Geldanamycin derivatives and neuroprotective effect on cultured P19-derived neurons

Article abstract of DOI:10.1016/j.bmcl.2006.12.041

Geldanamycin (1), an antifungal and anticancer ansamycin, was reported as a neurotrophic and neuroprotective substance against antineoplastic drugs, paclitaxel, vincristine, and cisplatin, on cultured dorsal root ganglion neurons from chick embryos. In this study, 1 in a large quantity, together with a known 17-O-demethylgeldanamycin (2), and a new 17-O-demethylgeldanamycin hydroquinone (3) were obtained from a mangrove Streptomyces sp. A series of O-alkyl and N-alkyl derivatives of 1 were prepared by modification of C-17 and/or C-19 on the quinone ring and were evaluated for in vitro activity against P19-derived neurons. Compound 1 and 19-O-methylgeldanamycin (7) at a very low dose (1 nM) enhanced survival and neurite outgrowth of P19-derived neurons and prevented neurotoxicity of paclitaxel and vinblastine. Compound 7, possessing the lowest cytotoxicity and neurotoxicity, is serving as the most promising candidate in neurodegenerative therapy against neurotoxic anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2006.12.041

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2939–2943
Geldanamycin derivatives and neuroprotective effect on
cultured P19-derived neurons
Sarin Tadtong,a Duangdeun Meksuriyen,b Somboon Tanasupawat,c Minoru Isobed
and Khanit Suwanboriruxa,*
aCenter for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Department of Pharmacognosy,
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
bDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
cDepartment of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
dLaboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601, Japan
Received 25 August 2006; revised 22 November 2006; accepted 12 December 2006
Available online 15 December 2006
Abstract—Geldanamycin (1), an antifungal and anticancer ansamycin, was reported as a neurotrophic and neuroprotective sub-
stance against antineoplastic drugs, paclitaxel, vincristine, and cisplatin, on cultured dorsal root ganglion neurons from chick
embryos. In this study, 1 in a large quantity, together with a known 17-O-demethylgeldanamycin (2), and a new 17-O-demethylgel-
danamycin hydroquinone (3) were obtained from a mangrove Streptomyces sp. A series of O-alkyl and N-alkyl derivatives of 1 were
prepared by modification of C-17 and/or C-19 on the quinone ring and were evaluated for in vitro activity against P19-derived neu-
rons. Compound 1 and 19-O-methylgeldanamycin (7) at a very low dose (1 nM) enhanced survival and neurite outgrowth of P19-
derived neurons and prevented neurotoxicity of paclitaxel and vinblastine. Compound 7, possessing the lowest cytotoxicity and
neurotoxicity, is serving as the most promising candidate in neurodegenerative therapy against neurotoxic anticancer drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Geldanamycin (1), a 17-substituted benzoquinoid ansa-
mycin possessing antifungal and anticancer activities,
was previously reported as the secondary metabolite pro-
duced by the terrestrial Streptomyces hygroscopicus var.
geldanus.1 Initially, 1 was thought to be a nonspecific
kinase inhibitor, however, was later found to bind to
the ATP binding site of the chaperone heat shock protein
90 (Hsp90), resulting in prevention of several signaling
proteins from reaching their mature form, inhibiting
their activity, and affecting their stability.2 The semi-syn-
thetic 17-allylamino-17-demethoxygeldanamycin has
been currently studied in phase II clinical trial for treat-
ment of solid tumors.3 Interestingly, 1 prevented neuro-
toxic effects of anticancer drugs on cultured dorsal root
ganglion neurons from chick embryo at low doses4 and
protected rat brain from focal ischemia.5 Moreover, 1
exhibited neuroprotective ability on a variety of in vivo
neurodegeneration models.6 In order to further investi-
gate the neuronal effect of other geldanamycin deriva-
tives, P19-derived neurons were used as a neuronal
model in our study. P19 embryonal carcinoma cell is a
pluripotent stem cell line which is differentiated into neu-
rons by retinoic acid.7 P19-derived neurons are irrevers-
ibly postmitotic and exhibit many characteristics of
mature CNS neurons containing particular neurotrans-
mitters such as c-aminobutyric acid8 and acetylcholine.9
The differentiated P19 neuroglial cultures have been used
for neurotoxicity evaluation of cysteinylcatechols.10 In
this study, we succeeded in isolating 1 in a large quantity
from a mangrove Streptomyces sp. The availability of 1
enabled us to prepare a number of geldanamycin analogs
for evaluating biological effects on P19 cells and P19-de-
rived neurons. In addition, paclitaxel and vinblastine, the
anticancer agents causing irreversibly peripheral neurop-
athy side effect, were used as neurotoxic substances for
evaluation of neuroprotective property.
