Bioactive prenylated phenyl derivatives derived from marine natural products: Novel scaffolds for the design of BACE inhibitors

Article abstract of DOI:10.1039/c3md00236e

Abnormal accumulation of neurotoxic beta-amyloid peptides (Aβ) is a key factor in the development of Alzheimer's disease (AD) and strategies to reduce Aβ production constitute an active field of research for the development of novel therapeutic agents for the treatment of AD. In particular, β-secretase-1 (BACE-1) has been a prime target for modulating Aβ production although obtaining drug-like BACE-1 inhibitors has proven to be highly challenging. Here we report the isolation and biochemical characterization of a marine natural product, the prenylated hydroxybenzoic acid 1, with BACE-1 inhibitory activity and ability to decrease Aβ production in cell-based assays. Synthesis and biological activity of a number of new synthetic analogues are also reported, as well as initial structure-activity relationship (SAR) analysis on this chemical family. Hence, these compounds constitute novel scaffolds from which more potent and selective BACE-1 inhibitors could be designed as potential therapeutic agents for the treatment of Alzheimer's disease.

Full text of DOI:10.1039/c3md00236e

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CONCISE ARTICLE
Bioactive prenylated phenyl derivatives derived
from marine natural products: novel scaffolds for
the design of BACE inhibitors†
Cite this: Med. Chem. Commun., 2014,
5, 474
Javier Lopez-Ogalla,a Esther Garcıa-Palomero,a Jorge Sanchez-Quesada,a
´
´
´
ac
Laura Rubio,a Elena Delgado,a Pablo Garcıa,a Miguel Medina,ab Ana Castro
*
´
a
*
and Pilar Munoz
˜
Abnormal accumulation of neurotoxic beta-amyloid peptides (Ab) is a key factor in the development of
Alzheimer's disease (AD) and strategies to reduce Ab production constitute an active field of research for
the development of novel therapeutic agents for the treatment of AD. In particular, b-secretase-1
(BACE-1) has been a prime target for modulating Ab production although obtaining drug-like BACE-1
inhibitors has proven to be highly challenging. Here we report the isolation and biochemical
characterization of a marine natural product, the prenylated hydroxybenzoic acid 1, with BACE-1
inhibitory activity and ability to decrease Ab production in cell-based assays. Synthesis and biological
activity of a number of new synthetic analogues are also reported, as well as initial structure–activity
relationship (SAR) analysis on this chemical family. Hence, these compounds constitute novel scaffolds
from which more potent and selective BACE-1 inhibitors could be designed as potential therapeutic
agents for the treatment of Alzheimer's disease.
Received 22nd August 2013
Accepted 2nd January 2014
DOI: 10.1039/c3md00236e
www.rsc.org/medchemcomm
plaques is involved in neuronal loss in brain regions (hippo-
campus and cortex) responsible for memory and cognitive
Introduction
functions. Clearly, strategies to reduce Ab production will
facilitate development of therapeutic agents for the treatment
of AD.5
Alzheimer's disease (AD) is a neurodegenerative disorder of the
central nervous system and the leading cause of dementia in the
elderly population. Its clinical symptoms include not only
impairment of memory, cognition, temporal and local orienta-
tion, judgment and reasoning but also severe emotional
disturbances. There are currently no treatments available which
can prevent or reverse the disease or its progression. Thus, AD
has become a major problem in all societies with high life
expectancies and also a signicant economic burden for their
health systems.
Although the cause of AD remains unknown, a large body of
evidence has accumulated suggesting that abnormal accumu-
lation of neurotoxic Ab peptides is a key factor in the develop-
ment of AD, and thought to be the likely cause of memory and
cognitive loss in this disease condition.1–4 Neuropathological
examination of brains from AD patients reveals that the accu-
mulation of secreted Ab peptides in extracellular amyloid
According to the amyloid hypothesis, the generation of Ab
peptides is a critical event in AD, and inhibiting this process
may affect disease progression. Ab peptides are generated in
neuronal secretory vesicles by proteolytic cleavage of the type I
transmembrane amyloid precursor protein (APP) by two aspar-
tyl-proteases, called b-secretase and g-secretase, that sequen-
tially cleave at the N terminus and variant C-termini of Ab
within APP, respectively, resulting in Ab of 40 or 42 amino acids
(Ab40 and Ab42, respectively).5 Both peptides are believed to
initiate aggregation and later deposition in the neuritic plaques
characteristic of AD.6
The importance of the therapeutic potential of inhibiting Ab
deposition has motivated many researchers to isolate and
characterize secretase enzymes and to identify potential inhib-
itors. BACE is a transmembrane aspartyl protease of type I
topology with b-secretase activity, in which the N-terminus and
catalytic site reside on the luminal or extracellular side of the
membrane. In fact, BACE is the major b-secretase responsible
for Ab generation in the brain. BACE catalyses the initial step in
the amyloidogenic metabolism of the large transmembrane
APP, releasing a soluble APPb (sAPPb) ectodomain by specic
hydrolysis at the b-site (Met671-Asp672) and simultaneously
generating a membrane-bound C-terminal fragment consisting
aNOSCIRA S.A., Avenida de la Industria 52, 28760 Tres Cantos, Madrid, Spain. E-mail:
acastro@iqm.csic.es; p_munozruiz@yahoo.com; Fax: +34 91 803 4660; Tel: +34 91
8061130
b
´
´
Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas
(CIBERNED), Nicolas Cabrera 1, 28049 – Madrid, Spain
c
´
´
Instituto de Quımica Medica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00236e
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of 99 amino acids (CTF99). The latter is then further processed
by the g-secretase enzyme complex which, in turn, generates the
APP intracellular domain and releases the 39–42-amino-acid
Ab-peptide. The identication of the Ab peptide as the main
constituent of the extracellular plaques which characterize AD
led to the formulation of the amyloid cascade hypothesis of AD.
Interruption of this metabolic cascade at one of several sites
could potentially reduce the amyloid burden, and slow or even
reverse the devastating consequences of the disease. b-Secretase
is particularly attractive in this context as it catalyses the rst
and rate-limiting step in the pathway. Moreover, the subtype
BACE-1 is the major b-secretase responsible for Ab generation in
the brain. Thus, b-secretase inhibition aimed at blocking what
is believed to be excessive activity or altered control of BACE-1 in
AD is a key target for therapeutic intervention.7,8
Therefore, BACE-1 inhibitors have been actively sought by
different drug discovery approaches as potential anti-AD drug
candidates.9 The rst generation of BACE-1 inhibitors included
peptide and peptidomimetic compounds acting as transition-
state analogs able to tightly interact with the BACE-1 aspartic
acids of the catalytic dyad. Unfortunately, as expected, despite
their nanomolar affinity for the enzyme, peptides and pepti-
domimetics do not have an optimal pharmacokinetic prole.10
Recently, based on the large amount of structural information,
Fig. 1 Chemical structure of the marine prototype 1 and its cell-based
assay activity.
structures as attractive scaffolds for the design of new
analogues. Consequently, we also report here on the synthesis
and biological activity of a number of new synthetic analogues
by incorporating different structural modications to initially
explore the importance of the substituents of the phenyl ring in
the inhibitory activity. Structure–activity relationship studies
were also conducted along this work and will be discussed
as well.
Results and discussion
Chemistry
different drug discovery approaches have been employed, and The total synthesis of the marine prototype 1 (Fig. 1) is per-
several non-peptidic BACE-1 inhibitors also with nanomolar formed in a similar manner to that previously reported by Lang
activity have been identied,11 including carbinamine,12 mac- et al.23 (Scheme 1), aiming to complete its biological charac-
rocycles,13 aminoimidazol14 or hydroxyethylamine moieties.15 terization and to synthesize derived compounds for further
Due to the involvement of b-amyloid in Alzheimer's disease and investigation of the inuence of both the carboxylic acid and
the need to develop therapeutic agents that effectively reduce hydroxyl groups present in the molecule in the inhibitory
b-amyloid deposition, increasing effort has been made in this activity.
area of research leading to the development of compounds that
have reached phase II clinical trials.16–18
The key step of the synthesis consisted of the coupling
between the phenyl derivative 4 and the required geranylgeranyl
Natural products have historically played a critical role in the bromide 6. The preparation of the appropriate phenyl derivative
discovery and development of new drugs. Despite the fact that 4 is successfully achieved by protection of the aldehyde group of
the marine environment remains largely unexploited, the the benzaldehyde 2 through formation of its corresponding
recognition that marine organisms are a rich source of potential acetal 3 followed by protection of the hydroxyl group with
drug candidates in recent years has sparkled the intensive methoxymethyl chloride to afford the diprotected derivative 4.
exploration of new substances from marine organisms.19 Thus, On the other hand, the synthesis of the geranylgeranyl bromide
marine organisms represent a diverse source of biologically 6 is quantitatively performed, employing the reported meth-
active compounds as drug leads and inspiration for the odology (Scheme 1).24
synthesis of non-natural molecules with improved drug-like
Therefore, the coupling between the phenyl derivative 4 and
properties. As part of our continuous screening program geranylgeranyl bromide 6 is performed employing n-butyl-
focused on the identication of novel marine origin compounds lithium and copper bromide through formation of its lithium
with therapeutic potential for neurodegenerative diseases, dicuprate derivative, followed by acid mediated MOM-depro-
bioassay guided-fractionation of active extracts from the sponge tection to obtain the aldehyde 8. Subsequent deprotection of the
Sarcotragus sp. (Ircinidae family) led to the isolation of bioactive aldehyde group with CSA (+/ꢀ)-camphor-10-sulfonic acid, fol-
prenylated phenyl derivatives as Ab peptide inhibitors.
lowed by oxidation of the aldehyde resulting group with sodium
In particular, by using an ELISA cell-based assay we have hydrogen phosphate and sodium chlorite, afforded the nal
identied the prenylated hydroxybenzoic acid 1 (Fig. 1), previ- synthetic prototype 1, with moderate yields. The chemical
ously isolated from other marine sponges,20–22 as an inhibitor of structure of the synthetic prototype 1 is assigned from detailed
Ab(1ꢀ40) peptide production in the micromolar range (10 ꢁ NMR spectroscopy data and HPLC-MS spectrometry data as well
7.5% (1 mM); 47 ꢁ 19% (10 mM)). Further studies on their as by comparison with data from the literature,21 conrming
mechanism of action showed that this compound also inhibited that of the marine prototype.
BACE-1 enzymatic activity (% BACE-1 activity: 45 ꢁ 21% at 1 mM).
Regarding the synthesis of compounds derived from preny-
In view of the biological activity of this family of marine- lated hydroxybenzoic acid 1 the prototype 1 is transformed into
derived compounds we considered these prenylated phenyl the corresponding benzyl esters 12 and 13, by treatment with
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Scheme 1 Synthesis of compounds 1 and 11. Reagents and conditions: (a) ethylene glycol, pTsOH, toluene; (b) MOMCl; (c) (1) n-BuLi, CuBr-DMS;
(2) HCl; (d) CSA, MeOH; (e) NaClO2, NaH2PO4, THF/H2O.
benzyl bromide in the presence of a base, and the hydroxyl relative disposition of the functional groups in the prototype 1,
group is acetylated with acetic anhydride in pyridine to afford is based on the alkylation of different anilines with farnesyl
the acetylated derivative 14 (Scheme 2).
bromide (5). Thus, alkylation of commercially available anilines
Preparation of synthetic analogues commenced by short- 15a–c, in the presence of K2CO3 in DMF or THF (Scheme 3),
ening the prenylated chain present in this molecule in order to resulted in the corresponding monoalkylated analogues 16a–c,
evaluate its importance in the inhibitory activity and also aim- in moderate yield. Subsequent hydrolysis of the methyl ester
ing to decrease lipophylicity. This new analogue 11 is prepared groups in compounds 16a,b afforded the corresponding
by coupling the aryl bromide 4 with the corresponding farnesyl carboxylic acid analogues 17a,b in quantitative yields. The same
bromide 5 following the same methodology as that for the methodology is employed for the preparation of the pyridine
preparation of the prototype 1 (Scheme 1).
analogue 19 from the protected nicotinic acid 18.
