Design, synthesis, and structure - Activity relationship of N-arylnaphthylamine derivatives as amyloid aggregation inhibitors

Article abstract of DOI:10.1021/jm301105m

Dyes like CR are able to inhibit the aggregation of Aβ fibrils. Thus, a screening of a series of dyes including ABBB (1) was performed. Its main component 2 tested in an in vitro assay (i.e., ThT assay) showed good potency at inhibiting fibrils associatio

Full text of DOI:10.1021/jm301105m

Article
pubs.acs.org/jmc
Design, Synthesis, and Structure−Activity Relationship of
N‑Arylnaphthylamine Derivatives as Amyloid Aggregation Inhibitors
Roberto Di Santo,*,† Roberta Costi,† Giuliana Cuzzucoli Crucitti,† Luca Pescatori,† Federica Rosi,†
Luigi Scipione,† Diana Celona,‡ Mario Vertechy,‡ Orlando Ghirardi,‡ Paola Piovesan,‡ Mauro Marzi,‡
Silvio Caccia,§ Giovanna Guiso,§ Fabrizio Giorgi,‡ and Patrizia Minetti*,‡
†Dipartimento di Chimica e Tecnologie del Farmaco, Istituto PasteurFondazione Cenci Bolognetti, “Sapienza” Universita
‡Sigma-Tau S.p.A., Via Pontina, Km 30.400, I-00040 Pomezia, Italy
§Dipartimento di Neuroscienze, Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa, 19, I-20156 Milano, Italy
̀
di Roma,
P.le A. Moro 5, I-00185 Roma, Italy
S
* Supporting Information
ABSTRACT: Dyes like CR are able to inhibit the aggregation
of Aβ fibrils. Thus, a screening of a series of dyes including
ABBB (1) was performed. Its main component 2 tested in an
in vitro assay (i.e., ThT assay) showed good potency at
inhibiting fibrils association. Congeners 4−9 have been
designed and synthesized as inhibitors of Aβ aggregation. A
number of these newly synthesized compounds have been
found to be active in the ThT assay with IC50 of 1−57.4 μM.
The most potent compound of this series, 4k, showed
micromolar activity in this test. Another potent derivative 4q
(IC50 = 5.6 μM) rapidly crossed the blood−brain barrier, achieving whole brain concentrations higher than in plasma. So 4q
could be developed to find novel potent antiaggregating βA agents useful in Alzheimer disease as well as other neurological
diseases characterized by deposits of amyloid aggregates.
The main constituents of Aβ fibrils, Aβ(1−40) and Aβ(1−42)
,
INTRODUCTION
■
normally present as soluble species in extracellular fluids,
twisted around each other, resulting in filamentous structures
with a diameter of 6−10 nm. Amyloid fibrils have been defined
as thermodynamically stable, structurally organized, and highly
insoluble, having affinity for the Congo Red (CR) stain8 and
filamentous protein aggregates being composed of repeating
units of β-sheets aligned perpendicular to the fiber axis, with
potentially parallel and antiparallel organization.9 In AD, the
amyloid fibers accumulate outside the cell in the extracellular
spaces of the brain and in the tunica media of the cortical and
meningeal arterioles, producing three different macroscopic
changes: senile plaques and diffuse plaques which can be
differentiated by the presence or absence of modifications in
the neuronal processes around the central amyloid deposit, and
amyloid angiopathy, which represents an infiltration of amyloid
fibers between the smooth muscle fibers and the internal elastic
lamina of the arterial wall.10 The clinical manifestations of this
progressive and irreversible dementia, in which are implicated
the Aβ(1−42) aggregated fibrils, are the progressive loss of
memory and cognitive functions.11 In general, the damage
caused by Aβ aggregation may be summarized by the following:
(i) changes in amyloidogenesis; (ii) increase in the vulnerability
Alzheimer’s disease (AD) is a degenerative brain disorder and a
major cause of dementia for which no cure is known. Currently,
more than 35 million people worldwide are believed to suffer
from this disease, and with the aging of the population this
number is expected to increase by a factor of 3−4 over the next
40 years.1
The presence of extracellular β-amyloid (Aβ) peptide fibrils
deposits and the presence of intracellular neurofibrillary tangles
(NFT) made by abnormally hyperphosphorylated protein tau
are among the clearest signs of AD. These two events, which
involve mainly the medial temporal lobe structures, in particular
the hippocampus and cortical areas of the brain, are necessary
conditions for the onset of the disease, even if the final
pathological picture of the disease involves the whole central
nervous system.2,3 These microscopic aggregates are still well
beyond the resolution of conventional neuroimaging techni-
ques (positron emission tomography, computerized tomog-
raphy, magnetic resonance imaging) used for the evaluation of
patients with AD. Selective tau imaging for in vivo NFT
quantification is still in the early stages of development.4 Since
Aβ is at the center of AD pathogenesis and given that several
pharmacological agents aimed at reducing Aβ levels in the brain
are being developed and tested, many efforts have focused on
generating radiotracers or agents that allow Aβ imaging in
vivo.5−7
Received: July 27, 2012
© XXXX American Chemical Society
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Figure 1. 1, 2 (main component of a commercial mixture of 1), and 3 (pamoic acid).
of neurons to excitotoxicity; (iii) increase in the vulnerability of
the neurons to hypoglycaemic damage; (iv) changes in the
homeostasis of calcium; (v) increase in damage by oxidation;
(vi) activation of inflammatory mechanisms; (vii) activation of
the microglia; (viii) induction of lysosomial proteases; (ix) changes
in the phosphorylation of the protein tau; (x) induction of
apoptosis; (xi) damage to membranes.12
However, 1 as sold by Aldrich was well-known to be a
mixture of anthraquinone derivatives that necessitated
purification for elucidation of the nature of the various
components. Within the commercial mixture of 1 we isolated
and identified the major component (∼40%) as the 3,4-
dihydroxy-1-[1-(4-sulfophenylamino)]anthracene-9,10-dione
(2) and tested it for its ability to inhibit the Aβ aggregation in
the ThT assay. Compound 2 was an efficient inhibitor showing
IC50 = 1.1 μM. In parallel, during research performed at our
company on negatively charged compounds as inhibitors of Aβ
aggregation, we discovered that pamoic acid (3) (Figure 1)
demonstrated some inhibiting properties with regard to Aβ
aggregation in in vitro assays, showing IC50 = 38.2 μM.
Since the toxicity and neurodegeneration in AD are
attributed to soluble intermediate oligomers and to a lesser
extent to amyloid fibrils,13 agents that prevent or reverse the
oligomerization and/or fibrillization of Aβ may have potential
therapeutic application in the treatment of AD.14 From a purely
theoretical point of view, the reduction of the damage caused
by Aβ can be addressed through different therapeutic
approaches: (i) reducing the production of Aβ using inhibitors
of the secretases to change the metabolism of the APP
(increasing the α or reducing the β and γ secretases); (ii)
preventing or blocking the aggregation of the Aβ; (iii)
increasing the clearance of the Aβ; (iv) blocking the neurotoxic
effects of Aβ restoring calcium homeostasis; (v) preventing the
toxicity due to the free radicals; (vi) preventing excitotoxicity;
(vii) reducing the damage caused by the inflammatory
response; (viii) correcting the imbalance between zinc and
copper; (ix) inhibiting neuronal apoptosis.15−17 Efforts at
searching for inhibitors of Aβ aggregation have demonstrated
that small molecules, containing single or multiple (hetero)-
aromatic rings often bearing hydroxyl group, can inhibit protein−
protein interaction.18 Several molecular dynamics studies suggest
that the driving forces governing the Aβ assembly might include
aromatic packing, hydrophobic forces, and electrostatic inter-
actions.19−21 The hydrophobic interaction between Aβ side chain
leading to Aβ fibrillization could be blocked by small molecules
competing with such an association, ultimately leading to
inhibition of fibril formation.22 These interactions could be
reinforced by hydrogen bond formation between hydroxyl or
methoxy groups on aromatic rings of the inhibitor and hydrogen
bond acceptor groups of Aβ.
To date, no specific therapy exists to prevent, slow down, or
arrest the amyloidogenic processes at the root of AD. Indeed the
treatments currently used for this disease are exclusively
symptomatic, and even if they act on various aspects, they
fundamentally only interfere with the neurotransmitter mecha-
nisms, which govern learning and memory. The substances mostly
used include the reversible inhibitors of acetylcholinesterase, such
as tacrine, donepezil, and rivastigmine.23
Our initial attempts at generating small molecule inhibitors of
Aβ aggregation were based on the knowledge that CR showed
some affinity for amyloid protein fibrils. Thus, we decided to
screen a few dyes as potential inhibitors of the Aβ aggregation,
and among them we found that alizarin blue-black B (ABBB) (1)
(Figure 1) (Sigma-Aldrich mordant black [1324-21-6]) showed
interesting potency in inhibiting the fibrils association in the ThT
assay.24,25
The above data stimulated us to design new compounds
structurally related to 2 and 3 as Aβ aggregation inhibitors. In
particular, N-arylnaphthylamine derivatives 4, in which the
anthraquinone moiety of 2 was replaced by naphthalene ring
typical of compound 3, were designed (Figure 2). This replacement
Figure 2. N-Arylnaphthylamines (4−7) and thio analogues 8 and 9
designed as Aβ aggregation inhibitors (for substituents R and R1, see
Table 1).
was motivated by the fact that 9,10-anthraquinone derivatives, such
as antitumor agent adriamycin, are known to generate in vivo toxic
metabolites that are particularly noxious for heart tissues.26
Some analogues of 4 such as compounds 5−8 were further
designed to better define the structure−activity relationship (SAR)
within this class of inhibitors (Figure 2). As a first step in these
preliminary SAR studies, N and S linkers between the two aromatic
rings were chosen essentially because of easier synthetic feasibility.
