Three-Component Aminoalkylations Yielding Dihydronaphthoxazine-Based Sirtuin Inhibitors: Scaffold Modification and Exploration of Space for Polar Side-Chains

Article abstract of DOI:10.1002/ardp.201700097

Nonpolar derivatives of heterocyclic aromatic screening hits like the non-selective sirtuin inhibitor splitomicin tend to be poorly soluble in biological fluids. Unlike sp³-rich natural products, flat aromatic compounds are prone to stacking and often difficult to optimize into leads with activity in cellular systems. The aim of this work was to identify anchor points for the introduction of sp³-rich fragments with polar functional groups into the newly discovered active (IC₅₀ = 5 μM) but nonpolar scaffold 1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione by a molecular modeling approach. Docking studies were conducted with structural data from crystallized human SIRT2 enzyme. Subsequent evaluation of the in silico hypotheses through synthesis and biological evaluation of the designed structures was accomplished with the aim to discover new SIRT2 inhibitors with improved aqueous solubility. Derivatives of 8-bromo-1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione N-alkylated with a hydrophilic morpholino-alkyl chain at the thiocarbamate group intended for binding in the acetyl-lysine pocket of the enzyme appeared to be promising. Both the sulfur of the thiocarbamate and the bromo substituent were assumed to result in favorable hydrophobic interactions and the basic morpholino-nitrogen was predicted to build a hydrogen bond with the backbone Ile196. While the brominated scaffold showed moderately improved activity (IC₅₀ = 1.8 μM), none of the new compounds displayed submicromolar activity. Synthesis and characterization of the new compounds are reported and the possible reasons for the outcome are discussed.

Full text of DOI:10.1002/ardp.201700097

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700097
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Full Paper
Three-Component Aminoalkylations Yielding
Dihydronaphthoxazine-Based Sirtuin Inhibitors: Scaffold
Modification and Exploration of Space for Polar Side-Chains
4
1
1
2
3
1
€
Steffen Vojacek , Katja Beese , Zayan Alhalabi , Soren Swyter , Anja Bodtke , Carola Schulzke ,
Manfred Jung3, Wolfgang Sippl2, and Andreas Link
1
1
Institute of Pharmacy, University of Greifswald, Greifswald, Germany
Institute of Pharmacy, Martin Luther University of Halle-Wittenberg, Halle/Saale, Germany
Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany
Institute of Biochemistry, University of Greifswald, Greifswald, Germany
2
3
4
Nonpolar derivatives of heterocyclic aromatic screening hits like the non-selective sirtuin inhibitor
splitomicin tend to be poorly soluble in biological fluids. Unlike sp3-rich natural products, flat aromatic
compounds are prone to stacking and often difficult to optimize into leads with activity in cellular
systems. The aim of this work was to identify anchor points for the introduction of sp3-rich fragments
with polar functional groups into the newly discovered active (IC50 ¼ 5 mM) but nonpolar scaffold 1,2-
dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione by a molecular modeling approach. Docking studies were
conducted with structural data from crystallized human SIRT2 enzyme. Subsequent evaluation of the in
silico hypotheses through synthesis and biological evaluation of the designed structures was accomplished
with the aim to discover new SIRT2 inhibitors with improved aqueous solubility. Derivatives of 8-bromo-
1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione N-alkylated with a hydrophilic morpholino-alkyl chain
at the thiocarbamate group intended for binding in the acetyl-lysine pocket of the enzyme appeared to
be promising. Both the sulfur of the thiocarbamate and the bromo substituent were assumed to result in
favorable hydrophobic interactions and the basic morpholino-nitrogen was predicted to build a hydrogen
bond with the backbone Ile196. While the brominated scaffold showed moderately improved activity
(IC50 ¼ 1.8 mM), none of the new compounds displayed submicromolar activity. Synthesis and characteriza-
tion of the new compounds are reported and the possible reasons for the outcome are discussed.
Keywords: Docking studies / Enzyme inhibitors / Epigenetics / Mannich reaction / Sirtuins
Received: March 20, 2017; Revised: April 10, 2017; Accepted: April 17, 2017
DOI 10.1002/ardp.201700097
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
nicotinamide adenine dinucleotide (NADþ) for deacetyla-
tion of substrates [1]. In spite of the name histone
Introduction
Human sirtuin-2 (SIRT2) is a ubiquitously expressed class III
histone deacetylase (HDAC). HDACs of this class require
deacetylases, sirtuins deacylate various other proteins than
histones [2]. In the case of SIRT2, important substrates
are transcription factors (FOXO1 [3] and FOXO3a [4]),
a-tubulin [5], and glucose-6-phosphate dehydrogenase
(G6PD) [6].
Even though the role of SIRT2 as a drug target is far from
being settled, a recent study revealed that regulation of
G6PD acetylation by SIRT2 is involved in the metabolic
reprogramming of acute myeloid leukemia (AML) cells.
Inhibition of SIRT2 through small-molecules or genetic
Correspondence: Prof. Andreas Link, Institute of Pharmacy,
University of Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17487
Greifswald, Germany.
E-mail: link@uni-greifswald.de
Fax: þ49 3834 4204895
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ablation seems beneficial for the treatment of a number of
neurodegenerative diseases and AML. The molecular basis
for interference with leukemia cell proliferation is based
upon the pronounced dependence on the oxidative branch
of the pentose phosphate pathway (PPP) in these malignant
cells. The first and rate-limiting enzyme of the PPP is G6PD. It
has been reported, that SIRT2 is overexpressed in clinical
AML samples, while acetylation at lysine 403 of G6PD is
downregulated and G6PD catalytic activity is increased in
comparison to normal controls. Because SIRT2 deacetylates
G6PD at K403, it promotes production of NADPH. Pharma-
cological inhibition of SIRT2 thus leads to reduced cell
proliferation of leukemia cells, leaving normal hematopoi-
etic stem and progenitor cells unaffected. For this and other
reasons, SIRT2 may serve as a promising target for further
therapeutic investigations [6].
Replacement of the hydrogen-bond acceptor oxygen atom of
the lactone series by a nitrogen atom resulted in non-
hydrolysable, biologically active lactames such as 4. Again,
separation of enantiomers was necessary in order to investi-
gate the activity of the racemic mixture in more depth. In a
different approach toward less hydrolysable analogs we made
use of a variant of the Betti reaction [1], a classical three-
component reaction. Instead of aromatic amines, urea or
thiourea were reacted with suitable b-naphthols and aro-
matic aldehydes. In an operationally simple one-pot reaction,
we were able to obtain compounds such as 5 and 6 in racemic
form. While both of these new splitomicin analogs displayed
the desired activity against the target enzyme in low
micromolar concentrations in a trypsin-coupled homoge-
neous assay (IC50 ¼ 2.6 ꢀ 0.3 mM for 5 and IC50 ¼ 6.7 ꢀ 0.9 mM
for 6), the molecules still require laborious chiral separation
and are characterized by pronounced lipophilicity. In order to
tackle both of these issues at the same time, we designed
achiral naphtho[1,3]oxazin-2-one 7 and naphtho[1,3]oxazine-
2-thione 8 derivatives (Fig. 1) and synthesized these two
compound series via solvent-free one-pot syntheses.
While these molecular frameworks are closely related to 3
in terms of hydrogen-bond-acceptor properties, the carba-
mate and thiocarbamate structures are much less sensitive to
hydrolysis than the lactone ring of splitomicin (1). The shift of
the substituent from the benzylic carbon atom to the nitrogen
atom resulted in a still non-flat pattern but without the
formation of a chiral center. The exchange of aromatic
substituents in position R2 for aliphatic side-chains with polar
hetero atoms as solubility enhancing motifs was intended to
cure the problems associated with poor solubility.
Results and discussion
The non-selective sirtuin inhibitor splitomicin [7] (1) is a
screening hit that was used as starting point for hit-to-lead
optimization seeking novel chemotypes inhibiting SIRT2.
Splitomicin (1) is a fragment-like, flat molecule with Mr <200.
The hydrolysis-sensitive lactone is inactive in cellular systems
and was analyzed in structure–activity studies, resulting in
b-phenyl-8-bromo derivatives such as compound 2 that shows
a manifest improvement in terms of stability and target
inhibition (Fig. 1). Because docking studies as well as
biological tests with recombinant human SIRT2 indicated
that the (R)-1-phenyl splitomicin derivative 3R was markedly
more active than its (S)-enantiomer 3S, the individual
investigation of enantiomers was regarded mandatory in
order to obtain valid SAR data within this series of
compounds [8].
We started our docking study using hSIRT2 in complex with
the inhibitor EX-243 ((1S)-6-chloro-2,3,4,9-tetrahydro-1H-
carbazole-1-carboxamide, PDB ID 5D7P). Interestingly, two
inhibitor molecules are interacting in the C- and extended-C
Figure 1. Splitomicin (1), hydrolysable derivatives (2–3) and less hydrolysable analogs (4–8).
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Figure 2. (a) Crystal structure of hSIRT2 complexed with the potent inhibitor EX-243 (green carbon atoms). Two inhibitor molecules
are observed in the crystal structure, one bound to the nicotinamide site and one bound in the extended-C site of hSIRT2. Hydrogen
bonds are shown as dashed lines. The cocrystallized ADPR molecule is colored in salmon. (b) Top-ranked docking solution for 8b (cyan
carbon atoms) in comparison to the cocrystallized inhibitor EX-243 (same coloring scheme as in a). (c) Docking solutions for 8b (cyan
carbon atoms) in comparison to the cocrystallized inhibitor EX-243 (same coloring scheme as in b).
site of hSIRT2 [9] (Fig. 2a). The docked 1,2-dihydro-3H-
naphth[1,2-e][1,3]oxazine-3-thiones 8a and 8b were found to
interact similarly as observed for Ex-243 (Fig. 2b). The sulfur
atom of the thiocarbamate makes favorable hydrophobic
interactions with Ile193 that might explain the loss of activity
for the more polar carbamate derivatives. Moreover, the
8-bromo substituent indicated good hydrophobic interactions
with the enzyme (Phe119, Leu134, Ile232). The tricyclic
moieties of both inhibitors are nicely superimposed. We also
observed that as in case of EX-243 (PDB ID 5D7P) and CHIC35
(PDB ID 5D7Q) two inhibitor molecules of 8a and 8b can be
bound simultaneously in the hSIRT2 pocket. The top-ranked
docking solution is found for the nicotinamide binding site
whereas the second molecule interacts at the extended C-site.
The high structural similarity to the cocrystallized EX-243 is
obvious (Fig. 2c).
the exit of the acetyl-lysine pocket of the enzyme (Fig. 3)
where the second inhibitor molecule of EX-243 is bound in the
hSIRT2 crystal structure.
Our initial synthetic strategy to access the target compound
was to prepare an unsubstituted naphthoxazine thione (e.g.,
8a) with subsequent alkylation of the nitrogen atom. Key
reaction of this approach was the formation of an electro-
philic aromatic quinone methide in situ and subsequent
oxazinthione-ring closure employing inorganic potassium
thiocyanate [10].
Dimethylmethylidene ammonium iodide, a dimethylami-
nomethylation agent known as Eschenmoser’s salt, was
reacted with 2-naphthol under basic conditions, resulting in
the formation of the corresponding Mannich bases 9 and 10,
respectively (Scheme 1). Using Eschenmoser’s salt, superior
regioselectivity and predominating mono-substitution com-
pared to the classical Mannich reaction with formaldehyde
and dimethyl amine, was anticipated [11]. In the next step, the
In case of the substituted derivatives, the hydrophilic
morpholino-alkyl-chain in position R2 was proposed to bind at
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Figure 3. Docking result for the substituted derivative 8d (orange carbon atoms). The two cocrystallized inhibitor molecules of EX-
243 are shown as green sticks. The molecular surface of the binding pocket is displayed and colored according to the hydrophobicity
(polar regions are colored magenta, hydrophobic regions are colored green).
resulting Mannich product was exhaustively alkylated with
methyl iodide, and subsequently reacted with potassium
thiocyanate [10]. Despite repeated attempts to purify the
quaternized intermediate resulting from exhaustive alkyl-
ation by established procedures, we were unable to obtain
the ammonium salts in pure form. While the progress of the
reaction could be deduced from the precipitation observed,
purification of the precipitate was neither possible via silica
gel chromatography on normal nor on reversed phase,
because of compound polarity. On normal silica gel phases,
the compounds could not be eluted, because of the very
strong interactions with the stationary phase. Quite to the
contrary, preparative endcapped RP-18 HPLC columns were
unsuitable, because of very weak retention of the com-
pounds. Due to a lack of thermal- and photo-stability of the
quaternary products, recrystallization was not effective,
either. So the intermediate was collected by filtration,
washed, dried, and used without further purifications for
the next step. A mechanistic reason for the failure to isolate
the quaternary compounds can be attributed to the possible
formation of quinone methide species. Most probably, highly
reactive and short-lived quinone methide intermediates were
generated. While these quinone methides formed in situ were
too unstable to be isolated, they could efficiently be trapped
by reaction with thiocyanate ions to yield the desired isolated
products [12]. In addition to analytical characterization by
Scheme 1. First synthetic strategy. Dimethylaminomethylation followed by exhaustive methylation and subsequent
thiocarbamylation.
