Design, Synthesis, and Structure–Activity Relationships of Azolylmethylpyrroloquinolines as Nonsteroidal Aromatase Inhibitors

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Article

Design, synthesis and SARs of azolylmethyl-pyrroloquinolines as non steroidal aromatase inhibitors

Maria Grazia Ferlin, Davide Carta, Roberta Bortolozzi, Razieh Ghodsi, Adele Chimento, VincenzoPezzi, Stefano Moro, Nina Hanke, Rolf W. Hartmann, Giuseppe Basso, and Giampietro Viola

J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400377z • Publication Date (Web): 12 Sep 2013

Downloaded from http://pubs.acs.org on September 27, 2013

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Design, synthesis and SARs of azolylmethyl-pyrroloquinolines as non steroidal

aromatase inhibitors.

Maria Grazia Ferlin†, Davide Carta†, Roberta Bortolozzi‡, Razieh Ghodsi¥, Adele Chimento±,

Vincenzo Pezzi±, Stefano Moro†, Nina Hanke≡, Rolf W. Hartmann«, Giuseppe Basso‡, Giampietro

Viola‡

†Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova,

Italy

‡Department of Woman and Child health, Laboratory of Oncohematology University of Padova,

Padova, Italy

¥Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical

Sciences, Mashhad, Iran

±Department of Pharmaco-Biology, University of Calabria, Calabria, Italy

≡ Pharmaceutical and Medicinal Chemistry, Campus C 2.3, Saarland University, D-66123

Saarbruecken, Germany

« Pharmaceutical and Medicinal Chemistry & Helmholtz Institute for Pharmaceutical Research

Saarland (HIPS), Campus C 2.3 P.O. D-66123 Saarbruecken, Germany

Keywords: pyrroloquinolines, CYP19, CYP11B1, CYP17, aromatase inhibitors, breast cancer,

molecular docking.

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ABSTRACT

A small library of both [2,3-h] and [3,2-f] novel pyrroloquinolines equipped with an azolylmethyl

group was designed and synthesized as non steroidal CYP19 aromatase inhibitors. The results

showed that azolylmethyl derivatives 11, 13, 14, 21 and 22 exhibited an inhibitory potency on

aromatase comparable to Letrozole chosen as a reference compound. When assayed on CYP11B1

(steroid-11β-hydroxylase) and CYP17 (17α-hydroxy/17,20-lyase), compound 22 was found to be

the best and most selective CYP19 inhibitor of them all.

In a panel of nine human cancer cell lines, all compounds were either slightly cytotoxic or not at all.

Docking simulations were carried out to inspect crucial enzyme/inhibitor interactions such as

hydrophobic interactions, hydrogen bonding and heme iron coordination. This study, along with the

prediction of the pharmacokinetics of compounds 11, 13, 14, 21 and 22, demonstrates that the

pyrroloquinoline scaffold represents a starting point for the development of new pyrroloquinoline-

based aromatase inhibitors.

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INTRODUCTION

Estrogens play a crucial role in the development and progression of breast cancer1. The biosynthesis

of estrogens from androgens is catalyzed by a complex enzyme “aromatase” CYP19. Aromatase

inhibitors (AIs) can be both steroidal and non steroidal compounds (Figure 1) and act by reducing

the biosynthesis of estrogens in both pre- and post-menopausal women2. The first non-steroidal

inhibitor was aminoglutethimide and some other more potent and selective derivatives like

cycloexyl-aminoglutethimide3,4. These compounds have been replaced by other more selective and

potent non-steroidal inhibitors like Fadrozole, Letrozole, or Anastrozole, that are currently being

compared with Tamoxifen in first-line metastatic, adjuvant, and neoadjuvant settings5. Although

Tamoxifen and AIs are both hormone therapies, Tamoxifen acts in different ways because it blocks

estrogen receptors on breast cancer cells and unfortunately has many drawbacks6. Thus, based

primarily on a superior side effect profile7, Letrozole has recently been approved as a first-line

therapy of metastatic breast cancer in several countries8. In seeking effective aromatase inhibitors,

two considerations are paramount: intrinsic biologic activity and specific inhibition9. Generally

speaking, non-steroidal inhibitors are more likely than steroidal compounds to lack specificity since

they have the potential to block several cytochrome P450-mediated steroid hydroxylations. On the

other hand, steroidal inhibitors or their metabolites have a greater potential to produce estrogen,

androgen, glucocorticoid or progestinic agonist or antagonistic effects through the inherent

properties of their structures. Although the third generation aromatase inhibitors such as Letrozole,

Anastrozole, and Exemestane (Figure 1) are now considered valid alternatives to Tamoxifen as first

line treatment of advanced breast cancer10, the search for potent and selective AIs still remains an

engaging subject11.

Moreover, AIs resistance has been confirmed as the major obstacle to optimal therapy management,

thus strategies such as the development of multipotent compounds are being evaluated by different

research groups12-15. From a molecular point of view, the structure of third generation non-steroidal

AIs (Figure 1) can be considered to consist of two parts: one is the azole ring with a nitrogen atom

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which interacts with the heme iron atom of the cytochrome P450 of aromatase. The other is the

bulky aryl part, which mimics the steroid ring of the substrate.

Some years ago, we discovered a new class of antimitotic phenyl-pyrroloquinolinone derivatives

(PPyQs), and in particular we showed that some of them, namely 2-phenyl-pyrrolo[2,3-h]quinolin-

4-ones, had an interesting dual activity, as both antimicrotubule and aromatase inhibitors16. It was

suggested that their anti-aromatase activity could be due to their structural similarity with potent AI

2-phenyl-7,8-benzoflavone,17-19 and in particular to the presence of the carbonyl group in position 4

like a structural element responsible for the interaction with the enzyme. At the same time, the

presence of the 4-carbonyl group was also shown essential to antitubulin activity of 2-PPyQs.

In this context, our strategy was to chemically modify 2-PPyQ by introducing an imidazolylmethyl

or triazolylmethyl group in position 4 (Figure 2), with the aim of obtaining more potent and specific

aromatase inhibitors. Owing to their structural similarities with the natural substrate

androstenedione, the resulting pyrrolequinoline derivatives (PyQs) are also endowed with an

imidazole or triazole ring allowing for a strong interaction with the heme iron atom of CYP19 and

the bulky tricycle structure (Figure 3). The replacement of the carbonyl function of flavanones by

an imidazolyl group led to a marked increase of inhibitory potency, probably due to a better

interaction with heme iron.20 Attaching methylimidazolyl or triazolyl groups to other known

heterocycles such as coumarins or indoles have produced CYP-19 or CYP-17 inhibitory activity21-

24. Moreover, the newly designed 2-phenyl-pyrroloquinolines present a side phenyl ring, which

might establish useful hydrophobic interaction in the active enzyme site (Figure 2).

In the present study, some new 4-imidazolylmethyl- and 4-triazolylmethyl-pyrrolo[2,3-h]quinoline

derivatives have been synthesized and evaluated as non-steroidal competitive inhibitors of CYP19

(aromatase) taking Letrozole as positive control, CYP11B1 (steroid-11β-hydroxylase) and CYP17

(17α-hydroxy/17,20-lyase). The inhibition of aromatase enzyme was also evaluated, as were all

tricyclic compounds both in vitro using a fluorimetric assay and in H295R cells, by means of the

tritiated water release assay taking the potent non-steroidal aromatase inhibitor Letrozole as

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reference. The cytotoxicity of the new 4-azolylmethyl-pyrrolo[2,3-h]quinoline derivatives 11-15 as

well as all intermediates like the esters 3-6 and alcohols 7-10 were evaluated by MTT assay against

a panel of both estrogen (MCF-7, IGROV-1, OVCAR-3 and H295R) and non-estrogen-sensitive

(RS 4;11, SEM, Jurkat, HeLa and HT-29) human tumor cell lines. In order to understand SARs on

new PyQ derivatives, we also prepared and tested some analogous isomeric 3H-pyrrolo[3,2-

f]quinoline compounds 19-23 and the 4-carboxylic acids 16, 17 and 23 of both [2.3-h] and [3,2-f]

geometries respectively. Docking studies and molecular dynamics simulations were also performed.

RESULTS and DISCUSSION

Chemistry.

Synthesis of Pyrrolo[2,3-h] and Pyrrolo[3,2-f]quinoline Inhibitors.

The mutually synthetic route to access of both pyrrolo[2,3-h] and pyrrolo[3,2-f]quinoline

derivatives bearing an ethyl carboxilate group at positions 4 and 9 respectively (key intermediates in

the synthesis of desired azolylmethyl compounds) is described in Schemes 1 and 2. As outlined in

Scheme 1, the key ethyl 4-carboxylate intermediates 3-6 were prepared by applying a one-step

Doebner-Von Miller quinoline synthesis25 to the 4-aminoindoles derivatives 1-2, obtained as

previously described.16,26 The appropriate 4-aminoindole 1 or 2 reacted with an aldehydic

compound and ethyl pyruvate in refluxing acidic ethanol (HCl 37%) to provide the ethyl

pyrroloquinoline 4-carboxylates 3-6 (20-50%). Due to the presence of pyruvic acid, the Doebner-

Miller reaction didn’t work well in any of the above mentioned cases, unlike those where quinoline

derivatives were used.27 However, the reaction workup was quite laborious due to the formation of

a disturbing and poorly identified green solid product (see Experimental section) to which the low

yields should be ascribed. The next step which consisted of a reduction reaction of the 4-ethyl esters

3-6 to the corresponding 4-methyl alcohols 7-10 was carried out with LiAlH4 in dry THF, resulting

in very high yields of precursor intermediates (80-95%). Hence, following a recognised

procedure28, the alcohols 7-10 were subjected to a reaction with N,N-carbonyldiimidazole (CDI) or

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N,N-carbonylditriazole (CTI) in N-methyl-pyrrolidone (NMP) at 170°C to produce the target 4-

imidazolylmethyl 11-14 and triazolylmethyl 15 substituted pyrroloquinilines in good yields (40-

73%). In addition, the 4-ethyl esters 3 and 6 were transformed into the corresponding 4-carboxylic

acids 16 and 17 with NaOH 20% in refluxing ethanol (>90%). Following the same synthetic route

of the 4-azolylmethyl-pyrrolo[2,3-h]quinolines and starting from 5-amino-1-ethyl-indole 1826, two

9-azolylmethyl-pyrrolo[2,3-f]quinolines were prepared (Scheme 2). In short, the key intermediate 9-

ethyl ester 19 was firstly obtained by way of the Doebner-Miller reaction (15.2%), which was then

reduced by LiAlH4 in dry THF into the corresponding alcoholic derivative 20 (62.8%). The latter

finally reacted with either CDI in NMP to give the 9-imidazolylmethyl, or with CDT to give 9-

triazolylmethyl 21 and 22 (82.8 and 34%) respectively. As in scheme 1, the 4-carboxylic acid 23

was also obtained from the ester 19 in alkaline ethanol (84.9%). All tricyclic compounds were

completely characterized and their chemical-physic and spectrometric properties are reported (see

Experimental section).

The exact structure of the newly synthesized PyQ derivatives was confirmed by experiments (see

the SI) 1-D and 2-D NMR. The 1H NMR (400 MHz, [D6]DMSO) spectrum of compound 12, taken

as a sample for the [2,3-h] angular series, showed a singlet at δ 5.83 integrating for 2 protons due to

the methylenic bridge between the imidazole ring and the tricyclic PyQ. The other signals

correspond with the anticipated structure. Both the confirmation of the angular [2,3-h] geometry

and the completion of structural assignments were realized by means of 2D NMR experiments like

NOESY, HSQC and HMBC. The HMBC experiments, used to reveal correlations within a distance

of 2-3 C-H bonds (Figure 4A, blue arrows), showed a significant correlation between the proton

signal at 5.83 ppm (s, 2H, -CH2-) and carbon signals at 113.88 (3-C), 119.37 (4-C), 120.63 (5’-C)

and 144.06 ppm (4a-C), corresponding with the expected molecular structure.