The rationale to design N-arylnaphthylamines as inhibitors
of Aβ aggregation has also been strengthened by reports on
N-phenylanthranilic derivatives, represented by compounds 10−12,
which have been recently described as Aβ aggregation inhibitors
(Figure 3).27
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Figure 3. N-Phenylanthranilates reported in literature that inhibit Aβ aggregation.
Thus, in the present study we describe a series of novel
N-arylnaphthylamines (4−7) and thio analogues 8 and 9
(Figure 2 and Table 1) as potential inhibitors of Aβ aggregation
in in vitro assays, using the ThT.24
route to obtain both hydroxy ester and hydroxy acid derivatives
was found by performing the reaction at room temperature
with a short reaction time (∼30 min). In such a way,
deprotection of 5c and 6f gave a mixture of 5a, 5b and 6b, 6c,
respectively, which were separated by chromatography (Schemes 2
and 3). Further, the deprotection of 4t at room temperature for
15 h gave only the salicylic derivative 4l (Scheme 2).
Carboxylic acids 4a,n,s and 6e were obtained by basic
hydrolysis of the corresponding esters 4b,o,t and 6f (Scheme 3).
Finally, the carbinol 4h was obtained starting from the corres-
ponding methyl ester 4j by reduction with LiAlH4 (Scheme 2).
Unfortunately, derivative 4h was unstable and degraded rapidly after
workup of the reaction.
Bis-naphthylamines 7 were obtained through a route
similar to that described for compounds 4 and 6. In particular,
N-arylation was performed starting from 1-bromo-2-methox-
ynaphthalene and 2-methoxy-1-naphthalenamine in the pres-
ence of Pd(OAc)2, BINAP, and Cs2CO3. The bis-naphthyl-
amine 7b that formed was then deprotected using BBr3 to
afford 7a, which proved to be unstable (Scheme 4).
The synthesis of thio derivatives 8 and 9 is reported in
Scheme 5. The arylation step was obtained by palladium
catalyzed reaction of 4-methoxythiophenol, using 1-iodo-4-
methoxynaphthalene as arylating agent in the presence of
tris(dibenzylideneacetone)dipalladium (Pd2dba3) and bis(2-
diphenylphosphinophenyl) ether (DPEphos) as catalysts and
t-BuOK (Scheme 5). This reaction afforded derivative 8b,
which was deprotected in the presence of BBr3 to furnish the
phenol 8a or oxidized to afford sulfone 9b, which was in turn
deprotected to give the phenol 9a.
Evaluation of Biological Activities. In Vitro Assays.
The antiaggregating activity of compounds 4−9 on the peptide
Aβ(1−42) has been determined via the binding of the ThT
according to the following procedure and is reported in more
detail in the Experimental Section. Test compounds were
added in the wells containing both the aggregate and the
nonaggregate Aβ(1−42), incubated at 37 °C under agitation for
24 h. ThT was added to each well, and the fluorescence was
measured. The assay was performed in triplicate in 96-well
plates, and the data obtained were expressed as the dose
reducing the aggregate formation by 50% (IC50) (Table 1).
Among the 36 newly synthesized arylamines 4−9, 29
compounds were tested as reported above. The remaining
compounds were not submitted to the ThT assay, since two
compounds (4h and 7a) were unstable and five derivatives (4m,o,
6c, 8b, 9b) had poor solubility in the assay conditions. Derivatives
4a−d,n,p,r−t, 5b,c, 6e−g, and 9a were found to be inactive when
tested at the highest concentration allowed by their solubility.
Conversely, the remaining 14 compounds showed IC50 ranging
from 1 to 57.4 μM. In particular, nine compounds (4e−g,j,k,q,
6a,b, and 7b) were active at micromolar concentrations (IC50
ranging from 1 to 5.7 μM), with the dihydroxy derivative 4k being
the most potent one within this series, with IC50 = 1 μM being
comparable to the ones of alizarin derivative 2 and antranilates 11
and 12 and 38 times more potent than pamoate 3.
As typical substituents R and R1 (see Table 1), hydroxyls and
carboxylic acids, including salicylic moieties, were chosen to
mimic substituents that characterize compounds 1−3 and 10−12.
Some replacement groups were also attempted to better define
the SAR within this class of inhibitors. Furthermore, since the
blood−brain barrier crossing always represents one of the main
problems for the compounds aimed at acting on the central
nervous system, we also performed in vivo preliminary studies
on mice and assessed the ability of the most interesting inhib-
itor to cross the blood−brain barrier.
RESULTS AND DISCUSSION
■
Chemistry. Analysis. Mixture 1 as furnished by Aldrich
(mordant black 1324-21-6), was analyzed by LC−MS using a
RP18 column and ESI-MS detector. Various peaks were found,
and the main one was quantified to represent around 40% of
the mixture based on UV detection. After separation by
semipreparative HPLC by means of a 1 cm RP18 column, this
component was unequivocally identified to be 4a,9a-dihydro-
3,4-dihydroxy-1-[1-(4-sulfophenylamino)]anthracene-9,10-
dione (2) by NMR and MS analyses.
Synthesis. Syntheses of compounds 4−7 have been made
through N-arylation of a substituted naphthylamine with the
appropriate aryl halide by means of the Buchwald palladium-
catalyzed amination reaction using a mixture of palladium
acetate (Pd(OAc)2) and racemic 2,2′-bis(diphenylphosphino)-
1,1′-binaphthyl (BINAP) as catalysts, in the presence of
Cs2CO3. Thus, N-arylnaphthylamines 4b,m,o−r,t, 5c, and 6d,f,g
have been synthesized in the cited conditions (Scheme 1). The
above-described reaction performed on 4-nitro-1-naphthalenamine
with 4-bromoanisole as electrophile agent gave very poor yield
(<5%) because of the low reactivity of the deactivated
naphthalenamine. Thus, we decided to invert the reactivity of
the reagents using 4-iodo-1-nitronaphthalene as electrophile and
4-methoxyaniline as nucleophile agents, obtaining nitro derivative
4e in good yields (Scheme 1). This approach was also used for
compounds 4c,d,f using the appropriate bromonaphthalene
derivative and the properly substituted aniline (Scheme 1).
Deprotection of methoxy groups of 4m,q, and 6d was
performed with BBr3 at −45 °C to furnish 4g,k and 6a, respectively
(Scheme 2).
The same reaction was used to deprotect methoxy and/or
carboxymethyl groups of 4o,t, 5c, and 6f to furnish 4i,j,l, 5a,b,
and 6b,c, respectively (Schemes 2 and 3).
Partial deprotection was obtained by performing the reaction
at a controlled temperature. In fact, the methoxy group of
compound 4o was selectively deprotected at −45 °C to give the
corresponding phenol 4j. Conversely, when the temperature of
the reaction was increased to room temperature with a longer
reaction time (15 h), the carboxymethyl group was deprotected
as well to obtain 4i (Scheme 3). However, a more convenient
C
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Table 1. Antiaggregating Activity of 4a−t, 5a−c, 6a−g, 7a,b, 8a,b, 9a,b, and Reference Compounds 2, 3, and 10−12 on the
Peptide Aβ
a
compd
R
R1
4-COOH
IC50 (μM)
4n
4o
4p
4q
4r
4s
4t
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
>60
ND
>5
4-COOCH3
2-OCH3
4-OCH3
2-OCH3-5-I
3-COOH-4-OCH3
3-COOCH3-4-OCH3
COOH
COOCH3
COOCH3
H
5.6
>5
>80
ND
14.1
>20
>5
5a
5b
5c
6a
6b
6c
6d
6e
6f
OH
OCH3
OH
2.4
OH
COOH
COOCH3
H
2.6
OH
ND
33.6
>20
>20
>5
OCH3
OCH3
OCH3
OCH3
OH
a
compd
R
R1
4-COOH
IC50 (μM)
COOH
COOCH3
I
4a
4b
4c
4d
4e
4f
H
H
F
>100
>20
>30
>5
6g
7a
7b
8a
8b
9a
9b
2
4-COOCH3
4-F
OH
ND
5.7
OCH3
OH
OCH3
F
4-SCH3
4-OCH3
4-SCH3
H
OH
10.9
ND
>100
ND
1.1
NO2
N(CH3)2
OH
2.7
OCH3
OH
OCH3
2.2
OH
4g
4h
4i
5.4
OCH3
OCH3
OH
4-CH2OH
4-COOH
4-COOCH3
4-OH
ND
57.4
5.1
OH
3
38.2
0.1
4j
OH
10
11
12
4k
4l
OH
1
1
OH
3-COOH-4-OH
H
46.1
ND
2
4m
OCH3
a
Inhibitory concentration, 50% (μM), determined from dose−response curves. Experiments were performed in triplicate.