As part of the medicinal chemistry strategy, new structural
The replacement of the methoxy group by other alkoxy
modications were explored such as incorporating an amino groups is conducted employing 3-amino-4-hydroxybenzoic acid
group as linkage between the aryl and the prenylated moieties (20) as the starting material as shown in Scheme 4. The
to see its inuence on potency. Furthermore, these analogues carboxylic acid group is protected prior to alkylation, employing
were more readily synthesized than those containing a C–C H2SO4 in MeOH, to give the methyl ester derivative 21, in
linkage. In general, the preparation of these new analogues, quantitative yield. Alkylation of 21 with farnesyl bromide (5) in
which maintained either two or three substituents in the same THF, in the absence of a base, produced selectively the alkylated
Scheme 2 Synthesis of compounds 12–14. Reagents and conditions: (a) BrBn, K2CO3, DMF, (b) Ac2O, pyridine, 0 ꢂC.
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aminophenol derivative 22 in 30% yield, the alkylation of the
aromatic hydroxyl group not being detected. Further hydrolysis
of its methyl ester group generated the carboxylic acid 23, in
quantitative yields. On the other hand, alkylation of the ami-
nophenol 22 with 1-bromopropyl, employing K2CO3 as base in
DMF, afforded the corresponding O-alkylated compound 24
together with the simultaneously O- and N-alkylated compound
26. Similarly, alkylation of the aminophenol 22 with ethyl bro-
moacetate employing NaH in THF, in the presence of a phase
transfer catalyst, produced a mixture of the simultaneously N-
and O-dialkylated analogue 27 together with the monoalkylated
intramolecular-cyclized analogue 28 (Scheme 4) in 1 : 7 ratio.
Employing the same conditions, alkylation of the aminophenol
22 with 1-propanosulfonyl chloride afforded mainly the
O-monoalkylated compound 25.
Subsequent hydrolysis of compound 25 and the cyclized
compound 30, under common basic conditions, yielded the
corresponding carboxylic acids 29 and 30.
Preparation of analogues, incorporating the ethanolamine
moiety by replacing a prenyl unit in the tether chain, is per-
formed employing the epoxide fragment 31 as depicted in
Scheme 5. The ethanolamine intermediate 33 is synthesized, in
moderate yield, by addition of the 3-amino-4-methoxybenzoic
acid methyl ester (15a) to the aminoepoxide 31, in i-PrOH at
high temperature, followed by quantitative deprotection of the
Boc-amino group with HCl gas at 0 ꢂC. Subsequent alkylation of
the aminoalcohol 33 with geranyl bromide, employing EtB3BN
Scheme 3 Synthesis of compounds 16a–c, 17a–b and 19. Reagents
and conditions: (a) trans,trans-farnesyl bromide, K2CO3, THF; (b)
LiOH$H2O, H2O/MeOH (3 : 2).
ꢂ
in DMF at 50 C, generated the analogue 35, in low yields.
Scheme 4 Synthesis of compounds 22–30. Reagents and conditions: (a) MeOH, H2SO4, 70 ꢂC; (b) trans,trans-farnesyl bromide (5), THF, 30%; (c)
LiOH$H2O, H2O/MeOH (3 : 2); (d) 1-bromopropyl, K2CO3, DMF; (e) 1-propanosulfonyl chloride, NaH, THF, 10 min, 0 ꢂC then INBu4 (0.25 eq.),
18-crown-6 (f) ethyl bromoacetate, NaH, THF, 10 min, 0 ꢂC then INBu4 (0.25 eq.), 18-crown-6; (g) LiOH$H2O, THF/H2O or THF/MeOH/H2O.
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Scheme 5 Synthesis of ethanolamine 35. Reagents and conditions: (a)
tert-butyl (1S)-1-[(2S)-2-oxiranyl]-2-phenylethylcarbamate, iPrOH,
50 ꢂC; (b) HCl (g), EtOAc/MeOH (4 : 1), 0 ꢂC; (c) geranyl bromide (34),
Et3N, DMF/THF (5 : 2), 70 ꢂC.
The analogue 38, incorporating the ethanolamino moiety as
an amide into the carboxylic acid group, is obtained in 47%
yield by coupling the ethanolamine 37 and the carboxylic acid
17a with HOBt, EDC and DIPEA in DMF (Scheme 6). The
required ethanolamine fragment 37 is synthesized following the
methodology reported by Stachel et al.25 This methodology
consisted of an epoxide ringꢂopening of the aminoepoxide 31
with cyclopropylamine at 50 C, followed by Boc-amino depro-
tection employing TFA in DCM.
Scheme 6 Synthesis of ethanolamine 38. Reagents and conditions: (a)
tert-butyl
(1S)-1-[(2S)-2-oxiranyl]-2-phenylethylcarbamate
(31),
i
cyclopropylamine, PrOH, 50 ꢂC, 7 h; (b) TFA, DCM; (c) 17a, 37, DMF,
DIPEA, HOBt and EDC.
tether chain a new analogue is synthesized shortening the
prenyl chain to 3 prenyl units. However, this new analogue 11
Biological activity
All compounds synthesized were assayed in both the BACE-1 results in lower potency than the natural prototype 1.
inhibitory assay and the Ab peptide cell-based assay (Table 1). The introduction of an amino group as a linkage between the
Due to intrinsic uorescence emission of some synthesized phenyl and the three prenyl unit tether led to new analogues. In
analogues several enzymatic assays were attempted. Biological an initial investigation of the inuence of the substituents of
evaluation of the synthetic natural product 1 is shown to be the phenyl ring in the activity, we focused our attention on
comparable with the natural prototype 1, in both assays. The methylated analogues, readily obtained in the synthetic route. It
analogue program is initiated preparing new semi-synthetic is evidenced that while simultaneous methylation at the
compounds by direct derivatization of the two functional carboxylic acid and the hydroxyl groups in compound 16a
groups present in the phenyl ring of the synthetic prototype 1, resulted in a decrease of inhibitory activity in comparison to
the hydroxyl and the carboxylic acid groups. Among the that of the marine prototype 1, the analogue 17a methylated
compounds synthesized 12, 13 and 14, only the acetyl derivative only at the hydroxyl group, exhibited equipotent BACE-1
14 maintained the same inhibition pattern of the prototype 1, in inhibitory activity and Ab production inhibitory activity
both assays. Considering the high lipophylicity of the prenyl compared to the natural prototype 1. These results suggested
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Table 1 Inhibitory activities of natural and synthetic prototype 1 as well as synthesized compoundse
BACE-1 activity (%)
bA production inhib. (%)
Compound
1 mM
10 mM
1 mM
10 mM
Natural 1
Synthetic 1
45 ꢁ 21
42 ꢁ 1
0
0
10 ꢁ 7
8 ꢁ 6
47 ꢁ 19
30 ꢁ 9
11
100
5 ꢁ 9
NA
NA
14c
87 ꢁ 14
17 ꢁ 1
24 ꢁ 13
16aa
16c
100
68 ꢁ 11
64 ꢁ 9
0
0
29 ꢁ 13
94 ꢁ 8
50 ꢁ 9
NA
0
17aa,c
31 ꢁ 4
17bd
19
89 ꢁ 9
100
25 ꢁ 6
70 ꢁ 2
36 ꢁ 1
27 ꢁ 9
NA
NA
0
0
0
0
23
100
24a
88 ꢁ 4
27a
100
0
14 ꢁ 4
36 ꢁ 5
28a
29
59 ꢁ 7
93 ꢁ 2
100
22 ꢁ 11
3 ꢁ 2
0
29 ꢁ 6
36 ꢁ 4
0
14 ꢁ 2
30
17 ꢁ 9
0
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Table 1 (Contd.)
BACE-1 activity (%)
bA production inhib. (%)
Compound
1 mM
10 mM
56 ꢁ 3
1 mM
10 mM
35b,c
100
3 ꢁ 4
38b
63 ꢁ 6
34 ꢁ 11
10 ꢁ 7
28 ꢁ 1
a
Autouorescence employing the FRET assay. Data obtained with kit assay. b Autouorescence employing both the FRET and the kit assay. Data
obtained with TRF assay. c Toxic in the bA production inhibition assay at 10 mM concentration (*). d Toxic in the bA production inhibition assay (*).
(*) Toxicity assays were independently conducted with human neuroblastoma cell line SH-SY5Y proving the safety of these compounds in this range
of concentration (1.0–10 mM), indicating that the toxicity observed with the CHO-7W cell line is more related to its particular sensitivity. e Reference
compound OM99-2: BACE-1 activity: IC50 ¼ 7 ꢁ 1 nM; bA production inhib. (%) 80 ꢁ 10 (3 mM).
that the substitution of the hydroxyl group in the phenyl ring result in any notable improved activity with respect to the pre-
lead to an increase of the inhibitory activity in the cell-based Ab nylated analogue 16a. When the ester group in 35 is trans-
production assay. This result is in accordance with the inhibi- formed into a carboxylic acid group a loss in the BACE-1 activity
tory activity pattern of the above acetylated compound 14, is obtained. These data evidenced that further investigation of
suggesting that the hydroxyl group could be further derivatized structural requirements rather than the incorporation of the
to investigate other types of substituents in this position.
hydroxyl-containing scaffold fragment should be explored in
In parallel, the inuence of the two substituents of the order to increase potency.
phenyl ring is also studied in analogues 17b and 16c, lacking
It has so far been proved that while more potent compounds
either the methyloxy group 17b or the carboxilic acid group 16c. in the BACE-1 assay incorporated the underivatized carboxylic
As their inhibition pattern indicates, both of them lose inhibi- acid group or the hydroxyethylamine fragment in the meta
tory activity with respect to their tri-substituted analogue 17a. position with respect to the amine (17 and 38 respectively),
Consequently, this suggested that the 2 functional groups are these analogues contained a methoxy group in the ortho posi-
required for activity.
tion with respect to the prenyl tether chain. As transformation
The replacement of the phenyl main core by a bioisosteric of the hydroxyl group into a methoxy group in this type of
ring, such as the pyridine moiety, led to a new analogue 19 analogues containing an amino liker had furnished the most
which exhibited comparable BACE-1 inhibitory activity to its potent compounds, a new series of analogues 24–30 incorpo-
closest analogue 16a but devoid of activity in the cell-based rating different alkoxy fragments were synthesized to further
assay.
explore the inuence of these fragments in the inhibitory
Incorporation of different moieties at the site of the activity. As shown in Table 1 this position allowed quite a variety
carboxylic acid group is evaluated to further investigate of fragments to be introduced, furnishing compounds with
the effect in the inhibitory activity of other moieties rather than BACE-1 inhibitory activities in the micromolar range (24, 27, 28,
the methyl group. The transformation of the carboxylic acid 29 and 30) and what is more encouraging is that some of them
group into a variety of esters and amides did not furnish active were found to be active in the cell-based assay (27–29).
compounds.