NOESY provides extra information about the connectivity allowing a full assignments based on

through-space correlations. As a result, the NOESY spectrum of 12 displays correlations between

the methylenic bridge and the ring system (Figure 4A, red arrows). The aromatic region shows three

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bond correlations that are used to determine which protons are neighbors of each other. For

example, in Figure 4B the proton at 11.68 ppm (s, 1H, NH) is next to the proton at 7.48 ppm on the

same ring (pyrrole ring C) and near the proton at 7.70 ppm (benzene ring B). The singlet proton

signal at 7.45 ppm of 3-H (pyridine ring A) correlates with that of 7.32 (t, J=1.02 Hz, 1H, 5’-H) and

also that of 8.16 ppm (m, 2H, 2’’- and 6’’-H), an occurrence which can not be understood from the

1D or COSY spectra. The singlet signal generated by the methylenic protons presents clear

correlations with the protons at 7.80 (5-H, benzene ring, B), 7.45 (3-H, pyridine ring A), 7.91 (2’-H,

imidazole ring) and 7.32 ppm (5’-H, imidazole ring). This corresponds wtih and strongly supports

the [2,3-h] geometry, elucidating the exact structure of 12 and of all the similar PyQ derivatives.

In vitro Evaluation of Inhibition of Aromatase (CYP19) Activity. All PyQs were evaluated for

their aromatase inhibitory activity, using a CYP19 high-throughput screening kit with 7-methoxy-4-

trifluoromethyl coumarin (MFC) as the substrate and Letrozole as the reference compound. In order

to get information regarding structure-activity relationships, in Table 1 the tested compounds are

grouped together according to the kind of substitution present in positions 4 or 9 of the two [h] and

[f] series respectively. As shown in Table 1, the ethyl ester 3-6 and 19 were all ineffective

(IC50>10000 nM). Among the 4-methyl alcohol derivatives, compound 8 was moderately active

(IC50=922 nM), followed in order by 7 (IC50=2112 nM) and 10 (IC50=4152 nM). The other two

derivatives, 9 and 20, were not active (IC50>10000 nM).

Imidazole derivatives were the most active compounds, with compounds 11, 13, 14 and 21 found to

be as potent (IC50=3.1-11.4 nM) as Letrozole (IC50=3.4 nM), while compound 12 was only

moderately active (454 nM) as well as the 2-PPyQ, which is the 2-(m-methoxy-phenyl)-

pyrrolo[2,3-h]quinoli-4-one previously described16 (IC50=590 nM). The two triazolylmethyl

derivatives 15 and 22 showed opposite effects: whereas 22 was strongly active (IC50=13.3 nM), 15

was ineffective (IC50>10000 nM).

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Finally, among the 4-carboxylic acids 16, 17 and 23 only the latter turned out to be any way active

(IC50=1472 nM).

Evaluation of Inhibition of CYP19 Activity in H295R Cell Line. The ability of the PyQ

derivatives 3-17 and 19-23 to inhibit aromatase enzyme activity was further evaluated at increasing

doses of 0.1, 1 and 10 µM in subconfluent H295R cells, a commonly used model for the study of

aromatase activity29, by way of the tritiated water release assay with [1-3H(N)]-androst-4-ene-3,17-

dione. Letrozole at the concentration of 5 µM used as the reference compound according to our

previously reported protocol.6 Results are reported in Figure 5 .

With regards to ethyl esters 3-6 and 19, only compounds 3 and 4 inhibited aromatase at a

concentration of 10 µM in comparison with 5 µM Letrozole, while the other derivatives were

ineffective, corresponding with the results obtained from the isolated enzyme (see below). All the 4-

methyl alcohol derivatives inhibited enzyme activity except for the 2-methyl substituted 10 (in the

same way as the corresponding ester 6), albeit on a different scale: at 10 µM, the level of inhibition

of 7 stood at about 40% (60.25% residual activity, r.a.) and 20 at 90% (9.64% r.a.), which shows the

same geometry of the ester 19. The more interesting compound 8 inhibited the enzyme in a dose-

dependant manner (31.8% at 0.1, 21.7 at 1 and 1.1 % r.a. at 10 µM) and already at a concentration

of 0.1 µM which was much more active than the reference drug (5 folds) (32.00% r.a.).

Regarding the imidazolylmethyl derivatives 11-14 and 21, we can note that they show very

different behaviour amongst each other. Interestingly, compound 14 of the 2-methyl series inhibited

aromatase in a dose-dependant manner: 86.23% r.a. at 0.1 µM, 26.67% r.a. at 1µM and 1.0 % at 10

µM. Compound 21 was found to be very active also at the lowest concentration used (0.1 µM)

while compound 13 with opposite geometry is active only at 10 µM. Unexpectedly, the designed

molecule 12 was found to be completely inactive. Note that some compounds (11, 13, 15, and 17)

increased the activity of the enzyme at low concentration. This paradoxical activity could be due to

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the activation of phosphorylative pathways that are able to activate the enzyme and to increase its

activity.30,31

Of the two triazolylmethyl derivatives 15 and 22, only the latter belonging to the [3,2-f] geometric

series was found to inhibit the enzyme at 10 µM (50.85% r.a.), though on a lesser scale than

Letrozole, at the concentration of 5 µM (32.00% r.a.). Finally, of the 4-carboxylic acids 16, 17 and

23, only compound 16 was found to inhibit strongly and much more than the reference compound,

and only when at a concentration of 1 µM (10.6 % r.a.).

Evaluation of Inhibition of CYP11B1 and CYP17 Activities

In order to understand the selectivity features of the novel azolylmethyl compounds 11-14, 21 and

22, which were all endowed with the highest activity for CYP19, they were also evaluated for

inhibitory activity with two other steroidogenic enzymes, CYP11B1 and CYP17 respectively.

CYP11B1 is essential for the biosynthesis of glucocorticoids while CYP17 plays an important role

in the biosynthesis of androgens. Moreover, because of high homology levels between CYP11B1

and B2 (aldosterone synthase) inhibition values would be expected to be very similar, and

accordingly we may also deduce the selectivity of CYP11B2. We also included compound 15,

which was structurally very similar to compound 12. The inhibition of CYP11B1 by selected

compounds was determined using a cellular assay, with hamster fibroblast cells V79MZh which

express human CYP11B1 and with [3H]-11-deoxycorticostesone as a substrate39. CYP17 activity

was determined in a cell-free assay40 in which human CYP17-expressing E. coli was used as the

enzyme source14,15.

As reported in Table 1, compounds 11-14 and 21 showed high inhibitory activities on CYP11B1

displaying IC50 values in the range of 75-1010 nM. In particular, compound 14 presents the highest

activity with an IC50 value of 75 nM, whilst being inactive on CYP17 (IC50 > 5000). Compounds

11-13 present a moderate inhibitory activity on CYP17 with IC50s in the range of 480-919 nM.

Compound 15, which didn’t inhibit CYP19, was also inactive on CYP11B1 and CYP17.

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Interestingly, the triazole derivative 22 was found to be very selective over CYP11B1 and CYP17,

as it showed low inhibition on both enzymes. In this context, it should be highlighted that letrozole

showed an IC50 value of 2620 nM and 7 nM on CYP11B1 and CYP17 respectively14, indicating that

compound 22 is an excellent and selective CYP19 inhibitor (13.3 nM).

Antiproliferative Activity. The antiproliferative activity of the PyQs 3-17 and 19-23 was evaluated

by MTT assay32 against a panel of five non estrogens (RS4;11, SEM, Jurkat, HeLa, and HT29) and

four estrogens sensitive tumor cell lines (MCF-7, IGROV-1, OVCAR-3 and H295R). GI50 values

are all reported in the SI. In Table 2, azole PQs are reported with 2-PPyQ which is the 2-(m-

methoxy-phenyl)-pyrrolo[2,3-h]quinoli-4-one previously described as a dual acting

antimitotic/aromatase inhibitor and taken as a reference compound.16 In general, all newly

synthesized compounds showed poor cytotoxic activity with GI50 ranging from low values up to

values of 100 µM and little selectivity towards the estrogen sensitive cell lines, in comparison with

the reference 2-PPyQ. As previously hypothesised, the latter showed a certain selective effect

toward estrogen cell lines as a consequence of the dual mechanism mentioned above, and thus we

expected the same with the new compounds. In our experimental conditions, Letrozole -which was

taken as a reference aromatase inhibitor- is inactive in all cell lines (GI50 >100 µM)33. Considering

only GI50s less than 20 µM as the significant values for a cytotoxic effect, we can observe that

among the ethyl 4-carboxylates 3-6 and 19, compounds 3-5 demonstrated good activity on HT29

cells (GI50s 17.2-4.3 µM) with the most active 2-(p-methoxy)-phenyl derivative 4 displaying a GI50

of 1.9 µM. Some activity on leukaemia cell lines has also been observed with an interesting value of

13.0 µM on SEM cells by 7-ethyl-2-phenyl derivative 5, which also showed certain cytotoxicity on

the MCF-7 cell line (GI50=18.5 µM). Among the 4-methylalcohol derivatives 7-10 and 20, the most

interesting data is that of the GI50 of 5.7 µM on MCF-7 by the 2-methyl 10. Some cytotoxicity was

also observed in the leukaemia cell line SEM by the alcoholic derivatives 10 (16.2 µM) and 20

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(13.4 µM) respectively. Among the 4-imidazolylmethyl PyQs 11-14 and 21, compound 13 was

found to be cytotoxic against both non-estrogens (GI50s: 19.2 µM RS4;11, 16.7 µM Jurkat, 19.5

µM Hela and 6.5 µM HT-29) and all the four estrogen cell lines (3.8 µM MCF-7, 18.9 µM IGROV-

1, 17.5 µM OVCAR-3, 19.0 µM H295R), whereas 21 was the most cytotoxic one with its GI50

values lower than 20 µM in almost all cell lines and with a value of 3.8 µM on the H295R line.

Compound 11 had a GI50 of 17.0 µM, 13.5 µM and 16.9 µM against SEM, HT-29 and OVCAR-3,

respectively. 4-Triazolylmethyl PyQs 15 and 22 were more cytotoxic than those of 4-

imidazolylmethyl, if we consider the pyrrolo[3,2-f]quinoline 22 dispalying two low GI50s of 2.7 and

5.5 µM on leukemic cells RS4;11 and SEM respectively. Furthermore, the latter was also found to

be active on OVCAR-3 cells (13.6 µM GI50). Finally, on our cell lines panel, the carboxylic acids

16, 17 and 23 were inactive.

Cell cycles analysis. The effect of compounds 8, 11, 21 and 22 on the cell cycle was evaluated in

the non-estrogens dependent cell line HeLa and in the estrogen-dependent cell line MCF7. As

shown in Figure 6, compounds 8, 11 increased the G1 phase in MCF-7 cells in a concentration

dependent manner, whilst reducing the S phase (for additional cell cycles refer to SI). In contrast,

we did not observe any significant modification of the Hela cell cycle. A completely different type

of behaviour was observed in compound 21 and 22, which caused a marked increased of the G2/M

phase along with a reduction of the G1 and S phases in both cell lines. Interestingly, Letrozole did

not modify the cell cycle in either the MCF-7 or HeLa cells. In this context, recent papers have

shown that treatment with Letrozole arrests the G1 phase of the cell cycle, but this occurs after

longer periods of incubation (6 days) compared to those used in both this body of work and in

MCF-7 breast cancer cells that have been transfected with the gene for aromatase34. It is important

to note that compound 21 belongs to the series of compounds characterised by the [f] geometry, a

typical feature of molecules endowed with strong anti-tubulinic activity16. Meanwhile, the other

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compounds (8 and 11) have a [h] geometry which presents lower cytotoxicity but higher

antiaromatase activity, as shown before.

