Dimerization of an inactive fragment of huperzine a produces a drug with twice the potency of the natural product

Full text of DOI:10.1002/(SICI)1521-3773(20000515)39:10<1775::AID-ANIE1775>3.0.CO;2-Q

COMMUNICATIONS
Huperzine A (À)-1, isolated from the clubmoss Huperzia
serrata, is a selective and potent reversible acetylcholinester-
ase (AChE) inhibitor that shows promise for the palliative
treatment of Alzheimerꢁs disease.[4] Asymmetric syntheses of
(À)-1 as short as 12 steps have been reported,[5] and
considerable effort has been directed towards the develop-
ment of more easily synthesized analogues.[6] To date 5-amino-
2(1H)-quinolinones which lack the C6 ± C8 bridge of 1 have
not significantly inhibited AChE (e.g. (Æ)-2, (Æ)-3a, (Æ)-3b,
IC50 > 100000 nm).[6] Yet, based on the X-ray crystal structure
analysis of the Torpedo AChE ´ (À)-1 complex,[7] analogue 3
appears to possess muchof the requisite functionality. In
particular 3 retains the pyridone oxygen atom and NH group
of 1, which form hydrogen bonds to Tyr-130 and to Gly-117/
Glu-199 (through a water molecule), respectively, and the
5-amino group of 1, which undergoes a cation ± p-interaction
with Trp-84/Phe-330. We conjectured that high affinity of
analogues like 3 would be possible, if the loss of hydrophobic
contact in the active site could be offset by additional
interactions at the AChE peripheral site (12 Š distant from
the active site)[8]. Our studies of alkylene-linked tacrine
homodimers[2a, 9] and heterodimers[10] have demonstrated that
this site binds a variety of protonated amine ligands, including
Dimerization of an Inactive Fragment of
Huperzine A Produces a Drug with Twice the
Potency of the Natural Product**
Paul R. Carlier,* Da-Ming Du, Yi-Fan Han,* Jing Liu,
Emanuele Perola, Ian D. Williams, and Yuan-Ping Pang
Bivalency is an effective strategy for improving drug
potency and selectivity.[1] When multiple recognition sites
for the same substrate exist, homodimers can exhibit dramatic
performance enhancements relative to the corresponding
monomers.[1a, 2] In this vein, homodimers of structurally
complex natural products have recently been investigated.[3]
Herein we demonstrate a new strategy in bivalent drug design
by dimerizing an easily synthesized but pharmacologically
inactive fragment of Huperzine A (À)-1: the optimum dimers
(S,S)-(À)-4h,i are more than twice as potent as the natural
product.
moieties related to 3.
Thus 5 and 6 (eachavailable in two steps)
[11]
were
condensed with a,w-diamines and reduced, affording dimers
4a ± f,h and 7c ± f,h as mixtures of the rac- and meso-
diastereomers (Scheme 1). These compounds were tested
[*] Prof. P. R. Carlier, Dr. D.-M. Du, Prof. I. D. Williams[‡]
Department of Chemistry and Molecular Neuroscience Center
Hong Kong University of Science and Technology
Clear Water Bay, Kowloon (Hong Kong)
Fax : (‡852)2358-1594
Scheme 1. Synthesis of dimeric inhibitors. a) 0.5 equiv NH2(CH2)nNH2,
cat. CH3CO2H, benzene reflux, 24 h; NaBH4, MeOH, room temperature,
E-mail: chpaul@ust.hk
12 h,
rac-/meso-4a ± f,h:
38 ± 58%,
rac-/meso-7c ± f,h:
22 ± 35%;
Prof. Y.-F. Han, J. Liu
b) nBuNH2, cat. CH3CO2H, benzene reflux, 24 h; NaBH4, MeOH, room
temperature, 12 h, 51%. Lettering scheme (see also Scheme 2): a, n ˆ 5; b,
n ˆ 6; c, n ˆ 7; d, n ˆ 8; e, n ˆ 9; f, n ˆ 10; g, n ˆ 11; h, n ˆ 12; i, n ˆ 13; j, n ˆ
14.