On the other hand, common hydroxyl-containing inhibitor
scaffolds, including hydroxyethylene, hydroxyethylamine core,
Conclusion
have been applied successfully to a number of BACE-1 inhibitor The prototype 1 has been successfully synthesized and its bio-
series. The hydroxyl groups of the cores engage in hydrogen logical activity conrmed to be consistent with that of the
bonds to the catalytic aspartic acids (Asp32 and Asp128) of the natural compound isolated from the marine organism. The
BACE-1 protease. Therefore the incorporation of these kinds of synthetic prototype 1 was indeed employed for an initial
fragments, around different positions of the initial scaffold, is exploration of the structural requirements for inhibitory
also examined. As result, the new inhibitor containing an activity. Since model compounds lacking the prenyl chain
hydroxyethylamide scaffold at the site of the carboxylic acid proved to be inactive in the enzymatic assay, the chemical
group 38 increased the potency with respect to the methyl ester modications were focused on the phenyl core ring. Further-
analogue 16a, in both enzyme (63.5% at 1 mM) and cell-based more, a new family of compounds, more readily synthesized
assays (10% at 1 mM), exhibiting similar potency to 1. The than the prototype, bearing an amino linker between the phenyl
hydroxyethylene fragment 35, also incorporated between the ring and the three prenyl unit tether chain furnished a
phenyl methyl ester unit and the two prenyl tether units, did not compound 17a of equal inhibitory prole to the marine
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prototype. However, synthesized analogues did not improve the 29.8 mmol) and p-toluenesulfonic acid monohydrate (473 mg,
inhibitory potency found in the marine prototype, probably due 2.49 mmol) are added. The resulting mixture is heated to
to the lack of specic interactions between the prenyl unit tether 135 ꢂC; preferably using a Dean-Stark apparatus, for 5 hours;
chain and BACE-1 active site cavity.
once this time has elapsed, the mixture is brought to room
There is evidence indicating that the carboxylic acid group in temperature. Triethylamine (5 mL) is added, and the solvent is
the meta-position with respect to the tether chain in both the eliminated under reduced pressure. A purication process was
natural product and synthesized analogues is relevant for the employed using a silica gel chromatographic column, with a
inhibitory activity of BACE-1, while the ortho-position with mixture of ethyl acetate–hexane in a ratio of 1 : 2 as an eluent,
respect to the tether chain seems to play an important role in obtaining 5.4 g of 3 as a white solid (yield: 90%). 1H NMR
the bA inhibitory activity. Furthermore, compound 38 including (400 MHz, CD3OD) d (ppm) 7.54 (d, 1H, J ¼ 2.0 Hz), 7.24 (dd, 1H,
the hydroxyethylamine moiety, known to favour the interaction J ¼ 2.0 Hz, 8.3 Hz), 6.88 (m, 1H), 5.63 (s, 1H), 4.02 (m, 5H). 13
C
with the BACE active site, displayed activity in both enzymatic NMR (100 MHz, CD3OD) d (ppm) 156.3, 132.6, 132.0, 128.3,
and cell-based assays. In conclusion, synthetic efforts have not 116.9, 110.5, 104.2, 66.3.
led to an improvement in the biological activity. Nevertheless,
2-(3-Bromo-4-methoxymethoxyphenyl)-[1,3]dioxolane (4).26
due to synthetic accessibility of the compounds, further inves- To a solution of dioxolan 3 (5.3 g, 22.0 mmol) in anhydrous THF
tigations on the synthesis of new analogues could be exploited (75 mL), cooled to 0 ꢂC and under a nitrogen atmosphere,
for the preparation of novel chemical entities targeted to other DIPEA (9.4 mL, 54.0 mmol) is slowly added. The resulting
biological proles.
mixture is stirred for 15 minutes at 0 ꢂC. Once this time has
elapsed, ClMOM (3.5 mL, 43.0 mmol) is added dropwise, and
the reaction mixture is stirred for 16 hours at room tempera-
ture. The mixture is dried under reduced pressure, and a puri-
cation process using a silica gel column is performed, using a
Experimental section
Chemistry
General methods. Chromatographic purity of all compounds mixture of ethyl acetate–hexane as the mobile phase in a ratio of
synthesized in this work is determined using HPLC chromato- 1 : 10, obtaining 6.0 g of 4 as transparent liquid (yield: 95%). 1H
graphic methods. To carry out the biological analysis it is NMR (400 MHz, CDCl3) d (ppm) 7.66 (s, 1H), 7.33 (dd, 1H, J ¼ 1.5
strongly required that the purity of the synthesized compounds Hz, 8.4 Hz), 7.12 (d, 1H, J ¼ 8.5 Hz), 5.72 (s, 1H), 5.20 (m, 2H),
exceeds 95%. HPLC-MS (electrospray): mass spectra were 4.04 (m, 4H), 3.48 (m, 3H). 13C NMR (100 MHz, CDCl3) d (ppm)
recorded on a spectrometer Micromass Quatro Micro API 154.3, 132.9, 131.5, 126.8, 115.6, 112.7, 102.7, 95.0, 65.3, 56.3.
ZQ2000. The purity of all compounds was measured using a
General procedure for the synthesis of compounds 7 and 8.
HPLC analytic Waters Delta, with PDA 2996. Sample prepara- To a solution of 2-(3-bromo-4-methoxymethoxy-phenyl)-[1,3]
tion is performed as follows: weigh about 1 mg of product in an dioxolane (4) (3.46 mmol) in anhydrous toluene (6 mL) and
Eppendorf, dissolve it in a sufficient volume of DMSO with a anhydrous diethyl ether (10 mL), a small quantity of molecular
concentration of 1 mg mLꢀ1, transfer 0.1 mL of this solution to sieves are added. To this solution, at room temperature and
an HPLC vial and dilute with 0.9 mL of mixture of 50% v/v under a nitrogen atmosphere, n-BuLi (4.50 mmol, 1.6 M solu-
acetonitrile–water. The resulting solution has a concentration tion in hexane) is added, and the mixture is stirred for 5
of 0.1 mg mLꢀ1. Symmetry column C18, 3.5 mm, 2.1 ꢃ 100 mm. minutes. Subsequently, CuBr$DMS (2.07 mmol) is added, and
Column temperature 30 ꢂC. Mobile phase: phase A: 0.1% formic the mixture is stirred for another 30 minutes; once the time has
acid in water, phase B: 0.1% formic acid in acetonitrile. Injec- elapsed, the corresponding prenyl bromide (3.80 mmol) is
tion volume: 10 mL. Flow: 0.3 mL minꢀ1, wavelength: 254 nm added. Aer 4 hours, an aqueous saturated solution of ammo-
(normally). Melting points were measured by a DSC technique, nium chloride (5 mL) is added. The resulting mixture is
using a TA Instruments model Q10. Experimental conditions extracted with diethyl ether (2 ꢃ 50 mL), and the organic phase
(general): weight of sample of 1 to 5 mg. Conditions: starting is acidied with a 1 N solution of hydrochloric acid (2 ꢃ 50 mL).
temperature 50 ꢂC. Final temperature: 300 ꢂC, if thermody- The organic phase is dried with sodium sulphate, ltered and
namic phenomenon is not observed the nalꢂtemperature is dried under reduced pressure. A purication process using a
increased to 400 C. Temperature gradient: 10 C minꢀ1. NMR silica gel column is performed, using a mixture of ethyl acetate–
ꢂ
spectra were recorded at room temperature (25 ꢂC) with a Varian hexane in a ratio of 1 : 10 as the mobile phase, obtaining the
spectrometer Mercury Plus 400 MHz using a probe type BBI. product as transparent oil.
Values of chemical shis (d) are expressed in ppm, using tet-
(2E,6E)-4-Methoxymethoxy-3-(3,7,11-trimethyldodeca-2,6,10-
1
ramethylsilane (TMS) as the reference. In the H NMR spectra trienyl)benzaldehyde (7). Reagents: 2-(3-bromo-4-methox-
are indicated: the multiplicity of signals (s, singlet; d, doublet; ymethoxyphenyl)-[1,3]dioxolane (4) (1.0 g, 3.46 mmol), n-BuLi
dd, doublet of doublet; t, triplet; q, quartet; m, multiplet), the (2.8 mL, 4.50 mmol, 1.6 M in hexanes), trans,trans-farnesyl
number of protons derived by integration and the value of the bromide (0.9 mL, 3.46 mmol), CuBr$DMS (355 mg, 1.73 mmol),
constants coupling (J) in Hz. To obtain the 13C NMR spectra diethyl ether anhydrous (10 mL) and toluene anhydrous (6 mL).
the same spectrometer at 100 MHz is employed.
Appearance: transparent oil. Yield: 60%. 1H NMR (400 MHz,
2-Bromo-4-[1,3]dioxolan-2-yl-phenol (3).26 To a solution of 3- CDCl3) d (ppm) 9.87 (s, 1H), 7.70 (m, 2H), 7.17 (d, 1H, J ¼ 8.7
bromo 3-bromo-4-hydroxybenzaldehyde (5.0 g, 24.8 mmol) in Hz), 5.33 (t, 1H, J ¼ 7.2 Hz), 5.29 (s, 3H), 5.10 (m, 2H), 3.49 (s,
anhydrous toluene (75 mL), ethylene glycol (1.66 mL, 3H), 3.39 (d, 2H, J ¼ 7.3 Hz), 2.05 (m, 8H), 1.73 (s, 3H), 1.67 (s,
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3H), 1.59 (s, 6H). 13C NMR (100 MHz, CDCl3) d (ppm) 191.2, 25.7, 17.7, 16.3, 16.1, 16.0. RP-HPLC: 12.6 min, purity: 94.5%,
159.9, 137.1, 135.1, 131.5, 131.3, 130.8, 130.4, 130.0, 124.3, ESI-MS[M]+ ¼ 395.25.
124.0, 121.2, 113.2, 94.0, 56.3, 39.8, 39.7, 28.4, 26.7, 26.6, 25.7,
General procedure for the synthesis of compounds 11 and 1.
17.7, 16.2, 16.0. RP-HPLC: 13.60 min, purity: 98.9%, ESI-MS[M]+ To a solution of aldehyde 9 and 10 (0.43 mmol) in a mixture of
¼ 371.08. THF–H2O (5 : 1, 2.5 mL/0.5 mL) and 2-methyl-2-butene (0.1 mL),
(2E,6E,10E)-4-Methoxymethoxy-3-(3,7,11,15-tetramethylhex- sodium dihydrogen phosphate (1.01 mmol) and sodium chlorite
adeca-2,6,10,14-tetraenyl) benzaldehyde (8). Reagents: 2-(3- (1.06 mmol) are added one aer the other. The reaction mixture
bromo-4-methoxymethoxyphenyl)-[1,3]-dioxolane (4) (500 mg, is stirred for 4 hours at room temperature; once the time has
1.73 mmol), n-BuLi (1.4 mL, 2.25 mmol, 1.6 M in hexanes), elapsed, the mixture is neutralized using a 1 N solution of
geranyl bromide (611 mg, 1.73 mmol), CuBr$DMS (178 mg, hydrochloric acid, until slight acidication occurs (pH ¼ 4–5).
0.86 mmol), diethyl ether anhydrous (5 mL) and anhydrous Water (20 mL) is added, and it is extracted with CH2Cl2 (2 ꢃ
toluene (3 mL). Appearance: transparent oil. Yield: 50%. 1H NMR 25 mL); the organic phase is dried with sodium sulphate, ltered
(400 MHz, CDCl3) d (ppm) 9.87 (s, 1H), 7.72–7.65 (m, 2H), 7.17 (d, and dried under reduced pressure. A purication process using a
J ¼ 9.0 Hz, 1H), 5.35–5.30 (m, 1H), 5.29 (s, 2H), 5.16–5.05 (m, chromatographic column is performed, using a mixture of
3H), 3.49 (s, 3H), 3.39 (d, J ¼ 7.30 Hz, 2H), 2.21–1.90 (m, 12H), dichloromethane with 3% of methanol as an eluent.