Removal of the phenolic OH group of 4k or its replacement
compounds 5 gave results similar to those obtained for series
4, giving inactive derivatives 5b,c.
with COOCH3 led to arylamines 4g and 4j, which were about
5-fold less potent than the parent compound (4g, IC50 = 5.4
μM; 4j, IC50 = 5.1 μM). Conversely, its replacement with a
COOH (4i) or transformation in a salycilic derivative (4l) gave
a more substantial drop in activity (4i, IC50 = 57.4 μM; 4l, IC50 =
46.1 μM).
Replacement of both hydroxyls of 4k with OCH3 groups led
to naphthylarylamine 4q, which was about 5-fold less potent
than the parent compound (IC50 = 5.6 μM). However,
generally the methylation of OH gave inactive compounds or
compounds with low activity (compare 4g,i−l with 4m−t).
Compounds with no hydroxyl groups such as 4a−d were
inactive. In particular, replacement of the OH of 4k with a fluorine
atom led to fluoroderivative 4c that showed IC50 > 30 μM.
The methylthiofluoro derivative 4d was inactive as well, while
the corresponding compound 4f, in which the fluorine atom
was replaced by a dimethylamino group, showed very good
potency (IC50 = 2.2 μM).
Although the presence of OH groups seems to be relevant
for activity, it is not vital because its replacement with groups
such as COOH, COOCH3, OCH3, NO2, N(CH3)2, SCH3 led
to compounds active in the micromolar range (compare 4k
with 4e−g,j,q, and 6a,b).
Shift of substituents of 4i from the 4- to 2-position of the
aromatic rings led to 5a, which was 4-fold more potent than 4i
(4i, IC50 = 57.4 μM; 5a, IC50 = 14.1 μM). However,
introduction of less polar groups within the series of
Compound 6a, the isomer of 4g in which the 4-hydro-
xyphenylamino moiety is transposed from the 4- to 2-position of
the naphthalene ring, was about 2 times more potent than the
parent compound (4g, IC50 = 5.4 μM; 6a, IC50 = 2.4 μM). An
even stronger effect was obtained for the corresponding acid
derivatives 4i and 6b. In fact, compound 6b showed IC50 = 2.6
μM and was 22 times more active than 4i (IC50 = 57.4 μM).
Interestingly, introduction of a carboxylic group in the 4-position
of the phenyl ring of 6a gave 6b, which was equipotent to the
parent compound (6a, IC50 = 2.4 μM; 6b, IC50 = 2.6 μM).
Conversely, a similar modification in the 4 series gave 4i that was
about 10-fold less potent than 4g (4i, IC50 = 57.4 μM; 4g, IC50 =
5.4 μM). As confirmation of a general rule, phenol derivatives of
the 6 series were more potent if compared to the less polar
counterparts (see 6a,b and 6d−g).
Replacement of the phenyl ring of 4q with a naphthalene
moiety gave the equipotent bis-naphthalenylamino derivative
7b (4q, IC50 = 5.6 μM; 7b, IC50 = 5.7 μM). No comparison was
possible between the corresponding dihydroxyderivatives 4k
and 7a, since the bis-naphtholamine derivative 7a was unstable.
Isosteric substitution of the linker of 4k with replacement of
NH with a sulfur atom gave compound 8a, which was 11 times
less active than the corresponding amine (4k, IC50 = 1 μM; 8a,
IC50 = 10.9 μM). The oxidation of the linker of 8a led to the
inactive sulphone 9a. Finally, both the dimethoxy derivatives 8b
and 9b were too scarcely soluble to be tested.
D
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a
Figure 4 shows the plasma and brain concentration−time
curves of compound 4q after subcutaneous dosing in mice.
Subcutaneous absorption was relatively rapid as indicated by
the tmax, which was 60 min after a 25 mg/kg dose (dissolved in
ethanol, PEG-400, saline, 10:40:50 v/v/v). The Cmax was 164
ng/mL (n = 3) and AUCt was 850 ng/(mL·h). Plasma
concentrations of 4q fell slowly thereafter, about 80% of the
plasma Cmax remaining after 6 h. However, in these preliminary
studies, plasma sampling was not sufficiently long for a proper
determination of half-life and of other conventional pharma-
cokinetic parameters.
Scheme 1
From the plasma concentration−time curve, the compound
achieved brain Cmax at 60 min. However, brain Cmax (410
ng/mL; n = 3) was 2.5 times that in plasma, indicating
concentration of this N-arylnaphthylamine derivative in brain
tissue.
After the peak, mean brain concentrations remained
consistently 1.7- to 2.5-fold the plasma concentrations within
6 h of dosing, yielding a mean brain-to-plasma AUCt ratio of
about 2. This led us to conclude that 4q, one of the most
potent derivatives of the present series, rapidly entered the
mouse central nervous system and equilibrated between plasma
and brain.
a
Reagents and conditions: (i) Pd(OAc)2, ( )-BINAP, Cs2CO3, 80 °C.
For substituents R and R1, see Table 1.
a
Scheme 2
Compound 4q was well tolerated, since no discomfort or
side effects were observed.
CONCLUSIONS
■
On the basis of the knowledge that a few dyes like CR were
able to inhibit the aggregation of Aβ fibrils, we perfomed a
screening of a series of dyes. Mixture 1 was found to be active
and its main component, anthraquinone derivative 2 which was
isolated and unequivocally identified, was tested for its
antiaggregating properties in in vitro assay. Interestingly,
compound 2 showed good potency in inhibiting the fibrils
association in the ThT assay (IC50 = 1 μM). So a series of
N-arylnaphthylamine derivatives (4−7) and thio analogues 8 and
9 were designed as Aβ aggregation inhibitors. A number of the
newly synthesized compounds have been found to be active
with IC50 ranging from to 1 to 57.4 μM. The most potent
compounds showed IC50 in the low micromolar range,
comparable to that of hit compound 2. Furthermore, these
novel N-arylnaphthylamine derivatives are potentially less toxic
than the anthraquinone derivative 2 and one of the more
interesting derivatives (4q) rapidly crossed the blood−brain
barrier and diffused within the central nervous system,
achieving concentrations about twice those in plasma, looking
at the whole brain exposure. This brain-to-plasma ratio was
similar to that of donezepil (1.74−2.24), slightly lower than
that of rivastagmine and tacrine in rats (about 3 and 5,
respectively),28−30 but it remains to be clarified whether such
observations reflect the true extent of brain distribution, since
the plasma protein binding of 4q was not evaluated in this
study. So 4q represents a very interesting compound that could
be developed with the aim to find new potent antiaggregating
Aβ agents useful for the treatment of Alzheimer disease as well
as other neurological diseases characterized by deposits of
amyloid aggregates.
a
Reagents and conditions: (i) BBr3, CH2Cl2, −45 °C; (ii) BBr3,
CH2Cl2, rt, 15 h; (iii) BBr3, CH2Cl2, rt, 30 min; (iv) LiAlH4, THF, rt.
For substituents R1, see Table 1.
Brain-to-Plasma Distribution Studies. The aim of these
animal model studies was to evaluate the distribution between
brain and plasma of N-arylnaphthylamines. Compound 4q was
selected as a representative derivative of the series because of a
good balance between activity and lipophilicity as well as
acceptable chemical stability. However, 4k, which was the most
potent compound within this series and proved to be 5−6
times more potent than 4q in in vitro assays, showed oxidative
degradation.
EXPERIMENTAL SECTION
■
Chemistry. General. Melting points were determined on a Bibby
Stuart Scientific SMP1 melting point apparatus and are uncorrected.
The purity of the compounds was always >95% as determined by high
pressure liquid chromatography (HPLC). HPLC analyses were carried
E
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a
Scheme 3
a
Reagents and conditions: (i) BBr3, CH2Cl2, −45 °C, 1 h; (ii) BBr3, CH2Cl2, rt, 15 h; (iii) NaOH, THF/MeOH 1:1, reflux; (iv) BBr3, CH2Cl2, rt,
35 min.
a
Scheme 4
a
Reagents and conditions: (i) Pd(OAc)2, ( )-BINAP, Cs2CO3, 80 °C; (ii) BBr3, CH2Cl2, −45 °C.
out with a pump/autosampler Waters (Alliance model 2695), a UV
photodiode array detector Waters (model 2996), and a system data
management Waters (Empower 2). The column used was generally
Suplex pkb-100 (250 mm × 4.6 mm, 5 μm). Infrared (IR) spectra
(Nujol mulls) were recorded on a Perkin-Elmer Spectrum-One
spectrophotometer. 1H NMR spectra were recorded at 400 MHz on a
Bruker AC 400 Ultrashield 10 spectrophotometer (400 MHz) and on
Varian Gemini 200 and Varian XL 300 spectrometers (200 and 300
MHz, respectively). The LC−MS analyses have been performed with
instrumentation equipped with HPLC−MS Thermo-Finnigan Sur-
veyor LC pump, PDA detector, and single quadrupole MSQ detector.
