Development of proneurogenic, neuroprotective small molecules

Article abstract of DOI:10.1021/ja108211m

Degeneration of the hippocampus is associated with Alzheimer's disease and occurs very early in the progression of the disease. Current options for treating the cognitive symptoms associated with Alzheimer's are inadequate, giving urgency to the search for novel therapeutic strategies. Pharmacologic agents that safely enhance hippocampal neurogenesis may provide new therapeutic approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apopotic cell death, and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile, and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or photo-cross-linking.

Full text of DOI:10.1021/ja108211m

ARTICLE
pubs.acs.org/JACS
Development of Proneurogenic, Neuroprotective Small Molecules
Karen S. MacMillan,† Jacinth Naidoo,† Jue Liang,† Lisa Melito,† Noelle S. Williams,† Lorraine Morlock,†
Paula J. Huntington,‡ Sandi Jo Estill,† Jamie Longgood,† Ginger L. Becker,‡ Steven L. McKnight,†
Andrew A. Pieper,*,†,‡ Jef K. De Brabander,† and Joseph M. Ready*,†
†Departments of Biochemistry and ‡Psychiatry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, Texas 75390-9038, United States
S Supporting Information
b
ABSTRACT: Degeneration of
the hippocampus is associated
with Alzheimer's disease and
occurs very early in the progres-
sion of the disease. Current op-
tions for treating the cognitive
symptoms associated with Alz-
heimer's are inadequate, giving
urgency to the search for novel
therapeutic strategies. Pharma-
cologic agents that safely en-
hance hippocampal neurogene-
sis may provide new therapeutic
approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apopotic cell death,
and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal
neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile,
and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward
increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in
mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar
concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or
photo-cross-linking.
’ INTRODUCTION
Disappointingly, the most recent data from late-stage trials of a
γ-secretase inhibitor, Eli Lilly's semagacestat, were sufficiently
discouraging to halt the trial.3 Currently, several biological and
small molecule drug candidates that target the production or
accumulation of Aβ42 plaques are in late-stage clinical trials.
Other therapies for AD include acetylcholinesterase inhibi-
tors (Aricept, Exelon, Razadyne, and Cognex) and an NMDA
antagonist (Namenda). These treatments offer some sympto-
matic relief, but no lasting benefit as they fail to address the
underlying biology of AD.
An alternative approach to treating AD focuses on promoting
hippocampal neurogenesis. AD is characterized by neurode-
generation in the cerebral cortex, hippocampus, and other sub-
cortical structures.4 Accordingly, we hypothesized that small
molecules that retard neuronal death or increase neurogenesis
may represent novel therapeutic agents. Currently, no drugs
operate through this mechanism. Nonetheless, groundbreaking
Alzheimer's disease (AD) accounts for about 80% of all cases
of dementia, and is the fifth leading cause of death in individuals
aged 65 or older. AD is the most prevalent and devastating
neurodegenerative disorder today. The greatest risk factor for
developing this disease is advancing age. By 2025, the majority of
baby boomers will have reached age 65, and the number of
people in this age group with AD is predicted to expand by about
50% to nearly 8 million. By 2050, the number of afflicted
individuals in the U.S. is expected to have risen to between 11
and 16 million. People with AD are high users of health care,
long-term care, and hospice services, and total payments for these
types of care from all sources currently exceed $177 billion per
year in the US.1
Pathologic changes in the brains of patients with AD include
the accumulation of extracellular neuritic plaques between
neurons, and neurofibrillary tangles within neurons.2 Numerous
clinical trials aimed at reducing these plaques in Alzheimer's
patients have failed to elicit cognitive improvement, even
when the brain tissue of patients has been cleared of plaques.
Received: September 11, 2010
Published: January 6, 2011
r
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in adult mice and humans,15 and allopregnanolone, a metabolite
of progesterone that is synthesized in the CNS, promotes
neurogenesis and learning in a mouse model of AD.16 In the
latter instance, enhancement of neurogenesis was associated with
amelioration of cognitive deficits.17 Finally, deficits in neurogen-
esis precede the formation of plaques and tangles associated with
AD in mouse models of that disease.18 Taken together, the
evidence suggests that drugs that promote hippocampal neuro-
genesis might provide effective treatment strategies for patients
with Alzheimer's disease.
To identify small molecules capable of promoting neurogen-
esis, we screened a collection of 2000 structurally unrelated
small molecules assembled from an in-house compound library
(Figure 1).19 Initially, molecules were screened by directly
injecting mixtures of 10 compounds intracerebroventricularly
(ICV) into the left lateral ventricle of living mice. Compounds
were introduced over 7 days at a concentration of 10 μM each
(12 μL/day at a constant rate). With this dosing, we estimated
that compounds would attain intracerebroventricular concentra-
tions in the low-micromolar to mid-nanomolar concentration
range. During compound infusion, the thymidine analogue
bromodeoxyuridine (BrdU, 50 mg/kg) was administered daily
to mark newly born hippocampal neural precursor cells. After
1 week of compound administration, the animals were sacrificed,
40 μm brain slices were prepared, and tissue was processed for
immunohistochemical staining with an antibody to BrdU to
identify newborn cells. Neurogenesis was quantified by counting
BrdUþ cells in the hippocampal dentate gyrus in the brain
hemisphere opposite to the side of compound infusion. The
number of positive cells was normalized against the volume of the
dentate gyrus.
