Design, synthesis, and photophysical properties of pyrroloquinoline-based compounds showing strong blue fluorescence as potential dyes for biomedical applications

DOI: 10.1002/cmdc.201500366

Full Papers

Design, Synthesis, and Photophysical Properties of

Pyrroloquinoline-Based Compounds Showing Strong Blue

Fluorescence as Potential Dyes for Biomedical Applications

[

a]

Davide Carta, Anna Balasso, Paolo Caliceti, and Maria Grazia Ferlin*

A small library of 3-ethylpyrrolo[3,2-f]quinoline derivatives was

synthesized to identify a novel class of dyes for use in biologi-

cal studies. According to the spectroscopic analyses performed

to evaluate the fluorimetric parameters of quantum yield and

brightness, 7-methyl- and 6,7-dimethylpyrroloquinolin(9)one

derivatives were found to be the best blue luminescent dyes

for biological applications. To enhance the luminescence pro-

files and to obtain probes that could be conjugated to func-

tional groups of supramolecular drug delivery systems, these

compounds were further modified at position 3 to obtain 3-

heptanoic acid and 3-aminohexylpyrroloquinolin(9)one methy-

lated derivatives. The most brilliant 6,7-dimethyl-3-aminohexyl-

pyrroloquinolinone hydrochloride was conjugated to pullulan,

a biocompatible polysaccharide used to produce colloidal sys-

tems for drug delivery. Comparative studies showed that this

compound can be properly exploited as a blue fluorescent

label in biological investigations, namely cell trafficking and

pharmacokinetics/biodistribution studies. These molecules pos-

sess higher fluorescence efficiency than commercial dyes in

biological media, making them suitable alternatives to com-

mercially available products in current use.

Introduction

Due to their high sensitivity, rapid response time, and easy

handling, fluorescent dyes are attractive and versatile labeling

agents for a variety of analytical purposes, including cell cul-

[1]

ture studies and pharmacokinetic investigations. These label-

ing agents are usually classified into four groups according to

their molecular structure: nucleic acids, peptides, polymers,

and small molecules. Small and simple chemical structures are

often more chemically stable and possess better cellular per-

meability than macromolecular probes. These properties make

them suitable for both in vitro and in vivo biological studies.

However, despite the advantages offered by small fluorescent

molecules over peptides and nucleic acids, only a few dyes are

thesis of non-cytotoxic and new fluorescent PQ and pyrrolo-

quinolinone (PQone) derivatives and on the evaluation of their

photophysical characteristics for potential use as labeling

agents in biological assays, namely those for cell trafficking,

biodistribution, and pharmacokinetics investigations. In the

first step of this study, a series of prototype compounds was

obtained by introducing small functional groups characterized

by various electronic properties at various positions on the 3-

ethyl-PQ scaffold. Spectrometric analyses enabled the identifi-

cation of the best candidates for further tailoring and fluores-

cent labeling. PQone derivatives bearing a linear carbon chain

at the 3N-position and terminated with an amine or carboxyl-

ate functionality were designed and synthesized. By virtue of

its excellent fluorescence properties, especially its brightness,

compound 38 (3-(6-aminohexyl)-6,7-dimethyl-PQ) was selected

as model blue fluorescent dye to investigate the analytical po-

tential of this class of compounds for biological and biomedi-

cal applications. Fluorescence studies in various biological

media, including cell uptake investigations, were performed by

conjugating compound 38 to pullulan, which was used as

a model macromolecule. Pullulan is a biocompatible polysac-

charide that has been widely investigated to produce innova-

tive colloidal systems for the delivery of both small drugs and

[

2]

currently available.

The coumarin bicyclic core is the scaffold used to synthesize

most of the commercial blue fluorescent dyes widely used in

chemical and biochemical applications, including fluorescent

labeling of biomolecules. For example, derivatives of aminome-

thylcoumarin acetate (AMCA), the lead molecule for this class

of fluorescent probes, have been activated and conjugated to

[

3]

macromolecules, namely antibodies and other proteins. The

tricyclic core of pyrrolo[3,2-f]quinoline (PQ) is also a blue fluo-

rochrome and could be a suitable scaffold for the synthesis of

small and highly fluorescent blue dyes.

Exploiting the experience acquired to date on the synthesis

[

4–9]

of PQ derivatives,

the present work was focused on the syn-

[

a] Dr. D. Carta, Dr. A. Balasso, Prof. P. Caliceti, Prof. M. G. Ferlin

Department of Pharmaceutical and Pharmacological Sciences

University of Padova, 35131 Padova (Italy)

E-mail: mariagrazia.ferlin@unipd.it

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201500366.

ChemMedChem 2015, 10, 1846 – 1862

1846

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

biological molecules. Pullulan-based drug delivery systems in-

clude macromolecular bioconjugates, nanoparticles, nanovesi-

were obtained by reacting the 5-aminoindoles 5 and 7 (synthe-

[

14]

sis in Figure 35 SI) with Meldrum’s reagent. Intermediates 8

and 9 were purified by silica gel chromatography and then

thermally cyclized in boiling diphenyl ether to yield the tricy-

clic PQone derivatives 10 and 11.

Scheme 2 shows the alkylation of compound 11 with methyl

iodide in the presence of sodium hydride. The reaction was

[10,12]

cles, and supramolecular assemblies.

Pullulan is a suitable

scaffold for chemical conjugation because it can be easily acti-

vated to covalently attach molecules, including labeling agents

along the backbone. Therefore, pullulan was considered a suita-

ble model colloid for labeling and biological studies.

Results and Discussion

Chemistry

PQ derivatives were synthesized according to Schemes 1–4.

Two chemical routes were followed, both leading to the pyrro-

lo[3,2-f]quinolin-9-one scaffold: 1) condensation of starting 5-

aminoindole derivatives with Meldrum’s acid (2,2-dimethyl-1,3-

dioxane-4,6-dione), which yielded the final tricycles that were

not substituted at positions 7 or 8 (Scheme 1B); 2) condensa-

tion of 5-aminoindole derivatives with a b-keto ester, with the

ethyl acetoacetate yielding 7-methyl-PQones according to the

[13]

Conrad–Limpach quinoline synthesis (Scheme 2).

Scheme 1A shows the conventional synthesis of intermedi-

ate 1-substituted 5-aminoindoles starting from the commercial

Scheme 2. Synthesis of PQone 12 and PQ 13. Reagents and conditions:

a) 60% NaH, CH

3

I, anhydrous DMF, RT, 2 h; b) 60% NaH, CH

3

I, anhydrous

THF, RT, 2 h; c) POCl

3

, reflux, 12 h; d) Na, anhydrous MeOH, reflux, 72 h.

performed in two different solvents: DMF and THF. The methyl-

ation of compound 11 in anhydrous DMF produced an irre-

solvable mixture of compounds 12 and 13, with 12 in excess

1

(

60%, as estimated by H NMR integrations). The methylation

of compound 11 in anhydrous THF yielded only compound 12

according to a regioselective reaction. Compound 13 was ob-

tained by the two-step synthetic protocol shown in Scheme 2

(

steps c and d). Chlorination of 11 with phosphoryl chloride

yielded the 9-chloro derivative 14, which was methoxylated by

[

15]

reaction with sodium methoxide.

The exact structures of compounds 12 and 13 were deter-

1

mined by H NMR analysis (Figure S1–S8, SI). The spectrum of

compound 12 showed a doublet at d=6.08 ppm and a singlet

at 3.89 ppm, corresponding to the 8-H and methyl group of

the 6N-methylquinolinone structure, respectively. The spectrum

of compound 13 showed the 8-H doublet and 9-methoxy

singlet at d=7.08 and 4.22 ppm, respectively, of 9-methoxy-

PQ. The coupling constants of 8-H with 7-H of compounds 12

and 13 were in agreement with the proposed structures, as

they were 7.49 Hz for the vinyl amine group of compound 12

and 5.26 Hz for the aromatic quinoline structure of compound

13.

Scheme 1. A) Synthesis of aminoindole derivatives 4–6 and B) synthetic

route to PQone derivatives 10 and 11. Reagents and conditions: a) 60% NaH,

ethyl 7-bromoheptanoate, 6-bromohexanenitrile and 3-bromopropionitrile,

anhydrous DMF, 5 h; b) H

methyl orthoformate, reflux, 4 h; d) Ph

2

, 10% Pd/C, EtOAc, 508C, 3–5 h; c) Meldrum’s acid,

O, 2508C, 15 min.

2

5

-nitroindole, which was firstly 1N-alkylated by the appropriate

halo-compound in the presence of sodium hydride to yield

compounds 1–3. These products were catalytically reduced

with H and Pd/C (10%) to obtain the corresponding amino

2

derivatives 4–6 (experimental details provided in Supporting

Information (SI) SI 42).

Scheme 3 shows the synthesis of PQone compounds 19–22.

According to the Conrad–Limpach quinoline synthesis, ethyl

acetoacetate reacted with the 1N-substituted-5-aminoindole

Aimed at obtaining fluorescent molecules, PQ derivatives

were synthesized by starting from two model indole deriva-

tives, compounds 5 and 7, which have a cyano group and an

alkyl group at position 1N, respectively. Scheme 1B illustrates

the synthesis of compounds 10 and 11 and their intermediates

[

7]

derivatives 4–7 to yield enamine intermediates 15–18, which

then underwent thermal cyclization in boiling diphenyl ether

to produce the PQone compounds 19–22.

Compounds 19, 21 and 22 were selectively methylated at

the 6N-position with methyl iodide/sodium hydride in THF to

give compounds 23–25. However, to avoid forming a mixture,

8

and 9 (experimental details in SI 42). The synthesis of com-

[7]

pound 7 was reported previously. Compounds 10 and 11

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1847

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

amine group with (Boc) O produced the protected

2

amino derivatives 33–35, which could be easily iso-

lated and then converted into the corresponding hy-

1

drochloride salts 36–38. H NMR analyses of com-

pounds 36–38 (Figures S23 and S24, SI) showed the

characteristic signals of 9-hydroxyquinolinic struc-

tures. The singlet at d~15 ppm, due to the 9-hydroxy

proton, and the signal from 8-H at d=7.04 confirmed

the hydroxypyrroloquinoline form of these com-

pounds. In all spectra, signals corresponding to pro-

tonated amino groups appeared at d~7.95 ppm. In

the case of compound 38, the presence of the singlet

at d=4.21 that integrated three protons corresponds

to the methyl group at position 6, suggesting that

this product is a dichloride salt. This result was con-

firmed by elemental analysis.

Scheme 3. Synthetic route to 7-methyl-PQone derivatives 19–25 and PQ derivative 26.

Reagents and conditions: a) ethyl acetoacetate, Drierite, glacial acetic acid, absolute EtOH,

2 3

18 h, reflux; b) Ph O, 2508C, 15 min, reflux; c) 60% NaH, CH I, anhydrous THF, 45 min;

d) POCl , reflux, 12 h; e) Na, anhydrous MeOH, 1 h, reflux.

3

compound 22 was transformed into the corresponding 9-me-

thoxy derivative 26 by the same reactions reported in

Scheme 2, steps c and d. The PQone derivatives were charac-

1

terized by H NMR (Figures S9–S14, SI), which showed a chemi-

cal shift of 8-H at d=6.09 for compound 23, 6.08 for com-

pound 24, 6.10 for compound 25, and 6.96 ppm for

the 9-methoxy quinoline derivative 26.

Scheme 4 shows the chemical modification of the

tricyclic derivatives that were synthesized according

to Schemes 1B–3. Scheme 4A shows the methylation

reaction of the 9-methoxylated derivative 26 per-

formed with neat methyl iodide to give the 6,7-di-

methyl iodide derivative 27, which was characterized

by NMR (Figures S15 and S16, SI). In particular, the

1

H NMR spectrum (Figure S15, SI) showed the pres-

ence of three singlets corresponding to the three

methyl groups of the molecule: 3.00 (7-CH ), 4.31 (6-

3

CH ), and 4.37 (9-OCH ). Scheme 4B shows the chlori-

3

nation of the PQone derivative 22 at position 9 by

phosphoryl chloride. The resulting compound 28 was

reacted with methylamine to yield compound 29. Al-

ternatively, compound 28 was subjected to hydroge-

nolysis [H , Pd/C (10%)] to give the PQ derivative 30.

2

Scheme 4C shows the alkaline hydrolysis of the ethyl

esters of compounds 19 and 23 along with the for-

mation of the corresponding PQ acids 31 and 32

upon treatment with 37% hydrochloric acid. The

1

H NMR spectrum of compound 31 (Figure S20, SI)

shows the characteristic signals of a quinoline struc-

ture, with singlets at d=14.37 and 7.04 ppm as-

signed to the 9-hydroxy group and 8-H, respectively.

The spectra of compounds 31 and 32 (Figures S21

and S22, SI) show a broad singlet at d=11.97 ppm,

which corresponds to the carboxylic proton. The

spectrum of the PQone derivative 32 shows a singlet

at d=4.11 ppm, corresponding to the 6-methyl

group. Finally, Scheme 4D shows the reduction of Scheme 4. A) Synthesis of the 7-methylpyrroloquinolinium iodide derivative 27; B) syn-

thesis of PQ derivatives 28–30; C) synthesis of the 7-methyl-PQ acids 31 and 32; D) syn-

thetic pathway to hydrochlorides 3-alkylamino-PQ derivatives 36–38. Reagents and condi-

the cyano group of compounds 10, 21, and 25 by

sodium borohydride and catalytic amounts of cobal-

tions: a) CH

reflux; d) H

·6H O, MeOH, 12 h; g) HCl (dry gas), EtOAc/EtOH, 48C, 12 h.

3

I, 4 h reflux, b) POCl

, Pd/C 10%, EtOAc, 12 h; e) 10% NaOH, MeOH, 1 h; f) NaBH

3

, 12 h reflux; c) CH

3

NH

2

, anhydrous DMF, 508C, 16 h

t(II) chloride hexahydrate in the presence of di-(tert-

2

4

, (Boc) O,

2

[16,17]

butoxycarbonyl) ether [(Boc) O].

Reaction of the CoCl

2

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1848

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

Spectrophotometric studies: UV/Vis and fluorescence

emission spectroscopy

UV/Vis absorption spectra of equimolar aqueous solutions

[

18]

[7]

(

15.07 mm) of PQ, Et-PQ, the 3-ethyl-PQ derivatives 13, 14,

2

6–30, and the 3-ethyl-PQone derivatives 11, 12, 20, 22, and

5 at pH 7 were collected in the 200–600 nm range and their

molar absorption coefficients (e) at l_m_a_xwere calculated. The

[

18]

e values of the unsubstituted pyrroloquinoline (PQ ) were

considered as reference (e=45056 at l_m_a_x219 nm, e=27869

at l_m_a_x259 nm, e=9090 at l_m_a_x333 nm). The spectroscopic

data obtained from the UV/Vis analyses of PQ and PQone de-

rivatives are summarized in Tables S1 and S2 (SI).

Compound 13, bearing an electron-donating methoxy

group at position 9, showed a slight l_m_a_xred shift and a signifi-

cant decrease in e value. The trisubstituted PQs displayed four

l_m_a_xvalues (compounds 26, 27, 29 and 30), and had similar

UV/Vis profiles and spectroscopic values (Table S1, SI).

UV/Vis analyses of PQones 11 and 22 were performed under

neutral (pH 7), alkaline (pH 11), and acidic (pH 1) conditions to

investigate the prototropic tau-

Figure 1. Absorption profiles of compounds 11 (blue), 12 (green), 22 (red),

and 25 (purple) obtained at 15.07 mm and pH 7.

tomerism of the 9-hydroxy-

[19]

9

(6H)-quinolinone moiety (UV/

Vis absorption spectra are pre-

sented in Figure S36, SI). At

pH 1, compounds 11 and 22 dis-

played similar absorption behav-

ior, although the 7-methyl-sub-

stituted compound 22 had

slightly higher absorption, espe-

cially at ~250 nm, relative to

compound 22, as expected. At

pH 7, the absorption profiles of

both compounds were slightly

different from those at pH 1 for

intensity. At pH 11, the UV/Vis spectra of compounds 11 and

Scheme 5. Tautomerism of the PQone derivatives 11 and 22 in the pH 4–9 range.