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Figure 4. X-ray crystallography confirmed structure of compound 8a. Ellipsoids are shown at the 50% probability level.
NMR and MS spectroscopy, the formation of the desired
product 8a was confirmed by X-ray crystallography (Fig. 4).
The ring system under investigation contains a thiocarbamate
with an exocyclic carbon sulfur double bond ROC(¼S)NR2,
which can be called O-organyl thiocarbamate or thionour-
ethane in order to indicate the difference to another isomeric
thiocarbamate, with an exocyclic carbonyl group RSC(¼O)NR2,
which could be called an S-organyl thiocarbamate or
thiolurethane.
similarities, Mannich and Betti reactions differ by the use of
a secondary amine in place of ammonia and an enolizable CH-
acidic compound instead of an electron-rich 2-naphthol [18].
In certain cases, however, for instance in the synthesis of the
alkaloid gramine by aminomethylation of indole, it could be
argued that this atypical Mannich reaction could as well or
even better be classified as atypical Betti reaction [19].
In order to synthesize compounds of classes 7 and 8,
benzaldehyde had to be replaced by paraformaldehyde in
order to obtain 1-aminomethyl-2-naphthols with two ben-
zylic protons as intermediates under comparable 3-compo-
nent Betti reaction conditions. However, in our case this did
not result in the anticipated formation of primary amines, but
yielded the bis-Betti-product 12. The attempted hydrolysis of
the N,O-acetals 12 would not cleave the C–N single bond to
the required primary amines. Thus the 1-aminomethyl-2-
naphthol intermediate 17 was synthesized by reaction of
2-naphthol with methenamine in the presence of acetic acid
with subsequent hydrolysis [20]. This so-called Duff reaction
can be regarded as related or similar to the observation
Because of the modest overall yield of the 1,2-dihydro-3H-
naphth[1,2-e][1,3]oxazine-3-thiones 8a and 8b of not more
than 10% (Scheme 1), an alternative route to the desired test
compounds 8d was investigated. In contrast to the strategy
described above, we pursued a synthesis with 1-aminomethyl-
2-naphthols as intermediates. It was known from the synthesis
of 6, that there are a number of suitable reagents, for instance
thiophosgene [13], 1,10-thiocarbonyldiimidazole [14] or car-
bon disulfide [15] available that are suited to transform the
synthesized intermediate aminomethyl-2-naphthols into the
desired heterocyclic carbamates 7 and thiocarbamates 8.
The electrophilic aromatic substitution of an in situ formed
benzaldimine with 2-naphthol was described by Betti even
before the related 3-component reaction was reported by
Mannich [16]. In a typical 3-component Betti reaction,
2-naphthol reacts in the presence of an ammonia source
with benzaldehyde derivatives to give 1,3-diphenyl-2,3-
dihydro-1H-naphth-[1,2-e][1,3]-oxazines. After hydrolysis of
the intermediately formed N,O-acetals, the racemic Betti
bases 11 with primary amino groups are formed
(Scheme 2) [17]. Cyclization of compounds of type 11 yielded
compounds 5 and 6. To our understanding this 3-component
aminoalkylation reaction can be interpreted as a Mannich
reaction in the broadest sense, or with respect to the historical
chronology, vice versa. However, despite mechanistic
€
published by Mannich and Krosche [18], that methenamine
reacted in acidic solution with antipyrine, an electron-rich
aromatic drug, which led Mannich to the development of the
synthetic use of this phenomenon. Whereas the unwanted
reaction of methenamine and antipyrine results in an
aminomethylene bonded trimeric pyrazolone compound [21],
in the Duff reaction the bis-naphtol-derivative 13 (respec-
tively, 14) with an iminomethyl group is formed.
In the first step of this reaction, protonated methenamine,
acting as an electrophile, forms the aminomethyl group as
condensation product with naphthol. Remarkably, in the
course of the reaction methenamine acts as an oxidizing
agent, as well [22]. The N,N-acetal-carbon of methenamine is
reduced to methylamine, meanwhile the compound is
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Scheme 2. Synthetic strategy. Betti reaction employing paraformadehyde instead of the benzaldehyde derivative.
oxidized to an imine [23]. In the next step, the imine
concurrently reacts with another methanal molecule and a
second molecule naphthol to form the anticipated dimer.
Hydrolysis of this intermediate results in two different
naphthol derivatives: the primary amine and the aldehyde
(Scheme 3). Consequently, the Duff synthesis suffers from the
fact, that only half of the starting 2-naphthol can be
converted into the aldehyde. On the other hand, it is reported
that the yield of the respective aldehyde could be improved by
hydrolysis of the intermediate imine 13 in aqueous acetic acid.
One hydrolysis product, the primary amine, becomes oxidized
by remaining methenamine yielding 1-iminomethyl-2-naph-
thol which is hydrolyzed to the aldehyde, directly. Although
the drawback of the Duff reaction to yield the aldehyde in
only 50% could thus be overcome by the above operation [23],
we pursued an alternate synthesis employing the Gatter-
mann–Adams reaction (Scheme 4) [24]. Hence, the 2-naphthol
derivative was stirred with zinc cyanide in diethyl ether.
Subsequent treatment with hydrochloric acid gas released
zinc chloride and likewise HCN, which was protonated to form
the intermediate iminium ion. Additionally, the zinc ions
catalyzed due to their Lewis acid character the aromatic
substitution of the electrophilic iminium yielding more than
85% of the aldehyde (based on recovered starting material).
In the second step, formation of the 1,2-dihydro-3H-
naphth[1,2-e][1,3]oxazine-3-thione ring was envisioned. In
order to achieve this goal, the thiocarbamate synthesis using
carbon disulfide under basic conditions seemed to be the most
promising strategy. The 1-aminomethyl-2-naphthol reacted
with CS2 in basified methanol to a dithiocarbamate anion. The
following addition of hydrogen peroxide caused the ring
closure reaction to the thiocarbamate, presumably via an
isothiocyanate intermediate [15]. Most likely, an intermediary
dithiocarbamate disulfide [25] species emerged and subse-
quently disintegrated by ring closure reaction to the cyclic
thiocarbamate.
In an analogous approach reported by Keck et al. [15],
2-hydroxymethyl aniline derivatives treated with one equiva-
lent of triethylamine, three equivalents of carbon disulfide
and two equivalents of hydrogen peroxide for disintegration
yielded the inverse thionourethane in 78% yield. Under the
same conditions but starting from aminomethyl-naphthol
instead of 2-hydroxymethyl aniline, we were only able to gain
the desired product in 24% yield despite performing several
attempts. Possibly, the reason for the much lower yield in our
case compared to the inverse oxazine-thione is the dissimilar-
ity in acidity of the different starting materials. In contrast to
the weakly basic aniline derivative with only one nucleophilic
center employed by Keck et al. [15], 1-aminomethyl-naphth-2-
ol possesses two potentially nucleophilic groups, a basic
aliphatic amino group and a phenolic OH-group. Most likely
in our hands the phenol was partly deprotonated, thus
forming a strong nucleophile giving rise to the formation of
xanthates as side-products and hampering the ring closure
reaction.
Once the envisioned thiocarbamate was synthesized in
moderate yield, the derivatization was to be conducted with a
strong base and subsequent addition of a alkyl halide in a
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Scheme 3. Third synthetic strategy. Duff reaction with different attempts toward derivatization.
third step [26]. In order to deprotonate the N–H group,
triethylamine was added [27]. Despite cooling or changes in
solvent properties, the addition of the alkyl halide (e.g.,
chloride, iodide, or bromide) caused decomposition of the
thiocarbamate in all attempts. Most of the degradation
products emerged as amorphous solid, insoluble in common
solvents like DMSO or DMF. Therefore only the supernatant
could be purified, yielding 5.5% of the 1,10-[thiobis-
(methylene)]bis(naphthalen-2-ol) as yellowish crystals. The
isolated by-product indicated that once the sulfur was
Scheme 4. Alternative synthesis route. Gattermann–Adams reaction.
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alkylated, the sulfonium ion forced the cleavage to occur.
Probably, a thiocyanante derivative was eliminated, yielding a
naphthoquinone methide. The intermediate quinone
methide as a strong nucleophile could react with another
thiocarbamate as sulfur donating molecule. Eventually an
intramolecular rearrangement caused the connection to the
accessible thiomethyl-naphthol as nucleophile with subse-
quent elimination of a cyano-group containing molecule,
explaining the formation of a thioether.
The rearrangement of substituted thiourea derivatives via
methyl mercaptan elimination is described in the litera-
ture [28]. According to these reports, the methyl iodide
reacts with the sulfur atom of thiourea derivatives. Upon
basification, methyl mercaptan can be eliminated. Depend-
ing on the nucleophile, the compounds under investigation
rearrange to cyclic isourea [13] or guanidine derivatives [29],
respectively. Although the transformations discussed here
are aimed at the formation of thionourethanes, from our
preliminary observations together with the knowledge
available from reports on thiourea rearrangement reactions,
we draw the conclusion, that a different route toward these
thiocarbamate derivatives had to be pursued. Based on our
analysis of this problem, derivatization of carbamates
appeared more suitable than alkylation of thiocarbonyl
containing compounds. Thus, step three was reformed and
divided into the synthesis of substituted carbamates and an
additional step four, where the oxo groups should be
exchanged for a thiono group with the help of Lawesson’s
reagent [30].
The conceptual basis for the intended carbamylation was
the successful model reaction of 17 and 18 with 1-chloro-1-(4-
nitrophenyl)formate (NPCF) in dioxane yielding 7a and 8a,
respectively [31]. This transformation had allowed us to avoid
highly toxic and hazardous reagents such as phosgene. On the
other hand, the reaction produced only small amounts of the
desired product, e.g., 7a in 7% yield (Scheme 3). Hence,
further experiments like derivatization or thionylation of 7a,
could not be pursued due to a lack of material.
Because the ring closure reaction of the 1-aminomethyl-2-
naphthol to the thiocarbamate was effective but derivatiza-
tion failed, and the alternative carbamylation route suffers
from poor yields, synthetic access was attempted via
derivatization of the 2-hydroxy-naphth-1-yl-methanal 15
using a modified reductive amination [32] with subsequent
thiocarbamylation of the secondary amine. The aldehyde 15
was stirred with 3-morpholinopropan-1-amine in methanolic
solution to form a yellowish imine. After a short reaction time,
the reducing agent sodium borohydride was added [32].
Addition of solid borohydride in portions to the cooled
solution led to a sluggish reaction with the formation of
multiple by-products lowering yield and thwarting purifica-
tion. With the aim of improving the outcome of this reaction,
NaBH4 was dissolved in a small amount of water and added as
aqueous solution. After extraction and subsequent addition
of HCl gas, the secondary amine could thus be obtained as its
hydrochloride salt in almost 70% yield.
In the next step, thiophosgene was used to acquire the desired
cyclic thiocarbamate in dichloromethane in good yields using
triethylamine to neutralize released HCl. Alternatively, 1,10-
thiocarbonyldiimidazole (TCDI) was used as solid reactant.
Advantageously, besides the low toxicity of TCDI, the released
imidazole molecules made the addition of triethylamine
unnecessary.
Overall, the synthesis of new test compounds with
naphthoxazinethione scaffold and a hydrophilic side chain
intended as water-soluble SIRT2 inhibitors was conducted and
reactions were optimized. Key of the most suitable reaction
was the formation of 2-hydroxy-1-naphthaldehyde by the
Duff reaction, yielding more than 75% product referring to
the use of two equivalents of 2-naphthol. Although the
Gattermann–Adams reaction led to even higher yields (based
on recovered starting material) of aldehyde 15 compared to
the Duff synthesis, the Duff reaction using comparatively
cheap and non-hazardous methenamine was advantageous
with respect to improved safety, reduced expenditure and
lower costs.
The following reductive amination was performed by
combining both the formation of the imine and the reduction
with sodium borohydride in one pot. The secondary amines 19
and 20 were gained as HCl-salt by treatment of extracted
organic phases with hydrochloric acid gas. The conversion to
the thionocarbamates was realized by the addition of
thiophosgene and an auxiliary base. Compound 8c was also
synthesized from 19 by an alternative route using TCDI.
Advantageously, the synthesis could be conducted without
adding further bases, because released imidazole accepts
protons from the hydrochloride salt 19.
The 4-step synthesis for the bromo-substituted product 8d
could be achieved in a satisfactory overall yield of 26.6% using
TCDI as ring closure reagent.