Keywords: Geldanamycin; 19-O-methylgeldanamycin; Neuroprotective
effect; Cultured P19-derived neurons.
*
The antifungal-guided fractionation of the EtOAc
extract obtained from the fermentation broth of the
Corresponding author. Tel.: +66 0 2218 8257; fax: +66 0 2254
5195; e-mail: skhanit@chula.ac.th
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.041

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S. Tadtong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2939–2943
Table 2. Percentages of cell viability of P19-derived neurons treated
with 1 and its derivatives at a concentration of 1 nM
mangrove Streptomyces sp. TRA98752 led to the isola-
tion of 1 in 1.2% yield, based on the EtOAc extract, to-
gether with a known 17-O-demethylgeldanamycin (2),11
and a new 17-O-demethylgeldanamycin hydroquinone
(3) in 0.04% yield, each. Their chemical structures were
determined by extensive analyses of UV, IR, 1H
NMR, 13C NMR, 2D NMR, and MS spectra, and com-
parison with those reported in the literatures.12–14 The
1H and 13C NMR spectra of all compounds showed al-
most identical characters of the macrolactam ring and
the carbamate moiety. Based on the exact mass of 3
from ESI-TOFMS which showed a pseudomolecular
ion peak at m/z 571.2649 [M+Na]+, its molecular for-
mula was determined as C28H40N2O9, which was
2 amu greater than that of 2. The 13C NMR spectrum
of 3 was different from that of 2 by the absence of two
equivalent quinone carbonyl carbons (d 183.16) and
the presence of two oxygenated aromatic carbons at d
156.18 (C-18) and d 143.82 (C-21). Therefore, 3 was de-
duced as 17-O-demethylgeldanamycin hydroquinone
which might be oxidized to 2 during the isolation pro-
cess. The 1H and 13C NMR spectral data of 3 are shown
in Table 1.
Compound
% Cell viabilitya
1
2
121
50
43
113
121
111
108
0
3
4
5
6
7
8
9
91
85
49
0
10
11
12
13
14
15
16
17
66
44
46
0
0
a % Cell viability for tested compounds versus control cells was cal-
culated by the absorbance at 450 nm from three independent
experiments and each experiment was run in triplicate.
All isolated geldanamycins (1–3) were primarily
screened for biological effects on P19-derived neurons15
at a very low dose of 1 nM by XTT reduction assay16 as
shown in Table 2. Only 1 promoted high cell viability of
the cultured neurons (% cell viability = 121), while 2 and
3 were toxic. The results suggested that the O-alkyl sub-
stituent at C-17 might play important roles for this
remarkable effect.