1

agree with the H NMR data obtained under strongly acidic

2

2 were significantly different from those recorded at pH 1

conditions for derivatives 31, 32, 37, and 38 (Figures S20–S24,

SI), which were found as 9-hydroxy-PQs. This finding is likely

due to a structural feature of the pyrrolo[3,2-f]quinoline core

(Figures S26–S29, SI).

and 7. The presence of another l_m_a_x(230 nm) suggests that

a new form of the PQone scaffold is prevalent under alkaline

conditions. To further investigate tautomerism of the PQone

derivatives, we compared the UV/Vis spectra of 11 and 22 with

those of compounds 22 and 25, which have a methyl group at

position 6 and do not undergo tautomerism. Figure 1 shows

the absorption profiles of compounds 11 and 22 and those of

analogues 12 and 25 recorded at pH 7. The four UV/Vis profiles

have the same behavior, which indicates that the 9-carbonylic

form of 11 and 22 is prevalent at neutral pH.

The pK of compounds 11, 12, and 13 confirmed the pres-

a

ence of the keto form in the pH 4–9 range. According to

a report that all 4-hydroxypyridines have a single pK value at

a

[

20]

~3, compounds 11 (6-NH) and 12 (6-NCH ) have a single pK

3

a

value at 3.84Æ0.01 and 3.90Æ0.01, respectively, verifying the

presence of the vinyl amine form. In the case of compound 11,

it should be noted that the NH group acquires strong aromatic

character and does not lose the proton within the examined

UV/Vis spectra of aqueous solutions of compounds 11 and

22 across the range of pH 1–14 were recorded to evaluate the

pH range (2–11). The slightly higher pK of compound 12 rela-

a

pH span over which the various molecular forms are present.

The UV/Vis profiles of these compounds significantly changed

as the pH increased to ~4 and then again at pH 9 (Figure S25,

SI). Therefore, according to Scheme 5, we can conclude that

PQone derivatives 11 and 22 undergo tautomerism in the

pH 4–9 range, where they exist in the keto form, whereas

below pH 4 and above pH 9 they are mainly in the non-dissoci-

ated and dissociated 9-enolic form, respectively. These data

tive to compound 11 is due to the electron-donating effect of

the N-methyl group, which stabilizes the positively charged

form. The pK of 7.35 for compound 13 is characteristic of an

a

aromatic pyridine ring bearing a methoxy group at position 4

and that does not show tautomeric equilibrium. The pK of

a

3.84 of compounds 11 and 12 confirmed the carbonyl form of

the pyrroloquinolin-9-ones at physiological pH.

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1849

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

Table 1 lists the fluorescence

Table 1. Fluorescence properties of pyrroloquinoline (PQ) derivatives PQ, Et-PQ, 13, 14 and 26–30, and PQone

derivatives 11, 12, 22, and 25.

[

18]

properties of PQ

(reference),

[

7]

Et-PQ, compounds 11–14, 22

and 26–30, and PQone deriva-

tives 11, 12, 22 and 25, obtained

from the emission spectra of

equimolar aqueous solutions of

these compounds at various pH

and recorded using l_e_x350 nm,

which was preliminarily found to

be close to the l_e_xmaxima. It

l

ex/lem

DlStokes

B350/B250

Compd

I

f

QY

À1

[nm]

[cm mol ]

1

R =H

[

18]

2

PQ

R =H

pH 7

350/456

350/495

350/497

350/478

6

0.02

0.03

0.19

–

106

145

–

128/435

1059/5131

–/–

3

R =H

1

should be noted that l 350 nm

R =C H

ex

2

5

[

7]

2

Et-PQ

R =H

is commonly used with commer-

cial blue fluorescent dyes,

3

R =H

namely

AMCA

and

Alexa

1

R =C

2

H

5

2

Fluor 350. Moreover, 350 nm is

the lowest l_e_xvalue used by

laser devices in optical microsco-

py for biological investigations.

All of the examined com-

pounds were found to be fluo-

13

R =OCH

520

–

3

R =H

1

R =C

2

H

5

2

1

2

4

6

R =Cl

3

R =H

1

R =C

2

H

5

rescent in the blue region (l_e_m

50–497 nm, Figure S30, SI). Qui-

nine sulfate in 0.1m H SO was

=

2

R =OCH

3

117

0.32

128

2717/6030

4

3

R =CH

3

2

4

1

R =C

2

H

5

used as a standard to determine

the quantum yield (QY) of the

novel fluorophores, because it

absorbs within the same UV/Vis

range as the PQ derivatives. At

2

R =OCH

27

pH 7

350/483

199

0.43

133

2938/7646

3

R =CH

3

4

R =CH

1

R =C

2

H

5

2

8

9

R =Cl

pH 7

350/495

350/465

350/453

6

115

6

0.038

0.24

0.03

–

273/1134

2213/3456

173/318

l =350 nm, quinine sulfate has

ex

3

R =CH

3

[

2]

a QY of 0.577.

The unsubstituted PQ

[18]

1

dis-

R =C H

2

5

2

R =NHCH

3

114

played a very low QY value. Simi-

larly, the derivative bearing an

ethyl group at position 3 (Et-

3

R =CH

3

1

R =C

2

H

5

[7]

2

PQ ) and the derivative bearing

an ethyl group at position 3 and

a methyl group at position 7

30

R =H

103

3

R =CH

3

1

R =C

2

H

5

pH 1

pH 7

pH 11

350/485

354/439

352/436

121

110

137

0.34

0.36

0.37

135

85

84

–

(

compound 30) showed low QY

11

2412/5016

–

3

4

R =R =H

values. Compounds 14 and 28,

which have a 9-chlorine atom,

showed relatively high emission

wavelengths, although their QY

was very low. In the tricyclic Et-

PQ derivatives, the electron-do-

nating methoxy group at posi-

tion9 had a significant and posi-

tive effect on the fluorescence

properties. Indeed, the values re-

ported in Table 1 show that

1

R =C

2

H

5

3

1

2

R =H

pH 7

357/435

161

0.31

78

3980/4690

4

R =CH

3

1

R =C

2

H

5

pH 1

350/478

221

0.44

128

–

3

R =CH

3

22

pH 7

pH 11

356 432

354 432

123

168

0.39

0.52

76

78

3519/9885

–

4

R =H

1

R =C

2

H

5

3

2

5

R =CH

3

pH 7

357/444

350/451

210

–

0.32

87

5181/7792

4

R =CH

compound 13 has a higher l

em

[18]

[

a]

and QY than those of PQ. The

-methoxy-PQ derivatives 26

Quinine

590

0.577

3170/17076

9

[2]

[

a] Quinine sulfate in 0.1m H SO .

2 4

and 27, which bear a methyl

group at position 7 and two

methyl groups at positions 6 and

7

, respectively, exhibit longer

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1850

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

emission wavelengths and up to

6- and 21-fold higher QY

1

values, respectively, relative to

those of PQ and Et-PQ. The 9-

methylamino derivative 29 ex-

hibited a higher QY both in

water and methanol than the

corresponding

unsubstituted

compound 30, but a lower QY

than that of 9-methoxy deriva-

tive 26. Stokes shifts ranged

from 106 nm for PQ to 145 nm

for compound 13. The most bril-

liant fluorophores were found to Figure 2. Emission spectra obtained at pH 7 (H O), concentration of 15.07 mm and at l_e_x=350 nm: A) PQs 13

2

be 26, 27, and 29, which dis- (green), 26 (blue), and 27 (yellow); B) PQones 11 (blue), 12 (green), 22 (yellow), and 25 (red); C) PQone 38 (red)

and AMCA (blue).

played high brightness values.

The fluorescence properties of

the PQone derivatives 11, 12, 22,

and 25 are summarized in Table 1 and show that, in general,

the keto form (9-C=O) of the quinoline core improves the

spectroscopic parameters of this compound class. Compounds

keto form because they are present in this form under the

analysis conditions, as described above. Compounds 31 and

32 have similar spectrometric profiles, except for 31, which has

four l_m_a_xvalues in water relative to the three in methanol and

PBS. Compound 32 has four l_m_a_xvalues in water and metha-

nol, but not in PBS. Compounds 36–38 and their acid counter-

parts showed similar UV/Vis profiles with four l_m_a_xvalues at

1

1 and 22 have higher QY values in water at all examined pH

[7]

values (1, 7, 11) than the analogues of Et-PQ and compound

0 bearing a methyl group at position 7, although slightly

3

lower l_e_mand Stokes shifts were observed. In water at pH 7,

the PQone derivatives 11, 12, 22, and 25 showed QY values

ranging from 0.31 for compound 12 to 0.39 for compound 22.

Compounds 11 and 22 had relatively constant parameters at

the three different pH values. Importantly, the QY data indicate

that the fluorophores are fairly stable in various chemical and

biological environments. At pH 7, compounds 22 and 25 ex-

hibited the greatest brightness at 350 nm.

pH 7. All five compounds in H O showed one more l

at

max

2

~213 nm with very high molar absorption coefficients of

40544, 35169, 35899, 36297 and 30676 for 31, 32, 36, 37,

and 38, respectively (Table S3, SI). Table 2 shows that the l_e_mof

compounds 31 and 32 were shorter than those of parent com-

pounds 22 and 25. The Stokes shifts in water maintained

roughly the same values, whereas QY values decreased slightly

relative to the parent compounds. The results listed in Table 2

show that the amino derivatives 36–38 have similar QYs but

Thus, in the considered series of fluorescent pyrrolo[3,2-

f]quinoline derivatives bearing small atomic groups at various

positions on the tricyclic scaffold, it was observed that CH₃,

C H , OCH , NHCH , and C=O groups enhanced the fluores-

slightly different brightness values at l =350 nm, with com-

ex

pound 38 being the most brilliant. However, they possess

lower Stokes shifts with respect to all the PQones described in

Table 1. In summary, these reactive compounds maintained

high QYs in both water and PBS, and their QYs were 1.5- to 2-

fold higher than the QY of AMCA.

2

5

3

cence of the PQ, whereas Cl did not affect its emission. The 7-

methyl- and 6,7-dimethyl-PQones 22 and 25, which showed

the best emission properties, namely high QY and brightness,

were chosen as lead molecules to obtain conjugatable fluores-

cent PQones bearing an amine- or carboxy-terminated six-

carbon alkyl chain bound to the pyrrole nitrogen atom. Fig-

ure 2A,B shows the emission spectra of the PQs and PQones

obtained in water at pH 7.

Figure 3C shows the emission spectra of compound 38 and

AMCA registered at pH 7 in water, with 38 showing a fluores-

cence profile similar to that of AMCA, which is the gold stan-

dard for labeling agents in in vitro studies. Having selected

pullulan as a model macromolecule, we chose the amino-func-

tionalized compound 38 for labeling with the polysaccharide,

thereby producing the conjugate 42 described below, used for

carrying out subsequent biological investigations.

Compounds 22 and 25 were functionalized with a carboxy-

terminated six-carbon 3-alkyl chain (31 and 32) and also with

an amino-terminated three-carbon 3-alkyl (36) and a six-

carbon 3-alkyl chain (37 and 38) to facilitate chemical labeling

of various substrates. The fluorescence properties of the car-

boxylic acid functionalized PQones 31 and 32 and the amino

derivatives 36–38 are listed in Table 2. Equimolar solutions of

compounds 31, 32, and 36–38 in water at pH 7, in phosphate-

buffered saline (PBS) at pH 7.4 and in methanol were analyzed

by UV/Vis spectroscopy (Table S3, SI). The UV/Vis behaviors at

the various pH values of compounds 31, 32, and 36–38 are

similar to those of the lead molecule 22. In Table 2, com-

pounds 31, 32, and 36–38 are shown in the corresponding

Spectrofluorimetric stability of compound 38

Spectrofluorimetric studies were performed to determine the

stability of compound 38 at pH 4.5, 5.5, and 7.2, which simu-

late the pH conditions of biological compartments—lyso-

somes, endosomes, and plasma, respectively—as well as those

of cell culture media and plasma. To simulate physiological

conditions and the conditions used in biological protocols, the

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1851

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

(

buffers, plasma, cell lysate, and

Table 2. Fluorescence properties of the PQone derivatives 31, 32, and 36–38.

Triton X-100). The fluorescence

efficiency, which was obtained

from the slope of the fluores-

cence/concentration plot, yield-

ed similar values for all of the

buffers and Triton X-100, but

lower slopes were obtained in

plasma and cell lysate. The

extent of fluorescence quench-

ing relative to buffer, calculated

as the ratio of slopes from the

fluorescence/concentration

l

ex/lem

DlStokes

B350/B250

Compd

I

f

QY

À1

[

nm]

[nm]

[cm mol ]

1

R =(CH

2

)

6

3

6

COOH

pH 7

PBS

MeOH

350/428

350/431

356/413

201

179

97

78

81

63

0.33

0.34

0.21

3065/8102

2707/7648

2229/6214

2

3

1

2

6

7

8

R =CH

3

R =H

1

R =(CH

2

COOH

pH 7

PBS

MeOH

350/431

350/430

350/416

195

235

243

81

80

66

0.29

0.27

0.23

3348/5927

3547/5715

4090/6227

2

R =CH

3

R =CH

3

1

R =(CH

2

NH

2

pH 7

MeOH

PBS

350/421

350/412

350/418

154

152

154

71

63

68

0.28

0.30

0.25

2489/6094

2767/6668

2422/7631

plots, was found to be 0.83 and

2

R =H

0

.093 for cell lysate and plasma,

3

R =H

respectively. The lowest detecta-

ble concentrations of compound

38 and AMCA in Triton X-100,

cell lysate, and plasma are listed

in Table 3, showing that in cell

lysate and plasma the lowest de-

tectable concentration of com-

pound 38 is ~4.8- to 3.2-fold

lower than AMCA in cell lysate

and plasma, respectively. There-

fore, compound 38 was found

to have very high fluorescence

efficiency, which makes it a suita-

1

R =(CH

2

pH 7

PBS

MeOH

350/428

350/430

350/416

156

161

209

78

80

66

0.30

0.32

0.30

2488/6270

2590/6625

2986/7146

2

R =CH

3

R =H

1

R =(CH

2

pH 7

PBS

MeOH

350/428

350/430

350/416

247

306

200

78

80

66

0.28

0.33

0.25

3901/6837

4379/8999

3317/6652

2

R =CH

3

R =CH

3

pH 7

PBS350/440

MeOH350/436

350/441

125

131

–

0.176

–

AMCA

0.171

0.169

3044/1207

120

[a]

Quinine

H

2

SO

4

350/451

–

590

0.577

3170/17076

[

2]

[

2 4

a] Quinine sulfate in 0.1m H SO .

Table 3. Lowest detectable concentrations of compound 38 and AMCA

[a]

in Triton X-100, cell lysate, and rat plasma.

Units

AMCA

38

T X-100 Lysate

Plasma

T X-100 Lysate Plasma

À1

ngmL

nm

2Æ0.1 16Æ0.2 94Æ1

11 91 535

4Æ0.1 6Æ0.5 53Æ0.9

14

19

170

[

a] Concentration values are the mean ÆSD of at least three independent

experiments.

Figure 3. Cytotoxicity of compound 38 in KB (^) and MCF7 (

) cell lines, rel-

&

ble alternative to compounds in current use for biological pro-

ative to that of AMCA in MCF7 (~) cells. Percentage viability values represent

[21]

tocols.

the mean ÆSD of at least three independent experiments.

