Photochemical reactivity of naphthol-naphthalimide conjugates and their biological activity

Article abstract of DOI:10.3390/molecules26113355

Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, F?rster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (Φ_F ≈ 10^?2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (Φ_R ≈ 10^?5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants K_a ≈ 10⁵-10⁶ M^?1, as well as with bovine serum albumin (BSA) K_a ≈ 10⁵ M^?1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

Full text of DOI:10.3390/molecules26113355

molecules
Article
Photochemical Reactivity of Naphthol-Naphthalimide
Conjugates and Their Biological Activity
3,4
1
5
Matija Sambol 1,2, Patricia Bencˇic´
, Antonija Erben 1, Marija Matkovic´ , Branka Mihaljevic´ ,
Ivo Piantanida 1 , Marijeta Kralj 3 and Nikola Basaric´
*
1,
1
Department of Organic Chemistry and Biochemistry, Rud¯er Boškovic´ Institute, Bijenicˇka cesta 54,
10000 Zagreb, Croatia; m.sambol01@gmail.com (M.S.); Antonija.Erben@irb.hr (A.E.);
Marija.Matkovic@irb.hr (M.M.); Ivo.Piantanida@irb.hr (I.P.)
Fidelta Ltd., Prilaz Baruna Filipovic´a 29, 10000 Zagreb, Croatia
Division of Molecular Medicine, Rud¯er Boškovic´ Institute, Bijenicˇka cesta 54, 10000 Zagreb, Croatia;
patricia.bencic@pharmazie.uni-freiburg.de (P.B.); marijeta.kralj@irb.hr (M.K.)
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25,
79104 Freiburg, Germany
2
3
4
5
Division of Material Chemistry, Rud¯er Boškovic´ Institute, Bijenicˇka cesta 54, 10000 Zagreb, Croatia;
branka.mihaljevic@irb.hr
*
Correspondence: nbasaric@irb.hr
Abstract: Quinone methide precursors 1a–e, with different alkyl linkers between the naphthol and the
naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical
reactivity were investigated and connected with biological activity. Upon excitation of the naphthol,
Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of
fluorescence are low (ΦF ≈ 10−2). Due to FRET, photodehydration of naphthols to QMs takes place
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Sambol, M.; Bencˇic´, P.;
Erben, A.; Matkovic´, M.; Mihaljevic´,
B.; Piantanida, I.; Kralj, M.; Basaric´, N.
Photochemical Reactivity of
inefficiently (ΦR
≈
10−5). However, the formation of QMs can also be initiated upon excitation of
naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron
transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e
form complexes with ct-DNA with moderate association constants Ka
≈
105–106 M−1, as well as with
Naphthol-Naphthalimide Conjugates
and Their Biological Activity.
−1
bovine serum albumin (BSA) Ka
≈
105 M (1:1 complex). The irradiation of the complex 1e@BSA
resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a–e against
Molecules 2021, 26, 3355. https://
doi.org/10.3390/molecules26113355
two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or
irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the
enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results
presented have importance in the rational design of new generations of anticancer phototherapeutics
that absorb visible light.
Academic Editors: Raul Perez and M.
Consuelo Jiménez
Received: 5 May 2021
Accepted: 28 May 2021
Published: 2 June 2021
Keywords: naphthols; naphthalimides; FRET; PET; quinone methides; antiproliferative activity
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Reagents that can alkylate and cross-link DNA molecules are among the most cyto-
toxic molecules, often employed in anticancer treatment [
have been intensively studied as DNA cross-linking agents are quinone methides (QMs),
reactive intermediates in phenol chemistry and photochemistry [ ]. The biological activity
of QMs [ ] has been connected to their reactivity with proteins [ ], nucleobases [ ],
DNA [ 12] and G-quadruplexes [13 16]. Moreover, the antiproliferative action of the anti-
cancer antibiotic mitomycin [17 19] is rationalized by the intracellular formation of QMs,
followed by reversible DNA cross-linking [20 22]. An important advantage in the use of
QMs in biological systems stems from the fact that QMs can be generated in photochemical
reactions under mild conditions [23 24], which allows for the spatial and temporal control
of their formation. However, QMs react efficiently with H2O molecules [25 27], which
competes with the reactivity with biological targets such as DNA. Therefore, for efficient
1]. Among the molecules that
2
3
,4
5–
7
8
Copyright:
© 2021 by the authors.
9–
–
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
–
–
,
–
Molecules 2021, 26, 3355. https://doi.org/10.3390/molecules26113355
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3355
2 of 26
DNA alkylation and cross-linking, it is important to attach to the QM precursor units to
groups that can bind to DNA through noncovalent interactions [10].
We have investigated the photoinduced antiproliferative effect of several classes
of QM-precursors, consisting of hydroxybiphenyls [28,29], naphthols [30,31], and an-
throls [32,33]. Some anthrol derivatives were particularly interesting since they did not
exhibit cytotoxicity in the dark, but upon exposure to near-visible light the cytotoxicity
was enhanced 100 times and showed selectivity against cancer stem cells [33]. However,
for the ultimate application of QM precursors in biological systems, it is important to
develop molecules that can generate QMs upon excitation with visible light [34], as well as
to increase their reaction selectivity with biological targets, which can be facilitated via the
substitution of QM precursors to DNA binding units [10]. In that context, we have explored
the application of QM precursors attached to phenanthridine [35]. Further modifications
are required, since in these examples QMs were generated by UV light, precluding their
application in living cells.
Herein we present an investigation of the photochemical reactivity and noncova-
lent and covalent binding of a series of naphthol-naphthalimide conjugates,
thol QM precursors are known to undergo efficient photodehydration and deliver QMs
Scheme 1) [36], which can be applied in biological systems and labelling [ 37 40]. Our
new QM precursors are connected by an alkyl linker of different length to 1,8-naphthalimide,
which is a known pharmacophore, exhibiting antiviral [41 43], analgesic [44], local anes-
thetic [45], antitrypanosomal [46] and antagonist activity against 5-HT3 and 5-HT4 recep-
tors [47 49]. Of particular interest is the ability of 1,8-naphthalimide derivatives to interca-
late into DNA [50 51], which was exploited in the development of anticancer agents [52 53],
initiating further clinical studies [54 58]. Furthermore, 1,8-naphthalimides undergo pho-
toinduced electron transfer with guanine-rich polynucleotides [59 60] and induce cleavage
of the chains [61 62]. Consequently, newly designed molecules contain two warheads for
their anticancer activity. We have investigated the photochemical reactivity of and showed
1. Naph-
(
6, –
–
–
,
,
–
,
,
1
that compounds undergo dehydration, delivering QMs, albeit inefficiently. Furthermore,
QMs can be formed upon the excitation of low-energy chromophore naphthalimide, in a
process that involves photoinduced electron transfer (PET), as has been demonstrated in a
similar system by Freccero et al. [63]. The photophysical properties of
with steady-state and time-resolved fluorescence, whereas reactive intermediates in the
photochemistry of were detected through laser flash photolysis (LFP). The biological
applicability of was demonstrated by the binding study of to calf thymus DNA (ct-DNA)
and bovine serum albumin (BSA). The antiproliferative activity for was investigated
1 were investigated
1
1
1
1
on human cancer carcinoma cells, with or without exposure to irradiation, showing an
enhancement of the effect upon irradiation.
Scheme 1. Photodehydration of Naphth to quinone methide.
2. Results and Discussion
2.1. Synthesis
The synthesis of naphthol-naphthalimide conjugates is based on a Grignard reac-
tion as the key step in connecting two molecular fragments. The key intermediates, 1,8-
naphthalimide derivatives
good to excellent yields. These steps involved the alkylation of the imide by dibromides to
afford 64], followed by hydrolysis by HMPA and H2O to alcohols 65], and oxidation
to furnish . We tried two oxidation protocols, those of Swern [66] and Dess-Martin [67],
both of which produced in good yields. However, the procedure of Dess-Martin is a more
elegant procedure, allowing for easier isolation of the products.
4, for the coupling, were prepared in three steps (Scheme 2) in
2
[
3 [
4
4

Molecules 2021, 26, 3355
3 of 26
Scheme 2. Synthesis of naphthalimide precursors 4.
Grignard reagent for the coupling was prepared in two steps (Scheme 3), according
6
to the known procedure from 3-bromo-2-naphthol, which was protected by a benzyl group
and then transformed to the organometallic reagent via treatment with Mg [30].
Scheme 3. Preparation of naphthol Grignard reagent 6 [30].
Naphthalimide aldehydes
4 were treated with Grignard reagent 6, and after hydroly-
sis, coupled products were isolated in moderate yields. We tried several deprotection
7
protocols, such as the usual hydrogenolysis, over Pd/C [30] or treatment with triethylsilane
and Pd/C [68], or cyclohexene and Pd/C [69], which were unsuccessful due to a compet-
ing elimination of the benzylic alcohol. However, we encountered a hydrogen transfer
procedure with ammonium formate on Pd/C [70] that gave the desired target molecules
in moderate yields (Scheme 4).
1
Scheme 4. Synthesis of target naphthol-naphthalimides 1a–1e.
2.2. Photophysical Properties
UV-vis and steady-state fluorescence measurements for
and CH3CN-H2O (4:1, v/v) solutions, where the difference was expected due to excited
state proton transfer (ESPT) pathways taking place in the aqueous solution only [71 74],
and because -naphthols are known to undergo ESPT [75]. However, molecule contains
1 were conducted in CH3CN
–
β
1
two chromophores, naphthol and naphthalimide. Figure 1 shows the absorption spectra
of 1c, together with the spectra of the corresponding naphthol (Naphth, Scheme 1) and
naphthalimide alcohol 3c (for UV-vis spectra of
1 see Figure S1). The absorption spectrum
of 1c corresponds to the sum of the absorption spectra of the two chromophores (Figure
S2 in the ESI), indicating that the two chromophores do not interact in the ground state.
On the contrary, the normalized emission spectra of Naphth, 3c and 1c (Figure 1, right),
show that the emission from 1c originates from the naphthalimide chromophore and not
from the naphthol. The same behavior was observed for all derivatives,
1 (Figure S3).
Since the emission spectrum of the naphthol overlaps with the absorption spectrum of the

Molecules 2021, 26, 3355
4 of 26
naphthalimide (Figure S4 in the ESI), upon excitation of the naphthol, an efficient Förster
resonance energy transfer (FRET) to the naphthalimide takes place (vide infra).
1.2x104
1.0x104
8.0x103
6.0x103
4.0x103
2.0x103
0.0
1c
3c
Naphth
8.0x104
Naphth
3c
1c
6.0x104
4.0x104
2.0x104
0.0
ε
250
300
350
Wavelength / nm
ε
200
250
300
350
325 350 375 400 425 450 475 500 525
Wavelength / nm
Wavelength / nm
(a)
(b)
Figure 1.
(a) Absorption spectra of Naphth, 3c and 1c in CH3OH-H2O (4:1 v/v), (b) normalized fluorescence spectra
(λex = 310 nm) of Naphth, 3c and 1c in CH3CN.
Quantum yields of fluorescence (Φf) were measured in CH3CN and CH3CN-H2O
(4:1 v/v) with the use of quinine sulfate in 0.5 M H2SO4 as a reference (Φf = 0.546) [76
(Table 1). The f values are generally low, in accordance with the emission properties of
1,8-naphthalimides, which are known to undergo efficient intersystem crossing ( ISC > 0.9
and populate triplet excited states [77]. The higher f measured in the aqueous solvent than
]
Φ
Φ
)
Φ
in the neat CH3CN is also in accord with the photophysical properties of naphthalimides.
Protic solvents form H-bonds with the imide chromophore, which induces bathochromic
shifts in the fluorescence spectra. Furthermore, protic solvents perturb the energy levels
of the n,π* and π,π* singlet and triplet excited states, and therefore affect the efficiency of
the ISC [74]. One generally sees an increase in the Φf by increasing the alkyl linker length
between the two chromophores. A mechanism that can involve depopulation of the imide
singlet or triplet excited state is photoinduced electron transfer (PET) from the naphthol to
the imide, since 1,8-naphthlimides are good oxidizing agents in the excited state [78–80].
a
Table 1. Quantum yields of fluorescence Φf and efficiencies of FRET (ΦFRET) b.
Φf (CH3CN) × 103
Φf (CH3CN-H2O) × 103 c
Φ
(CH3CN) b
Comp.
FRET
1a
1b
1c
1d
1e
12 ± 1
15 ± 2
16 ± 2
17 ± 2
18 ± 2
0.70
0.79
0.82
0.71
0.69
14.3 ± 0.7
32.6 ± 0.5
37.4 ± 0.7
40 ± 1
48.2 ± 0.9
a
Quantum yield of fluorescence was measured using quinine sulfate in 0.5 M H2SO4
(
Φf = 0.546) [76]. The
fluorescence spectra were measured by exciting at 300, 310 and 320 nm, and the average values of the quantum
yields (Equation (S1) in the ESI) were calculated. The measurement was performed three times and the average
b
values are reported. The associated errors correspond to the maximum absolute deviation. The efficiency of
the Förster resonance energy transfer was determined by shortening of the average decay time of the donor
(naphthol) detected at 340 nm, compared to the decay time of Naphth, according to Equations (S8) and (S9) in the
ESI. c CH3CN-H2O (4:1, v/v).
Singlet excited state lifetimes for 1a–1e in CH3CN were measured by means of time-
correlated single photon counting (TC-SPC; for all data see Figures S5–S10 and Table S4 in
the ESI). The samples were excited at 340 nm where almost only naphthalimide absorbs
light, and the emission was detected at 390 nm. Fluorescence decays were fit to single
exponential functions, giving lifetimes in the range 80–130 ps, at the limits of detection of
the setup used. For some derivatives, fluorescence decay was best described as sum of

Molecules 2021, 26, 3355
5 of 26
two exponentials, with a small contribution of a longer decay component ascribed to the
emission from the naphthol.
Normalized fluorescence spectra of 1a
350 and 425 nm corresponding to the emission from the naphthalimide, and very weak
shoulders at 340 nm corresponding to the emission from naphthol, quenched by FRET
–1e in CH3CN show the strong bands between
≈
to the naphthalimide (Figure S4 in the ESI). To measure the efficiency of FRET, we per-
formed TC-SPC measurements by exciting samples at 280 nm, where both naphthol and
naphthalimide absorb light, and by collecting decays at 340 nm, where only naphthol
emits. These decays were compared to that of Naphth in CH3CN. Fluorescence decays of
1a
significantly shorter compared to that of Naphth (
FRET to be between 69% and 82% (Table 1). The three-exponential decays were explained
by the conformational mobility of molecules 1a 1e, resulting in a distribution of distances
–1e at 340 nm were all fit to a sum of three exponents, and the average decay time was
τ
= 9.07 ns), revealing the efficiency of
–
between the donor and the acceptor and different rates of FRET.
2.3. Photochemistry
Irradiation of Naphth in aqueous CH3OH leads to the dehydration and formation of
QM, which reacts with nucleophiles, delivering the photomethanolysis product, methyl
ether (Naphth-OMe Scheme 1) [36]. To probe for the methanolysis reaction with naphthol-
naphthalimide conjugates, 1, we performed irradiations in CH3OH-H2O (4:1 v/v), where
the anticipated methyl ethers would indicate the formation of QMs as intermediates.
Irradiations were performed at 254 nm, exciting mostly naphthol, or at 350 nm, where the
naphthalimide was only excited. In both cases, photolyses were not efficient, 1 h irradiation
gave photoproducts in 1–2% yield, detected using UPLC-MS. In addition to the anticipated
ethers, some unidentified products were also formed. To characterize the anticipated
ethers, they were prepared from
1 by means of H2SO4-catalyzed thermal methanolysis. By
comparing UPLC-MS chromatograms of the synthesized ethers and irradiated mixtures, we
have shown that the same ethers were formed in the photolyses. Preparative irradiations
of 1a, 1c and 1e were performed at 350 nm, since photolyses were more efficient than
those conducted at 254 nm. After the irradiations, the mixtures were separated by means
of UPLC-MS, and enriched fractions in photoproducts were analyzed using NMR and
UPLC-MS. In addition to the anticipated ethers, we detected 1,8-naphthalimide, formed
via the fragmentation of 1.
The quantum yields of the photomethanolysis reaction were estimated upon excitation
at 254 nm in CH3OH-H2O (4:1 v/v), which is a monochromatic irradiation source, and
KI/KIO3 was used as an actinometer (ΦR = 0.74) [81]. The results are compiled in Table 2.
One generally sees very low values for
ΦR, which are due to FRET from the naphthol to
the naphthalimide. Such a result was not anticipated in the first instance, since it is known
that photodehydration takes place in an ultrafast photochemical reaction [82], which in
principle could be in competition with FRET.
Table 2. Quantum yields (ΦR) of the photomethanolyses of 1 in CH3OH-H2O (4:1 v/v) a.
Comp.
Φ
× 105
R
1a
1b
1c
1d
1e
3.0 ± 0.3
2.6 ± 0.8
2.8 ± 0.5
3.5 ± 0.9
3.6 ± 0.8
a
KI/KIO3 (ΦR = 0.74) [81] was used as an actinometer and the samples were excited at 254 nm and analyzed
using UPLC-MS. The measurement was performed in triplicate and the average value is reported. The quoted
error corresponds to the maximum standard deviation.