Selected compounds were tested for inhibition of human
NAD-dependent deacetylase hSIRT2 in a fluorescence assay,
with IC50 determined for the best inhibitors (Table 1,
Fig. 5) [33]. Unexpectedly the thionocarbamates 8c and 8d,
the most promising compounds revealed by prior docking
studies on hSIRT2, showed only moderate inhibition. To the
contrary, both the non-derivatized naphtho[1,3]oxazine-2-
thione 8a and its bromo derivative 8b exhibited good
inhibition. An explanation for the only moderate inhibition
of 8c/8d is the high hydrophobicity of the extended C-site
(Leu138, Ile169, Phe190, Leu206). We calculated the hydro-
phobic interaction between a methyl probe and the extended
C-site using the GRID approach [34]. The hydrophobicity of
the extended-C site is also favorable for the binding of the
lipophilic myristoyl chain of a substrate peptide (PDB ID
4Y6L) [35] (Fig. 6a and b).
The results of the biochemical assay revealed that both
small molecules inhibited the enzyme with IC50 values of 5.0
and 1.8 mM, respectively. Apparently, the hydrophilic sp3-rich
side chain elongations hinder binding in the catalytic cleft,
which was not expected from the anticipated docking pose.
Nevertheless, the sulfur appeared to be important for
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Table 1. Biological test data of some selected compounds
against hSIRT2 in an assay with a fluorescence-labeled
N-acetyl-lysine derivative (ZMAL) [36] trypsin-coupled
homogeneous assay.
these derivatives with potential chelating properties were
able to complex the allosteric zinc ion of the enzyme forcing a
change in conformation resulting in decreasing its efficacy.
Entry
IC50 or percentual
inhibition of hSirt2
Conclusion
Nicotinamidea)
rac-5
rac-6
7a
7b
7c
7d
8a
8b
8c
8d
19
49.8 ꢀ 4.6 mM
2.6 ꢀ 0.3 mM
6.7 ꢀ 0.9 mM
n.i.b) @ 25 mM
n.i. @ 25 mM
n.i. @ 25 mM
n.s.c)
5.0 ꢀ 0.4 mM
1.8 ꢀ 0.3 mM
11% @ 25 mM
n.i. @ 25 mM
33% @ 25 mM
33% @ 25 mM
n.i.% @ 25 mM
19% @ 25 mM
The synthesis of 1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-
thiones 8 intended as intermediate led to the discovery of
achiral splitomicin analogs with improved stability and
significant biological activity. The most active new compound
was the brominated scaffold 8-bromo-1,2-dihydro-3H-
naphth[1,2-e][1,3]oxazine-3-thione 8b [IC50 ¼ 1.8 mM]. Be-
cause these novel scaffolds for diversity showed promising
results, the designed N-alkylated derivatives with a hydro-
philic morpholino-alkyl chain at the thiocarbamate that
appeared to be promising were synthesized and tested. A
thorough investigation of classical chemical name reactions
was necessary in order to obtain the test candidates in
practical yields for biological evaluation. While the predicted
favorable hydrophobic interactions of the sulfur of the
thiocarbamate and the bromo substituent resulted in
the good activity of 8b, the anticipated hydrogen bond of
the basic morpholino-nitrogen with the backbone Ile196 did
not, most likely due to the high hydrophobicity of this region.
Soaking experiments with 8b should give further insights and
guide the way for an alternative position for the connection
of additional binding motifs necessary for improved solubility
and affinity.
20
22
23
a) Nicotinamide is shown as a reference inhibitor.
n.i. ¼ no
b)
inhibition (<10% inhibition). c) n.s. ¼ not sufficiently soluble
in DMSO.
inhibition whereas the oxo-derivatives, e.g., 7a and 7b
showed no inhibition under the same conditions. Most likely
the more space-filling and lipophilic sulfur-containing com-
pounds could fit more suitably in the binding pocket with
increased efficacy by interacting with Ile93 which is mainly
driven by van-der-Waals interaction. In addition, the sulfur
atom is known to be much more polarizable than the oxygen
atom, which may account for the observed differences of
closely related analogs as well.
Experimental
Chemistry
General remarks
Interestingly the amino-naphthol derivatives 19 and 20
appeared to be also moderate inhibitors of Sirt2. Possibly
All chemicals and solvents were purchased from commercial
suppliers and used without further purification. Melting
Figure 5. Additional target compounds which were tested in the biological assay.
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Figure 6. (a) Docking result of compound 8d (orange carbon atoms) in comparison to the co-crystallized myristoyl-lysine peptide
(magenta carbon atoms) which was found to be a substrate of hSIRT2 (PDB ID 4 ꢁ 3O). The distance between the thione sulfur and
̊
Ile93 is given in A (same coloring scheme as in Fig. 3). (b) Hydrophobic interaction calculated for the extended C-site (shown as green
contour plot, C3 probe, contour level ꢂ2.2 kcal/mol) (same coloring scheme as in panel a).
€
points were determined with a Buchi “Schmelzpunkt M-565”
apparatus and are uncorrected. Microwave-assisted synthesis
was performed using a microwave synthesis reactor Mono-
wave 300 (“closed vessel” mode, G30-vials: 20 mL total
capacity vessel, temperature control via IR sensor) from
Anton Paar, while stirring at 600 rpm. NMR spectroscopic
measurements were recorded with a Bruker Biospin Avance III
Ultrashield 400 instrument (1H: 400.2 MHz, 13C: 100.6 MHz).
Samples were dissolved in deuterated solvents, and chemical
shifts (d) in 1H and 13C NMR spectra are given in parts per
million (ppm) with tetramethylsilane (TMS) signals as refer-
ence. Abbreviations are defined as follows: s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet. Mid-infrared spectra were
recorded on an ALPHA FT-IR instrument from Bruker Optics
with diamond ATR accessory. Elemental analyses (C, H, N, and
S) were carried out with an Elementar Vario micro elemental
analyzer. TLC was performed using pre-coated aluminum foil
sheets silica gel 60 F354 provided by Merck. Flash chromatog-
raphy and medium pressure liquid chromatography (MPLC)
were performed using silica gel 60 (particle size 50–100 mm,
was carried out using the MMFF94ꢁ force field implemented
in Molecular Operating Environment System (MOE) 2012.10
(Chemical Computing Group, Montreal, Canada). All com-
pounds were used in their neutral form. A maximum of 100
conformations were generated for each ligand using the
Conformational Search module implemented in MOE.
Sirt2 protein structures in complex with small molecule
inhibitors were downloaded from the Protein Data Bank (PDB
ID 5D7P and 5D7Q) [9]. Currently there are 21 crystal
structures of human Sirt2 stored in the PDB. We took the
protein structures cocrystallized with inhibitors structurally
similar to the compounds under study. All protein structures
were prepared by using the Structure Preparation module in
MOE. Hydrogen atoms were added, for titratable amino acids
the protonation state was calculated using the Protonate 3D
module in MOE. Protein structure was energy minimized
using the AMBER99 force field [37] using a tethering force
̊
constant of (3/2) kT/2 (s ¼ 0.5 A) for all atoms during the
minimization. AM1-BCC charges [38] were used for ligands.
All molecules except the zinc ion were removed from the
structures.
€
140–270 mesh provided by Macherey-Nagel) with Buchi
devices C-630, C-601, C-615, and C-660 (column length
40 cm, column diameter 3.5 cm). High performance liquid
chromatography (HPLC) was performed using Shimadzu
devices CBM-20A, LC-20A P, SIL-20A, and FRC-10A with a
SPD 20A UV-Vis detector and LiChrospher100 RP-18 end-
capped (250 ꢁ 25 mm) HPLC columns.
Protein-ligand docking was performed using program
GOLD 5.2 [39]. Phe96 was used to define the size of the grid
box (15 A radius). The ligand was treated as flexible and 20
̊
docking poses were calculated for each inhibitor. All other
options were left at their default values. The top-ranked pose
from each docking run was included in the final analysis and
viewed graphically together with the protein structure using
the program. Using the docking setup, the two cocrystallized
inhibitors EX-243 and CHIR35 could be correctly docked into
The NMR spectra as well as the InChI codes of the
investigated compounds together with some biological
activity data are provided as Supporting Information.
̊
its crystal structure with RMSD values below 0.8 A (PDB ID,
Computational methods
5D7P, and 5D7Q).
3D structures of all compounds under study were generated
from SMILES strings, and a subsequent energy minimization
The hydrophobic interaction at the extended C-site was
calculated using the GRID approach [34] implemented in the
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Table 2. Crystal data and structure refinement parame-
ters for 8a.
MOE program. The interaction was calculated for the region
around the docked inhibitor 8d using the hydrophobic methyl
probe (C3) and was contoured at an interaction energy level
of ꢂ2.2 kcal/mol. The protein structure PDB ID 5D7P was used
for the calculation.
Binding free energies for the inhibitors under study were
calculated using the top-ranked docking poses. Structurally
conserved water molecules included for docking studies were
maintained during the geometry optimization of the com-
plexes. The protein–inhibitor complexes were energy mini-
mized using the AMBER PFROSST force field and the GBSA
solvation model implemented in MOE 2012.10.
Parameters
8a
Formula
C12H9NOS
215.26
Monoclinic
P21/n
Formula weight
Crystal system
Space group
Z
4
̊
a, A
5.0351 (10)
10.062 (2)
19.736 (4)
90.10 (3)
999.9 (3)
293 (2)
0.71073
0.291
1.430
̊
b, A
̊
c, A
b, deg
3
̊
V, A
T, K
X-ray crystallography
̊
A suitable single crystal of compound 8a was mounted on a
thin glass fiber coated with paraffin oil. X-ray single-crystal
structural data were collected at room temperature using a
STOE-IPDS 2T diffractometer equipped with a normal-focus,
2.4 kW, sealed-tube X-ray source with graphite-monochro-
l(MoKa), A
m, mmꢂ1
Dcalcd, g/cm3
F (000)
448
Collected reflections
Unique reflections
Rint
9967
2690
0.0840
0.971
̊
mated Mo Ka radiation (l ¼ 0.71073 A). The program XArea
was used for integration of diffraction profiles; a numerical
absorption correction was applied with the programs X-Shape
and X-Red32; all from STOE © 2010. The structure was solved
by SIR92 [40] and refined by full-matrix least-squares methods
using SHELXL-2013 [41]. The non-hydrogen atoms were
refined anisotropically. The hydrogen atoms were refined
isotropically on calculated positions using a riding model with
their Uiso values constrained to 1.2 Ueq of their pivot atoms.
All calculations were carried out using SHELXL-2013 and the
WinGX system, Ver1.70.01.61. Basic crystallographic data and
structure refinement parameters are summarized in Table 2.
Crystallographic data have been deposited with the Cam-
bridge Crystallographic Data Centre with deposition number
CCDC 1538210. Copies of the data can be obtained free of
charge by contacting the CCDC via e-mail: deposit@ccdc.cam.-
ac.uk or its webpage https://www.ccdc.cam.ac.uk/.
GOF on F2
b)
R1a), Rw [I > 2s (I)]
0.0467, 0.1067
0.1004, 0.1307
0.260, ꢂ0.204
R1, Rw (all data) ꢂ3
̊
Dr max/min (e A
)
h n
o
n
oi
1ꢂ2
ꢀ
ꢁ
ꢀ
ꢁ
2
2
a) R1 ¼ S||Fo| ꢂ |Fc||/S|Fo|. b)Rw ¼ S w F2o ꢂ F2c =S w F2o
:
(C12, C120), 130.1 (C7), 130.4 (C5), 131.7 (C4a),ꢃ137.4 (C13),
139.7 (C10), 147.7 (C2), 149.0 (C15) ppm; FT-IR (n): 3149 cmꢂ1
(m), 1747 cmꢂ1 (s), 1582 cmꢂ1 (m), 1499 cmꢂ1 (m), 1219 cmꢂ1
(s), 1109 cmꢂ1 (m); HRMS (ESI): found 368.0273, calcd. for
C
19H14NO2Br [MþH]þ m/z ¼ 368.0281.