Additionally, the QYs of 38 and AMCA at the same concen-

tration (15.07 mm) were measured in cell lysate and cell culture

medium. By comparing the QYs, it was observed that AMCA

was quenched to a greater extent than 38: 14.4 vs. 1% in cell

lysate, and 35.33 vs. 26.3% in cell culture medium (Figure S34,

SI). Spectrofluorimetric studies showed that compound 38 is

very stable under various conditions typically used in biological

assays. No significant differences were observed at various pH

values, indicating that the fluorescence efficiency is not affect-

ed by the cellular microenvironment to which the probe may

localize, namely lysosomes, endosomes, and cytosol. Further-

more, although the fluorescence of this PQone derivative was

partially quenched by biological media, specifically cell lysates

and plasma, the lowest detectable concentration was

experiments were performed at 378C, and the stability of

AMCA was investigated for comparison (Figure S31, SI). The

fluorescence of AMCA remained unchanged after 72 h incuba-

tion at pH 5.5 and 7.5 in cell culture medium and plasma, but

it decreased by ~10% at pH 4.5. In contrast, the fluorescence

of compound 38 did not change after 72 h incubation in all

media.

Fluorescence studies were also carried out to evaluate the

lowest detectable concentration of 38 in various media as well

as its fluorescence efficiency and fluorescence/concentration

correlation (Figure S32, SI). A linear fluorescence/concentration

correlation was observed in all the media conditions tested

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1852

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

<

200 nm. In both media, the QY

values indicated that the extent

of quenching was greater for

AMCA than 38 relative to QYs

measured in water. The compari-

son of QYs for 38 and AMCA in

cell lysate and cell culture

medium showed that AMCA is

quenched to a greater extent

than 38.

Biological properties of

compound 38

To verify the potential utility of

compound 38 as

a labeling

agent in biological studies, ex-

periments were carried out to in-

vestigate the cytotoxicity, stabili-

ty, and fluorescence efficiency of

this molecule in different media.

The cytotoxicity of 38 was pre-

liminarily tested using two cell

lines: human cervical carcinoma

(

KB) and human breast adeno-

carcinoma (MCF7) cells. The cy-

totoxicity of AMCA was also as-

sessed for comparison using

MCF7 cells. Figure 3 shows that

compound 38 and AMCA have

Figure 4. Confocal microscopy images of MCF7 cell uptake of a) AMCA at 171 mm and compound 38 at b) 86.5

similar cytotoxicity profiles and and c) 865 mm. Images were acquired using a blue channel for detection of compound 38 (panels 1) and a red

channel for membrane detection (panels 2). Panels at right (panels 3) show merged images of the blue and red

channels.

that both compounds have low

toxicity on the tested cell lines.

Only high concentrations of 38

Bioconjugtion of compound 38 to pullulan

and AMCA (8.6 and 17.1 mm, respectively) were found to pro-

duce a cytotoxic effect. Notably, no differences were observed

between KB and MCF7 cytotoxicity profiles, suggesting that

the products do not have cell-specific cytotoxicity at concen-

trations typically used in biological investigations. These results

demonstrate that compound 38 possesses the prerequisites

for cell culture studies as well as for pharmacokinetics and bio-

distribution investigations.

Scheme 6 shows the conjugation of compound 38 to pullulan,

which was preliminarily oxidized with sodium periodate ac-

[

22]

cording to a reported protocol in order to introduce alde-

hyde groups along the polysaccharide chain 39 (Scheme 6A).

Potentiometric analyses showed that the oxidation reaction

yielded 27Æ1.9% oxidized glucose units. The amino PQone 38

was conjugated to the aldehyde groups of the oxidized pullu-

lan to form Schiff bases (compound 40), which were stabilized

under amino-reductive conditions using sodium cyanoborohy-

dride to obtain compound 41. Finally, derivative 41 was treat-

ed with sodium borohydride to quench the unreacted alde-

hyde groups and yield the desired conjugate compound 42

(Pull–38).

Cell uptake of compound 38 and AMCA was investigated

and compared by fluorescence confocal microscopy using

MCF7 cells. The results obtained by fluorescence microscopy

show that compound 38 is taken up by cells to a greater

extent than AMCA (Figure S33, SI). These results were con-

firmed by confocal microscopy. The images presented in

Figure 4 show that a significant amount of compound 38 is

taken up by MCF7 cells, and the extent of cell labeling was

higher than that obtained using equivalent concentrations of

AMCA. Furthermore, the confocal images presented in Figure 4

show that both AMCA and compound 38 were distributed in

the cytosol.

After purification by precipitation and dialysis, spectrometric

analysis was performed to determine the degree of pullulan

derivatization with compound 38. The reaction yield was

À1

found to be 86 mol%mol corresponding to 37.2 mg of com-

pound 38 per milligram of conjugate and 3.5% of the alde-

hydes being derivatized with compound 38. Therefore, consid-

ering that the molecular weight of the oxidized pullulan was

8

5 kDa, 10 molecules of compound 38 were conjugated per

pullulan chain.

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1853

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

Scheme 6. Functionalization of pullulan with the conjugate amino compound 38 to yield compound 42. A) pullulan oxidation; B) conjugation to oxidized pul-

lulan. Reagents and conditions: a) NaIO , H O, 1 h, then mannitol, RT; b) 38, H O, 1n HCl, 24 h, RT; c) NaCNBH , 24 h, RT; d) NaBH , 1 h, RT.

4 2 2 3 4

Figure 6. Uptake of Pull–38 in MCF7 cells at various concentrations. Values

represent the mean ÆSD of at least three independent experiments.

Figure 5. Cytotoxicity of Pull–38 (^), native pullulan (

) and PullOx (~) in

&

MCF7 cells assessed by MTT assay. Percentage viability values represent the

mean ÆSD of at least three independent experiments.

38 is localized in the cytosol. The discrete fluorescent spots

seem to indicate that the conjugate concentrated in specific

subcellular sites. The cell uptake studies performed by fluores-

cence and confocal microscopy showed that similar to AMCA,

compound 42 can easily diffuse into the cell and distribute in

the cytosol.

Biological properties of Pull–38

The cytotoxicity of Pull–38, native pullulan, and oxidized pullu-

lan (PullOx) was comparatively investigated using MCF7 cells.

The results presented in Figure 5 show that all pullulan deriva-

tives have low cytotoxicity, even at high concentrations. Solu-

tions of 29.4 mm resulted in ~70% cell viability.

Cell uptake studies were performed by incubating MCF7

cells with varying concentrations of 42. The results shown in

Figure 6 indicate that the cell uptake of Pull–38 increased as

the bioconjugate concentration increased. The experiments

performed with pullulan showed that compound 38 can also

be successfully used to label macromolecules, namely poly-

mers or supramolecular systems, to enable monitoring of cell

trafficking and quantitative analysis.

Conclusions

In this study, a group of pyrrolo[3,2-f]quinoline fluorochrome-

based derivatives was synthesized and systematically charac-

terized to identify compounds with spectral properties superior

[

18]

to that of the parent PQ

for the subsequent design of

probes that better match the instrumental and analytical re-

quirements of biological applications (e.g., optimal excitation

wavelength, efficient fluorescence detection).

Confocal microscopy confirmed the high degree of uptake

of Pull–38. The images presented in Figure 7 show that Pull–

PQ and Et-PQ compounds did not show interesting spectro-

metric properties. Therefore, the study was focused on the syn-

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1854

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

H, N elemental analyzer model

2

40B, and analyses indicated by

the symbols of the elements were

within Æ0.4% of the theoretical

values. Mass spectra were ob-

tained on a Mat 112 Varian Mat

Bremen (70 eV) mass spectrometer

and Applied Biosystems Mariner

System 5220 LC–MS (nozzle poten-

tial 140 eV). Column flash chroma-

tography was performed on Merck

silica gel (250–400 mesh ASTM); re-

actions were monitored by analyti-

cal thin-layer chromatography

Figure 7. Confocal microscopy images of a) untreated MCF7 cells b) MCF7 cells treated with wheat germ aggluti-

nin–Alexa Fluor 633 conjugate for membrane staining, and c) cells treated with the membrane stain and incubat-

ed for 24 h with Pull–38.

(

TLC) on Merck silica gel 60 F₂₅₄

glass plates. Solutions were con-

centrated by rotary evaporation

thesis of novel PQ derivatives and evaluation of their photo-

physical properties. The introduction of small functional

groups on the PQ scaffold led to new derivatives with im-

proved QYs and brightness over the parent molecule. PQones

under reduced pressure. Starting materials were purchased from

Sigma–Aldrich and Alfa Aesar, and solvents from Carlo Erba, Fluka,

and Lab-Scan. DMSO was obtained anhydrous by distillation under

vacuum and stored on molecular sieves. AMCA was purchased

from Sigma–Aldrich. The purity of new tested compounds was

checked by HPLC using a Varian ProStar model 210 instrument

equipped with a DAD Varian ProStar 335 detector. Analyses were

22 and 25, which showed high QY and brightness, were further

modified at position 3 by the attachment of a carboxyl- (31

and 32) or amino- (36–38) terminated six-carbon chain suitable

for chemical conjugation and labeling purposes. The amine de-

rivative 38 was subsequently chosen to label pullulan, a macro-

molecule used in drug delivery selected to investigate the use

of this dye in biological studies. The results indicated that due

to its very high blue fluorescence efficiency, stability, bright-

ness and with the presence of an amine group suitable for

facile chemical coupling to a variety of chemical moieties, com-

pound 38 might be an alternative to conventional small-mole-

cule fluorescent labels. Moreover, it should be emphasized that

the synthesis of the compounds described herein involves con-

ventional laboratory reactions that require mild operation con-

ditions, inexpensive and readily available reagents of technical

grade, and give high yields of reasonably pure products.

In conclusion, we have demonstrated that the PQ core,

when appropriately tailored, is a useful and adaptable scaffold

for easily obtaining small molecules that can be exploited as

blue fluorescent dyes and could have broad applicability in

biological studies.

À1

performed at a flow rate of 1 mLmin , with a C₁₈column (250

4

.6 mm, particle size: 5 mm) and with a loop of 10 mL. The detector

was operated at l 300 nm. The mobile phase consisted of phase A

(Milli-Q H O 18.0 MW, 0.05% TFA), and phase B (95% MeCN, 5%

2

phase A). Gradient elution was performed as follows: 0 min, 10%

B; 0–20 min, 90% B; 20–25 min, 90% B; 25–26 min, 10% B; 26–

3

1 min, 10% B. Pullulan (100 kDa, M /M 2.03), sodium borohy-

w n

dride (NaBH ), sodium cyanoborohydride (NaCNBH ), mannitol,

4

3

sodium periodate (NaIO ), and all solvents were purchased from

4

Sigma Chemical Co. (St. Louis, MO, USA). Tissue culture products

and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) were supplied from Sigma Chemical Co., and wheat germ

agglutinin–Alexa Fluor 633 conjugate was purchased from Life

Technologies (Carlsbad, CA, USA).

Synthesis of fluorophores

General procedure for the synthesis of 1N-substituted 5-nitro-

indoles 1–3: In a dried 100 mL flask, excess NaH (60% dispersion

in mineral oil) was placed and washed three times with toluene.

Via a funnel, commercial 5-nitroindole dissolved in 5 mL anhydrous

DMF was added dropwise, and the mixture changed from light

yellow to red with the development of hydrogen fumes. The reac-

tion mixture was left stirring for 1 h at room temperature. Then,

the halo-compound (twofold molar excess over starting indole) dis-

solved in 1 mL anhydrous DMF was added, and the solution was

left stirring at room temperature for 5 h, monitoring reaction prog-

Experimental Section

Materials and methods

Melting points were determined on a Büchi Melting Point M-560

capillary melting point apparatus, and are uncorrected. Infrared

spectra were recorded on a PerkinElmer 1760 FTIR spectrometer

ress by TLC (toluene/nHex/EtOAc, 1:1:1). At the end, H O (20–

2

5 mL) was added to the reaction mixture, which was then extract-

À1

with KBr pressed disks; all values are expressed in cm . UV/Vis

ed with n-butanol. The combined extracts were washed several

spectra were recorded on a Thermo Helyos a spectrometer.

times with H O, dried with anhydrous MgSO , filtered and finally

1

2

4

H NMR spectra were determined on Bruker 300 and 400 MHz

evaporated to dryness by rotary evaporation, obtaining a yellow

semisolid residue which was then triturated with cyclohexane. The

powdery solid was collected and dried under vacuum at room

temperature.

spectrometers, with the solvents indicated; chemical shifts are re-

ported in d (ppm) downfield from tetramethylsilane as internal ref-

erence. Coupling constants are given in Hertz. In the case of mul-

tiplets, chemical shifts were measured starting from the approxi-

mate center. Integrations were satisfactorily in line with those ex-

pected on the basis of compound structure. Elemental analyses

were performed in the Microanalytical Laboratory, Department of

Pharmaceutical Sciences, University of Padova, on a PerkinElmer C,

Ethyl 7-(5-nitro-1H-indol-1-yl)heptanoate (1): Following the gen-

eral procedure, reacting 5-nitroindole (1.5 g, 9.25 mmol), NaH

(0.666 g, 27 mmol), ethyl 7-bromoheptanoate (3.658 mL,

À1

18.51 mmol, d=1.217 gmL ), 1 was obtained as a yellow solid

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1855

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

(

2.5 g, 85%); R =0.75 (EtOAc/nHex/toluene, 1:1:1); mp: 89–908C;

(dd, J=2.95 Hz and J=0.54 Hz, 1H, H-1), 7.95 (dd, J=8.96 Hz and

J=0.71 Hz, 1H, H-4), 7.95 (m, 1H, H-7), 11.71 ppm (d, J=4.64 Hz,

1H, NH); C{ H} NMR (101 MHz, [D ]DMSO): d=19.52 (NCH CH CN),

6 2 2

f

1

H NMR (400 MHz, [D ]DMSO): d=8.57 (d, J=2.20 Hz, 1H, H-4),

6

13

1

8

7

4

2

.02 (dd, J=9.06, 2.34 Hz, 1H, H-6), 7.70 (d, J=9.16 Hz, 1H, H-7),

.66 (d, J=3.24 Hz, 1H, H-2), 6.75 (dd, J=3.31, 0.76 Hz, 1H, H-3),

.25 (t, J=7.08 Hz, 2H, N-CH -), 4.02 (q, J=7.12 Hz, 2H, O-CH CH ),

41.81 (NCH CH CN), 104.83 (C1), 110.08 (C8), 112.53 (C5), 115.89

2

(C4), 119.19 (CN), 119.24 (C9a), 123.95 (C9b), 128.99 (C2), 131.40

2

3

.23 (t, J=7.34 Hz, 2H, -CH -COO-), 1.76 (quint, J=7.16 Hz, 2H,

(C3a), 136.53 (C5a), 139.09 (C7), 178.28 ppm (C9); UV/Vis (H O),

2

NCH -CH -), 1.46 (quint, J=7.64 Hz, 2H, CH -CH COO-), 1.25 (m, 4H,

l_m_a_x=330.09 (A=0.121), 280.86 (A=0.173), 250.47 (A=0.209),

210.25 nm (A=0.3368); l_m_i_n=297.94 (A=0.053), 260.60 (A=0.103),

2

N(CH ) -CH -CH (CH ) COO-), 1.15 ppm (t, J=7.12 Hz, 3H,

2

1

2

1

2 2

3

OCH CH ); C{ H} NMR (101 MHz, [D ]DMSO): d=14.6 (OCH CH ),

231.05 nm (A=0.166); IR (KBr): n˜ =3422.51 (NÀH), 2247.89 (CꢀN),

2

3

6

2

3

À1

2

4.7 (CH -CH COO-), 26.2 (N(CH ) -CH CH (CH ) COO-), 28.4

1617 cm

(C=O); Fluorescence (H O): l =212.03 nm, l =

2 exc em

2

2 2

2-

2

2 2

+

(

N(CH ) CH CH -(CH ) COO-), 30.1 (NCH -CH -), 33.8 (-CH -COO-),

426.06 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]

calcd for

2

2-

2

2 2

2

+

4

6.3 (NCH -), 60.1 (OCH CH ), 104.0 (C3), 110.8 (C7), 116.7 (C6),

C H N O : 238.0980, found: 238.1002.

2

3

14 12

3

1

18.0 (C4), 127.73 (C3a), 133.0 (C2), 139.1 (C7a), 141.1 (C5),

73.3 ppm (COO-Et).