Docking studies of pyrrolo[2,3-h] and pyrrolo[3,2-f]quinoline inhibitors and molecular

dynamic simulations of compound 21

Molecular docking studies were performed to elucidate the possible binding mode of aromatase and

its inhibitors and to rationalize the observed SARs.

Starting from the crystallographic structure of the placental aromatase cytochrome P450 in complex

with 4-androstene-3-17-dione (PDB code: 3EQM)35, the catalytic site of aromatase is located at the

juncture of the I and F helices, β-sheet 3, and as the B-C loop. The substrate, Androstenedione,

binds into the steroid binding pocket so that its β-face orientates towards the heme group of the

aromatase, placing C19 within 4.0 Å of the iron atom35. This active site is highly hydrophobic and

is dominated by aliphatic amino acid residues, and it has been observed that all aromatase inhibitors

engage hydrophobic interactions with the following residues: F134, W224, T310 and V37336.

Hence, inhibitors with alkyl or aromatic groups are expected to bind with much higher affinity.

Another very important key interaction is mediated by the presence of a basic nitrogen atom

(usually an imidazole or triazole moiety) in the structure of aromatase inhibitors that allows them to

apically coordinate the iron atom of the heme prosthetic group of the enzyme35,36.

Our docking studies confirmed that all novel synthesized pyrrolo[2,3-h] and pyrrolo[3,2-f]quinoline

inhibitors can be accommodated inside the catalytic site of aromatase surrounded by the following

amino acids: R115, I133, F134, F221, W224, L228, I305, A306, D309, T310, V370, L372, V373,

M374, R435 and L477. However, their specific inhibition is strongly determined by the peculiar

structural aspects of the binders that allow a close fit to the substrate-binding site of aromatase. In

particular, the replacement of a methyl group at position 2 of pyrrolo[2,3-h]quinoline moiety by a

phenyl, such as in compounds 7 and 14 (IC50 = 2112 and 6.0 nM, respectively), appreciably

increased the inhibitory potency in comparison to their analogs 10 and 11 (IC50 = 4153 and 11.4

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nM, respectively). In fact, the phenyl group at position 2 of the pyrrolo[2,3-h]quinoline nucleus is

positioned into a hydrophobic pocket surrounded by F221, T310, V313 and V369. The same

interactions profile can also be proposed for pyrrolo[3,2-f]quinoline analogs, as shown in Figure 8

with compound 21 as a possible prototype. The same trend is also observed when analysing the

presence of the ethyl substituent on the pyrrolic NH position of pyrrolo[2,3-h]quinoline moiety, as

indicated by the increased inhibitory potency, comparing the compound 11 with its analogue 13

(IC50 = 11.4 and 5.3 nM respectively). Also, the pyrrolic NH position is surrounded by several

hydrophobic amino acids such as F134, L372, V373 and M374. As anticipated, the coordination of

the inhibitor with the iron atom of the heme moiety is an important feature of potent and selective

aromatase inhibitors36. Functional groups such as imidazole, triazole and pyridine are able to forge

nitrogen heme iron coordination, and others such as phenol or thiophene may coordinate with the

heme iron. In particular, almost all pyrrolo-quinolines bearing either the imidazolylmethyl or

triazolylmethyl group in positions 4 or 9 (11-15, 21 and 22) are able to interact well with the iron

atom of the heme prosthetic group of aromatase, and this interaction can significantly improve the

binding affinity with the enzyme. In conclusion, of this novel class of aromatase inhibitors,

derivatives 21 and 22 ought to be considered the best prototypes. The hypothetical binding mode of

derivative 21 obtained by molecular docking simulation is shown in Figure 7. Moreover, molecular

dynamic simulations of the best docked pose of compound 21 in complex with aromatase support

the time-dependent stability of this complex. This is demonstrated by the low perturbation of the

imidazole basic nitrogen atom position which apically coordinates the iron atom of the CYP19

heme prosthetic group (details of molecular dynamics simulations are collected in SI).

Calculated physicochemical and ADME properties.

The calculated physicochemical and ADME properties of all pyrrolo[2,3-h] and pyrrolo[3,2-

f]quinoline inhibitors together with those of Letrozole are listed in Table 2 of SI. In particular,

lipophilicity (clogP), aqueous solubility (logS), human Ether-à-go-go Related Gene (hERG) channel

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inhibition, cytochrome P450 metabolism (2D6 and 2C9), P-glycoprotein binding (P-gp), blood

brain barrier (BBB) penetration, plasma protein binding (PPB90) and human intestinal absorption

(HIA) have been predicted using the StarDrop software37.

Among the most active PyQs bearing an imidazolylmethyl or triazolylmethyl group, only derivative

14 presents suitable values of both lipophilicity (which is expressed as a logarithm of partition

coefficient between n-octanol and water, clogP < 3.6) and aqueous solubility (logS > 1, µM). In

contrast, in-silico predictions suggested that all the compounds included in this study may have a

good human intestinal absorption at least higher than 30%, but among the most active compounds

there is a risk of high plasma protein binding probably higher than 80%. BBB penetration, P-

glycoprotein binding and hERG channel inhibition all remain difficult tasks for several of these

compounds (refer to SI). The prediction of cytochrome metabolism for two of the most predominant

drug metabolizing P450 enzymes, i.e. CYP2D6 and CYP2C9, has also been performed, suggesting

a potentially moderate metabolic stability of almost all PyQs.

CONCLUSION

Nowadays, aromatase CYP19 competitive inhibitors are the first choice as adjuvant therapeutics for

postmenopausal breast cancer patients. However, charged with overcoming the low specificity and

the resistance drawback of the clinically used inhibitors, the search for potent and selective AIs still

remains an attractive topic. Our design strategy was to combine the angular tricyclic PyQ core of

antimitotic PPyQs and in particular of 2-PPyQs endowed with a certain aromatase inhibitory

activity with a determinant structural element derived from non-steroidal AIs of the third

generation, ie the azole ring. Evaluation of the CYP19 inhibitory activity of derivatives belonging to

both the [2,3-h] and [3,2-f] series demonstrates that the PyQ geometry is not an absolute

requirement and identifies the unique structural characteristic was as the appended azolylmethyl

group in positions 4 and 9 of the two PyQ series. Indeed, the azolylmethyl substitution present in

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11-14, 21 and 22 strongly affected the enzymatic inhibitory activity of PyQ derivatives, since the

corresponding esters 3-19, alcohols 7-10 and 20, and acids 16, 17 and 23, were either inactive or

almost inactive. As expected, the IC50 value of 590 nM displayed by the parent 2-PPyQ decreased

to 11.4 nM for the 4-imidazolylmethyl analogue 11, most likely due to the stronger interaction

established by the azole with the heme of CYP19.

More specifically, compounds 11, 13, 14, 21 and 22 (with the exception of 15) showed high

inhibitory activity with IC50 values in the range of 13.3-3.1 nM and their inhibition profiles were

comparable with that of Letrozole (3.4 nM). It’s interesting to note that the introduction of a

methoxylic group in position 4 of the side phenyl ring of compound 11 led to a marked decrease of

affinity (12, IC50 454.4 nM), and this may indicate a somewhat steric clash. The presence of the

methoxylic group on the phenyl ring in the methyltriazolyl derivative 15 might also explain its lack

of CYP19 affinity (IC50>10000 nM). No significant difference of CYP19 inhibitory activity

ascribed to imidazolyl or triazolyl groups was observed, and nor was there to the presence of an

ethyl at pyrrolic N (11, 13) or either to a side phenyl or a methyl group (11, 14). We may deduce

that such substitutions are well tolerated and not crucial for the enzymatic activity, while the 4’-

methoxyl group is not tolerated probably due to steric hindrance.

Results from the CYP19 inhibitory activity in the H295R cell line assay were corresponded with

those of the enzymatic inhibition. Compound 21 proved to be the most active inhibitor among all

imidazolyl and triazolyl derivatives (with the exception of 15 in correspondence with the enzymatic

test), which were however found to be potent inhibitors and importantly, in a dose dependent

manner. Some little differences among them, which were most likely due to the specific lipophilic

characteristics and cellular metabolism, were observed. Indeed, among the alcohol derivatives 7-10

and 20, it is interesting to note the good profiles of 7 and 8 that inhibited the aromatase activity in a

dose dependent manner and at similar concentrations to those of the imidazolylmethyl analogues

which do not correspond with the enzymatic results.

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In the field of aromatase inhibitors, it is of fundamental importance to realize the selectivity of CYP

inhibition (see Introduction). Thus, a further evaluation of CYP11B1 and CYP17 inhibition was

carried out with the most active aromatase inhibitors, which revealed that compound 22 is endowed

with high selectivity for CYP19 as it showed very poor inhibitory activity against CYP11B1 and

CYP17. Considering that compounds 21 and 22 display the same [3,2-f] geometry, it is both

surprising and interesting to note how the diverse selectivity behavior against the two enzymes is

solely due to the azole ring: imidazole in 21 and triazole in 22, the elements responsible for heme

interactions. Moreover, it is interesting to note the structure activity-relationship which exists

between the imidazole derivatives of [2,3-h] geometry 11-14 and the inhibition of both CYP11B1

and CYP17: the sole difference to be found occurs in position 2. Whilst 2-(4’-methoxy-phenyl)-

substituted 12, 2-phenyl-substituted 11 and 13 inhibited the two enzymes in just a moderate fashion,

the 2-methyl-substituted 14 inhibited completely CYP11B1 (IC50=75 nM), it didn’t inhibit CYP17

whatsoever. This suggests that for the pyrrolo[2,3-h]quinolines hereby studied, the size of the 2-

substitution affects both the potency and the selectivity (14 ≥ 11-13). Accordingly, (interestingly)

the 2-methyl derivative 14, which strongly inhibited CYP19 and CYP11B1 with IC50 values of 6

nM and 75 nM respectively, may be considered a dual inhibitor and might be useful in the treatment

of breast cancer of postmenopausal women as proposed recently by some authors14,15.

With regards to the cytotoxicity of the tested tumor cell lines, setting the azolylmethyl group on the

tricyclic PyQ core led to derivatives with poor antiproliferative activity compared to the parent

pyrroloquinolinone compounds. Letrozole also proved to be inactive. Of particular interest is the

significant decrease of cytotoxic activity of PyQs displaying [f] geometry from nanomolar26 up to

high micromolar GI50s, although with some exceptions as in the case of 21 which showed low

micromolar GI50s.

A sequence of cell cycle analyses performed with representative compounds revealed a different

behaviour, causing the [3,2-f] derivatives 21 and 22 to act by significantly increasing the G2/M

phase whilst reducing the G1 and S phases. Moreover, the [2,3-h] 8 and 11-14 led to a concentration

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dependent increase of the G1 phase in MCF-7 cells, as well as a reduction of the S phase. This

different behaviour merits further investigations.

Docking studies showed that all novel synthesized PyQs fit well inside the catalytic site of

aromatase and interact effectively with the iron atom of the heme prosthetic group with derivative

21 being the best prototype, which all corresponds with the aromatase activity assays.

In conclusion, we have shown in this work that PyQs can be used as AIs by means of appropriate

structural modulation. For the first time, a small set of pyrrolo[2,3-h] and pyrrolo[3,2-f]quinolines

bearing either an imidazolylmethyl or triazolylmethyl group in positions 4 and 9 respectively was

synthesized and tested for its aromatase inhibitory effects. Even if their pharmacokinetic profiles

need to be more finely tuned, compounds 11, 13, 14, 21 and 22 all showed potency against

aromatase similar to that of Letrozole. 11 and 13 were equally potent against the three enzymes

respectively, whereas 14 didn’t inhibit CYP17 and 22 neither CYP11B1 nor CYP17. This

demonstrates that the pyrroloquinoline scaffold can be further optimized for the development of

new potent and selective therapeutic agents for hormone-dependent breast cancer.