8-Bromo-1-(p-tolyl)-1,2-dihydro-3H-naphth[1,2-e][1,3]-
oxazine-3-thione (rac-6)
8-Bromo-1-(p-tolyl)-1,2-dihydro-3H-naphtho[1,2-e][1,3]-
oxazin-3-one (rac-5)
To a suspension of 11 (0.20 g, 0.5 mmol) in 10 mL tetrahydro-
furan, 1,10-thiocarbonyldiimidazole (0.13 g, 0.74 mmol, 1.4
equiv.) was added in a G30 vial. The vial cap was closed and
the vial was placed in the reaction chamber of the microwave
reactor. The reaction was conducted at 250 W maximum to
reach the reaction temperature of 100°C within 5 min and
temperature was held for 10 min. Afterwards the solvent was
evaporated and residue was purified by column chromatog-
raphy (n-hexane/ethyl acetate/acetic acid; 10:10:1). Product
was crystallized from ethyl acetate/n-hexane, yielding com-
pound as colorless needles. Yield: 0.08 g (38.6%); mp: 207.5°C;
1H-NMR (DMSO-d6, 400 MHz): d ¼ 2.23 (s, 3H, C(14)H3), 6.20 (s,
1H, C(9)H), 7.14 (d, 2H, C(12)H, C(120)H, J ¼ 8.8 Hz), 7.17 (d, 2H,
C(11)H, C(110)H, J ¼ 8.8 Hz), 7.53 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.61
(d, 1H, C(7)H, J ¼ 8.8 Hz), 7.74 (d, 1H, C(8)H, J ¼ 8.8 Hz), 8.02 (d,
1H, C(4)H, J ¼ 9.2 Hz), 8.28 (s, 1H, C(5)H), 11.11 (s, 1H, NH) ppm;
13C-NMR (DMSO-d6, 100 MHz): d ¼ 20.6 (C14), 53.5 (C9), 119.8
(C1), 117.5 (C3), 118.7 (C6), 125.4 (C8), 127.2 (C11, C110), 127.3
(C8a), 129.5 (C12, C120), 129.8 (C4), 130.3 (C7), 130.5 (C5), 132.2
To a suspension of 11 (0.57 g, 1.5 mmol) in 10 mL tetrahydro-
furan, carbonyl diimidazole (0.24 g, 1.5 mmol, 1.0 equiv.) was
added in a G30 vial. The vial cap was closed and the vial was
placed in the reaction chamber of the microwave reactor. The
reaction was conducted at 250 W maximum to reach the
reaction temperature of 100°C. Reaction temperature was
held at 100°C for 10 min. Afterwards the solvent was
evaporated and residue was crystallized from ethyl acetate/
n-hexane, yielding compound rac-5 as colorless needles. Yield:
0.34 g (62.3%); mp: 200.5°C; 1H-NMR (DMSO-d6, 400 MHz):
d ¼ 2.23 (s, 3H, C(14)H3), 6.16 (s, 1H, C(9)H), 7.13 (d, 2H, C(12)H,
C(120)H, J ¼ 8.0 Hz), 7.18 (d, 2H, C(11)H, C(110)H, J ¼ 8.0 Hz),
7.44 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.59 (d, 1H, C(7)H, J ¼ 9.2 Hz),
7.75 (d, 1H, C(8)H, J ¼ 9.2 Hz), 7.98 (d, 1H, C(4)H, J ¼ 8.8 Hz),
8.24 (s, 1H, C(5)H), 8.86 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 20.6 (C14), 53.3 (C9), 114.5 (C1), 118.2 (C6), 118.2
(C3), 125.4 (C8), 126.8 (C11, C110), 127.6 (C8a), 129.4 (C4), 129.5
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(C4a), 137.8 (C13), 138.3 (C10), 146.3 (C2), 180.0 (C15) ppm; FT-
3234 cmꢂ1 (w), 3117 cmꢂ1 (w), 2956 cmꢂ1 (w), 1726 cmꢂ1 (s),
1498 cmꢂ1 (m), 1220 cmꢂ1 (m), 808 cmꢂ1 (s); C12H8BrNO2
(276.97): calcd.: C, 51.83; H, 2.90; N, 5.04; found C, 51.82; H,
2.78; N, 5.43 [42].
ꢃ
IR (n): 3198 cmꢂ1 (m), 1584 cmꢂ1 (m), 1496 cmꢂ1 (s), 1145 cmꢂ1
(s), 1081 cmꢂ1 (m); HRMS (ESI): found 384.0066, calcd. for
C
19H14NOSBr [MþH]þ m/z ¼ 384.0052.
1,2-Dihydro-3H-naphtho[1,2-e][1,3]oxazin-3-one (7a)
To a solution of 17 (2.26 g, 10.8 mmol) in dioxane (20 mL) was
added triethylamine (1.65 mL, 1.20 g, 11.8 mmol, 1.1 equiv.)
while stirring under nitrogen atmosphere at room tempera-
ture. Subsequently, 4-nitrophenyl chloroformate (1.94 g,
9.6 mmol, 1.0 equiv.) pre-dispersed in dioxane (10 mL) was
added dropwise. After addition was complete, the reaction
was stirred for further 10 min and then solid was collected by
filtration. Solid was dissolved in 50 mL dichloromethane and
extracted 3 times with 30 mL ammonia-ammonium chloride
buffer (pH 9). Addition of 5 mL acetic acid to organic phase
caused a precipitation. Solid was recrystallized from toluene
yielding product as white needle-shaped crystals. Yield:
0.138 g (7.2%); mp: 193.7°C (degrad.); 1H-NMR (DMSO-d6,
400 MHz): d ¼ 4.80 (s, 2H, C(9)H2), 7.23 (d, 1H, C(3)H, J ¼ 9.2 Hz),
7.52 (t, 1H, C(6)H, J ¼ 7.6 Hz), 7.60 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.75
(d, 1H, C(8)H, J ¼ 8.2 Hz), 7.91 (d, 1H, C(4)H, J ¼ 9.2 Hz), 7.96 (d,
1H, C(5)H, J ¼ 8.0 Hz), 8.23 (s, 1H, N(10)H) ppm; 13C-NMR
(DMSO-d6, 100 MHz): d ¼ 39.9 (C9), 110.0 (C1), 116.6 (C3), 122.4
2-(3-Morpholinopropyl)-1,2-dihydro-3H-naphtho[1,2-e]-
[1,3]oxazin-3-one (7c)
To an emulsion of 19 (1.12 g, 3.0 mmol) in 40 mL toluene/
water (1:1) was added triethylamine (2.51 mL, 1.82 g,
18.0 mmol) while stirring under argon atmosphere. To the
ice-cooled reaction mixture, a 20% solution of phosgene in
toluene (2.33 mL, 4.5 mmol) was added. Reaction was stirred
for 6 h at room temperature and quenched by addition of
ethyl acetate (40 mL) and 0.1 M hydrochloric acid solution
(40 mL). The phases were separated and the organic phase
was dried with sodium sulfate and concentrated in vacuo.
Meanwhile, the aqueous phase was pH-adjusted to pH 9 with
sodium carbonate and extracted with ethyl acetate
(3 ꢁ 50 mL). Ethyl acetate phase was dried with sodium sulfate
and hydrochloric acid gas was injected. The solid formed was
collected by filtration, dissolved in 50 mL 1 M sodium
hydroxide solution and extracted with ethyl acetate
(3 ꢁ 50 mL). Organic phase was dried over sodium sulfate
and concentrated in vacuo. Both residues were combined,
purified by column chromatography (diethyl ether/isopropa-
nol/ammonia, 10:3:0.1) and recrystallized from ethyl acetate/
isopropanol (5:1) yielding product as colorless crystals. Yield:
0.11 g (11.0%); mp: 98.5°C; 1H-NMR (DMSO-d6, 400 MHz):
d ¼ 1.83–1.90 (m, 2H, C(12)H2), 2.35 (t, 6H, C(13,15,18)H2,
J ¼ 6.8 Hz), 3.50–3.54 (m, 6H, C(11,16,17)H2), 4.93 (s, 2H, C(9)
H2), 7.24 (d, 1H, C(3)H, J ¼ 9.2 Hz), 7.52 (t, 1H, C(6)H, J ¼ 7.6 Hz),
7.63 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.80 (d, 1H, C(8)H, J ¼ 8.0 Hz),
7.92 (d, 1H, C(4)H, J ¼ 8.8 Hz), 7.97 (d, 1H, C(5)H, J ¼ 8.0 Hz)
ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 22.8 (C12), 45.5 (C9),
47.3 (C11), 53.3 (C15,18), 55.5 (C13), 66.1 (C16,17), 110.3 (C1),
116.3 (C3), 122.4 (C8), 125.1 (C6), 127.3 (C7), 128.5 (C5), 128.9
(C8), 125.1 (C6), 127.3 (C7), 128.4 (C5), 129.1 (C8a), 129.3 (C4),
ꢃ
129.9 (C4a), 147.0 (C2), 149.4 (C19) ppm; FT-IR (n): 3241 cmꢂ1
(w), 3150 cmꢂ1 (w), 1732 cmꢂ1 (s), 1634 cmꢂ1 (w), 1380 cmꢂ1
(m), 1215 cmꢂ1 (m), 1171 cmꢂ1 (s), 743 cmꢂ1 (s); C12H9NO2
(199.06): calcd.: C, 72.35; H, 4.55; N, 7.03; found C, 72.54; H,
4.32; N, 7.41 [28].
8-Bromo-1,2-dihydro-3H-naphtho[1,2-e][1,3]oxazin-3-one
(7b)
To a solution of 18 (1.13 g, 3.91 mmol) in dioxane (30 mL) was
added triethylamine (2.44 mL, 1.77 g, 17.5 mmol, 4.5 equiv.)
while stirring under nitrogen atmosphere at room tempera-
ture. Further 20 mL dioxane and 10 mL dimethylformamide
were added because of poor solubility. Subsequently,
4-nitrophenyl chloroformate (0.75 g, 3.7 mmol, 1.0 equiv.)
pre-dispersed in dioxane (10 mL) was added dropwise. After
addition was complete, the reaction mixture was stirred for a
further 10 min at room temperature and then heated up to
reflux for 1 h. While heating reaction color had changed to
orange. After reaction was stirred for further 10 h at room
temperature, dioxane was removed in rotary evaporator. A
solid precipitated when 30 mL dichloromethane and 30 mL
aqueous sodium carbonate solution (8%) were added to oily
residue. The solid was collected by filtration off and was
recrystallized from toluene yielding product as beige needle-
shaped crystals. Yield: 0.183 g (17.8%); mp: 231.9°C (degrad.);
1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.78 (s, 2H, C(9)H2), 7.29 (d,
1H, C(3)H, J ¼ 9.2 Hz), 7.68– 7.73 (m, 2H, C(7,8)H), 7.91 (d, 1H,
C(4)H, J ¼ 8.8 Hz), 8.24 (s, 1H, C(5)H), 8.25 (s, 1H, N(10)H) ppm;
13C-NMR (DMSO-d6, 100 MHz): d ¼ 39.8 (C9), 110.5 (C1), 118.0
(C8a), 129.3 (C4), 129.8 (C4), 129.8 (C4a), 146.6 (C2), 149.1
ꢃ
(C19) ppm; FT-IR (n): 3083 cmꢂ1 (w), 2947 cmꢂ1 (w), 1699 cmꢂ1
(m), 1225 cmꢂ1 (m), 822 cmꢂ1 (m), 744 cmꢂ1 (m); HRMS (ESI):
found 327.1703, calcd. for
327.1703.
C
19H22N2O3 [MþH]þ m/z ¼
8-Bromo-2-(3-morpholinopropyl)-1,2-dihydro-3H-
naphtho[1,2-e][1,3]oxazin-3-one (7d)
To an emulsion of 20 (0.45 g, 1.0 mmol) in 40 mL toluene/
water (1:1) was added triethylamine (0.84 mL, 0.61 g,
6.0 mmol) while stirring under argon atmosphere. To the
ice-cooled reaction mixture, a 20% solution of phosgene in
toluene (0.77 mL, 1.5 mmol) was added and reaction was
stirred for 4 h at room temperature. The emerged product as
white solid was collected by filtration. More product was
obtained by addition of 0.1 N hydrochloric acid solution
(40 mL) to filtrate and following filtration. Then filtrate
was pH adjusted to pH 11 and subsequently extracted with
ethyl acetate (40 mL). Organic phase was dried with sodium
sulfate and hydrochloric acid gas was injected. The emerged
(C3), 118.2 (C6), 124.9 (C8), 127.8 (C8a), 128.6 (C4), 130.1 (C7),
ꢃ
130.2 (C5), 131.2 (C4a), 147.4 (C2), 149.2 (C19) ppm; FT-IR (n):
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white solid was collected by filtration. The solids were
combined yielding product as white crystals. Yield: 0.17 g
(38.4%); mp: 257.9°C (degrad.); 1H-NMR (DMSO-d6, 400 MHz):
d ¼ 1.82–1.89 (m, 2H, C(12)H2), 2.35 (t, 6H, C(13,15,18)H2,
J ¼ 6.8 Hz), 3.50–3.53 (m, 6H, C(11,16,17)H2), 7.29 (d, 1H, C(3)H,
J ¼ 8.8 Hz), 4.91 (s, 2H, C(9)H2), 7.43 (d, 1H, C(7)H, J ¼ 9.2 Hz),
7.76 (d, 1H, C(8)H, J ¼ 8.8 Hz), 7.91 (d, 1H, C(4)H, J ¼ 8.8 Hz),
8.26 (s, 1H, C(5)H) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 22.7
(C12), 45.4 (C9), 47.4 (C11), 53.3 (C15,18), 55.6 (C13), 66.2
(C16,17), 110.8 (C1), 117.6 (C3), 118.2 (C6), 124.8 (C8), 127.6
(2 ꢁ 50 mL) and was recrystallized from ethyl acetate, yielding
product 8a as colorless crystals. Yield: 0.18 g (9.8%); mp:
181°C (decomp.); 1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.80 (s, 2H,
C(9)H2), 7.,32 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.55 (t, 1H, C(6)H,
J ¼ 7.6 Hz), 7.63 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.74 (d, 1H, C(8)H,
J ¼ 8.4 Hz), 7.95 (d, 1H, C(4)H, J ¼ 9.2 Hz), 7.98 (d, 1H, C(5)H,
J ¼ 8.0 Hz), 10.56 (s, 1H, N(10)H) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 40.1 (C9), 109.4 (C1), 116.0 (C3), 122.6 (C8), 125.6
(C6), 127.5 (C7), 128.5 (C5), 128.9 (C8a), 129.6 (C4), 130.4 (C4a),
ꢃ
145.3 (C2), 180.9 (C19) ppm; FT-IR (n): 3164 cmꢂ1 (w),
(C8a), 128.7 (C4), 130.1 (C7), 130.1 (C7), 130.3 (C5), 131.2 (C4a),
3085 cmꢂ1 (w), 2993 cmꢂ1 (w), 1636 cmꢂ1 (w), 1580 cmꢂ1
(m), 1175 cmꢂ1 (s), 1149 cmꢂ1 (s), 806 cmꢂ1 (s), 746 cmꢂ1 (s).