3

-Ethyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (11): Following the

general procedure, reacting compound 9 (0.745 g, 2.12 mmol) and

3

-(5-Nitro-1H-indol-1-yl)propanenitrile (2): Following the general

after purification (liquid column chromatography) gave compound

procedure, reacting 5-nitroindole (621 g, 10 mmol), NaH (0.720 g,

11 as a light-brown solid (0.626 g, 84%); R =0.35 (high blue fluo-

rescent spot, EtOAc/MeOH, 8:2); mp: 3008C (dec); H NMR

f

1

3

1

0

0 mmol),

3-bromopropionitrile

(1.24 mL,

15 mmol,

d=

À1

.615 gmL ), 2 was obtained as a yellow solid (2.12 g, 99%); R =

.27 (toluene/nHex/EtOAc, 1:1:1); mp: 97–988C; H NMR (400 MHz,

(400 MHz, [D ]DMSO): d=1.39 (t, J=7.10 Hz, 3H, -CH CH ), 4.30 (q,

f

6

2

3

1

J=7.10 Hz, 2H -CH CH ), 6.08 (d, J=7.24 Hz, 1H, H-8), 7.31 (d, J=

2 3

[

D ]DMSO): d=3.10 (t, 2H, J=6.60 Hz, N-CH -CH -CN), 4.61 (t, 2H,

8.82 Hz, 1H, H-5), 7.49 (d, J=2.92 Hz, 1H, H-2), 7.54 (dd, J=2.92 Hz

and J=0.67 Hz, 1H H-1), 7.82 (d, J=7.24 Hz, 1H H-7), 7.85 (dd, J=

6

2

J=6.52 Hz, N-CH -CH -CN), 6.81 (dd, 1H, J=3.24, 0.84 Hz, H-3), 7.72

2

(

0

d, 1H, J=3.31 Hz, H-2), 7.83 (dt, 1H, J=9.13, 0.79 Hz, H-7), 8.06

ddd, 1H, J=9.08, 2.29, 0.25 Hz, H-6), 8.59 ppm (dd, 1H, J=2.24,

8.82 Hz and J=0.67 Hz, 1H, H-4), 11.72 ppm (s , 1H, NH);

C{ H} NMR (101 MHz, [D ]DMSO): d=16.37 (-CH CH ), 40.97

6 2 3

(CH CH ), 104.05 (C1), 109.97 (C8), 112.14 (C5), 115.77 (C4), 119.26

2 3

b

13

1

3

1

.25 Hz, H-4); C{ H} NMR (101 MHz, [D ]DMSO): d=19.1 (NCH -

6

2

CH -CN), 42.1 (NCH -CH -CN), 104.8 (C3), 111.1 (C7), 117.0 (C6),

(C9a), 123.78 (C9b), 128.44 (C2), 131.22 (C3a), 136.30 (C5a), 136.90

2

1

118.0 (C4), 119.1 (CN), 132.8 (C2), 132.8 (C3a), 139.1 (C7a),

(C7), 178.30 ppm (C9); H NMR (400 MHz, [D ]DMF): d=1.47 (t, J=

7

1

41.5 ppm (C5).

7.25 Hz, 3H, -CH CH ), 4.43 (q, J=7.25 Hz, 2H, -CH CH ), 6.46 (d, J=

2

3

2

3

7

1

8

.13 Hz, 1H, H-8), 7.52 (d, J=8.89 Hz, 1H, H-5), 7.60 (d, J=2.91 Hz,

6

-(5-Nitro-1H-indol-1-yl)hexanenitrile (3): Following the general

H, H-2), 7.70 (d, J=2.90 Hz, 1H, H-1), 8.01 (d, J=8.82 Hz, 1H, H-4),

procedure, reacting 5-nitroindole (1.6 g, 9.8 mmol), NaH (0.696 g,

13

1

.11 (d, J=7.19 Hz, 1H, H-7), 12.49 ppm (s , 1H, NH); C{ H} NMR

b

2

1

0

9 mmol), 6-bromohexanenitrile (1.69 mL, 12.8 mmol, d=

.328 gmL ), 3 was obtained as a yellow solid (2.47 g, 98%); R =

.37 (toluene/nHex/EtOAc, 1:1:1); mp: 918C; H NMR (400 MHz,

(

101 MHz, [D ]DMF): d=15.61 (-CH CH ), 40.92 (CH CH ), 104.29

7 2 3 2 3

C1), 108.98 (C8), 111.96 (C5), 116.10 (C4), 123.21 (C9b), 128.26 (C2),

À1

f

1

30.08 (C9a), 131.49 (C3a), 136.65 (C5a), 137.53 (C7), 178.21 ppm

[

D ]DMSO): d=8.57 (d, J=2.16 Hz, 1H, H-4), 8.02 (dd, J=9.10,

À1

6

(

C9); IR (KBr): n˜ =3430 (NÀH), 1610 cm (C=O); Fluorescence (H O):

2

1

2

7

.34 Hz, 1H, H-7), 7.71 (d, J=9.16 Hz, 1H, H-6), 7.67 (d, J=3.20 Hz,

H, H-2), 6.76 (dd, J=3.21, 0.77 Hz, 1H, H-3), 4.28 (t, J=7.08 Hz,

H, N-CH -), 2.46 (t, J=7.08 Hz, 2H, CH -CN), 1.80 (quint, J=

+

l =217.96 nm, l =436.96 nm; HRMS (ESI, 140 eV), m/z [M+H]

ex

em

+

calcd for C H N O : 213.1028, found: 213.0997.

13

2

.46 Hz, 2H, NCH CH CH CH CH CN), 1.56 (quint, J=7.68 Hz, 2H,

Synthesis of 3-ethyl-6-methyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-

one (12): Method A. In a 50 mL two-neck flask, 0.113 g (4.71 mmol)

of NaH were placed and activated with toluene (three times). Then

3-ethylpyrroloquinolinone 11 (0.25 g, 1.18 mmol) in 15 mL anhy-

drous DMF was dropped into the flask. After 45 min the flask was

2

NCH CH CH CH CH CN), 1.32 ppm (quint, J=7.76 Hz, 2H,

NCH CH CH CH CH CN); C{ H} NMR (101 MHz, [D ]DMSO): d=16.5

2

1

3

1

2

6

(

NCH CH CH CH CH CN), 24.8 (NCH CH CH CH CH CN), 25.7

2 2 2 2 2 2 2 2 2 2

NCH CH CH CH CH CN), 29.5 (NCH CH CH CH CH CN), 46.1

NCH CH CH CH CH CN), 104.1 (C3), 110.83 (C6), 116.8 (C7), 118.04

C4), 121.1 (CN), 127.7 (C3a), 133.0 (C2), 139.10 (C7a), 141.1 ppm

C5).

2

placed in an ice bath and at 08C CH I (0.110 mL, 4.71 mmol) in

2

3

1 mL anhydrous DMF was added to the mixture. The reaction mix-

ture was stirred at room temperature, monitoring the course of

the reaction by TLC analysis (EtOAc/MeOH, 8:2). At the end of reac-

tion (1 h), the solution was cooled, treated with 15 mL H O and ex-

tracted with EtOAc (310 mL). The combined extracts were

washed with H O, dried with anhydrous Na SO and finally the sol-

General procedure for the synthesis of 3N-substituded pyrrolo-

quinolinones 10 and 11: Boiling Ph O (~50 mL) was quickly added

to a flask containing indole derivative 8 or 9 (3.50 mmol). The reac-

tion mixture was maintained at boiling for ~15 min and then

cooled to room temperature. Subsequently, Et O (20 mL) was

added to the mixture and the formation of a precipitate was ob-

served which was allowed to deposit at room temperature over-

night. It was then collected, washed several times with Et O and

dried in a vacuum oven.

2

4

vent was removed by rotary evaporation, yielding 0.075 g of

a dark-brown solid of a two-compound mixture: 12 (60%) and 13

2

(

40%). Method B. The same procedure as for the method A was fol-

lowed using THF as solvent: 0.250 g was reacted and 0.213 g of

a yellow solid was obtained that was a single compound only.

2

3

-Ethyl-6-methyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (12): Yield:

3

-(9-Oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)propanenitrile (10):

Following the general procedure, reacting compound 8 (0.632 g,

.07 mmol) and after purification (liquid column chromatography)

gave compound 10 as a light-brown solid (0.393 g, 63%); R =0.70

8

1

0%; R =0.7 (high blue fluorescent spot, EtOAc/MeOH 8:2); mp:

388C; H NMR (400 MHz, [D ]DMSO): d=1.40 (t, J=7.21 Hz, 3H,

f

1

6

2

CH CH ), 3.89 (s, 3H, N(quinolinic)CH ), 4.34 (q, J=7.21 Hz, 2H,

CH CH ), 6.08 (d J=7.57 Hz, 1H, H-8), 7.43 (d, J=9.14 Hz, 1H, H-5),

2

3

f

2

3

(

high blue fluorescent spot, EtOAc/MeOH, 8:2); mp: 2648C;

H NMR (400 MHz, [D ]DMSO): d=3.07 (t, J=6.51 Hz, 2H,

7

.55 (d, J=2.92 Hz, 1H, H-2), 7.64 (dd, J=2.92, 0.83 Hz, 1H, H-1),

.91 (d, J=7.57 Hz, 1H, H-7), 7.94 ppm (dd, J=9.14, 0.83 Hz, H-4);

1

6

NCH CH CN), 4.61 (t, J=6.53 Hz, 2H, NCH CH CN), 6.09 (m, 1H, H-

13

1

2

C{ H} NMR (101 MHz, [D ]DMSO): d=16.4 (CH CH ), 41.0 (CH CH ),

6

2

3

2

3

8

), 7.33 (d, J=8.87 Hz, 1H, H-5), 7.54 (d, J=3.00 Hz, 1H, H-2), 7.58

4

1.4 (N(quinolinic)CH ), 104.2 (C1), 109.9 (C8), 110.2 (C5), 116.0 (C4),

3

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1856

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

1

19.9 (C9a), 124.4 (C9b), 129.2 (C2), 132.0 (C3a), 136.9 (C5a), 142.6

column chromatography) 15 was obtained as a dark-brown oil

À1

(

C7), 177.9 ppm (C9); IR (KBr): n˜ =3419.96 (OÀH), 1616.60 cm (C=

(0.530 g, 62%); R_f=0.74 (toluene/EtOAc/cyclohexane, 1:1:1);

+

1

O); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O :

H NMR (400 MHz, [D ]DMSO): d=10.25 (s, 1H, NH), 7.45 (d, J=

1

4

15

2

6

2

27.1184, found: 227.1120.

8.79 Hz, 1H, H-7), 7.40 (d, J=3.08 Hz, 1H, H-2), 7.35 (d, J=1.89 Hz,

1

H, H-4), 6.94 (dd, J=8.64, 2.05 Hz, 1H, H-6), 6.39 (d, J=3.06 Hz,

H, H-3), 4.60 (d, J=0.51 Hz, 1H, C=C-H), 4.14 (t, J=6.96 Hz, 2H,

Synthesis of 3-ethyl-9-methoxy-3H-pyrrolo[3,2-f]quinoline (13):

0

In a 50 mL flask, excess Na (0.200 mg, 8.70 mmol) was dissolved in

NCH -), 4.03 (q, J=7.27 Hz, 2H, COOCH CH ), 2.23 (t, J=7.30 Hz,

2

CH CH -), 1.47 (quint, J=7.11 Hz, 2H, CH -CH -COO-), 1.24 (m, 4H,

N-(CH ) -CH CH -(CH ) -COO-),

0

2

3

1

5 mL anhydrous MeOH and kept in an ice bath until the Na was

H, CH -COOEt), 1.89 (s, 3H, C-CH ), 1.72 (quint, J=6.90 Hz, 2H, N-

2 3

fully dissolved. Then, 9-chloropyrroloquinoline 14 (0.148 g,

.46 mmol) in anhydrous MeOH (10 mL) was added dropwise, and

2

0

1.18

(t,

J=7.03 Hz,

3H,

2

2 2

the solution was maintained at reflux for 48 h, checking reaction

progress by TLC (EtOAc/MeOH, 8:2). As the reaction did not reach

CH COOCH CH ), 1.15 ppm (t, J=7.03 Hz, 3H, OCH CH );

2

3

2

3

13

1

C{ H} NMR (101 MHz, [D ]DMSO): d=14.6 (OCH CH ), 15.0

6

2

3

completion, H O was added, MeOH was removed by rotary evapo-

2

(

OCH CH ), 20.3 (CCH ), 24.9 (CH -CH -COO-), 26.3 (N(CH ) -CH CH -

2 3 3 2 2 2 2 2 2

ration, and the residue was extracted with EtOAc. The combined

extracts were dried over anhydrous Na SO and concentrated to

(

CH ) -COO-), 28.4 (N(CH ) -CH CH -(CH ) -COO-), 30.1 (NCH CH -),

2 2 2 2 2 2 2 2 2 2

2

4

3

8

3.8 (-CH -COO-), 46.0 (NCH -), 58.4 (OCH CH ), 60.1 (OCH CH ),

2 2 2 3 2 3

4.0 (C=C-H), 101.0 (C3), 110.6 (C7), 117.61 (C4), 119.7 (C6), 128.7

dryness obtaining a crude yellow oil (0.151 g) that was purified by

silica gel column chromatography (eluent EtOAc/MeOH, 9.5:0.5),

(

C3a), 130.3 (C2), 130.8 (C7a), 134.2 (C5), 161.0 (C=C-H), 170.0 (C=C-

yielding 0.089 g of pure yellow oil. Yield: 85%; R =0.51 (green flu-

f

1

COOEt), 173.3 ppm (COO-Et).

orescent spot, EtOAc/MeOH, 8:2); mp: 1958C; H NMR (400 MHz,

[

4

(

9

D ]DMSO): d=1.40 (t, J=7.12 Hz, 3H, CH CH ), 4.22 (s, 3H, OCH ),

.34 (q, J=7.12 Hz, 2H, CH CH ), 7.08 (d, J=5.24 Hz, 1H, H-8), 7.21

2 3

(E,Z)-Ethyl 3-(1-(2-cyanoethyl)-1H-indol-5-ylamino)but-2-enoate

(16): Following the general procedure, reacting compound 5

(1.180 g, 6 mmol), after purification (liquid column chromatogra-

phy) compound 16 was obtained as a brown semisolid product

6

2

3

dd, J=2.94, 0.71 Hz, 1H, H-1), 7.52 (m, 1H, H-2), 7.67 (d, J=

.06 Hz, 1H, H-5), 7.96 (dd, J=9.06, 0.71 Hz, 1H, H-4), 8.61 ppm (d,

1

3

1

J=5.24 Hz, 1H, H-7); C{ H} NMR (101 MHz, [D ]DMSO): d=16.5

(

(0.900 g, 50%); R_f=0.43 (toluene/EtOAc/cyclohexane, 1:1:1);

6

1

CH CH ), 39.8 (CH CH ), 56.2 (OCH ), 101.8 (C8), 105.35 (C1), 115.7

H NMR (400 MHz, [D ]DMSO): d=10.27 (s, 1H, NH), 7.57 (d, J=

2

3

2

3

6

C9a), 115.8 (C4), 122.7 (C9b), 122.71 (C5), 129.0 (C2), 131.6 (C3a),

3.27 Hz, 1H, H-2), 7.47 (d, J=9.14 Hz, 1H, H-7), 7.38 (d, J=1.68 Hz,

1H, H-4), 6.98 (dd, J=9.14, 2.02 Hz, 1H, H-6), 6.46 (d, J=3.15 Hz,

1H, H-3), 4.62 (s, 1H, C=C-H), 4.49 (t, J=6.49 Hz, 2H, NCH CH CN),

À1

1

46.59 (C5a), 148.0 (C7), 163.2 ppm (C9); IR (KBr): n˜ =2929.41 cm

;

+

HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O : 227.1184,

1

4

15

2

found: 227.1117.