Dimethylsulfoxide-d6, 99.9% (code 44,139-2), and deuterochloroform,
98.8% (code 41,675-4), of isotopic purity (Aldrich) were used as NMR
solvents. Column chromatographies were performed on silica gel
(Merck, 70−230 mesh). All compounds were routinely checked by
TLC by using aluminum-baked silica gel plates (Fluka DC-Alufolien
Kieselgel 60 F254). Plates were visualized by UV light. Solvents were
reagent grade and, when necessary, purified and dried by standard
methods. Concentration of solutions after reactions and extractions
involved the use of rotary evaporator (Buchi) operating at a reduced
̈
pressure (∼20 Torr). Organic solutions were dried over anhydrous
sodium sulfate (Merck).
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a
Scheme 5
a
Reagents and conditions: (i) Pd2dba3, DPEphos, t-BuOK, 100 °C; (ii) BBr3, THF, −45 °C, 2 h; (iii) Oxone, water, rt, 16 h; (iv) BBr3, THF,
20 h, rt.
flask was then purged with argon for 30 s. After the mixture was stirred
at room temperature for 1 min, a solution of substituted naphthyl-
amine (3.5 mmol) and appropriate aryl halide in toluene (1.5 mL) was
added. Then Cs2CO3 (4.0 mmol) was introduced within the mixture
which was then diluted with toluene (7 mL) and then purged with
argon again. The mixture was heated to 80 °C under stirring and was
cooled to room temperature before being diluted with ether, filtered,
and concentrated. The crude product obtained was purified by column
chromatography. Reagents, reaction time, eluent of chromatography,
1
yield (%), melting point (°C), recrystallization solvent, IR, H NMR,
and analytical data are reported for each of the following compounds.
Methyl 4-(1-Naphthylamino)benzoate (4b). 1-Naphthalenamine
and methyl 4-bromobenzoate; 16 h; chloroform/petroleum ether 3:1;
1
96%; 130−132 °C; toluene. IR ν 3340 (NH), 1694 (CO) cm−1. H
NMR (DMSO-d6) δ 3.79 (s, 3H, CH3), 6.95 (m, 2H, benzene C3−H
and C5−H), 7.45−7.60 (m, 4H, naphthalene H), 7.73 (m, 1H,
Figure 4. Mean plasma and brain concentration−time curves of
naphthalene H), 7.79 (m, 2H, benzene C2−H and C6−H), 7.98 (m,
compound 4q after 25 mg/kg subcutaneous injection in mice. Each
1H, naphthalene H), 8.06 (m, 1H, naphthalene H), 8.88 (s broad, 1H,
NH). Anal. (C18H15NO2) C, H, N, O.
value is the mean
SD of three determinations for plasma (plain
circles) and brain (empty circles).
4-Methoxy-N-phenyl-1-naphthalenamine (4m). 4-Methoxy-1-
naphthalenamine31 and bromobenzene; 21 h; ethyl acetate/n-hexane
1:1; 70%; 141−143 °C; cyclohexane/n-hexane. IR ν 3400 (NH) cm−1.
1H NMR (CDCl3) δ 4.07 (s, 3H, CH3), 5.75 (s broad, 1H, NH),
6.75−6.90 (m, 4H, naphthalene H and benzene C2−H and C6−H),
7.15−7.25 (m, 3H, benzene C3−H, C4−H, and C5−H), 7.50−7.60
(m, 2H, naphthalene H), 8.04 (m, 1H, naphthalene H), 8.33 (m, 1H,
naphthalene H). Anal. (C17H15NO) C, H, N.
Methyl 4-[(4-Methoxynaphthalen-1-yl)amino]benzoate (4o).
4-Methoxy-1-naphthalenamine31 and methyl 4-bromobenzoate; 24 h;
ethyl acetate/n-hexane 1:1; 77%; 168−170 °C; benzene. IR ν 3300
(NH) cm−1. 1H NMR (CDCl3) δ 3.89 (s, 3H, COOCH3), 4.08 (s, 3H,
OCH3), 6.03 (s broad, 1H, NH), 6.45−6.71 (m, 2H, benzene C3−H
and C5−H), 6.85 (d, Jo = 8.1 Hz, naphthalene H), 7.39 (d, Jo = 8.1 Hz,
naphthalene H), 7.48−7.61 (m, 2H, naphthalene H), 7.85−7.96 (m,
3H, naphthalene H and benzene C2−H and C6−H), 8.35−8.41 (m,
1H, naphthalene H). Anal. (C19H17NO3) C, H, N, O.
N-(4-Methoxy-1-naphthyl)-N-(2-methoxyphenyl)amine (4p). 4-
Methoxy-1-naphthalenamine31 and 2-methoxy-1-bromobenzene; 39 h;
ethyl acetate/n-hexane 1:2; 70%; 108−110 °C; cyclohexane. IR ν 3395
(NH) cm−1. 1H NMR (CDCl3) δ 3.98 and 4.02 (2s, 6H, CH3), 6.60 and
6.91 (2 m, 2H, benzene C3−H and C6−H), 6.73 (m, 2H, benzene C4−
H and C5−H), 6.80 (d, 1H, Jo = 8.1 Hz, naphthalene C2−H), 7.35 (d,
1H, Jo = 8.1 Hz, naphthalene C3−H), 7.48 (m, 2H, naphthalene C6−H
and C7−H), 7.98 and 8.03 (2 m, 2H, naphthalene C5−H and C8−H).
Anal. (C18H17NO2) C, H, N.
Analyses. 4a,9a-Dihydro-3,4-dihydroxy-1-[1-(4-sulfophenylamino)]-
anthracene-9,10-dione (2) isolated by mixture 1 (Aldrich, mordant black
1324-21-6) was analyzed by LC−MS. The major component showed
negative ion ESI-MS: calcd for C20H12NO7S [M − 1] 410.4, found
409.6.
A chromatographic separation was done on a semipreparative
column Waters Xterra C18, 10 mm × 100 mm, 5 μm, under reversed
phase conditions with a mobile phase consisting of A (AcONH4 10 mM/
CH3CN 80/20), B (AcONH4 10 mM/CH3CN 60/40) under gradient
conditions from 100% to 0% of A in 20 min, delivered at a flow rate of
1.00 mL/min, with ultraviolet detection at 280 nm. The column
temperature was set at 25 °C, and the main component of the mixture
1
(C20H12NO7S [M − 1] 410.4) was isolated, 2. H NMR (DMSO-d6) δ
6.58 (s, 1H, anthracene C2−H), 7.31 (d, 2H, Jo = 10.8 Hz, benzene H),
7.63 (d, 2H, Jo = 10.8 Hz, benzene H), 7.89 (t, 2H, Jo = 9.6 Hz,
anthracene C6−H and anthracene C7−H), 8.25 (dd, 2H, Jo = 9.6 Hz,
anthracene C5−H and anthracene C8−H), 9.19 (s, 1H, NH). Anal.
(C20H13NO7S) C, H, N, S.
Syntheses. General Procedure for the Synthesis of N-Arylna-
phthylamines 4b,m,o−r,t, 5c, 6d,f,g, and 7b. A dried flask was
purged with argon, charged with ( )-BINAP (0.11 mmol), and
capped with rubber septum. The flask was purged with argon again,
and toluene (9.7 mL) was added. The mixture was heated to 80 °C
under stirring until BINAP dissolved (1 min). The solution was cooled
to room temperature, and Pd(OAc)2 (0.07 mmol) was added. The
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4-Methoxy-N-(4-methoxyphenyl)-1-naphthalenamine (4q). 4-
Methoxy-1-naphthalenamine31 and 4-methoxy-1-bromobenzene; 21 h;
ethyl acetate/n-hexane 1:1; 96%; oil. IR ν 3380 (NH) cm−1. 1H NMR
(CDCl3) δ 3.81 and 4.04 (2s, 6H, CH3), 5.90 (s broad, 1H, NH),
6.74−6.85 (m, 6H, naphthalene C2−H and C3−H and benzene H),
7.51−7.58 (m, 2H, naphthalene H), 8.04 (m, 1H, naphthalene H),
8.35 (m, 1H, naphthalene H). Anal. (C18H17NO2) C, H, N.
mixture was heated to 80 °C under stirring until the BINAP was
dissolved (1 min). The solution was cooled to room temperature, and
Pd(OAc)2 (0.07 mmol) was added. The flask was then purged with
argon for 30 s. After stirring of the mixture at room temperature for
1 min, a solution of 4-halo-1-substituted naphthalene (3.5 mmol) with
appropriate 4-substituted aniline (2.9 mmol) in toluene (1.5 mL) was
added. Cs2CO3 (4.0 mmol) was introduced within the mixture.