1.73 (s, 3H), 1.68 (s, 3H), 1.59 (s, 6H), 1.58 (s, 3H). 13C NMR
(2E,6E)-4-Hydroxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyl)
(100 MHz, CDCl3) d (ppm) 191.2, 159.9, 137.1, 135.1, 134.9, benzoic acid (11). Reagents: (2E,6E)-4-hydroxy-3-(3,7,11-trime-
131.5, 131.2, 130.8, 130.4, 130.0, 124.4, 124.2, 124.0, 121.2, 113.2, thyldodeca-2,6,10-trienyl) benzaldehyde (9) (100 mg,
94.0, 56.2, 39.8, 39.7, 28.4, 26.7, 26.6, 25.7, 17.6, 16.2, 16.0, 16.0. 0.24 mmol), NaH2PO4 (80 mg, 0.58 mmol), NaClO2 (55 mg,
RP-HPLC: 14.10 min, purity: 95%, ESI-MS[M]+ ¼ 438.61.
0.60 mmol), 2-methyl-2-butene (0.1 mL), THF (2.5 mL) and
General procedure for the synthesis of compounds 9 and 10. water (0.5 mL). Appearance: transparent oil. Yield: 87%. 1H
To a solution of the products 7 and 8 (0.24 mmol), dissolved in NMR (400 MHz, CDCl3) d (ppm) 7.95 (dd, 1H, J ¼ 1.2 Hz, J ¼ 8.5
methanol (10 mL), (+/ꢀ)-camphor-10-sulfonic acid (0.26 mmol) Hz), 7.92 (d, 1H, J ¼ 1.9 Hz), 7.38 (m, 5H), 6.94 (d, 1H, J ¼ 8.5
ꢂ
is added. The resulting solution is heated to 70 C for 4 hours. Hz), 5.34 (t, 1H, J ¼ 7.2 Hz), 5.17 (s, 2H), 5.11 (m, 2H), 3.42 (d,
Aer 4 hours, an aqueous saturated solution of ammonium 2H, J ¼ 7.2 Hz), 2.04 (m, 8H), 1.67 (s, 6H), 1.59 (s, 3H), 1.59 (s,
chloride (5 mL) is added. The resulting mixture is extracted 3H). 13C NMR (100 MHz, CDCl3) d (ppm) 177.7, 160.8, 136.8,
using diethyl ether (2 ꢃ 25 mL), and acidied with water (1 ꢃ 136.5, 135.0, 131.6, 131.2, 130.6, 130.1, 128.5, 128.0, 127.1,
25 mL) and brine (1 ꢃ 25 mL). The organic phase is dried with 124.4, 124.1, 121.5, 121.4, 110.8, 70.0, 39.7, 39.6, 28.5, 26.7, 26.6,
sodium sulphate, ltered and dried under reduced pressure. A 25.6, 17.6, 16.1, 16.0. RP-HPLC: 10.62 min, purity: 97.8%, ESI-
purication process with a silica gel column is performed, using MS[M]+ ¼ 433.12.
a mixture of ethyl acetate–hexane in a ratio of 1 : 4 as the mobile
phase, obtaining a transparent, oily product.
(2E,6E,10E)-4-Hydroxy-3-(3,7,11,15-tetramethylhexadeca-
2,6,10,14-tetraenyl) benzoic acid (1). Reagents: (2E,6E,10E)-4-
(2E,6E)-4-Hydroxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyl) hydroxy-3-(3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl)
benzaldehyde (9). Reagents: (2E,6E)-4-methoxymethoxy-3- benzaldehyde (10) (66 mg, 0.17 mmol), NaH2PO4 (48 mg,
(3,7,11-trimethyldodeca-2,6,10-trienyl) benzaldehyde (7) (100 0.40 mmol), NaClO2 (38 mg, 0.42 mmol), 2-methyl-2-butane
mg, 0.24 mmol), (+/ꢀ)-camphor-10-sulfonic acid (62 mg, 0.26 (0.05 mL), THF (2 mL) and water (0.5 mL). Appearance: white
1
mmol) and methanol (10 mL). Appearance: transparent oil. solid. Yield: 44%. M.p.: 143 ꢂC. H NMR (400 MHz, CDCl3) d
Yield: 76%. 1H NMR (400 MHz, CDCl3) d (ppm) 9.85 (s, 1H), 7.67 (ppm) 7.88 (m, 2H), 6.83 (d, J ¼ 8.6 Hz, 1H), 5.34 (t, J ¼ 7.0 Hz,
(m, 2H), 6.91 (d, 1H, J ¼ 8.8 Hz), 5.78 (s, 1H), 5.33 (t, 1H, J ¼ 7.4 1H), 5.11 (m, 3H), 3.41 (d, J ¼ 7.1 Hz, 2H), 2.61–1.87 (m, 12H),
Hz), 5.08 (m, 2H), 3.43 (d, 2H, J ¼ 7.1 Hz), 2.07 (m, 8H), 1.79 (s, 1.79 (s, 3H), 1.68 (s, 3H), 1.60 (s, 6H), 1.60 (s, 6H). 13C NMR (100
3H), 1.67 (s, 3H), 1.59 (s, 6H). 13C NMR (100 MHz, CDCl3) d MHz, CDCl3) d (ppm) 159.5, 139.6, 135.8, 135.1, 132.8, 131.2,
(ppm) 191.0, 160.1, 139.9, 135.7, 131.9, 131.3, 130.4, 130.0, 130.6, 127.1, 124.7, 124.5, 123.8, 122.2, 121.0, 115.9, 39.9, 39.8,
127.4, 124.3, 123.4, 121.1, 120.4, 116.3, 39.6, 29.6, 26.6, 26.2, 39.7, 29.8, 27.0, 26.9, 26.7, 25.7, 17.7, 16.4, 16.2, 16.1. RP-HPLC:
25.6, 17.6, 16.3, 16.0. RP-HPLC: 9.42 min, purity: 98.7%, ESI-MS 11.3 min, purity: 91.4%, ESI-MS[M]+ ¼ 411.28.
[M]+ ¼ 327.02.
(2E,6E,10E)-4-Acetoxy-3-(3,7,11,15-tetramethylhexadeca-
solution of
(2E,6E,10E)-4-Hydroxy-3-(3,7,11,15-tetramethylhexadeca- 2,6,10,14-tetraenyl) benzoic acid (14). To
a
2,6,10,14-tetraenyl) benzaldehyde (10). Reagents: (2E,6E,10E)-4- (2E,6E,10E)-4-hydroxy-3-(3,7,11,15-tetramethylhexadeca-2,6,10,14-
methoxymethoxy-3-(3,7,11,15-tetramethylhexadeca-2,6,10,14- tetraenyl) benzoic acid (1) (100 mg, 0.25 mmol) in pyridine
tetraenyl) benzaldehyde (8) (152 mg, 0.35 mmol), anhydrous (3 mL) is added acetic anhydride (0.045 mL,
(+/ꢀ)-camphor-10-sulfonic acid (88 mg, 0.38 mmol) and meth- 0.50 mmol) at 0 ꢂC. The reaction mixture is stirred for
anol (10 mL). Appearance: transparent oil. Yield: 56%. 1H NMR 15 minutes at 0 C and then further stirred for 16 hours at room
(400 MHz, CDCl3) d (ppm) 9.84 (s, 1H), 7.67 (m, 2H), 6.92 (d, J ¼ temperature. The reaction mixture is dried under reduced
8.34 Hz, 1H), 5.33 (t, J ¼ 6.59 Hz, 1H), 5.20–5.02 (m, 3H), 3.43 (d, pressure, obtaining the product as a white solid (113 mg). Yield:
J ¼ 6.70 Hz, 2H), 2.30–1.87 (m, 12H), 1.79 (s, 3H), 1.67 (s, 3H), 100%. M.p.: 122 ꢂC. 1H NMR (400 MHz, CDCl3) d (ppm) 8.02 (d,
1.60 (s, 9H). 13C NMR (100 MHz, CDCl3) d (ppm) 191.2, 139.7, 1H), 7.99 (dd, J ¼ 8.4 Hz, 1H), 7.14 (d, J ¼ 8.34 Hz, 1H), 5.26 (dd,
135.7, 134.9, 132.0, 131.2, 130.5, 129.9, 127.6, 124.4, 124.2, J ¼ 7.2 Hz, 6.1 Hz, 1H), 5.11 (tt, J ¼ 8.3 Hz, 4.2 Hz, 3H), 3.31 (d, J
123.5, 121.2, 120.5, 116.2, 39.7, 39.6, 29.5, 26.7, 26.6, 26.3, 26.3, ¼ 7.1 Hz, 2H), 2.33 (s, 3H), 2.20–1.91 (m, 12H), 1.72 (s, 3H), 1.68
482 | Med. Chem. Commun., 2014, 5, 474–488
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(d, J ¼ 0.8 Hz, 3H), 1.61 (s, 3H), 1.59 (s, 6H). 13C NMR (100 MHz, 2H), 4.15 (s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.76 (d, 2H, J ¼ 6.7
CDCl3) d (ppm) 168.7, 153.3, 137.7, 135.2, 134.9, 134.1, 132.3, Hz), 2.06 (m, 8H), 1.74 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.60 (s,
131.2, 129.3, 124.4, 124.2, 123.9, 122.5, 120.6, 39.7, 28.7, 26.8, 3H). 13C NMR (100 MHz, CDCl3) d (ppm) 167.5, 150.5, 139.3,
26.6, 26.6, 25.6, 20.8, 17.6, 16.3, 16.0, 16.0. RP-HPLC: 10.27 min, 137.9, 135.2, 131.2, 124.3, 123.8, 122.9, 121.0, 119.2, 110.4,
purity: 95.35%, ESI-MS[M]ꢀ ¼ 451.07.
108.2, 55.5, 51.7, 41.5, 39.6, 39.5, 26.7, 26.3, 25.6, 17.6, 16.4,
General procedure for the synthesis of compounds 12 and 16.0. RP-HPLC: 13.50 min, purity: 97.9%, ESI-MS[M]+ ¼ 386.7.
13. Benzyl bromide (43 mg, 0.25 mmol) is added portion wise to
(2E,6E)-3-(3,7,11-Trimethyldodeca-2,6,10-trienylamino)ben-
a
suspension of (2E,6E,10E)-4-hydroxy-3-(3,7,11,15-tetrame- zoic acid methyl ester (16b). Reagents: methyl 3-aminobenzoate
thylhexadeca-2,6,10,14-tetraenyl) benzoic acid (1) (100 mg, (500 mg, 3.3 mmol), K2CO3 (1.1 g, 8.3 mmol), trans,trans-far-
0.25 mmol) and K2CO3 (34 mg, 0.25 mmol) in DMF (1.2 mL) and nesyl bromide (0.9 mL, 3.6 mmol), THF (25 + 5 mL). Eluent:
the mixture is stirred for four hours. Aer two hours the amber AcOEt–hexane 1 : 30. Appearance: transparent oil. Yield: 58%.
solution with K2CO3 in suspension turned to a colorless solu- 1H NMR (400 MHz, CDCl3) d (ppm) 7.36 (td, 1H, J ¼ 1.2 Hz, J ¼
tion with a white suspension. Water is added and the mixture is 7.4 Hz), 7.27 (t, 1H, J ¼ 2.0 Hz), 7.22 (t, 1H, J ¼ 7.9 Hz), 6.77 (ddd,
extracted with ethyl ether (25 mL). The ether phase is washed 1H, J ¼ 0.9 Hz, J ¼ 2.6 Hz, J ¼ 8.2 Hz), 5.32 (dt, 1H, J ¼ 1.1 Hz, J ¼
eight times with water (10 mL) and once with brine, and the 6.6 Hz), 5.09 (m, 2H), 3.89 (s, 3H), 3.74 (d, 2H, J ¼ 6.7 Hz), 2.06
solvent was evaporated. The purication process is performed (m, 8H), 1.73 (s, 3H), 1.68 (s, 3H), 1.60 (s, 6H). 13C NMR (100
with radial chromatography 10 : 1 (hexane–ethyl acetate).