Molecules 2021, 26, 3355
6 of 26
2.4. Laser Flash Photolysis (LFP)
To detect intermediates in the photochemistry of naphthol-naphthalimides
1, LFP was
used. The samples were excited with a Nd:YAG laser at 266 or 355 nm. The measurements
were performed in Ar- and O2-purged CH3CN solution, where O2 was expected to quench
triplets and radicals, but not QMs (for all transient absorption spectra and the associated
decay kinetics, see Figures S11–S16).
Upon excitation at 266 nm, in both Ar- and O2-purged solution, a strong transient
was detected, with a maximum at 350 nm, with weaker absorption extending over the
visible region. Thus, the transient absorption at 400 nm was fit to a sum of two exponents
with lifetimes of
τ
= 2.6
±
0.1 ns and
τ
= 23.1
±
0.8 ns. The transients were tentatively
assigned to the naphthol radical cation (λmax = 360 and 460 nm) [83] and the naphthalimide
radical anion (λmax = 410 nm) [84], based on comparison with the published spectra in the
preceding literature, and the known photochemistry of the phenol-imide conjugates [63].
The short lifetime was tentatively associated to the naphthol radical cation, prone to fast
decay via deprotonation to a naphthoxyl radical. The species with longer lifetime probably
corresponds to the naphthalimide radical anion, for which the main decay pathway is
probably back electron transfer, taking place more slowly than protonation.
LFP measurements allowed for the detection of additional weak transient absorption
with a maximum at 470 nm. It decayed with unimolecular kinetics, with the following
lifetimes: τAr = 460
sient was quenched by O2, kq = (8.7
with literature [63 79], it was assigned to the naphthalimide triplet excited state. The
±
20 ns; τair = 290
±
20 ns and τO2 = 100
×
±
3 ns. Since the tran-
±
0.7)
108 M−1 s−1, and based on the comparison
,
naphthalimide triplet was formed using FRET from the naphthol, followed by ISC. Upon
excitation at 355 nm, only one strong transient was detected, absorbing with a maximum at
470 nm (Figure 2), decaying with unimolecular kinetics with the lifetimes; τAr = 340
±
2 ns;
)
τair = 245 ± 3 ns and τO2 = 105
±
2 ns. Based on the quenching by O2, kq = (7.3 ± 0.3 ×
−1
108 M
s
−1; and its maximum of absorption [63
,
79], it was assigned to the naphthalimide
triplet excited state. Note the much shorter decay time of the naphthalimide triplet in
Ar-purged solution than the published value ( = 0.25 0.12 ms) [79], which can be ex-
plained by the intramolecular PET between the naphthol and the naphthalimide, although
characterized with a slow process and the rate constant of k = 2.9
106 s−1. Based on the
τ
±
×
proposed PET between the naphthol and the naphthalimide, the corresponding radical
cation and the radical anion should have been detected. However, their fast decay and low
absorptivity precluded their detection. Thus, after the decay of the triplet, due to the low
intensity of the transient absorption signal, additional transients were not detected.
0.08
0.10
0 ns
160 ns
0.08
0.06
0.04
0.02
0.00
310 ns
470 ns
620 ns
780 ns
940 ns
1.1 μs
Ar
air
O2
0.06
0.04
0.02
0.00
300
400
500
600
700
0
500
1000
1500
2000
Wavelength / nm
Time / ns
(a)
(b)
Figure 2.
excited at 355 nm, laser power
O2-purged CH3CN solution.
(
a
) Transient absorption spectra of N2-purged solution of 1a in CH3CN (c = 7.23
×
10−5 M) The samples were
20 mJ/pulse. (b) Decay of transient absorbance at 470 nm in Ar-purged, not purged and
≈

Molecules 2021, 26, 3355
7 of 26
2.5. Photochemical Reaction Mechanism
A plausible mechanism for the formation of ethers, as well as for the cleavage photo-
products upon excitation at 350 nm, is shown in Scheme 5. The mechanism involves PET
from the naphthol to the naphthalimide as the key step, which was indicated from the LFP
measurements, and is in accordance with the mechanism proposed by Freccero et al. [63].
Thus, the formation of the QM and methanolysis product upon excitation at 254 nm may
proceed via the dehydration mechanism proposed by Popik et al. [36]. However, due to
efficient FRET from the naphthol to the naphthalimide, the methanolysis upon excitation at
254 nm is not efficient. On the other hand, excitation at 350 nm leads to naphthalimide S1,
which, after an efficient ISC, leads to a naphthalimide triplet excited state. The naphthalim-
ide triplet may undergo PET, whereupon the naphthol radical cation and the naphthalimide
radical anion are formed (1(CT) in Scheme 5). Efficient back electron transfer (BET) gives 1
in the ground state, and it is probably the main deactivation pathway resulting in the low
efficiency of the photochemical processes. In a protic solvent, the acidic radical cation is
deprotonated to the phenoxyl radical
is protonated to a ketyl radical (BR). The formation of radicals most probably leads to
combinations of disproportionation and cleavage reactions, whereupon naphthalimide is
formed as a detectable product. Note that the formation of biradicals such as (BR) may
1(BRA), whereas the naphthalimide radical anion
1
1
occur via photoinduced H-transfer reactions. Although such processes are not ubiquitous
in the naphthalimide photochemistry [77], photochemical hydrogen abstractions are well
documented for imide derivatives such as phthalimides [85]. Furthermore, as suggested by
Freccero [63], the naphthalimide radical anion may be protonated before the BET, where-
upon naphtholate
1
is formed. A subsequent loss of OH− leads to the formation of QM.
Note that the QM formation, in this case, was initiated by excitation of the lower-energy
chromophore naphthalimide, at 350 nm. Furthermore, the formation of QMs takes place
more efficiently upon excitation at 350 nm, although excitation of the naphthol at 254 nm is
followed by FRET to the naphthalimide. However, the FRET is not 100% efficient.
Scheme 5. Plausible photochemical processes upon excitation at 254 nm or 350 nm.

Molecules 2021, 26, 3355
8 of 26
The irradiation experiments and mechanistic investigations indicated the formation
of QMs from
photolysis of
1
1
, which can induce biological effects, but the reaction is inefficient. However,
gives rise to a plethora of other reactive intermediates, such as free radicals
that can engage in reactions with biomacromolecules and induce cell damage. Most
importantly, upon excitation at 350 nm, the naphthalimide triplet excited state is populated,
which is quenched by O2, giving rise to singlet O2 and increased ROS, which is probably
the main species responsible for the cytotoxic effect.
2.6. Noncovalent Binding to ct-DNA
Since naphthalimide derivatives are known to bind to polynucleotides [50,51], we
investigated the non-covalent binding of 1 with naturally occurring calf thymus DNA (ct-
DNA), characterized by the typical B-helical secondary structure and the almost equimolar
ratio of AT- and GC-basepairs. The effect of binding was investigated via thermal denatu-
ration experiments, whereas binding constants were determined by fluorescence titration.
To probe for the binding mode (intercalation vs groove binding), we conducted CD mea-
surements. Due to low solubility in H2O, stock solutions of
diluted with cacodylate buffer solutions.
1 were prepared in DMSO and
2.6.1. Thermal Denaturation
The binding of small molecules to double stranded (ds) polynucleotides affects the
stability of the ds-helix, usually by stabilizing its structure. Thus, thermal denaturation
and unwinding of the ds-helix into the single stranded (ss) form takes place at a higher or
lower temperature, known as the melting temperature (Tm) [86]. The difference between
the Tm value of a free ds-polynucleotide and a complex with a small molecule (∆Tm
value) provides information about the interaction between the small molecule and the ds-
◦
polynucleotides. Moderate to strong stabilization (
∆
T
m > 5 C) supports the intercalative or
◦
minor groove binding interaction [87], whereas weak stabilization (
∆
T
m = 0–5 C) suggests
a binding process driven by hydrophobic effects, accompanied by weak H-bonding or
electrostatic interactions.
The thermal denaturation experiments were conducted with 1a and 1f and ct-DNA
(Tables S5 and S6 and Figures S17 and S18 in the ESI). However, the compounds in the
concentration corresponding to the ratio r = (1)/(ct-DNA) = 0.3 did not affect the thermal
stability of the helices, suggesting non-specific binding along the DNA double helix.
2.6.2. UV-Vis and Fluorescence Titrations
The binding of
cacodylate buffer at pH 7. However, UV-vis titrations did not show applicability in
determining the association constants due to the low solubility of molecules in the
aqueous cacodylate buffer (containing less than 1% DMSO). In the measurement conducted
with the use of a 5 cm pathlength probe, allowing titrations at lower concentrations of and
DNA, 1a precipitated immediately, whereas 1e precipitated after the addition of ct-DNA
at the ratio r = ( )/(ct-DNA) = 0.5, precluding estimation of the constant. However, even
three additions of DNA at r = 2–0.5 revealed a weak hypochromic effect at λ < 370 nm, and
a hyperchromic effect at > 370 nm (Figure S19 in the ESI), suggesting the formation of
a complex. Fortunately, fluorescence of allowed fluorescence titrations at much lower
concentrations. The addition of polynucleotide to the solution generally induced weak
emission changes in , allowing the estimation of the binding constants (Ka) only. The
1 to ct-DNA was assayed via UV-vis and fluorescence titrations in
1
1
1
λ
1
1
samples were excited at 320 nm, where the hypochromic effect in UV spectrum was
negligible (<7%). The titration with ct-DNA induced weak emission quenching of 1a
(Figure 3, top) but an increase in the emission of 1e (Figure 3, bottom).

Molecules 2021, 26, 3355
9 of 26
300
280
260
240
300
250
200
150
100
50
2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-5 1.0x10-4
350
400
450
0.0
c (ct-DNA) / mol dm−3
Wavelength / nm
(a)
(b)
210
200
190
180
170
160
200
150
100
50
0
350
3.0x10-5 6.0x10-5 9.0x10-5 1.2x10-4 1.5x10-4
0.0
400
450
c (ct-DNA) / mol dm−3
(d)
Wavelength / nm
(c)
Figure 3.
of ct-DNA. (
(
a
,
c
b
) Fluorescence spectra (λexc = 320 nm) of 1a
(
top) and 1e
(
bottom) (c = 2.0
×
10−6 M) at different concentrations
) and 1e ); the black dots
,
d) Dependence of the fluorescence intensity at 390 nm for the solution of 1a
(a
,b
(c,d
are experimental values, whereas the red line is a fitted value◦according to the Scatchard model [88]. The measurements
were performed in Na cacodylate buffer (pH 7.0, 50 mM at 25 C, containing <1% DMSO).
The quenching of fluorescence by ct-DNA may be rationalized by PET between the
naphthalimide and guanine, which is the nucleobase with the lowest oxidation poten-
tial [59,60,89], and in principle, it can be static (due to the formation of a nonfluorescent
complex) or dynamic (due to diffusion). To check if the quenching is static, we performed
TC-SPC measurement for 1a and a mixture of 1a and ct-DNA, in the same concentrations
as in the fluorescence titration experiments (see Figures S20–S21 and Table S7 in the ESI).
The same decay time in the presence of ct-DNA would indicate the static quenching. How-
ever, the short decay times at the limits of the detection of the setup used precluded their
precise determination. Thus, although shortening of the main decay component from 210
to 170 ps was observed, these values are within experimental error. Consequently, we
cannot completely rule out a dynamic quenching mechanism, but the short decay times
and quenching in the concentration of DNA at 10−4 M is in favor of the static quenching
mechanism. Namely, the Stern–Volmer analysis of the quenching by DNA would pro-
vide the quenching constant kq in the order of 1012 M−1 −1, which is higher than the
s
diffusion limit.
Non-linear fitting of the fluorescence dependence on the ct-DNA concentration using
the Scatchard model [88] gave a binding constant logKa = 5.8, for the fixed value of the
Scatchard ratio n[1a]/[ct-DNA] = 0.2. On the contrary, the addition of polynucleotide to
the solution of 1e resulted in an increase in the fluorescence (Figure 3, bottom), further sup-
porting the possibility that the changes in fluorescence are not due to dynamic quenching.
A tentative explanation for the fluorescence enhancement is the aggregation of the larger
molecule 1e in the aqueous medium, yielding a lower intensity in respect to 1a (Figure 3,

Molecules 2021, 26, 3355
10 of 26
see starting intensities of free dyes). Thus, upon the addition of the DNA, de-aggregation
of 1e takes place, resulting in the fluorescence increase. The association constant logKa = 6.3
(fixed value n = 0.2) was estimated through the non-linear fitting of data to the Scatchard
model [88].
2.6.3. CD Experiments
Although the thermal denaturation experiments indicated that derivatives 1 do not
intercalate or bind to grooves of ct-DNA, binding was further investigated by means of
circular dichroism (CD) spectroscopy. Namely, CD spectroscopy is a powerful analytical
tool for the binding studies of small molecules to chiral macromolecules such as DNA [90],
giving rise to the distinctive spectral differences for intercalators and groove binding
derivatives [91,92]. Compounds 1 are chiral, but they are present in a racemic mixture, and
therefore they do not have measurable CD spectra. However, upon their addition to the
solution of ct-DNA, they can affect the helical chirality of DNA, or if they form complexes
with the characteristic organization of the chromophores along the chiral axes of DNA, an
induced CD signal can show up in the region where molecules 1 absorb light [91,92].
The addition of 1a or 1e to the solution of ct-DNA induced only small changes in the
DNA CD signal, decreasing the intensity of the positive band at 275 nm, and that of the
negative band at 245 nm (Figures S22 and S23 in the ESI). The lack of measurable ICD
bands >300 nm suggests a non-uniform orientation of the chromophores of 1e in respect
to the DNA chiral axis, which is in agreement with the non-specific binding of the small
molecule along DNA double helix.
The binding studies with ct-DNA have shown that compounds 1a and 1e form com-
plexes with polynucleotides with moderate binding constants. However, the negligible
effect of 1a and 1e in the thermal denaturation of ct-DNA, as well as the absence of ICD
bands in the CD experiments, suggest that the studied naphthol-naphthalimides bind to
ds-DNA in a nonspecific way, most probably by agglomeration along the double helix,
caused by hydrophobic effects, accompanied potentially by weak H-bonding interactions.
2.7. Noncovalent and Covalent Binding to BSA Protein
Naphthol naphthalimide derivatives are non-charged species and not very polar, being
good substrates for noncovalent binding to serum albumin proteins. We took advantage
of the intrinsic fluorescence of 1a and 1e to study their binding to BSA, presuming that
the insertion of small molecules into a BSA binding site would change the hydration of
chromophores, consequently yielding a measurable change in their emission. The dynamic
quenching mechanism was again dismissed due to the short singlet excited state lifetimes
of 1a–1e and the low concentration range of protein, which affected the fluorescence.
Accordingly, upon the addition of BSA, the fluorescence of 1a was quenched until
a ratio of 1:1 between 1a and BSA was reached (Figure 4, arrow A). Further additions of
BSA induced a fluorescence increase in 1a and a considerable broadening of the emission
spectrum, accompanied by a significant hypsochromic shift of the emission maximum
(Figure 4, arrow B). Such opposite changes clearly indicate the formation of two types of
complexes. The fluorescence quenching upon the addition of BSA to 1a is assigned to the
aggregation of two molecules within the BSA binding site, whereas further additions of
BSA allowed each molecule of 1a to bind to an independent binding site, thus resulting
in the formation of a 1:1 complex (denoted as 1a@BSA) and a consequent fluorescence
increase. Multicomponent nonlinear regression analysis of the fluorescence data using
HypSpec2014 software [93] gave the best fit to the formation of 1a@BSA complexes in the
stoichiometries 2:1 and 1:1, with the association constants log
and logβ (1a@BSA) = 5.24 ± 0.05, respectively.
β
(
1a2@BSA) = 11.38
±
0.04

