Diterpenoid pyrones, novel blockers of the voltage-gated potassium channel Kv1.3 from fungal fermentations

Article abstract of DOI:10.1016/S0040-4039(00)02258-9

The isolation, structure elucidation and chemical modification of nalanthalide, a novel diterpenoid pyrone blocker of the voltage-gated potassium channel Kv1.3 are reported. The structure-activity relationship of the derivatives with respect to various associated biological activities is also discussed.

Full text of DOI:10.1016/S0040-4039(00)02258-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1255–1257
Diterpenoid pyrones, novel blockers of the voltage-gated
potassium channel Kv1.3 from fungal fermentations
Michael A. Goetz,* Deborah L. Zink, Gabe Dezeny, Anne Dombrowski, Jon D. Polishook,
John P. Felix, Robert S. Slaughter and Sheo B. Singh*
Merck Research Laboratories, RY80Y-355, PO Box 2000, Rahway, NJ 07065-0900, USA
Received 2 October 2000; revised 6 December 2000; accepted 7 December 2000
Abstract—The isolation, structure elucidation and chemical modification of nalanthalide, a novel diterpenoid pyrone blocker of
the voltage-gated potassium channel Kv1.3 are reported. The structure–activity relationship of the derivatives with respect to
various associated biological activities is also discussed. © 2001 Elsevier Science Ltd. All rights reserved.
The human lymphocyte specific voltage-gated potas-
sium channel, Kv1.3, has a central role in the control of
membrane potential in human lymphocytes where it
sets the resting potential. The depolarization that
results from blockade of the channel causes a reduction
in calcium entry and consequently a decrease in
lymphokine release and synthesis from calcium-depen-
dent pathways. Thus, blockers of the channel suppress
activation and proliferation of human T cells. Kv1.3 is
therefore thought to represent a novel target for
immunosuppression. Peptide inhibitors [charybdotoxin
Rainin Dynamax C18 column with 75% acetonitrile in
water. On a larger scale, 1 could be more conveniently
prepared by repeating the silica gel step and triturating
the resulting enriched material with methanol to give an
6
amorphous powder of pure compound. 7Another
source of 1 was Chaunopycnis alba, MF 6799.
(
ChTX), margatoxin (MgTX) and noxiustoxin] have
been shown to block Kv1.3 and thereby inhibit
lymphokine production and the rise in intracellular
calcium. Our laboratories have recently reported on
1
2
the discovery of correolide, which potently inhibits
Kv1.3, and as expected, depolarizes T cells and inhibits
3
,4
T cell activation events.
Continued screening of natural products led to the
discovery of nalanthalide (1), a diterpenoid pyrone, as a
novel inhibitor of Kv1.3. We now report on the discov-
ery including the isolation, structure elucidation, chemi-
cal modifications and structure–activity relationship of
the diterpenoid pyrone class of inhibitors.
Structure elucidation: High resolution EIMS analysis of
6
nalanthalide (1) showed a molecular ion at m/z
4
84.3197 (calcd 484.3189 for C H O ) and suggested
30 44 5
the presence of nine degrees of unsaturation. The for-
13
mula was corroborated by analysis of the C NMR
spectrum, which displayed 30 carbons. Of these, eight
were methyls, eight were methylenes, and four were
methines as determined by the APT spectrum. Of the
Isolation: Nalanthalide (1) was produced in submerged
fermentations by the fungal culture MF 5638, a Nalan-
5
thamala sp. Yields of compound 1 ranged from 80 to
1
40 mg/l broth after a purification scheme which
3
remaining quaternary carbons, two were of sp type
included extraction with methyl ethyl ketone, column
chromatography on silica gel and finally HPLC on a
and the rest were sp type, including two carbonyl
carbons assigned to a conjugated ketone and an ester.
These carbon types were confirmed by the analysis of
1
1
1
*
0
Corresponding authors.
the H NMR spectrum together with H– H COSY and
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02258-9