Department of Biochemistry and Molecular Neuroscience Center
Hong Kong University of Science and Technology
Clear Water Bay, Kowloon (Hong Kong)
Fax : (‡852)2358-1552
E-mail: bcyfhan@ust.hk
Prof. Y.-P. Pang, Dr. E. Perola
for AChE and butyrylcholinesterase (BChE) inhibition.[12] As
expected, monomer (Æ)-3c proved an extremely weak AChE
inhibitor (IC50 ꢀ 500000 nm, Table 1, entry 1). However
corresponding dimers rac-/meso-4a ± f, h showed dramatically
enhanced potency, with the highest potency observed at a
tether length of 12 methylene groups (rac-/meso-4h, 159 nm,
Table 1, entry 2). Dimers rac-/meso-7c ± f, h were similarly
optimized at n ˆ 12, but were less potent than rac-/meso-4h
(cf. Table 1, entries 2, 3; data for 4a ± f, 7c ± f not shown).
As a test of the design strategy, 2-methoxypyridine ana-
logue rac-/meso-8h was prepared from 5 (Scheme 2). The 31-
Mayo Clinic Cancer Center, Department of Molecular Pharmacology
and Experimental Therapeutics, Tumor Biology Program, Molecular
Neuroscience Program, Mayo Clinic, 200 First Street SW, Rochester,
MN 55905 (USA)
‡
[ ] X-ray crystallography
[**] This work was supported by the Research Grants Council of Hong
Kong (HKUST6156/97M), the Biotechnology Research Institute
(HKUST), the Mayo Foundation, and the Istituto Pasteur Fondazione
Cenci Bolognetti (E.P.). We thank Prof. X. C. Tang (Shanghai Institute
of Materia Medica) for a gift of (À)-1.
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/angewandte/ or from the author.
Angew. Chem. Int. Ed. 2000, 39, No. 10
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0570-0833/00/3910-1775 $ 17.50+.50/0
1775

COMMUNICATIONS
Table 1. Cholinesterase inhibition by huperzine A fragment dimers and
controls.
tether length of 12 ± 13 methylene groups is consistent with
simultaneous binding of these drugs to the catalytic and
peripheral sites of AChE.[9, 10] Remarkably the optimum drugs
(S,S)-(À)-4h ± i are nearly 10000-fold more potent than the
related monomer (Æ)-3c, and are twice as potent as (À)-1, th e
natural product which inspired their synthesis. Enzyme
kinetics confirmed the superior potency of (S,S)-(À)-4h
(KI ˆ 19.6 nm) relative to (À)-1 (KI ˆ 47.1 nm). Although
(S,S)-(À)-4h,i are not as potent as 10-axial-methyl-huper-
zine[4] or tacrine/huperzine hybrids,[10b, 14] they are easily
synthesized and are superior to the latter in terms of
selectivity for AChE.
As a prelude to computational and experimental evalua-
tions of the binding of these dimers to AChE, studies of the
docking[15] of monomers (R)- and (S)-3d (protonated at N5)
into the active site of AChE were carried out. Both
enantiomers were found to bind in a similar way to (À)-1,
and binding of (R)-3d was 3 kcalmolÀ1 more exothermic than
binding of (S)-3d. However, examination of all the computed
binding orientations of (R)-3d reveals that the 5-amino group
does not easily accommodate a C12-alkylene tether leading
upwards towards the peripheral site (Figure 1, magenta); in
contrast (S)-3d encounters no suchproblem (Figure 1, cyan).
We therefore attribute the observed 60-fold enantio-prefer-
ence for (S,S)-(À)-4h to the disposition of the tether leading
to the peripheral site.