Therefore, we prepared O-alkyl and N-alkyl derivatives
(4–17) by modifications of the C-17 and/or C-19 on the
quinone moiety of 1 (Figure 1). Three 17-O-alkyl-17-O-
demethyl-geldanamycins (4–6) were obtained in approx-
imately 70% yields from treatment of 1 with 1.4 equiv of
the alkoxides generated from NaH or Na metal and the
corresponding alcohols. The reaction between 1 and 3
equiv of the corresponding alkoxides gave the 17,19-di-
O-alkyl-17-O-demethylgeldanamycins (7–8) in 73 and
15% yields, respectively. Addition of ammonia to the
methanolic solution of 1 gave 19-aminogeldanamycin
(9) in 73% yield. Treatment of 1 with ammonia or primary
alkylamines17 in DMF at room temperature provided the
17-alkylamino (10–14) and 17,19-di-alkylamino-17-
demethoxygeldanamycins (15–17) in 43–99% yields. All
Table 1. NMR data of 3 in CDCl3:DMSO-d6 (1:1)
Position
dC
dH, mult., (J in Hz)
1
167.81
134.00
126.82
125.98
136.05
81.38
80.51
132.84
132.47
31.91
71.76
81.38
29.30
27.73
33.22
113.57
143.82
156.18
106.36
132.84
143.82
—
—
—
2
3
6.98, d (11)
6.51, t (11, 11)
5.77, t (11, 10)
4.25, d (10)
4.93, br s
—
4
1
compounds were characterized by UV, H NMR, 13C
5
6
NMR, 2D NMR, and ESI-Q-TOFMS measurements.
7
8
All synthesized geldanamycin derivatives at 1 nM were
evaluated for their effects on P19-derived neurons by
XTT reduction assay to compare with 1 as shown in Ta-
ble 2. All of the alkylamino derivatives (9–17) exhibited
less % cell viability, indicating their neurotoxicity, while
the O-alkyl derivatives (4–7), except the di-O-ethyl 8,
promoted viability of the neurons at more than 100%
cell viability comparable to 1. Therefore, the morphol-
ogy of P19-derived neurons in the presence of 1 and
4–7 was further observed under a phase-contrast micro-
scope. Interestingly, the neurites of these neurons pos-
sessed more branching than those in the absence of the
compounds (data not shown).
9
10
5.73, d (9)
2.59, m
3.41, m
11
12
3.23, m
0.99, 2H, br s
1.70, m
13
14
15
16
2.24, 2H, m
—
—
17
18
—
6.89, s
19
20
—
—
21
1-NH
2-CH3
6-OCH3
7-OCONH2
8-CH3
10-CH3
12-OCH3
14-CH3
9.58, br s
1.92, 3H, s
3.14, 3H, s
6.09, NH2, br s
1.66, 3H, s
0.81, 3H, d (7)
3.22, 3H, s
0.83, 3H, d (7)
12.43
56.39
156.18
12.84
12.43
56.21
23.68
As a result, 1 along with 4–7 were further comparatively
evaluated for their neurotoxicity against P19-derived
neurons and cytotoxicity against P19 cells as shown in
Figures 2 and 3, respectively. Compounds 1, 4, 5, 6,
and 7 exhibited neurotoxicity at IC50 of 1.8, 1.6, 6.7,
>10, and >10 lM, and showed cytotoxicity at IC50 of
0.1, 0.1, 0.2, 0.5, and >10 lM, respectively. To examine
mult. = multiplicity.

S. Tadtong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2939–2943
2941
Figure 3. Cytotoxicity of 1 and 4–7 on cultured P19 cells incubated for
18 h. % Cell viability for tested compounds versus control cells was
calculated by the absorbance at 450 nm. Each point represents the
average of three independent experiments SEM (bar) and each
experiment was run in triplicate.
Figure 1. Structures of 1 and its derivatives.
(0.39 lM) compared to that of 1 (TI 1800). Both com-
pounds showed similar effects and only the data of 7
are shown in Figure 4. Under the pre-treatment with 7
(1 nM) for 18 h and then with paclitaxel or vinblastine
for further 18 h, the neurons showed impressive survival
and the neurite outgrowth was found to be similar to
both the co-treatment experiment as described above
and the post-treatment experiment.
The above results led us to define the structure–activity
relationships for neuroactivity and cytotoxicity. Four
important points have been made: (i) substitutions on
the quinone ring with O-alkyl groups at C-17 provided
neuroprotective activity; (ii) the decreases in neurotox-
icity and cytotoxicity were realized in substitution at
C-17 with larger O-alkyl groups (1 and 4–6); (iii) di-
O-alkyl substituents larger than the methyl on the
quinone ring (7–8) increased neurotoxicity; and (iv)
substitutions on the quinone ring with N-alkyl groups
at C-17 and/or C-19 (9–17) resulted in potent
neurotoxicity.