One of the molecules that we discovered with this approach
was named P7C3, as it was identified as Compound 3 (C3) from
Pool 7 (P7). Mice treated with P7C3 had approximately twice
the number of BrdUþ cells as mice treated with vehicle alone
(artificial cerebral spinal fluid, CSF), and a similar number as
mice treated with a positive control, fibroblast growth factor
2 (FGF-2). Unlike some antidepressant drugs that have been
reported to enhance neurogenesis after two to three weeks of
treatment,20 P7C3 affects neurogenesis within days of adminis-
tration. Additional studies have revealed that P7C3 maintains
activity when a 1 μM solution was infused ICV (12 μL/day for
7 days); it displays maximal neurogenesis activity with an oral
dose of 5 mg/kg and measurable activity at 1 mg/kg. The
molecular target of P7C3 is not known. However, time-course
experiments revealed that P7C3 does not stimulate proliferation
of neural precursor cells. Rather, P7C3 increases the number of
newborn neurons that survive the month-long maturation pro-
cess. The mature neurons express neuron-specific proteins and
appear fully functional.19 Accordingly, P7C3 enhances neuro-
genesis, that is, is proneurogenic, by virtue of its neuroprotective
activity. P7C3 was found to repair the structure and function of
the dentate gyrus in mice deficient in the npas3 gene, a genotype
characterized by an absence of hippocampal neurogenesis.21
Furthermore, it also enhanced neurogenesis in the dentate gyrus,
impeded neuron cell death, and preserved cognitive capacity as a
function of terminal aging in rats. In contrast, no effect was
observed on neurogenesis in middle-aged rats, which do not
display impaired neurogenesis.22 Finally, P7C3 is not toxic to
embryonic, weaning, or adult mice.
Figure 1. In vivo screen for pro-neurogenic compounds. Drugs were
infused directly into the brain of ive mice over 7 days. Subsequent
sectioning and immunohistochemical staining revealed newly formed
neurons (black cells on right section).
studies starting in the 1960s demonstrated that neurogenesis in
adult mammals occurs in two regions of the brain, the subven-
tricular zone (SVZ) and the subgranular zone (SGZ) of the
dentate gyrus within the hippocampus.5-7 This process is
important for maintenance of the normal structure and function
of the hippocampus, and is thus essential to hippocampal-
dependent memory and learning.8 In particular, neurogenesis
in the dentate gyrus serves to produce neurons that integrate
locally in the granular layer of the dentate gyrus, which displays
lifelong structural and functional plasticity. This process ap-
pears to be active within all mammalian species, including
humans.9 In adult mice, neurogenesis is a month-long process
involving proliferation of neuronal precursor cells and subse-
quent maturation. During this process, the vast majority of the
newly formed hippocampal neural precursor cells undergo
apoptosis prior to maturation, whereas the surviving cells
mostly become incorporated as fully functional neurons.
The process of neurogenesis in adult mice can be affected by
both environmental and chemical stimuli. Neurogenesis in-
creases in adult mice when they are housed in an enriched
environment containing toys, nesting materials, and exercise
equipment.10,11 Similarly, neurogenesis is enhanced when mice
are free to exercise voluntarily.12,13 Provocatively, engagement in
enriching educational activities and exercise also delays the onset
and progression of AD.14 Antidepressants enhance neurogenesis
Here we describe efforts to understand the structural require-
ments for activity and to improve P7C3's potency, stability, and
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discovered that methyl (3-5) or ortho-methoxy substitutions
appeared to decrease activity in our assay. In contrast, a methoxy
group was well tolerated in the meta or para position (7, 8). A
long-term objective is to identify the molecular target of P7C3,
and to this end we planned to synthesize active derivatives that
could be used as reagents for affinity chromatography. In that
context, the observation that an O-alkyl group did not compro-
mise activity was encouraging. Indeed, many of the synthetic
analogues (see below) were prepared from m-anisidine with this
consideration in mind. We tested two analogues possessing satu-
rated heterocyclic rings rather than an aniline (9, 10). Previous
work from a group at Serono Pharmaceuticals revealed that sev-
eral carbazoles inhibited Bid-mediated release of cytochrome c,
and, as a consequence, were antiapoptotic.23 The most active
compound from the Serono study (9) proved moderately active
in our assay, while a less active Serono compound (10) was also
less active in our experiments.19
Finally, we evaluated the activity of dimebon (11), a γ-
tetrahydrocarboline originally used as an antihistamine in Russia.
This drug has recently been reported to block apoptosis of
cortical neurons exposed to amyloid-beta peptide.24 Anecdotal
evidence of its efficacy against AD and age-related cognitive
decline25 prompted Medivation and Pfizer to conduct clinical
trials in the U.S. to determine dimebon's efficacy in treating
several forms of neurodegenerative disease. While phase II data
appeared exceptionally encouraging,26 unpublished results from
a phase III study showed that dimebon offered no benefit for
patients suffering from AD.27 Provocatively, dimebon is active in
our neurogenesis assay, but substantially less so than P7C3.