Molecules 2021, 26, 3355
11 of 26
160
140
120
100
80
180
150
120
90
B
A
r [1a]/[BSA] = 1
60
60
30
40
0
350
2.0x10-6
4.0x10-6
6.0x10-6
0.0
400
450
500
550
600
c (BSA) / mol dm−3
Wavelength / nm
(a)
(b)
−6
10 M) in the presence of different concentrations of BSA.
) Dependence of the fluorescence intensity at 390 nm on the BSA concentration. The black dots are experimental values
Figure 4.
(a
) Fluorescence spectra (λexc = 320 nm) of 1a (c = 1.0
×
(
b
and the red line is the fit to the model, involving 1a@BSA in the stoichiometric ratios of 1:1 and 2:1. The measurement was
◦
conducted in aqueous solution, containing 0.05 M sodium cacodylate buffer at pH = 7.0, at 25 C (containing <1% DMSO).
Contrary to 1a, the addition of BSA to the solution of 1e led to a fluorescence increase
only (Figure S23). Nonlinear regression analysis using HypSpec2014 software revealed the
formation of one complex with the stoichiometric ratio of 1:1 and the association constant
logKa (1e@BSA) = 5.05
BSA with similar binding constants, but only the smallest 1a forms a 2:1 complex within a
BSA binding site.
±
0.05. It should be noted that 1a and 1e form 1:1 complexes with
Our preliminary binding experiments with BSA undoubtedly indicate the formation
of complexes 1a@BSA and 1e@BSA. However, the exact binding site of
remained undetermined and is not in the scope of this report. To demonstrate that the
irradiation of complexes of with BSA leads to the alkylation of the protein, we performed
1 in the protein
1
irradiations of the 1a@BSA and 1e@BSA complexes. The aqueous buffered solutions of
the complexes were irradiated at 350 nm, followed by desalting of the protein and analysis
by means of MALDI-MS. Naphthol-naphthalimide with the shortest linker, 1a, did not
alkylate the protein, whereas the one with the longest linker, 1e, covalently attached to
BSA. The molecular weight of 67.1–67.2 kDa pointed to the addition of 1e to the protein
(see Figures S25–S27 and Table S8 in the ESI). It is plausible to assume that the photoin-
duced mechanism of protein alkylation involves QM intermediates, as seen in the prior
literature [6,7], although the photodehydration to QMs from 1 is inefficient.
2.8. Antiproliferative Activity
We have demonstrated that compounds 1a–1e bind to a model DNA and protein
by means of noncovalent interactions, where, after photochemical excitation, they may
induce damage. Therefore, we investigated the antiproliferative activity of compounds
1a–1e on two human cancer cell lines, H-460 (lung) and MCF-7 (breast), with the cells
kept in the dark, or irradiated at 350 nm (for all data, see Table S9 in the ESI). Control
experiments were performed with a psoralene derivative (trioxsalen), which is known
to induce photoactivable cross-linking [12,94] and Naphth (Scheme 1). The analysis of
results in Table S9 showed decreasing cytotoxicity under dark conditions (expressed by
IC50 values) proportional to the linker length between the two chromophores, with 1e
bearing the longest linker, being non-toxic for the H-460 cell line even at the highest
tested concentration. However, the enhancement of the antiproliferative effect upon
UV irradiation (Table S9, 350 nm) shows the opposite trend—the strongest effects were
observed for the compounds with longer linkers (1d, 1e). This result suggests that UV
irradiation of 1e inside the cell leads to the production of a higher amount of cytotoxic agent

Molecules 2021, 26, 3355
12 of 26
(possibly QMs, as well as singlet oxygen/reactive oxygen species (ROS)) in comparison to
the smallest, 1a (showing an order of magnitude weaker effect). Furthermore, compounds
1a–e exhibit higher cytotoxicity than Naphth, indicating that the naphthalimide moiety
is important for this activity. Although we do not have clear evidence as to which of the
reactive species formed in the photochemical reactions led to the observed photoinduced
antiproliferative effect, it is plausible that singlet O2 and subsequent ROS formed by
the quenching of the naphthalimide triplet played an important role, as well as QMs
and reactive radicals formed in the photoinduced processes. It should be noted that the
investigated molecules cannot have therapeutic value, since irradiation with UV light
was used. However, this proof of principle is important for the future rational design of
photoactivable compounds with antiproliferative activity.
3. Conclusions
Naphthol-naphthalimide conjugates, QM precursors
1 undergo photodehydration to
the corresponding QMs very inefficiently due to FRET from the naphthol to the naphthal-
imide. In addition to the elimination of H2O, QMs can also be formed upon excitation of
the naphthalimide in a process that involves PET from the naphthol to the naphthalimide.
The linker length variation between the naphthol and the naphthalimide in the 1a–1e
series mildly affects their photophysical properties and the photochemical reactivity, but
had a much more obvious impact on bio-relevant properties, DNA-fluorimetric response,
protein-binding stoichiometry and cytotoxicity on cell lines. The naphthol-naphthalimide
conjugates showed moderate affinity toward ds-DNA (Ka
≈
105–106 M−1), due to their
non-specific agglomeration along the DNA helix. Comparison of the shortest (1a) and the
longest (1e) derivative revealed opposite fluorimetric responses, attributed to 1e aggre-
gation in a free state and de-aggregation upon DNA binding. Furthermore, the studied
compounds also efficiently bind to the BSA protein, whereby the shortest analogue, 1a
,
can form dimers inside a BSA binding site, at variance to the longest, 1e, which binds
only as a monomer. Upon irradiation of the complex 1e@BSA, photoinduced alkylation
of the protein takes place, probably via the QM. Derivatives 1a–e exhibit cytotoxic effects
against cancer cell lines H-460 and MCF-7 that is reversely proportional to the linker length
and correlated to the aggregation propensity of the longest derivative, 1e. All compounds
showed enhanced cytotoxicity upon exposure to irradiation at 350 nm, and this was partic-
ularly pronounced for the longest analogue, 1e, which not only increased by two orders of
magnitude in H-460 cells but also was an order of a magnitude stronger than the shortest
analogue, 1a. The enhancement of this activity probably stems from the formation of singlet
oxygen (or ROS-related species) but the effect of QMs and reactive radicals formed in the
photoinduced processes cannot be disregarded. The particularly promising theragnostic
properties of 1e, unifying photoinduced-bioactivity and fluorescence enhancement upon
binding to biorelevant targets, encourage the further study of its close analogues.
4. Materials and Methods
General: 1H and 13C NMR spectra were recorded at 300, 400, 500 or 600 MHz at
rt, using TMS as a reference, and chemical shifts were reported in ppm. Melting points
were determined using a Mikroheiztisch apparatus and were not corrected. IR spectra
were recorded on an ATR spectrometer and the characteristic peak values were given in
cm−1. HRMS was carried out on a MALDI TOF/TOF instrument. Details on semiprepara-
tive MPLC and UPLC separations and analyses are reported in the ESI. Chemicals were
purchased from the usual commercial sources and were used as received. Solvents for
conducting moisture-sensitive reactions were further dried over sodium metal. Solvents
for chromatographic separations were used as delivered from the supplier (p.a. or HPLC-
grade). Preparations of known compounds
was dissolved in Na-cacodylate buffer, I = 0.05 mol dm−3, pH 7.0, and it was additionally
sonicated and filtered through a 0.45- m filter. The polynucleotide concentration was
2, 3, 5 and 6 are described in the ESI. ct-DNA
µ

Molecules 2021, 26, 3355
13 of 26
determined spectroscopically as the concentration per nucleotide/phosphate, using molar
absorption coefficient for calf thymus DNA of 6600 M−1 cm−1 at 260 nm.
•
General procedure for the preparation of N-(ω-formylalkyl)-1,8-naphthalimide 4—
Swern oxidation
A dry two-neck round-bottom flask, equipped with a dropping funnel and a septum,
under Ar inert atmosphere, was charged with oxalyl chloride (5 mmol) and dry CH2Cl2
(1 mL/mmol oxalyl chloride). The reaction mixture was cooled down to
−
78 ◦C, and a
solution of DMSO (10 mmol) in dry CH2Cl2 (0.2 mL/mmol DMSO) was added dropwise.
◦
After 1 h of stirring at
CH2Cl2 (1 mL/mmol
−
78 C to the reaction mixture, a solution of
3
(1 mmol) in dry
3
) was added dropwise. The reaction mixture was stirred at
−
78 ◦C
for 2 h. Dry t◦riethylamine (15 mmol) was then added dropwise, and the reaction was
stirred at
−
78 C for 1 h and then was left to spontaneously reach rt over 17 h. The reaction
mixture was transferred to a separation funnel, diluted with H2O and extracted with
CH2Cl2 (3 20 mL). The combined organic layers were washed with saturated aqueous
NH4Cl (3 20 mL) and brine (2 20 mL). The solution was dried over anhydrous Na2SO4,
filtered and the solvent was removed on a rotary evaporator. The residue was purified on a
silica gel column using EtOAc (0 to 50%)/cyclohexane as eluent to afford the pure product.
×
×
×
•
General procedure for the preparation of N-(ω-formylalkyl)-1,8-naphthalimide—
Dess–Martin oxidation [95]
A round-bottom flask, equipped with a dropping funnel, was charged with Dess–
Martin periodinane (DMP, 1.2 mmol) and CH2Cl2 (2.5 mL/mmol DMP). A solution of
3
(1 mmol) in CH2Cl2 (2.5 mL/mmol ) was added dropwise at rt. The reaction mixture was
3
stirred at rt for 1.5 h, and then a solution of Na2S2O3 (9.5 mmol) in a saturated aqueous
solution of NaHCO3 (1 mL/mmol Na2S2O3) was added. The resulting suspension was
stirred for 20 min after it was transferred to a separation funnel. The layers were separated
and the aqueous layer was extracted with CH2Cl2 (3
layers were washed with saturated aqueous NaHCO3 (3
The solution was dried over anhydrous Na2SO4, filtered and the solvent was removed on
a rotary evaporator. The residue was purified on a silica gel column using EtOAc (0 to
50%)/cyclohexane as eluent to afford the pure product.
×
20 mL). The combined organic
10 mL) and brine (2 10 mL).
×
×
N-(3-formylprop-1-yl)-1,8-naphthalimide (4a) was prepared according to the general
procedure for Swern oxidation from alcohol 3a (2.15 g, 7.9 mmol). The reaction after
work-up and chromatography furnished 1.17 g (55%) of the pure product in the form of
a colorless solid. The NMR characterization was in accordance with the data from the
preceding literature [96].
mp 78–80 ◦C; IR (ATR) νe/cm−1: 3063 (Ar C-H), 2952, 2729 (C-H), 2830 (C-H ald.), 1698
1
(C=O, ald.), 1660 (C=O), 1586 (C–N amide), 1341 (C=C); H NMR (CDCl3, 400 MHz)
δ
/ppm: 9.81 (t, 1H, J = 1.4 Hz), 8.59 (dd, 2H, J = 7.3 Hz, J = 1.1 Hz), 8.22 (dd, 2H, J = 8.3 Hz,
J = 1.0 Hz), 7.76 (dd, 2H, J = 8.2 Hz, J = 7.2 Hz), 4.25 (t, 2H, J = 7.0 Hz), 2.59 (td, 2H, J = 7.3 Hz,
J = 1.4 Hz), 2.11 (quin, 2H, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz)
δ
/ppm: 201.4 (d), 164.2
(s, 2C), 134.0 (d, 2C), 131.5 (s), 131.3 (d, 2C), 128.1 (s), 126.9 (d, 2C), 122.5 (s, 2C), 41.3 (t), 39.4
+
2
ꢀ
(t), 20.7 (t); UPLC-MS/UV: method
, tR = 0.96 min, m/z = 268.10 [M + H] , found 268.14;
HRMS (MALDI TOF/TOF): calculated for C16H13NO3 [M + H]+ 268.0974; found 268.0984.
N-(4-formylbut-1-yl)-1,8-naphthalimide (4b) was prepared according to the general
procedure for Swern oxidation from alcohol 3b (1.93 g, 6.8 mmol). The reaction after
work-up and chromatography furnished 1.22 g (55%) of the pure product in the form of a
colorless solid.
The Dess–Martin oxidation method with alcohol 3b (4.86 g, 17.2 mmol) with DMP
(8.73 g, 20.6 mmol) after the work-up and chromatography gave 4.25 g (88%) of the pure
product in the form of a colorless solid.
mp 94–95 ◦C; IR (ATR) νe/cm−1: 2942, 2732 (C-H), 2831 (C-H ald.), 1690 (C=O, ald.), 1656
(C=O), 1586 (C–N amide), 1339 (C=C); 1H NMR (CDCl3, 300 MHz)
δ/ppm: 9.79 (t, 1H,