4.05 (q, J=7.08 Hz, 2H, OCH CH ), 3.03 (t, J=6.51 Hz, 2H,

2 3

NCH CH CN), 1.91 (s, 3H, CH ), 1.19 ppm (t, J=7.06 Hz, 3H,

2

3

Synthesis of 9-chloro-3-ethyl-3H-pyrrolo[3,2-f]quinoline (14): In

a 10 mL flask pyrroloquinolinone 11 (0.200 g, 0.94 mmol) and

OCH CH ).

2

3

POCl (3 mL) were mixed. The solution was heated (oil bath) and

maintained at reflux for 12 h, checking the progress of the reaction

(E,Z)-Ethyl 3-(1-(5-cyanopentyl)-1H-indol-5-ylamino)but-2-enoate

(17): Following the general procedure, reacting compound 6

(1.890 g, 8.32 mmol), after purification (liquid column chromatogra-

3

by TLC (EtOAc/MeOH, 8:2). At the end of reaction, 10 mL H O were

2

added, and the solution was made alkaline with 20% NH OH and

phy) compound 17 was obtained as a brown semisolid product

4

1

then extracted with EtOAc. The organic phase was dried over

Na SO and evaporated to dryness under vacuum, obtaining

(1.97 g, 70%); R =0.66 (toluene/EtOAc/cyclohexane, 1:1:1); H NMR

f

(400 MHz, [D ]DMSO): d=10.26 (s, 1H, NH), 7.47 (d, J=8.67 Hz, 1H,

2

4

6

0

.148 g of a dark oil. Yield 68%; R =0.91 (green fluorescent spot,

H-7), 7.41 (d, J=3.07 Hz, 1H, H-2), 7.35 (d, J=1.77 Hz, 1H, H-4),

6.95 (dd, J=8.28, 1.92 Hz, 1H, H-6), 6.40 (d, J=2.98 Hz, 1H, H-3),

4.61 (s, 1H, C=C-H), 4.17 (t, J=7.03 Hz, 2H, NCH CH CH CH CH CN),

f

1

EtOAc/MeOH, 8:2); mp: 1548C; H NMR (400 MHz, [D ]DMSO): d=

6

1

7

.42 (t, J=7.22 Hz, 3H, CH CH ), 4.40 (q, J=7.20 Hz, 2H, CH CH ),

2 3 2 3

.54 (dd, J=3.17, 0.77 Hz, 1H, H-1), 7.63 (d, J=3.17 Hz, 1H, H-2),

.69 (d, J=4.80 Hz, 1H, H-8), 7.80 (d, J=9.12 Hz, 1H, H-5), 8.09 (dd,

2

4.07 (q, J=6.97 Hz, 2H, OCH CH ), 2.46 (t, J=7.09 Hz, 2H,

2 3

NCH CH CH CH CH CN), 1.89 (s, 3H, C-CH ), 1.77 (quint, J=7.35 Hz,

2

3

J=9.17, 0.77 Hz, 1H, H-4), 8.68 ppm (d, J=4.80 Hz, 1H, H-7);

2H, NCH CH CH CH CH CN), 1.56 (quint, J=7.51 Hz, 2H,

2 2 2 2 2

13

1

C{ H} NMR (101 MHz, [D ]DMSO): d=16.4 (CH CH ), 41.3 (CH CH ),

NCH CH CH CH CH CN),

1.34

(quint,

J=7.05 Hz,

2H,

6

2

3

2

3

2

1

05.3 (C1), 116.9 (C4), 127.7 (C2), 120.2 (C9b), 121.2 (C9a), 122.2

NCH CH CH CH CH CN), 1.18 ppm (t, J=7.08 Hz, 3H, OCH CH );

C{ H} NMR (101 MHz, [D ]DMSO): d=14.5 (OCH CH ), 16.0

6 2 3

(NCH CH CH CH CH CN), 19.8 (CCH ), 24.3 (NCH CH CH CH CH CN),

2 2 2 2 2 3 2 2 2 2 2

2

3

13

1

(

C8), 123.6 (C5), 132.8 (C3a), 139.7 (C9), 146.6 (C7), 147.2 ppm

+

(

C5a); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H ClN

:

1

3

12

2

31.0689, found: 231.0696.

25.4 (NCH CH CH CH CH CN), 29.0 (NCH CH CH CH CH CN), 45.3

2 2 2 2 2 2 2 2 2 2

(

1

NCH CH CH CH CH CN), 60.4 (OCH CH ), 83.5 (C=C-H), 100.5 (C3),

2 2 2 2 2 2 3

10.2 (C7), 116.7 (C4), 119.2 (C6), 120.6 (CN), 128.2 (C3a), 130.4 (C2),

33.7 (C7a), 139.6 (C5), 160.5 (C=C-H), 169.5 ppm (COOEt).

General procedure for the synthesis of acrylate derivatives 15–

8: In a 100 mL round-bottomed flask, a mixture of 5-aminoindole

derivative (2 mmol) dissolved in 30 mL of absolute EtOH, five drops

1

of glacial acetic acid, ~0.100 g Drierite and ethyl acetoacetate

(E,Z)-Ethyl 3-(1-ethyl-1H-indol-5-ylamino)but-2-enoate (18): Fol-

lowing the general procedure, reacting compound 7 (1.570 g,

9 mmol), after purification (liquid column chromatography) gave

(

3 mmol) was heated at reflux and stirred for 18 h, checking the

progress of the reaction by TLC (nHex/toluene/EtOAc, 1:1:1). At the

end of the reaction, the mixture was filtered through a Celite pad,

and the filtrate was then evaporated to dryness by rotary evapora-

tion. Generally, a tarry dark liquid was obtained which was then

purified by silica gel column chromatography (eluent: nHex/tolu-

ene/EtOAc, 1:1:1).

compound 18 as a brown semisolid product (1.60 g, 60%); R =

f

1

0.76 (toluene/EtOAc/cyclohexane, 1:1:1);

H NMR (400 MHz,

[D ]DMSO): d=10.30 (s, 1H, NH), 7.44 (d, J=8.64 Hz, 1H, H-7), 7.40

6

(d, J=3.08 Hz, 1H, H-2), 7.36 (d, J=2.08 Hz, 1H, H-4), 6.95 (dd, J=

8.64, 2.12 Hz, 1H, H-6), 6.41 (dd, J=3.06, 0.76 Hz, 1H, H-3), 4.63 (d,

J=0.52 Hz, 1H, C=CH), 4.18 (q, J=7.21 Hz, 2H, CH CH ), 4.06 (q,

2

3

Ethyl

7-(5-((E,Z)-1-(ethoxycarbonyl)prop-1-en-2-ylamino)-1H-

J=7.08 Hz, 2H, OCH CH ), 1.90 (s, 3H, CH ), 1.35 (t, J=7.22 Hz, 3H,

2

3

indol-1-yl)heptanoate (15): Following the general procedure, re-

acting compound 4 (0.620 g, 2.15 mmol), after purification (liquid

13

1

CH CH ), 1.20 ppm (t, J=7.10 Hz, 3H, OCH CH ); C{ H} NMR

2

3

2

3

(

101 MHz, [D ]DMSO): d=15.0 (OCH CH ), 15.9 (CH CH ), 20.2

6 2 3 2 3

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1857

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

(

CCH ), 40.9 (CH CH ), 58.4 (OCH CH ), 84.1 (C=CH), 101.1 (C3),

5.8 mmol), after purification (liquid column chromatography) gave

3

2

3

2

3

110.4 (C6), 117.2 (C4), 119.7 (C7), 128.8 (C2), 129.5 (C3a), 130.9 (C8),

compound 22 as a yellowish solid (0.98 g, 74%); R =0.60 (high

f

1

33.9 (C7a), 160.9 (C), 170.0 ppm (COOEt).

blue fluorescent spot, CHCl /MeOH, 8:2); mp: 1388C (1518C dec);

3

1

H NMR (400 MHz, [D ]DMSO): d=11.46 (s, 1H, NH), 7.80 (dd, J=

6

General procedure for the synthesis of 7-methyl-pyrroloquinoli-

nones derivatives 19–22: Boiling Ph O (~15 mL) was quickly put

into a flask containing indole derivatives 19–20 (2.0–3.50 mmol).

The reaction mixture was maintained at boiling for ~15 min and

then cooled to room temperature. As a dark oily precipitate

formed, supernatant Ph O was removed, and the oily product was

dissolved in a minimal amount of MeOH and then added to 20 mL

Et O. The compound precipitated as a yellow powder that was col-

lected, washed several times with Et O and dried under vacuum.

8

2

.88, 0.7 Hz, 1H, H-4), 7.49 (dd, J=2.9, 0.7 Hz, 1H, H-1), 7.45 (d, J=

.9 Hz, 1H, H-2), 7.28 (d, J=8.88 Hz, 1H, H-5), 5.93 (s, 1H, H-8), 4.28

2

(

7

q, J=7.25 Hz, 2H, CH CH ), 2.35 (s, 3H, C-CH ), 1.38 ppm (t, J=

2

13

3

1

3

.25 Hz, 3H, CH CH ); C{ H} NMR (101 MHz, [D ]DMSO): d=16.3

2

3

6

(

CH CH ), 19.6 (CH ), 103.9 (C1), 109.8 (C8), 111.8 (C5), 115.4 (C4),

2 3 3

2

117.8 (C9a), 124.0 (C9b), 128.3 (C2), 131.2 (C3a), 136.3 (C5a), 146.8

(

1

C7), 178.3 ppm (C9); IR (KBr): n˜ =3401 (NÀH), 2865 (CÀH),

2

À1

+

616 cm (C=C); HRMS (ESI, 140 eV), m/z [M+H] calcd for

2

+

C H N O : 227.1184, found: 227.1178.

14

15

2

Ethyl 7-(7-methyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)hepta-

noate (19): Following the general procedure, reacting compound

General procedure for the synthesis of 6,7-dimethylpyrroloqui-

nolinone derivatives 23–25: To obtain the 6-methyl derivatives

1

6 (1.31 g, 3.27 mmol), after purification (liquid column chromato-

23–25, the same procedure described for compound 12 (Method B)

graphy) gave compound 19 as a beige solid (0.810 g, 70%); R =

f

was followed, using THF as solvent.

0

.23 (high blue fluorescent, spot CHCl /MeOH, 9:1); mp: 2788C;

3

1

H NMR (400 MHz, [D ]DMSO): d=11.44 (s, 1H, NH), 7.79 (dd, J=

Ethyl 7-(6,7-dimethyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)-

heptanoate (23): Following the general procedure, reacting com-

pound 19 (0.730 g, 2.0 mmol), after purification (liquid column

chromatography) compound 23 was obtained as a yellow solid

6

8

.90, 0.58 Hz, 1H, H-4), 7.48 (dd, J=2.89, 0.60 Hz, 1H, H-1), 7.43 (d,

J=2.92 Hz, 1H, H-2), 7.27 (d, J=8.84 Hz, 1H, H-5), 5.93 (d, J=

1

7

.04 Hz, 1H, H-8), 4.23 (t, J=6.98 Hz, 2H, N-CH -), 4.02 (q, J=

2

.12 Hz, 2H, O-CH CH ), 2.35 (s, 3H, C-CH ), 2.23 (t, J=7.32 Hz, 2H,

(0.530 g, 72%); R =0.54 (high blue fluorescent spot, EtOAc/MeOH,

2

3

f

1

-

CH -COO-), 1.76 (quint, J=7.26 Hz, 2H, N-CH -CH -CH CH CH -CH -

8:2); mp: 220–2228C; H NMR (400 MHz, [D ]DMSO): d=7.90 (d,

2

6

COO), 1.46 (quint, J=7.28 Hz, 2H, N-CH CH CH CH CH -CH -COO-),

J=9.30 Hz, 1H, H-4), 7.60 (d, J=2.88 Hz, 1H, H-2), 7.53 (d, J=

9.33 Hz, 1H, H-5), 7.40 (d, J=2.94 Hz, 1H, H-1), 6.09 (s, 1H, H-8),

4.32 (t, J=5.25 Hz, 2H, NCH (CH ) COOEt), 4.01 (q, J=7.12 Hz, 2H,

2

1

7

.24 (m, 4H, N-CH CH CH CH CH CH -COO-), 1.15 ppm (t, J=

2

3

2

1

.12 Hz, 3H, OCH CH ); C{ H} NMR (101 MHz, [D ]DMSO): d=14.6

2

3

6

2

2 5

(

OCH CH ), 19.6 (CH ), 24. (NCH CH CH CH CH -CH -COO-), 26.3

OCH CH ), 3.78 (s, 3H, N-CH ), 2.51 (s, 3H, C-CH ), 2.22(t, J=

2 3 3 3

7.24 Hz, 2H, (CH ) CH COOEt), 1.76 (quint, J=7.20 Hz, 2H,

2 5 2

2

3

2

NCH CH -CH CH CH CH -COO-), 28.4 (NCH CH -CH CH CH CH -

2

COO-),

30.5

(NCH -CH CH CH CH CH -COO),

33.8

CH CH (CH ) COOEt), 1.51–1.21 (m, 6H, (CH ) CH CH CH CH COOEt),

2 2 2 4 2 2 2 2 2 2

2

(

NCH CH CH CH CH CH -COO-), 46.0 (NCH -CH CH CH CH CH -

1.14 ppm (t, J=7.08 Hz, 3H, COOCH CH ); HRMS (ESI, 140 eV), m/z

2 3

[M+H] calcd for C H N O : 369.2178, found: 369.2154.

22 29 2 3

2

+

COO), 60.1 (OCH CH ), 103.8 (C1), 109.8 (C8), 111.8 (C5), 115.5 (C4),

2

3

1

17.7 (C9a), 123.7 (C9b), 129.0 (C2), 131.5 (C3a), 136.3 (C5a), 146.8

6

-(6,7-Dimethyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)hexane-

nitrile (24): Following the general procedure, reacting compound

1 (0.450 g, 1.53 mmol), after purification (liquid column chroma-

(

C7), 173.3 (COO-Et), 178.3 ppm (C9); HRMS (ESI, 140 eV), m/z [M+

+

H] calcd for C H N O : 355.2022, found: 355.1997.

2

1

27

2

3

2

3

-(7-Methyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)propaneni-

trile (20): Following the general procedure, reacting compound 16

0.74 g, 2.49 mmol), gave a mixture of the desired compound and

a compound lacking the nitrogen side chain as a beige solid

0.50 g, 40%).

tography) gave compound 24 as a yellow solid (0.460 g, 97%); R =

f

0.71 (high blue fluorescent spot, CHCl /MeOH, 8:2); mp: 208–

3

1

(

2098C; H NMR (400 MHz, [D ]DMSO): d=7.81 (d, J=9.27 Hz, 1H,

6

H-4), 7.61 (d, J=2.82 Hz, 1H, H-3), 7.54 (d, J=9.39 Hz, 1H, H-5),

7.50 (d, J=2.91 Hz, 1H, H-2), 6.10 (s, 1H, H-8), 4.29 (quint, J=

(

6

3

1

7

2

1

.96 Hz, 2H, NCH CH CH CH CH CN), 3.79 (s, 3H, N-CH ) 2.49 (s,

2 2 2 2 2 3

6

-(7-Methyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)hexaneni-

trile (21): Following the general procedure, reacting compound 17

1.5 g, 4.4 mmol), after purification (liquid column chromatography)

H, C-CH ), 2.44 (quint, J=7.05 Hz, 2H, NCH CH CH CH CH CN),

3

2

.80 (quint, J=7.47 Hz, 2H, NCH CH CH CH CH CN), 1.55 (quint, J=

2

(

.30 Hz, 2H, NCH CH CH CH CH CN), 1.31 ppm (quint, J=6.52 Hz,

2

13

2

1

gave compound 21 as a beige solid (0.815 g, 63%); R =0.34 (high

blue fluorescent spot, CHCl /MeOH, 9:1); mp: 2658C; H NMR

f

H, NCH CH CH CH CH CN); C{ H} NMR (101 MHz, [D ]DMSO): d=

1

2

6

3

6.5

(NCH CH CH CH CH CN),

21.9

(CCH₃),

24.8

2

(

400 MHz, [D ]DMSO): d=11.46 (s, 1H, NH), 7.81 (d, J=8.91 Hz, 1H,

6

(

NCH CH CH CH CH CN), 25.8 (NCH CH CH CH CH CN), 29.9

2 2 2 2 2 2 2 2 2 2

H-4), 7.48 (d, J=2.82 Hz, 1H, H-3), 7.44 (d, J=2.9.1 Hz, 1H, H-2),

(

NCH CH CH CH CH CN), 35.8 (NCH ), 45.8 (NCH CH CH CH CH CN),

2 2 2 2 2 3 2 2 2 2 2

7

6

.27 (d, J=8.88 Hz, 1H, H-5), 5.93 (s, 1H, H-8), 4.26 (quint, J=

.90 Hz, 2H, NCH CH2CH CH CH CN), 2.45 (quint, J=7.05 Hz, 2H,

1

04.3 (C1), 110.4 (C5), 111.9 (C8), 115.6 (C4), 119.0 (C9a), 121.1 (CN),

24.3 (C9b), 129.8 (C2), 130.5 (C3a), 131.7 (C5a), 149.9 (C7),

77.4 ppm (C9); HRMS (ESI, 140 eV), m/z [M+H] calcd for

2

CH -CN), 2.34 (s, 3H, C-CH₃), 1.79 (quint, J=7.40 Hz, 2H,

NCH CH CH CH CH CN),

NCH CH CH CH CH CN), 1.32 ppm (quint, J=7.76 Hz, 2H,

NCH CH CH CH CH CN); C{ H} NMR (101 MHz, [D ]DMSO): d=16.0

+

2

1.55

(quint,

J=7.59 Hz,

2H,

+

2

C H N O : 308.1763, found: 308.1758.