Entry Drug[a]
n[b] AChE
IC50 [nm][c]
BChE
IC50 [nm][d]
Selectivity
for AChE[e]
1
2
3
4
5
6
7
8
9
(Æ)-3c
na 500000[f]
500000[f]
24400 Æ 910
1
153
16.6
15.8
12.1
13.8
185
321
248
94.9
rac-/meso-4h 12
rac-/meso-7h 12
rac-/meso-8h 12
159 Æ 26
3620 Æ 110 60000 Æ 3230
5000 Æ 190 78900 Æ 14500
(S,S)-(À)-4 f
10
151 Æ 36
84 Æ 5
1820 Æ 70
1160 Æ 80
(S,S)-(À)-4g 11
(S,S)-(À)-4h 12
52 Æ 8
9600 Æ 300
16700 Æ 650
59500 Æ 10100
(S,S)-(À)-4i
(S,S)-(À)-4j
13
14
52 Æ 9
240 Æ 50
10
11
12
(R,R)-(‡)-4h 12
3130 Æ 790 297000 Æ 78400
(À)-1[g]
na
na
115 Æ 1
135000 Æ 6000 1200
tacrine
231 Æ 16
77.2 Æ 5.8
0.3
[a] Drugs 3c, 4 were assayed as the hydrochloride salts; drugs rac-/meso-7
and rac-/meso-8 were assayed as the fumaric acid salts. Elemental analyses
matched the proposed salt formulations (C, H, N, Cl Æ 0.4%). [b] na ˆ not
applicable. [c] Assay performed using rat cortex homogenate, in the
presence of ethopropazine as
a specific BChE inhibitor. [d] Assay
performed using rat serum, in the presence of BW284c51 as a specific
AChE inhibitor. [e] Selectivity for AChE is defined as IC50(BChE)/
IC50(AChE). [f] Estimated values based on ꢀ50% inhibition of AChE
and BChE at 0.5 mm, the highest drug concentration tested. [g] [a]D20
À1498 (c ˆ 0.17, CHCl3).
ˆ
Scheme 2. Synthesis of enantiomerically pure dimers. a) MeI, Ag2CO3,
CHCl3, room temperature, 48 h, 81%; b) 0.5 equiv NH2(CH2)12NH2, cat.
CH3CO2H, benzene, reflux, 24 h; NaBH4, MeOH, room temperature, 12 h,
57%; c) BnBr, Ag2CO3, toluene, 48 h, 80%; d) NH2OBn ´ HCl, pyridine,
room temperature, 16 h, 95%; e) BH3 ´ THF, room temperature, 12 h,
reflux 2 h; 20% KOH, reflux 1.5 h, 66%; f) ( R)-(À)-mandelic acid, MeOH,
two
recrystallizations;
CH2Cl2/aq.
NaOH,
29%;
g) 0.5 equiv
À
ClC(O) (CH2)nÀ2C(O)Cl, Et3N, benzene, reflux 4 h, 80 ± 91%; h) 6 equiv
BH3 ´ THF, room temperature 12 h, reflux 2 h; 20% KOH, reflux 1.5 h, 87 ±
92%; i) H2, 10% Pd/C, EtOH, 24 h, 86 ± 91%. Lettering scheme (see
Scheme 1).
Figure 1. Overlay of the energetically favorable binding orientations of
‡
‡
(R)-3d ´ H (magenta) and (S)-3d ´ H (cyan) at the catalytic site of
Torpedo AChE (white); in each the nitrogen and oxygen atoms are green
and red, respectively. F ˆ Phe; E ˆ Glu; Yˆ Tyr ; W ˆ Trp. The perspective
shows a cross-section of the catalytic site.
fold lower potency relative to rac-/meso-4h (Table 1, en-
tries 2, 4) suggests that, as hoped, hydrogen bonding of the
pyridone with the active site contributes to affinity. The
synthesis of pure enantiomers of 4 f ± j was then undertaken
(Scheme 2). Pyridone 5 was converted to (Æ)-9 in 50% yield
over three steps, and resolved with (R)-(À)-mandelic acid; an
X-ray crystal structure analysis of the salt established the S
configuration for (‡)-9.[13] The enantiomeric free bases were
transformed to (S,S)-(À)-4 f ± j and (R,R)-(‡)-4h in 62 ± 74%
yield over three steps. The 60-fold greater potency of (S,S)-
(À)-4h relative to its enantiomer (Table 1, entries 7 and 10) is
suggestive of a multipoint interaction with the AChE active
site. The optimization of AChE IC50 in the S,S series at a
We have demonstrated that dimerization of an easily
synthesized but inactive fragment of Huperzine A can pro-
duce a drug with greater potency than the natural product
itself. Given the importance of structurally complex natural
products in drug discovery, we anticipate that the simple
approachdescribed here will be useful in developing new
leads for other biological targets.