1
1
256
M. A. Goetz et al. / Tetrahedron Letters 42 (2001) 1255–1257
13
H– C COSY (HETCOR). The carbons bearing pro-
tons were assigned by analysis of the latter spectrum.
The COSY spectrum of 1 revealed the presence of four
isolated spin systems consisting of C1–C3, C5–C7,
C11–C13, and C9–C19. The H –20 and H–13 showed
2
long range allylic couplings to H-7 (l 2.35, m) and the
allylic methyl groups, respectively. These fragments
were connected to each other and to the remainder of
the molecule with the help of a long range HETCOR
spectrum. The H -18 methyl group showed two- and
3
three-bond proton–carbon correlations to C-1, C-9 and
C-10; H -17 methyl group produced correlations to
3
C-3, C-4, C-5 and C-11; H -15 and H -16 methyl
3
3
groups to C-13, C-14 and to each other; H -20 (the
2
exocyclic methylene protons) showed correlations to
C-7, C-8 and C-9; and H -19 exhibited correlations to
Figure 2. Selected NOE of 1.
2
C-3% of the pyrone ring. The methyl and the methoxy
groups of the pyrone ring were accordingly assigned by
the long-range HETCOR correlations.
C-18 angular methyl groups gave NOE enhancements
to both H-2 and H-6 axial protons thus establishing the
respective 1,3-diaxial positions. Direct NOE effects of
H -17 and H -18 could not be accomplished due to
partial saturation of each other during irradiation
experiments. These measurements together with others
established the relative stereochemistry and conforma-
tion of nalanthalide as shown in Fig. 2, which was
supported by a model generated from the ChemDraw
The mass spectrum of 1 produced two major fragment
ions at m/z 343.2262 (C H O ) and m/z 167.0707
3
3
22
31
3
(
C H O ). The former ion is produced as a result of
9 11 3
the concomitant loss of the acetate group at C-3 and
the side-chain at C-4, and the latter fragment is derived
from the pyrone ring as shown in Fig. 1.
3
D minimized structure shown in Fig. 3. The stereo-
Stereochemistry: The relative stereochemistry of nalan-
thalide (1) was elucidated by measurements of the
scalar couplings and by NOE difference spectroscopy.
The methine proton at C-3 appeared as a doublet of
doublets at l 4.85 ppm and showed a 9 Hz coupling
with the axial proton at C-2, confirming its axial orien-
chemistry and the solution conformation of 1 appears
to be similar to the stereochemistry and X-ray derived
solid state conformation of viridoxins. Structurally
related pyrones, subglutinols and sesquicillin have
been reported to show immuosuppressive and glucocor-
ticoid mediated signal transduction inhibitory activities,
respectively.
8
9
10
tation in a chair conformation. The H -19 (l 2.42) and
a
H -19 (l 2.62) showed a large (J=ꢀ12 Hz) and small
b
couplings (J=ꢀ3.5 Hz) with H-9 (l 1.95), respectively,
indicating a anti and syn geometric relationship, which
was confirmed by the observation of NOE effect from
H -19 to H-9 (Fig. 2). The H -19 also gave NOE
enhancements to the terminal methyl groups (C-15 and
C-16) of the side-chain suggesting the spatial proximity
Chemical modification: To explore the chemistry and
evaluate structure–activity relationship, a few deriva-
tives of nalanthalide were prepared by chemical modifi-
cations. Mild basic hydrolysis with potassium
carbonate in a mixture of THF and methanol gave 90%
yield of the hydroxy compound 2. The reaction of
compound 2 with p-bromophenyl isocyanate in methyl-
ene chloride quantitatively produced the desired ure-
thane 3. Epoxidation of 1 with m-CPBA exclusively
produced mono epoxide 4, leaving the exocyclic methyl-
ene group intact. Zone’s oxidation of 2 cleanly afforded
the 3-keto derivative 5. Selective hydrogenation with
b
b
of these groups to H -19, at least in solution. Irradia-
b
tion of H-3 produced NOE effects to H-5 suggesting
their 1,3-diaxial disposition. Similarly the C-17 and
Figure 1.
Figure 3. ChemDraw 3D minimized model of 1.