Toluene (7 mL) was added and then the flask purged with argon
again. The mixture was heated to 80 °C under stirring, then was
cooled to room temperature, diluted with ether, filtered, and
concentrated. The crude product that was obtained was purified by
column chromatography to obtain the desired adduct. Reagents,
reaction time, eluent of chromatography, yield (%), melting point
N-(5-Iodo-2-methoxyphenyl)-N-(4-methoxy)-1-naphthylamine
(4r). 4-Methoxy-1-naphthalenamine31 and 2,4-diiodoanisole; 48 h;
flash chromatography ethyl acetate/n-hexane 1:20; 18%; oil. IR ν 3390
1
(NH) cm−1. H NMR (CDCl3) δ 3.95 and 4.03 (2s, 6H, CH3), 6.61
(d, 1H, Jo = 8.1 Hz, benzene C3−H), 6.78 (d, 1H, Jm = 2.0 Hz,
benzene C6−H), 6.82 (d, 1H, Jo = 8.1 Hz, naphthalene C3−H), 7.01
(dd, 1H, Jo = 8.3 Hz, Jm = 2.0 Hz benzene C4−H), 7.34 (d, 1H, Jo =
8.1 Hz, naphthalene C2−H), 7.50 (m, 2H, naphthalene C6−H and
C7−H), 7.92 and 8.31 (2 m, 2H, naphthalene C5−H and C8−H).
Anal. (C18H16NO2I) C, H, N, I.
1
(°C), recrystallization solvent, IR, H NMR, and analytical data are
reported for each of the following compounds.
4-Fluoro-N-(4-fluorophenyl)naphthalen-1-amine (4c). 4-Fluoro-
aniline and 1-bromo-4-nitronaphthalene; 5 h and 45 min; chloroform;
91%; 62−64 °C; n-hexane. IR ν 3395 (NH) cm−1. 1H NMR (CDCl3)
δ 5.55 (s broad, 1H, NH), 6.89−6.90 (m, 2H, benzene H), 6.98−7.03
(m, 2H, benzene H), 7.11−7.17 (m, 1H, naphthalene C2−H), 7.24
(m, 1H, naphthalene C3−H), 7.57−7.66 (m, 2H, naphthalene C6−H
and C7−H), 8.05 and 8.20 (2 m, 2H, naphthalene C5−H and C8−H).
Anal. (C16H11NF2) C, H, N, F.
Methyl 2-(Methoxymethyl)-5-[(4-methoxynaphthalen-1-yl)-
amino]benzoate (4t). 4-Methoxy-1-naphthalenamine31 (9.1 mmol)
and methyl 2-methoxy-5-bromobenzoate using ( ) BINAP (0.76 mmol),
Pd(OAc)2 (0.51 mmol); 120 °C; 24 h; ethyl acetate/n-hexane 1:1; 97%;
1
oil. IR ν 3320 (NH), 1695 (CO) cm−1. H NMR (CDCl3) δ 3.88 (2s,
3H, CH3), 3.89 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 5.60 (s broad, 1H,
NH), 6.79 (d, 1H, J = 8.2 Hz, naphthalene H), 6.88 (m, 2H, benzene
C5−H and C6−H), 7.22 (d, 1H, J = 8.2 Hz, naphthalene H), 7.34 (m,
1H, benzene C2−H), 7.50−7.58 (m, 2H, naphthalene H), 7.99 and 8.34
(2 m, 2H, naphthalene H).
4-Fluoro-N-[4-(methylthio)phenyl]naphthalen-1-amine (4d).
1-(Methylthio)aniline and 1-bromo-4-fluoronaphthalene; 17 h; chloro-
form/petroleum ether 1:1; 94%; 71−72 °C; n-hexane. IR ν 3337
(NH) cm−1. 1H NMR (DMSO-d6) δ 2.44 (s, 3H, SCH3), 6.93 (d, 2H,
Jo = 8.7 Hz, benzene C3−H and C5−H), 7.20−7.33 (d, 2H, Jo = 8.7
Hz, benzene C2−H and C3−H), 7.63−7.71 (m, 2H, naphthalene
C6−H and C7−H), 8.07 and 8.18 (2 m, 2H, naphthalene C5−H and
C8−H), 8.21 (s broad, 1H, NH). Anal. (C17H14NFS) C, H, N, F, S.
N-(4-Methoxyphenyl)-4-nitronaphthalen-1-amine (4e). 4-Iodo-1-
nitronaphthalene34 and 4-methoxyaniline; chloroform; 15 h; 38%; oil.
Methyl 2-[(2-Methoxy-1-naphthyl)amino]benzoate (5c). 2-Me-
thoxy-1-naphthalenamine32 and methyl-2-bromobenzoate; 15.5 h;
ethyl acetate/n-hexane 1:5; 100%; 144−146 °C. IR ν 3321 (NH),
1
1681 (CO) cm−1. H NMR (DMSO-d6) δ 3.86 (s, 3H, CH3), 3.89 (s,
3H, CH3), 6.09 (m, 1H, benzene H), 6.66 (m, 1H, benzene H), 7.18
(m, 1H, naphthalene H), 7.35−7.45 (m, 2H, benzene H and
naphthalene H), 7.57 (m, 1H, naphthalene H), 7.68 (m, 1H, benzene
H), 7.88−7.95 (m, 3H, naphthalene H), 9.17 (s broad, 1H, NH). Anal.
(C19H17NO3) C, H, N.
1
IR ν 3365 (NH) cm−1. H NMR (DMSO-d6) δ 3.93 (s, 3H, CH3),
6.85 (s broad, 1H, NH), 6.80 (m, 1H, naphthalene H), 7.05 (d, 2H,
Jo = 8.8 Hz, benzene C3−H and C5−H), 7.30 (d, 2H, Jo = 8.8 Hz,
benzene C2−H and C6−H), 7.67−7.81 (2 m, 2H, naphthalene C6−H
and C7−H), 8.08, 8.39, and 9.07 (3 m, 3H, naphthalene C2−H, C5−
H, and C8−H). Anal. (C17H14N2O3) C, H, N.
(1-Methoxy-2-naphthyl)phenylamine (6d). 1-Methoxy-2-naphtha-
lenamine33 and bromobenzene; 16 h; ethyl acetate/n-hexane 1:1; 83%;
1
43−45 °C; n-hexane. IR ν 3395 (NH) cm−1. H NMR (DMSO-d6) δ
3.80 (s, 3H, CH3), 6.85 (m, 1H, benzene H), 7.07−7.65 (m, 8H,
naphthalene H and benzene H), 7.84 (m, 1H, naphthalene H), 7.93 (s
broad, 1H, NH), 7.99 (m, 1H, naphthalene H). Anal. (C17H15NO) C,
H, N.
N,N-Dimethyl-N′-[4-(methylthio)phenyl]naphthalene-1,4-dia-
mine (4f). 4-(Methylthio)aniline and 1-bromo-4-(dimethylamino)-
1
naphthalene; 16 h; chloroform; 81%; oil. IR ν 3381 (NH) cm−1. H
NMR (DMSO-d6) δ 2.42 (s, 3H, SCH3), 2.84 (s, 6H, NCH3), 6.86 (d,
2H, Jo = 8.8 Hz, benzene C2−H and C6−H), 7.14−7.20 (m, 3H, Jo =
8.8 Hz, naphthalene H and benzene C3−H and C5−H), 7.56 (m, 2H,
naphthalene C2−H and C3−H), 8.07−8.09 (m, 2H, NH and
naphthalene H), 8.23 (m, 1H, naphthalene H). Anal. (C19H20NS)
C, H, N, S.
Methyl 4-(1-Methoxy-2-naphthylamino)benzoate (6f). 1-Me-
thoxy-2-naphthalenamine33 and methyl 4-bromobenzoate; 4 h and
10 min; chloroform/ethyl acetate 9:1; 97%; 153−154 °C; ligroin. IR ν
1
3327 (NH), 1691 (CO) cm−1. H NMR (CDCl3) δ 3.95 (s, 3H,
CH3), 7.14 (d, 2H, Jo = 8.8 Hz, benzene C2−H and C6−H), 7.44−
7.48 (m, 2H, naphthalene H), 7.55−7.59 (m, 1H, naphthalene H),
7.64−7.69 (m, 2H, naphthalene H), 8.03 (d, 2H, Jo = 8.8 Hz, benzene
C3−H and C5−H), 8.10 (m, 1H, naphthalene H). Anal. (C19H17NO3)
C, H, N.