MHz, CDCl3) d (ppm) 167.4, 135.3, 131.3, 131.0, 129.1, 124.2,
(2E,6E,10E)-4-Benzyloxy-3-(3,7,11,15-tetramethylhexadeca- 123.7, 120.4, 119.1, 119.1, 118.1, 118.0, 114.0, 52.0, 42.3, 39.6,
2,6,10,14-tetraenyl) benzoic acid benzyl ester (12). Appearance: 39.5, 26.7, 26.3, 25.6, 17.6, 16.4, 16.0. RP-HPLC: 11.57 min,
transparent oil. Yield: 44%. 1H NMR (400 MHz, CDCl3) d (ppm) purity: 99.5%, ESI-MS[M]+ ¼ 356.36.
7.94–7.90 (m, 2H), 7.47–7.30 (m, 10H), 6.91 (d, J ¼ 9.2 Hz, 1H),
(2E,6E)-(2-Methoxyphenyl)-(3,7,11-trimethyldodeca-2,6,10-
5.34 (s, 2H), 5.34–5.30 (m, 1H), 5.15 (s, 2H), 5.14–5.07 (m, 3H), trienyl)amine (16c). Reagents: 2-methoxyaniline (500 mg,
3.41 (d, J ¼ 7.2 Hz, 2H), 2.16–1.92 (m, 12H), 1.69 (d, J ¼ 1.1 Hz, 4.1 mmol), K2CO3 (1.3 g, 10.0 mmol), trans,trans-farnesyl
3H), 1.67 (d, J ¼ 0.9 Hz, 3H), 1.60 (s, 3H), 1.59 (s, 6H). 13C NMR bromide (1.2 mL, 4.5 mmol), THF (25 + 5 mL). Eluent: CH2Cl2–
1
(100 MHz, CDCl3) d (ppm) 166.4, 160.3, 136.7, 136.6, 136.4, hexane 1 : 2. Appearance: transparent oil. Yield: 32%. H NMR
135.0, 134.9, 131.2, 130.5, 129.4, 128.6, 128.5, 128.0, 128.0, (400 MHz, CDCl3) d (ppm) 6.88 (tt, 1H, J ¼ 1.4 Hz, J ¼ 7.6 Hz),
127.2, 124.4, 124.3, 124.2, 122.4, 121.7, 110.8, 70.0, 66.2, 39.8, 6.77 (dt, 1H, J ¼ 1.3 Hz, J ¼ 7.9 Hz), 6.68 (td, 1H, J ¼ 1.4 Hz, J ¼
39.7, 28.6, 26.8, 26.7, 26.7, 25.7, 17.7, 16.2, 16.0. RP-HPLC: 11.59 2.3 Hz), 6.62 (dt, 1H, J ¼ 1.4 Hz, J ¼ 7.8 Hz), 5.38 (dt, 1H, J ¼ 1.3
min, purity: 100%, ESI-MS[M]ꢀ ¼ 589.87.
Hz, J ¼ 6.6 Hz), 5.11 (m, 2H), 4.14 (s, 1H), 3.84 (s, 3H), 3.73 (d,
(2E,6E,10E)-4-Hydroxy-3-(3,7,11,15-tetramethylhexadeca- 2H, J ¼ 6.6 Hz), 2.07 (m, 8H), 1.72 (s, 3H), 1.69 (s, 3H), 1.61 (s,
2,6,10,14-tetraenyl) benzoic acid benzyl ester (13). Appearance: 6H). 13C NMR (100 MHz, CDCl3) d (ppm) 146.8, 138.7, 138.4,
transparent oil. Yield: 36%. 1H NMR (400 MHz, CDCl3) d (ppm) 135.2, 131.2, 124.3, 123.8, 121.7, 121.2, 116.2, 109.9, 109.2, 55.3,
7.89–7.85 (m, 2H), 7.46–7.30 (m, 5H), 6.83 (d, J ¼ 8.9 Hz, 1H), 41.6, 39.6, 39.5, 26.7, 26.3, 25.6, 17.6, 16.3, 16.0. RP-HPLC: 10.43
5.86 (s, 1H), 5.35 (s, 2H), 5.32 (m, 1H), 5.14–5.07 (m, 3H), 3.40 (d, min, purity: 99.0%, ESI-MS[M]+ ¼ 327.56.
J ¼ 7.2 Hz, 2H), 2.18–1.94 (m, 14H), 1.79 (s, 3H), 1.69 (s, 3H),
General procedure for the synthesis of compounds 17a–b.
1.62–1.59 (m, 9H). 13C NMR (100 MHz, CDCl3) d (ppm) 166.7, The obtained methyl ester derivative (75 mg, 0.19 mmol) is
159.2, 139.5, 136.6, 135.8, 135.1, 132.3, 131.4, 130.1, 128.8, dissolved in a mixture of THF (2.5 mL), MeOH (1 mL) and water
128.3, 128.3, 127.0, 124.6, 124.5, 123.8, 122.7, 121.1, 115.9, 66.6, (1.5 mL), and lithium hydroxide monohydrate (82 mg,
39.9, 39.9, 39.9, 29.9, 27.0, 26.8, 26.6, 25.9, 17.9, 16.5, 16.3, 16.2. 1.90 mmol) is added. The reaction mixture is stirred for 48
RP-HPLC: 11.43 min, purity: 99.4%, ESI-MS[M]ꢀ ¼ 499.13.
hours and the reaction mixture is neutralized to pH ¼ 4 with 1
General procedure for the synthesis of compounds 16a–c. To M HCl solution. The resulting mixture is extracted with CH2Cl2
a solution of 3-amino-4-methoxy-benzoic acid methyl ester (3 ꢃ 25 mL). The combined extracts were washed with water
(500 mg, 2.8 mmol) in THF anhydrous (25 mL) powder potas- (25 mL), saturated NaCl solution (25 mL) and dried with Na2SO4
sium carbonate (1100 mg, 8.3 mmol) is added and the resulting anhydrous. Evaporation of the solvent under reduced pressure
mixture is stirred for 10 minutes. Trans,trans-farnesyl bromide gave a residue which is puried by column ash chromatog-
(0.8 mL, 3.0 mmol) in THF (5 mL) is added and the resulting raphy to give the desired compound.
mixture is stirred for a further 16–18 hours. The solvent is
evaporated under reduced pressure and the resulting residue is mino)benzoic acid (17a). Reagents: (2E,6E)-4-methoxy-3-(3,7,11-
puried by ash column chromatography. trimethyldodeca-2,6,10-trienylamino) benzoic acid methyl ester
(2E,6E)-4-Methoxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla-
(2E,6E)-4-Methoxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla- (16a) (75 mg, 0.19 mmol), LiOH$H2O (82 mg, 1.9 mmol), THF
mino) benzoic acid methyl ester (16a). Reagents: methyl 3- (2.5 mL), MeOH (1 mL), H2O (1.5 mL). Appearance: transparent
amino-4-methoxybenzoate (500 mg, 2.8 mmol), K2CO3 (1.1 g, oil. Rto.: 97%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.52 (td, 1H, J
8.3 mmol), trans,trans-farnesyl bromide (0.8 mL, 3.0 mmol), ¼ 1.8 Hz, J ¼ 8.3 Hz), 7.31 (d, 1H, J ¼ 1.7 Hz), 6.79 (d, 1H, J ¼ 8.4
THF (25 + 5 mL). Eluent: AcOEt–hexane 1 : 4. Appearance: Hz), 5.36 (dt, 1H, J ¼ 1.0 Hz, J ¼ 6.7 Hz), 5.10 (m, 2H), 3.91 (s,
transparent oil. Yield: 30%. 1H NMR (400 MHz, CDCl3) d (ppm) 3H), 3.78 (d, 2H, J ¼ 6.7 Hz), 2.06 (m, 8H), 1.75 (s, 3H), 1.68 (s,
7.43 (dd, 1H, J ¼ 2.1 Hz, J ¼ 8.3 Hz), 7.25 (d, 1H, J ¼ 2.0 Hz), 6.75 3H), 1.61 (s, 3H), 1.60 (s, 3H). 13C NMR (100 MHz, CDCl3) d
(d, 1H, J ¼ 8.4 Hz), 5.36 (dt, 1H, J ¼ 1.3 Hz, J ¼ 6.7 Hz), 5.09 (m, (ppm) 172.1, 151.1, 139.4, 138.0, 135.3, 131.2, 124.3, 123.8,
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122.0, 120.9, 120.1, 110.7, 108.3, 55.5, 41.5, 39.6, 39.5, 26.7, 26.3, (1 ꢃ 25 mL), dried with Na2SO4 anhydrous, ltered and the
25.6, 17.6, 16.4, 16.0. RP-HPLC: 12.15 min, purity: 100%, ESI-MS solvent is evaporated under reduced pressure. The mixture is
[M]+ ¼ 372.03.
puried by ash column chromatography, using AcOEt–
(2E,6E)-3-(3,7,11-Trimethyldodeca-2,6,10-trienylamino)ben- hexanes 1 : 10 as an eluent, to give the desired compound as an
zoic acid (17b). Reagents: (2E,6E)-3-(3,7,11-trimethyldodeca- orange solid. M.p.: 191 ꢂC. Yield: 30%. 1H NMR (400 MHz,
2,6,10-trienylamino) benzoic acid methyl ester (16b) (576 mg, CDCl3) d (ppm) 7.39 (dd, 1H, J ¼ 1.8 Hz, J ¼ 8.1 Hz), 7.36 (d, 1H, J
1.6 mmol), LiOH$H2O (680 mg, 16.2 mmol), THF (2.5 mL), ¼ 1.9 Hz), 6.72 (d, 1H, J ¼ 8.0 Hz), 5.36 (t, 1H, J ¼ 6.7 Hz), 5.10
MeOH (1 mL), H2O (1.5 mL). Appearance: white solid. M.p.: 75 (m, 2H), 3.87 (s, 3H), 3.75 (d, 2H, J ¼ 6.7 Hz), 2.06 (m, 8H), 1.73
ꢂC. Yield: 31%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.44 (d, 1H, J (s, 3H), 1.68 (s, 3H), 1.60 (s, 6H). 13C RMN (100 MHz. CDCl3) d
¼ 7.6 Hz), 7.32 (s, 1H), 7.25 (t, 1H, J ¼ 7.8 Hz), 6.82 (dd, 1H, J ¼ (ppm) 167.5, 147.9, 139.5, 135.3, 131.3, 124.3, 123.8, 123.3,
2.1 Hz, J ¼ 8.0 Hz), 5.33 (t, 1H, J ¼ 6.9 Hz), 5.10 (q, 2H, J ¼ 5.7 120.9, 120.4, 118.0, 113.5, 113.3, 51.8, 42.2, 39.6, 39.5, 26.7, 26.3,
Hz), 3.75 (d, 2H, J ¼ 6.6 Hz), 2.06 (m, 8H), 1.73 (s, 3H), 1.68 (s, 25.6, 17.6, 16.4, 16.0. RP-HPLC: 8.93 min, purity: 99.0%, ESI-MS
3H), 1.60 (s, 6H). 13C NMR (100 MHz, CDCl3) d (ppm) 172.2, [M]+ ¼ 372.03.