Product confirmed by X-ray crystallography.
ꢃ
147.0 (C2), 148.9 (C19) ppm; FT-IR (n): 3075 cmꢂ1 (w),
2981 cmꢂ1 (w), 2548 cmꢂ1 (w), 2468 cmꢂ1 (w), 1716 cmꢂ1
(m), 1108 cmꢂ1 (m), 821 cmꢂ1 (w); HRMS (ESI): found
405.0808, calcd. for C19H21BrN2O3 [MþH]þ m/z ¼ 405.0808.
8-Bromo-1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-
thione (8b)
1,2-Dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione (8a)
A suspension of 17 (3.13 g, 15.0 mmol) in methanol (40 mL)
was stirred at room temperature. Successively triethylamine
(3.1 mL, 2.25 g, 22.5 mmol, 1.5 equiv.) and carbon disulfide
(2.72 mL, 3.43 g, 45.0 mmol, 3.0 equiv.) were added. The pale
yellow solution was stirred for 1 h at room temperature. Via
dropping funnel hydrogen peroxide (6.0 mL, 58.7 mmol, 3.9
equiv.) was added in such a manner as the solution boiled
slightly. After addition was completed, the solution was
allowed cooling to room temperature. After 1 h the formed
precipitate was collected by filtration and washed with
methanol (3 ꢁ 20 mL). Colorless solid was used in next step
without further purification. Yield: 0.789 g (24.4%); mp:
181°C (degrad.); 1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.80 (s, 2H,
C(9)H2), 7.,32 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.55 (t, 1H, C(6)H,
J ¼ 7.6 Hz), 7.63 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.75 (d, 1H, C(8)H,
J ¼ 8.0 Hz), 7.96 (d, 1H, C(4)H, J ¼ 8.8 Hz), 7.99 (d, 1H, C(5)H,
J ¼ 8.8 Hz), 10.54 (s, 1H, N(10)H) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 40.06 (C9), 109.4 (C1), 116.0 (C3), 122.6 (C8),
To a solution of 10 (3.08 g, 11.0 mmol) in dichloromethane
(20 mL) was added methyl iodide (2.06 mL, 4.68 g, 33.0 mmol,
3.0 equiv.). After 7 h stirring at room temperature the solid
was collected by filtration and washed with dichloromethane
(2 ꢁ 50 mL). Solid was used directly in the crude form for next
step. So a suspension of crude intermediate product (2.988 g,
7.1 mmol) and potassium thiocyanate (1.37 g, 14.1 mmol) in
butanone (60 mL) was heated to reflux. After 24 h, reaction
mixture was allowed to cool down to room temperature.
Afterwards 10 mL 1 M aqueous hydrochloric acid solution
were added and solid was collected by filtration. Crude
product
was
washed
with
acetone
(2 ꢁ 20 mL),
H2O (2 ꢁ 50 mL), methanol (2 ꢁ 50 mL) and was recrystallized
from ethyl acetate, yielding product 8b as colorless crystals.
Yield: 0.31 g (9.4%); mp: 255°C (decomp.); 1H-NMR (DMSO-d6,
400 MHz): d ¼ 4.79 (s, 2H, C(9)H2), 7.38 (d, 1H, C(3)H, J ¼ 8.8 Hz),
7.72 (m, 2H, C(4)H, C(8)H), 7.95 (d, 1H, C(7)H, J ¼ 9.2 Hz), 8.28
(s, 1H, C(5)H), 10.58 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 40.1 (C9), 110.0 (C1), 117.4 (C3), 118.7 (C6), 125.0
125.6 (C6), 127.5 (C7), 128.5 (C5), 128.9 (C8a), 129.6 (C4), 130.4
(C8), 127.6 (C8a), 128.9 (C7), 130.3 (C4, C5), 131.7 (C4a), 145.6
ꢃ
ꢃ
(C4a), 145.3 (C2), 180.9 (C19) ppm; FT-IR (n): 3164 cmꢂ1 (w),
(C2), 180.7 (C¼S) ppm; FT-IR (n): 3343 cmꢂ1 (m), 3065 cmꢂ1 (w),
3085 cmꢂ1 (w), 2993 cmꢂ1 (w), 1636 cmꢂ1 (w), 1580 cmꢂ1 (m),
1175 cmꢂ1 (s), 1149 cmꢂ1 (s), 806 cmꢂ1 (s), 746 cmꢂ1 (s). Product
was confirmed by X-ray crystallography.
1635 cmꢂ1 (m), 1502 cmꢂ1 (s), 1173 cmꢂ1 (s), 1139 cmꢂ1 (s);
HRMS (ESI): found 293.9569, calcd. for C12H8BrNOS [MþH]þ
m/z ¼ 293.9583.
Alternative synthesis of 1,2-dihydro-3H-naphth[1,2-e]
[1,3]-oxazine-3-thione (8a)
2-(3-Morpholinopropyl)-1,2-dihydro-3H-naphth[1,2-e]-
[1,3]oxazine-3-thione (8c)
To a solution of 9 (1.71 g, 8.5 mmol) in dichloromethane
(35 mL) was added methyl iodide (1.59 mL, 3.61 g, 25.4 mmol,
3.0 equiv.). After 7 h stirring at room temperature, the solid
was collected by filtration and washed with dichloromethane
(2 ꢁ 50 mL). Solid was used directly in the crude form for next
step. So a suspension of crude intermediate product (1.722 g,
5.0 mmol) and potassium thiocyanate (0.99 g, 10.3 mmol) in
butanone (50 mL) was heated to reflux. After 24 h, reaction
mixture was allowed to cool down to room temperature and
solvent was removed in rotary evaporator. Afterwards 100 mL
0.25 M aqueous hydrochloric acid solution was added and
solid was collected by filtration. Crude product was washed
with acetone (2 ꢁ 20 mL), H2O (2 ꢁ 50 mL), methanol
To a suspension of 19 (0.75 g, 2.0 mmol) in dichloromethane
(40 mL) triethylamine (1.39 mL, 1.01 g, 10.0 mmol) was added
under argon atmosphere. While stirring in an ice bath,
thiophosgene (0.25 mL, 0.37 g, 3.0 mmol) dissolved in 10 mL
dichloromethane was added dropwise. After addition was
complete, ice bath was removed and reaction was stirred for
2 h at room temperature. Reaction mixture was extracted
with water (2 ꢁ 20 mL) and finally organic phase was
extracted with 0.5 N hydrochloric acid (20 mL). Dichloro-
methane phase was dried with anhydrous sodium sulfate
and concentrated in vacuo. Residue was diluted in less
dichloromethane and extracted with aqueous sodium hy-
droxide solution (2 ꢁ 10 mL). Organic phase was dried with
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sodium sulfate and concentrated in vacuo. Solid residue was
recrystallized from ethyl acetate/isopropanol (5:1) yielding
pale brownish crystals. Yield: 0.32 g (42.3%); mp: 144°C; 1H-
NMR (DMSO-d6, 400 MHz): d ¼ 1.99–2.07 (m, 2H, C(12)H2),
2.35–2.41 (m, 6H, C(13,15,18)H2), 3.48 (t, 4H, C(16,17)H2,
J ¼ 4.6 Hz), 4.03 (t, 2H, C(11)H2, J ¼ 7.2 Hz), 4.54 (s, 2H, C(9)H2),
7.32 (d, 1H, C(3)H, J ¼ 9.2 Hz), 7.56 (t, 1H, C(6)H, J ¼ 7.6 Hz),
7.65 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.82 (d, 1H, C(8)H, J ¼ 8.0 Hz),
7.96 (d, 1H, C(4)H, J ¼ 8.8 Hz), 7.99 (d, 1H, C(5)H, J ¼ 8.0 Hz)
ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 21.3 (C12), 46.3 (C9),
53.2 (C15,18), 53.9 (C11), 55.4 (C13), 66.1 (C16,17), 110.2 (C1),
115.5 (C3), 115.5 (C3), 122.4 (C8), 125.6 (C6), 127.5 (C7), 128.6
dichloromethane phase was extracted with aqueous sodium
hydroxide solution (2 ꢁ 10 mL), dried with anhydrous sodium
sulfate and concentrated in vacuo. Residue was purified with
column chromatography (diethyl ether/isopropanol/25% am-
monia solution, 10:3:0.1) yielding product as pale brownish
platelets. Yield: 0.11 g (12.9%); mp: 167°C (degrad.); 1H-NMR
(DMSO-d6, 400 MHz): d ¼ 1.99–2.06 (m, 2H, C(12)H2), 2.35–2.41
(m, 6H, C(13,15,18)H2), 3.47 (t, 4H, C(16,17)H2, J ¼ 4.4 Hz), 4.01
(t, 2H, C(11)H2, J ¼ 7.4 Hz), 5.07 (s, 2H, C(9)H2), 7.38 (d, 1H, C(3)
H, J ¼ 8.8 Hz), 7.78 (s, 1H, C(7,8)H), 7.95 (d, 1H, C(4)H,
J ¼ 8.8 Hz), 8.30 (s, 1H, C(5)H) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 21.3 (C12), 46.1 (C9), 53.2 (C15,18), 54.0 (C11),
55.4 (C13), 66.1 (C16,17), 110.7 (C1), 116.9 (C3), 118.7 (C6),
(C8a,5), 129.6 (C4), 130.3 (C4a), 144.8 (C2), 179.9 (C19) ppm;
ꢃ
FT-IR (n): 3054 cmꢂ1 (w), 2976 cmꢂ1 (w), 1200 cmꢂ1 (m),
124.8 (C8), 127.3 (C8a), 128.9 (C4), 130,3 (C7), 130.4 (C5), 131.6
ꢃ
1110 cmꢂ1 (s), 811 cmꢂ1 (m), 745 cmꢂ1 (m); HRMS (ESI): found
(C4a), 145.1 (C2), 179.7ꢂ(1C19) ppm; FT-IR (n): 3068 cmꢂ1 (w),
343.1475, calcd. for C19H22N2O2S [MþH]þ m/z ¼ 343.1475.
2950 cmꢂ1 (w), 1536 cm (m), 1353 cmꢂ1 (m), 1204 cmꢂ1 (m),
1109 cmꢂ1 (m), 808 cmꢂ1 (m); HRMS (ESI): found 443.0399,
calcd. for C19H21BrN2O2S [MþNa]þ m/z ¼ 443.0399.
Alternative synthesis of 2-(3-morpholinopropyl)-1,2-
dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione (8c)
To a suspension of 19 (1.12 g, 3.0 mmol) in tetrahydrofuran
(10 mL) was added 1,10-thiocarbonyldiimidazole (0.54 g,
3.0 mmol) pre-dissolved in 5 mL tetrahydrofuran while stirring
under argon atmosphere. Reaction mixture was stirred 43 h at
room temperature, and then solvent was removed in rotary
evaporator. Aqueous sodium hydroxide solution (50 mL, 1 N)
was added to residue and solution was extracted with ethyl
acetate (3 ꢁ 50 mL). Organic phases were combined and
washed with water (2 ꢁ 30 mL). Ethyl acetate phase was dried
over anhydrous sodium sulfate and concentrated in vacuo.