Received: November 11, 1999 [Z14260]
1776
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0570-0833/00/3910-1776 $ 17.50+.50/0
Angew. Chem. Int. Ed. 2000, 39, No. 10

COMMUNICATIONS
[1] a) P. S. Portoghese, J. Med. Chem. 1992, 35, 1927 ± 1937; b) S. B.
Shuker, P. J. Hajduk, R. P. Meadows, S. W. Fesik, Science 1996, 274,
Naked-Eye Detection of Anions in
Dichloromethane: Colorimetric Anion Sensors
Based on Calix[4]pyrrole**
Â
1531 ± 1534; c) S. Hanessian, P.-P. Lu, J.-Y. Sanceau, P. Chemla, K.
Gohda, R. Fonne-Pfister, L. Prade, S. W. Cowan-Jacob, Angew. Chem.
1999, 111, 3354 ± 3356; Angew. Chem. Int. Ed. 1999, 38, 3160 ± 3162.
[2] a) Y.-P. Pang, P. Quiram, T. Jelacic, F. Hong, S. Brimijoin, J. Biol.
Chem. 1996, 271, 23646 ± 23649; b) L. E. Burgess, B. J. Newhouse, P.
Ibrahim, J. Rizzi, M. A. Kashem, A. Hartman, B. J. Brandhuber, C. D.
Wright, D. S. Thomson, G. P. A. Vigers, K. Koch, Proc. Natl. Acad. Sci.
USA 1999, 96, 8348 ± 8352.
[3] a) P. A. Wender, M. F. T. Koehler, D. L. Wright, K. Irie, Synthesis
1999, 1401 ± 1406; b) G. H. Posner, P. Ploypradith, M. H. Parker, H.
OꢁDowd, S.-H. Woo, J. Northrop, M. Krasavin, P. Dolan, T. W.
Kensler, S. Xia, T. A. Shapiro, J. Med. Chem. 1999, 42, 4275 ± 4280.
[4] A. P. Kozikowski, W. Tückmantel, Acc. Chem. Res. 1999, 32, 641 ± 650.
[5] S. Kaneko, T. Yoshino, T. Katoh, S. Terashima, Tetrahedron 1998, 54,
5471 ± 5484.
[6] a) A. P. Kozikowski, C. P. Miller, F. Yamada, Y.-P. Pang, J. H. Miller,
M. McKinney, R. G. Ball, J. Med. Chem. 1991, 34, 3399 ± 3402; b) D.
Bai, Pure Appl. Chem. 1993, 65, 1103 ± 1112; c) D. M. Fink, G. M.
Bores, R. C. Effland, F. P. Huger, B. E. Kurys, D. K. Rush, D. E. Selk,
J. Med. Chem. 1995, 38, 3645 ± 3651.
[7] M. L. Raves, M. Harel, Y.-P. Pang, I. Silman, A. P. Kozikowski, J. L.
Sussman, Nat. Struct. Biol. 1997, 4, 57 ± 63.
[8] M. Harel, I. Schalk, L. Ehret-Sabatier, F. Bouet, M. Goeldner, C.
Hirth, P. H. Axelsen, I. Silman, J. L. Sussman, Proc. Natl. Acad. Sci.
USA 1993, 90, 9031 ± 9035.