148.4, 139.6, 135.3, 131.3, 130.1, 129.1, 124.2, 123.7, 120.8,
(2E,6E)-4-Hydroxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla-
119.0, 118.1, 113.8, 41.9, 39.6, 39.5, 26.7, 26.3, 25.6, 17.6, 16.4, mino)benzoic acid (23). A solution of (2E,6E)-4-hydroxy-3-
16.0. RP-HPLC: 4.88 min, purity: 96.4%, ESI-MS[M]+ ¼ 342.10.
(3,7,11-trimethyldodeca-2,6,10-trienylamino)
benzoic acid
(2E,6E)-2-(3,7,11-Trimethyldodeca-2,6,10-trienylamino)iso- methyl ester (22) (100 mg, 0.27 mmol) is dissolved in a mixture
nicotinic acid methyl ester (19). To a solution of methyl 2- of THF (2.5 mL), MeOH (1 mL) and water (1.5 mL), and lithium
aminoisonicotinate (265 mg, 1.7 mmol) in DMF (5 mL) an hydroxide monohydrate (114 mg, 2.70 mmol) is added. The
aqueous solution of NaHCO3 (365 mg, 4.3 mmol) is added, and reaction mixture is stirred for 24 hours and the reaction mixture
the resulting mixture is stirred for 5 minutes. Trans,trans-far- is neutralized to pH ¼ 4 with 1 M HCl solution. The resulting
nesyl bromide (0.5 mL, 1.9 mmol) in DMF (1 mL) is added and mixture is extracted with CH2Cl2 (3 ꢃ 25 mL). The combined
ꢂ
the resulting mixture is stirred for a further 16 hours at 60 C. extracts were washed with water (25 mL), saturated NaCl solu-
The solvent is evaporated under reduced pressure and the tion (25 mL) and dried with Na2SO4 anhydrous. Evaporation of
resulting residue is puried by ash column chromatography. the solvent under reduced pressure gave a residue which is
Eluent: AcOEt–hexane 1 : 15. Appearance: transparent oil. Yield: puried by column ash chromatography to give the desired
18%. 1H NMR (400 MHz, CDCl3) d (ppm) 8.18 (d, J ¼ 6.0 Hz, 1H), compound as a yellow pale oil. Yield: 36%. 1H NMR (400 MHz,
7.07 (dd, J ¼ 5.2 Hz, J ¼ 1.4 Hz, 1H), 6.95 (d, J ¼ 0.9 Hz, 1H), 5.32 CDCl3) d (ppm) 7.30 (m, 1H), 7.26 (d, 1H, J ¼ 2.0 Hz), 6.71 (d, 1H,
(t, J ¼ 6.8, J ¼ 6.8 Hz, 1H), 5.10 (d, J ¼ 5.3 Hz, 2H), 4.70 (s, 1H), J ¼ 8.1 Hz), 5.33 (m, 1H), 5.09 (m, 2H), 3.76 (d, 2H, J ¼ 6.6 Hz),
3.91 (s, 3H), 3.89 (d, J ¼ 1.9 Hz, 2H), 2.41–1.85 (m, 8H), 1.73 (s, 2.06 (m, 8H), 1.77 (s, 3H), 1.66 (s, 3H), 1.59 (s, 3H), 1.59 (s, 3H).
3H), 1.68 (s, 3H), 1.60 (s, 6H). 13C NMR (100 MHz, CDCl3) d 13C NMR (100 MHz, CDCl3) d (ppm) 171.0, 150.8, 139.8, 138.3,
(ppm) 166.5, 159.5, 149.1, 140.1, 138.9, 135.6, 131.5, 124.52, 136.2, 132.0, 125.5, 125.2, 123.3, 123.2, 121.5, 113.6, 113.5, 42.8,
123.9, 120.7, 111.8, 106.8, 52.6, 40.5, 39.9, 39.8, 26.9, 26.6, 25.9, 40.8, 40.7, 27.8, 27.4, 25.9, 17.8, 16.5, 16.1. RP-HPLC: 11.5 min,
17.9, 16.6, 16.2. RP-HPLC: 9.4 min, purity: 99.6%, ESI-MS[M]+ ¼ purity: 97.9%, ESI-MS[M]+ ¼ 358.16.
357.38.
General procedure for the synthesis of compounds 24 and
3-Amino-4-hydroxybenzoic acid methyl ester (21).27 To a 26. To a solution of (2E,6E)-4-hydroxy-3-(3,7,11-trimethyldo-
solution of 3-amino-4-hydroxybenzoic acid (25 g, 16.0 mmol) in deca-2,6,10-trienylamino) benzoic acid methyl ester (22) (200
ꢂ
MeOH (15 mL), H2SO4 conc. (2 mL) is added dropwise at 0 C. mg, 0.54 mmol) in DMF anhydrous (8 mL) under an inert
The mixture is stirred for 8 hours at 70 ꢂC, and then the mixture atmosphere is added K2CO3 (148 mg, 0.81 mmol) and the
is neutralized with aqueous solution of NaOH 5 N to pH 8–9. mixture is stirred for 40 minutes at room temperature. Aer 1-
The mixture is extracted with AcOEt (2 ꢃ 200 mL) and the iodopropane is added (63 mL, 0.64 mmol) the mixture is stirred
organic phase dried with Na2SO4 anhydrous, ltered and the for 4 hours. The mixture is concentrated to dryness and the
solvent is evaporated under reduced pressure, to give residue is puried by column chromatography, using as an
the desired compound as a white solid. M.p.: 183 ꢂC. Yield: eluent hexane–AcOEt 10 : 1 to obtain the compounds 22 (45 mg,
93%. 1H NMR (400 MHz, DMSO-d6) d (ppm) 9.90 (s, 1H), 7.25 (d, yield 20%) and 24 (10 mg, yield 4%), both as transparent oil.
J ¼ 2.1 Hz, 1H), 7.10 (dd, J ¼ 8.2 Hz, J ¼ 2.1 Hz, 1H), 6.71 (d, J ¼
(2E,6E)-4-Propoxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla-
8.2 Hz, 1H), 4.77 (s, 2H), 3.74 (s, 3H). 13C NMR (100 MHz, mino) benzoic acid methyl ester (24). 1H NMR (400 MHz,
DMSO-d6) d (ppm) 166.5, 148.5, 136.5, 120.6, 118.7, 114.6, 113.6, CDCl3) d (ppm) 7.40 (dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, 1H), 7.25 (d, J ¼
51.3. RP-HPLC: 1.46 min, purity: 100%, ESI-MS[M]+ ¼ 167.51.
2.0 Hz, 1H), 6.74 (d, J ¼ 8.3 Hz, 1H), 5.35 (dt, J ¼ 6.6 Hz, J ¼ 1.2
(2E,6E)-4-Hydroxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla- Hz, 1H), 5.1–5.0 (m, 2H), 4.18 (s broad, 1H), 4.00 (t, J ¼ 6.6 Hz,
mino) benzoic acid methyl ester (22). To a solution of 3-amino- 2H), 3.87 (s, 3H), 3.78 (d, J ¼ 6.9 Hz, 2H), 2.24–1.93 (m, 8H), 1.79
4-hydroxybenzoic acid methyl ester (21) (12.0 mmol, 2.0 g) in (sext, J ¼ 6.6 Hz, 2H), 1.75 (d, J ¼ 1.1 Hz, 3H), 1.68 (d, J ¼ 1.1 Hz,
THF anhydrous (40 mL) is added trans,trans-farnesyl bromide 3H), 1.60 (s, 6H), 1.05 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz,
(3.2 mL, 1.8 mmol) and the mixture is stirred for 16 hours at CDCl3) d (ppm) 167.6, 149.9, 139.2, 138.0, 135.2, 131.2, 124.3,
room temperature, aer the mixture is neutralized with 123.8, 122.7, 121.2, 119.2, 110.5, 109.1, 69.8, 51.7, 41.6, 39.7,
aqueous solution of NaOH 1 M to pH 6–7 and extracted with 39.6, 26.7, 26.4, 25.7, 22.7, 17.6, 16.4, 16.0, 10.5. RP-HPLC: 11.02
CH2Cl2 (3 ꢃ 25 mL). The organic phase is washed with water min, purity: 87.1%, ESI-MS[M]+ ¼ 413.6.
484 | Med. Chem. Commun., 2014, 5, 474–488
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5.05–4.94 (m, 2H), 4.65 (s, 4H), 4.18 (q, J ¼ 7.1 Hz, 4H), 4.08 (s,
2H), 4.07–4.00 (m, 4H), 3.92 (d, J ¼ 6.1 Hz, 2H), 3.80 (s, 3H),
2.10–1.81 (m, 8H), 1.62 (s, 3H), 1.60 (d, J ¼ 1.1 Hz, 3H), 1.52 (s,
3H), 1.51 (d, J ¼ 0.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d (ppm)
168.4, 167.0, 153.9, 135.5, 131.5, 124.9, 124.6, 124.0, 120.3,
112.4, 105.0, 65.8, 61.7, 60.7, 52.2, 52.1, 50.4, 40.0, 39.9, 27.0,
26.7, 25.9, 17.9, 16.5, 16.2, 14.4, 14.3. RP-HPLC: 11.6 min,
purity: 95.8%, ESI-MS[M]+ ¼ 544.05.
(2E,6E)-4-Propoxy-3-[propyl-(3,7,11-trimethyldodeca-2,6,10-
trienyl)amino] benzoic acid methyl ester (26). 1H NMR (400
MHz, CDCl3) d (ppm) 7.63 (dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, 1H), 7.56
(d, J ¼ 1.9 Hz, 1H), 6.82 (d, J ¼ 8.5 Hz, 1H), 5.24 (dt, J ¼ 6.5 Hz, J
¼ 0.9 Hz, 1H), 5.12–5.04 (m, 2H), 4.01 (t, J ¼ 6.6 Hz, 2H), 3.86 (s,
3H), 3.74 (d, J ¼ 6.5 Hz, 2H), 3.10–3.03 (m, 2H), 2.10–1.92 (m,
8H), 1.87 (td, J ¼ 13.9, J ¼ 6.9 Hz, 2H), 1.67 (d, J ¼ 1.1 Hz, 3H),
1.64 (d, J ¼ 0.5 Hz, 3H), 1.59 (d, J ¼ 0.5 Hz, 3H), 1.57 (d, J ¼ 0.7
Hz, 3H), 1.48 (qd, J ¼ 14.9 Hz, J ¼ 7.6 Hz, 2H), 1.07 (t, J ¼ 7.4 Hz,
3H), 0.85 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) d (ppm)
167.3, 156.5, 140.0, 137.7, 135.0, 131.2, 124.4, 124.3, 124.0,
122.2, 122.0, 121.7, 111.3, 70.0, 53.1, 51.7, 50.0, 39.7, 39.7, 26.7,
26.5, 25.7, 22.6, 20.3, 17.6, 16.2, 15.9, 11.7, 10.7. RP-HPLC: 12.49
min, purity: 97.9%, ESI-MS[M]+ ¼ 456.
(2E,6E)-3-Oxo-4-(3,7,11-trimethyldodeca-2,6,10-trienyl)-3,4-
dihydro-2H-benzo-[1,4]oxazine-6-carboxylic acid methyl ester
(28). Reagent: see compound 27. Eluent: AcOEt–hexane 1 : 4.