Residue was recrystallized from ethyl acetate/isopropanol
(5:1) yielding desired product as pale brownish crystals. Yield:
0.52 g (50.0%); mp: 144°C; 1H-NMR (DMSO-d6, 400 MHz):
d ¼ 1.99–2.07 (m, 2H, C(12)H2), 2.35–2.41 (m, 6H, C(13,15,18)
H2), 3.48 (t, 4H, C(16,17)H2, J ¼ 4.6 Hz), 4.03 (t, 2H, C(11)H2,
J ¼ 7.2 Hz), 4.54 (s, 2H, C(9)H2), 7.32 (d, 1H, C(3)H, J ¼ 9.2 Hz),
7.56 (t, 1H, C(6)H, J ¼ 7.6 Hz), 7.65 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.82
(d, 1H, C(8)H, J ¼ 8.0 Hz), 7.96 (d, 1H, C(4)H, J ¼ 8.8 Hz), 7.99 (d,
1H, C(5)H, J ¼ 8.0 Hz) ppm; 13C-NMR (DMSO-d6, 100 MHz):
d ¼ 21.3 (C12), 46.3 (C9), 53.2 (C15,18), 53.9 (C11), 55.4 (C13),
66.1 (C16,17), 110.2 (C1), 115.5 (C3), 115.5 (C3), 122.4 (C8),
1-[(Dimethylamino)methyl]naphthalen-2-ol (9)
To a suspension of 2-naphthol (5.85 g, 40.2 mmol), dimethyl-
methylideneammonium iodide (97%, 7.58 g, 39.7 mmol, 1.0
equiv.) and potassium carbonate (8.31 g, 60.1 mmol, 1.5
equiv.) in toluene (20 mL) was added 30 mL dichloromethane.
After stirring for 24 h at room temperature, the reaction
mixture was extracted with 3 ꢁ 60 mL 1 M aqueous hydro-
chloride acid solution. Phases were separated and potassium
carbonate was added to the aqueous phase adjusting the pH
to 8, whereby product precipitation emerged. The aqueous
phase was stored at 5°C for 5 h and the white solid was
collected by filtration, yielding 9 as white crystals. Yield: 7.98 g
(98.7%); mp: 74.5°C; 1H-NMR (DMSO-d6, 400 MHz): d ¼ 3.98 (s,
2H, C(9)H2), 7.06 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.27 (t, 1H, C(6)H,
J ¼ 8.0 Hz), 7.43 (t, 1H, C(7)H, J ¼ 8.4 Hz), 7.70 (d, 1H, C(8)H,
J ¼ 8.8 Hz), 7.77 (d, 1H, C(4)H, J ¼ 8.0 Hz), 7.94 (d, 1H, C(5)H,
J ¼ 8.4 Hz) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 44.3 (N
(CH3)2), 55.4 (C9), 113.0 (C8a), 118.5 (C3), 122.2 (C5, C6), 126.1
(C7), 127.9 (C1), 128.3 (C4), 128.7 (C8), 132.9 (C4a), 155.4 (C2)
ꢃ
ppm; FT-IR (n): 3380 cmꢂ1 (w), 3006 cmꢂ1 (w), 2950 cmꢂ1 (w),
1626 cmꢂ1 (s), 1439 cmꢂ1 (m), 1322 cmꢂ1 (m), 1245 cmꢂ1 (s);
HRMS (ESI): found 202.1226, calcd. for C13H15NO [MþH]þ m/
z ¼ 202.1226 [12].
125.6 (C6), 127.5 (C7), 128.6 (C8a,5), 129.6 (C4), 130.3 (C4a),
ꢃ
144.8 (C2), 179.9 (C19) ppm; FT-IR (n): 3054 cmꢂ1 (w),
2976 cmꢂ1 (w), 1200 cmꢂ1 (m), 1110 cmꢂ1 (s), 811 cmꢂ1 (s),
745 cmꢂ1 (s).
6-Bromo-1-[(dimethylamino)methyl]naphthalen-2-ol (10)
To a suspension of 6-bromo-2-naphthol (9.20 g, 40.0 mmol),
dimethylmethylideneammonium iodide (97%, 7.62 g,
40.0 mmol, 1.0 equiv.) and potassium carbonate (8.30 g,
60.0 mmol, 1.5 equiv.) in toluene (30 mL) was added 20 mL
dichloromethane. The suspension was stirred 16 h at room
temperature. Product was gained as a solid by filtering the
reaction mixture. In addition 0.2 M hydrochloride acid
solution (50 mL) was added to the filtrate. Phases were
separated and 2.0 g potassium carbonate was added to the
aqueous phase, whereby product 10 emerged as a voluminous
precipitate. Solids were combined and washed with water
(3 ꢁ 50 mL) yielding 10 as colorless solid. Yield: 10.35 g
8-Bromo-2-(3-morpholinopropyl)-1,2-dihydro-3H-naphth
[1,2-e][1,3]oxazine-3-thione (8d)
To a suspension of 20 (0.90 g, 2.0 mmol) in dichloromethane
(15 mL), triethylamine (1.39 mL, 1.01 g, 10.0 mmol) was added
under argon atmosphere. While stirring in an ice bath,
thiophosgene (0.25 mL, 0.37 g, 3.0 mmol) dissolved in 10 mL
dichloromethane was added dropwise. After addition was
complete, ice bath was removed and reaction was stirred for
3 h at room temperature. Reaction mixture was extracted
with water (2 ꢁ 20 mL) and afterwards organic phase was
extracted with 0.5 M hydrochloric acid (20 mL). Finally the
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(92.4%); mp: 178°C; 1H-NMR (DMSO-d6, 400 MHz): d ¼ 2.28 (s,
6H, N(CH3)2), 3.96 (s, 2H, C(9)H2), 7.12 (d, 1H, C(3)H, J ¼ 8.8 Hz),
7.53 (d, 1H, C(4)H, J ¼ 9.2 Hz), 7.71 (d, 1H, C(8)H, J ¼ 8.8 Hz),
7.91 (d, 1H, C(9)H, J ¼ 8.8 Hz), 8.04 (s, 1H, C(6)H), 11.33 (s, 1H,
OH) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 44.3 (N(CH3)2),
55.0 (C9), 113.5 (C1), 115.0 (C6), 119.7 (C3), 124.9 (C8), 128.0
(70)H), 7.78 (d, 1H, C(80)H, J ¼ 8.4 Hz), 7.74 (dd, 2H, C (40)H, C(4)
H, J ¼ 8.8 Hz), 7.79 (d, 1H, C(50)H, J ¼ 8.0 Hz), 7.84 (d, 1H, C(5)H,
J ¼ 8.0 Hz), 8.06 (d, 1H, C(8)H, J ¼ 8.4 Hz), 9.63 (s, 1H, OH) ppm;
13C-NMR (DMSO-d6, 100 MHz): d ¼ 45.1 (C90), 46.0 (C9), 82.0
(C10), 112.4 (C1), 114.7 (C10), 118.0 (30), 118.4 (C3), 121.3 (C8),
122.4 (C6), 123.2 (C60), 123.7 (C80), 126.1 (C70), 126.4 (C7), 127.6
(C7), 128.8 (C4), 129.3 (C4a), 129.9 (C5), 131.7 (C8a), 155.7 (C2)
(C4), 128.1 (C50), 128.2 (C4a), 128.3 (C5), 129.1 (C4a0), 131.7
ꢃ
ꢃ
ppm; FT-IR (n): 3195 cmꢂ1 (m), 3036 cmꢂ1 (w), 2955 cmꢂ1 (w),
(C8a), 134.0 (C8a0), 151.5 (C2), 153.8 (C20) ppm; FT-IR (n):
1357 cmꢂ1 (m), 1270 cmꢂ1 (s); HRMS (ESI): found 280.0332,
3059 cmꢂ1 (w), 2996 cmꢂ1 (w), 1593 cmꢂ1 (m), 1213 cmꢂ1 (s),
807 cmꢂ1 (s); HRMS (ESI): found 342.1491, calcd. for C23H19NO2
[MþH]þ m/z ¼ 342.1489.
calcd. for C13H14BrNO [MþH]þ m/z ¼ 280.0332.
(6-Bromo-2-hydroxynaphthalen-1-yl)(p-tolyl)-
methanaminium chloride (11)
1-({[(E)-(2-Hydroxynaphthalen-1-yl)methyl]imino}methyl)-
naphthalen-2-ol (13)
To a flask containing 6-bromo-2-naphthol (2.23 g, 10.0 mmol)
and ammonium formate (1.1 g, 22.3 mmol, 2.2 equiv.) was
added 4-methylbenzaldehyde (3.36 mL, 2.40 g, 2.0 eqiv).
Reaction mixture was heated to 110°C for 15 min and was
allowed to cool down to room temperature. To the solid
residue 50 mL water and 2 mL HCl (36%) were added and the
slurry was heated for 2 h to 100°C. The solid was collected by
filtration and was subsequently diluted again in 50 mL water
and 1 mL H2SO4 (96%) and then heated for 15 h to 70°C. Slurry
was stored at 5°C for 1 h. Solid was collected by filtration and
was refluxed in ethyl acetate. The product was collected by
filtration of hot slurry. Procedure was repeated for the
insoluble precipitate yielding product as colorless crystals.
Yield: 1.15 g (30.4%); mp: 188°C (degrad.); 1H-NMR (DMSO-d6,
400 MHz): d ¼ 2.26 (s, 3H, C(14)H3), 6.20 (s, 1H, C(9)H), 7.16 (d,
2H, C(12)H, C(120)H, J ¼ 8.0 Hz), 7.37 (d, 2H, C(11)H, C(110)H,
J ¼ 8.4 Hz), 7.52 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.56 (d, 1H, C(7)H,
J ¼ 8.8 Hz), 7.87 (d, 1H, C(4)H, J ¼ 8.8 Hz), 7.97 (d, 1H, C(8)H,
J ¼ 9.2 Hz), 8.14 (s, 1H, C(5)H), 8.91 (s, 3H, NþH3), 11.22 (s, 1H,
OH) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 20.6 (C14), 50.5
(C9), 114.3 (C1), 115.7 (C6), 119.9 (C3), 124.4 (C8), 127.1 (C11,
110), 129.0 (C12, 120), 129.4 (C4a), 129.7 (C4), 129.8 (C7), 130.4
General procedure: To a mixture of 2-naphthol (10.0 g,
69.4 mmol) and methenamine (10.0 g, 71.3 mmol) acetic acid
(40 mL) was added. The suspension was stirred at 100°C for
1 h. The yellow precipitate formed was collected by filtration
and washed with ethanol (3 ꢁ 20 mL). The amorphous yellow
solid was used in the next step without further purification.
Yield: 8.55 g (75.3%); mp: 211°C (degrad.); 1H-NMR (DMSO-d6,
400 MHz): d ¼ 5.28 (s, 2H, C(11)H2), 6.63 (d, 1H, C(3)H,
J ¼ 9.6 Hz), 7.19 (t, 1H, C(6)H, J ¼ 8.0 Hz), 7.29 (d, 1H, C(14)H,
J ¼ 8.8 Hz), 7.34 (t, 1H, C(17)H, J ¼ 8.0 Hz), 7.46 (t, 1H, C(7)H,
J ¼ 7.6 Hz), 7.55 (t, 1H, C(18)H, J ¼ 7.6 Hz), 7.61 (d, 1H, C(5)H,
J ¼ 8.0 Hz), 7.68 (d, 1H, C(4)H, J ¼ 9.6 Hz), 7.87–7.83 (2d, 2H,
C(15, 16)H), 8.09 (d, 1H, C(8)H, J ¼ 8.4 Hz), 8.22 (d, 1H, C(19)H,
J ¼ 8.4 Hz), 9.39 (d, 1H, C(9)H, J ¼ 10.4 Hz), 14.27 (s, 1H,
¼Nþ(10)H), 10.34 (s, 1H, OH) ppm; 13C-NMR (DMSO-d6,
100 MHz): d ¼ 44.8 (C11), 105.5 (C1), 113.8 (C12), 118.0
(C14), 118.2 (C8), 122.1 (C19), 122.1 (C6), 122.8 (C17), 125.1
(C4a), 125.5 (C3), 127.0 (C18), 127.9 (C7), 128.1 (C15a), 128.6
(C16), 128.9 (C5), 130.0 (C15), 132.7 (C19a), 134.3 (C8a),137.0
ꢃ
(C4), 15ꢂ31.8 (C13), 158.3 (C9), 177.3 (C2) ppm; FT-IR (n):
3050 cm
(w, nCꢂH, , sat.),
unsat.), 2957 cmꢂ1 (w, nC–H
(C5), 130.5 (C8a), 134.3 (C13), 137.4 (C10), 154.3 (C2) ppm; FT-
2497 cmꢂ1 (w, nO–H), 1639 cmꢂ1 (s, nC¼N); HRMS (ESI): found
328.1332, calcd. for C22H17NO [MþH]þ m/z ¼ 328.1332 [43].
ꢃ
IR (n): 3059 cmꢂ1 (m), 2903 cmꢂ1 (m), 1517 cmꢂ1 (s), 1503 cmꢂ1
(s), 1273 cmꢂ1 (s), 455 cmꢂ1 (s); HRMS (ESI): found 325.0209,
calcd. for C18H13OBr [MþH]þ m/z ¼ 325.0223.