Experimental Section

Chemistry

Melting points were determined on a Buchi Melting Point M-560 capillary melting point apparatus,

and are uncorrected. Infrared spectra were recorded on a Varian 670-IR/Pike MIRacle ATR

instrument; all values are expressed in cm-1. UV-Vis spectra were recorded on a Thermo Helyos α

spectrometer.

1H NMR spectra were determined on Bruker 300 and 400 MHz spectrometers, with the solvents

indicated; chemical shifts are reported in δ (ppm) downfield from tetramethylsilane as internal

reference. Coupling constants are given in Hertz. In the case of multiplets, chemical shifts were

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measured starting from the approximate centre. Integrals were satisfactorily in line with those

expected on the basis of compound structure. Elemental analyses were performed in the

Microanalytical Laboratory, Department of Pharmaceutical Sciences, University of Padova, on a

Perkin-Elmer C, H, N elemental analyzer model 240B, and analyses indicated by the symbols of the

elements were within ± 0.4% of the theoretical values. Analytical data are presented in detail for

each final compound. Mass spectra were obtained on a Mat 112 Varian Mat Bremen (70 ev) mass

spectrometer and Applied Biosystems Mariner System 5220 LC/Ms (nozzle potential 140.00).

Column flash chromatography was performed on Merck silica gels (250-400 mesh ASTM);

chemical reactions were monitored by analytical thin-layer chromatography (TLC) on Merck silica

gel 60 F-254 glass plates with a 9:1 dichloromethane/methanol mixture as eluant, unless otherwise

specified.

Solutions were concentrated on a rotary evaporator under reduced pressure. Starting materials were

purchased from Aldrich Chimica and Acros, and solvents from Carlo Erba, Fluka and Lab-Scan.

DMSO was obtained anhydrous by distillation under vacuum and stored on molecular sieves.

The purity of new tested compounds was checked by HPLC using the instrument HPLC VARIAN

ProStar model 210, with detector DAD VARIAN ProStar 335, confirming to be ≥ 95%. The

analysis was performed with a flow of 1 mL / min, a C-18 column of dimensions 250x4.6 mm, a

particle size 5 µm and with a loop of 10 µL. The detector worked at 300 nm. The mobile phase

consisted of phase A (milliQ H2O, 18.0 MΩ, TFA 0.05%), and the phase B (95% MeCN, 5% of

phase A, TFA 0.1%). The gradient elution was performed as reported: 0 min, %B =10; 0-20 min,

%B =90; 20-25 min, %B=90; 25-26 min, %B=10; 26-31 min, %B=10.

General procedure for the synthesis of ethyl pyrrolo[2,3-h[quinolin-4-carboxylates 3-6 and 19.

In a 50 mL two-necked flask, 5 mL of absolute ethanol and 2-3 drops of 37% HCl were placed. To

this solution, in the order equimolar amounts of ethyl pyruvate and aldehydic compound (3.2-5.4

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mmol) were added and the mixture heated at 80°C (oil bath) under inert atmosphere (N2) for about

1 h. After on, the ethanolic solution took a slight yellowish colour, in equal molar amounts with the

previous reagents, the aminoindole derivative 1 or 2, dissolved in 10 mL of absolute ethanol, was

slowly added drop wise, and the heating increased up to 100°C. The reaction was monitored by

TLC analysis (eluent ethyl acetate/n-hexane 1:1). At the end of the reaction (2-10 hours), the

solvent was eliminated by evaporation and to the remained residue water was added, neutralized

with 20% NH4OH and then an exhaustive extraction with ethyl acetate of the resulted suspension

was accomplished. After several washings of the total extract with water and brine, it was dried

with anhydrous sodium sulphate and the organic solvent evaporated on a evaporator. From the raw

residue of reaction the desired product was isolated and purified by proper crystallization and/or

separation on column chromatography as reported below for each compound.

Ethyl 2-phenyl-7H-pyrrolo[2,3-h]quinoline-4-carboxylate (3). It was followed the general

procedure for the Doebner-Miller reaction previously described by reacting 0.730 mL (6.55 mmol,

d: 1.45 g⋅ml-1) of ethyl pyruvate, 0.633 mL (6.55 mmol, d: 1.54 g⋅ml-1) of benzaldehyde and 0.866 g

(6.55 mmol) of 4-amino indole 1. Instantly after adding the amino-indole 1 to the ethanolic solution

inside the reaction flask, it was observed the formation of a yellow solid and the presence of a spot

with yellow fluorescence was noted on TLC already after 1.5 h. After the workup of the reaction

mixture as above, a crude brown/green semisolid residue was obtained (2.688 g). The isolation of

the reaction product required an early treatment of the residue with ethanol 96% in order to

eliminate the insoluble green solid, a by-product not well identified. The next purification on

column chromatography (l =30 cm, Ø =3 cm, 60 Ǻ, 230-400 mesh silica gel, EtOAC/n-Hex, 1:1) of

the yellow solid product (1.073 g) obtained on evaporating the ethanolic solution gave compound 3

as a yellow powder (0.397 g, 19.15%): Rf =0.77 (EtOAc/n-Hex, 1:1); mp: 180 °C; 1H NMR (300

MHz, [D6]DMSO) δ=11.81 (s, 1H, NH), 8.37 (m, 2H, 2’- and 6’-H), 8.32 (s, 1H, 3-H), 8.15 (d,

J=9.00 Hz, 1H, 5-H), 7.80 (d, 1H, J=9.00 Hz, 1H, 6-H), 7.59 (m, 2H, 3’- and 5’-H), 7.53 (m, 1H, 8-

H), 7.53 (m, 1H, 4’-H), 7.29 (m, 1H, 9-H), 4.50 (q, J=7.10 Hz, 2H, -OCH2CH3), 1.44 ppm (t,

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J=7.10 Hz, 3H, -OCH2CH3); 13C NMR (75 MHz, [D6]DMSO) δ=13.51 (-OCH2CH3), 61.10 (-

OCH2CH3), 101.99 (9-C), 114.77 (5-C), 115.24 (6-C), 116.80 (3-C), 117.61 (4-C), 123.22 (9a-C),

126.98 (8-C), 126.37 (2’- and 6’-C), 128.33 (3’- and 5’-C), 128.91 (4’-C), 134.28 (1’-C), 136.34

(6a-C), 137.81 (4a-C), 143.77 (9b-C), 152.98 (2-C), 166.21 ppm (-COO-); IR (ATR ZnSe): ν

=3373, 3000, 1700, 1555 cm-1; UV/Vis (MeOH): λmax (ε)=213 (643), 234 (643), 288 (550), 363 nm

(190); Fluorescence (MeOH): λexc =286, λem =441 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd

for C20H17N2O2: 317.1255, found: 317.1264. Purity (RP-C18 HPLC): 98.02%.

Ethyl 2-(4-methoxyphenyl)-7H-pyrrolo[2,3-h]quinoline-4-carboxylate (4).

It was followed the general procedure for the Doebner-Miller reaction previously described by

adding 1.089g (8.24 mmol) of aminoindole 1 to the acidic ethanol solution containing 0.918 mL

(8.24 mmol) of ethyl pyruvate and 0.786 mL (8.24 mmol) of p-anisaldehyde. After a few time, the

formation of a yellow-orange solid was noted and about after 1.5 h a slight orange spot, intensely

yellow fluorescent appeared on TLC (EtOAC/n-Hex, 1:1). By working up the reaction mixture as

for compound 3, a crude brown-green solid of the weight of 2.917 g was obtained, which was

treated and purified as before to finally yield the pure compound 4 (0.583 g, 20%): Rf =0.70

(EtOAc/n-Hex, 1:1); mp: 155 °C; 1H NMR (300 MHz, [D6]DMSO) δ=11.81 (s, 1H, NH), 8.33 (m,

2H, 2’- and 6’-H), 8.25 (s, 1H, 3-H), 8.10 (d, J=9.00 Hz, 1H, 5-H), 7.75 (d, 1H, J=9.00 Hz, 1H, 6-

H), 7.50 (m, J=2.73 Hz, 1H, 8-H), 7.25 (m, J=2.73 Hz, 1H, 9-H), 7.13 (m, 2H, 3’- and 5’-H), 4.49

(q, J=7.10 Hz, 2H, -OCH2CH3), 3.86 (s, 3H, -OCH3), 1.43 ppm (t, J=7.10 Hz, 3H, -OCH2CH3); 13C

NMR (75 MHz, [D6]DMSO) δ=14.04 (-OCH2CH3), 55.22 (-OCH2CH3), 61.57 (-OCH3), 101.97 (9-

C), 114.22 (3’- and 5’-C), 114.74 (6-C), 115.23 (5-C), 117.35 (3-C), 117.61 (4-C), 123.63 (9a-C),

124.22 (8-C), 128.31 (2’- and 6’-C), 130.81 (6a-C), 134.79 (1’-C), 136.79 (4a-C), 144.23 (9b-C),

153.27 (2-C), 160.49 (4’-C), 166.81 ppm (-COO-); IR (ATR ZnSe): ν =3400, 3200, 3178, 1702,

1555 cm-1; UV/Vis (MeOH): λmax (ε)=210 (664), 231 (564), 295 (557), 366 nm (215); Fluorescence

(MeOH): λexc =286, λem =542, 318 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C21H19N2O3:

347.1381, found: 347.1393. Purity (RP-C18 HPLC): 97.84%.

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Ethyl 7-ethyl-2-phenyl-7H-pyrrolo[2,3-h]quinoline-4-carboxylate (5).

It was followed the general procedure of Doebner-Miller by reacting 0.725 g (6.24 mmol) of ethyl

pyruvate, 0.662 g (6.24 mmol) of benzaldehyde and 1.000 g (6.24 mmol) of 1-ethyl-4-aminoindole

2. Since the beginning a yellow solid formed and after about 1.5 h the TLC analysis showed the

presence of a yellow fluorescent spot indicative of the formation of the tricyclic compound. At the

end, after working up of the reaction mixture a brown semi-solid was obtained (2.445 g). After

purification on column chromatography as described before (eluent EtOAC/n-Hex 4:6), compound

5 was obtained as a yellow solid (1.085 g, 50.47%): Rf =0.76 (EtOAc/n-Hex, 4:6); mp: 107 °C; 1H

NMR (400 MHz, [D6]DMSO) δ=8.37 (m, 2H, 2’- and 6’-H), 8.33 (s, 1H, 3-H), 8.20 (d, J=9.15 Hz,

1H, 5-H), 7.93 (dd, J=9.15 Hz and J=0.66 Hz, 1H, 6-H), 7.60 (m, 2H, 2’- and 5’-H), 7.59 (d, J=3.00

Hz, 1H, 8-H), 7.52 (m, 1H, 4’-H), 7.29 (dd, J=3.00 Hz and J=0.63 Hz, 1H, 9-H), 4.51 (q, J=7.25 Hz,

2H, -NCH2CH3), 4.39 (q, J=6.98 Hz, 2H, -OCH2CH3), 1.45 (t, J=7.25 Hz, 3H, -NCH2CH3), 1.44

ppm (t, J=6.98 Hz, 3H, -OCH2CH3); 13C NMR (101 MHz, [D6]DMSO) δ=14.58 (-OCH2CH3),

16.52 (-NCH2CH3), 41.30 (-NCH2CH3), 62.20 (-OCH2CH3), 102.06 (9-C), 114.54 (5-C), 116.06 (3-

C), 117.89 (6-C), 118.62 (4-C), 124.73 (9a-C), 127.47 (2’- and 6’-C), 127.71 (8-C), 129.42 (3’- and

5’-C), 130.05 (4’-C), 134.80 (1’-C), 137.37 (6a-C), 138.82 (4a-C), 144.80 (9b-C), 154.24 (2-C),

167.22 ppm (-COO-); IR (ATR ZnSe): ν =3025, 1697, 1578 cm-1; UV/Vis (MeOH): λmax (ε)=208

(606), 239 (663), 285 (503), 369 nm (190); Fluorescence (MeOH): λexc =286, λem =544 nm; HRMS

(ESI, 140 eV): m/z [M+H+] calcd for C22H21N2O2: 345.1518, found: 345.1500. Purity (RP-C18

HPLC): 98.46%.