Synthetically, P7C3 can emerge from a union of a carbazole,
an aniline, and an epoxide (Scheme 2). This convergent synthetic
approach allows the independent variation of the two rings.
Furthermore, additional processing of P7C3 and its derivatives
provided access to a range of derivatives. Throughout the
manuscript, compounds with numbers highlighted in green
showed activity as good as or better than our positive control,
FGF-2 (see below). As shown in Scheme 2, a wide range of
analogues was prepared by first condensing 2- or 3-ring hetero-
cycles with epibromohydrin.28 The resulting epoxides 13 were
then opened with various anilines, thiols, or heterocycles. We
found that ring-opening with anilines generally required Lewis
Figure 2. Analysis of commercial analogues. In vivo analysis of com-
mercially available analogues of P7C3. Data are expressed as mean (
SEM, 4 mice/compound. Compounds were infused ICV as a 10 μM
solution introduced at a constant rate (12 μL/day) over 7 days.
Scheme 1. P7C3 and Commercially Available Analogues
29
acid activation of the epoxides (BiCl3 or LiBr30), whereas
alcohols, indoles, and carbazoles required deprotonation of the
nucleophile with NaH. Stronger nucleophiles such as sodium
azide, hydrazine, ammonia, thiophenol, or aminopyridines re-
acted with the terminal epoxides upon simple heating. In a slight
modification of this sequence, dibromocarbazole was allowed
to react with arylglycidyl ethers to provide aryl ethers 42 and
43 directly. Similarly, m-anisidine was first condensed with
epoxy-2-methyl-propyl chloride and then exposed to the car-
bazole to yield the tertiary alcohol 46.31 This synthetic approach
provided access to a wide range of structural diversity, although
the overall yields ranged from rather abysmal to nearly quanti-
tative. However, none of the syntheses has been optimized.
Complete experimental details are provided as Supporting In-
formation.
Several of the analogues prepared according to Scheme 2 were
subject to additional transformations. For instance, the thio-
ethers 24 and 44 were oxidized with either NaIO4 or m-CPBA
to provide either a diastereomeric mixture of sulfoxides (25)
or the sulfones (26, 45), respectively. Phthalimide 39 was
deprotected and acylated to provide derivative 41. Similarly,
toxicity profile. To the best of our knowledge, this is the first class
of small molecules designed to promote neurogenesis, and as
such may represent lead compounds for the treatment of
debilitating neurodegenerative diseases.
’ RESULTS
P7C3 contains a dibromocarbazole connected to an aniline
with a 2-hydroxypropyl linker. We set out to determine which
positions of P7C3 would tolerate modification, systematically
optimize each subsection of P7C3, and identify derivatives that
could be used for affinity chromatography. Several commercially
available analogues of P7C3 provided early indications of the
structure/activity relationship. The bromines on the carbazole
appear particularly important, as the derivatives with dichloro (1)
and parent (2) carbazole did not appear active at the concentra-
tions tested (Scheme 1, Figure 1). We tested derivatives posses-
sing methyl or methoxy substitution on the aniline ring, and
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Scheme 2. Synthesis of Deriviatives of P7C3
a Dibromocarbazole was added to aryl glycidyl ether rather than epibromohydrin.
b Dibromocarbazole was added to corresponding epoxyaniline rather than epibromohydrin.
Scheme 3. Modification of Analogue 7
P7C3 analogue 7 was subject to further processing (Scheme 3).
For example, methylation was O-selective at low conversion,
yielding ether 47. Urea formation and acylation were facile,
providing 48 and 49 in quantitative yield. Oxidation with
SO3 Py produced ketone 50 and, unexpectedly, thioether 51.
3
A potential mechanism for this unusual and, to our knowledge,
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Table 1. Fluorination of Alcohols
Scheme 5. Synthesis of Analogues with Modified Linkers
a Isolated yield over one or two steps. b Ns = nitrobenzene sulfonyl.
c After oxidation with m-CPBA.
aminopyridines 64-66. Five-membered heterocyclic rings were
incorporated as shown in Scheme 4. Thus, hydrazine 30 was
condensed with a diketone to yield pyrazole 67, while azide 31
underwent Cu-catalyzed cycloaddition to yield the correspond-
ing triazole (68).34
Scheme 4a
a (a) 2,4-Pentanedione, EtOH, 70 °C (45%) and (b) 4-pentynol,
Cu(SO4), NaAscorbate, H2O/tBuOH (54%).
unprecedented, oxidation is provided in the Supporting Informa-
tion. In brief, an R-ketoimine may be trapped by methanethiol
released from DMSO.
Several analogues were prepared with modified linking groups
between the carbazole and aniline. As shown in Scheme 5,
epoxide openings and alkylations provided derivatives without
the aniline NH (69), without the central OH (70), or with
additional methylenes in the linker (71, 72). Similarly, the
secondary aniline was replaced with a secondary amide (73)
through the following sequence: ring-opening of methyl glyci-
date with dibromocarbazole, hydrolysis of the ester, and coupling
to aniline by way of the acid chloride (eq 3).
Fluorination appeared to improve activity (see below), so a
series of fluorinated analogues were prepared, as shown in
Table 1. We used either DAST (Et2NSF3) or its morpholino
analogue. Reactions were generally cleaner using the latter
reagent. For aniline-containing starting materials, the NH proved
problematic. In these cases, protection of the nitrogen with a
4-nitrobenzene sulfonyl (4-Ns) group32 greatly improved the
fluorination. Similarly, the difluorides 61 and 62 were prepared
from the corresponding ketones (eq 1).