6-Bromo-1-({[(6-bromo-2-hydroxynaphthalen-1-yl)-
methyl]imino}methyl)naphthalen-2-ol (14)
1-([1H-Naphtho[1,2-e][1,3]oxazin-2(3H)-yl]methyl)-
naphthalen-2-ol (12)
To a mixture of 6-bromo-naphth-2-ol (15.9 g, 71.3 mmol) and
methenamine (10.0 g, 71.3 mmol) acetic acid (40 mL) was
added. The suspension was stirred at 100°C for 2 h. The yellow
precipitate formed was collected by filtration and washed with
acetic acid (2 ꢁ 20mL) and finally with hot ethanol (3 ꢁ 30mL).
The amorphous yellow solid was used in the next step without
furtherpurification.Yield:15.72 g(90.7%);mp:222°C(degrad.);
1H-NMR (DMSO-d6, 400MHz): d ¼ 5.25 (s, 2H, C(11)H2), 6.67 (d,
1H, C(3)H, J ¼ 9.2 Hz), 7.32 (d, 1H, C(14)H, J ¼ 8.8 Hz), 7.58 (d, 1H,
C(18)H,J ¼ 8.8 Hz),7.64(d, 1H,C(7)H,J ¼ 8,8 Hz),7.68(d, 1H,C(4)
H, J ¼ 9,2 Hz), 7.83 (d, 1H, C(15)H, J ¼ 8.8 Hz), 7.87 (s, 1H, C(5)H),
8.04 (d, 1H, C(8)H, J ¼ 9.2 Hz), 8.13 (s, 1H, C(16)H), 8.19 (d, 1H,
C(19)H, J ¼ 9.2 Hz), 9.39 (s, 1H, C(9)H), 10.55 (s, 1H, OH), 14.23 (s,
1H, ¼Nþ(10)H) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 44.7
(C11),105.3(C1),114.1(C12),114.4(C6),115.6(C17),119.2(C14),
120.6(C8), 124.6(C19), 126.7(C4a), 126.9(C3),129.3(C15), 129.4
A suspension of 2-naphthol (7.26 g, 50.4 mmol), paraformal-
dehyde (3.07 g, 102.1 mmol, 2.0 equiv.) and ammonium
formate (4.98 g, 78.9 mmol, 1.5 equiv.) in dimethylformamide
(30 mL) was heated to 120°C for 20 min. Within 15 min mixture
turned into a homogeneous yellow solution. Then reaction
mixture was allowed to cool down to room temperature and
was poured into 600 mL water. The mixture was stored 1 h at
5°C and precipitate was collected by filtration. The collected
solid was recrystallized from ethanol, yielding product 12 as
pale pink needle-shaped crystals. Yield: 2.76 g (32.1%); mp:
162°C; 1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.25 (s, 2H, C(9)H2),
4.35 (s, 2H, C(90)H2), 5.02 (s, 2H, C(10)H2), 7.11 (d, 1H, C(3)H,
J ¼ 8.8 Hz), 7.15 (d, 1H, C(30)H, J ¼ 8.8 Hz), 7.30 (t, 1H, C(60)H,
J ¼ 7.6 Hz), 7.36 (t, 1H, C(6)H, J ¼ 7.6 Hz), 7.44 (m, 2H, C(7)H, C
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(C15a), 129.8(C7), 130.3 (C16,C18), 133.2 (C8a), 136.0 (C4), 154.3
and was collected by filtration, yielding product as yellow
solid. The filtrates were allowed to cool to 5°C and formed
precipitations were combined recovering 22.98 g starting
material 6-bromo-2-naphthol. Yield: 7.49 g (21.6%, 85.3%
based on recovered starting material); mp: 150°C (degrad.).
Spectroscopic data were consistent with those reported in
literature.
(C13), 130.6 (C5), 131.3 (C19a), 158.6 (C9), 177.5 (C2) ppm; FT-IR
ꢃ
(n): 3052 cmꢂ1 (w), 2582 cmꢂ1 (m), 1627 cmꢂ1 (s), 1534 cmꢂ1 (w),
1494 cmꢂ1 (s), 1345 cmꢂ1 (s), 1165 cmꢂ1 (s), 804cmꢂ1 (s); HRMS
(ESI): found 481.9401, calcd. for C22H15NO2Br2 [MꢂH]ꢂ m/
z ¼ 481.9397.
2-Hydroxy-1-naphthaldehyde (15)
To a suspension of 13 (12.07 g, 36.85 mmol) in ethanol (200 mL)
was added 6 N hydrochloric acid (40mL) via dropping funnel
with continuous stirring. After adding was completed, mixture
was refuxed for 2 h. Subsequently suspension was cooled down
on an ice bath. Solid was filtered off. Hereafter cold water
(400 mL) was added to filtrate, whereby a yellow precipitate
was formed immediately. Suspension was stored at 5°C for 2 h
and then precipitate was collected by filtration. A pale yellow
solid was obtained by recrystallization in 2-propanol. Yield:
5.03 g (79.3%); mp: 82°C (degrad.). Spectroscopic data were
consistent with those reported in the literature [44].
(2-Hydroxynaphthalen-1-yl)methanaminium chloride (17)
To a suspension of 13 (12.07 g, 36.85 mmol) in ethanol
(200 mL) was added dropwise 6 M hydrochloric acid (40 mL)
with stirring. After addition was completed, mixture was
refluxed for 2 h. Subsequently, the suspension was cooled
down on an ice bath. The precipitate was collected by
filtration and was washed thoroughly with cold ethanol
(3 ꢁ 20 mL). A colorless solid was recrystallized from 2-
propanol. Yield: 6.43 g (83.2%); mp: 207°C (degrad.); 1H-
NMR (DMSO-d6, 400 MHz): d ¼ 4.39 (s, 2H, C(9)H2), 7.33–7.40
(m, 2H, C(3, 6)H), 7.53 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.85 (d, 2H, C(5,
4)H, J ¼ 8.8 Hz), 8.03 (d, 1H, C(8)H, J ¼ 8.4 Hz), 8.25 (s, 3H, N(10)
H3), 10.71 (s, 1H, OH) ppm; 13C-NMR (DMSO-d6, 100 MHz):
d ¼ 32.7 (C9), 111.0 (C1), 118.0 (C3), 122.4 (C8), 122.8 (C6),
6-Bromo-2-hydroxy-1-naphthaldehyde (16)
To a suspension of 14 (12.20 g, 25.0 mmol) in ethanol
(150 mL) was added 6 N hydrochloric acid (37.5 mL) via
dropping funnel with continuous stirring. After adding
was completed, mixture was refuxed for 2.5 h. Subsequently
suspension was cooled down on an ice bath. Solid was
filtered off. Hereinafter cold water (300 mL) was added to
filtrate, whereby a bluish precipitate was formed immedi-
ately. Suspension was stored at 5°C for 2 h and then
precipitate was collected by filtration. Solid was dissolved
in diethyl ether (100 mL), insoluble materials were collected
by filtration. Filtrate was dried with sodium sulfate and
evaporated to dryness, by means of a rotary evaporator. Yield
wasimprovedbyextractinghydrolysisfiltratewithdiethylether
(3 ꢁ 30mL). Organic phase was filtered and desiccated with
sodium sulfate and evaporated to dryness, by means of a rotary
evaporator. Yielding product as yellow crystals. Yield: 5.30g
(83.9%); mp: 150°C (degrad.); 1H-NMR (DMSO-d6, 400MHz):
d ¼ 7.31 (d, 1H, C(3)H, J ¼ 9.2 Hz), 7.73 (d, 1H, C(7)H, J ¼ 9.2 Hz),
8.11 (d, 1H, C(4)H, J ¼ 8.8 Hz), 8.17 (s, 1H, C(5)H), 8.93 (d, 1H, C(8)
H, J ¼ 9.2 Hz), 10.77 (s, 1H, C(9)H), 11.91 (s, 1H, OH) ppm; 13C-
NMR (DMSO-d6, 100MHz): d ¼ 112.6 (C1), 116,9 (C6), 120.1 (C3),
126.9 (C7), 127.9 (C4a), 128.5 (C5), 130.6 (C4), 132.7 (C8a),
ꢃ
154.6 (C2) ppm; FT-IR (n): 3020 cmꢂ1 (m), 2923 cmꢂ1(m),
1597 cmꢂ1 (m), 1517 cmꢂ1 (m), 1286 cmꢂ1 (s), 813 cmꢂ1 (s),
746 cmꢂ1 (s); HRMS (ESI): found 174.0913, calcd. for C11H12NO
[M]þ m/z ¼ 174.0913 [45].
(6-Bromo-2-hydroxynaphthalen-1-yl)methanaminium
chloride (18)
To a suspension of 14 (12.20 g, 25.0 mmol) in ethanol (150 mL)
was added 6 M hydrochloric acid (37.5 mL) via dropping
funnel with continuous stirring. After addition was com-
pleted, the mixture was refluxed for 2.5 h. Subsequently the
resulting suspension was cooled down on an ice bath for 2 h.
Formed solid was collected by filtration and was washed with
hot ethanol (2 ꢁ 20 mL). Yield was improved by treatment of
filtrate with cold water (300 mL), whereby a bluish precipitate
was formed immediately. Suspension was stored at 5°C for 2 h
and then bluish precipitate was collected by filtration. The
solid was dissolved in diethyl ether (100 mL), thereby an
insoluble solid was collected by filtration and washed with
ethanol (2 ꢁ 20 mL). Both the solid of the hydrolysis and the
diethyl ether precipitate were combined yielding product as
pale yellow crystals. Yield: 6.91 g (95.2%); mp: 223°C (degrad.);
1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.37 (s, 2H, C(9)H2), 7.41 (d,
1H, C(3)H, J ¼ 8.8 Hz), 7.63 (d, 1H, C(7)H, J ¼ 9.2 Hz), 7.86 (d, 1H,
C(4)H, J ¼ 9.2 Hz), 8.01 (d, 1H, C(8)H, J ¼ 9.2 Hz), 8.12 (s, 1H, C(5)
H), 8.20 (s, 3H, Nþ(10)H3), 10.85 (s, 1H, OH) ppm; 13C-NMR
(DMSO-d6, 100 MHz): d ¼ 32.7 (C9), 111.4 (C1), 115.6 (C6), 119.1
125.0 (C8), 129.0 (C4a), 130.2 (C8a), 130.5 (C5), 131.9 (C7), 137.2
ꢃ
(C4), 164.1 (C2), 192.1 (C9) ppm; FT-IR (n): 2983 cmꢂ1 (w),
1630 cmꢂ1 (m), 1458 cmꢂ1 (m), 1302 cmꢂ1 (m), 1163 cmꢂ1 (m),
809cmꢂ1 (m); HRMS (ESI): found 248.9565, calcd. for C11H7O2Br
[MꢂH]ꢂ m/z ¼ 248.
Alternative synthesis of 6-bromo-2-hydroxy-1-
naphthaldehyde (16)
To a suspension of 6-bromo-naphthol (30.78 g, 138,0 mmol)
and zinc cyanide (20.76 g, 176.8 mmol) in 150 mL diethyl ether
was injected continuously hydrochloride acid gas at room
temperature. After 2 h, yellowish solid was collected by
filtration and was refluxed two times in 250 mL water. The
insoluble solid was refluxed in 240 mL water/methanol (4:1)
(C3), 125.0 (C8), 129.1 (C4a), 129.5 (C7), 129.9 (C4), 130.1 (C5),
ꢃ
131.4 (C8a), 155.1 (C2) ppm; FT-IR ꢂ(1n): 3198 cmꢂ1 (m),
3022 cmꢂ1 (m), 2925 cmꢂ1 (m), 1578 cm (m), 1496 cmꢂ1 (s),
1271 cmꢂ1 (s), 1053 cmꢂ1 (w), 879 cmꢂ1 (s), 811 cmꢂ1 (s);
HRMS (ESI): found 252.0019, calcd. for C11H11BrNO [M]þ
m/z ¼ 252.0019 [45].
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Dihydronaphthoxazine-Based Sirtuin Inhibitors
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4-(3-{[(2-Hydroxynaphthalen-1-yl)methyl]ammonio}-
propyl)morpholin-4-ium chloride (19)
ethyl acetate (3 ꢁ 50 mL). Both organic phases were dried with
anhydrous sodium sulfate and then hydrochloric acid gas was
introduced. The resulting precipitate was collected by
filtration after storage at ꢂ20°C for 12 h. Solid was recrystal-
lized from ethyl acetate/methanol/isopropanol yielding prod-
uct as pale rose crystals. Yield: 3.16 g (69.9%); mp:
184°C (degrad.); 1H-NMR (DMSO-d6, 400 MHz): d ¼ 2.21–2.26
(m, 2H, C(12)H2), 3.05–3.18 (m, 6H, C(11,13,15/18)H2), 3.37 (s,
2H, C(15/18)H), 3.85–3.97 (m, 4H, C(16,17)H2), 4.52 (s, 2H, C(9)
H2), 7.47 (d, 1H, C(3)H, J ¼ 8.8 Hz), 7.63 (d, 1H, C(7)H,
J ¼ 8.8 Hz), 7.89 (d, 1H, C(4)H, J ¼ 8.8 Hz), 8.11 (d, 1H, C(8)H,
J ¼ 9.2 Hz), 9.13 (s, 1H, Nþ(10)H2), 11.03 (s, 1H, OH), 11.62 (s,
1H, Nþ(14)H) ppm; 13C-NMR (DMSO-d6, 100 MHz): d ¼ 19.6
(C12), 40.6 (C9), 44.3 (C11), 50.9 (C15,18), 53.0 (C13), 63.0
(C16,17), 109.5 (C1), 115.7 (C6), 119.1 (C6), 125.3 (C8), 129.2
To a solution of 15 (3.44 g, 20.0 mmol) in methanol (50 mL) 3-
(morpholino-4-yl)propan-1-amine (2.90 g, 20.1 mmol) was
added under a nitrogen atmosphere. After 3 h stirring at
room temperature, mixture was cooled in an ice-bath.