Molecules 2021, 26, 3355
14 of 26
J = 1.6 Hz), 8.60 (dd, 2H, J = 7.3 Hz, J = 1.0 Hz), 8.22 (dd, 2H, J = 8.3 Hz, J = 0.9 Hz), 7.76
(dd, 2H, J = 8.2 Hz, J = 7.3 Hz), 4.22 (t, 2H, J = 6.9 Hz), 2.59–2.48 (m, 2H), 1.88–1.70 (m, 4H);
13C NMR (CDCl3, 100 MHz)
δ/ppm: 202.2 (d), 164.2 (s, 2C), 133.9 (d, 2C), 131.5 (s), 131.2
(d, 2C), 128.1 (s), 126.9 (d, 2C), 122.5 (s, 2C), 43.4 (t), 39.7 (t), 27.5 (t), 19.5 (t); UPLC-MS/UV:
+
2
ꢀ
method
, tR = 1.03 min, m/z = 282.11 [M + H] , found 282.10; HRMS (MALDI TOF/TOF):
calculated for C17H15NO3 [M + H]+ 282.1130; found 282.1140.
N-(5-formylpent-1-yl)-1,8-naphthalimide (4c) was prepared according to the general
procedure for Swern oxidation from alcohol 3c (5.24 g, 17.6 mmol). The reaction after
work-up and chromatography furnished 4.65 g (89%) of the pure product in the form of a
colorless solid.
mp 76–77 C; IR (ATR) νe/cm−1: 3060 (Ar C-H), 2927, 2851 (C-H), 2699 (C-H ald.), 1690 (C=O,
◦
ald.), 1649 (C=O), 1584 (C–N amide), 1343 (C=C); 1H NMR (CDCl3, 400 MHz)
δ
/ppm: 9.76
(t, 1H, J = 1.8 Hz), 8.59 (dd, 2H, J = 7.3 Hz, J =1.0 Hz), 8.20 (dd, 2H, J = 8.3 Hz, J = 1.0 Hz),
7.74 (dd, 2H, J = 8.2 Hz, J = 7.3 Hz), 4.18 (t, 2H, J = 7.5 Hz), 2.45 (td, 2H, J = 7.3 Hz
,
J = 1.7 Hz), 1.82–1.66 (m, 4H), 1.52–1.42 (m, 2H); 13C NMR (CDCl3, 100 MHz)
δ
/ppm: 202.5
(d), 164.1 (s, 2C), 133.9 (d, 2C), 131.5 (s), 131.2 (d, 2C), 128.1 (s), 126.9 (d, 2C), 122.6 (s,
2
2C), 43.7 (t), 40.0 (t), 27.7 (t), 26.5 (t), 21.5 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.11 min,
m/z = 296.13 [M + H]+, found 296.16; HRMS (MALDI TOF/TOF): calculated for C18H17NO3
[M + H]+ 296.1287; found 296.1291.
N-(6-formylhex-1-yl)-1,8-naphthalimide (4d) was prepared according to the general
procedure for Dess–Martin oxidation from alcohol 3d (3.68 g, 11.8 mmol) and DMP (6.02 g,
14.2 mmol). The reaction after work-up and chromatography furnished 2.82 g (77%) of the
pure product in the form of a colorless solid.
mp 83–84 ◦C; IR (ATR) νe/cm−1: 2931, 2857 (C-H), 2710 (C-H ald.), 1693 (C=O, ald.), 1657
(C=O), 1586 (C–N amide), 1343 (C=C); 1H NMR (CDCl3, 400 MHz)
δ/ppm: 9.75 (t, 1H,
J = 1.9 Hz), 8.59 (dd, 2H, J = 7.4 Hz, J = 0.9 Hz), 8.20 (dd, 2H, J = 8.2 Hz, J = 0.9 Hz), 7.74
(dd, 2H, J = 8.2 Hz, J = 7.3 Hz), 4.17 (t, 2H, J = 7.4 Hz), 2.43 (td, 2H, J = 7.3 Hz, J = 1.8 Hz),
1.75 (quin, 2H, J = 7.4 Hz), 1.65 (quin, 2H, J = 7.3 Hz), 1.50–1.36 (m, 4H); 13C NMR (CDCl3,
100 MHz) δ/ppm: 202.7 (d), 164.1 (s, 2C), 133.8 (d, 2C), 131.5 (s), 131.1 (d, 2C), 128.1 (s),
126.9 (d, 2C), 122.6 (s, 2C), 43.7 (t), 40.2 (t), 28.8 (t), 27.8 (t), 26.8 (t), 21.9 (t); UPLC-MS/UV:
+
2
ꢀ
method
, tR = 1.19 min, m/z = 310.14 [M + H] , found 310.18; HRMS (MALDI TOF/TOF):
calculated for C19H19NO3 [M + H]+ 310.1443; found 310.1438.
N-(7-formylhept-1-yl)-1,8-naphthalimide (4e) was prepared according to the general
procedure for Swern oxidation from alcohol 3e (5.85 g, 17.9 mmol). The reaction after
work-up and chromatography furnished 3.57 g (61%) of the pure product in the form of a
colorless solid.
mp 71–73 ◦C; IR (ATR) νe/cm−1: 2928, 2727 (C-H), 2850 (C-H ald.), 1695 (C=O, ald.), 1655
(C=O), 1590 (C–N amide), 1342 (C=C); 1H NMR (CDCl3, 400 MHz)
δ/ppm: 9.75 (t, 1H,
J = 1.8 Hz), 8.59 (dd, 2H, J = 7.2 Hz, J = 0.8 Hz), 8.20 (dd, 2H, J = 8.2 Hz, J = 0.7 Hz), 7.74
(t, 2H, J = 7.8 Hz), 4.17 (t, 2H, J = 7.5 Hz), 2.41 (td, 2H, J = 7.3 Hz, J = 1.7 Hz), 1.73 (quin,
2H, J = 7.4 Hz), 1.62 (quin, 2H, J = 7.3Hz), 1.49–1.28 (m, 6H); 13C NMR (CDCl3, 100 MHz)
δ
/ppm: 202.8 (d), 164.1 (s, 2C), 133.8 (d, 2C), 131.5 (s), 131.1 (d, 2C), 128.1 (s), 126.9 (d, 2C),
2
,
122.7 (s, 2C), 43.8 (t), 40.3 (t), 29.0 (t, 2C), 27.9 (t), 26.8 (t), 22.0 (t); UPLC-MS/UV: method
ꢀ
tR = 1.26 min, m/z = 324.16 [M + H]+, found 324.20; HRMS (MALDI TOF/TOF): calculated
for C20H21NO3 [M + H]+ 324.1600; found 324.1598.
•
Grignard reaction of N-(ω-formylalkyl)-1,8-naphthalimide 4 and 6—general
procedure
A well-dried round-bottom flask equipped with a septum under Ar inert atmosphere
was charged with N-(
ω
-formylalkyl)-1,8-naphthalimide (
4
, 1.0 mmol) and anhydrous THF
(7 mL). The solution was cooled to 0 ◦C, and a solution of Grignard reagent
6
(1.1 mmol)

Molecules 2021, 26, 3355
15 of 26
was added dropwise via a syringe. The reaction mixture was stirred at 0 ◦C for 30 min,
and then it was allowed to reach rt over 5 h. To the reaction mixture, a saturated aqueous
solution of NH4Cl (10 mL) was added, and after 15 min of stirring, the mixture was
transferred to a separation funnel. Extraction with EtOAc (3
×
20 mL) was carried out. The
combined organic layers were dried over anhydrous MgSO4 and filtered and the solvent
was removed on a rotary evaporator. The residue was purified on a silica gel column using
EtOAc (0 to 30%)/cyclohexane as eluent to afford the pure product.
N-{4-[3-(benzyloxy)naphthalen-2-yl]-4-hydroxybut-1-yl}-1,8-naphthalimide (7a). Pre-
pared according to the general procedure from aldehyde 4a (920 mg, 3.44 mmol) and the
Grignard reagent, 6. After chromatography, the reaction furnished 385 mg (22%) of the
pure product in the form of a pale yellow solid.
◦
mp 156–157 C; IR (ATR) νe/cm−1: 3031 (Ar C-H), 2954, 2923 (C-H), 1655 (C=O), 1591 (C–N
amide), 1341 (C=C), 1254 (C–O), 1061 (C–O–C); 1H NMR (CDCl3, 400 MHz)
δ
/ppm: 8.57
(d, 2H, J = 7.3 Hz), 8.19 (d, 2H, J = 8.2 Hz), 7.81 (s, 1H), 7.78–7.69 (m, 3H), 7.67 (dd, 1H,
J = 8.2 Hz, J = 0.3 Hz), 7.49–7.42 (m, 2H), 7.42–7.35 (m, 3H), 7.35–7.27 (m, 2H), 7.17 (s, 1H),
5.19 (s, 2H), 5.15 (q, 1H, J = 6.0 Hz), 4.25 (t, 2H, J = 7.2 Hz), 2.85 (d, 1H, J = 6.7 Hz), 2.14–1.78
(m, 4H); 13C NMR (CDCl3, 100 MHz)
δ/ppm: 164.2 (s, 2C), 154.3 (s), 136.5 (s), 133.8 (d, 2C),
133.6 (s), 131.5 (s), 131.2 (d, 2C), 128.79 (s), 128.73 (d, 2C), 128.1 (s), 128.0 (d), 127.7 (d), 127.3
(d, 2C), 126.9 (d, 2C), 126.3 (d), 126.2 (d), 126.1 (d), 123.9 (d), 122.6 (s, 2C), 106.6 (d), 71.2
(d), 70.0 (t), 40.1 (t), 34.6 (t), 24.8 (t), a signal of one quaternary C-atom was not observed;
2
UPLC-MS/UV: method
ꢀ
, tR = 1.42 min, m/z = 484.19 [M
−
OH]+, found 484.15; HRMS
(MALDI TOF/TOF): calculated for C33H27NO4 [M + Na]+ 524.1838; found 524.1852.
N-{4-[3-(benzyloxy)naphthalen-2-yl]-4-hydroxypent-1-yl}-1,8-naphthalimide (7b) was
prepared according to the general procedure from aldehyde 4b (968 mg, 3.44 mmol) and
Grignard reagent 6. After chromatography, the reaction furnished 381 mg (21%) of the pure
product in the form of a pale yellow solid.
◦
mp 155–157 C; IR (ATR) νe/cm−1: 3047 (Ar C-H), 2955, 2925, 2854 (C-H), 1649 (C=O),
1591 (C–N amide), 1340 (C=C), 1237 (C–O), 1091 (C–O–C); 1H NMR (CDCl3, 400 MHz)
δ
/ppm: 8.58 (dd, 2H, J = 7.3 Hz, J = 0.9 Hz), 8.20 (dd, 2H, J = 8.3 Hz, J = 0.8 Hz), 7.81
(s, 1H), 7.79–7.66 (m, 4H), 7.49–7.43 (m, 2H), 7.43–7.30 (m, 4H), 7.25–7.18 (m, 2H), 5.21 (s,
2H), 5.10 (q, 1H, J = 6.2 Hz), 4.16 (t, 2H, J = 7.5 Hz), 2.60 (d, 1H, J = 6.2 Hz), 2.06–1.88 (m,
2H), 1.84–1.70 (m, 2H), 1.69–1.58 (m, 1H), 1.56–1.44 (m, 1H); 13C NMR (CDCl3, 100 MHz)
δ
/ppm: 164.1 (s, 2C), 154.4 (s), 136.5 (s), 134.2 (s), 133.8 (d, 2C), 133.5 (s), 131.5 (s), 131.2 (d,
2C), 128.8 (s), 128.6 (d, 2C), 128.1 (s), 128.0 (d), 127.7 (d), 127.4 (d, 2C), 126.9 (d, 2C), 126.3
(d), 126.1 (d), 126.0 (d), 123.9 (d), 122.7 (s, 2C), 106.6 (d), 70.9 (d), 70.1 (t), 40.2 (t), 37.1 (t),
2
27.8 (t), 23.5 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.45 min, m/z = 498.21 [M
−
OH]+, found
498.25; HRMS (MALDI TOF/TOF): calculated for C34H29NO4 [M + Na]+ 538.1994; found
538.1970.
N-{4-[3-(benzyloxy)naphthalen-2-yl]-4-hydroxyhex-1-yl}-1,8-naphthalimide (7c) was
prepared according to the general procedure from aldehyde 4c (930 mg, 3.15 mmol) and
Grignard reagent 6. After chromatography the reaction furnished 662 mg (40%) of the pure
product in the form of a pale yellow solid.
◦
mp 146–149 C; IR (ATR) νe/cm−1: 3053, 3027 (Ar C-H), 2926, 2858 (C-H), 1648 (C=O), 1590
(C–N amid), 1345 (C=C), 1239 (C–O), 1074 (C–O–C); 1H NMR (CDCl3, 400 MHz)
δ
/ppm:
8.58 (dd, 2H, J = 7.2 Hz, J = 0.7 Hz), 8.19 (dd, 2H, J = 8.3 Hz, J = 0.6 Hz), 7.82–7.66 (m, 5H),
7.48–7.27 (m, 7H), 7.19 (s, 1H), 5.20 (s, 2H), 5.11–5.02 (m, 1H), 4.20–4.10 (m, 2H), 2.00–1.82
(m, 2H), 1.79–1.67 (m, 2H), 1.65–1.43 (m, 4H); 13C NMR (CDCl3, 100 MHz)
δ/ppm: 164.1 (s,
2C), 154.4 (s), 136.5 (s), 134.2 (s), 133.8 (d, 2C), 133.5 (s), 131.5 (s), 131.2 (d, 2C), 128.8 (s),
128.6 (d, 2C), 128.1 (s), 128.0 (d), 127.7 (d), 127.3 (d, 2C), 126.9 (d, 2C), 126.3 (d), 126.1 (d),
126.0 (d), 123.9 (d), 122.7 (s, 2C), 106.5 (d), 70.9 (d), 70.0 (t), 40.3 (t), 37.3 (t), 28.0 (t), 26.9 (t),
2
25.7 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.50 min, m/z = 512.22 [M
−
OH]+, found 512.69;
HRMS (MALDI TOF/TOF): calculated for C35H31NO4 [M + Na]+ 552.2151 found 552.2131.

Molecules 2021, 26, 3355
16 of 26
N-{4-[3-(benzyloxy)naphthalen-2-yl]-4-hydroxyhept-1-yl}-1,8-naphthalimide (7d) was
prepared according to the general procedure from aldehyde 4d (1064 mg, 3.44 mmol) and
Grignard reagent 6. After chromatography the reaction furnished 508 mg (27%) of the pure
product in the form of a pale yellow solid.
◦
mp 122–123 C; IR (ATR) νe/cm−1: 3048, 3023 (Ar C-H), 2938, 2925, 2851 (C-H), 1645 (C=O),
1588 (C–N amide), 1347 (C=C), 1237 (C–O), 1054 (C–O–C); 1H NMR (CDCl3, 400 MHz)
δ/ppm: 8.58 (dd, 2H, J = 7.2 Hz, J = 0.9 Hz), 8.19 (dd, 2H, J = 8.3 Hz, J = 0.7 Hz), 7.81–7.66
(m, 5H), 7.49–7.36 (m, 5H), 7.36–7.29 (m, 2H) 7.20 (s, 1H), 5.21 (s, 2H), 5.10–5.00 (m, 1H), 4.15
(t, 2H, J = 7.6 Hz), 2.59 (d, 1H, J = 6.1 Hz), 1.97–1.79 (m, 2H), 1.77–1.62 (m, 2H), 1.56–1.33 (m,
6H); 13C NMR (CDCl3, 100 MHz)
δ/ppm: 164.1 (s, 2C), 154.4 (s), 136.5 (s), 134.3 (s), 133.8
(d, 2C), 133.5 (s), 131.5 (s), 131.2 (d, 2C), 128.8 (s), 128.6 (d, 2C), 128.1 (s), 128.0 (d), 127.7 (d),
127.4 (d, 2C), 126.9 (d, 2C), 126.3 (d), 126.1 (d), 126.0 (d), 123.9 (d), 122.7 (s, 2C), 106.6 (d),
71.1 (d), 70.1 (t), 40.3 (t), 37.4 (t), 29.1 (t), 28.0 (t), 27.0 (t), 25.9 (t); UPLC-MS/UV: method
OH]+, found 526.16; HRMS (MALDI TOF/TOF):
2
ꢀ
, tR = 1.55 min, m/z = 526.24 [M
−
calculated for C36H33NO4 [M + Na]+ 566.2307; found 566.2295.
N-{4-[3-(benzyloxy)naphthalen-2-yl]-4-hydroxyoct-1-yl}-1,8-naphthalimide (7e) was
prepared according to the general procedure from aldehyde 4e (1113 mg, 3.44 mmol) and
Grignard reagent 6. After chromatography the reaction furnished 542 mg (28%) of the pure
product in the form of a pale yellow solid.
◦
mp 111–112 C; IR (ATR) νe/cm−1: 3060, 3032 (Ar C-H), 2931, 2925 (C-H), 1663 (C=O), 1590
(C–N amide), 1341 (C=C), 1232 (C–O), 1072 (C–O–C); 1H NMR (CDCl3, 400 MHz)
δ
/ppm:
8.59 (dd, 2H, J = 7.2 Hz, J = 0.9 Hz), 8.19 (dd, 2H, J = 8.3 Hz, J = 1.0 Hz), 7.82–7.66 (m, 5H),
7.49–7.36 (m, 5H), 7.36–7.28 (m, 2H), 7.21 (s, 1H), 5.21 (s, 2H), 5.10–4.97 (m, 1H), 4.15 (t, 2H,
J = 7.5 Hz), 2.55 (d, 1H, J = 5.9 Hz), 1.97–1.79 (m, 2H), 1.77–1.63 (m, 2H), 1.56–1.27 (m, 8H);
13C NMR (CDCl3, 100 MHz)
δ/ppm: 164.1 (s, 2C), 154.4 (s), 136.5 (s), 134.3 (s), 133.8 (d, 2C),
133.5 (s), 131.5 (s), 131.1 (d, 2C), 128.8 (s), 128.7 (d, 2C), 128.16 (s), 128.13 (d), 127.7 (d), 127.4
(d, 2C), 126.9 (d, 2C), 126.3 (d), 126.1 (d), 126.0 (d), 123.9 (d), 122.7 (s, 2C), 106.6 (d), 71.2 (d),
70.1 (t), 40.4 (t), 37.4 (t), 29.4 (t), 29.2 (t), 28.0 (t), 27.0 (t), 26.0 (t); UPLC-MS/UV: method
OH]+, found 540.17; HRMS (MALDI TOF/TOF):
2
ꢀ
, tR = 1.59 min, m/z = 540.25 [M
−
calculated for C37H35NO4 [M + Na]+ 580.2464; found 580.2460.
•
Removal of the benzyl group—general procedure
A round-bottom flask, equipped with a septum, was charged with benzyl ether
7
(1 mmol), THF (5 mL/mmol 7), methanol (5 mL/mmol 7), Pd/C (10%, 0.1 mmol) and
ammonium formate (5.0 mmol). The reaction mixture was heated at 65 ◦C for 2–6 h and
2
the progress of the reaction was monitored by means of UPLC-MS/UV (method
ꢀ
, see the
ESI). After all the benzyl ether was consumed, the reaction mixture was cooled down to rt,
filtered through a celite plug, and the solvent was removed on a rotary evaporator. The
residue was purified by means of preparative HPLC-MS/UV or on a column of silica gel
using EtOAc (0 to 30%)/cyclohexane as eluent to afford the pure product.
N-{4-[3-hydroxynaphthalen-2-yl]-4-hydroxybut-1-yl}-1,8-naphthalimide (1a) was pre-
pared according to the general procedure from ether 7a (350 mg, 0.70 mmol), Pd/C (10%,
75 mg, 70 µmol) and ammonium formate (220 mg, 3.49 mmol). After chromatography, the
reaction furnished 113 mg (39%) of the pure product in the form of a pale yellow solid.
◦
mp 205–207 C; IR (ATR) νe/cm−1: 3425 (O–H alcohol), 3207 (Ar O–H), 3045 (Ar C-H), 2953,
2916 (C-H), 1652 (C=O), 1585 (C–N amide), 1347 (C=C), 1239 (C–O); 1H NMR (DMSO-d6,
400 MHz)
δ
/ppm: 9.78 (br. s, 1H), 8.46 (dd, 2H, J = 7.2 Hz, J = 1.1 Hz), 8.44 (dd, 2H,
J = 8.4 Hz, J = 1.0 Hz), 7.90–7.78 (m, 3H), 7.72 (dd, 1H, J = 7.9 Hz, J = 0.5 Hz), 7.62 (dd, 1H,
J = 8.4 Hz, J = 0.5 Hz), 7.36–7.28 (m, 1H), 7.26–7.18 (m, 1H), 7.08 (s, 1H), 5.21 (br. s, 1H), 5.04–
4.91 (m, 1H), 4.16–3.99 (m, 2H), 1.91–1.56 (m, 4H); 13C NMR (DMSO-d6, 100 MHz)
δ/ppm:
163.3 (s, 2C), 152.6 (s), 135.3 (s), 134.2 (d, 2C), 133.2 (s), 131.2 (s), 130.7 (d, 2C), 127.6 (s), 127.4
(d), 127.3 (s), 127.2 (d, 2C), 125.4 (d), 125.3 (d), 125.0 (d), 122.6 (d), 122.0 (s, 2C), 108.3 (d),