Ethyl 2-methyl-7H-pyrrolo[2,3-h]quinoline-4-carboxylate (6).

Following the procedure described for the reaction of Doebner-Miller, to the ethanolic solution of

1.109 g (9.55 mmol) of ethyl pyruvate and 0421 g (9:55 mmol) of acetaldehyde, 1.052 g (7.96

mmol) of 4-amino-indole 1 were added. The course of the reaction was monitored by TLC (eluent

EtOAc/n-Hex, 1:1). At the end of the reaction lasting about 10 hours, after the removal of reaction

solvent the solid crude residue was extracted with ethyl acetate and the insoluble solid separated by

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filtration under vacuum. After washing the organic phase with water and dried with anhydrous

sodium sulphate, the solvent was evaporated under vacuum obtaining a brown solid of 1.151 g. The

purification consisted of a direct separation on chromatographic column (l =30 cm, Ø =2 cm, silica

gel 60 Ǻ, 230-400 mesh) using EtOAc/n-Hex, 1:1 as eluent. A beige solid corresponding to

compound 6 was retrieved (0.332 g, 16.40%): Rf =0.61 (EtOAc/n-Hex, 1:1); mp: 130 °C; 1H NMR

(400 MHz, [D6]DMSO) δ=11.72 (m, 1H, NH), 8.14 (d, J=9.09 Hz, 1H, 5-H), 7.73 (dd, J= 9.07 Hz

and J= 0.60 Hz, 1H, 6-H), 7.65 (s, 1H, 3-H), 7.47 (t, J=2.68 Hz, 1H, 8-H), 7.13 (m, J=2.52 Hz and

J=0.79 Hz, 1H, 9-H), 4.46 (q, J=7.13 Hz, 2H, -OCH2CH3-), 2.75 (s, 3H, -CH3), 1.40 ppm (t, J=7.13

Hz, 3H, -OCH2CH3-); 13C NMR (101 MHz, [D6]DMSO) δ=14.58 (-OCH2CH3), 25.26 (-CH3),

61.97 (-OCH2CH3), 102.35 (9-C), 115.31 (6-C), 117.66 (4-C), 117.93 (5-C), 119.40 (3-C), 123.76

(9a-C), 124.59 (8-C), 135.17 (6a-C), 136.23 (4a-C), 144.65 (9b-C), 156.97 (2-C), 167.25 ppm (-

COO-); IR (ATR ZnSe): ν =3404, 3100, 1699, 1525 cm-1; UV/Vis (MeOH): λmax (ε)=204 (628),

221 (866), 274 (511), 346 nm (131); Fluorescence (MeOH): λexc =261, λem =546 nm; HRMS (ESI,

140 eV): m/z [M+H+] calcd for C15H15N2O2: 255.1139, found: 255.1153. Purity (RP-C18 HPLC):

97.91%.

Ethyl 3-ethyl-7-phenyl-3H-pyrrolo[3,2-f]quinoline-9-carboxylate (19). For the reaction of

Doebner-Miller, to the ethanolic solution of 0.550 g (4.72 mmol) of ethyl pyruvate and 0. 210 g

(4.72 mmol) of acetaldehyde, 0.760 g (4.72 mmol) of 1-ethyl-5-amino-indole 2 [ ] were added. The

mixture was then refluxed for 2 h. The course of the reaction was monitored by TLC (eluent

EtOAc/n-Hex, 9:1). At the end of the reaction, after the removal of reaction solvent, the crude

residue was dissolved in EtOAc and washed with brine. The organic phases were dried over sodium

sulphate and evaporated. The purification consisted of a direct separation in a chromatographic

column (l =30 cm, Ø =2 cm, silica gel 60 Ǻ, 230-400 mesh) using EtOAc/n-Hex, 9:1 as eluent

obtaining compound 19 as a yellow-brown oil which became solid at RT (0.115 g, 15.2%): Rf =0.83

(EtOAc/n-Hex, 4:6); mp: 118 °C; 1H NMR (400 MHz, CD3OD) δ=8.12 (m, 2H, 2’- and 6’-H), 7.99

(dd, J=9.20 Hz and J=0.72 Hz, 1H, 4-H), 7.98 (s, 1H, 8-H), 7.91 (dd, J=9.20 Hz and J= 0.31 Hz,

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1H, 5-H), 7.54 (m, 2H, 3’- and 5’-H), 7.47 (m, 1H, 4’-H), 7.41 (d, J=3.18 Hz, 1H, 2-H), 6.81 (dd,

J=3.18 Hz and J=0.79 Hz, 1H, 1-C), 4.61 (q, J=6.94 Hz, 2H, -OCH2CH3), 4.38 (q, J=7.26 Hz, 2H, -

NCH2CH3), 1.51 (t, J=7.26 Hz, 3H, -NCH2CH3), 1.47 ppm (t, J=6.94 Hz, 3H, -OCH2CH3); 13C

NMR (101 MHz, CD3OD) δ=13.04 (-OCH2CH3), 14.94 (-NCH2CH3), 40.91 (-NCH2CH3), 62.04 (-

OCH2CH3), 102.74 (1-C), 115.77 (8-C), 116.31 (4-C), 117.54 (9-C), 120.07 (9b-C), 122.64 (5-C),

126.53 (2-C), 127.02 (2’- and 6’-C), 128.52 (3’- and 5’-C), 128.87 (4’-C), 132.73 (3a-C), 138.17

(9a-C), 139.03 (1’-C), 145.70 (5a-C), 153.44 (7-C), 169.63 ppm (-COO-); IR (ATR ZnSe): ν

=2975, 2920, 1725, 1562, 1496, 1451, 1347, 1243, 1021, 743, 692 cm-1; UV/Vis (MeOH): λmax

(ε)=204 (758), 242 (940), 291 (200), 359 nm (508); Fluorescence (MeOH): λexc =260, λem =553

nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C22H21N2O2: 345.1524, found: 345.1585. Purity

(RP-C18 HPLC): 98.08%.

General procedure for the synthesis of 4-methylalcohol-substituted pyrroloquinoline-

derivatives 7-10 and 20.

In a 250 mL flask, based on the starting material, a double molar amount of LiAlH4 was suspended

in 5 mL of anhydrous THF. The ethyl pyrroloquinoline 4-carboxylate derivative (3-6 and 19) (3 - 5

mmol) dissolved in 10 mL of THF was slowly dropped under inert atmosphere (N2) and at room

temperature. The course of the reaction was monitored by TLC, in all cases noting the appearance

of an intensely fluorescent spot with a Rf lower than that of starting ethyl ester.

At the end of the reaction lasting about 3 h, the excess of LiAlH4 was turned off with a saturated

aqueous solution of NH4Cl and the solid was separated by filtration and washed with ethyl acetate

some times. The total filtrate was dried with anhydrous Na2SO4 and evaporated under vacuum

yielding the crude reaction product which was purified by liquid chromatography.

(2-Phenyl-7H-pyrrolo[2,3-h]quinolin-4-yl)methanol (7). The general procedure was followed by

adding drop wise a solution of 2.445 g (3.77 mmol) of the ethyl ester derivative 3 in 10 mL of THF

anhydrous to the suspension of 0.539 g (7.54 mmol) of LiAlH4 in 5 mL of anhydrous THF. At the

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end of the reaction (2 h), by working up the reaction mixture a crude yellow-orange solid was

obtained (1.024 g). After purification in a chromatographic column (l =25 cm, Ø =2 cm, with silica

gel 60 Ǻ, 230-400 mesh, eluent EtOAc/n-Hex, 6:4) a yellow solid corresponding to compound 7

was obtained (0.953 g, 92.20%): Rf =0.37 (EtOAc/n-Hex, 1:1); mp: 206 °C; 1H NMR (300 MHz,

[D6]DMSO) δ=11.62 (s, 1H, NH), 8.32 (m, 2H, 2’- and 6’-H), 8.07 (s, 1H, 3-H), 7.66 (s, 2H, 5- and

6-H), 7.57 (m, 2H, 3’- and 5’-H), 7.47 (m, 1H, 4’-H), 7.45 (m, J=2.61 Hz, 1H, 8-H), 7.22 (m,

J=2.49 Hz, 1H, 9-H), 5.59 (t, J=5.31 Hz, 1H, -OH), 5.10 ppm (d, J=5.31 Hz, 2H, -CH2-); 13C NMR

(75 MHz, [D6]DMSO) δ=60.98 (-CH2OH), 102.42 (9-C), 112.98 (6-C), 114.44 (5-C), 116.47 (3-C),

119.74 (4-C), 124.30 (8-C), 124.59 (9a-C), 127.34 (2’- and 6’-C), 129.29 (3’- and 5’-C), 129.47

(4’-C), 135.26 (1’-C), 140.04 (6a-C), 143.75 (4a-C), 148.96 (9b-C), 154.19 ppm (2-C); IR (ATR

ZnSe): ν =3600, 3403, 1573 cm-1; UV/Vis (MeOH): λmax (ε)=205 (879), 230 (858), 276 (743), 345

nm (287); Fluorescence (MeOH): λexc =286, λem =460 nm; HRMS (ESI, 140 eV): m/z [M+H+]

calcd for C18H15N2O: 275.1210, found: 275.1224. Purity (RP-C18 HPLC): 97.54%.

(2-(4-Methoxyphenyl)-7H-pyrrolo[2,3-h]quinolin-4-yl)methanol (8). Following the general

procedure, to a suspension of 0.275 g (7.23 mmol) of LiAlH4 in 5 mL of anhydrous THF, 1.253 g

(3.62 mmol) of the ethyl ester 4 in 10 mL of THF anhydrous was slowly added. At the end of the

reaction (2 h), on TLC a yellow spot with intense blue fluorescence appeared, having a Rf lower

than that of the starting compound. By working up the reaction mixture a crude yellow-beige solid

residue was obtained (1.050 g), which was purified on chromatographic column (l =25 cm, Ø =2

cm, with silica gel 60 Ǻ, 230-400 mesh, eluent EtOAc/n-Hex, 6:4) yielding compound 8 as a beige

solid residue (0.950 g, 86.50%): Rf =0.34 (EtOAc/n-Hex, 1:1); mp: 202 °C; 1H NMR (300 MHz,

[D6]DMSO) δ=11.60 (s, 1H, NH), 8.29 (m, 2H, 2’- and 6’-H), 8.02 (s, 1H, 3-H), 7.64 (s, 2H, 5- and

6-H), 7.47 (m, 1H, 4’-H), 7.44 (m, J=2.49 Hz, 1H, 9-H), 7.21 (m, J=2.54 Hz, 1H, 8-H), 7.12 (m,

2H, 3’- and 5’-H), 5.57 (t, J=5.51 Hz, 1H, -OH), 5.09 (d, J=5.51 Hz, 2H, -CH2-), 3.85 ppm (s, 3H, -

OCH3); 13C NMR (75 MHz, [D6]DMSO) δ=55.17 (-CH2OH), 60.45 (-OCH3), 101.80 (9-C), 111.88

(6-C), 113.38 (5-C), 114.09 (3’- and 5’-C), 115.93 (3-C), 118.75 (4-C), 123.58 (8-C), 123.94 (9a-

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C), 128.10 (2’- and 6’-C), 131.96 (1’-C), 134.69 (6a-C), 143.16 (4a-C), 148.14 (9b-C), 153.58 (2-

C), 160.08 ppm (4’-C); IR (ATR ZnSe): ν =3645, 3404, 1578 cm-1; UV/Vis (MeOH): λmax (ε)=206

(868), 227 (895), 283 (952), 348 nm (449); Fluorescence (MeOH): λexc =286, λem =453 nm; HRMS

(ESI, 140 eV): m/z [M+H+] calcd for C19H17N2O2: 305.1245, found: 305.1255. Purity (RP-C18

HPLC): 98.65%.