19

22

3

2

1

3

1

3-Ethyl-6,7-dimethyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one

(25):

2

6

(

NCH CH CH CH CH CN),

19.1

(CCH₃),

24.31

29.

Following the general procedure, reacting compound 22 (0.278 g,

1.2 mmol), and after purification (liquid column chromatography)

2

NCH CH CH CH CH CN),

25.4

(NCH CH CH CH CH CN),

2

(

1

NCH CH CH CH CH CN), 45.3 (NCH CH CH CH CH CN), 103.3 (C1),

09.3 (C8), 111.3 (C5), 115.0 (C4), 117.2 (C9a), 120.6 (CN), 123.2

gave compound 25 as a yellow solid (0.165 g, 56%); R =0.31 (high

2

f

1

blue fluorescent spot, CHCl /MeOH, 9:1); mp: 223.58C; H NMR

3

(

2

C9b), 128.5 (C2), 131.0 (C3a), 135.8 (C5a), 146.3 (C7), 177.8 ppm

(400 MHz, [D ]DMSO): d=7.90 (dd, J=9.26, 0.74 Hz, 1H, H-4), 7.62

(dd, J=2.95, 0.75 Hz, 1H, H-1), 7.54 (d, J=9.27 Hz, 1H, H-5), 7.51

(d, J=2.95 Hz, 1H, H-2), 6.06 (d, J=0.56 Hz, 1H, H-8), 4.32 (q, J=

6

+

C9); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O :

1

8

20

3

94.1606, found: 294.1599.

7

1

.21 Hz, 2H, CH CH ), 3.79 (s, 3H, N-CH ), 2.49 (s, 3H, C-CH ),

2 3 3 3

3

-Ethyl-7-methyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (22): Fol-

13

1

.39 ppm (t, J=7.22 Hz, 3H, CH CH ); C{ H} NMR (101 MHz,

2

3

lowing the general procedure, reacting compound 18 (1.60 g,

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1858

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

[

D ]DMSO): d=16.2 (CH CH ), 21.8 (CCH₃), 35.6 (NCH₃) 40.8

a brown solid (0.280 g, 76%); R =0.76 (green fluorescent spot,

EtOAc/MeOH, 8:2); mp: 252.28C (2808C dec); H NMR (400 MHz,

6

2

3

f

1

(CH CH ), 104.2 (C1), 110.2 (C4), 111.8 (C8), 115.3 (C5), 119.2 (C9a),

2

3

1

24.3 (C9b), 129.0 (C2), 131.3 (C3a), 137.8 (C5a), 149.2 (C6),

[D ]DMSO): d=8.46 (d, J=9.24 Hz, 1H, H-4), 8.15 (s, 1H, H-8), 8.07

6

À1

77.3 ppm (C9); IR (KBr): n˜ =2980 (CÀH), 1615 cm (C=C); HRMS

(d, J=9.08 Hz, 1H, H-5), 7.90 (d, J=3.20 Hz, 1H, H-2), 7.66 (dd, J=

3.14, 0.74 Hz, 1H, H-1), 4.49 (q, J=7.20 Hz, 2H, CH CH ), 2.89 (s,

+

(

ESI, 140 eV), m/z [M+H] calcd for C H N O : 241.1341, found:

15 17 2

2

3

1

2

41.1350.

3H, C-CH ), 1.45 ppm (t, J=7.20 Hz, 3H, CH CH ); C{ H} NMR

3 2 3

(

1

(

101 MHz, [D ]DMSO): d=16.4 (CH CH ), 31.1 (CCH ), 41.6 (CH CH ),

6 2 3 3 2 3

05.2 (C1), 112.1 (C9a), 112.1 (C5), 120.3 (C4), 120.3 (C9b), 123.9

Synthesis of 3-ethyl-9-methoxy-7-methyl-3H-pyrrolo[3,2-f]quino-

line (26): In a 10 mL flask compound 22 (0.498 g, 2.2 mmol) was

C8), 130.4 (C2), 132.2 (C3a), 133.1 (C5a), 152.6 (C7), 169.1 ppm

added to 3 mL POCl , and the solution was maintained at reflux

À1

3

C9); IR (KBr): n˜ =2971 (CÀH), 1369 cm (CꢀN); HRMS (ESI, 140 eV),

(

1308C) in an oil bath for 12 h, checking reaction progress by TLC

+

m/z [M+H] calcd for C H ClN : 245.0846, found: 245.0864.

14

2

EtOAc/MeOH, 8:2). At the end, H O (10 mL) was added to the re-

2

action mixture and the obtained solution, made basic with concen-

trated ammonia (27%), was extracted with EtOAc (325 mL). The

organic phase was dried over Na SO and then evaporated to dry-

Synthesis of 3-ethyl-N,7-dimethyl-3H-pyrrolo[3,2-f]quinolin-9-

amine (29): In a 50 mL flask, a mixture of 0.280 g (1.14 mmol) of

28 in anhydrous DMF and 0.21 mL methylamine (33 wt% in abso-

lute EtOH) was heated at reflux (oil bath) for 16 h, monitoring the

course of the reaction by TLC (EtOAc/MeOH, 8:2). At the end of

2

4

ness to give 9-chloro derivative as a dark oil. Then in a 50 mL flask,

0

.200 g Na (8.70 mmol) was dissolved in 15 mL anhydrous cold

MeOH. To the NaOMe solution, the chloro derivative dissolved in

the minimum amount of anhydrous MeOH was dropped and the

mixture was refluxed for 48 h, checking the progress of the reac-

tion by TLC (EtOAc/MeOH, 8:2). As the reaction did not reach com-

the reaction, the solution was treated with H O and then extracted

with EtOAc (325 mL). The organic phase was washed several

2

times with H O and then brought to dryness by rotary evaporation,

2

obtaining a dark solid which was purified by a silica gel column

chromatography using EtOAc/MeOH 8:2 as eluent. A dark solid

pletion, excess NaOMe was deactivated with H O (3 mL), MeOH

2

was removed under reduced pressure, and the resulting residue

was extracted with EtOAc (430 mL). The extracts were combined,

dried with anhydrous Na SO and evaporated to dryness by rotary

(0.270 g) was obtained. Yield: 98%; R =0.56 (EtOAc/MeOH, 8:2);

f

1

mp: 86.58C; H NMR (400 MHz, [D ]DMSO): d=8.20 (d, J=9.03 Hz,

6

1H, H-4), 7.86 (d, 3.06 Hz, 1H, H-2), 7.68 (d, J=9.09 Hz, 1H, H-5),

2

4

evaporation, obtaining a yellow oil. The crude product was purified

by silica gel column chromatography (eluent EtOAc/MeOH, 9.5:0.5)

7.55 (q, J=4.59 Hz, 1H, NH-CH ), 7.54 (d, J=3.21 Hz, 1H, H-1), 6.80

3

(s, 1H, H-8), 4.42 (q, J=7.21 Hz, 2H, CH CH ), 3.18 (d, J=4.62 Hz,

2

3

yielding 0.344 g of a yellow solid. Yield: 64.9%; R =0.23 (green flu-

3H, NH-CH ), 2.68 (s, 3H, C-CH ), 1.43 ppm (t, J=7.14 Hz, 3H,

3 3

CH CH ); C{ H} NMR (101 MHz, [D ]DMSO): d=16.3 (CH CH ), 20.2

2 3 6 2 3

(CCH ), 31.2 (NH-CH ), 41.5 (CH CH ), 99.5 (C8), 102.4 (C1), 109.9

3 3 2 3

f

13

1

orescent spot, CHCl /MeOH, 9:1), 0.64 (green fluorescent spot,

3

1

CHCl /MeOH, 8:2); mp: 1458C; H NMR (400 MHz, [D ]DMSO): d=

3

6

7

6

2

.87 (dd, J=9.04, 0.68 Hz, 1H, H-4), 7.57 (d, J=8.88 Hz, 1H, H-5),

.48 (d, J=2.92 Hz, 1H, H-2), 7.14 (dd, J=2.97, 0.73 Hz, 1H, H-1),

.96 (s, 1H, H-8), 4.34 (q, J=7.21, 2H, CH CH ), 4.09 (s, 3H, OCH ),

(C9a), 113.0 (C4), 118.4 (C5), 119.5 (C9b), 130.1 (C2), 132.2 (C3a),

145.9 (C5a), 150.5 (C7), 155.9 ppm (C9); IR (KBr): n˜ =3422 (NÀH),

À1

2971 (CÀH), 1617 (C=C), 1364 cm (CꢀN); HRMS (ESI, 140 eV), m/z

2

3

+

.61 (s, 3H, C-CH₃), 1.41 ppm (t, J=7.22 Hz, 3H, CH CH );

[M+H] calcd for C H N =240.1501, found: 240.1518.

15 18 3

2

3

1

3

1

C{ H} NMR (101 MHz, [D ]DMSO): d=16.4 (CH CH ), 25.5 (CCH ),

6

2

3

Synthesis of 3-ethyl-7-methyl-3H-pyrrolo[3,2-f]quinoline (30): A

50 mL dry two-neck flask containing 0.200 g Pd/C (10%) and

4

1.0 (CH CH ), 56.1 (OCH ), 101.8 (C8), 105.0 (C1), 113.4 (C9a), 115.1

2 3 3

2

(

C4), 120.1 (C9b), 122.3 (C5), 126.9 (C2), 131.6 (C3a), 146.1 (C5a),

À1

60 mL EtOAc was connected to a balloon containing hydrogen.

The reaction was allowed to stir at room temperature for 1 h so as

to saturate the suspension with hydrogen. Then, a solution of

1

55.9 (C7), 162.9 ppm (C9); IR (KBr): n˜ =2939 (CÀH), 1578 cm (C=

+

C); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O :

2

1

5

17

2

41.1341, found: 241.1312.

0

.791 g (3.2 mmol) of 28 in 10 mL EtOAc was added dropwise. The

Synthesis of 3-ethyl-9-methoxy-6,7-dimethyl-3H-pyrrolo[3,2-

f]quinolinium iodide (27): In a 50 mL flask, 0.300 g of 26 and 5 mL

reaction mixture was stirred in the presence of hydrogen at atmos-

pheric pressure and heated at 50–608C by means of an oil bath for

of CH I (1:25 molar ratio) were heated at reflux for 4 h, checking

12 h, monitoring the progress of the reaction by TLC (CHCl /MeOH,

3

the progress of the reaction by TLC (CHCl /MeOH, 1:1). At the end,

95:5). At the end of the reaction, the mixture was filtered through

a Celite pad to remove the catalyst, and the solution was evaporat-

ed to dryness by rotary evaporation, thereby obtaining 0.660 g of

orange–yellow solid. Yield: 97%; R =0.55 (CHCl /MeOH, 95:5);

3

the reaction mixture was concentrated to dryness and the ob-

tained solid residue (0.477 g) was recrystallized from a mixture of

nHex/toluene (80:20) to give bright-yellow needles (0.468 g). Yield:

f

3

1

9

2

0

1

8%; R =0.15 (high green fluorescent spot, CHCl /MeOH, 1:1); mp:

H NMR (400 MHz, [D ]DMSO): d=9.32 (d, J=8.60 Hz, 1H, H-9),

6

f

3

1

108C; H NMR (400 MHz, [D ]DMSO): d=8.44 (dd, J=9.46,

8.35 (d, J=9.00 Hz, 1H, H-4), 8.01 (d, J=9.24 Hz, 1H, H-5), 7.91 (d,

J=8.56 Hz, 1H, H-8), 7.80 (d, J=3.08 Hz, 1H, H-2), 7.36 (d, J=

3.04 Hz, 1H, H-1), 4.44 (q, J=7.18 Hz, 2H, CH CH ), 2.94 (s, 3H, C-

6

.74 Hz, 1H, H-4), 8.09 (d, J=9.56 Hz, 1H, H-5), 7.89 (d, J=3.00 Hz,

H, H-2), 7.69 (s, 1H, H-8), 7.38 (dd, J=3.01, 0.76 Hz, 1H, H-1), 4.49

2

3

+

13

1

(

q, J=7.28 Hz, 2H, CH CH ), 4.37 (s, 3H, OCH ), 4.31 (s, 3H, N

CH ), 1.44 ppm (t, J=7.28 Hz, CH CH ); C{ H} NMR (101 MHz,

3 2 3

2

3

-

CH ), 3.00 (s, 3H, C-CH ), 1.44 ppm (t, J=7.22 Hz, 3H, CH CH );

[D ]DMSO): d=16.5 (CH CH ), 20.6 (CCH ), 41.5 (CH CH ), 102.0 (C1),

6 2 3 3 2 3

3

1

3

2

3

13

C{ H} NMR (101 MHz, [D ]DMSO): d=16.5 (CH CH ), 23.2 (CCH ),

113.1 (C5), 120.0 (C4), 122.3 (C9b), 122.7 (C8), 122.8 (C9a), 130.40

(C2), 132.30 (C3a), 135.45 (C5a), 140.29 (C9), 153.12 ppm (C7); IR

6

2

3

+

3

1

9.4 (N -CH ), 41.5 (CH CH ), 58.6 (OCH ), 105.1 (C8), 105.6 (C1),

3 2 3 3

À1

11.2 (C5), 114.4 (C9a), 120.0 (C4), 120.7 (C9b), 131.3 (C2), 132.5

(KBr): n˜ =2962 (CÀH), 1638 (C=C), 1371 cm (CꢀN); HRMS (ESI,

+

(

C3a), 138.0 (C5a), 157.0 (C7), 167.5 ppm (C9); IR (KBr): n˜ =3428 (CÀ

140 eV), m/z [M+H] calcd for C H N : 211.1235, found:

14 15 2

À1

+

H), 1591 (C=C), 1355 cm (CꢀN); HRMS (ESI, 140 eV), m/z [M+H]

211.1286.

+

calcd for C H N O : 255.1492, found: 255.1500.