Molecules 2021, 26, 3355
17 of 26
67.0 (d), 35.2 (t), 24.2 (t), one CH2 signal is cove−red by the DMSO-signal; UPLC-MS/UV:
3
,
method
ꢀ
, tR = 1.34 min, m/z = 410.14 [M
−
H] , found 410.48; m/z = 394.14 [M − OH]+
found 394.66; HRMS (MALDI TOF/TOF): calculated for C26H21NO4 [M + H]+ 412.1549;
found 412.1542.
N-{4-[3-hydroxynaphthalen-2-yl]-4-hydroxypent-1-yl}-1,8-naphthalimide (1b) was
prepared according to the general procedure from ether 7b (330 mg, 0.64 mmol), Pd/C
(10%, 68 mg, 64 µmol) and ammonium formate (214 mg, 3.39 mmol). After chromatography,
the reaction furnished 121 mg (45%) of the pure product in the form of a pale yellow solid.
◦
mp 167–168 C; IR (ATR) νe/cm−1: 3423 (O–H alcohol), 3231 (Ar O–H), 3042 (Ar C-H), 2948,
2866 (C-H), 1663 (C=O), 1591 (C–N amide), 1342 (C=C), 1242 (C–O); 1H NMR (DMSO-d6,
600 MHz)
δ
/ppm: 9.76 (s, 1H), 8.45 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.42 (dd, 2H, J = 8.3 Hz
,
J = 1.0 Hz), 7.86–7.79 (m, 3H), 7.72 (d, 1H, J = 8.1 Hz), 7.61 (d, 1H, J = 7.9 Hz), 7.33–7.29 (m,
1H), 7.23–7.19 (m, 1H), 7.08 (s, 1H), 5.16 (d, 1H, J = 4.6 Hz), 4.95 (quin, 1H, J = 3.9 Hz), 4.02 (t,
2H, J = 7.4 Hz), 1.83–1.76 (m, 1H), 1.72–1.54 (m, 3H), 1.53–1.41 (m, 2H); 13C NMR (DMSO-d6,
150 MHz) δ/ppm: 163.3 (s, 2C), 152.8 (s), 135.6 (s), 134.2 (d, 2C), 133.2 (s), 131.2 (s), 130.6
(d, 2C), 127.6 (s), 127.4 (d), 127.3 (s), 127.2 (d, 2C), 125.4 (d), 125.3 (d), 124.9 (d), 122.5 (d),
122.0 (s, 2C), 108.3 (d), 67.1 (d), 37.5 (t), 27.6 (t), 23.1 (t), one CH2 signal is covered by the
3
DMSO-signal; UPLC-MS/UV: method
ꢀ
, tR = 1.41 min, m/z = 424.16 [M − H]−, found
424.09; m/z = 408.16 [M
−
OH]+, found 408.17; HRMS (MALDI TOF/TOF): calculated for
C27H23NO4 [M + H]+ 426.1705; found 426.1709.
N-{4-[3-hydroxynaphthalen-2-yl]-4-hydroxyhex-1-yl}-1,8-naphthalimide (1c) was pre-
pared according to the general procedure from ether 7c (350 mg, 0.66 mmol), Pd/C (10%,
70 mg, 66 µmol) and ammonium formate (208 mg, 3.30 mmol). After chromatography the
reaction furnished 169 mg (58%) of the pure product in the form of a pale yellow solid.
◦
mp 145–146 C; IR (ATR) νe/cm−1: 3521 (O–H alcohol), 3264 (Ar O–H), 3052 (Ar C-H), 2939,
2928 (C-H), 1645 (C=O), 1589 (C–N amide), 1342 (C=C), 1238 (C–O); 1H NMR (DMSO-d6,
400 MHz) δ/ppm: 9.75 (br. s, 1H), 8.54–8.40 (m, 4H), 7.86 (dd (t), 2H, J = 7.8 Hz), 7.84
(s, 1H), 7.73 (d, 1H, J = 7.9 Hz), 7.62 (d, 1H, J = 8.1 Hz), 7.36–7.28 (m, 1H), 7.26–7.18
(m, 1H), 7.08 (s, 1H), 5.15 (br. s, 1H), 4.99–4.90 (m, 1H), 4.03 (t, 2H, J = 7.4 Hz), 1.82–
1.27 (m, 8H); 13C NMR (DMSO-d6, 100 MHz)
δ/ppm: 163.3 (s, 2C), 152.8 (s), 135.7 (s),
134.2 (d, 2C), 133.2 (s), 131.2 (s), 130.7 (d, 2C), 127.7 (s), 127.4 (d), 127.3 (s), 127.1 (d, 2C),
125.4 (d), 125.3 (d), 124.9 (d), 122.5 (d), 122.0 (s, 2C), 108.2 (d), 67.0 (d), 37.6 (t), 27.6 (t),
26.6 (t), 25.2 (t), one CH2 signal is covered by the DMSO-signal; UPLC-MS/UV: method
3
ꢀ,
tR = 1.49 min
,
m/z = 438.17 [M − H]−, found 438.76; m/z = 422.18 [M − OH]+, found
422.71; HRMS (MALDI TOF/TOF): calculated for C28H25NO4 [M + Na]+ 462.1681; found
462.1678.
N-{4-[3-hydroxynaphthalen-2-yl]-4-hydroxyhept-1-yl}-1,8-naphthalimide (1d) was
prepared according to the general procedure from ether 7d (350 mg, 0.64 mmol), Pd/C (10%,
69 mg, 65 µmol) and ammonium formate (203 mg, 3.22 mmol). After chromatrography the
reaction furnished 95 mg (33%) of the pure product in the form of a pale yellow solid.
mp 75–76 ◦C; IR (ATR) νe/cm−1: 3314 (O–H), 3056 (Ar C-H), 2929, 2855 (C-H), 1648 (C=O),
1589 (C–N amide), 1343 (C=C), 1234 (C–O); 1H NMR (DMSO-d6, 600 MHz)
δ/ppm: 9.74
(s, 1H), 8.46 (dd, 2H, J = 7.3 Hz, J = 1.1 Hz), 8.42 (dd, 2H, J = 8.3 Hz, J = 1.0 Hz), 7.84 (dd
(t), 2H, J = 7.6 Hz), 7.80 (s, 1H), 7.73 (d, 1H, J = 7.9 Hz), 7.61 (d, 1H, J = 7.9 Hz), 7.33–7.28
(m, 1H), 7.23–7.18 (m, 1H), 7.07 (s, 1H), 5.12 (d, 1H, J = 4.6 Hz), 4.94 (quin, 1H, J = 3.9 Hz),
4.01 (t, 2H, J = 7.5 Hz), 1.74–1.68 (m, 1H), 1.64–1.57 (m, 2H), 1.56–1.49 (m, 1H), 1.45–1.32 (m,
6H); 13C NMR (DMSO-d6, 150 MHz)
δ/ppm: 163.3 (s, 2C), 152.8 (s), 135.7 (s), 134.2 (d, 2C),
133.2 (s), 131.2 (s), 130.6 (d, 2C), 127.7 (s), 127.4 (d), 127.3 (s), 127.1 (d, 2C), 125.4 (d), 125.3
(d), 125.0 (d), 122.5 (d), 122.0 (s, 2C), 108.3 (d), 67.1 (d), 37.7 (t), 28.8 (t), 27.5 (t), 26.6 (t), 25.3
3
(t), one CH2 signal is covered by the DMSO-signal; UPLC-MS/UV: method
ꢀ
, tR = 1.56
−
min, m/z = 452.19 [M
−
H] , found 452.14; m/z = 436.19 [M
−
OH]+, found 436.22; HRMS
(MALDI TOF/TOF): calculated for C29H27NO4 [M+K]+ 492.1577; found 492.1592.

Molecules 2021, 26, 3355
18 of 26
N-{4-[3-hydroxynaphthalen-2-yl]-4-hydroxyoct-1-yl}-1,8-naphthalimide (1e) was pre-
pared according to the general procedure from ether 7e (350 mg, 0.63 mmol), Pd/C (10%,
67 mg, 63 µmol) and ammonium formate (198 mg, 3.14 mmol). After chromatography the
reaction furnished 45 mg (15%) of the pure product in the form of a pale yellow solid.
mp 69–70 ◦C; IR (ATR) νe/cm−1: 3423 (O–H alcohol), 3301 (Ar O–H), 3053 (Ar C-H), 2930,
2855 (C-H), 1657 (C=O), 1590 (C–N amide), 1346 (C=C), 1236 (C–O); 1H NMR (DMSO-d6,
600 MHz) δ/ppm: 9.74 (s, 1H), 8.46 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.41 (dd, 2H, J = 8.2 Hz,
J = 0.7 Hz), 7.84 (dd (t), 2H, J = 7.6 Hz), 7.80 (s, 1H), 7.73 (d, 1H, J = 8.0 Hz), 7.61 (d, 1H,
J = 8.1 Hz), 7.32–7.28 (m, 1H), 7.23–7.18 (m, 1H), 7.07 (s, 1H), 5.11 (d, 1H, J = 4.6 Hz), 4.94
(quin, 1H, J = 3.9 Hz), 4.00 (t, 2H, J = 7.5 Hz), 1.76–1.67 (m, 1H), 1.64–1.48 (m, 3H), 1.45–1.16
(m, 8H); 13C NMR (CDCl3, 150 MHz)
δ/ppm: 163.3 (s, 2C), 152.8 (s), 135.7 (s), 134.2 (d,
2C), 133.2 (s), 131.2 (s), 130.6 (d, 2C), 127.7 (s), 127.4 (d), 127.3 (s), 127.1 (d, 2C), 125.4 (d),
125.3 (d), 125.0 (d), 122.5 (d), 122.0 (s, 2C), 108.3 (d), 67.2 (d), 37.7 (t), 28.9 (t), 28.8 (t), 27.4 (t),
3
26.8 (t), 25.3 (t), one CH2 signal is covered by the DMSO-signal; UPLC-MS/UV: method
ꢀ
,
tR = 1.64 min, m/z = 466.20 [M
−
H]−, found 466.09; m/z = 450.21 [M
−
OH]+, found
450.22; HRMS (MALDI TOF/TOF): calculated for C30H29NO4 [M+K]+ 506.1734; found
506.1751.
•
General procedure for the preparation of 1-OMe ethers using the acid-catalyzed
thermal method
A round-bottom flask (10 mL) was charged with a derivative
(3.0 mL) and concentrated sulfuric acid (50 L). The reaction mixture was stirred at rt for
15 h and then diluted with H2O (10 mL). The reaction mixture was neutralized with a
saturated aqueous solution of NaHCO3 ( 2 mL), transferred to a separation funnel and
extracted with diethyl ether (3 5 mL). The combined organic layers were dried over
1
(
≈
10 mg), methanol
µ
≈
×
anhydrous MgSO4 and filtered and the solvent was removed on a rotary evaporator. The
residue was chromatographed on a TLC using cyclohexane-EtOAc (7:3 v/v) as eluent to
afford the pure product.
N-{4-[3-hydroxynaphthalen-2-yl]-4-methoxybut-1-yl}-1,8-naphthalimide (1a-OMe
)
was prepared according to the general procedure from alcohol 1a (10 mg, 24 mol). After
µ
chromatrographic separation the reaction afforded 5 mg (44%) of the pure product in the
form of a pale yellow solid.
◦
mp 64–65 C; IR (ATR) νe/cm−1: 3229 (Ar O–H), 3054 (Ar C-H), 2948, 2918 (C-H), 1646
1
(C=O), 1588 (C–N amide), 1344 (C=C), 1233 (C–O), 1105 (C–O–C); H NMR (CDCl3,
600 MHz)
δ
/ppm: 8.58 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.19 (dd, 2H, J = 8.2 Hz,
J = 1.0 Hz), 8.02 (s, 1H), 7.74 (dd (t), 2H, J = 7.7 Hz), 7.69 (dd, 1H, J = 8.1 Hz, J = 0.6 Hz),
7.66 (dd, 1H, J = 8.2 Hz, J = 0.4 Hz), 7.52 (s, 1H), 7.39–7.35 (m, 1H), 7.29–7.26 (m, 1H), 7.20
(s, 1H), 4.58–4.53 (m, 1H), 4.31–4.19 (m, 2H), 3.42 (s, 3H), 2.17–2.10 (m, 1H), 2.00–1.86 (m,
2H), 1.83–1.75 (m, 1H); 13C NMR (CDCl3, 150 MHz)
δ/ppm: 164.2 (s, 2C), 153.4 (s), 134.4
(s), 133.9 (d, 2C), 131.5 (s), 131.2 (d, 2C), 128.15 (s), 128.13 (s), 127.9 (d), 127.4 (d), 127.2 (s),
126.9 (d, 2C), 126.2 (d, 2C), 123.4 (d), 122.6 (s, 2C), 111.4 (d), 85.5 (d), 57.4 (−q), 39.7 (t), 33.3
2
H] , found 424.08;
(t), 24.6 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.30 min, m/z = 424.16 [M
−
m/z = 394.14 [M
−
OCH3]+, found 394.16; HRMS (MALDI TOF/TOF): calculated for
C27H23NO4 [M + Na]+ 448.1525; found 448.1520.
N-{4-[3-hydroxynaphthalen-2-yl]-4-methoxypent-1-yl}-1,8-naphthalimide (1b-OMe
)
was prepared according to the general procedure from alcohol 1b (10 mg, 24 µmol). After
chromatographic separation the reaction afforded 6 mg (60%) of the pure product in the
form of a pale yellow solid.
◦
mp 58–60 C; IR (ATR) νe/cm−1: 3320 (Ar O–H), 3056 (Ar C-H), 2931, 2863 (C-H), 1657
1
(C=O), 1589 (C–N amide), 1342 (C=C), 1234 (C–O), 1169 (C–O–C); H NMR (CDCl3,
600 MHz)
δ
/ppm: 8.58 (dd, 2H, J = 7.3 Hz, J = 1.0 Hz), 8.20 (dd, 2H, J = 8.3 Hz,
J = 0.9 Hz), 8.03 (s, 1H), 7.74 (dd (t), 2H, J = 7.7 Hz), 7.69 (t, 2H, J = 7.6 Hz), 7.47 (s, 1H),