(7-Ethyl-2-phenyl-7H-pyrrolo[2,3-h]quinolin-4-yl)methanol (9). Following the general

procedure, in a 250 mL flask, to a suspension of 0.218 g (5.73 mmol) of LiAlH4 in 5 mL of

anhydrous THF, a solution of 0.987 g (2.87 mmol) of the ethyl ester derivative 5 in 7 mL of

anhydrous THF was added. At the end of the reaction (2 h), on TLC a yellow spot with intense blue

fluorescence appeared, having a Rf lower than that of the starting compound. By working up the

reaction mixture, a crude clear brownish solid residue was obtained (0.900 g), which was purified

on chromatographic column (l =25 cm, Ø =2 cm, with silica gel 60 Ǻ, 230-400 mesh, eluent

EtOAc/n-Hex, 6:4) yielding a yellow brownish solid residue characterized as compound 9 (0.793 g,

91.50%): Rf =0.38 (EtOAc/n-Hex, 1:1); mp: 148 °C; 1H NMR (300 MHz, [D6]DMSO) δ=8.32 (m,

2H, 2’- and 6’-H), 8.08 (s, 1H, 3-H), 7.80 (d, J=9.01 Hz, 1H, 5-H), 7.71 (d, J=9.01 Hz, 1H, 6-H),

7.57 (m, 2H, 3’- and 5’-H), 7.51 (m, J=3.00 Hz, 1H, 8-H), 7.49 (m, 1H, 4’-H), 7.22 (m, J=3.00 Hz,

1H, 9-H), 5.59 (t, J=5.59 Hz, 1H, -OH), 5.12 (d, J=5.59 Hz, 2H, -CH2-), 4.32 (q, J=7.21 Hz, 2H, N-

CH2CH3), 1.44 ppm (t, J=7.21 Hz, 3H, N-CH2CH3); 13C NMR (75 MHz, [D6]DMSO) δ=16.00 (N-

CH2CH3), 40.49 (N-CH2CH3), 60.43 (-CH2OH), 101.31 (9-C), 112.10 (6-C), 112.60 (5-C), 116.02

(3-C), 118.07 (4-C), 124.52 (8-C), 126.54 (9a-C), 126.81 (2’- and 6’-C), 128.76 (3’- and 5’-C),

128.99 (4’-C), 134.20 (1’-C), 139.43 (6a-C), 143.18 (4a-C), 148.49 (9b-C), 152.78 ppm (2-C); IR

(ATR ZnSe): ν =3605, 1576 cm-1; UV/Vis (MeOH): λmax (ε)=208 (714), 234 (810), 273 (633), 351

nm (273); Fluorescence (MeOH): λexc =286, λem =461 nm; HRMS (ESI, 140 eV): m/z [M+H+]


(2-Methyl-7H-pyrrolo[2,3-h]quinolin-4-yl)methanol (10). Following the general procedure, into

a 250 mL flask, to a suspension of 0.239 g (6.29 mmol) of LiAlH4 in 5 mL of anhydrous THF, a

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solution of 0.799 g (3.14 mmol) of the ethyl ester derivative 6 in 10 mL of anhydrous THF was

added. At the end of the reaction (3 h), on TLC a yellow spot with intense blue-green fluorescence

appeared, having a Rf lower than that of the starting compound. By working up the reaction

mixture, a crude clear brownish solid residue was obtained 0.632 g, which was purified on

chromatographic column (l =25 cm, Ø =2 cm, with silica gel 60 Ǻ, 230-400 mesh, eluent

EtOAc/MeOH, 95:5) yielding 10 as a yellow solid residue (0.457 g, 68.51%): Rf =0.53 (EtOAc/n-

Hex, 9.5:0.5); mp: 212 °C; 1H NMR (400 MHz, [D6]DMSO) δ=11.54 (m, 1H, NH), 7.59 (s, 2H, 5-

H), 7.59 (s, 2H, 6-H), 7.40 (m, J=2.77 Hz, 1H, 8-H), 7.38 (s, 1H, 3-H), 7.08 (m, J=2.69 Hz, 1H, 9-

H), 5.48 (t, J=4.56 Hz, 1H, -CH2OH), 5.01 (d, J=4.56 Hz, 2H, -CH2OH), 2.69 ppm (s, 3H, -CH3);

13C NMR (101 MHz, [D6]DMSO) δ=25.57 (-CH3), 60.68 (-CH2OH), 102.19 (9-C), 113.38 (6-C),

116.43 (5-C), 116.44 (3-C), 118.55 (4-C), 123.94 (8-C), 124.06 (9a-C), 135.07 (6a-C), 143.48 (4a-

C), 147.97 (9b-C), 156.84 ppm (2-C); IR (ATR ZnSe): ν =3609, 3407, 1532 cm-1; UV/Vis

(MeOH): λmax (ε)=223 (828), 262 (697), 332 nm (178); Fluorescence (MeOH): λexc =261, λem =520

nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C13H13N2O: 213.0983, found: 213.1072. Purity

(RP-C18 HPLC): 97.87%.

(3-ethyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9-yl)methanol (20). According to the general

procedure, to 0.740 g (19.47 mmol) of LiAlH4 suspended in 10 mL of anhydrous THF a solution of

0.745 g (2.16 mmol) of the ethyl ester derivative 19 in in 10 mL of THF anhydrous was added

dropwise and the mixture stirred for 2 h at RT. At the end of the reaction (TLC, EtOAc/n-Hex, 1:1),

after working-up the reaction mixture, a raw green solid was obtained. Purification in a

chromatographic column (l =25 cm, Ø =2 cm, with silica gel 60 Ǻ, 230-400 mesh, eluent EtOAc/n-

Hex, 1:1) gave compound 20 as a light brown crystalline solid (0.494 g, 62.80%): Rf =0.35

(EtOAc/n-Hex, 4:6); mp: 176.5 °C; 1H NMR (300 MHz, CD3OD) δ=8.31 (t, J=1.10 Hz, 1H, 8-H),

8.20 (m, 2H, 2’- and 6’-H), 8.07 (d, J=9.24 Hz, 1H, 4-H), 8.02 (d, J=9.24 Hz, 1H, 5-H), 7.65 (m,

2H, 3’- and 5’-H), 7.59 (d, J=3.26 Hz, 1H, 2-H), 7.57 (m, 1H, 4’-H), 7.10 (d, J=3.26 Hz, 1H, 1-H),

5.50 (d, J=1.10 Hz, 2H, -CH2OH), 4.52 (q, J=7.22 Hz, 2H, -NCH2CH3), 1.63 ppm (t, J=7.22 Hz, -

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NCH2CH3); 13C NMR (75 MHz, CD3OD) δ=16.75 (-NCH2CH3), 42.70 (-NCH2CH3), 64.52 (-

CH2OH), 106.29 (1-C), 116.56 (4-C), 116.66 (5-C), 121.83 (9-C), 122.45 (9b-C), 124.40 (8-C),

128.12 (2-C), 129.05 (2’- and 6’-C), 130.20 (3’- and 5’-C), 130.27 (4’-C), 134.09 (1’-C), 141.96

(3a-C), 146.95 (9a-C), 150.31 (5a-C), 155.85 ppm (7-C); IR (ATR ZnSe): ν =3173, 2973, 1570,

1450, 1361, 1093, 1028, 705, 689 cm-1; UV/Vis (MeOH): λmax (ε)=211 (404), 236 (346), 283 (748),

326 nm (120); Fluorescence (MeOH): λexc =284, λem =525 nm; HRMS (ESI, 140 eV): m/z [M+H+]


General procedure for the synthesis of 4-methylimidazolyl- and 4-methyltriazolyl substituted

pyrroloquinolines 11-15, 21 and 22. In a 100 mL flask, the 4-methylalcohol pyrroloquinoline

derivative (0.99-1.49 mmol) was dissolved in 7 mL of NMP (N-methyl pyrrolidone) and to this

solution a five-fold molar amount of carbonyl-di-imidazole (CDI) or carbonyl-di-triazole (CDT)

was added, and the solution was heated at 180° C by an oil bath. The course of the reaction was

monitored by TLC analysis (eluent EtOAc/MeOH, 8:2). At the end of the reaction (2-10 h) an

intensely fluorescent blue spot on the TLC plate was evident. After cooling the mixture at room

temperature, 10 mL of water was added and an exhaustive extraction with either EtOAc or CH2Cl2

was made. The organic extract was repeatedly washed with a saturated NaCl aqueous solution and

water and finally evaporated to dryness to give a brown semi-solid or oily residue which was then

purified on column chromatography (EtOAc/MeOH, 8:2).

4-((1H-imidazol-1-yl)methyl)-2-phenyl-7H-pyrrolo[2,3-h]quinoline (11). Following the general

procedure, to a solution of 0.400 g (1.46 mmol) of the methylalcohol derivative 8 in 7 mL of NMP

were added 1.182 g (7:29 mmol) of CDI. After 1 h the solution became deep brown coloured and on

a TLC plate a blue fluorescent spot appeared. By working up the reaction mixture, a brown crude

oil was obtained (1.503 g), which was purified on column chromatography (l =25 cm, Ø =2 cm with

silica gel 60 Ǻ, 230-400 mesh) using EtOAC/MeOH, 8:2. as eluent. A brown oil (0.607 g) which

became a powdery solid by treatment with cold CH3CN (3 mL) was obtained yielding compound 11

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as a white powder (0.297 g, 62.80%): Rf =0.38 (EtOAc/MeOH, 9:1); mp: 260 °C; 1H NMR (300

MHz, [D6]DMSO) δ=11.73 (s, 1H, NH), 8.20 (m, 2H, 2’’- and 6’’-H), 8.06 (s, 1H, 3-H), 7.83 (d,

J=9.00 Hz, 1H, 5-H), 7.74 (dd, J=9.00 Hz and J=0.80 Hz, 1H, 6-H), 7.56 (m, 2H, 3’’- and 5’’-H),

7.53 (m, J=1.10 Hz, 1H, 5’-H), 7.50 (m, J=2.10 Hz, 1H, 8-H), 7.48 (m, 1H, 4’’-H), 7.39 (m, J=1.17

Hz, 1H, 5’-H), 7.24 (m, J=2.10 Hz and J=0.70 Hz, 1H, 9-H), 7.07 (m, J=1.07 Hz, 1H, 4’-H), 5.87

ppm (s, 2H, -CH2-); 13C NMR (75 MHz, [D6]DMSO) δ=47.15 (-CH2-), 101.72 (9-C), 113.77 (6-C),

114.39 (3-C), 115.42 (5-C), 119.02 (4-C), 120.12 (5’-C), 123.83 (9a-C), 123.99 (2’’- and 6’’-C),

124.20 (8-C), 126.51 (4’-C), 127.59 (3’’- and 5’’-C), 128.53 (4’’-C), 134.65 (6a-C), 137.58 (2’-C),

138.72 (1’’-C), 143.34 (4a-C), 144.47 (9b-C), 153.68 ppm (2-C); IR (ATR ZnSe): ν =3543, 1492

cm-1; UV/Vis (MeOH): λmax (ε)=205 (720), 230 (741), 278 (665), 350 nm (267); Fluorescence

(MeOH): λexc =286, λem =464 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C21H17N4:

325.1509, found: 355.1511, m/2z [M+2H2+] calcd for C21H18N4: 163.0781, found: 163.0796. Purity

(RP-C18 HPLC): 99.16%.