Afterwards sodium borohydride (1.51 g, 39.9 mmol) pre-
dissolved in 20 mL water was added. After removing the
ice-bath, solution was stirred 1 h at room temperature.
Reaction was quenched by slow addition of 4 M hydrochloric
acid (25 mL). After addition was completed, methanol was
removed by means of a rotary evaporator and aqueous
residue was extracted with diethyl ether (3 ꢁ 30 mL). Subse-
quently aqueous phase was extracted with ethyl acetate
(3 ꢁ 50 mL) after pH-adjustment to pH 7–8 with ammonium
chloride and sodium carbonate. Immediately aqueous phase
was adjusted to pH 9 with sodium hydroxide and extracted
again with ethyl acetate (3 ꢁ 50 mL). Both organic phases
were dried with anhydrous sodium sulfate and then hydro-
chloric acid gas was introduced. The resulting precipitate was
collected by filtration after storage at ꢂ20°C for 12 h. Solid
was recrystallized from ethyl acetate/methanol/isopropanol
yielding product as pale rose crystals. Yield: 5.19 g (69.7%);
mp: 169°C (degrad.); 1H-NMR (DMSO-d6, 400 MHz): d ¼ 2.20–
2.27 (m, 2H, C(12)H2), 3.03–3.19 (m, 6H, C(11,13,15/18)H2), 3.36
(s, 2H, C(15/18)H), 3.85–3.97 (m, 4H, C(16,17)H2), 4.54 (s, 2H,
C(9)H2), 7.36 (t, 1H, C(6)H, J ¼ 7.4 Hz), 7.44 (d, 1H, C(3)H,
J ¼ 8.8 Hz), 7.54 (t, 1H, C(7)H, J ¼ 7.6 Hz), 7.89 (d, 1H, C(4)H,
J ¼ 9.2 Hz), 8.12 (d, 1H, C(8)H, J ¼ 8.4 Hz), 9.14 (s, 1H, Nþ(10)H2),
10.87 (s, 1H, OH), 11.70 (s, 1H, Nþ(14)H) ppm; 13C-NMR (DMSO-
d6, 100 MHz): d ¼ 19.6 (C12), 40.7 (C9), 44.3 (C11), 50.9
(C15,18), 53.0 (C13), 63.0 (C16,17), 109.0 (C1), 117.9 (C3),
(C4a), 129.6 (C7), 130.1 (C5), 130.4 (C4), 131.7 (C8a), 155.7 (C2)
ꢃ
ppm; FT-IR (n): 3444 cmꢂ1 (w), 3058 cmꢂ1 (w), 2935 cmꢂ1 (w),
2465 cmꢂ1 (w), 1595 cmꢂ1 (w), 1269 cmꢂ1 (m), 1087 cmꢂ1 (m),
816 cmꢂ1 (s); HRMS (ESI): found 449.0404, calcd. for
C
18H25BrCl2N2O2 [MꢂH]ꢂ m/z ¼ 449.0404.
1,10-[Thiobis(methylene)]bis(naphthalen-2-ol) (21)
To a suspension of 8a (1.076 g, 5.0 mmol) in 30mL dry
tetrahydrofuran was added triethylamine (1.50 mL, 1.10 g,
10.8 mmol, 2.2 equiv.). After 25 min stirring under nitrogen
atmosphere, ethyl-3-bromopropionate (0.64 mL, 0.90 g,
5.0 mmol, 1.0 equiv.) was added to the yellowish solution at
room temperature. Within few minutes the reaction mixture
became turbid. After 9 days stirring at room temperature (TLC
indicated that after 30 min the reaction was ended, composi-
tion does not change within the long reaction time) the
insoluble solid was filtered off. To the filtrate 10mL n-hexane
was added. The slurry was stored at ꢂ20°C for 10 h. The flaky
precipitation was collected by filtration and was subsequently
purified by column chromatography (n-hexane/ethyl acetate/
acetic acid; 10:5:1) yielding product as colorless crystals.
Crystallization for X-ray diffraction measurement was con-
ducted via vapor diffusion with a solution of the product in
dichloromethane inn-hexaneatmosphere.Yield:0.10 g(5.5%);
mp: 255°C (decomp.); 1H-NMR (DMSO-d6, 400 MHz): d ¼ 4.29 (s,
4H, C(9)H2, C(90)H2), 7.17(d, 2H, C(3)H, C(30)H, J ¼ 9.2 Hz), 7.25(t,
2H, C(6)H, C(60)H, J ¼ 8.0 Hz), 7.31 (t, 2H, C(7)H, C(70)H, J ¼ 8.0
Hz), 7.38(d, 2H, C(4)H, C(40)H, J ¼ 8.8 Hz), 7.76(d, 4H, C(5)H, C(8)
H, C(50)H, C(80)H, J ¼ 9.2 Hz), 9.66 (s, 2H, OH) ppm; 13C-NMR
(DMSO-d6, 100 MHz):d ¼ 26.3 (C9,90), 115.2(C1,10), 117.9 (C3,30),
122.4 (C6,60), 123.0 (C8,80), 126.0 (7,70), 128.2 (C4a, C4a0), 128.2
122.6 (C8), 122.9 (C6), 127.0 (C7), 127.9 (C4a), 128.5 (C5), 131.1
ꢃ
(C4), 133.0 (C8a), 155.3 (C2) ppm; FT-IR (n): 3429 cmꢂ1 (w),
3358 cmꢂ1 (w), 3135 cmꢂ1 (w), 2934 cmꢂ1 (w), 2590 cmꢂ1 (w),
1626 cmꢂ1 (w), 1264 cmꢂ1 (m), 1136 cmꢂ1 (w), 819 cmꢂ1 (m),
751 cmꢂ1 (m); HRMS (ESI): found 371.1299, calcd. for
C
18H26Cl2N2O2 [MꢂH]ꢂ m/z ¼ 371.1299.
4-(3-{[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-
ammonio}propyl)morpholin-4-ium chloride (20)
To a solution of 18 (2.51 g, 10.0 mmol) in methanol (50 mL) 3-
(morpholino-4-yl)propan-1-amine (2.90 g, 20.1 mmol) was
added under a nitrogen atmosphere. After 2 h stirring at
room temperature, mixture was cooled in an ice-bath.
Afterwards sodium borohydride (1.0 g, 26.4 mmol) pre-dis-
solved in 20 mL water was added. After removing the ice-
bath, solution was stirred 1 h at room temperature. Reaction
was quenched by slow addition of 4 M hydrochloric acid
(30 mL). After addition was completed, methanol was
removed on a rotary evaporator and aqueous residue was
extracted with diethyl ether (3 ꢁ 30 mL). Subsequently,
aqueous phase was extracted with ethyl acetate (3 ꢁ 50 mL)
after pH-adjustment to pH 7–8 with sodium carbonate.
Immediately aqueous phase was adjusted to pH 9 with
ammonia and sodium carbonate and extracted again with
(C5, C50), 128.4 (C4, C40), 132.8 (C8a, C8a0), 152.8 (C2, C20) ppm;
ꢃ
FT-IR (n): 3526 cmꢂ1 (w), 3056 cmꢂ1 (w), 1626 cmꢂ1 (m),
1356 cmꢂ1 (m), 1213 cmꢂ1 (m), 809cmꢂ1 (s). Product structure
was confirmed by X-ray crystallography.
Biochemistry
Expression and purification of recombinant protein
The expression and purification of Sirt256–356 was performed
as described previously with minor modifications [46]. The
enzyme was resuspended in lysis buffer (25 mM KH2PO4,
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Archiv der Pharmazie
25 mM NaH2PO4, 400 mM NaCl, 5% (v/v) glycerol, 5 mM 2-
mercaptoethanol, pH 8.0) and finally purified with a Superdex
S75 26/60 gel filtration column (25 mM Hepes, 200 mM NaCl,
5% (v/v) glycerol, pH 8.0). Via SDS-PAGE the identity and
purity of the produced enzymes was verified [47] and the
protein concentration was determined by Bradford assay [48].
The catalytic reaction of hSirt2 was dependent on NADþ and
was able to inhibit by nicotinamide.
S. Coppola, L. Sansone, G. Passarino, D. Bellizzi,
M. A. Russo, M. Fini, M. Tafani, J. Cell. Physiol. 2017,
232, 1835–1844.
[3] E. Jing, S. Gesta, C. Ronald Kahn, Cell Metab. 2007, 6,
105–114.
[4] F. Wang, M. Nguyen, F.X. -F. Qin, Q. Tong, Aging Cell
2007, 6, 505–514.
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[6] S.-N. Xu, T.-S. Wang, X. Li, Y.-P. Wang, Sci. Rep. 2016, 6,
32734.
Biochemical assay
For the activity testing of hSirt2 an established high-
throughput (96-well plate) fluorescence-based assay was
used [33]. hSirt256–356 was added to a solution of NADþ (final
assay concentration 500 mM), the substrate Z-(Ac)Lys-AMC
(ZMAL, final assay concentration 10.5 mM), DMSO as a control
or the inhibitor in several concentrations dissolved in DMSO
(final DMSO concentration 5% (v/v)), filled up with assay
buffer (50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8.0) to 60 mL.
The enzyme concentration was adjusted to a final substrate
conversion of 15–30% to stay in a linear range. The incubation
time was 4 h at 37°C and 150 rpm. The catalytic reaction was
stopped by adding 60 mL of a developer solution (50 mM Tris,
100 mM NaCl, 6.7% (v/v) DMSO, trypsin 16.5 U/mL, 8 mM
nicotinamide, pH 8.0) and incubated 20 min at 37°C and
150 rpm. Fluorescence intensity was measured in a microplate
reader (BMG Polarstar, lex ¼ 390 nm, lem ¼ 460 nm). All
compounds were pretested on auto-fluorescence, amino-
methylcoumarin (AMC) quenching, and trypsin inhibition
under assay conditions. No interferences were observed. The
inhibitory effect was determined by using the DMSO-controls
as a reference. Graphpad Prism software (La Jolla, CA) was
used to calculate IC50 values.
[7] A. Bedalov, T. Gatbonton, W. P. Irvine, D. E. Gottschling,
J. A. Simon, Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
15113–15118.
[8] R. C. Neugebauer, U. Uchiechowska, R. Meier, H. Hruby,
V. Valkov, E. Verdin, W. Sippl, M. Jung, J. Med. Chem.
2008, 51, 1203–1213.
[9] T. Rumpf, S. Gerhardt, O. Einsle, M. Jung, Acta
Crystallogr. F 2015, 71, 1498–1510.
[10] J. Arct, E. Jakubska, G. Olszewska, Synthesis 1977, 1977,
314–315.
[11] A. Pochini, G. Puglia, R. Ungaro, Synthesis 1983, 1983,
906–907.
[12] A. Blade-Font, T. de Mas Rocabayera, J. Chem. Soc.,
Perkin Trans. 1982, 1, 841–848.
€ €
[13] M. Heydenreich, A. Koch, I. Szatmari, F. Fulop,
E. Kleinpeter, Tetrahedron 2008, 64, 7378–7385.
[14] T. Meyer, D. Geffken, Pharmazie 2003, 59, 593–596.
€
[15] D. Keck, S. Vanderheiden, S. Brase, Eur. J. Org. Chem.
2006, 2006, 4916–4923.
[16] C. Cardellicchio, M. A. M. Capozzi, F. Naso, Tetrahedron-
Asymmetry 2010, 21, 507–517.
ꢀ
ꢀ
€ €
[17] D. Toth, I. Szatmari, F. Fulop, Eur. J. Org. Chem. 2006,
2006, 4664–4669.
The Jung group thanks the Deutsche Forschungsgemeinschaft
(DFG, within GRK1976) for support.
[18] S. B. Sapkal, K. F. Shelke, A. H. Kategaonkar,
M. S. Shingare, Green Chem. lett. Rev. 2009, 2, 57–60.
[19] J.-T. Li, S.-F. Sun, M.-X. Sun, Ultrason. Sonochem. 2011,
18, 42–44.
[20] J. C. Duff, E. J. Bills, J. Chem. Soc. 1934, 282, 1305–1308.
Author contributions
€
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