Molecules 2021, 26, 3355
19 of 26
7.40–7.36 (m, 1H), 7.29–7.26 (m, 1H), 7.21 (s, 1H), 4.49–4.45 (m, 1H), 4.17 (t, 2H, J = 7.6 Hz),
3.40 (s, 3H), 2.15–2.06 (m, 1H), 1.90–1.82 (m, 1H), 1.82–1.73 (m, 2H), 1.65–1.57 (m, 1H),
1.50–1.42 (m, 1H); 13C NMR (CDCl3, 150 MHz)
δ/ppm: 164.1 (s, 2C), 153.5 (s), 134.4 (s),
133.8 (d, 2C), 131.5 (s), 131.2 (d, 2C), 128.15 (s), 128.11 (s), 127.7 (d), 127.4 (s), 127.3 (d), 126.9
(d, 2C), 126.2 (d, 2C), 123.4 (d), 122.6 (s, 2C), 111.3 (d), 85.8 (d), 57.3 (q), 40.1 (t), 35.5 (t), 27.6
−
H] , found 438.61;
2
(t), 23.4 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.35 min, m/z = 438.17 [M
−
m/z = 408.16 [M
−
OCH3]+, found 408.64; HRMS (MALDI TOF/TOF): calculated for
C28H25NO4 [M + Na]+ 462.1681; found 462.1671.
N-{4-[3-hydroxynaphthalen-2-yl]-4-methoxyhex-1-yl}-1,8-naphthalimide (1c-OMe
)
was prepared according to the general procedure from alcohol 1c (10 mg, 23 µmol). After
chromatographic separation the reaction afforded 6 mg (53%) of the pure product in the
form of pale yellow solid.
◦
mp 55–56 C; IR (ATR) νe/cm−1: 3319 (Ar O–H), 3056 (Ar C-H), 2933, 2858 (C-H), 1658
1
(C=O), 1589 (C–N amide), 1341 (C=C), 1236 (C–O), 1106 (C–O–C); H NMR (CDCl3,
600 MHz)
δ
/ppm: 8.58 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.19 (dd, 2H, J = 8.3 Hz, J = 1.0 Hz),
8.03 (s, 1H), 7.74 (dd (t), 2H, J = 7.7 Hz), 7.69 (d, 1H, J = 8.1 Hz), 7.67 (d, 1H, J = 8.2 Hz), 7.45
(s, 1H), 7.40–7.36 (m, 1H), 7.30–7.26 (m, 1H), 7.21 (s, 1H), 4.43 (t, 1H, J = 7.0 Hz), 4.19–4.13
(m, 2H), 3.39 (s, 3H), 2.08–1.98 (m, 1H), 1.83–1.67 (m, 3H), 1.59–1.51 (m, 1H), 1.49–1.41 (m,
2H), 1.41–1.33 (m, 1H); 13C NMR (CDCl3, 150 MHz)
δ/ppm:164.1 (s, 2C), 153.5 (s), 134.4 (s),
133.8 (d, 2C), 131.5 (s), 131.1 (d, 2C), 128.1 (s), 128.0 (s), 127.7 (d), 127.5 (s), 127.3 (d), 126.9 (d,
2C), 126.2 (d, 2C), 123.4 (d), 122.7 (s, 2C), 111.3 (d), 86.0 (d), 57.2 (q), 40.2 (t), 35.8 (t), 27.9 (t),
2
26.8 (t), 25.6 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.41 min, m/z = 452.19 [M − H]−, found
452.23; m/z = 422.18 [M
−
OCH3]+, found 422.26; HRMS (MALDI TOF/TOF): calculated
for C29H27NO4 [M + Na]+ 476.1838; found 476.1815.
N-{4-[3-hydroxynaphthalen-2-yl]-4-methoxyhept-1-yl}-1,8-naphthalimide (1d-OMe
)
was prepared according to the general procedure from alcohol 1d (10 mg, 22 µmol). After
chromatographic separation the reaction afforded 8 mg (80%) of the pure product in the
form of a pale yellow solid.
◦
mp 38–40 C; IR (ATR) νe/cm−1: 3343 (Ar O–H), 3055 (Ar C-H), 2930, 2856 (C-H), 1657
1
(C=O), 1589 (C–N amide), 1342 (C=C), 1233 (C–O), 1107 (C–O–C); H NMR (CDCl3,
600 MHz)
δ
/ppm: 8.59 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.19 (dd, 2H, J = 8.2 Hz, J = 0.9 Hz),
8.05 (s, 1H), 7.74 (dd (t), 2H, J = 7.7 Hz), 7.71 (d, 1H, J = 8.1 Hz), 7.67 (d, 1H, J = 8.1 Hz), 7.46
(s, 1H), 7.40–7.36 (m, 1H), 7.30–7.23 (m, 1H), 7.22 (s, 1H), 4.42 (t, 1H, J = 7.0 Hz), 4.16 (t, 2H,
J = 7.6 Hz), 3.40 (s, 3H), 2.05–1.97 (m, 1H), 1.81–1.68 (m, 3H), 1.51–1.26 (m, 6H); 13C
NMR (CDCl3, 150 MHz) δ/ppm:164.1 (s, 2C), 153.5 (s), 134.3 (s), 133.8 (d, 2C), 131.5 (s),
131.1 (d, 2C), 128.1 (s), 128.0 (s), 127.7 (d), 127.5 (s), 127.3 (d), 126.9 (d, 2C), 126.2 (d, 2C),
123.4 (d), 122.7 (s, 2C), 111.3 (d), 86.0 (d), 57.2 (q), 40.3 (t), 35.8 (t), 29.0 (t), 28.0 (t), 26.9
2
(t), 25.8 (t); UPLC-MS/UV: method
ꢀ
, tR = 1.47 min, m/z = 466.20 [M
−
H]−, found
466.19; m/z = 436.19 [M − OCH3]+, found 436.27; HRMS (MALDI TOF/TOF): calculated
for C30H29NO4 [M + Na]+ 490.1994; found 490.1983.
N-{4-[3-hydroxynaphthalen-2-yl]-4-methoxyoct-1-yl}-1,8-naphthalimide (1e-OMe) was
prepared according to the general procedure from alcohol 1e (10 mg, 21 µmol). After
chromatographic separation the reaction afforded 6 mg (53%) of the pure product in the
form of a pale yellow solid.
◦
mp 38–40 C; IR (ATR) νe/cm−1: 3343 (Ar O–H), 3055 (Ar C-H), 2928, 2855 (C-H), 1658
1
(C=O), 1589 (C–N amide), 1342 (C=C), 1233 (C–O), 1167 (C–O–C); H NMR (CDCl3,
600 MHz)
δ
/ppm: 8.59 (dd, 2H, J = 7.2 Hz, J = 1.0 Hz), 8.20 (dd, 2H, J = 8.2 Hz, J = 0.9 Hz),
8.06 (s, 1H), 7.74 (dd (t), 2H, J = 7.7 Hz), 7.71 (dd, 1H, J = 8.3 Hz, J = 0.6 Hz), 7.68 (dd, 1H,
J = 8.2 Hz, J = 0.6 Hz), 7.46 (s, 1H), 7.40–7.36 (m, 1H), 7.30–7.27 (m, 1H), 7.22 (s, 1H), 4.26
(t, 1H, J = 7.0 Hz), 4.16 (t, 2H, J = 7.6 Hz), 3.40 (s, 3H), 2.04–1.95 (m, 1H), 1.80–1.67 (m,
3H), 1.49–1.35 (m, 4H), 1.33–1.23 (m, 4H); 13C NMR (CDCl3, 150 MHz)
δ
/ppm: 164.2 (s,
2C), 153.5 (s), 134.3 (s), 133.8 (d, 2C), 131.5 (s), 131.1 (d, 2C), 128.16 (s), 128.10 (s), 127.7 (d),

Molecules 2021, 26, 3355
20 of 26
127.6 (s), 127.3 (d), 126.9 (d, 2C), 126.2 (d, 2C), 123.4 (d), 122.7 (s, 2C), 111.2 (d), 86.1 (d),
2
,
57.3 (q), 40.4 (t), 35.8 (t), 29.1 (t, 2C), 28.0 (t), 26.9 (t), 25.8 (t); UPLC-MS/UV: method
ꢀ
tR = 1.52 min, m/z = 480.22 [M
−
H]−, found 480.26; m/z = 450.21 [M
−
OCH3]+, found
450.23; HRMS (MALDI TOF/TOF): calculated for C31H31NO4 [M + Na]+ 504.2151; found
504.2133.
•
Irradiation experiments—general
A quartz test tube (20 mL) was filled with a solution of
1 in CH3OH (7 mL/1 mg of 1),
and the solution was diluted with H2O. The final CH3OH-H2O ratio was 4:1 (v/v). The
solution was sealed with a septum and purged with Ar for 15 min and irradiated in a
reactor equipped with 8 or 14 lamps (1 lamp = 8 W) with the output at 254 nm, or
≈
350 nm.
CH3OH for the irradiations was of HPLC purity, and Milli-Q H2O was used. The solutions
were irradiated from 60 to 180 min, and the composition of the irradiated solutions was
analyzed using UPLC-MS/UV.
•
Irradiation of 1a
According to the general procedure, 1a (9 mg, 22 µmol) was dissolved in CH3OH-H2O
(4:1 v/v) (20 mL) and irradiated at 350 nm (14 lamps) over 180 min. After the irradiation,
1a-OMe and 1,8-naphthalimide were detected in a complex mixture of photoproducts
determined, by analysis of the solution using UPLC/MS-UV.
OH]+, found 394.26;
2
UPLC-MS/UV: method
ꢀ
, tR (1a) = 1.16 min, m/z = 394.14 [M
−
tR (1a-OMe) = 1.32 min, m/z = 394.14 [M
−
OCH3]+, found 394.31; tR (1,8-naphthalimide)
= 0.74 min, m/z = 198.05 [M + H]+, found 198.21.
•
Irradiation of 1c
According to the general procedure, 1c (7 mg, 13 µmol) was dissolved in CH3OH-H2O
(4:1 v/v) (20 mL) and irradiated at 254 nm or 350 nm (8 lamps) over 60 min or at 350 nm
(14 lamps) over 180 min. After the irradiation, 1c-OMe was detected in a complex mixture
of photoproducts, determined by analysis of the solution using UPLC/MS-UV.
2
(at 254 nm, 60 min) tR (1c) = 1.27 min, m/z = 438.17
UPLC-MS/UV: method
ꢀ
[M − H]−, found 438.55; tR
(
1c-OMe) = 1.42 min, m/z = 422.18 [M
−
OCH3]+, found
H]−, found 438.55; tR
422.66; [at 350 nm, 60 min] tR (1c) = 1.25 min, m/z = 438.17 [M
−
(1c-OMe) = 1.42 min, m/z = 422.18 [M − OCH3]+, found 422.60.
•
Irradiation of 1e
According to the general procedure, 1e (8 mg, 22 µmol) was dissolved in CH3OH-H2O
(4:1 v/v) (20 mL) and irradiated at 350 nm (14 lamps) over 180 min. After the irradiation,
1e-OMe and 1,8-naphthalimide were detected in a complex mixture of photoproducts,
determined by analysis of the solution using UPLC/MS-UV.
2
UPLC-MS/UV: method
ꢀ
, tR (1e) = 1.39 min, m/z = 450.21 [M
−
OH]+, found 450.36;
tR (1e-OMe) = 1.55 min, m/z = 450.36 [M
−
OCH3]+, found 450.41; tR (1,8-naphthtalimide)
= 0.74 min, m/z = 198.05 [M + H]+, found 198.16.
•
Quantum yield of methanolysis
Quantum yields for the photomethanolysis reactions for
1
were determined using a
in
KI/KIO3 (Φ254 = 0.74) actinometer [81], as recently described by us [97]. Solutions of
1
CH3OH-H2O (4:1 v/v) and the actinometer were freshly prepared and their concentrations
were adjusted to have absorbances of 0.4–0.8 at 254 nm. After the adjustment of the
concentrations and measurement of the corresponding UV-vis spectra, the solutions were
purged with a stream of Ar (20 min), and then, sealed with a cap. The cells were placed
in a holder, which ensured an equal distance of all samples from the lamp, and were
irradiated at the same time in the reactor with 1 lamp at 254 nm for 6 h (1 min for the
actinometer). Before and after the irradiation, the samples were taken from the cells with
the use of a syringe and were analyzed using UPLC-MS/UV to determine photochemical
conversions. The conversion did not exceed 30% to avoid a change in the absorbance or

Molecules 2021, 26, 3355
21 of 26
filtering of the light by the product. Based on the actinometer conversion, irradiance was
calculated according to Equations (S1)–(S5), reported in the ESI. The average value of three
measurements was reported.
•
Absorption and fluorescence measurements
Absorption spectra were recorded on a PG T80/T80+ or a Varian Cary 100 Bio spec-
trophotometer at rt. Fluorescence measurements were performed on an Agilent Cary
Eclipse fluorimeter with slits corresponding to the bandpass of 10 nm for the excitation
and 20 nm for the emission. The samples were dissolved in CH3CN or CH3CN-H2O (4:1
v/v) and the concentrations were adjusted to absorbances of less than 0.1 at the excitation
wavelengths of 300, 310 and 320 nm. Fluorescence quantum yields were determined
through the comparison of the integral of the emission bands with that of quinine sulfate in
0.05 M H2SO4 (Φf = 0.546) [76]. One fluorescence measurement was performed by exciting
sample at three different wavelengths and the average value was calculated (Equation (S6)
in the ESI). Prior to the measurements, the solutions were purged with Ar for 15 min. The
measurement was performed at rt (25 ◦C). Fluorescence decays were measured using the
TC-SPC method on an Edinburgh FS5 spectrometer equipped with pulsed LEDs at 280 or
340 nm. The duration of the pulse was
1023 channels with the time increment of
they reached 1000 counts in the peak channel. The histograms were analyzed by means of
a nonlinear least-squares deconvolution method using Equation (S7) in the ESI.
≈
800 ps. Fluorescence signals were monitored over
20 ps/channel. The decays were collected until
≈
•
Thermal denaturation experiments with ct-DNA [86]
A stock solution of 1a or 1e was prepared in DMSO (c = 2.0
×
10−2 M), whereas the
stock solution of ct-DNA was prepared in aqueous cacodylate buffer (pH = 7.0, 50 mM)
c(ct-DNA) = 1.1
10−3 M. In the denaturation experiments, the ct-DNA solution was
diluted in a quartz UV-vis cell (with the optical path of 1.0 cm) with cacodylate buffer to
the concentration of c = 3.0
10−5 M, and the appropriate amount of the solution of 1a or
1e was added to reach the desired ratio r (( )/(ct-DNA)) = 0.3 (containing <1% DMSO). The
×
×
1
dependence of the absorbance at 260 nm as a function of temperature was measured on a
◦
Cary 100 Bio (Agilent Varian) UV-vis spectrometer. The temperature was varied from 25 C
to 98 ◦C in intervals of 0.5 ◦C. The denaturation temperature Tm values are the midpoints
of the transition curves, determined from the maximum of the first derivative.
∆Tm values
were calculated by subtracting the Tm of the free nucleic acid from that of the respective
complex, with ∆Tm values being the average of at least two independent measurements
and the error in ∆Tm being ca. ±0.5 ◦C.
•
Fluorescence titrations with ct-DNA
Stock solutions of solution 1a or 1e were prepared in DMSO (c = 1.0
×
10−3 M). For
the titrations, the stock solutions were diluted in a fluorescence cell (3 mL) with cacody-
late buffer (pH 7.0, 50 mM) to reach the concentration c = 2.0
×
10−6 M (containing <1%
DMSO). The fluorescence spectra were measured on a Cary Eclipse (Agilent Technologies,
Santa Clara, CA, USA) at 25 ◦C. The samples were excited at 320 nm, and the emission
was recorded in the range of 340–650 nm. The excitation slit was set to the bandpass
of 10 nm, and the emission slit to 20 nm. Small aliquots of the solutions of the ct-DNA
(
c = 5.0 × 10−3
M) were added to the solution and after an incubation time of 2 min, fluo-
rescence spectra were measured. Data obtained by means of fluorescence titrations were
processed using nonlinear regression analysis according to the Scatchard model (McGhee,
von Hippel formalism) [88].
•
Circular dichroism spectroscopy with ct-DNA
Circular dichroism spectra were measured on a Jasco J-815 spectrometer in quartz
◦
cells with an optical path of 1 cm at 25 C. To the polynucleotide solutions in cells
(
c = 3.0 × 10−5 M) in cacodylate buffer (pH = 7.0, 50 mM), aliquots of the solution of 1a or 1e
in buffer (c = 1.0
×
10−3 M) were added to reach the concentration ratio r(
1)/(polynucleotide)

