J. Med. Chem. 1997, 40, 1863-1869
1863
Neurotrophic 3,9-Bis[(alkylthio)methyl]- and -Bis(alkoxymethyl)-K-252a Derivatives Masami Kaneko,† Yutaka Saito,† Hiromitsu Saito,† Tadashi Matsumoto,† Yuzuru Matsuda,† Jeffry L. Vaught,‡ Craig A. Dionne,§ Thelma S. Angeles,§ Marcie A. Glicksman,§ Nicola T. Neff,§ David P. Rotella,| James C. Kauer,| John P. Mallamo,| Robert L. Hudkins,*,| and Chikara Murakata*,⊥ Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka 411, Japan, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahicho, Machida, Tokyo 194, Japan, and Departments of Medicinal Chemistry, Cell Biology, and Discovery Research, Cephalon Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380 Received January 14, 1997X
A series of 3,9-disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C, protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons. Introduction Neurotrophism may be defined as the ability to slow or prevent compromised neurons from undergoing necrotic or apoptotic cell death and to assist compromised neurons to maintain or express a functional phenotype. The classical example of neurotrophic activity is that displayed by the protein growth factors such as nerve growth factor (NGF), neurotrophin-3 (NT-3), and brainderived neurotrophic factor (BDNF) which mediate neuronal survival in a variety of models in vitro and in vivo.1 NGF has distinct, selective survival-promoting activities for cholinergic neurons in the central nervous system (CNS), as well as neurite outgrowth-promoting properties on sympathetic and sensory neurons of the dorsal root ganglia (DRG).2 Septal forebrain cholinergic neurons respond to NGF by increasing choline acetyltransferase (ChAT) activity.3 Insulin-like growth factor I (IGF-I), another neurotrophic factor, prevents the loss of ChAT activity in rat embryonic spinal cord cultures and reduces the programmed cell death of motoneurons in vivo during normal development or following axotomy or spinal transection.4 Although positive clinical results have been reported with several neurotrophic proteins such as NGF5 and IGF-I,6 the therapeutic potential of these polypeptides remains limited due to their size and pharmacokinetic characteristics, which prevents their systemic administration for treatment of central neuro* Address corespondence to: Dr. Robert L. Hudkins, Dept of Medicinal Chemistry, Cephalon Inc., 145 Brandywine Parkway, West Chester, PA 19380. E-mail:
[email protected]. † Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. ‡ Discovery Research, Cephalon Inc. § Cell Biology, Cephalon Inc. | Medicinal Chemistry, Cephalon Inc. ⊥ Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(97)00031-9 CCC: $14.00
degenerative diseases. Clearly, the discovery and development of low molecular weight, CNS permeable molecules with neurotrophic activity represents a valuable therapeutic opportunity. The development of small molecule therapeutics will be greatly aided by the understanding of intracellular pathways which mediate neurotrophic activity. Neurotrophic proteins bind to specific cell surface receptors, initiating intracellular signaling cascades which result in the regulation of specific gene transcription and neuronal survival.7 Despite the initial focus on promotion of neuronal survival by protein growth factors, it is now evident that several pathways may regulate survival in a manner dependent upon the activation state of survival-promoting or death/injury-activated signaling pathways.8 Thus, neurotrophism may result from regulation of mechanisms in addition to, and distinct from, growth factor-activated survival pathways.
K-252a (1, R ) H), an indolocarbazole alkaloid isolated from Nocardiopsis sp.,9 originally identified as a protein kinase C (PKC) inhibitor and subsequently found to inhibit a number of serine/threonine protein kinases,10 has been reported to possess neurotrophic© 1997 American Chemical Society
1864
Journal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Kaneko et al.