4-((1H-imidazol-1-yl)methyl)-2-(4-methoxyphenyl)-7H-pyrrolo[2,3-h]quinoline (12). Following

the general procedure, to a solution of 0.350 g (1.15 mmol) of the methylalcohol derivative 9 in 8

mL of NMP were added 0932 g (5.75 mmol) of CDI. The reaction was completed in 1 h: the

solution became deep brown coloured and on a TLC plate a blue fluorescent spot appeared. By

working up the reaction mixture, a brown crude oil was obtained 1.459 g, which was purified on

column chromatography (l =25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400 mesh) using

EtOAc/MeOH, 8:2. as eluent. It was obtained a brown oil (0.634 g), which became a powdery solid

by treatment with cold CH3CN (3 mL). Compound 12 was obtained as a beige solid product (0.257

g, 63%): Rf =0.49 (EtOAc/MeOH, 8:2); mp: 282 °C; 1H NMR (400 MHz, [D6]DMSO) δ=11.68 (m,

1H, NH), 8.16 (m, 2H, 2’’- and 6’’-H), 7.91 (s, 1H, 2’-H), 7.80 (d, J=8.96 Hz, 1H, 5-H), 7.70 (dd,

J=8.96 Hz and J=0.77 Hz, 1H, 6-H), 7.48 (m, J=2.61 Hz, 1H, 8-H), 7.45 (s, 1H, 3-H), 7.33 (t,

J=1.02 Hz, 1H, 5’-H), 7.22 (m, J=2.61, 1H, 9-H), 7.11 (m, 2H, 3’’- and 5’’-H), 6.99 (t, J=1.02 Hz,

1H, 4’-H), 5.83 (s, 2H, -CH2-), 3.85 ppm (s, 3H, -OCH3); 13C NMR (101 MHz, [D6]DMSO)

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δ=47.71 (-CH2-), 55.75 (-OCH3), 102.46 (9-C), 113.89 (3-C), 114.62 (6-C), 114.71 (2’’- and 6’’-C),

116.26 (5-C), 119.38 (4-C), 120.64 (5’-C), 124.52 (8-C), 124.59 (9a-C), 128.62 (3’’- and 5’’-C),

129.20 (4’-C), 131.98 (1’’-C), 135.42 (6a-C), 138.55 (2’-C), 144.07 (4a-C), 144.25 (9b-C), 154.21

(2-C), 160.82 ppm (4’’-C); IR (ATR ZnSe): ν =3400, 1555 cm-1; UV/Vis (MeOH): λmax (ε)=353

(380), 288 (769), 229 (798), 216 nm (699); Fluorescence (MeOH): λexc =286 nm, λem = 455.00 and

309.70 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C22H19N4O: 355.1714, found: 355.1750;

Purity (RP-C18 HPLC): 98.22%.

4-((1H-imidazol-1-yl)methyl)-7-ethyl-2-phenyl-7H-pyrrolo[2,3-h]quinoline (13). Following the

general procedure, to a solution of 0.450 g (1.49 mmol) of the methylalcohol derivative 10 in 10 mL

of NMP were added 1.207 g (7.44 mmol) of CDI. The reaction was completed in 1 h; the solution

became deep brown coloured and a blue fluorescent spot appeared on a TLC plate. By working up

the reaction mixture, a brown crude oil was obtained (2.083 g) which was purified on column

chromatography (l =25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400 mesh) using EtOAc/MeOH, 8:2.

as eluent. A brown oil (0.974 g) which became a powdery solid by treatment with cold CH3CN (4

mL) gave compound 13 as a beige solid product (0.375 g, 71.50%): Rf =0.40 (EtOAc/MeOH, 9:1);

mp: 180 °C; 1H NMR (400 MHz, [D6]DMSO) δ=8.20 (m, 2H, 2’’- and 6’’-H), 7.92 (m, J=1.04 Hz,

1H, 2’-H), 7.87 (s, 2H, 5- and 6-H), 7.56 (m, 2H, 3’’- and 5’’-H), 7.55 (m, 1H, 8-H), 7.54 (s, 1H, 3-

H), 7.48 (m, 1H, 4’’-H), 7.32 (m, J=1.17 Hz, 1H, 5’-H), 7.25 (d, J=3.01 Hz, 1H, 9-H), 6.99 (m,

J=1.07 Hz, 1H, 4’-H), 5.85 (s, 2H, -CH2-), 4.38 (q, J=7.18 Hz, 2H, -NCH2CH3), 1.43 ppm (t, J=7.18

Hz, 3H, -NCH2CH3); 13C NMR (101 MHz, [D6]DMSO) δ=16.52 (N-CH2CH3), 41.23 (N-CH2CH3),

47.67 (-CH2-), 101.96 (9-C), 113.35 (6-C), 114.67 (3-C), 116.31 (5-C), 119.80 (4-C), 120.61 (5’-C),

125.04 (9a-C), 127.30 (2’’- and 6’’-C), 127.48 (8-C), 129.22 (4’-C), 129.32 (3’’- and 5’’-C), 129.75

(4’’-C), 134.90 (6a-C), 138.53 (2’-C), 139.45 (1’’-C), 144.07 (4a-C), 144.47 (9b-C), 154.61 ppm (2-

C); IR (ATR ZnSe): ν =1509 cm-1; UV/Vis (MeOH): λmax (ε)=208 (487), 236 (588), 274 (514), 355

nm (229); Fluorescence (MeOH): λexc =286, λem =463 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd

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for C23H21N4: 353.1822, found: 353.1822, m/2z [M+2H2+] calcd for C23H22N4: 177.0978, found:

177.0973. Purity (RP-C18 HPLC): 99.29%.

4-((1H-imidazol-1-yl)methyl)-2-methyl-7H-pyrrolo[2,3-h]quinoline (14). Following the general

procedure, to a solution of 0.250 g (1.18 mmol) of the methylalcohol derivative 11 in 5 mL of NMP

were added 0.955 g (5899 mmol) of CDI. The reaction was completed in 2 h: the solution became

deep brown coloured and on a TLC plate a blue fluorescent spot appeared. By working up the

reaction mixture, a brown crude oil was obtained 0.703 g, which was purified on column

chromatography (l =25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400 mesh) using as eluent

EtOAc/MeOH, 8:2. A brown oil (0.307 g), which became a powdery solid by treatment with cold

water (3 mL), gave compound 14 as a yellow coloured product (0.189 g, 61.20%): Rf =0.38

(EtOAc/MeOH, 8:2); mp: 244 °C; 1H NMR (400 MHz, [D6]DMSO) δ=11.63 (s, 1H, NH), 7.84 (m,

J=0.97 Hz, 1H, 2’-H), 7.75 (d, J=8.99 Hz, 1H, 5-H), 7.67 (dd, J=8.99 Hz and J=0.56 Hz, 1H, 6-H),

7.45 (m, J=2.64 Hz, 1H, 8-H), 7.25 (t, J=1.20 Hz, 1H, 5’-H), 7.09 (m, J=2.64 Hz and J=0.69 Hz, 1H,

9-H), 6.98 (t, J=1.07 Hz, 1H, 4’-H), 6.70 (s, 1H, 3-H), 5.76 (s, 2H, -CH2-), 2.61 ppm (s, 3H, -CH3);

13C NMR (101 MHz, [D6]DMSO) δ=25.50 (-CH3), 47.37 (-CH2-), 102.29 (9-C), 114.09 (6-C),

116.18 (5-C), 117.25 (3-C), 118.61 (4-C), 120.67 (5’-C), 124.06 (9a-C), 124.40 (8-C), 129.15 (4’-

C), 135.26 (6a-C), 138.51 (2’-C), 143.66 (4a-C), 143.77 (9b-C), 157.08 ppm (2-C); IR (ATR ZnSe):

ν =3393, 1487 cm-1; UV/Vis (MeOH): λmax (ε)=222 (775), 264 (657), 336 nm (176); Fluorescence

(MeOH): λexc =261, λem =442 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C16H14N4:

263.1252, found: 263.1270; Purity (RP-C18 HPLC): 98.78%.

9-((1H-imidazol-1-yl)methyl)-3-ethyl-7-phenyl-3H-pyrrolo[3,2-f]quinoline (21). Following the

general procedure, to a solution of 0.450 g (1.49 mmol) of the methylalcohol derivative 20 in 15 mL

of NMP were added 1.21 g (7.44 mmol) of CDI. The mixture was heated to reflux at 170 °C. The

reaction progress was monitored by TLC, EtOAc/MeOH, 9:1. After 1 h the solution was poured

with water and extracted with EtOAc. Tre resulting organic phases were dried over sodium sulfate

and rotary evaporated to give a brown crude oil, which was purified on column chromatography (l

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=25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400 mesh) using EtOAC/MeOH, 9:1 as eluent to yield

compound 21 as a beige crystalline solid (0.372 g, 82.80%): Rf =0.68 (EtOAc/MeOH, 1:9); mp:

182.5 °C; 1H NMR (400 MHz, [D6]DMSO) δ=8.09 (d, J=8.93 Hz, 1H, 4-H), 7.99 (m, 2H, 2’’- and

6’’-H), 7.93 (m, J=1.05 Hz, 1H, 2’-H), 7.88 (d, J=8.93 Hz, 1H, 5-H), 7.68 (d, J=2.95 Hz, 1H, 2-C),

7.51 (m, 2H, 3’’- and 5’’-H), 7.44 (m, 1H, 4’’-H), 7.41 (m, J=1.05 Hz, 1H, 5’-H), 7.15 (d, J=2.95

Hz, 1H, 1-H), 7.12 (m, J=1.05 Hz, 1H, 4’-H), 7.03 (s, 1H, 8-H), 6.04 (s, 2H, -CH2-), 4.43 (q, J=7.11

Hz, 2H, N-CH2CH3), 1.45 ppm (t, J=7.11 Hz, 3H, N-CH2CH3); 13C NMR (101 MHz, [D6]DMSO)

δ=16.35 (N-CH2CH3), 41.14 (N-CH2CH3), 49.75 (-CH2-), 105.00 (1-C), 114.30 (8-C), 116.14 (4-

C), 120.08 (9-C), 120.40 (9b-C), 121.00 (5’-C), 124.04 (5-C), 126.77 (2’’- and 6’’-C), 127.72 (2-

C), 129.15 (4’-C), 129.20 (3’’- and 5’’-C), 129.30 (4’’-C), 132.44 (3a-C), 138.75 (2’-C), 139.26

(1’’-C), 144.59 (9a-C), 145.65 (5a-C), 152.38 ppm (7-C); IR (ATR ZnSe): ν =3144, 3121, 3052,

2973, 2881, 1567, 1498, 1448, 1355, 1030, 694, 664 cm-1; UV/Vis (MeOH): λmax (ε)=205 (442),

236 (317), 285 nm (686); Fluorescence (MeOH): λexc =264, λem =440 nm; HRMS (ESI, 140 eV):

m/z [M+H+] calcd for C23H21N4: 353.2059, found: 353.2395. Purity (RP-C18 HPLC): 97.90%.

4-((1H-1,2,4-triazol-1-yl)methyl)-2-(4-methoxyphenyl)-7H-pyrrolo[2,3-h]quinoline (15).

