Photochemical reactivity of naphthol-naphthalimide conjugates and their biological activity

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 ³and 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 (Φ_F≈ 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 (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

≈

10⁵–10⁶M⁻¹, as well as with

Naphthol-Naphthalimide Conjugates

and Their Biological Activity.

−1

bovine serum albumin (BSA) K_a

≈

10⁵M (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

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This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

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Molecules 2021, 26, 3355. https://doi.org/10.3390/molecules26113355

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 3355

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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-HT₃and 5-HT₄recep-

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

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

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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

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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

(a)

(b)

Figure 1.

(a) Absorption spectra of Naphth, 3c and 1c in CH₃OH-H₂O (4:1 v/v), (b) normalized ﬂuorescence spectra

(λ_ex= 310 nm) of Naphth, 3c and 1c in CH₃CN.

Quantum yields of ﬂuorescence (Φ_f) were measured in CH₃CN and CH₃CN-H₂O

(4:1 v/v) with the use of quinine sulfate in 0.5 M H₂SO₄as a reference (Φ_f= 0.546) [76

(Table 1). The _fvalues 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 _fmeasured in the aqueous solvent than

]

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 Φ_fby 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 Φ_fand efﬁciencies of FRET (Φ_FRET) ^b.

Φ_f(CH₃CN) × 10³

Φ_f(CH₃CN-H₂O) × 10^{3 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

(

Φ_f= 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. ^cCH₃CN-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

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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/KIO₃was used as an actinometer (Φ_R= 0.74) [81]. The results are compiled in Table 2.

One generally sees very low values for

Φ_R, 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 (Φ_R) 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(Φ_R= 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.

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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 O₂was 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 (λ_max= 360 and 460 nm) [83] and the naphthalimide

radical anion (λ_max= 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: τ_Ar= 460

sient was quenched by O₂, k_q= (8.7

with literature [63 79], it was assigned to the naphthalimide triplet excited state. The

±

20 ns; τ_air= 290

±

20 ns and τ_O2= 100

×

±

3 ns. Since the tran-

±

0.7)

10⁸M⁻¹s⁻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; τ_Ar= 340

±

2 ns;

)

τ_air= 245 ± 3 ns and τ_O2= 105

±

2 ns. Based on the quenching by O₂, k_q= (7.3 ± 0.3 ×

−1

10⁸M

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

10⁶s⁻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

×

10⁻⁵M) The samples were

20 mJ/pulse. (b) Decay of transient absorbance at 470 nm in Ar-purged, not purged and

≈

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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

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.

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The irradiation experiments and mechanistic investigations indicated the formation

of QMs from

photolysis of

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 O₂and 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 (T_m) [86]. The difference between

the T_mvalue of a free ds-polynucleotide and a complex with a small molecule (∆T_m

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 (K_a) only. The

1 to ct-DNA was assayed via UV-vis and ﬂuorescence titrations in

1

λ

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

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300

280

260

240

300

250

200

150

100

50

2.0x10^-54.0x10^-56.0x10^-58.0x10^-51.0x10^-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^-56.0x10^-59.0x10^-51.2x10^-41.5x10^-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 (λ_exc= 320 nm) of 1a

(

top) and 1e

(

bottom) (c = 2.0

×

10⁻⁶M) 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 10⁻⁴M is in favor of the static quenching

mechanism. Namely, the Stern–Volmer analysis of the quenching by DNA would pro-

vide the quenching constant k_qin the order of 10¹²M^{−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 logK_a= 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

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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 logK_a= 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

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160

140

120

100

80

180

150

120

90

B

A

r [1a]/[BSA] = 1

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 (λ_exc= 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

logK_a(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

IC₅₀values) 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

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(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 O₂and 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 (K_a

≈

10⁵–10⁶M⁻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

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determined spectroscopically as the concentration per nucleotide/phosphate, using molar

absorption coefﬁcient for calf thymus DNA of 6600 M⁻¹cm⁻¹at 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

, t_R= 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

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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

, t_R= 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

ꢀ

, t_R= 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

, t_R= 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

ꢀ

t_R= 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

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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 MgSO₄and 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

ꢀ

, t_R= 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

ꢀ

, t_R= 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

ꢀ

, t_R= 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

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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

ꢀ

, t_R= 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

ꢀ

, t_R= 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

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67.0 (d), 35.2 (t), 24.2 (t), one CH₂signal is cove₋red by the DMSO-signal; UPLC-MS/UV:

3

,

method

ꢀ

, t_R= 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 CH₂signal is covered by the

3

DMSO-signal; UPLC-MS/UV: method

ꢀ

, t_R= 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 CH₂signal is covered by the DMSO-signal; UPLC-MS/UV: method

3

ꢀ,

t_R= 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 CH₂signal is covered by the DMSO-signal; UPLC-MS/UV: method

ꢀ

, t_R= 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.

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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 CH₂signal is covered by the DMSO-signal; UPLC-MS/UV: method

ꢀ

,

t_R= 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 MgSO₄and 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

ꢀ

, t_R= 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

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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

ꢀ

, t_R= 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

ꢀ

, t_R= 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

ꢀ

, t_R= 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

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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

ꢀ

t_R= 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

ꢀ

, t_R(1a) = 1.16 min, m/z = 394.14 [M

−

t_R(1a-OMe) = 1.32 min, m/z = 394.14 [M

−

OCH₃]⁺, found 394.31; t_R(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) t_R(1c) = 1.27 min, m/z = 438.17

UPLC-MS/UV: method

ꢀ

[M − H]⁻, found 438.55; t_R

(

1c-OMe) = 1.42 min, m/z = 422.18 [M

−

OCH₃]⁺, found

H]⁻, found 438.55; t_R

422.66; [at 350 nm, 60 min] t_R(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

ꢀ

, t_R(1e) = 1.39 min, m/z = 450.21 [M

−

OH]⁺, found 450.36;

t_R(1e-OMe) = 1.55 min, m/z = 450.36 [M

−

OCH₃]⁺, found 450.41; t_R(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

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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(Φ_f= 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

×

10⁻²M), whereas the

stock solution of ct-DNA was prepared in aqueous cacodylate buffer (pH = 7.0, 50 mM)

c(ct-DNA) = 1.1

10⁻³M. 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

10⁻⁵M, 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 T_mvalues are the midpoints

of the transition curves, determined from the maximum of the ﬁrst derivative.

∆T_mvalues

were calculated by subtracting the T_mof the free nucleic acid from that of the respective

complex, with ∆T_mvalues being the average of at least two independent measurements

and the error in ∆T_mbeing ca. ±0.5 ^◦C.

•

Fluorescence titrations with ct-DNA

Stock solutions of solution 1a or 1e were prepared in DMSO (c = 1.0

×

10⁻³M). 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

×

10⁻⁶M (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 × 10⁻⁵M) in cacodylate buffer (pH = 7.0, 50 mM), aliquots of the solution of 1a or 1e

in buffer (c = 1.0

×

10⁻³M) were added to reach the concentration ratio r(

1)/(polynucleotide)

Molecules 2021, 26, 3355

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= 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

×

10⁻³M). 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].

×

10⁻⁶M (containing <1%

×

10⁻³M. The

◦

•

Photochemical alkylation of BSA

A ﬂuorescence cell containing an aqueous solution of BSA (c = 1.4

×

10⁻⁵M) 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

×

10⁻⁶M) in DMSO (total

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 O₂prior 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.

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Photochemical reactivity of naphthol-naphthalimide conjugates and their biological activity

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