Scheme 1a
a (a) Ac O, DMAP, THF, room temperature, 93%; (b) Cl CHOCH , TiCl , CH Cl , 66%; (c) NaBH CH OH, CHCl , 65%; (d) NaOCH , 2 2 3 4 2 2 4 3 3 3 CH3OH, ClCH2CH2Cl, room temperature, 90%; (e) ROH, CSA, CH2Cl2; (f) RSH, CSA, CH2Cl2.
like properties.11 K-252a has been demonstrated to promote neurite outgrowth in human SH-SY5Y neuroblastoma cells.12 In primary cultures of embryonic neurons, K-252a induced survival of dorsal root ganglion and ciliary ganglion neurons,13 enhanced ChAT activity in spinal cord14 and basal forebrain cultures,15 and promoted survival and ChAT activity in striatal cultures.15 The neurotrophic activity demonstrated by K-252a for enhancing ChAT activity in spinal cord cultures was comparable to responses elicited by CNTF, BDNF, and IGF-I. In addition, studies have shown that K-252a protects neurons against glucose deprivation,16 free-radical-mediated injury, and amyloid β-peptide toxicity.17 These results suggest that the development of compounds from the K-252a class as neurotrophic agents may be effective therapy for the treatment of peripheral and central neurodegenerative diseases. In addition to neurotrophic properties, K-252a has a number of biochemical properties which limit its utility as a therapeutic agent. K-252a inhibited NGF-induced neuronal differentiation and survival18a-c and specifically inhibited the autophosphorylation of the highaffinity NGF receptor trk A, as well as related neurotrophin receptors trk B, trk C, and trk oncogenes at low nanomolar concentrations.19 A medicinal chemistry effort was undertaken to enhance and separate neurotrophic activity from the undesirable NGF (trk A kinase) and PKC inhibitory properties of K-252a. This paper reports on the selective neurotrophic activity of a series of 3,9-bis-(alkoxymethyl) and bis[(alkylthio)methyl] derivatives of K-252a as assessed by enhancement of
ChAT activity in rat embryonic spinal cord and basal forebrain cultures, and the subsequent identification of 3,9-bis[(ethylthio)methyl]-K-252a (8), which exhibited greater neurotrophic potency and efficacy with low kinase inhibitory properties compared to K-252a. Chemistry The preparation of the 3,9-bis(alkoxymethyl)- and bis[(alkylthio)methyl]-K-252a analogs required the synthesis of 3,9-bis(hydroxymethyl)-K-252a as the key intermediate (Scheme 1). K-252a was protected as its diacetyl derivative 2 in high yield. Diacetyl-K-252a (2) was converted to 3,9-dialdehyde 3 (66% yield) by treatment with 10 equiv of TiCl4 and 20 equiv of R,Rdichloromethyl methyl ether in CH2Cl2. Reducing the quantity of R,R-dichloromethyl methyl ether in the reaction yielded higher amounts of the undesired C-3 monoaldehyde product. The position of the formyl groups was readily assigned from 1H-NMR spectra. Aryl protons H-4 and H-8, which appear on 2 as a set of doublets at δ 9.09 and 8.07, respectively, appear as singlets at δ 9.53 and 8.66 on dialdehyde 3. Diacetyldialdehyde 3 was reduced to diacetyldiol 4 (65%, NaBH4, CHCl3-methanol), followed by deprotection to diol 5 in 90% yield using catalytic NaOMe in methanol. Treatment of intermediate diol 5 with various alcohols or thiols in the presence of camphorsulfonic acid in CH2Cl2 yielded the desired bis(alkoxymethyl)- (6, 7) and bis[(alkylthio)methyl]-K-252a derivatives (8-13) in good yield.
K-252a Derivatives
Journal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1865
Table 1. Neurotrophic Activity and trk A Inhibitory Activity of K-252a Derivatives
% of controla spinal cord ChAT
basal forebrain ChAT
compound
R
30 nM
300 nM
5 0 nM
250 nM
trk A IC50 (nM)c
1 (K-252a) 5 6 7 8 9 10 11 12 13
H CH2OH CH2OMe CH2OEt CH2SEt CH2SnPr CH2SiPr CH2SCH2CHdCH2 CH2SnBu CH2SCH2CH2NMe2