1

6

19

2

Synthesis of 7-(7-methyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-3(9H)-

yl)heptanoic acid (31): In a 50 mL flask containing 0.143 g

(0.4 mmol) of 19, 10 mL of a solution of NaOH 10% in MeOH (1:1)

was introduced, and the solution was stirred at room temperature

for 1 h, monitoring the course of the reaction by TLC (EtOAc/

Synthesis of 9-chloro-3-ethyl-7-methyl-3H-pyrrolo[3,2-f]quino-

line (28): Following the same procedure as for compound 14, re-

acting compound 22 (0.350 g, 1.5 mmol), after purification (recrys-

tallization from Et O/MeOH) compound 28 was obtained as

2

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1859

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

MeOH, 8:2). At the end of reaction, the MeOH was evaporated and

then a solution of HCl (10m) was added dropwise to pH 1. A pre-

cipitate formed which was collected by filtration and washed with

pound 33 was obtained as a yellow solid (0.635 g, 70.5%); R =0.24

f

(high blue fluorescent spot, CHCl /MeOH, 9:1); mp: 180–1818C;

3

1

H NMR (400 MHz, [D ]DMSO): d=11.40 (s, 1H, NH), 8.14 (d, J=

6

H O to give 0.131 g of a yellow powder after drying. Yield: 98%;

5.94 Hz, 1H, H-7), 7.75 (d, J=9.14 Hz, 1H, H-4), 7.67 (d, J=2.89 Hz,

1H, H-2), 7.44 (d, J=2.94 Hz, 1H, H-1), 7.26 (d, J=9.10 Hz, 1H, H-5),

6.76 (t, J=5.14 Hz, 1H, NHCO), 6.14 (d, J=5.98 Hz, 1H, H-8), 4.34 (t,

J=6.97 Hz, 2H, CH (CH ) NH-), 2.86 (t, J=6.03 Hz, 2H, (CH ) CH NH-

2

R =0.34 (high blue fluorescent spot, CHCl /MeOH, 8:2); mp:

f

3

1

9

6.48C; H NMR (400 MHz, [D ]DMSO): d=14.37 (s, 1H, OH), 11.97

6

(

s, 1H, COOH), 8.27 (dd, J=9.08, 0.65 Hz, 1H, H-4), 7.74 (d, J=3.00,

2

2 3

2

1

2

H, H-2), 7.71 (d, J=9.08 Hz, 1H, H-5), 7.35 (dd, J=3.10, 0.65 Hz,

H, H-1), 7.04 (s, 1H, H-8), 4.38 (t, J=7.10 Hz, 2H, CH (CH ) COOH),

), 1.74 ppm (quint, J=6.55 Hz, 2H, CH CH CH NH-).

2 2 2

2

2 5

3

9

-(6-tert-Butylaminohexyl)-7-methyl-3H-pyrrolo[3,2-f]quinolin-

(6H)-one (34): Following the general procedure, reacting com-

.74 (s, 3H, C-CH ), 2.16 (t, J=7.26 Hz, 2H, (CH ) CH COOH), 1.80

3

2 5

2

(

quint, J=7.18 Hz, 2H, CH CH (CH ) COOH), 1.44 (quint, J=7.40 Hz,

2 2 2 4

pound 21 (0.15 g, 3.9 mmol), di-tert-butyl dicarbonate (2.56 g,

11 mmol), CoCl ·6H (0.180 g, 0.78 mmol), NaBH (0.147 g,

9 mmol), compound 34 was obtained as a yellow solid (1.03 g,

6.5%); R =0.17 (high blue fluorescent spot, CHCl /MeOH, 9:1);

2

H,

(CH ) CH (CH ) COOH),

1.25 ppm

(m,

4H,

2

3

2

2 2

1

3

1

2

O

4

(

CH ) CH CH (CH ) COOH); C{ H} NMR (101 MHz, [D ]DMSO): d=

2 2 2 2 2 2 6

3

6

1

2

9.6 (CCH ), 24.3 ((CH ) CH CH COOH), 25.8 ((CH ) CH (CH ) COOH),

3 2 4 2 2 2 3 2 2 2

f

3

8.0 ((CH ) CH (CH ) COOH), 30.2 (CH CH (CH ) COOH), 33.4

2

2 3

2

2 4

1

mp: 175.58C; H NMR (400 MHz, [D ]DMSO): d=11.45 (s, 1H, NH),

6

(

(CH ) CH COOH), 45.9 (CH (CH ) COOH), 104.3 (C1), 106.1 (C8),

2 5 2 2 2 5

7

.78 (d, J=8.88 Hz, 1H, H-4), 7.47 (d, J=2.73 Hz, 1H, H-2), 7.42 (d,

1

2

11.5 (C5), 119.0 (C4 and C-9a), 120.3 (C9b), 130.3 (C2), 136.4 (C5a),

J=3.60 Hz, 1H, H-1), 7.26 (d, J=8.85 Hz, 1H, H-5), 6.75 (t, J=

5.09 Hz, 1H, NHCO), 5.92 (s, 1H, H-8), 4.23 (t, J=6.78 Hz, 2H,

51.8 (C7), 169.4 (C9), 174.4 ppm (COOH); IR (KBr): n˜ =3402 (NÀH),

À1

937 (CÀH), 1624 (C=C), 1379 cm (O-H); HRMS (ESI, 140 eV), m/z

+

CH (CH ) NH-), 2.86 (t, J=6.03 Hz, 2H, (CH ) CH NH-), 2.34 (s, 3H, C-

2 2 5 2 5 2

[M+H] calcd for C H N O : 327.1709, found: 327.1699; C₁₈RP-

19 23 2 3

CH ), 1.74 (quint, J=6.55 Hz, 2H, CH CH (CH ) NH-), 1.35 (s, 9H,

3

2

2 4

HPLC: 98.76%, t =7.54 min.

R

C(CH ) ), 1.29 ppm (m, 6H, (CH ) CH CH CH CH NH-).

3

2 2

2

Synthesis of 7-(6,7-dimethyl-9-oxo-6H-pyrrolo[3,2-f]quinolin-

(9H)-yl)heptanoic acid (32): Following the same procedure as for

compound 31, reacting compound 23 (0.530 g, 1.4 mmol), after

3

-(6-tert-Butylaminohexyl)-6,7-dimethyl-3H-pyrrolo[3,2-f]quino-

3

lin-9(6H)-one (35): Following the general procedure, reacting com-

pound 24 (0.460 g, 1.49 mmol), di-tert-butyl dicarbonate (0.997 g,

purification (precipitation from acidic H O), compound 32 was ob-

2

4

.48 mmol), CoCl ·6H O (0.070 g, 0.3 mmol), NaBH₄(0.757 g,

2 2

tained as a yellow powdery solid (0.466 g, 98%); R =0.42 (high

f

1

20 mmol), compound 35 was obtained as a yellow solid (0.380 g,

blue fluorescent spot, CHCl /MeOH, 8:2); mp: 2808C (dec); H NMR

3

6

2

2%); R =0.65 (high blue fluorescent spot, CHCl /MeOH, 9:1); mp:

018C; H NMR (400 MHz, [D ]DMSO): d=7.89 (d, J=9.09 Hz, 1H,

f

3

(

400 MHz, [D ]DMSO): d=8.26 (d, J=9.45 Hz, 1H, H-4), 7.89 (d, J=

6

1

6

9

1

4

.51 Hz, 1H, H-5), 7.72 (d, J=3.00 Hz, 1H, H-2), 7.43 (d, J=2.82 Hz,

H, H-1), 6.96 (s, 1H, H-8), 4.37 (t, J=6.73H, 2H, CH (CH ) COOH),

H-4), 7.60 (d, J=2.85 Hz, 1H, H-2), 7.53 (d, J=9.36 Hz, 1H, H-5),

.48 (d, J=2.88 Hz, 1H, H-1), 6.73 (t, J=5.09 Hz, 1H, NHCO), 6.09

s, 1H, H-8), 4.27 (t, J=6.96 Hz, 2H, CH (CH ) NH-), 3.78 (s, 3H, N-

2

2 5

7

(

.11 (s, 3H, N-CH ), 2.77 (s, 3H, C-CH ), 2.13 (t, J=7.23 2H,

3

2

2 5

(

CH ) CH COOH), 1.78 (quint, J=6.81 Hz, 2H, CH CH (CH ) COOH),

2 5 2 2 2 2 4

CH ), 2.84 (t, J=6.33 Hz, 2H, ((CH ) CH NH-), 1.98 (s, 3H, C-CH ),

1.76 (quint, J=7.08 Hz, 2H, CH

3

2 5

2

3

1

.41 (quint, J=7.52 Hz, 2H, (CH ) CH CH COOH), 1.21 ppm (m, 4H,

2 4 2 2

1

3

1

2

CH

2

(CH

2

)

4

NH-), 1.35 (s, 9H, C(CH

)

3

),

(

CH ) CH CH CH CH COOH); C{ H} NMR (101 MHz, [D ]DMSO): d=

2 2 2 2 2 2 6

1

.17 ppm (m, 6H, (CH ) CH CH CH CH NH-).

2 2 2 2 2 2

2

3

2.6 (CCH ), 24.7 ((CH ) CH CH COOH), 30.6 (CH CH (CH ) COOH),

3 2 4 2 2 2 2 2 4

3.9 ((CH ) CH COOH), 38.2 (NCH ), 46.3 (CH (CH ) COOH), 105.1

General procedure for the synthesis of 3N-alkylaminopyrrolo-

quinolinone hydrochloride derivatives 36–38: In a 100 mL flask,

tert-butyl derivatives 33–35 were dissolved in ~20 mL of absolute

EtOH, and dry HCl gas was bubbled into the solution until the for-

mation of a yellow precipitate. The mixture was cooled at 48C

overnight and then the product collected by filtration.

2

5

2

3

2

2 5

(

C1), 108.8 (C8), 110.9 (C9a), 114.1 (C5), 115.3 (C4), 121.9 (C9b),

1

32.3 (C2), 138.5 (C3a), 151.0 (C5a), 154.4 (C7), 170.0 (C9),

75.1 ppm (COOH); IR (KBr): n˜ =3431 (OÀH), 2928 (CÀH), 1725 (C=

À1

+

O), 1603 (C=C), 1363 cm (CꢀN); HRMS (ESI, 140 eV), m/z [M+H]

+

calcd for C H N O : 341.1865, found: 341.1856; C RP-HPLC:

2

0

25

2

3

18

9

9.02%, t =6.24 min.

R

3-(3-Aminopropyl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one

hydro-

General procedure for the synthesis of N-Boc-protected 3N-

alkylaminepyrroloquinolinone derivatives 33–35: In a 100 mL

flask, a solution of nitrile derivative 10, 21 or 24 dissolved in hot

MeOH was cooled to 08C (ice bath) before adding di-tert-butyldi-

carbonate (threefold molar excess) and a catalytic amount of

CoCl ·6H O. Excess NaBH was subsequently added portionwise

chloride (36): Yield: 87%; R =0.89 (high blue fluorescent spot,

f

1

EtOAc/MeOH, 8:2); mp: 1238C; H NMR (400 MHz, [D ]DMSO): d=

6

15.62 ppm (s, 1H, OH), 8.75 (d, J=6.28 Hz, 1H, H-7), 8.30 (d, J=

+

9.06 Hz, 1H, H-4), 8.25 (brs, 3H, NH₃),7.95 (d, J=9.01 Hz, 1H, H-5),

7.86 (d, J=2.70 Hz, H-2), 7.54 (d, J=6.57 Hz, 1H, H-8), 7.39 (d, J=

+

2

4

2.69 Hz, 1H, H-1), 4.58 (t, J=7.05 Hz, 2H, CH CH CH NH ), 2.76 (m,

2

3

+

with the development of H gas and formation a black precipitate.

2H, CH CH CH NH )

and 2.15 ppm (t, J=7.10 Hz, 2H,

C{ H} NMR (101 MHz, [D ]DMSO): d=28.9

(CH CH CH NH ), 36.7 (CH CH CH NH ), 43.8 (CH CH CH NH ),

2 2 2 3 2 2 2 3 2 2 2 3

2

3

+

13

1

The mixture was left at room temperature for 12 h, and the reac-

CH CH CH NH );

2 2 2 3

6

+

tion was monitored by TLC (CHCl /MeOH, 9:1). At the end of reac-

3

tion, an equimolar amount of diethylenetriamine was added, and

after ~1 h the mixture was concentrated to dryness by rotary evap-

oration. The yellow solid residue was dissolved in EtOAc and the

organic solution was washed with a saturated NaHCO₃solution

105.5 (C1), 106.03 (C8), 112.6 (C5), 114.7 (C9a), 120.3 (C9b), 120.5

(C4), 130.8 (C2), 132.5 (C3a), 137.2 (C5a), 140.9 (C7), 169.6 ppm

+

(C9); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O :

14

16

3

242.1293, found: 242.1265.

(

330 mL). The separated organic phase was dried with anhydrous

3

-(6-Aminohexyl)-7-methyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one

Na SO and evaporated to dryness, furnishing the desired Boc-pro-

tected amine.

2

4

hydrochloride (37): Yield: 98%; R =0.47 (high blue fluorescent

spot, CHCl /MeOH, 1:1); mp: 56.38C; H NMR (400 MHz, [D ]DMSO):

f

1

3

6

3

-(3-tert-Butylaminopropyl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one

d=14.93 (s, 1H, OH), 8.30 (d, J=9.06 Hz, 1H, H-4), 7.86 (brs, 3H,

NH₃), 7.84 (d, J=9.06 Hz, 1H, H-5), 7.76 (d, J=2.97 Hz, 1H, H-2),

+

(

2

33): Following the general procedure, reacting 10 (0.686 g,

.66 mmol), di-tert-butyl dicarbonate (1.742 g, 7.98 mmol),

7.32 (d, J=2.82 Hz, 1H, H-1), 7.18 (s, 1H, H-8), 4.40 (t, J=6.82 Hz,

+

CoCl ·6H O (0.143 g, 0.52 mmol), NaBH (0.201 g, 20 mmol), com-

2H, NCH CH CH CH CH CH NH ), 2.77 (s, 3H, C-CH ), 2.71 (t, J=

2

4

2

3

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1860

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

+

7

2

.08 Hz, 2H, NCH CH CH CH CH CH NH ), 1.80 (quint, J=7.68 Hz,

Assays

Absorbance and fluorescence measurements: Stock solutions

2

+

2

3

H, NCH CH CH CH CH CH NH ), 1.48 (quint, J=7.20 Hz, 2H,

NCH CH CH CH CH CH NH ), 1.31 (quint, J=7.83 Hz, 2H,

2

+

3

2

3

+

(25 mL 1.507 mm in MeOH/H O 4:21) of all compounds except the

2

(

NCH CH CH CH CH CH NH ),

1.22 ppm

(m,

2H,

2

3

+

13

1

hydrochloride salts 32 and 36–38, were prepared and then diluted

to 15.07 mm concentration with the appropriate solvent, cell lysate,

and cell culture medium. Stock solutions of compounds 32 and

36–38, which were soluble in aqueous media, were directly pre-

pared by dissolution in buffer at a concentration of 1.507 mm. Ab-

NCH CH CH CH CH CH NH ); C{ H} NMR (101 MHz, [D ]DMSO):

d=20.00 (CCH₃), 25.7 (NCH CH CH CH CH CH NH ), 26.0

2

3

6

+

2

3

+

(

3

(

1

(

2

1

NCH CH CH CH CH CH NH ), 27.2 (NCH CH2CH CH CH CH NH ),

2

3

2

3

+

0.6

(NCH CH CH CH CH CH NH ),

39.1

2

3

+

NCH CH CH CH CH CH NH ), 46.3 (NCH CH CH CH CH CH NH ),

2

3

2

3

sorption spectra were determined in H O (pH 7) for compounds

2

04.8 (C1), 106.4 (C8), 112.1 (C5), 113.3 (C9a), 119.8 (C4), 120.6

C9b), 130.8 (C2), 132.4 (C3a), 152.3 (C5a), 157.1 (C7), 169.1 ppm

1

3, 14, 25–30, in H O (pH 7, 1, and 11) for compounds 11 and 22,

2

À1

in H O (pH 7), MeOH and PBS for compounds 20, 31, 32, 36–38

2

C9); IR (KBr): n˜ =3401 (OÀH), 2954 (CÀH), 1605 cm (C=C); HRMS

+

and 43. Absorbance was kept <0.05 AU to avoid inner filter dis-

tortion. Samples were measured in a 10 mm quartz cell. For accu-

rate measurement of the fluorescence, the concentration of the

samples were in the range in which the Beer–Lambert law applies,

ESI, 140 eV), m/z [M+H] calcd for C H N O : 298.1919, found:

18 24 3

2

+

2+

98.2016; m/2z [M+2H] calcd for C H N O : 149.5999, found:

18 25 3

49.5063; C₁₈RP-HPLC: 97.44%, t =9.74 min.