Ester 73 was originally conceived as a precursor to optically
active derivatives of P7C3. Our plan was to perform the ring-
opening with enantioenriched methyl glycidate, and, following
amidation, reduce scalemic 73 to yield optically active 7. In the
event, however, we found that the ester generated in this manner
was racemic, with the optical purity presumably being a casualty
of the basic reaction conditions. Similarly, ring-opening of
optically active epichlorohydrin yielded racemic epoxide 13
(heterocycle = Br2-carbazole). In this case, epichlorohydrin
was likely racemized by adventitious chloride ion.35 Ultimately,
using the known Mitsunobu reaction of dibromocarbazole with
optically active glycidol,23 we were able to prepare both enantio-
mers of epoxide 13 in >99% ee, and from them access the single
enantiomers of both 7 and the sulfone 26.36 Likewise, both
enantiomers of the aryl ether 42 were prepared from optically
active phenyl glycidyl ether. Interestingly, when these alcohols
To explore the SAR on the right-hand fragment, we replaced
the N-phenyl ring with heterocyclic rings as shown in eq 2. The
primary amine 28 condensed with 2-chloropyrimidine to provide
analogue 63. Introduction of pyridine rings required initial
conversion to a urea. Subsequent Cu-catalyzed cross coupling
with iodo-pyridines33 and hydrolysis of the cyclic urea yielded
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Scheme 6. Synthesis of a Coumarin-Tagged Derivative
compound prepared (53) is the fluorinated anisidine derivative.
Similar trends were observed with the N-methyl derivative (35 vs
56) and the sulfoxide-containing analogues (26 vs 58). However,
the effect of fluorines appears dependent on context. For
example, while the activities of the m- and p-anisidine-containing
analogues are improved by fluorination (7 vs 53; 8 vs 54), the
parent compound with an aniline ring gains little from fluorina-
tion (P7C3 vs 52). Similarly, the derivative featuring a phenol is
highly active with a 2-hydroxypropyl linker, but less active with a
2-fluoropropyl linker (42 vs 57).
Two additional changes to the linker region were well
tolerated. First, conversion to a tertiary alcohol (46) improved
activity. Second, extending to a hydroxy butyl linker maintained
high activity, so long as the extra methylene was inserted between
the hydroxy group and the aniline (72). In contrast, inclusion of
an additional methylene between the hydroxyl and the carbazole
(71) essentially abrogated activity. Intermediate activity was ob-
served for analogues possessing O- or N-methyl groups (35, 47), a
ketone (50), an R-thioketone (51), an N-acyl group (49), a cyclic
urea (48), an amide (73), or an unsubstituted propyl chain (70).
P7C3 features a nitrogen linking the propyl chain to the right-
hand aromatic ring. We synthesized several congeners to explore
the effectiveness of alternative heteroatoms. Encouragingly, the
aniline could be replaced with an aryl ether (42, 43), an aryl
thioether (24), or an aryl sulfone (26) and retain superior
activity. Interestingly, while both the thioether and sulfone were
highly active, the mixture of diastereomeric sulfoxides was
equivalent to vehicle. Because C-S bonds are longer than
C-N bonds, we speculated that smaller groups than Br might
be tolerated on the carbazole. While not confirming our reason-
ing, it was heartening to observe substantial neuroprotective
activity from a dichlorocarbazole possessing a sulfone linker
(45). While several heteroatoms appear acceptable at this posi-
tion, an analogue with only a hydrocarbon linker (69) did not
appear neuroprotective at the concentration tested.
The right-hand aromatic fragment tolerates substantial varia-
tion, although some surprising observations were made. Modest
activity is retained even with simple NH2- or O-alkyl-terminated
compounds (28, 29), that is, compounds lacking a right-hand
aromatic ring. As described above, a piperazine group is less
effective than an aniline, although significant neuroprotective
activity is maintained. Heteroaromatic rings have proved less
effective with two notable exceptions. The 2-aminopyridine (64)
displays activity nearly as high as P7C3 itself, and the azapyridone
(32) maintains robust activity. In contrast, the other pyridines,
pyrazines, indoles, carbazoles, pyrazoles, and triazoles were
substantially less active in our assays. Substitution around the
aniline ring of P7C3 was somewhat enigmatic. For instance, a
3-OMe (7) group had little effect on activity, but a minor
extension to an OEt (21) group23 essentially abrogated activity.
Likewise, a 3-N3 group (22) substantially decreased activity. None-
theless, substantial activity was observed for a derivative designed
to facilitate affinity chromatography and localization studies, the
coumrain-linked analogue (76),38 Meanwhile, the 4-OMe (8),
4-OEt,39 and 4-N3 (23) derivatives were all highly active.
Finally, several enantiomeric pairs were tested. Specifically, we
tested the anisidine analogue of P7C3 (7), the aryl ether 42, and
the sulfoxide 26. In all three cases evaluated, a significant
difference in activity was noted between the two enantiomers.
The same enantiomer displayed higher activity in all three cases,
although the R/S designation varies due to differences in priority
among the substituents.
were subjected to the fluorination reaction conditions shown in
Table 1, the resulting fluorides were found to have <20% ee.