N-(4-Iodophenyl)-1-methoxynaphthalen-2-amine (6g). 1-Me-
thoxy-2-naphthalenamine33 and 1,4-diiodobenzene; 19 h; 37%; 83−
84 °C; n-hexane. IR ν 3327 (NH) cm−1. 1H NMR (CDCl3) δ 3.96 (s,
3H, CH3), 6.97 (d, 2H, Jo = 8.8 Hz, benzene C2−H and C6−H),
7.40−7.43 (m, 1H, naphthalene H), 7.53−7.57 (d, 2H, Jo = 8.8 Hz,
benzene C3−H and C5−H), 7.61−7.74 (m, 3H, naphthalene H),
7.83−7.85 (m, 1H, naphthalene H), 8.06−8.08 (m, 1H, naphthalene
H). Anal. (C19H14NIO) C, H, N, I.
2-Methoxy-N-(2-methoxy-1-naphthyl)naphthalen-1-amine (7b).
2-Methoxy-1-naphthalenamine32 and 2-methoxy-1-bromonaphthalene;
ethyl acetate/n-hexane 1:5; 59%; oil. IR ν 3380 (NH) cm−1. 1H NMR
(DMSO-d6) δ 3.55 (s, 6H, CH3), 7.05 (s broad, 1H, NH), 7.20−7.38
(m, 6H, naphthalene H), 7.58 (m, 2H, naphthalene H), 7.81−7.93 (m,
4H, naphthalene H). Anal. (C22H20NO2) C, H, N.
General Procedure for the Synthesis of N-Arylnaphthyl-
amines 4c−f. A dried flask was purged with argon, charged with
( )-BINAP (0.11 mmol), and capped with rubber septum. The flask
was purged with argon again, and toluene (9.7 mL) was added. The
1-Methoxy-4-[(4-methoxyphenyl)thio]naphthalene (8b). A dried
flask was charged with Pd2dba3 (130 mg, 0.141 mmol), dissolved in
degassed toluene (115 mL), treated with DPEphos (150 mg, 0.28
mmol), and purged with argon. The mixture was stirred at room
temperature for 3 min. Then 1-methoxy-4-iodonaphthalene (4.0 g,
14.1 mmol) and 4-methoxythiophenol (2.0 g, 14.1 mmol) were added
under argon atmosphere. t-BuOK (1.7 g, 15.5 mmol) was added and
the flask purged with argon. The mixture was stirred for 2 h at 100 °C,
cooled to room temperature, and filtered on a Celite cake, and the
filtrate was concentrated in vacuo. The crude product (6.2 g) was
purified by column chromatography (n-hexane/acetone 10:1) to
obtain the desired adduct as a slightly colored (3.6 g) solid, which was
purified by recrystallization (n-hexane) to obtain 2.7 g of pure 8b
(65%): mp 83−85 °C. IR ν 2937 (CH) cm−1. 1H NMR (acetone-d6) δ
3.78 (s, 3H, CH3), 4.10 (s, 3H, CH3), 6.88 (d, 2H, Jo = 8.86 Hz,
benzene H), 7.03 (d, 1H, Jo = 8.01 Hz, naphthalene C2−H), 7.20 (d,
2H, Jo = 8.86 Hz, benzene H), 7.57−7.64 (m, 2H, naphthalene C6−H
and C7−H), 7.75 (d, 1H, Jo = 8.01 Hz, naphthalene C3−H), 8.34−
8.40 (m, 2H, naphthalene C5−H and C8−H). Anal. (C18H16O2S) C,
H, N, S.
General Procedure for the Synthesis of Acids 4a,n,s and 6e.
A solution of the appropriate methyl ester (1.5 mmol) and 1 N NaOH
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(3.7 mL) in THF/ethanol 1:1 (20 mL) was refluxed for 3 h for 4a, 3 h
and 30 min for 4n,t, and 1 h and 40 min for 6e, while stirring. Then
the mixture was poured into crushed ice and extracted with ethyl
acetate (30 mL). The aqueous layer was treated with 1 N HCl until
pH 3 and then extracted with ethyl acetate (3 × 50 mL). The organic
phases were collected, washed with brine (3 × 100 mL), dried, and the
solvent was removed under reduced pressure to yield the final product.
Reagents, reaction time, eluent of chromatography, yield (%), melting
8.45 (s broad, 1H, NH), 10.20 (s broad, 1H, OH), 12.15 (s broad, 1H,
COOH). Anal. (C17H13NO3) C, H, N.
Methyl 4-(4-Hydroxy-1-naphthylamino)benzoate (4j). 4o; −45 °C;
1 h; ethyl acetate/n-hexane 1:2; 58%; 188−190 °C; benzene/n-hexane.
IR ν 3377 (OH, NH), 1680 (CO) cm−1. 1H NMR (DMSO-d6) δ 3.73
(s, 3H, CH3), 6.60−6.67 (m, 2H, benzene C3−H and C5−H), 6.86−
6.88 (d, 1H, J = 8.0 Hz, naphthalene H), 7.21−7.23 (d, 1H, J = 8.0 Hz,
naphthalene H), 7.44−7.48 (m, 2H, naphthalene H), 7.65−7.67 (m,
2H, benzene C2−H and C6−H), 7.82 (m, 1H, naphthalene H), 8.17
(m, 1H, naphthalene H), 8.50 (s broad, 1H, NH), 10.18 (s broad, 1H,
OH). Anal. (C18H15NO3) C, H, N.
4-(4-Hydroxy-phenylamine)naphthalen-1-ol (4k). The solvent
was purged with argon. 4q; −45 °C; 15 h; ethyl acetate/n-hexane 1:1;
80%; 74 °C (dec). IR ν 3375 (OH, NH) cm−1. 1H NMR (DMSO-d6) δ
6.55−7.00 (m, 7H, naphthalene H, benzene H and NH), 7.38−7.47 (m,
2H, naphthalene H), 7.95−8.13 (m, 2H, naphthalene H), 8.67 and 9.67
(2s broad, 2H, OH). Anal. (C16H13NO2) C, H, N.
1
point (°C), recrystallization solvent, IR, H NMR, and analytical data
are reported for each of the following compounds
4-(1-Naphthylamino)benzoic Acid (4a). 4b; 3 h; 59%; 227−229 °C;
1
toluene. IR ν 3390 (NH), 2900 (OH), 1670 (CO) cm−1. H NMR
(DMSO-d6) δ 6.95 (m, 2H, benzene C3−H and C5−H), 7.46−7.60
(m, 4H, naphthalene H), 7.72 (m, 1H, naphthalene H), 7.78 (m, 2H,
benzene C2−H and C6−H), 7.96 (m, 1H, naphthalene H), 8.07 (m,
1H, naphthalene H), 8.81 (s broad, 1H, NH), 12.29 (s broad, 1H,
OH). Anal. (C17H13NO2) C, H, N.
4-(4-Methoxy-1-naphthylamino)benzoic Acid (4n). 4o; 3.5 h;
50%; 153−154 °C; isopropanol. IR ν 3400 (NH), 3000 (OH), 1650
2-Hydroxy-5-(4-hydroxy-1-naphthylamino)benzoic Acid (4l). 4t;
rt; 15 h; ethyl acetate; 17%; 175 °C (dec). IR ν 3350 (OH, NH), 3000
(COOH), 1635 (CO) cm−1. 1H NMR (DMSO-d6) δ 6.73 (d, 1H, Jo =
8.7 Hz, benzene C3−H), 6.82 (d, 1H, J = 8.0 Hz, naphthalene H),
6.97 (dd, 1H, Jo = 8.7 Hz, Jm = 2.7 Hz, benzene C4−H), 7.05 (d, 1H,
J = 8.0 Hz, naphthalene H), 7.20 (d, 1H, Jm = 2.7 Hz, benzene C6−
H), 7.44−7.49 (m, 2H, naphthalene C6−H and C7−H), 7.99 (m, 1H,
naphthalene H), 8.15 (m, 1H, naphthalene H), 9.85 (3s broad, 3H,
OH, COOH and NH). Anal. (C17H13NO4) C, H, N.
1
(CO) cm−1. H NMR (DMSO-d6) δ 4.01 (s, 3H, CH3), 6.70 (d, 2H,
Jo = 8.5 Hz, benzene C3−H and C5−H), 7.01 (d, 1H, J = 8.0 Hz,
naphthalene C3−H), 7.39 (d, 1H, J = 8.0 Hz, naphthalene C2−H),
7.55 (m, 2H, naphthalene C6−H and C7−H), 7.71 (d, 2H, Jo = 8.5
Hz, benzene C2−H and C6−H), 7.90 (m, 1H, naphthalene H), 8.20
(m, 1H, naphthalene H), 8.54 (s broad, 1H, NH). Anal. (C18H15NO3)
C, H, N.
2-Methoxy-5-(4-methoxy-1-naphthylamino)benzoic Acid (4s).