Molecules 2021, 26, 3355
22 of 26
= 0.1–0.7 (containing <1% DMSO). The CD spectra were recorded in the wavelength range
220–400 nm with a scanning rate of 200 nm/min and with 2 accumulations.
•
Fluorescence titrations with BSA
Stock solutions of solution 1a or 1e were prepared in DMSO (c = 1.0
×
10−3 M). For
the titrations, the stock solutions were diluted in a fluorescence cell (3 mL) with cacodylate
buffer (pH 7.0, 50 mM) to reach the concentration c = 1.0
DMSO). A stock solution of BSA in cacodylate buffer was prepared c = 1.0
fluorescence spectra were measured on a Cary Eclipse (Agilent Technologies) at 25 C. The
samples were excited at 320 nm, and the emission was recorded in the range 340–700 nm.
The excitation slit was set to the bandpass of 10 nm, and the emission slit to 20 nm. Small
aliquots of the solutions of BSA were added to the solution and, after an incubation time of
2 min, fluorescence spectra were measured. Data obtained via fluorescence titrations were
processed by means of multicomponent nonlinear regression analysis in HypSpec2014
software [93].
×
10−6 M (containing <1%
×
10−3 M. The
◦
•
Photochemical alkylation of BSA
A fluorescence cell containing an aqueous solution of BSA (c = 1.4
×
10−5 M) in
cacodylate buffer (2 mL, 50 mM, pH 7.0) and 1a or 1e (c = 1.0
amount < 1%) was irradiated in a Luzchem photoreactor with 8 lamps at 350 nm over
5 min. Prior to the MS analysis, 100 L of the irradiated solution was purified using Aspire
RP30 Desalting Tips (ThermoFisher Scientific, MA, USA). After the purification, the solvent
was evaporated in Speed-Vac (Eppendorf, Switzerland) and re-constituted in 10 L of
the sinapinic acid matrix (5 mg/mL of sinapinic acid in 1:1, CH3CN:0.1% TFA, v/v). The
mixture of the analyte and the matrix was deposited onto the MALDI plate (1 L) and
×
10−6 M) in DMSO (total
µ
µ
µ
dried slowly in a stream of cold air. MALDI mass spectra were recorded on an AB SCIEX
MALDI-TOF/TOF 4800+ (MA, USA).
•
Laser flash photolysis (LFP)
The measurements were performed on a LP980 Edinburgh Instruments spectrometer.
For the excitation, a Qsmart Q450 Quantel YAG laser was used, the fourth at 266 nm or
the third harmonic at 355 nm. The energy of the laser pulse was set to 20 mJ and the
pulse duration was 7 ns. Absorbances at the excitation wavelength were set to 0.3–0.4.
The static cells were used and they were frequently exchanged to assure no absorption of
light by photoproducts. The solutions were purged for 15 min with Ar or O2 prior to the
measurements, which were conducted at 25 ◦C.
Supplementary Materials: The following are available online. Synthetic procedures for the prepara-
tion of known precursors, fluorescence spectra, LFP data and non-covalent binding to ct-DNA and
BSA, as well as copies of 1H and 13C NMR spectra.
Author Contributions: M.S. was the main author involved in the conceptualization, investigation
and formal analysis (synthesis, photophysics, photochemistry). The investigation and formal analysis
were partly conducted by P.B. (biology), A.E. (DNA study) and M.M. (BSA study). I.P. and M.K.
were involved in supervision and validation of the biological part and B.M. provided LFP resources.
Conceptualization and original draft writing were performed by N.B. and M.S., and review and
editing were performed by all authors. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Croatian Science Foundation (HRZZ grant no. HRZZ
IP-2014-09-6312 and HRZZ-IP-2019-04-8008 to NB and HRZZ-IP-2018-01-5475 to IP).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Molecules 2021, 26, 3355
23 of 26
Data Availability Statement: Electronic supporting information (ESI) of this article contains synthetic
procedures for the preparation of known compounds, UV-vis and fluorescence data, noncovalent
binding to ct-DNA, noncovalent and covalent binding to BSA, data on antiproliferatve activity and
copes of 1H and 13C NMR spectra of all new compounds.
Acknowledgments: The authors acknowledge generous support from Fidelta Ltd.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.
References
1.
2.
3.
4.
Rajski, S.R.; Williams, R.M. DNA Cross-Linking Agents as Antitumor Drugs. Chem. Rev. 1998, 98, 2723–2796. [CrossRef]
Rokita, S.E. (Ed.) Quinone Methides; Wiley: Hoboken, NJ, USA, 2009.
Frecero, M. Quinone Methides as Alkylating and Cross-Linking Agents. Mini Rev. Org. Chem. 2004, 1, 403–415. [CrossRef]
Wang, P.; Song, Y.; Zhang, L.; He, H.; Zhou, X. Quinone Methide Derivatives: Important Intermediates to DNA Alkylating and
DNA Cross-linking Actions. Curr. Med. Chem. 2005, 12, 2893–2913. [CrossRef]
5.
McCracken, P.G.; Bolton, J.L.; Thatcher, G.R.J. Covalent Modification of Proteins and Peptides by the Quinone Methide from
2-tert-Butyl-4,6-dimethylphenol: Selectivity and Reactivity with Respect to Competitive Hydration. J. Org. Chem. 1997, 62,
1820–1825. [CrossRef]
6.
7.
8.
9.
Arumugam, S.; Guo, J.; Mbua, N.E.; Friscourt, F.; Lin, N.; Nekongo, E.; Boons, G.-J.; Popik, V.V. Selective and reversible
photochemical derivatization of cysteine residues in peptides and proteins. Chem. Sci. 2014, 5, 1591–1598. [CrossRef] [PubMed]
Pérez-Ruiz, R.; Molins-Molina, O.; Lence, E.; González-Bello, C.; Miranda, M.A.; Jiménez, M.C. Photogeneration of Quinone
Methides as Latent Electrophiles for Lysine Targeting. J. Org. Chem. 2018, 83, 13019–13029. [CrossRef] [PubMed]
Pande, P.; Shearer, J.; Yang, J.; Greenberg, W.A.; Rokita, S.E. Alkylation of Nucleic Acids by a Model Quinone Methide. J. Am.
Chem. Soc. 1999, 121, 6773–6779. [CrossRef]
Veldhuyzen, W.F.; Shallop, A.J.; Jones, R.A.; Rokita, S.E. Thermodynamic versus Kinetic Products of DNA Alkylation as Modeled
by Reaction of Deoxyadenosine. J. Am. Chem. Soc. 2001, 123, 11126–11132. [CrossRef]
10. Veldhuyzen, W.F.; Pande, P.; Rokita, S.E. A Transient Productof DNA Alkylation Can Be Stabilized by Binding Localization. J. Am.
Chem. Soc. 2003, 125, 14005–14013. [CrossRef] [PubMed]
11. Richter, S.N.; Maggi, S.; Mels, S.C.; Palumbo, M.; Freccero, M. Binol Quinone Methides as Bisalkylating and DNA Cross-Linking
Agents. J. Am. Chem. Soc. 2004, 126, 13973–13979. [CrossRef]
12. Verga, D.; Nadai, M.; Doria, F.; Percivalle, C.; Di Antonio, M.; Palumbo, M.; Richter, S.N.; Freccero, M. Photogeneration and
Reactivity of Naphthoquinone Methides as Purine Selective DNA Alkylating Agents. J. Am. Chem. Soc. 2010, 132, 14625–14637.
[CrossRef] [PubMed]
13. Di Antonio, M.; Doria, F.; Richter, S.N.; Bertipaglia, C.; Mella, M.; Sissi, C.; Palumbo, M.; Freccero, M. Quinone Methides Tethered
to Naphthalene Diimides as Selective G-Quadruplex Alkylating Agents. J. Am. Chem. Soc. 2009, 131, 13132–13141. [CrossRef]
[PubMed]
14. Nadai, M.; Doria, F.; Di Antonio, M.; Sattin, G.; Germani, L.; Percivalle, C.; Palumbo, M.; Richter, S.N.; Freccero, M. Naph-thalene
Diimide Scaffolds with Dual Reversible and Covalent Interaction Properties towards G-Quadruplex. Biochimie 2011, 93, 1328–1340.
[CrossRef]
15. Doria, F.; Nadai, M.; Folini, M.; Di Antonio, M.; Germani, L.; Percivalle, C.; Sissi, C.; Zaffaroni, N.; Alcaro, S.; Artese, A.; et al.
Hybrid ligand–alkylating agents targeting telomeric G-quadruplex structures. Org. Biomol. Chem. 2012, 10, 2798–2806. [CrossRef]
[PubMed]
16. Doria, F.; Nadai, M.; Folini, M.; Scalabrin, M.; Germani, L.; Sattin, G.; Mella, M.; Palumbo, M.; Zaffaroni, N.; Fabris, D.; et al.
Targeting Loop Adeninesin G-Quadruplexby a Selective Oxirane. Chem. Eur. J. 2013, 19, 78–81. [CrossRef] [PubMed]
17. Li, V.S.; Kohn, H. Studies on the bonding specificity for mitomycin C-DNA monoalkylation processes. J. Am. Chem. Soc. 1991
113, 275–283. [CrossRef]
,
18. Han, I.; Russell, D.J.; Kohn, H. Studies on the Mechanism of Mitomycin C(1) Electrophilic Transformations: Structure-Reactivity
Relationships. J. Org. Chem. 1992, 57, 1799–1807. [CrossRef]
19. Tomasz, M.; Das, A.; Tang, K.S.; Ford, M.G.J.; Minnock, A.; Musser, S.M.; Waring, M.J. The Purine 2-Amino Group as the Critical
Recognition Element for Sequence-Specific Alkylation and Cross-Linking of DNA by Mitomycin C. J. Am. Chem. Soc. 1998, 120,
11581–11593. [CrossRef]
20. Wang, H.; Wahi, M.S.; Rokita, S.E. Immortalizing a Transient Electrophile for DNA Cross-Linking. Angew. Chem. Int. Ed. 2008, 47,
1291–1293. [CrossRef]
21. Wang, H.; Rokita, S.E. Dynamic Cross-Linking is Retained in Duplex DNA after Multiple Exchange of Strands. Angew. Chem. Int.
Ed. 2010, 49, 5957–5960. [CrossRef]
22. Rossiter, C.S.; Modica, E.; Kumar, D.; Rokita, S.E. Few Constraints Limit the Design of Quinone Methide-Oligonucleotide
Self-Adducts for Directing DNA Alkylation. Chem. Commun. 2010, 47, 1476–1478. [CrossRef]

Molecules 2021, 26, 3355
24 of 26
23. Basaric´, N.; Mlinaric´-Majerski, K.; Kralj, M. Quinone Methides: Photochemical Generation and its Application in Biomedicine.
Curr. Org. Chem. 2014, 18, 3–18. [CrossRef]
24. Percivalle, C.; Doria, F.; Freccero, M. Quinone Methides as DNA Alkylating Agents: An Overview on Efficient Activation
Protocols for Enhanced Target Selectivity. Curr. Org. Chem. 2014, 18, 19–43. [CrossRef]
25. Chiang, Y.; Kresge, A.J.; Zhu, Y. Kinetics and Mechanisms of Hydration of o-Quinone Methides in Aqueous Solution. J. Am. Chem.
Soc. 2000, 122, 9854–9855. [CrossRef]
26. Chiang, Y.; Kresge, A.J.; Zhu, Y. Flash photolytic generation of ortho-quinone methide in aqueous solution and study of its
chemistry in that medium. J. Am. Chem. Soc. 2001, 123, 8089–8094. [CrossRef]
27. Toteva, M.M.; Richard, J.P. The Generation and Reactions of Quinone Methides. Adv. Phys. Org. Chem. 2011, 45, 39–91. [CrossRef]
[PubMed]
28. Basaric´, N.; Cindro, N.; Bobinac, D.; Mlinaric´-Majerski, K.; Uzelac, L.; Kralj, M.; Wan, P. Sterically Congested Quinone Methides
in Photodehydration Reactions of 4-Hydroxybiphenyl Derivatives and Investigation of their Antiproliferative Activity. Photochem.
Photobiol. Sci. 2011, 10, 1910–1925. [CrossRef]
29. Basaric´, N.; Cindro, N.; Bobinac, D.; Uzelac, L.; Mlinaric´-Majerski, K.; Kralj, M.; Wan, P. Zwitterionic Biphenyl Quinone Methides
in Photodehydration Reactions of 3-Hydroxybiphenyl Derivatives: Laser Flash Photolysis and Antiproliferation Study. Photochem.
Photobiol. Sci. 2012, 11, 381–396. [CrossRef]
30. Veljkovic´, J.; Uzelac, L.; Molcˇanov, K.; Mlinaric´-Majerski, K.; Kralj, M.; Wan, P.; Basaric´, N. Sterically Congested Adamantyl-
naphthalene Quinone Methides. J. Org. Chem. 2012, 77, 4596–4610. [CrossRef]
31. Sambol, M.; Ester, K.; Landgraf, S.; Mihaljevic´, B.; Cindric´, M.; Kralj, M.; Basaric´, N. Competing Photochemical Reactions of
bis-Naphthols and Their Photoinduced Antiproliferative Activity. Photochem. Photobiol. Sci. 2019, 18, 1197–1211. [CrossRef]
32. Škalamera, Ð.; Mlinaric´-Majerski, K.; Martin Kleiner, I.; Kralj, M.; Oake, J.; Wan, P.; Bohne, C.; Basaric´, N. Photochemical
Formation of Anthracene Quinone Methide Derivatives. J. Org. Chem. 2017, 82, 6006–6021. [CrossRef]
33. Uzelac, L.; Škalamera, Ð.; Mlinaric´-Majerski, K.; Basaric´, N.; Kralj, M. Selective Photocytotoxicity of Anthrols on Cancer Stem-like
Cells: The Effect of Quinone Methides or Reactive Oxygen Species. Eur. J. Med. Chem. 2017, 137, 558–574. [CrossRef] [PubMed]
34. Basaric´, N.; Kralj, M.; Mikecin, A.M.; Cindric´, M. Quinone Methide Precursors with BODIPY Chromophore, Method of
Preparation, Biological Activity and Application in Fluorescent Labeling. PCT/HR2017/000005, 15 May 2017.
35. Erben, A.; Matic´, J.; Basaric´, N.; Piantanida, I. The Phenanthridine-modified Tyrosine Dipeptide: Synthesis and Non-covalent
Binding to DNA and RNA. Croat. Chem. Acta. 2019, 92, 249–258. [CrossRef]
36. Arumugam, S.; Popik, V.V. Photochemical Generation and the Reactivity of o-Naphthoquinone Methides in Aqueous Solu-tions.
J. Am. Chem. Soc. 2009, 131, 11892–11899. [CrossRef] [PubMed]
37. Arumugam, S.; Popik, V.V. Attach, Remove, or Replace: Reversible Surface Functionalization Using Thiol–Quinone Methide
Photoclick Chemistry. J. Am. Chem. Soc. 2012, 134, 8408–8411. [CrossRef] [PubMed]
38. Arumugam, S.; Popik, V.V. Light-Induced Hetero-DielsAlder Cycloaddition: A Facile and Selective Photoclick Reaction. J. Am.
Chem. Soc. 2011, 133, 5573–5579. [CrossRef]
39. Arumugam, S.; Popik, V.V. Patterned Surface Derivatization Using Diels–Alder Photoclick Reaction. J. Am. Chem. Soc. 2011, 133,
15730–15736. [CrossRef]
40. Arumugam, S.; Orski, S.V.; Locklin, J.; Popik, V.V. Photoreactive Polymer Brushes for High-Density Patterned Surface Derivatiza-
tion Using a Diels–Alder Photoclick Reaction. J. Am. Chem. Soc. 2012, 134, 179–182. [CrossRef]
41. Chang, S.-C.; Archer, B.J.; Utecht, R.E.; Lewis, D.E.; Judy, M.M.; Matthews, J.L. 4-Alkylamino-3-bromo-N-alkyl-1,8-naphthalimides:
New Photochemically Activatable Antiviral Compounds. Bioorganic Med. Chem. Lett. 1993, 3, 555–556. [CrossRef]
42. Chanh, T.C.; Lewis, D.E.; Judy, M.M.; Sogandares-Bernal, F.; Michalek, G.R.; Utecht, R.E.; Skiles, H.; Chang, S.-C.; Matthews,
J.L. Inhibition of Retrovirus-Induced Syncytium Formation by Photoproducts of a Brominated 1,8-Naphthalimide Compound.
Antivir. Res. 1994, 25, 133–146. [CrossRef]
43. Chanh, T.C.; Lewis, D.E.; Allan, J.S.; Sogandares-Bernal, F.; Judy, M.M.; Utecht, R.E.; Matthews, J.L. Neutralization of HIV-1 and
Inhibition of HIV-1-Induced Syncytia by 1,8-Naphthalimide Photoactive Compound. Aids Res. Hum. Retrovir. 1993, 9, 891–896.
[CrossRef] [PubMed]
44. Andricopulo, A.D.; A Müller, L.; Filho, V.C.; Cani, G.S.; Roos, J.F.; Corrêa, R.; Santos, A.R.S.; Nunes, R.J.; Yunes, R.A. Analgesic
Activity of Cyclic Imides: 1,8-Naphthalimide and 1,4,5,8-Naphthalenediimide Derivatives. Il Farm. 2000, 55, 319–321. [CrossRef]
45. Mattocks, A.M.; Hutchison, O.S. Local Anesthetics: N-Dialkylaminoalkylimides of Naphthalic and Diphenylmaleic Acids. J. Am.
Chem. Soc. 1948, 70, 3474–3475. [CrossRef] [PubMed]
46. Muth, M.; Hoerr, V.; Glaser, M.; Ponte-Sucre, A.; Moll, H.; Stich, A.; Holzgrabe, U. Antitrypanosomal Activity of Quaternary
Naphthalimide Derivatives. Bioorganic Med. Chem. Lett. 2007, 17, 1590–1593. [CrossRef] [PubMed]
47. Langlois, M.; Soulier, J.; Rampillon, V.; Gallais, C.; Brémont, B.; Shen, S.; Yang, D.; Giudice, A.; Sureau, F. Synthesis of Quinazoline-
2,4-dione and Naphthalimide Derivatives as New 5-HT3 Receptor Antagonists. Eur. J. Med. Chem. 1994, 29, 925–940. [CrossRef]
48. Langlois, M.; Soulier, J.L.; Brémont, B.; Shen, S.; Rampillon, V.; Giudice, A. Derivatives of Naphthalimide: New Potent Con-
formationally Restricted Antagonists of 5-HT3 Receptors. Bioorg. Med. Chem. Lett. 1992, 2, 691–694. [CrossRef]
49. Berque-Bestel, I.; Soulier, J.-L.; Giner, M.; Rivail, L.; Langlois, M.; Sicsic, S. Synthesis and Characterization of the First Fluo-rescent
Antagonists for Human 5-HT4 Receptors. J. Med. Chem. 2003, 46, 2606–2620. [CrossRef]