Appearance: transparent oil. Rto.: 67%. 1H NMR (400 MHz,
CDCl3) d (ppm) 7.69 (dd, J ¼ 8.3 Hz, J ¼ 1.9 Hz, 1H), 7.66 (d, J ¼
1.9 Hz, 1H), 6.99 (d, J ¼ 8.3 Hz, 1H), 5.12 (dt, J ¼ 6.5 Hz, J ¼ 1.1
Hz, 1H), 5.07–4.99 (m, 2H), 4.67 (s, 3H), 4.59 (d, J ¼ 6.6 Hz, 2H),
3.89 (s, 2H), 2.13–1.92 (m, 8H), 1.88 (d, J ¼ 0.7 Hz, 3H), 1.66 (d, J
¼ 1.0 Hz, 3H), 1.56 (s, 3H), 1.55 (d, J ¼ 0.5 Hz, 3H). 13C NMR (100
MHz, CDCl3) d (ppm) 168.4, 167.0, 153.9, 153.2, 135.5, 131.5,
124.9, 124.6, 124.5, 124.0, 122.9, 120.3, 112.4, 105.0, 65.8, 61.7,
60.7, 52.1, 40.0, 27.0, 26.7, 25.9, 17.9, 16.5, 14.3. RP-HPLC: 11.86
min, purity: 95.3%, ESI-MS[M]+ ¼ 412.43.
General procedure for the synthesis of compounds 25, 27
and 28. To a solution of (2E,6E)-4-hydroxy-3-(3,7,11-trime-
thyldodeca-2,6,10-trienylamino) benzoic acid methyl ester (22)
(1.0 eq.) in THF anhydrous at 0 ꢂC is added NaH (60% in
mineral oil, 1.2 eq.). The mixture is stirred for 10 minutes and
then NBu4I (0.25 eq.), 18-crown-6 (0.01 eq.) and alkylating agent
(1.1 eq.) are added. The mixture is stirred for 2 hours at 70 ꢂC, in
an inert atmosphere, and then cooled at room temperature and
water (25 mL) is added. The mixture is extracted with CH2Cl2 (2
ꢃ 25 mL) and the organic phase is washed with brine (1 ꢃ
25 mL). The combined organic extract is dried with Na2SO4
anhydrous, ltered and the solvent is evaporated under reduced
pressure. The residue is puried by column ash chromatog-
raphy to give the desired compounds.
(2E,6E)-4-(Propane-1-sulfonyloxy)-3-(3,7,11-trimethyldodeca-
2,6,10-trienylamino) benzoic acid methyl ester (25). Reagent:
(2E,6E)-4-hydroxy-3-(3,7,11-trimethyldodeca-2,6,10-trieny-
lamino) benzoic acid methyl ester (22) (0.2 g, 0.54 mmol), NaH
(26 mg, 0.64 mmol), NBu4I (50 mg, 0.13 mmol), 18-crown-6 (1.4
mg, 0.005 mmol), 1-propanesulfonyl chloride (66 mL, 0.59
mmol) and THF anhydrous (1 + 4 mL). Eluent: AcOEt–hexane
1 : 10. Appearance: transparent oil. Yield: 44%. 1H NMR (400
MHz, CDCl3) d (ppm) 7.39 (d, J ¼ 2.0 Hz, 1H), 7.36 (dd, J ¼ 8.3
Hz, J ¼ 2.0 Hz, 1H), 7.21 (d, J ¼ 8.4 Hz, 1H), 5.35 (dt, J ¼ 6.5 Hz, J
¼ 1.1 Hz, 1H), 5.14–5.02 (m, 2H), 4.31 (s, 1H), 4.12–3.98 (m, 2H),
3.92 (s, 3H), 3.79 (d, J ¼ 7.2 Hz, 2H), 3.25 (t, J ¼ 8.8 Hz, 2H), 2.18–
1.87 (m, 10H), 1.77 (s, 3H), 1.72 (s, 3H), 1.64 (s, 6H), 1.13 (t, J ¼
8.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d (ppm) 167.5, 140.8,
140.5, 140.2, 137.9, 130.9, 130.0, 122.5, 122.3, 121.2, 120.5,
119.2, 112.0, 53.3, 52.5, 42.2, 40.3, 40.3, 27.4, 27.4, 27.0, 18.8,
18.7, 17.5, 17.3, 13.8. RP-HPLC: 12.12 min, purity: 98.8%, ESI-
MS[M]+ ¼ 478.1.
General procedure for the synthesis of compounds 29 and
30. Example, compound (2E,6E)-4-(propane-1-sulfonyloxy)-3-
(3,7,11-trimethyl-dodeca-2,6,10-trienylamino) benzoic acid (29).
A solution of (2E,6E)-4-(propane-1-sulfonyloxy)-3-(3,7,11-trime-
thyldodeca-2,6,10-trienylamino) benzoic acid methyl ester (25)
(95 mg, 0.19 mmol) is dissolved in a mixture of THF (2.0 mL)
and water (2.0 mL), and lithium hydroxide monohydrate
(105 mg, 2.50 mmol) is added. The reaction mixture is stirred
for 24 hours and the reaction mixture is neutralized to pH ¼ 4
with 1 M HCl solution. The resulting mixture is extracted with
CH2Cl2 (3 ꢃ 25 mL). The combined extracts were washed with
water (25 mL), saturated NaCl aqueous solution (25 mL) and
dried (Na2SO4 anhydrous). Evaporation of the solvent under
reduced pressure gave a residue which is puried by column
ash chromatography using AcOEt–hexane 1 : 4 as an eluent, to
give the desired compound as a transparent oil. Yield: 41%. 1H
NMR (400 MHz, CDCl3) d (ppm) 7.40–7.35 (m, 2H), 7.23–7.19
(m, 1H), 5.24 (dt, J ¼ 6.5 Hz, J ¼ 1.0 Hz, 1H), 5.02 (ddd, J ¼
8.0 Hz, J ¼ 7.0 Hz, J ¼ 4.1 Hz, 2H), 3.72 (d, J ¼ 6.7 Hz, 2H), 3.29–
3.16 (m, 2H), 2.13–1.81 (m, 10H), 1.69 (d, J ¼ 0.8 Hz, 3H), 1.60 (d,
J ¼ 0.9 Hz, 3H), 1.53 (s, 3H), 1.53 (d, J ¼ 0.5 Hz, 3H), 1.05 (t,
J ¼ 7.5 Hz, 3H). 13C NMR (100 MHz, CDCl3) d (ppm) 171.1, 141.3,
140.7, 140.3, 135.6, 131.5, 128.8, 124.5, 123.9, 122.3, 120.3,
114.2, 105.0, 53.2, 41.7, 39.9, 39.8, 26.9, 26.6, 25.9, 17.9, 17.6,
16.7, 16.2, 13.1. RP-HPLC: 11.25 min, purity: 97.3%, ESI-MS
[M]ꢀ ¼ 462.10.
(2E,6E)-4-Ethoxycarbonylmethoxy-3-[ethoxycarbonylmethyl-
(3,7,11-trimethyl-dodeca-2,6,10-trienyl)amino]benzoic acid
methyl ester (27). Reagent: (2E,6E)-4-hydroxy-3-(3,7,11-trime-
thyldodeca-2,6,10-trienylamino) benzoic acid methyl ester (22)
(284 mg, 0.76 mmol), NaH (30 mg, 0.84 mmol), NBu4I (70.5 mg,
0.19 mmol), 18-crown-6 (2.0 mg, 0.008 mmol), ethyl bromoa-
cetate (93 mL, 0.84 mmol), THF anhydrous (1 + 1 mL). Eluent:
(2E,6E)-3-Oxo-4-(3,7,11-trimethyldodeca-2,6,10-trienyl)-3,4-
dihydro-2H-benzo-[1,4]oxazine-6-carboxylic acid (30). Reagent:
(2E,6E)-3-oxo-4-(3,7,11-trimethyldodeca-2,6,10-trienyl)-3,4-dihy-
dro-2H-benzo-[1,4]oxazine-6-carboxylic acid methyl ester (28)
(24 mg, 0.052 mmol), LiOH$H2O (29 mg, 0.68 mmol), THF (1.0
mL), MeOH (0.5 mL), H2O (1.0 mL). Appearance: transparent
oil. Yield: 92%. M.p.: 169 ꢂC 1H NMR (400 MHz, CDCl3) d (ppm)
7.78 (dd, J ¼ 8.4 Hz, J ¼ 1.9 Hz, 1H), 7.73 (d, J ¼ 1.9 Hz, 1H), 7.03
(d, J ¼ 8.4 Hz, 1H), 5.14 (dd, J ¼ 6.6 Hz, J ¼ 5.6 Hz, 1H), 5.07–4.99
(m, 2H), 4.71 (s, 2H), 4.62 (d, J ¼ 6.5 Hz, 2H), 2.15–1.91 (m, 8H),
1
AcOEt–hexane 1 : 4. Appearance: transparent oil. Yield: 8%. H
NMR (400 MHz, CDCl3) d (ppm) 7.68 (d, J ¼ 1.9 Hz, 1H), 7.58 (d, J
¼ 8.4 Hz, 1H), 6.68 (d, J ¼ 8.5 Hz, 1H), 5.22 (t, J ¼ 6.6 Hz, 1H),
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1.90 (s, 3H), 1.65 (d, J ¼ 0.9 Hz, 3H), 1.56 (s, 3H), 1.55 (s, 3H). 13
C
eluent to give the desired compound as a pale yellow oil. Yield:
NMR (100 MHz, CDCl3) d (ppm) 170.9, 163.3, 149.8, 140.9, 135.4, 13%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.45 (dd, J ¼ 8.3 Hz, J ¼
131.2, 128.3, 126.5, 124.3, 123.6, 123.5, 117.8, 117.5, 116.9, 67.5, 2.0 Hz, 1H), 7.35–7.16 (m, 6H), 6.77 (d, J ¼ 8.4 Hz, 1H), 5.15–4.84
39.7, 39.6, 39.5, 26.7, 26.2, 25.6, 17.6, 16.6, 16.0. RP-HPLC: 10.9 (m, 2H), 3.99 (td, J ¼ 8.6 Hz, J ¼ 3.4 Hz, 1H), 3.91 (s, 1H), 3.90 (s,
min, purity: 94.6%, ESI-MS[M]+ ¼ 398.17, [M]+ + Na ¼ 420.16.
3H), 3.88 (s, 3H), 3.37 (dd, J ¼ 12.1 Hz, J ¼ 3.3 Hz, 1H), 3.22 (dd, J
¼ 12.1 Hz, J ¼ 8.7 Hz, 1H), 3.12 (t, J ¼ 6.2 Hz, 2H), 3.05–2.99 (m,
1H), 2.88 (dd, J ¼ 14.0, J ¼ 4.9 Hz, 1H), 2.72 (dd, J ¼ 13.9 Hz, J ¼
9.6 Hz, 1H), 2.06–1.97 (m, 2H), 1.97–1.89 (m, 3H), 1.67 (s, 3H),
Synthesis of compound 35
(2S,3R)-3-(3-tert-Butoxycarbonylamino-2-hydroxy-4-phenyl- 1.59 (s, 3H), 1.46 (s, 3H). 13C NMR (100 MHz, CDCl3) d (ppm)
butylamino)-4-methoxybenzoic acid methyl ester (32). To a 167.6, 150.9, 138.3, 138.0, 131.6, 129.2, 129.2, 129.1, 128.8,
solution of (2S,3S)-1,2-epoxy-3-(Boc-amino)-4-phenylbutane 128.7, 126.6, 123.9, 122.9, 121.8, 119.6, 110.4, 108.4, 68.8, 61.0,
(200 mg, 0.76 mmol) in iPrOH (5 mL) is added methyl 3-amino- 55.6, 51.8, 45.8, 44.9, 39.5, 35.3, 26.4, 25.7, 17.7, 16.1. RP-HPLC:
4-methoxybenzoate (206 mg, 1.1 mmol). The mixture is stirred 8.03 min, purity: 95.1%, ESI-MS[M]+ ¼ 481.93.