M. A. Goetz et al. / Tetrahedron Letters 42 (2001) 1255–1257
1257
Table 1. Biological activities of nalanthalide (1) and its
derivatives
References
1
. Lin, C. S.; Boltz, R. C.; Blake, J. T.; Nguyen, M.;
Talento, A.; Fischer, P. A.; Springer, M. S.; Sigal, N. H.;
Slaughter, R. S.; Garcia, M. L.; Kaczorowski, G. J.;
Koo, G. C. J. Exp. Med. 1993, 177, 637.
2
. Goetz, M. A.; Hensens, O. D.; Zink, D. L.; Borris, R. P.;
Morales, F.; Tamayo-Castillo, G.; Slaughter, R. S.; Felix,
J. P.; Ball, R. G. Tetrahedron Lett. 1998, 39, 2895.
. Felix, J. P.; Bugianesi, R. M.; Schmalhofer, W. A.;
Borris, R. P.; Goetz, M. A.; Hensens, O. D.; Bao, J.-M.;
Kayser, F.; Parsons, W. H.; Rupprecht, K.; Garcia, M.
L.; Kaczorowski, G. J.; Slaughter, R. S. Biochemistry
1999, 38, 4922.
3
Compound
125IChTX binding to
Jurkat membranes,
IC50 (mM)
86Rb+ reflux in
CHO-Kv1.3 cells
IC50 (mM)
4. Koo, G. C.; Blake, J. T.; Shah, K.; Staruch, M. J.;
Dumont, F.; Wunderler, D.; Sanchez, M.; McManus, O.
B.; Sirotina-Meisher, A.; Fischer, P. A.; Boltz, R. C.;
Goetz, M. A.; Baker, R.; Bao, J.-M.; Kayser, F.; Rup-
precht, K. M.; Parsons, W. H.; Tong, X.-C.; Ita, I. E.;
Pivnichny, J.; Vincent, S.; Cunningham, P.; Hora, D.;
Feeney, W.; Kaczorowski, G. J.; Springer, M. S. Cell
Immun. 1999, 197, 99.
1
2
3
4
5
6
7
3
11
>30
6
>10
5
3.9
3
25
5.1
3
31
16
6
5. Frozen vegetative mycelium was inoculated into a seed
medium containing yeast extract, malt extract, glucose
and junlon; after 3 days at 25°C, 220 rpm, a vermiculite
base mixed with a solution containing glucose, fructose,
sucrose, casamino acids, asparagine, yeast extract, dibasic
sodium phosphate, magnesium sulfate, calcium chloride
and trace elements was inoculated with vegetative seed.
Incubation at 25°C in bottles rolling on a Wheaton
machine at 4 rpm was carried out for 20 days.
Wilkinson’s catalyst in benzene at 40 psi furnished a 9:1
mixture of dihydro compounds 6 and 7. As expected,
the hydrogenation was stereospecific and hydrogen was
introduced from the less hindered pseudo equatorial
bottom face and thus produced an axial methyl at C-8.
It was interesting to note that a small amount of the
compound went through olefin isomerization to give
compound 7.
2
5
6
. Physical data of nalanthalide (1): mp 96.5–98°C; [h]
D
−
58.2 (c 0.275, CHCl ); UV (CH OH) umax: 216 (3.7),
3 3
2
1
60.5 (3.8); IR (ZnSe) w : 2900–3000, 1732, 1671, 1601,
252, 1241 cm , H and C NMR (300 and 400 MHz,
max
13
−
1 1
Biological activity and SAR: Nalanthalide (1) inhibits
the binding of ChTX to Jurkat membranes with an IC50
of 3 mM. It blocked Rb efflux in CHO-Kv1.3 cells with
an IC value of 3.9 mM. In electrophysiological mea-
surements it depolarizes human T cells to the same
extent as MgTX with an EC50 of 500 nM. The biologi-
cal properties of 1 and its derivatives are summarized in
Table 1. It appears that the acetate group at C-3 plays
a role in the ChTX binding activity and has less of an
CDCl ), position (lC; lH, m, J in Hz): C-1 (33.8; 1.98,
3
+
m; 1.35, m), C-2 (24.1; 1.95, m; 1.35, m); C-3 (76.2; 4.82,
dd, J=9, 7 Hz); C-4 (40.0), C-5 (39.1; 1.72, dd, J=12.3,
5
0
2
2
.9 Hz); C-6 (22.7; 1.45, m; 1.30, m); C-7 (30.7; 2.10, m;
.35, m); C-8 (148.7); C-9 (55.6; 1.95, dd, J=12, 3.5 Hz);
C-10 (37.4); C-11 (37.8; 1.15, m; 1.35, m); C-12 (21.7;
.90, m); C-13 (124.6; 5.06, t, J=7.2 Hz); C-14 (131.3);
C-15 (17.5; 1.59, brs); C-16 (25.7; 1.67, brs); C-17 (18.1;
1
0
3
.85, s); C-18 (22.9; 0.95, s); C-19 (19.9; 2.67, dd, J=12.7,
.7 Hz [H ]; 2.42, t, J=12.0 Hz [H ]); C-20 (109.5; 4.51,
+
effect on Rb efflux activity. For example, the hydroly-
b
a
sis of acetate (2) and subsequent oxidation to C-3
t, J=2.3 Hz [H ]; 4.19, t, J=2.3 Hz [H ]); C-2% (162.8);
+
b
a
ketone (5) did not affect the Rb efflux activity, but
C-3% (103.2); C-4% (180.3); C-5% (118.6); C-6% (154.9); C-7%
17.0; 2.23, s); C-8% (10.0; 1.89, s); C-9% (55.3; 3.84, s);
reduced the binding activity by 3-fold. Substitution of
(
acetate with bulky p-bromophenylurethane group
C-1%% (176.7); C-2%% (21.3; 2.03, s).
(
compound 3) led to significant reduction in both activ-
7
8
. Same as Ref. 5, except production medium contained
glucose, urea, NZ amine type A, dibasic sodium phos-
phate, magnesium sulfate, potassium chloride, zinc sul-
fate and calcium carbonate.
ities. Epoxidation (compound 4) of the side-chain olefin
caused some reduction in both activities. The reduction
of exocyclic methylene group (compounds 6 and 7) had
little (2-fold reduction) effect on the binding activity,
. Gupta, S.; Krasnoff, S. B.; Renwick, J. A. A.; Roberts,
+
but had a greater impact (4–8-fold reductions) on Rb
efflux activity. These compounds also blocked L-type
Ca channels in GH3 cells with the general same
potency.
D. W.; Steiner, J. R.; Clardy, J. J. Org. Chem. 1993, 58,
1062. Compound 1 was reported as semisynthetic deriva-
2
+
tive derived from viridoxin A while our studies were in
progress, but no spectroscopic data was reported.
. Lee, J. C.; Lobkovsky, E.; Pliam, N. B.; Strobel, G.;
Clardy, J. J. Org. Chem. 1995, 60, 7076.
9
Nalanthalide and derivatives represent a novel class of
voltage-gated potassium (Kv1.3) channel blockers and
are potential immunosuppressants.
.
10. Engel, B.; Erkel, G.; Anke, T.; Sterner, O. J. Antibiot.
1998, 51, 518.
.