(35) Yamato, M. Preparation of griseofulvin derivatives. Jpn. Kokai
Tokkyo Koho . JP 03255081, 1991.
(36) Rønnest, M. H.; Nielsen, M. T.; Leber, B.; Mortensen, U. H.;
Kramer, A.; Clausen, M. H.; Larsen, T. O.; Harris, P. (+)-Geodin from
̈
Aspergillus terreus. Acta Crystallogr., Sect. C: Cryst. Struct. Commun.
2011, 67, O125−O128.
660
dx.doi.org/10.1021/jm200835c | J. Med. Chem. 2012, 55, 652−660