4t; 3.5 h; 50%; 153−154 °C; isopropanol. IR ν 3300 (NH), 3160
(OH), 1690 (CO) cm−1. 1H NMR (DMSO-d6) δ 3.76 (s, 3H, OCH3),
3.97 (s, 3H, OCH3), 6.91−6.98 (m, 3H, naphthalene H and benzene
C3−H and C4−H), 7.16 (m, 1H, benzene C6−H), 7.21 (d, 1H,
naphthalene H), 7.53 (m, 2H, naphthalene H), 7.76 (s broad, 1H,
NH), 8.05 and 8.20 (2 m, 2H, naphthalene H), 12.50 (s broad, 1H,
OH). Anal. (C19H17NO4) C, H, N.
2-(2-Hydroxy-1-naphthylamino)benzoic Acid (5a) and Methyl
2-(2-Hydroxy-1-naphthylamino)benzoate (5b). 5c; rt; 23 min; ethyl
acetate/n-hexane 1:2. 5a: 55%; 215−216 °C; toluene. IR ν 3361 (OH
and COOH), 3325 (NH), 1659 (CO) cm−1. 1H NMR (DMSO-d6) δ
6.09 (m, 1H, benzene H), 6.62 (m, 1H, benzene H), 7.14 (m, 1H,
naphthalene H), 7.28−7.31 (m, 2H, naphthalene H), 7.39 (m, 1H
benzene H), 7.62 (m, 1H, benzene H), 7.75 (m, 1H, naphthalene H),
7.83−7.89 (m, 2H, naphthalene H), 9.28 (s broad, 1H, NH), 12.86 (s
broad, 1H, COOH). Anal. (C17H13NO3) C, H, N. 5b: 45%; 157−158 °C;
cyclohexane. IR ν 3407 (OH and COOH), 3319 (NH), 1681
(CO) cm−1. 1H NMR (DMSO-d6) δ 3.88 (s, 3H, CH3), 6.10 (m, 1H,
benzene H), 6.64 (m, 1H, benzene H), 7.17 (m, 1H, naphthalene H),
7.28−7.31 (m, 2H, naphthalene H), 7.39 (m, 1H benzene H), 7.62
(m, 1H, benzene H), 7.77 (m, 1H, naphthalene H), 7.84−7.95 (m,
2H, naphthalene H), 9.05 (s broad, 1H, NH), 9.78 (s broad, 1H, OH).
Anal. (C18H15NO3) C, H, N.
4-(1-Methoxy-2-naphthylamino)benzoic Acid (6e). 6f; 1 h and 40
min; 100%; 233−235 °C; ethanol. IR ν 3403 (OH and NH), 1691
1
(CO) cm−1. H NMR (DMSO-d6) δ 3.80 (s, 3H, CH3), 7.01 (d, 2H,
Jo = 8.6 Hz, benzene C2−H and C6−H), 7.46−7.59 (m, 3H,
naphthalene H), 7.72 (m, 1H, naphthalene H), 7.82 (d, 2H, Jo = 8.6
Hz, benzene C3−H and C5−H), 7.93 (m, 1H, naphthalene H), 8.08
(m, 1H, naphthalene H), 8.59 (s broad, 1H, NH), 12.32 (s broad, 1H,
COOH). Anal. (C18H15NO3) C, H, N.
1-Hydroxy-N-phenylnaphthalen-2-amine (6a). 6d; −45 °C; 30
General Procedure for the Synthesis of Compounds 4g,i−l,
5a,b, 6a−c, 7a, 8a, and 9a. A solution of the proper N-phenyl-1-
naphthalenamine derivative 4m,o,q,t, 5c, 6d,f, 7b, 8b, or 9b (2.4
mmol) in dichloromethane (27 mL) was added dropwise to 1 M BBr3
(12.6 mL, 12.6 mmol) in the same solvent at −45 °C under argon
atmosphere while stirring. After being diluted with water (50 mL), the
mixture was extracted with ethyl acetate (3 × 50 mL) and the organic
phases were collected, washed with brine (3 × 100 mL), and dried.
Evaporation of the solvent gave a crude product, which was
chromatographed to afford pure product(s). Substrate, reaction
temperature, reaction time, eluent of chromatography, yield (%),
melting point (°C), recrystallization solvent, IR, 1H NMR, and
analytical data are reported for each of the following compounds.
4-Phenylamino-naphthalen-1-ol (4g). 4m; −45 °C under argon
atmosphere; 15 h; ethyl acetate/n-hexane 1:3; 88%; oil. IR ν 3350
min; ethyl acetate/n-hexane 1:3; 88%; >300 °C. IR ν 3150 (NH and
1
OH) cm−1. H NMR (DMSO-d6) δ 6.70 (m, 1H, benzene H), 6.81−
6.88 (m, 2H, benzene H), 7.07−7.16 (m, 2H, naphthalene H), 7.31−
7.42 (m, 4H, benzene H and naphthalene H), 7.77 (m, 1H,
naphthalene H), 8.11 (m, 1H, naphthalene H). Anal. (C16H13NO)
C, H, N.
4-(1-Hydroxy-2-naphthylamino)benzoic Acid (6b) and Methyl
4-(1-Hydroxy-2-naphthylamino)benzoate (6c). 6f; rt; 35 min; ethyl
acetate/n-hexane 1:1. 6b: 45%; 210 °C (dec); methanol. IR ν 3426
(OH and COOH), 3353 (NH), 1654 (CO) cm−1. 1H NMR (DMSO-
d6) δ 6.78−6.80 (m, 2H, benzene C2−H and C6−H), 7.33−7.36 (m,
1H, naphthalene H), 7.42−7.50 (m, 3H, naphthalene H), 7.74−7.77
(m, 2H, benzene C3−H and C5−H), 7.84−7.86 (m, 1H, naphthalene
H), 8.18 (s broad, 1H, NH), 8.19−8.21 (m, 1H, naphthalene H), 9.40
(s broad, 1H, OH), 12.20 (s broad, 1H, COOH). Anal. (C17H13NO3)
C, H, N. 6c: acetone/n-hexane 1:4; 55%;175−176 °C; toluene. IR: ν
3334 (OH and NH) cm−1. 1H NMR (DMSO-d6) δ 3.79 (s, 3H, CH3),
7.28−7.31 (m, 2H, naphthalene H), 7.35 (m, 1H, naphthalene H),
7.39 (m, 2H, Jo = 8.8 Hz, benzene C2−H and C6−H), 7.45−7.51 (m,
3H, naphthalene H), 7.77 (m, 2H, Jo = 8.8 Hz, benzene C3−H and
C5−H), 8.25 (s broad, 1H, NH), 9.40 (s broad, 1H OH). Anal.
(C18H15NO3) C, H, N.
1
(OH, NH) cm−1. H NMR (CDCl3) δ 5.30 and 5.65 (2s,broad, 2H,
OH and NH), 6.75−6.87 (m, 4H, naphthalene H and benzene C2−H
and C6−H), 7.15−7.30 (m, 3H, benzene C3−H, C4−H, and C5−H),
7.50−7.57 (m, 2H, naphthalene H), 8.50 (m, 1H, naphthalene H),
8.25 (m, 1H, naphthalene H). Anal. (C16H13NO) C, H, N.
4-(4-Hydroxy-1-naphthylamino)benzoic Acid (4i). 4o; rt; 15 h;
ethyl acetate/n-hexane 9:2; 45%; 214−217 °C; toluene. IR ν 3360
1
(OH, NH), 2800 (COOH) cm−1. H NMR (DMSO-d6) δ 6.63 (d,
2H, Jo = 8.7 Hz, benzene C3−H and C5−H), 6.92 (d, 1H, J = 8.0 Hz,
naphthalene H), 7.27 (d, 1H, J = 8.0 Hz, naphthalene H), 7.50 (m,
2H, naphthalene H), 7.69 (d, 2H, Jo = 8.7 Hz, benzene C2−H and
C6−H), 7.85 (m, 1H, naphthalene H), 8.20 (m, 1H, naphthalene H),
2-Hydroxy-N-(2-hydroxy-1-naphthyl)naphthalen-1-amine (7b).
7a; −45 °C, rt; 15 h, ethyl acetate/n-hexane 1:2. After isolation the
compound 7a rapidly degraded so that no analytical and structural data
are available.
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4-[(4-Hydroxyphenyl)thio]-1-naphthol (8a). 8b; −45 °C, 15.5 h,
then rt; 7 h; ethyl acetate/n-hexane 2:5; 61%; 161−163 °C; toluene.
IR ν 3255 (OH) cm−1. 1H NMR (DMSO-d6) δ 6.70 (d, 2H, Jo = 8.69
Hz, benzene H), 6.93 (d, 1H, naphthalene C2−H), 7.06 (d, 2H, Jo =
8.69 Hz, benzene H), 7.51−7.62 (m, 3H, naphthalene C3−H, C6−H,
and C7−H), 8.23 and 8.27 (2 m, 2H, naphthalene C5−H and C8−H),
9.52 and 10.61 (2s broad, 2H, OH). Anal. (C16H12O2S) C, H, S.