Molecules 2021, 26, 3355
25 of 26
50. Tian, Z.; Zhao, Z.; Zang, F.; Wang, Y.; Wang, C. Spectroscopic Study on the Interaction Between Naphthalimide–Polyamine
Conjugates and DNA. J. Photochem. Photobiol. B Biol. 2014, 138, 202–210. [CrossRef]
51. Tian, Z.-Y.; Li, J.-H.; Li, Q.; Zang, F.-L.; Zhao, Z.-H.; Wang, C.-J. Study on the Synthesis, Biological Activity and Spectroscopy of
Naphthalimide-Diamine Conjugates. Molecules 2014, 19, 7646–7668. [CrossRef]
52. Braña, M.; Ramos, A. Naphthalimides as Anticancer Agents: Synthesis and Biological Activity. Curr. Med. Chem. Agents 2001, 1,
237–255. [CrossRef]
53. Brana, M.F.; Cacho, M.; Gradillas, A.; De Pascual-Teresa, B.; Ramos, A. Intercalators as Anticancer Drugs. Curr. Pharm. Des. 2001
7, 1745–1780. [CrossRef]
,
54. Rosell, R.; Carles, J.; Abad, A.; Ribelles, N.; Barnadas, A.; Benavides, A. Phase I Study of Mitonafide in 120 Hour Continuous
Infusion in Non-small Cell Lung Cancer. Investig. New Drugs 1992, 10, 171–175. [CrossRef]
55. Llombart, M.; Forner, E.; Olmos, T.; Ruiz, A.; Soriano, V.; Benavides, A.; Martín, M.; Schlick, E.; Guillém, V. Phase I Study of
Mitonafide in Solid Tumors. Investig. New Drugs 1992, 10, 177–181. [CrossRef]
56. Gesme, D.H.; Jett, J.R.; Schreffler, D.D.; Su, J.Q.; Mailliard, J.A.; Foley, J.F.; Krook, J.E.; Maksymiuk, A.W.; Hatfield, A.K.; Ebbert,
L.P.; et al. A Randomized Phase II Trial of Amonafide or Tri-metrexate in Patients with Advanced non-Small Cell Lung Cancer. A
Trial of the North Central Cancer Treatment Group. Cancer 1993, 71, 2723–2726. [CrossRef]
57. Kornek, G.; Raderer, M.; Depisch, D.; Haider, K.; Fazeny, B.; Dittrich, C.; Scheithauer, W. Amonafide as First-Line Chemo-therapy
for Metastatic Breast Cancer. Eur. J. Cancer 1994, 30, 398–400. [CrossRef]
58. Ratain, M.J.; Rosner, G.; Allen, S.L.; Costanza, M.; Van Echo, D.A.; Henderson, I.C.; Schilsky, R.L. Population Pharmaco-dynamic
Study of Amonafide: A Cancer and Leukemia Group B Study. J. Clin. Oncol. 1995, 13, 741–747. [CrossRef]
59. Rogers, J.E.; Weiss, S.J.; Kelly, L.A. Photoprocesses of Naphthalene Imide and Diimide Derivatives in Aqueous Solutions of DNA.
J. Am. Chem. Soc. 2000, 122, 427–436. [CrossRef]
60. Rogers, J.E.; Le, T.P.; Kelly, L.A. Nucleotide Oxidation Mediated by Naphthalimide Excited States with Covalently Attached
Viologen Cosensitizers. Photochem. Photobiol. 2001, 73, 223–229. [CrossRef]
61. Saito, I.; Takayama, M.; Sugiyama, H.; Nakatani, K. Photoinduced DNA Cleavage via Electron Transfer: Demonstration That
Guanine Residues Located 5’ to Guanine Are the Most Electron-Donating Sites. J. Am. Chem. Soc. 1995, 117, 6406–6407. [CrossRef]
62. Saito, I.; Saito, I.; Takayama, M. Photoactivatable DNA-Cleaving Amino Acids: Highly Sequence-Selective DNA Photocleavage
by Novel L-Lysine Derivatives. J. Am. Chem. Soc. 1995, 117, 5590–5591. [CrossRef]
63. Percivalle, C.; La Rosa, A.; Verga, D.; Doria, F.; Mella, M.; Palumbo, M.; Di Antonio, M.; Freccero, M. Quinone Methide Gen-eration
via Photoinduced Electron Transfer. J. Org. Chem. 2011, 76, 3096–3106. [CrossRef]
64. Ramchander, J.; Rameshwar, N.; Reddy, T.S.; Raju, G.; Reddy, A.R. Synthesis and Photophysical Properties of 1,4-Disubstituted
Naphthyloxymethyl-N-alkyl Naphthimido-1,2,3-triazole. J. Chem. Sci. 2014, 126, 1063–1074. [CrossRef]
65. Yaqiu, L.; Bin, C. Multi-substituted Amine Compound, as well as Preparation Method and Use Thereof. CN103570683, 30 July
2012.
66. Spallarossa, M.; Wang, Q.; Riva, R.; Zhu, J. Synthesis of Vinyl Isocyanides and Development of a Convertible Isonitrile. Org. Lett.
2016, 18, 1622–1625. [CrossRef]
67. Reggelin, M.; Junker, B.; Heinrich, T.; Slavik, S.; Bühle, P. Asymmetric Synthesis of Highly Substituted Azapolycyclic Com-pounds
via 2-Alkenyl Sulfoximines: Potential Scaffolds for Peptide Mimetics. J. Am. Chem. Soc. 2006, 128, 4023–4034. [CrossRef]
68. Mandal, P.K.; McMurray, J.S. Pd
6599–6601. [CrossRef]
−
C-Induced Catalytic Transfer Hydrogenation with Triethylsilane. J. Org. Chem. 2007, 72,
69. Anantharamaiah, G.M.; Sivanandaiah, K.M. Transfer Hydrogenation; a Convenient Method for Removal of Some Commonly
Used Protecting Groups in Peptide Synthesis. J. Chem. Soc. Perkin Trans. 1977, 1, 490–491. [CrossRef]
70. Bieg, T.; Szeja, W. Removal of O-Benzyl Protective Groups by Catalytic Transfer Hydrogenation. Synthesis 1985, 1985, 76–77.
[CrossRef]
71. Lee, J.; Robinson, G.W.; Webb, S.P.; Philips, L.A.; Clark, J.H. Hydration Dynamics of Protons from Photon Initiated Acids. J. Am.
Chem. Soc. 1986, 108, 6538–6542. [CrossRef]
72. Robinson, G.W. Proton Charge Transfer Involving the Water Solvent. J. Phys. Chem. 1991, 95, 10386–10391. [CrossRef]
73. Tolbert, L.M.; Haubrich, J.E. Photoexcited Proton Transfer from Enhanced Photoacids. J. Am. Chem. Soc. 1994, 116, 10593–10600.
[CrossRef]
74. Solntsev, K.M.; Huppert, D.; Agmon, A.N.; Tolbert, L.M. Photochemistry of “Super” Photoacids. 2. Excited-State Proton Transfer
in Methanol/Water Mixtures. J. Phys. Chem. A 2000, 104, 4658–4669. [CrossRef]
75. Laws, W.R.; Brand, L. Analysis of Two-State Excited-State Reactions. The Fluorescence Decay of 2-Naphthol. J. Phys. Chem. 1979
,
83, 795–802. [CrossRef]
76. Montalti, M.; Credi, A.; Prodi, L.; Gandolfi, M.T. Handbook of Photochemistry; CRC Taylor and Francis: Boca Raton, FL, USA, 2006.
77. Oelgemöller, M.; Kramer, W.H. Synthetic Photochemistry of Naphthalimides and Related Compounds. J. Photochem. Photobiol. C
Photochem. Rev. 2010, 11, 210–244. [CrossRef]
78. Wintgens, V.; Valat, P.; Kossanyi, J.; Biczok, L.; Demeter, A.; Bérces, T. Spectroscopic Properties of Aromatic Dicarboximides. Part
1—N–H and N-methyl-substituted Naphthalimides. J. Chem. Soc. Faraday Trans. 1994, 90, 411–421. [CrossRef]
79. Demeter, A.; Biczok, L.; Berces, T.; Wintgens, V.; Valat, P.; Kossanyi, J. Laser Photolysis Studies of Transient Processes in the
Photoreduction of Naphthalimides by Aliphatic Amines. J. Phys. Chem. 1993, 97, 3217–3224. [CrossRef]

Molecules 2021, 26, 3355
26 of 26
80. Kubo, Y.; Imaoka, T.; Shiragami, T.; Araki, T. A Photoallylation of N-Methylarenedicarboximides by Allylsilanes. Chem. Lett. 1986
,
15, 1749–1752. [CrossRef]
81. Goldstein, S.; Rabani, J. The ferrioxalate and Iodide–Iodate Actinometers in the UV Region. J. Photochem. Photobiol. A Chem. 2008
193, 50–55. [CrossRef]
,
82. Škalamera, Ð.; Antol, I.; Mlinaric´-Majerski, K.; Vancˇik, H.; Phillips, D.L.; Ma, J.; Basaric´, N. Ultrafast Adiabatic Photodehy-dration
of 2-Hydroxymethylphenol and the Formation of Quinone Methide. Chem. Eur. J. 2018, 24, 9426–9435. [CrossRef]
83. Mohan, H.; Hermann, R.; Naumov, S.; Mittal, J.P.; Brede, O. Two Channels of Electron Transfer Observed for the Reaction
of n-Butyl Chloride Parent Radical Cations with Naphthols and Hydroxybiphenyls. J. Phys. Chem. A. 1998, 102, 5754–5762.
[CrossRef]
84. Cho, D.W.; Fujitsuka, M.; Yoon, U.C.; Majima, T. Intermolecular Photoinduced Electron-Transfer of 1,8-Naphthalimides in Protic
Polar Solvents. Phys. Chem. Chem. Phys. 2008, 10, 4393–4399. [CrossRef]
85. Horvat, M.; Mlinaric´-Majerski, K.; Basaric´, N. Photochemistry of N-alkyl and N-aryl Substituted Phthalimides: H-Abstractions,
Single Elelctron Transfer and Cycloadditions. Croat. Chem. Acta 2010, 83, 179–188.
86. Mergny, J.-L.; Lacroix, L. Analysis of Thermal Melting Curves. Oligonucleotides 2003, 13, 515–537. [CrossRef]
87. Wilson, W.D.; Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin, D. The Search for Structure-Specific Nucleic Acid-Interactive Drugs:
Effects of Compound Structure on RNA versus DNA Interaction Strength. Biochemistry 1993, 32, 4098–4104. [CrossRef]
88. McGhee, J.D.; von Hippel, P.H. Theoretical Aspects of DNA-Protein Interactions: Co-operative and Non-co-operative Binding of
Large Ligands to a one-Dimensional Homogeneous Lattice. J. Mol. Biol. 1974, 86, 469–489. [CrossRef]
89. Steenken, S.; Jovanovic, S.V. How Easily Oxidizable Is DNA? One-Electron Reduction Potentials of Adenosine and Guanosine
Radicals in Aqueous Solution. J. Am. Chem. Soc. 1997, 119, 617–618. [CrossRef]
90. Rodger, A.; Norden, B. Circular Dichroism and Linear Dichroism; Oxford University Press: New York, NY, USA, 1997; Chapter 2.
91. Šmidlehner, T.; Piantanida, I.; Pescitelli, G. Polarization Spectroscopy Methods in the Determination of Interactions of Small
Molecules with Nucleic Acids—Tutorial. Beilstein J. Org. Chem. 2018, 14, 84–105. [CrossRef] [PubMed]
92. Eriksson, M.; Nordén, B. Linear and Circular Dichroism of Drug-Nucleic Acid Complexes. Met. Enzymol. 2001, 340, 68–98.
93. Gans, P.; Sabatini, A.; Vacca, A. Investigation of Equilibria in Solution. Determination of Equilibrium Constants with the
HYPERQUAD Suite of Programs. Talanta 1996, 43, 1739–1753. [CrossRef]
94. Doria, F.; Richter, S.N.; Nadai, M.; Colloredo-Mels, S.; Mella, M.; Palumbo, M.; Freccero, M. BINOL Amino Acid Conjugates as
−
Triggerable Carriers of DNA-Targeted Potent Photocytotoxic Agents. J. Med. Chem. 2007, 50, 6570–6579. [CrossRef]
95. Duncan, E.J.; Chao-Pin, L. Endhothelin Converting Enzyme Inhibitors. WO9513817 (A1) (1995), 16 November 1994.
96. Hunter, D.J.; Markwell, R.E.; Ward, R.W. Phosphonopeptides with Collagenase Inhibiting Activity. WO9309136 (A1) (1993),
5 April 1991.
97. Škalamera, Ð.; Mlinaric´-Majerski, K.; Martin-Kleiner, I.; Kralj, M.; Wan, P.; Basaric´, N. Near-Visible Light Generation of a Quinone
Methide from 3-Hydroxymethyl-2-anthrol. J. Org. Chem. 2014, 79, 4390–4397. [CrossRef]