R

[

1]

3

-(6-Aminohexyl)-6,7-dimethyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-

and the optical density was <0.05 to avoid the inner filter effect.

one hydrochloride (38): Yield: 94%; R =0.55 (high blue fluores-

Emission spectra of all compounds were determined in the same

medium as for absorption. The concentration of samples was

1.5 mm. Samples were measured in a 10 mm quartz cell.

f

1

cent spot, CHCl /MeOH, 1:1); mp: 558C; H NMR (400 MHz,

3

[

D ]DMSO): d=8.38 (d, J=9.33 Hz, 1H, H-4), 7.99 (d, J=9.54 Hz,

6

1

7

H, H-5), 7.81 (d, J=2.91 Hz, 1H, H-2), 7.41 (d, J=2.88 Hz, 1H, H-1),

.32 (s, 1H, H-8), 4.43 (t, J=6.76 Hz, 2H,

Quantum yield and brightness measurements: The quantum

yield of each compound was calculated from the observed absorb-

ance and the area of the fluorescence emission band. For accurate

measurement of the fluorescence, the concentration of the sam-

ples were in the range in which the Beer–Lambert law applies, and

the optical density was <0.05 to avoid the inner filter effect. The

fluorescence quantum yield (QY) was calculated based on a known

+

NCH CH CH CH CH CH NH ), 4.21 (s, 3H, N-CH ) 2.85 (s, 3H, C-

CH ), 2.70 (t, J=6.03, 2H, NCH CH CH CH CH CH NH ), 1.81 (quin,

J=6.85 Hz, 2H, NCH CH CH CH CH CH NH ), 1.48 (quint, J=

6

NCH CH CH CH CH CH NH ),

NCH CH CH CH CH CH NH ); C{ H} NMR (101 MHz, [D ]DMSO):

d=22.8

2

3

+

3

2

+

2

3

2

+

2

3

.81 Hz, 2H, NCH CH CH CH CH NH ), 1.31 (quint, J=8.34, 2H,

2 2 2 2 2 3

+

1.23 ppm

(m,

2H,

2

3

+

13

1

2

3

6

+

reference, quinine sulfate (QY=0.577; labs =350 nm, lem =446 nm

⁽^C^C^H3⁾^,

25.8 (NCH CH CH CH CH CH NH ), 26.0

2 2 2 2 2 2 3

[2]

+

in 0.1m H SO ), according to Equation (1), and labs and lem values

2 4

(

NCH CH CH CH CH CH NH ), 27.2 (NCH CH CH CH CH CH NH ),

2 2 2 2 2 2 3 2 2 2 2 2 2 3

+

of the reference compound were in the same range as those of

evaluated compounds:

3

0.6

(NCH CH CH CH CH CH NH ),

38.8

(NCH₃),

39.0

2

+

2

3

+

(

NCH CH CH CH CH CH NH ), 46.2 (NCH CH CH CH CH CH NH ),

2

3

2

3

1

05.3 (C1), 108.13 (C8), 111.1 (C5), 119.88 (C4), 121.4 (C9a), 131.6

2

QY ¼ ðA =A ÞðF =F Þðn =n Þ QY

ð1Þ

R

u

R

u

R

(

C2), 132.5 (C9b), 138.6 (C3a), 152.2 (C5a), 155.2 (C7), 168.5 ppm

À1

(

3

1

7

9

C9); IR (KBr): n˜ =3411 (OÀH), 2940 (CÀH), 1604 (C=C), 1367 cm

+

CꢀN); HRMS (ESI, 140 eV), m/z [M+H] calcd for C H N O :

in which A is the absorbance at the excitation l, F is the area

under the corrected emission curve, n is the refractive index of sol-

vents, and the subscripts u and R refer to unknown and reference,

respectively. Brightness (B) [cm mol ], at the two excitation

l values of 250 and 350 nm, was calculated by multiplying QY and

the molar absorption coefficient value (e) according to Equa-

tion (2):

1

9

26

3

2

+

2+

12.2076, found: 312.2062; m/2z [M+2H] calcd for C H N O :

19 27 3

56.6077, found: 156.6053; Anal. calcd for C H C N O: C 59.37, H

1

9

27 l2

3

[2]

À1

.08, N 10.93, found: C 59.07, H 7.04, N 10.87; C₁₈RP-HPLC:

8.12%, t =10.65 min.

R

Synthesis of Pull–38 (42): Pullulan was oxidized with NaIO ac-

cording to a reported protocol. Briefly, pullulan (1 g, 6.17 mmol

4

[22]

glucopyranose units) was dissolved in 100 mL distilled H O, and

2

B ¼ QY Â e

ð2Þ

NaIO (0.53 g, 2.47 mmol) was added. After 1 h stirring in the dark

4

at room temperature the reaction was quenched with mannitol

Gel permeation chromatography: Gel permeation chromatogra-

phy was performed using Yarra columns SEC-2000 and SEC-3000

(

0.67 g, 3.7 mmol). The product was purified by ultrafiltration using

a 10 kDa cutoff membrane and finally by precipitation in H O/EtOH

2

(

Phenomenex, Torrance, CA, USA) serially operated on a Jasco

(

5:1). The product was lyophilized and analyzed by potentiometric

LC2000 HPLC system equipped with a refractive index (RI) detector.

[22]

titration to determine the degree of oxidation. Oxidized pullulan

500 mg, 3.07 mmol glucopyranose units) was dissolved in 10 mL

distilled H O, and a solution of 38 (1 mL) in MeOH (36 mg,

The columns were isocratically eluted with 0.1m phosphate buffer,

(

À1

0

.15m Na SO , 10% MeOH, pH 7.4, at a flow rate of 0.8 mLmin .

2

4

2

The apparent molecular weights of the oxidized pullulan reduced

0

.1 mmol) was added. The solution was acidified to pH 5.0 with 1n

with NaBH and the bioconjugate were calculated on the basis of

4

HCl and maintained under stirring at room temperature for 24 h.

Then, NaCNBH₃(8 mg, 0.12 mmol) was added, and the solution

was basified to pH 8.0 with 1n NaOH. After 24 h, NaBH₄(5 mg,

a standard curve obtained by eluting 400, 200, 110, 50, 22, 12 and

6

kDa standard pullulan samples.

0

.12 mmol) was added and after 1 h the reaction mixture was dia-

Spectrofluorimetric analysis: Solutions of compound 38 (3.2 mm)

lyzed against H O with a Float-a-Lyzer (Spectrum Labs, Houston,

in deionized H O at pH 4.5, 5.5, and 7.4, cell culture medium, Triton

2

TX, USA) with a molecular weight cutoff of 14 kDa and lyophilized

to obtain a yellow powder (yield 86%). The amount of 38 in the

final bioconjugate was calculated by spectrofluorimetric analysis

X-100, cell lysate, and rat plasma were serially diluted with the

same dissolution medium and spectrofluorimetrically analyzed

using a Jasco FP-6500 (Kyoto, Japan) spectrofluorimeter. The fluori-

metric profiles were registered in the lem range of 355–500 nm

(lex =350 nm). Volumes (5 mL) of stock solutions of 38 (3.2 mm)

were incubated at 378C and spectrofluorimetrically analyzed at

scheduled times as reported above. The solutions were also ana-

(l_e_x=350, l_e_m=428) on the basis of a titration curve obtained with

fixed concentrations of 38 in H O.

2

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1861

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

lyzed by ESI-ToF mass spectrometry. AMCA solutions were analyzed

38; absorption and emission spectra of compound 11 and 22 tau-

tomers; fluorescence/concentration correlation of compound 38

and AMCA at various concentrations in different media and cell

lines; tables with all photophysical parameters of fluorescent com-

pounds; QY evaluation of AMCA and compound 38 in cell lysate

and in cell culture medium and emission quenching determina-

tions; simplified molecular-input line-entry (SMILES, see .csv file) of

all final compounds 10–14 and 19–38.

at l =266 nm, and the spectra were collected in the range of

ex

2

80–500 nm.

Cell culture: Human cervical carcinoma KB and human breast ade-

nocarcinoma MCF7 cells were grown at 378C under a 5% CO at-

2

mosphere using RPMI-1640 medium supplemented with 10% (v/v)

heat-inactivated fetal bovine serum (FBS), 2 mm l-glutamine,

À1

1

00 IUmL penicillin, 100 mgmL streptomycin, and 0.25 mgmL

amphotericin B.

Cell viability studies: KB and MCF7 cells were plated in a 96-well

microplate at a density of 510 cells per well. After 24 h incuba-

4

Acknowledgements

tion at 378C under a 5% CO atmosphere, the culture medium was

2

replaced with 100 mL medium containing increasing concentrations

of 38 (0.86–865 mm) or pullulan bioconjugate (0.012–29.4 mm).

After 24 h incubation at 378C, MTT (20 mL of a 5 mgmL solution

in PBS) was added to each well. The plates were incubated for 5 h

and then the medium was replaced with 200 mL DMSO. The plates

were kept under gentle stirring for 1 h, and the optical density at

l 570 nm was measured using an EL311SK plate reader (Biotek In-

struments Inc., Highland, VT, USA).

This work was funded by a grant from Progetto Strategico Cari-

paro 2012 NAMECA. We are grateful to Professor Valerio Di Mar-

co (Department of Chemical Sciences, University of Padova) for

À1

his contributions to the pK potentiometric determinations.

a

Keywords: fluorescence · drug delivery · fluorescent labeling ·

pullulan · pyrroloquinolines

Cell uptake: MCF7 cells were seeded in 24-well culture plates at

5

a density of 210 cells per well. After 24 h incubation at 378C

[

1] J. Liu, C. Liu, W. He, Curr. Org. Chem. 2013, 17, 564–579.

2] B. Valeur, M. Nuno Berberan-Santos, Molecular Fluorescence: Principles

and Applications, Wiley 2012 , p. 592.

under a 5% CO atmosphere, the culture medium was replaced

2

with 500 mL medium containing increasing concentrations (2.35–

2

9.4 mm) of pullulan–38 bioconjugate. After 24 h incubation, the

[3] G. M. Eldridge, G. A. Weiss, Bioconjugate Chem. 2011 , 22 , 2143–2153.

medium was removed, and 600 mL Triton X-100 (0.1% v/v) was

added to each well. After 30 min, the cell lysate was collected, so-

nicated for 15 min and centrifuged for 5 min at 8000 rpm. The

[4] M. G. Ferlin, G. Chiarelotto, V. Gasparotto, L. Dalla Via, V. Pezzi, L. Barzon,

G. Palu, I. Castagliuolo, J. Med. Chem. 2005 , 48 , 3417–3427.

[5] V. Gasparotto, I. Castagliuolo, G. Chiarelotto, V. Pezzi, D. Montanaro, P.

Brun, G. Pal, G. Viola, M. G. Ferlin, J. Med. Chem. 2006 , 49 , 1910–1915.

samples were analyzed by fluorescence spectroscopy at l =350

ex

[

6] M. G. Ferlin, M. T. Conconi, L. Urbani, B. Oselladore, D. Guidolin, R. Di

Liddo, P. P. Parnigotto, Bioorg. Med. Chem. 2011 , 19 , 448–457.

7] V. Gasparotto, I. Castagliuolo, M. G. Ferlin, J. Med. Chem. 2007 , 50 ,

and l_e_m=428. The results were normalized by cell number, which

was estimated on the basis of a titration curve obtained by BCA

assay.

5509–5513.

8] M. G. Ferlin, R. Bortolozzi, P. Brun, I. Castagliuolo, E. Hamel, G. Basso, G.

Viola, ChemMedChem 2010 , 5 , 1373–1385.

9] G. Viola, R. Bortolozzi, E. Hamel, S. Moro, P. Brun, I. Castagliuolo, M. G.

Ferlin, G. Basso, Biochem. Pharmacol. 2012 , 83 , 16–26.

Confocal microscopy: Confocal microscopy analyses were per-

5

formed with MCF7 cells that were seeded at a density of 110 on

BD Falcon chamber slides. After 24 h the medium was removed,

and the cells were incubated with AMCA (171 mm), compound 38

[

10] R. S. Singh, G. K. Saini, J. F. Kennedy, Carbohydr. Polym. 2008 , 73 , 515–

(

86.5 and 865 mm) and conjugate Pull–38 (3.5 mm) dissolved in

531.

complete medium. After 24 h the medium was removed, the cells

were washed three times with PBS and fixed with 0.5 mL 1% w/v

paraformaldehyde in PBS for 1 h at 48C. The fixed cells were

washed twice with PBS and then treated for 10 min with 200 mL of

[11] T. D. Leathers, Appl. Microbiol. Biotechnol. 2003 , 62 , 468–473.

[12] G. Bonzi, S. Salmaso, A. Scomparin, A. Eldar-Boock, R. Satchi-Fainaro, P.

Caliceti, Bioconjugate Chem. 2015 , 26 , 489–501.

[13] M. Conrad, L. Limpach, Ber. Dtsch. Chem. Ges. 1891 , 24 , 2990–2992.

À1

[14] R. Cassis, R. Tapia, J. A. Valderrama, Synth. Commun. 1985 , 15 , 125–133.

a 5 mgmL solution of wheat germ agglutinin–Alexa Fluor 633

[15] S. Goodwin, A. F. Smith, E. C. Horning, J. Am. Chem. Soc. 1957 , 79 ,

conjugate in PBS. Finally, the cells were washed with PBS and cov-

ered with a glass slide using Mowiol (Sigma) as mounting medium.

The samples were kept in the dark at 48C before starting micro-

scopic examinations. The images were obtained with a Leica TCS

SP5 confocal laser-scanning microscope (Leica microsystems,

Mannheim, Germany) equipped with a Leica HCX PL APO 63

2239–2241.

[16] C. Wu, F. Wu, Y. Bai, B. Yi, H. Zhang, Mater. Lett. 2005 , 59 , 1748–1751.

[17] P. Sahakitpichan, S. Ruchirawat, Tetrahedron Lett. 2003 , 44 , 5239–5241.

[18] M. G. Ferlin, B. Gatto, G. Chiarelotto, M. Palumbo, Bioorg. Med. Chem.

2001 , 9 , 1843–1848.

[19] H. R. Nasiri, M. Bolte, H. Schwalbe, Heterocycl. Commun. 2006, 12, 319–

/

1.40 oil-immersion objective lens. Laser irradiation at 351 nm was

used to detect the blue fluorescence from AMCA and compound

8, whereas laser at 561 nm was used to detect the red fluores-

322.

[

20] A. R. Katritzky, M. Karelson, P. A. Harris, Heterocycles 1991, 32, 329–369.

21] A. Scomparin, S. Salmaso, S. Bersani, R. Satchi-Fainaro, P. Caliceti, Eur. J.

Pharm. Sci. 2011 , 42 , 547–558.

3

cence from Alexa Fluor.

[

22] D. Bruneel, E. Schacht, Polymer 1993 , 34 , 2628–2632.

Supporting information: Experimental protocols and characteriza-

1

tion of compounds 4–9; synthesis of 1-ethyl-5-nitroindole (7); H

Received: August 10, 2015

1

3

and C NMR spectra of compounds 11–13, 19–27, 31, 32, 37, and

Published online on October 8, 2015

ChemMedChem 2015, 10, 1846 – 1862

www.chemmedchem.org

1862

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Article Doi

Design, synthesis, and photophysical properties of pyrroloquinoline-based compounds showing strong blue fluorescence as potential dyes for biomedical applications

DOI: 10.1002/cmdc.201500366

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cmdc.201500366

Full text of DOI:10.1002/cmdc.201500366

Products guided by the article

R&D Labs maybe for 15568-85-1

Relevant to this article

Hot Product