Partial reaction flux through an aziridinium ion likely provides a
mechanism for retention of configuration that competes with the
usual invertive pathway.
Finally, to provide a reagent for mechanism-of-action studies,
the coumarin-tagged37 derivative 76 was prepared from anisidine
derivative 7. Thus, cleavage of the methyl ether (f 74),
alkylation (f 75), and conjugation to a coumarin derivative
provided the desired reagent in acceptable yield (Scheme 6).
’ BIOLOGICAL DATA
All of the analogues were tested in live mice for their
proneurogenic effect. Following the procedure described above,
10 μM solutions were administered continuously for 7 days (12
μL/day) by means of subcutaneous osmotic minipumps that
delivered compound through a canula implanted into the left
lateral ventricle. We estimate that the in vivo concentrations were
in the low-micromolar to mid-nanomolar range.19 The results are
shown in Figure 3 grouped by the region of P7C3 that each
analogue modifies. Thus, derivatives of the carbazole, the propyl
linker, the heteroatom or the aromatic ring were tested (Scheme 7).
Some compounds represent multiple changes from P7C3.
The bromines on P7C3 are particularly important: the di-
methyl (14), dichloro (17), and diiodo (20) versions appear to
have little activity, and the monobromo derivative (16) appears
substantially less active in our assays. Nonetheless, dibromina-
tion is not required as the bromo-methyl-substituted compound
(15) is highly active. Likewise, in a promising result for the
synthesis of photo-cross-linking agents, the azido-bromo-carba-
zole (18) retains significant activity. The substantial activity of
the bis-nitrile (19) indicates that the bromines can be replaced
entirely and provides a lead compound with substantially
reduced lipophilicity.
The carbazole ring system does not appear to be strictly
required. For example, the β-carboline (38) is as active as P7C3.
The tetrahydro-γ-carboline 33, a structural hybrid between
dimebon and P7C3, shows intermediated activity, as does the
dimethyl indole 27.
In the linker region, several compounds were improved by
replacing the hydroxyl with a fluorine. Indeed, the most active
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Figure 3. Analysis of synthetic analogues. In vivo analysis of synthetic analogues of P7C3. Data are expressed as mean ( SEM, 4 mice/compound except
compound 56 (1 mouse). Compounds were infused ICV as a 10 μM solution introduced at a constant rate (12 μL/day) over 7 days. Brackets indicate
portion of analogue that differs from P7C3.
Scheme 7. Regions of P7C3 Subject to Modification As
Labeled in Figure 3
Tertiary alcohol 46, fluoro-aniline 53, and fluoro-sulfone 58
were evaluated at a range of doses. In each instance, 10-0.01 μM
solutions were infused over a 7-day period at a constant rate (12
μL/day) concurrent with IV administration of BrdU. The tertiary
alcohol (46) roughly paralleled P7C3, showing substantial
Figure 4. Dose-response of selected analogues. In vivo analysis of
activity when a 1 μM solution was infused, but showing little
synthetic analogues of P7C3. Data are expressed as mean ( SEM,
effect from a 0.1 μM solution. In contrast, both fluorides
4 mice/compound. Compounds were infused ICV at a constant rate
maintained activity at 0.1 μM. Indeed, our most potent analogue
(12 (μL/day) over 7 days as a solution of the indicated concentration.
(53) remained active when 0.01 μM solution was infused, which
corresponds to a total of 0.43 mg added over one week's time.
Several of our most potent compounds were analyzed for
pharmacokinetic properties and hallmarks of toxicity (Table 2).
A competition assay was performed to determine if P7C3 or its
derivatives bound to the hERG channel, as interference with
hERG function can cause cardiac toxicity.40 Specifically, we
measured what percentage of [3H]astemizole (Kd = 6.8 nM)
was displaced with a 10 μM concentration of drug.41 P7C3 and
(R)-7 showed significant displacement of the radioligand, and
(S)-42 showed modest binding. Encouragingly, the other ana-
logues tested appear to bind hERG only weakly.
Accordingly, we performed a competition assay using [125I]ami-
nopotentidine, a potent (Kd = 0.45 nM) ligand for the histamine
H2 receptor.41 Using this assay, dimebon displaced 99% of the
aminopotentidine at 10 μM. P7C3 and (R)-7 also display modest
binding to this receptor. However, histamine binding and
neuroprotection activity are clearly unrelated because several of
our most potent analogues display weak or no binding. No
analogue showed significant binding to the histamine H1, H3, or
H4 receptor.
To determine metabolic stability, compounds were incubated
with either mouse or human hepatocytes at 2 μM. HPLC/MS/
MS analysis of the culture media at several time points provided
measurements of half-life. Gratifyingly, of the analogues tested,
most were extremely stable. Compound 58, a fluorosulfone, had
The tetrahydrocarboline dimebon (11) had been used in
Russia as an antihistamine prior to engendering enthusiasm
regarding its potential as a treatment for Alzheimer's disease.