4-[(4-Hydroxyphenyl)sulfonyl]-1-naphthol (9a). 9b; −45 °C, 20
min, then rt; 20 h; ethyl acetate/chloroform 1:2; 58%; 203−205 °C;
inferior to 10%. The assays were performed in triplicate in 96-well
plates. The compounds to be tested were added in wells containing the
aggregate Aβ(1−42). Then after 15 min, the nonaggregate Aβ(1−42) was
added. The 96-well plates were incubated at 37 °C under agitation for
24 h. The following day 200 μL of a solution containing 10 μM ThT
and 50 μM Na2HPO4, pH 6.5, was added to each well. Fluorescence
was measured in a VICTOR 2 (WALLAC) fluorescence spectropho-
tometer (λex = 450 nm, λem = 486 nm).33
The data are expressed as percent of residual aggregated Aβ, and the
dose reducing the aggregate formation of the 50% (IC50) was estimated.
The % of aggregation was determined by the following formula:
1
toluene. IR ν 3300 (OH) cm−1. H NMR (DMSO-d6) δ 7.90 (d, 2H,
Jo = 8.77 Hz, benzene H), 7.07 (d, 1H, Jo = 8.29 Hz, naphthalene C2−
H), 7.58 and 7.67 (2 m, 2H, naphthalene C6−H and C7−H), 7.77 (d,
2H, Jo = 8.77 Hz, benzene H), 8.28−8.31 (m, 2H, naphthalene C5−H
and C8−H), 8.48 (d, 1H, Jo = 8.29 Hz, naphthalene C3−H), 10.54
and 11.51 (2s broad, 2H, OH). Anal. (C16H12O4S) C, H, S.
[(Aβ + test compound) − (blank + test compound)]
× 100/[(control + Aβ) − blank]
1-Methoxy-4-[(4-methoxyphenyl)sulfonyl]naphthalene (9b). 8b
(1 g, 3.4 mmol) was dissolved in methanol (84 mL). At 0 °C a
solution of Oxone (6.3 g, 10.2 mmol) in water (20 mL) was added.
The mixture was stirred for 16 h at room temperature. The mixture
was poured into water, extracted with ethyl acetate (3 × 100 mL), and
the collected organic layers were washed with brine (3 × 100 mL) and
dried over anhydrous Na2SO4. The solvent was evaporated in vacuum,
obtaining a crude product which was purified by column
chromatography (chloroform as eluent) affording the pure product
9b (82%): mp 165−167 °C; toluene. IR ν 2900 (CH) cm−1. 1H NMR
(DMSO-d6) δ 3.82 (s, 3H, CH3), 4.12 (s, 3H, CH3), 7.11 (d, 2H, Jo =
8.69 Hz, benzene H), 7.25 (d, 1H, Jo = 8.47 Hz, naphthalene C2−H),
7.64 and 7.72 (2 m, 2H, naphthalene C6−H and C7−H), 7.90 (d, 2H,
Jo = 8.69 Hz, benzene H), 8.30 (d, 1H, Jo = 8.47 Hz, naphthalene C3−
H), 8.46−8.52 (m, 2H, naphthalene C5−H and C8−H). Anal.
(C18H16O4S) C, H, S.
4-(4-Hydroxymethyl)phenylamino)naphthalen-1-ol (4h). A sol-
ution of 4j (350 mg, 1.2 mmol) in anhydrous THF (40 mL) was
added dropwise into a 0 °C suspension of LiAlH4 (230 mg, 6.0 mmol)
in the same solvent (40 mL). After the addition, the mixture was
stirred at room temperature for 15 h. The excess of LiAlH4 was
carefully destroyed with crushed ice, and the precipitate that formed
was filtered on a Buchner apparatus. The solution was concentrated,
extracted with ethyl acetate (3 × 50 mL). The combined organic layers
were washed with brine (3 × 100 mL) and dried over anhydrous
Na2SO4. The solvent was evaporated under vacuum, obtaining 320 mg
of crude product. Because it was unstable, it was immediately
solubilized in 10 mL of methanol and added dropwise to a solution of
HCl in methanol cooled at 0 °C, prepared by adding dropwise acetyl
chloride (2.2 mmol) in methanol (10 mL). The mixture was stirred at
0 °C for 5 min after which anhydrous diethyl ether (100 mL) was
added and the resulting precipitate was filtered to obtain 220 mg
(33%) of hydrochloride 4h, with mp > 300 °C.
Blood−Brain Barrier Crossing. Ten mice were given subcuta-
neously compound 4q dissolved in 10% ethanol, 40% PEG-400, and
50% saline and were killed by decapitation under deep anesthesia in
order to obtain information on the concentration achieved in brain
and its relationship with plasma concentrations. Mixed arteriovenous
trunk blood was collected at 0, 15, 30, 60, 120, 180, and 240 min after
dosing and centrifuged at 3000g for 10 min, and the plasma was stored
at −20 °C. Brain was removed immediately, blotted with paper to
remove excess surface blood, and quickly frozen in dry ice.
Compound 4q was extracted from plasma, and brain homogenate
was obtained by a solid−liquid extraction procedure and quantified by
reverse phase HPLC with UV detection (224 nm). To 0.3 mL of plasma
were added 0.3 mL of CH3CN/0.01 N phosphate buffer, pH 7.4 (70:30,
v/v), and 0.1 mL of a solution of the internal standard. After
centrifugation, the supernatants were added to Oasis HLB 1 cc cartridges
which were prewetted with methanol and distilled water. Cartridges were
washed with methanol−water (15:85 v/v) and then methanol only,
interrupting the vacuum before completely drying the column. The com-
pound was removed by eluting the cartridges with methanol (2.5 mL),
and the collected fractions evaporated to dryness under nitrogen. The
residue was dissolved in the mobile phase, centrifuged, and analyzed by
HPLC. Brain tissue was homogenized (1 g/5 mL) in CH3CN/0.001 M
phosphate buffer, pH 7.4 (70:30, v/v), and a volume containing
approximately 100 mg of tissue was centrifuged and processed as for
plasma. Separation was done on a μBondapak C18 column protected by a
LiChrosphere RP8 precolumn at room temperature. The mobile phase
was H2O/CH3CN/CH3OH/n-butanol (43:42:14:1, v/v), containing
65 mM ammonium acetate. The retention times were 10.5 min for 4q
and 20.5 for the internal standard.
Mean brain and plasma area under the concentration−time curve
over the dosing interval (AUCt) were determined using the linear
trapezoidal rule. The maximum concentration (Cmax) and the time (tmax
)
were read directly from the plasma and brain concentration time data.
Biological Assays. In Vitro Assays. Compounds 4a−t, 5a−c,
6a−g, 7a,b, 8a,b, and 9a,b (Table 1) were tested for their ability to
inhibit aggregation of Aβ(1−42), via the binding of the ThT.
Preparation of the Nonaggregate Aβ(1−42). The Aβ(1−42) was
dissolved in a mixture of acetonitrile and distilled water (CH3CN/H2O
1:1) to a final concentration of 1 mg/mL. The solution was divided in
aliquots of 2 mL and stored at −80 °C until use. The working solution
was prepared, diluting the stock solution with H2O to get a final
concentration of 44 μM.
Preparation of the Aggregate Aβ(1−42). The Aβ(1−42) was
dissolved in a mixture of acetonitrile and distilled water (CH3CN/H2O
1:1) to the final concentration of 1 mg/mL. An aliquot of 2 mL was
freeze-dried to eliminate the trifluoroacetic acid resulting from the
peptide synthesis. The Aβ(1−42) peptide was subsequently dissolved in
0.1 mL of DMSO and 5.0 mL of 2× PBS, pH 7.4. Once dissolved, the
Aβ(1−42) was incubated at 37 °C for 8 days. After sonication, it was
diluted with 2× PBS to get a final concentration 17.4 μM. The
aggregate Aβ(1−42) was divided in 20 aliquots and stored at −80 °C.
Screening of Inhibitory Activity of Aβ Aggregation (ThT).
The drugs to be tested as fibrillogenesis inhibitor were dissolved in a
minimum amount of DMSO and then diluted with water to the final
concentrations of 10 or 100 μM, with a total percentage of DMSO
ASSOCIATED CONTENT
* Supporting Information
Results from elemental analyses of compounds 2, 4a−g,i−s,
5a−c, 6a−g, 7b, 8a,b, and 9a,b. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For R.D.S.: phone, +39-06-49913150; fax, +39-06-49913133;
e-mail, roberto.disanto@uniroma1.it. For P.M.: phone, +39-06-
91393906; fax, +39-06-91393638; e-mail, Patrizia.Minetti@
sigma-tau.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Dr. Gilles Pain for his help in reviewing the
manuscript.
■
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molecule interfere with Abeta-peptide amyloid fibrillation? Protein Sci.
2009, 18, 792−800.
ABBREVIATIONS USED
■
CR, Congo Red; IL, interleukin; ABBB, alizarin blue-black B;
ThT, thioflavin T; BINAP, 2,2′-bis(diphenylphosphino)-1,1′-
binaphthyl; Pd2dba3, tris(dibenzylideneacetone)dipalladium;
DPEphos, bis(2-diphenylphosphinophenyl) ether
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