Hidekazu Miyaji, Wataru Sato, and
Jonathan L. Sessler*
The development of chemosensors for specific chemical
species is emerging as a researcharea of considerable
importance within the generalized field of supramolecular
chemistry.[1] One of the more appealing approaches in this
context involves the construction of optical sensors.[2±6] Such
systems generally contain some combination of substrate-
recognition functionality (receptor) and optical-signaling
capacity (chromophore), either directly linked[2, 3, 5] or appro-
priately associated in a noncovalent manner,[4] and are
designed to permit the detection of substrates by binding-
induced changes in absorption or emission properties (termed
colorimetric and fluorescent sensors, respectively). While the
utility of these approaches are becoming increasingly appre-
ciated in terms of bothqualitative and quantitative analysis, [5]
the number of colorimetric sensors available at present for
anionic substrates remains quite limited.[6] Indeed, only a few
systems are known that undergo color changes of sufficient
magnitude that they can be used for the direct ªnaked-eyeº
sensing of anions.[4b, 6b, c] Here we report the synthesis of a new
class of covalently linked calix[4]pyrrole ± anthraquinone
conjugates and show that they act as powerful naked-eye
[9] P. R. Carlier, Y. F. Han, E. S.-H. Chow, C. P.-L. Li, H. Wang, T. X.
Lieu, H. S. Wong, Y.-P. Pang, Bioorg. Med. Chem. 1999, 7, 351 ± 357.
[10] a) P. R. Carlier, E. S.-H. Chow, Y. Han, J. Liu, J. E. Yazal, Y.-P. Pang, J.
Med. Chem. 1999, 42, 4225 ± 4231; b) P. R. Carlier, D.-M. Du, Y.-F.
Han, J. Liu, Y.-P. Pang, Bioorg. Med. Chem. Lett. 1999, 9, 2335 ± 2338.
[11] a) 5: M. A. T. Dubas-Sluyter, W. N. Speckamp, H. O. Huisman,
Recueil 1972, 91, 157 ± 160; b) 6: G. Zacharias, O. S. Wolfbeis, H.
Junek, Monatsh. Chem. 1974, 105, 1283 ± 1291.
À
sensors for selected anions (namely, FÀ, ClÀ, H2PO4 ) in
dichloromethane.
[12] G. L. Ellman, K. D. Courtney, V. J. Andres, R. M. Featherstone,
Biochem. Pharm. 1961, 7, 88 ± 95.
The calix[4]pyrroles,[7] colorless macrocycles richin pyrrole
NH hydrogen bond donor functionality, are an easy-to-make
class of uncharged anion receptors that show considerable
promise in the area of anion sensing. In previous work we
demonstrated that anion sensors could be generated from
calix[4]pyrroles either by attaching a fluorescent reporter
group to the basic tetrapyrrolic skeleton[7d] or through the use
of a displacement process involving competition with para-
nitrophenolate anions.[4b] Unfortunately, we were unable to
produce inherently colored calix[4]pyrrole derivatives that
functioned as naked-eye anion sensors. Recently, however, we
discovered that calix[4]pyrroles functionalized at the b-
pyrrolic positions[8] may be made using a modified Sonoga-
shira coupling procedure.[9] By taking advantage of this
approach as well as the current availability of the ethynyl-
substituted calix[4]pyrrole 1,[8] we have now succeeded in
preparing (in 73% yield) an anthraquinone-functionalized
system (2) that bears an appended chromophore directly
[13] Crystallographic data (excluding structure factors) for the structures
reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC-137010. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax:
(‡44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
Ä
[14] P. Camps, R. E. Achab, D. M. Görbig, J. Morral, D. Munoz-Torrero, A.
Ä
Badia, J. E. Banos, N. M. a. Vivas, X. Barril, M. Orozco, F. J. Luque, J.
Med. Chem. 1999, 42, 3227 ± 3242.
[15] The docking study was performed with the SYSDOC program by
employing the AMBER 95 force field and the RESP charge model, as
described in the Supporting Information.
[*] Prof. J. L. Sessler, Dr. H. Miyaji, Dr. W. Sato
Department of Chemistry and Biochemistry
and Institute for Cellular and Molecular Biology
The University of Texas at Austin
Austin, TX 78712-1167 (USA)
Fax : (‡1)512-471-7550
E-mail: sessler@mail.utexas.edu
[**] This research was supported by the National Institutes of Health
(GM 58907) and the Texas Advanced Research Program.
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/angewandte/ or from the author.
Angew. Chem. Int. Ed. 2000, 39, No. 10
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0570-0833/00/3910-1777 $ 17.50+.50/0
1777