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Table 2. Off-Target, Stability and Toxicity Data for Highly Active Compounds
toxicity toward HeLa cells
half life in cell culture (min)
hERG binding
(% displacement @ 10 μM)a
histamine H2 binding
(% displacement @ 10 μM)b
compound
GI50 (μM)c
LC50 (μM)d
mouse hepatocytes
human hepatocytes
P7C3
(R)-7
26
92
75
24
52
36
19
38
63
88
5
5.1
23.5
16.1
10.3
10.9
>30
9.1
21.4
>30
>30
>30
>30
>30
20.3
>240
>240
224
>240
>240
43
(S)-42
46
52
52
21
-3
>240
>240
>240
141
>240
>240
>240
178
53
58
a % displacement of [3H]astemizole (1.5 nM; Kd = 6.8 nM) from hERG with 10 μM drug. b % displacement of nM [125I]aminopotentidine (0.1 nM; Kd =
0.45 nM) from histamine H2 receptor with 10 μM drug. c Concentration at which growth in cell number is 50% of control at 48 h. d Concentration at
which cell numbers at 48 h equal 50% of cell number at t = 0.
a half-life of 2-3 h. Compound 26, a β-hydroxy sulfoxide, was
quite stable toward mouse hepatocytes, but was processed by
human hepatocytes with a t1/2 < 1 h. All other analogues dis-
playing high activity showed only marginal levels of metabolism
after 4 h. While we did not determine the metabolites of the two
sulfoxides, it is interesting to note that during their syntheses
these compounds displayed a tendency to undergo β-elimination
to yield vinyl sulfones. We conjecture that this reactivity profile in
the flask is mirrored by their reactivity in cell culture.
As previously reported, P7C3 is orally available and displays
favorable distribution into the brain (AUC brain/AUC plasma
ratio of 3.7 for oral delivery). For comparison, brain exposure to
53 was determined after intraperitoneal (IP) and oral gavage
(PO) administration. It was found to have good penetration,
with a AUC brain/AUC plasma of 0.61 and 0.82 by IP and oral
gavage administration, respectively. Treating at 20 mg/kg PO
with fluoride 53 resulted in approximately 0.9 μM concentration
in the brain (450 ng/g brain tissue) 4 h after dosing.
xybutyl linker, or the aniline had been replaced with an amino-
pyridine, a phenol, a thiophenol or an aryl sulfone. This SAR
should allow the identification of safe, effective and bioavailable
analogues for further development.
Second, we have shown that the neuoroprotective activity of
P7C3 and its analogues is unrelated to histamine receptor
binding. No mechanism of action has been suggested for
dimebon's putative anti-AD activity, but (a) it is active in our
assay and (b) it is a potent antagonist of the histamine receptor.
We cannot claim with certainty that dimebon and the P7C3 class
of molecules work by the same mechanism, but we can exclude
the possibility that binding to the histamine receptor is required
for proneuorogenic activity.
Third, we have prepared analogues of P7C3 that we anticipate
will facilitate mechanism-of-action studies. Specifically, we have
prepared two aryl azides (18, 23) that could be used for
photoaffinity studies. Likewise, we have prepared an active
derivative containing a coumarin moiety for use in pull-down
and localization experiments. More broadly, we have revealed
which sections of P7C3 accommodate alteration; this knowledge
is expected to aid the preparation of other derivatives that will be
useful in mode-of-action studies.
Finally, we have identified analogues displaying no observed
toxicity, no binding of the hERG channel, good blood/brain
distribution, oral availability, and excellent metabolic stability.
We are optimistic that this information will enable the discovery
of the molecular target of P7C3 and will continue to facilitate the
identification of compounds suitable for further development.
P7C3 was discovered using an unusual approach. Modern
drug discovery relies on high-throughput screening against
defined receptors or enzymes using in vitro or cell-based assays.
In connection with neurodegenerative diseases, however, little
consensus exists regarding which receptors or enzymes represent
ideal targets. Furthermore, these diseases likely involve pathol-
ogies of multiple cell types in the complex setting of the central
nervous system. Accordingly, devising simple, predictive assays
has proven difficult, a challenge which manifests itself in the
paucity of available therapeutics. We opted for an alternative
strategy wherein we identified and optimized molecules capable
of affecting a desired function, neurogenesis, within a living
mammal. The risks of this approach were clear, among
which were low throughput, an inability to separate binding to
a target from pharmacokinetic effects, and ignorance of the
molecular target. Nonetheless, the benefits of this experimental
’ DISCUSSION
Neurodegenerative diseases remain an area of unmet medical
need. The poignancy of medicine's deficiency in this arena will
only become more evident in the near future. Aging populations
in most industrialized countries, and many developing ones,
will confront an increasing incidence of neurodegenerative
disorders.1 In that context, small molecules capable of fostering
the growth of new neurons could facilitate the development of
novel molecular therapeutics. P7C3 possesses this ability and has
been found to repair damaged neural circuitry in mice and
improve cognition in aged rats. Accordingly, we became inter-
ested in improving the activity of P7C3 in terms of potency and
toxicity as well as identifying reagents to aid studies of its mode of
action.
We modified nearly every atom of P7C3 during this study. Our
investigation has revealed several important characteristics of
P7C3 and has led to the discovery of improved analogues. First,
we found that P7C3 displays an encouraging combination of
sensitivity and flexibility. That is, while effects are evident from
modification to all sections of the molecule - the carbazole, the
linker region, the aryl ring; none of these subunits is sacrosanct.