Figure 2. Neurotoxicity of 1 and 4–7 on cultured P19-derived neurons
incubated for 18 h. % Cell viability for tested compounds versus
control cells was calculated by the absorbance at 450 nm. Each point
represents the average of three independent experiments SEM (bar)
and each experiment was run in triplicate.
the neuroprotective activities of 1 and 4–7 at the concen-
tration of 1 nM on cultured P19-derived neurons, the
compounds were co-treated with paclitaxel at its toxic
dose (IC50 0.65 lM). In the presence of only paclitaxel,
the neurons were remarkably degenerated and possessed
shrinking neurites when observed under a phase-con-
trast microscope. All of the paclitaxel-treated neurons
in the presence of the compounds showed 100% cell via-
bility except that of 7, which showed better cell viability
at 120%. When observed under a phase-contrast micro-
scope, these neurons were found survived and exhibited
long-branched neurites.
The mechanism(s) of action in neuroprotective activity
of these compounds is not easily explained. Recently,
neuroprotective activity of 1 was proposed as a conse-
quence of the heat–shock response protecting against
neurotoxic substances6 and the suppression of the sig-
nal transduction pathway(s) involving Hsp90 that led
to apoptosis.4 The other study reported that the cancer
cells maintaining Hsp90 in an activated conformation
were different from the normal cells.18 The activated
Hsp90 might possibly occur in neuronal cells. There-
fore, neuroprotective effects of 1 and derivatives at a
low dose on neuronal cells might be a consequence of
inhibition of this activated Hsp90. However, interac-
tions of the geldanamycins at a limited dose with other
specific target molecule(s) to enhance neurons’ survival
cannot be omitted. The mechanism(s) of the geldanam-
ycins for neuroprotective activity need further
investigation.
Compound 7, the 19-O-methylgeldanamycin which
showed the highest cell viability and possessed very wide
therapeutic index (TI) between neuroprotective and neu-
rotoxic activities (TI > 10,000), was further evaluated
for its neuroprotective ability by pre- and post-adminis-
tration with paclitaxel (0.65 lM) and vinblastine

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S. Tadtong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2939–2943
Figure 4. Neuroprotective effect of 7 on cultured P19-derived neurons. Phase-contrast micrographs of cultured P19-derived neurons incubated for
18 h and treated with: (a) no compounds, (b) 0.65 lM paclitaxel, (c) 1 nM 7, (d) 1 nM 7 in the presence of 0.65 lM paclitaxel, (e) 1 nM 7 for 18 h and
then 0.65 lM paclitaxel, and (f) 0.65 lM paclitaxel for 18 h and then 1 nM 7. Bar = 5 lm at 100·.
In summary, 1 and its 17-O-alkyl derivatives at a very
low dose (1 nM) enhanced the survival and neurite out-
growth of P19-derived neurons, similar to the effect of 1
on cultured dorsal root ganglion sensory neurons from
chick embryos.4 In contrast, the 17-alkylamino and
17,19-dialkylamino derivatives at the same dose caused
neurotoxicity on the P19-derived neurons. The present
results strongly suggest that 19-O-methylgeldanamycin
(7), which has dramatically wide therapeutic index be-
tween neuroprotective and neurotoxic activities, remains
the most promising for further development into neuro-
degenerative therapy against neurotoxic anticancer
agents.
can be found, in the online version, at doi:10.1016/
j.bmcl.2006.12.041.
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Acknowledgments
BNPME is supported by a grant for centers of excel-
lence, Chulalongkorn University, and Commission of
Higher Education’s Strategic Consortia for Human Re-
source Development, Thailand. S. Tadtong is supported
by grants from Royal Golden Jubilee (RGJ) Ph.D. pro-
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and JASSO short-term student exchange promotion
program (inbound) scholarship 2004, Japan. We thank
the National Science and Technology Development
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