at 50 ꢂC, under a nitrogen atmosphere for 16 hours, and then is
cooled at room temperature. The reaction mixture is concen-
trated under reduced pressure and puried by ash column
Synthesis of compounds 36–38
chromatography using AcOEt–hexane 1 : 2 to give the desired
(1S,2R)-(1-Benzyl-3-cyclopropylamino-2-hydroxypropyl) car-
1
compound as a white solid. Yield: 41%. M.p.: 162 C. H NMR bamic acid tert-butyl ester (36).23 To a solution of (2S,3S)-1,2-
(400 MHz, CDCl3) d (ppm) 7.46 (dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, 1H), epoxy-3-(Boc-amino)-4-phenylbutane (10 g, 37.0 mmol) in iPrOH
7.36–7.20 (m, 6H), 6.77 (d, J ¼ 8.4 Hz, 1H), 4.89–4.36 (m, 2H), (100 mL) is added cyclopropylamine (22.6 mL, 326.5 mmol) and
3.96 (dd, J ¼ 11.2 Hz, J ¼ 6.2 Hz, 2H), 3.90 (s, 3H), 3.88 (s, 3H), the mixture is stirred for 7 hours at 50 ꢂC. The reaction mixture
3.49 (d, J ¼ 4.1 Hz, 1H), 3.45–3.33 (m, 1H), 3.27–3.14 (m, 1H), is concentrated to dryness to give the desired compound 12.05 g
3.00 (dd, J ¼ 14.1 Hz, J ¼ 5.1 Hz, 1H), 2.95–2.82 (m, 1H), 1.38 (s, as a white solid. Yield: 99%. M.p. ¼ 233 ꢂC. 1H NMR (400 MHz,
9H). 13C NMR (100 MHz, CDCl3) d (ppm) 167.7, 151.2, 138.2, CDCl3) d (ppm) 7.29–7.08 (m, 6H), 6.63 (d, J ¼ 9.0 Hz, 1H), 4.73
137.8, 129.5, 128.8, 126.8, 123.1, 120.2, 120.0, 111.0, 108.8, 80.3, (d, J ¼ 5.2 Hz, 1H), 3.60–3.37 (m, 1H), 2.99 (m, 1H), 2.67 (m, 1H),
77.4, 72.7, 55.8, 52.0, 46.5, 36.6, 28.5. RP-HPLC: 9.91 min, 2.51 (m, 1H), 2.08 (m, 2H), 1.26 (s, 9H), 1.13 (s, 1H), 0.34 (m,
ꢂ
purity: 99.4%, ESI-MS[M]+ ¼ 444.82, [M]+ + Na ¼ 467.02.
2H), 0.2 (m, 2H). ESI-MS[M]+ ¼ 321.21.
(2S,3R)-3-(3-Amino-2-hydroxy-4-phenylbutylamino)-4-methoxy-
(2R,3S)-3-Amino-1-cyclopropylamino-4-phenylbutan-2-ol-
benzoic acid methyl ester (33). To a solution of 3-((2S,3R)-3-tert- ditriuoroacetate salt (37).23 To a suspension of (1S,2R)-(1-
butoxycarbonylamino-2-hidroxy-4-fenylbutylamino)-4-metox- benzyl-3-cyclopropylamino-2-hydroxypropyl) carbamic acid tert-
ybenzoic acid methyl ester (32) (100 mg, 0.22 mmol) of AcOEt– butyl ester (36) (11.8 g, 37.0 mmol) in CH2Cl2 (120 mL) is added
ꢂ
methanol 4 : 1 (5 mL) at 0 C is slowly passed a stream of HCl TFA (11.0 mL, 147.9 mmol) and the reaction mixture is stirred
gas for 5 minutes. The mixture is stirred at 0 ꢂC for 3 hours, and for 24 hours at room temperature. Aerwards, 4 eq. of TFA is
then the reaction mixture is dried under reduced pressure. The added and stirred for an additional 7 hours. The mixture is
solid obtained is washed with Et2O (10 mL) and is ltered to give concentrated to dryness and the oil obtained is washed with
1
the compound as a white solid. Yield: 99%. M.p.: 263 ꢂC. H CH2Cl2 (3 ꢃ 20 mL). The compound is puried by precipitation
NMR (400 MHz, CDCl3) d (ppm) 8.02–7.89 (m, 2H), 7.42–7.16 with Et2O (50 mL) and is washed with Et2O (2 ꢃ 25 mL) to give
(m, 5H), 7.04 (d, J ¼ 8.6 Hz, 1H), 4.27 (d, J ¼ 8.3 Hz, 1H), 4.22– 12 g of the compound as a white solid. Yield: 72%. M.p. ¼ 181
4.03 (m, 3H), 3.98 (s, 3H), 3.87 (s, 3H), 3.70 (t, J ¼ 7.7 Hz, J ¼ 7.7 ꢂC. 1H NMR (400 MHz, CDCl3) d (ppm) 7.34 (m, 5H), 6.4 (m, 1H),
Hz, 1H), 3.52–3.35 (m, 1H), 3.34–3.20 (m, 2H), 2.98 (d, J ¼ 7.0 4.08 (m, 1H), 3.55 (m, 1H), 3.29 (m, 1H), 3.10–2.75 (m, 3H), 2.69
Hz, 2H). 13C NMR (100 MHz, CDCl3) d (ppm) 166.1, 155.2, 135.1, (m, 1H), 0.95–0.59 (m, 4H). ESI-MS[M]+ ¼ 221.16.
131.2, 131.1, 129.3, 129.2, 127.7, 123.5, 123.2, 111.8, 66.3, 56.7,
N-((1S,2R)-1-Benzyl-3-cyclopropylamino-2-hydroxypropyl)-
55.8, 52.5, 51.4, 33.5. RP-HPLC: 6.75 min, purity: 98.5%, ESI-MS (2E,6E)-4-methoxy-3-(3,7,11-trimethyldodeca-2,6,10-trienyla-
[M]+ ¼ 346.14.
mino)benzamide (38). To a solution of (2R,3S)-3-amino-1-
3-[(2S,3R)-3-[(2E,6E)-(3,7-Dimethylocta-2,6-dienylamino)]-2- cyclopropylamino-4-phenylbutan-2-ol ditriuoro-acetate salt
hydroxy-4-phenyl-butylamino]-4-methoxy benzoic acid methyl (37) (310 mg, 0.69 mmol) in DMF anhydrous (2 mL) is sequen-
ester (35). To a solution of (2S,3R)-3-(3-amino-2-hydroxy-4-phe- tially added a solution of (17a) (2E,6E)-4-methoxy-3-(3,7,11-tri-
nylbutylamino)-4-methoxy-benzoic acid methyl ester (33) methyldodeca-2,6,10-trienylamino) benzoic acid (234 mg,
(100 mg, 0.29 mmol) in DMF anhydrous (3 mL) is added geranyl 0.63 mmol) in DMF anhydrous (1 mL), DIPEA (0.7 mL,
bromide (0.8 mL, 0.58 mmol) and Et3N (0.08 mL, 0.58 mmol). 3.80 mmol), HOBt (1.0 mL, 1.00 mmol) and EDC (181 mg,
ꢂ
The mixture is stirred for 16 hours at 70 C under a nitrogen 1.00 mmol). The mixture is stirred, under a nitrogen atmo-
atmosphere, and then the reaction mixture is cooled at room sphere, for 16 hours at room temperature, and then HCl 1 N is
temperature. The reaction mixture is diluted with CH2Cl2 (25 added until pH 7. Aerwards H2O (50 mL) is added and the
mL) and is washed with H2O (2 ꢃ 25 mL) and the organic phase mixture is extracted with AcOEt (3 ꢃ 50 mL). The combined
is dried with anhydrous Na2SO4, ltered and concentrated organic extracts are dried with MgSO4 anhydrous, ltered and
under reduced pressure to give a residue which is puried by concentrated under reduced pressure to give a residue which is
column ash chromatography using CH2Cl2/10% MeOH as the puried by ash column chromatography using CH2Cl2/10%
486 | Med. Chem. Commun., 2014, 5, 474–488
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MeOH as an eluent to give the desired compound as a white cDNA). Cells were grown in a culture medium consisting of
1
ꢂ
solid. Yield: 47%. M.p. ¼ 205 C. H NMR (400 MHz, CDCl3) d DMEM supplemented with 2% foetal bovine serum, 1% peni-
(ppm) 7.38–7.12 (m, 5H), 6.89 (dd, J ¼ 8.1 Hz, J ¼ 2.1 Hz, 1H), cillin-streptomycin, 1% L-glutamine and 200 mg mLꢀ1 G418.
6.87 (d, J ¼ 1.7 Hz, 1H), 6.69 (d, J ¼ 8.1 Hz, 1H), 6.39 (d, J ¼ 8.3 Cells were seeded in a 96-well culture microplate, at 5000 cells
Hz, 1H), 5.35 (dt, J ¼ 6.5 Hz, J ¼ 0.8 Hz, 1H), 5.17–5.04 (m, 2H), per well and treatment with different compounds, at 10ꢀ5 and
4.41–4.20 (m, 1H), 4.14 (s, 1H), 3.89 (t, J ¼ 6.2 Hz, 1H), 3.85 (s, 10ꢀ6 M concentrations, was performed 24 hours aer seeding.
3H), 3.75–3.63 (m, 4H), 3.10 (d, J ¼ 6.8 Hz, 2H), 3.07–2.81 (m, 24 hours later culture media were collected and analyzed by the
3H), 2.28 (d, J ¼ 4.6 Hz, 1H), 2.20–1.91 (m, 8H), 1.72 (s, 3H), 1.68 ELISA method. OM99-2, 3 mM, (Bachem, ref.: H-5108) a BACE
(d, J ¼ 0.8 Hz, 3H), 1.61 (d, J ¼ 0.6 Hz, 3H), 1.60 (s, 3H), 0.60 (d, J inhibitor, was used as the Ab(1–40) secretion reduction positive
¼ 10.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) d (ppm) 169.0, 149.9, control.
139.9, 138.6, 137.4, 135.6, 131.5, 129.8, 129.4, 128.9, 127.0,
124.5, 124.0, 120.9, 115.6, 108.6, 108.3, 70.3, 68.8, 55.8, 53.3,
Acknowledgements
51.9, 41.71 39.9, 39.8, 36.6, 31.0, 27.0, 26.6, 25.9, 17.9, 16.7, 16.3,
6.0, 5.5. RP-HPLC: 9.4 min, purity: 98.3%, ESI-MS[M]+ ¼ 574.3. The authors acknowledge grant support from Comunidad de
Madrid (CAM-38/2008), CDTI's CENIT program (DENDRIA,
CEN-20101023) and the Spanish MINECO (CTQ2010-19690).
Enzyme assay: BACE-1 inhibition
The assay is based on FRET technology, and is performed in a
96 well black microplate. The nal concentration of the
substrate (Fluorogenic Peptide Substrate IV, R&D Systems, ref.:
ES004) was 3.5 mM, and the enzyme concentration (rhBACE-1
b-secretase recombinant human, R&D Systems, ref.: 931-AS) was
0.5 mg mLꢀ1. The nal volume of the assay was 100 mL per well
and all reagents were diluted in reaction buffer (sodium acetate
50 mM, pH 4.5). The compounds were tested at a concentration
of 10ꢀ5 and 10ꢀ6 M. The control in the assay was the commer-
cial b-secretase inhibitor OM99-2, from BACHEM, which was
tested at 300 nM. All the samples and controls were studied by
duplicate. The plate was mixed gently and incubated for one
hour in the dark. Changes in the uorescence were measured
using a uorimeter plate reader (FLUOstar OPTIMA, BMG
LABTECH), with 320 nm excitation lter and 405 nm emission
lter.
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