For instance, highly active analogues were identified in which the
carbazole had been replaced with a carboline, the hydroxy-propyl
linker had been replaced either a fluoropropyl linker or a hydro-
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Journal of the American Chemical Society
ARTICLE
design appear to outweigh these limitations as we were able
to select simultaneously for molecules that possessed activity
within live mice and rats, desirable toxicity profiles, and accep-
table physical characteristics. It may be that this atavistic
approach to drug discovery deserves consideration for other
complex diseases.
control (Cep). The cells were then placed in a 37 °C, 5% CO2 incubator.
At the time points indicated, the well contents were harvested and a
2-fold volume of methanol added to lyse the cells and precipitate
proteins. The samples were incubated 10 min at RT and then spun at
15000g for 5 min in a microcentrifuge. The supernatant was analyzed by
LC/MS/MS. Metabolism of 7-ethoxycoumarin was used to monitor
hepatocyte performance.
Pharmacokinetic Analysis of P7C3 and 53. P7C3 was pre-
pared for dosing by dissolving the compound in DMSO at 50 mg/mL.
The compound was diluted to a final formulation of 3% DMSO/10%
cremophor EL (Sigma, St. Louis, MO)/87.5% D5W (5% dextrose in
water, pH 7.2). Adult mice were dosed IV, IP, or via oral gavage (PO) in
a total volume of 0.2 mL. Compound 53 was prepared in a similar
fashion in 5% DMSO/10% cremophor EL/85% D5W and dosed either
IP or PO. Animals were sacrificed at varying times after dosing by
inhalation overdose of CO2. Whole blood was collected with an ACD
solution (sodium citrate) coated syringe and needle. The blood was
subsequently centrifuged at 9300g for 10 min to isolate plasma. Plasma
was stored at -80 °C until analysis. Brains were isolated from mice
immediately after sacrifice, rinsed extensively with PBS, blotted dry,
weighed, and snap frozen in liquid nitrogen. Lysates were prepared by
homogenizing the brain tissue in a 3-fold volume of PBS (weight of brain
in g ꢀ 3 = volume of PBS in ml added). Total lysate volume was
estimated as volume of PBS added þ volume of brain in ml. One
hundred microliters of either plasma or brain was processed by addition
of 200 μL of acetonitrile to precipitate plasma or tissue protein and
release bound drug. In some experiments, this mixture was centrifuged at
16100g for 5 min and the supernatant was analyzed directly by HPLC/
MS/MS, while in other experiments the sample was additionally
processed by passage over a solid phase extraction column. An additional
700 μL of PBS was added to the plasma or lysate acetonitrile mixture and
the sample was centrifuged at 16100g for 5 min. Nine hundred
microliters of supernatant was mixed with 1 mL of PBS and passed
over a Waters (Milford, MA) OASIS HLB solid phase extraction column
primed by addition of 2 mL of acetonitrile followed by 2 mL of water.
The column was washed twice with 2 mL of 5% acetonitrile in H20 and
compound was eluted by addition of 2 mL of acetonitrile. 500 μL of the
eluant was added to 500 μL of H20 containing 0.2% formic acid (final
formic acid 0.1%) and analyzed by HPLC/MS/MS as described above.
Standard curves were prepared by addition of P7C3 to blank plasma or
blank brain lysate. A value of 3-fold above the signal obtained from blank
plasma or brain lysate was designated the limit of detection (LOD). The
limit of quantitation (LOQ) was defined as the lowest concentration at
which back calculation yielded a concentration within 20% of theoretical.
The LOQ for both plasma and brain was 5 ng/mL. In general, back
calculation of points on both curves yielded values within 25% of
theoretical over 4 orders of magnitude (10000 to 5 ng/mL). Pharmaco-
kinetic parameters were calculated using the noncompartmental analysis
’ EXPERIMENTAL DETAILS
Neuroprotection Assay. Test compounds were evaluated as
described previously.19 Compounds were infused intracerebroventricu-
larly (i.c.v.) into the left lateral ventricle of four adult (12 week old) wild-
type C57BL/J6 mice by means of surgically implanted Alzet osmotic
minipumps that delivered solution into animals at a constant rate of 0.5
μL/hour for 7 days. Bromodeoxyuridine (BrdU) was injected intraper-
itoneally at 50 mg/kg/day for six days during pump infusion. Twenty-
four hours after the final BrdU administration, mice were sacrificed by
transcardial perfusion with 4% paraformaldehyde at pH 7.4, and their
brains were processed for immunohistochemical detection of incorpo-
rated BrdU in the SGZ. Dissected brains were immersed in 4%
paraformaldehyde overnight at 4 °C, then cryoprotected in sucrose
before being sectioned into 40 μm thick free-floating sections. Unmask-
ing of BrdU antigen was achieved through incubating tissue sections for
two hours in 50% formamide/2X SSC at 65 °C, followed by a 5-min
wash in 2ꢀ SSC and subsequent incubation for 30 min in 2 M HCl at
37 °C. Sections were processed for immunohistochemical staining with
mouse monoclonal anti-BrdU (1:100). The number of BrdUþ cells in
the entire dentate gyrus SGZ in the contralateral hemisphere (opposite
side of surgically implanted pump) was quantified by counting BrdUþ
cells within the SGZ and dentate gyrus in every fifth section throughout
the entire hippocampus and then normalizing for dentate gyrus volume.
Binding Assays. Binding to hERG and histamine receptors was
performed by MDS Pharma Services, now Ricera. Briefly, a competition
binding assay was performed in which the level of specific binding of a
radioligand, used at a low concentration (eKd), was determined in the
presence of 10 μM test compound. Specific radioligands, the concentra-
tion utilized, and historical Kd values as measured by Ricera for each
receptor and radioligand are as follows: HERG: 1.5 nM [3H]astemizole
(Kd 6.8 nM); histamine H1: 1.2 nM [3H]pyrilamine (Kd 1.1 nm);
histamine H2: 0.1 nM [125I]aminopotentidine (Kd 0.45 nM); histamine
H3: 3 nM [3H] (R)-(-)-R-methylhistamine (Kd 2.4 nM); histamine
H4: 8.2 nM [3H]histamine (Kd 5.7 nM).
Cell Viability Assays. HeLa cells were plated at optimally deter-
mined densities for either 96- or 384-well plates, and compounds were
added in serial 5-fold dilutions the following day. Forty-eight hours later,
cell viability was assessed by addition of CellTiter-Glo Reagent
(Promega, Madison, WI). Growth inhibitory 50 (GI50) values were
calculated according to the formula 100 ꢀ (T - T0)/(C - T0) = 50,
where T = optical density of test well after 48 h exposure to compound,
T0 = optical density at time zero, and C = control optical density. The
LC50 (concentration of drug resulting in a 50% reduction in the
measured protein/ATP at the end of the drug treatment as compared
to that at the beginning) indicating a net loss of cells following treatment
is calculated from [(T - T0)/T0] ꢀ 100 = -50.
toolofWinNonLin (Pharsight). Bioavailability was calculated asAUCoral
/
AUCiv ꢀ Doseiv/Doseoral ꢀ 100 (AUC is area under the concentration
time curve). The brain/17blood ratio was calculated using AUC values.
’ ASSOCIATED CONTENT
S
Hepatocyte Metabolism Assays42. Male ICR/CD-1 mouse
hepatocytes, InVitroGRO HI and HT Medium, and Celsis Torpedo
Antibiotic Mix were purchased from Celsis/In Vitro Technologies
(Baltimore, MD). Cryopreserved hepatocytes were thawed in HT Media
containing antibiotics, resuspended in HI media at 2 ꢀ 106/mL and
plated in 96-well plates at 0.05 mL (105 cells)/well. Compounds to be
tested were dissolved in DMSO at 2 mM, further diluted to 4 μM in HI
media, and added to the cells in 50 μL so that the final compound
concentration was 2 μM. Two additional wells containing compound
and no cells were plated to serve as time 0 (C0) and end-point solvent
Supporting Information. Complete experimental details
b
and characterization data for new compounds; full citations for
abbreviated references. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
Andrew.Pieper@utsouthwestern.edu; Joseph.ready@utsouthwestern.
edu.
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Journal of the American Chemical Society
ARTICLE
’ ACKNOWLEDGMENT
(24) (a) Bachurin, S.; Bukatina, E.; Lermontova, N.; Tkachenko, S.;
Afanasiev, A.; Grigoriev, V.; Grigorieva, I.; Ivanov, Y.; Sablin, S.; Zefirov,
N. Ann. N.Y. Acad. Sci. 2001, 939, 425. (b) Bachurin, S. O.; Shevtsova,
E. P.; Kireeva, E. G.; Oxenkrug, G. F.; Sablin, S. O. Ann. N.Y. Acad. Sci.
2003, 993, 334.
(25) (a) O'Brien, J. T. Lancet Neurol. 2008, 7, 768. (b) Burns, A.;
Jacoby, R. Lancet 2008, 372, 179.
(26) Doody, R. S.; Gavrilova, S. I.; Sano, M.; Thomas, R. G.; Aisen,
P. S.; Bachurin, S. O.; Seely, L.; Hung, D. Lancet 2008, 372, 207.
(27) Miller, G. Science 2010, 327, 1309.
(28) Asso, V.; Ghilardi, E.; Bertini, S.; Digiacomo, M.; Granchi, C.;
Minutolo, F.; Rapposelli, S.; Bortolato, A.; Moro, S.; Macchia, M.
ChemMedChem 2008, 3, 1530.
(29) McCluskey, A.; Leitch, S. K.; Garner, J.; Caden, C. E.; Hill,
T. A.; Odell, L. R.; Stewart, S. G. Tetrahedron Lett. 2005, 46, 8229.
(30) Chakraborti, A. K.; Rudrawar, S.; Kondaskar, A. Eur. J. Org.
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Funding is provided by The Hartwell Foundation, Staglin
Family Fund, UT Southwestern High Risk/High Impact Re-
search Fund, National Alliance for Research on Schizophrenia
and Depression, Morton H. Meyerson Family Tzedakah Fund
(A.A.P), NIH (1R01 MH087986, 5DP1OD000276, RO1M-
N59388, 5PO1CA95471), a Simons Foundation Autism Re-
search Initiative Grant, the Welch foundation (I-1612, I-1422).
J.M.R. is a fellow of the Alfred P. Sloan Foundation.
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