Following the general procedure, to a solution of 0.350 g (1.15 mmol) of the methylalcohol

derivative 9 in 10 mL of NMP were added 0.944 g (5.75 mmol) of CDT. The reaction was

completed in 10 h: the solution became deep brown coloured and a blue fluorescent spot appeared

on a TLC plate. By working up the reaction mixture, a brown crude oil was obtained 1.683 g.,

which was purified on column chromatography (l =25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400

mesh) using as eluent EtOAc/MeOH, 8:2. A brown oil (0.454 g), which became a powdery solid by

treatment with cold water (2 mL), yield compound 15 as a beige solid product (0.195 g, 47.70%): Rf

=0.37 (EtOAc/MeOH, 8:2); mp: 292 °C; 1H NMR (400 MHz, [D6]DMSO) δ=11.71 (s, 1H, NH),

8.75 (s, 2H, 2’- and 5’-H), 8.20 (m, 2H, 2’’- and 6’’-H), 7.80 (d, J=9.00 Hz, 1H, 5-H), 7.71 (dd,

J=9.00 Hz and J=0.60 Hz, 1H, 6-H), 7.56 (s, 1H, 3-H), 7.49 (m, J=2.65 Hz, 1H, 8-H), 7.23 (m,

J=2.11 Hz, 1H, 9-H), 7.12 (m, 2H, 3’’- and 5’’-H), 5.90 (s, 2H, -CH2-), 3.85 ppm (s, 3H, -OCH3);

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13C NMR (101 MHz, [D6]DMSO) δ=45.90 (-CH2-), 55.76 (-OCH3), 102.47 (9-C), 114.38 (3-C),

114.70 (3’’- and 5’’-C), 114.84 (6-C), 116.09 (5-C), 119.21 (4-C), 124.55 (9a-C), 124.72 (8-C),

128.74 (2’’- and 6’’-C), 131.89 (1’’-C), 135.47 (6a-C), 142.38 (4a-C), 144.14 (9b-C), 144.19 (2’-

and 5’-C), 160.87 ppm (4’’-C); IR (ATR ZnSe): ν =3473, 1495 cm-1; UV/Vis (MeOH): λmax

(ε)=204 (786), 229 (644), 287 (546), 355 nm (260); Fluorescence (MeOH): λexc =286, λem =458,

310 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C21H18N5O: 356.1467, found: 356.1466.


9-((1H-1,2,4-triazol-1-yl)methyl)-3-ethyl-7-phenyl-3H-pyrrolo[3,2-f]quinoline (22). According

to the general procedure, to a solution of 0.290 g (0.95 mmol) of the methylalcohol derivative 20 in

15 mL of NMP were added 0.78 g (4.77 mmol) of CDT. The mixture was heated to reflux at 170

°C. The reaction progress was monitored by TLC, EtOAc/MeOH, 9:1. After 48 h the solution was

poured with water and extracted with EtOAc. The resulting organic phases were dried over sodium

sulfate and rotary evaporated. The resulting crude residue was purified on column chromatography

(l =25 cm Ø =2 cm with silica gel 60 Ǻ, 230-400 mesh) using EtOAC/MeOH, 9:1 as eluent to yield

compound 22 as a ivory crystalline solid (0.264 g, yield 34%): Rf =0.36 (MeOH/EtOAc, 1:9); mp:

263 °C; 1H NMR (400 MHz, [D6]DMSO) δ=8.75 (s, 2H, 2’- and 5’-H), 8.12 (dd, J=9.07 Hz and

J=0.47 Hz, 1H, 4-H), 8.02 (m, 2H, 2’’- and 6’’-H), 7.90 (d, J=9.07 Hz, 1H, 5-H), 7.69 (d, J=3.24

Hz, 1H, 2-H), 7.52 (m, 2H, 3’’- and 5’’-H), 7.45 (m, 1H, 4’’-H), 7.13 (dd, J=3.24 Hz and J=0.58

Hz, 1H, 1-H), 7.10 (s, 1H, 8-H), 6.12 (s, 2H, -CH2-), 4.43 (q, J=7.14 Hz, 2H, N-CH2CH3), 1.45 ppm

(t, J=7.14 Hz, 3H, N-CH2CH3); 13C NMR (101 MHz, [D6]DMSO) δ=16.33 (N-CH2CH3), 41.16 (N-

CH2CH3), 47.88 (-CH2-), 104.82 (1-C), 114.48 (8-C), 116.31 (4-C), 119.96 (9-C), 120.22 (9b-C),

124.05 (5-C), 126.88 (2’’- and 6’’-C), 127.88 (2-C), 129.21 (3’’- and 5’’-C), 129.38 (4’’-C), 132.51

(3a-C), 139.16 (1’’-C), 143.02 (9a-C), 144.93 (2’- and 5’-C), 145.72 (5a-C), 152.41 ppm (7-C); IR

(ATR ZnSe): ν =3088, 2964, 1566, 1521, 1450, 1352, 769, 693, 634 cm-1; UV/Vis (MeOH): λmax

(ε)=204 (359), 238 (322), 286 (721), 326 (103), 345 nm (288); Fluorescence (MeOH): λexc =286,

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λem =441 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for C22H20N5: 354.1674, found: 354.1660.


General procedure for the synthesis of 4-carboxilyc acid pyrroloquinoline derivatives 16, 17

and 23.

In a 50 mL flask, a solution of the ester derivative 4, 6 or 7 (0.43-0.59 mmol) in 10 mL of methanol

was added of 20% NaOH aqueous solution (3 mL) and heated at refluxing. The reaction ended after

about 2 h (TLC, EtOAc/MeOH, 8:2), when only a intensely fluorescent spot on the starting line was

observed. The reaction solvent was then removed, 5 mL of water added to the remained aqueous

solution (alkaline pH) and an extraction was carried out with ethyl acetate to remove any impurities

and un-reacted starting product. The aqueous phase was made acidic with 3 drops of 37% HCl to

precipitate the desired product, which was obtained pure by collecting it by filtration, washing with

water and drying it under vacuum.

2-(4-Methoxyphenyl)-7H-pyrrolo[2,3-h]quinoline-4-carboxylic acid (16). It was followed the

general procedure, starting from 0.150 g (0.43 mmol) of ester 4, leading to compound 16 as an

intense yellow solid that did not require further purification (0.125 g, 90.70%): mp: 203 °C; 1H

NMR (400 MHz, [D6]DMSO) δ=11.80 (m, 1H, NH), 8.32 (m, 2H, 2’- and 6’-H), 8.26 (s, 1H, 3-

H), 8.23 (d, J=9.10 Hz, 5-H), 7.64 (dd, J=9.10 Hz and J=0.55 Hz, 1H, 6-H), 7.51 (m, J=2.66 Hz,

1H, 8-H), 7.29 (m, J=2.11 Hz, 1H, 9-H), 7.13 (m, 2H, 3’- and 5’-H), 3.86 ppm (2, 3H, -OCH3); 13C

NMR (101 MHz, [D6]DMSO) δ=55.78 (-OCH3), 102.60 (9-C), 114.78 (3’- and 5’-C), 115.47 (3-

C), 116.68 (6-C), 118.34 (5-C), 118.50 (4-C), 123.33 (9a-C), 124.76 (8-C), 129.04 (2’- and 6’-C),

131.11 (1’-C), 135.44 (6a-C), 138.75 (4a-C), 144.41 (9b-C), 153.78 (2-C), 161.09 (4’-C), 169.01

ppm (-COOH); UV/Vis (MeOH): λmax (ε)=205 (837), 228 (695), 286 (663), 353 nm (268);

Fluorescence (MeOH): λexc =286, λem =503, 310 nm; HRMS (ESI, 140 eV): m/z [M+H+] calcd for

C19H15N2O3: 319.1038, found: 319.1024. Purity (RP-C18 HPLC): 98.04%.

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2-Methyl-7H-pyrrolo[2,3-h]quinoline-4-carboxylic acid (17). It was followed the general

procedure, starting from 0.133 g (0.59 mmol) of ester 6, obtaining an intense orange solid

corresponding to compound 17 that did not require further purification (0.123 g, 92.20%): mp: >

290 °C; 1H NMR (400 MHz, [D6]DMSO) δ=12.64 (m, 1H, NH), 8.19 (d, J=9.10 Hz, 1H, 5-H),

8.09 (m, 1H, 9-H), 8.01 (d, J=9.10 Hz, 1H, 6-H), 8.01 (s, 1H, 3-H), 7.69 (m, 1H, 8-H), 3.10 ppm

(s, 3H, -CH3); 13C NMR (101 MHz, [D6]DMSO) δ=20.69 (-CH3), 104.39 (9-C), 116.99 (9a-C),

117.78 (6-C), 118.49 (5-C), 119.55 (4-C), 120.20 (3-C), 126.64 (8-C), 136.25 (4a-C), 137.28 (6a-

C), 145.08 (9b-C), 155.67 (2-C), 167.29 ppm (-COOH); UV/Vis (MeOH): λmax (ε)=221 (646), 279

(462), 348 nm (150); Fluorescence (MeOH): λexc =261, λem =521 nm; HRMS (ESI, 140 eV): m/z

[M+H+] calcd for C13H11N2O2: 227.0876, found: 227.0926. Purity (RP-C18 HPLC): 98.94%.

3-ethyl-7-phenyl-3H-pyrrolo[3,2-f]quinoline-9-carboxylic acid (23). According to the general

procedure, starting from 0.640 g (1.87 mmol) of ester 19 and obtaining an intense orange solid which

corresponded to compound 23 as an orange crystalline solid (0.169 g, 84.90%): Rf =0.55

(EtOAc/MeOH, 7:3); mp: > 280 °C; 1H NMR (300 MHz, CD3OD) δ=8.42 (dd, J=9.28 Hz and J=0.60

Hz, 1H, 4-H), 8.33 (s, 1H, 8-H), 8.11 (d, J=9.28 Hz, 1H, 5-H), 8.09 (m, 2H, 2’- and 6’-H), 7.73 (m,

3H, 3’-, 4’- and 5’-H), 7.71 (d, J=3.25 Hz, 1H, 2-H), 7.23 (dd, J=3.25 Hz and J=0.72 Hz, 1H, 1-H),

4.50 (q, J=7.32 Hz, 2H, -NCH2CH3), 1.55 ppm (t, J=7.32 Hz, 3H, -NCH2CH3); 13C NMR (75 MHz,

CD3OD) δ=16.74 (-NCH2CH3), 43.18 (-NCH2CH3), 105.34 (1-C), 114.88 (8-C), 119.41 (4-C), 120.50

(5-C), 122.18 (9b-C), 122.52 (9-C), 130.35 (2’- and 6’-C), 131.24 (3’- and 5’-C), 131.51 (4’-C),

133.51 (2-C), 133.86 (3a-C), 135.05 (9a-C), 139.12 (1’-C), 148.97 (5a-C), 152.67 (7-C), 170.77 ppm

(-COO-); IR (ATR ZnSe): ν =3322-3232, 3082, 2979, 1647, 1601, 1538, 1495, 1447, 1362, 1212,

1028, 745, 670 cm-1; UV/Vis (MeOH): λmax (ε)=204 (630), 242 (499), 287 (858), 350 (176), 254

(450), 217 nm (299); HRMS (ESI, 140 eV): m/z [M+H+] calcd for C20H17N2O2: 317.1245, found:

317.2121. Purity (RP-C18 HPLC): 98.80%.

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Biology

Determination of aromatase inhibition

Inhibition of aromatase (CYP19) by synthesized compounds was determined using the P450

Inhibition Kit CYP19/MFC (BD Biosciences, Milano Italy) according to the manufacturer’s

instructions. Briefly, 100 ml of each compound was diluted in NADPH-cofactor mix for every

concentration tested (10000, 3333, 1111, 370, 123, 41, 13, 4 and 0 nM) and placed in duplicate on a

96-well plate. The plate was then incubated at 37 °C for 10 min. After incubation, 100 µl of the

enzyme/substrate mix was added to the treated conditions and the plate was then incubated at 37°C

for 30 min. After incubation, 75 µl of Stop Reagent was added to the entirety of the plate and 100

ml of the enzyme/substrate mix was added in the blank columns. Thereafter, the plate was subjected

to a fluorimetric analysis and read on a fluorescence microplate reader (Victor3 Perkin –Elmer

λex=410 nm λem=530 nm).

Aromatase activity assay in H295R cell line.

Aromatase activity in subconfluent H295R cells was measured by the tritiated water release assay29.

Briefly, H295R cells were cultured in DMEM-F12 (1:1) and seeded at 106 cells/well on six-well

plates and treated with different concentrations of the test compounds for 24 h. Letrozole was added

as reference compound at the concentration of 5 µM. At the end of the treatment the cells were

incubated with 0.5 µM [1β-3H(N)]-androst-4-ene-3,17-dione (25.3 Ci/mmol; DuPont NEN, Boston,

MA) as substrate. The results were expressed as percentage of decrease (or increase) of 3H2O

release, respect to the untreated cells.

Inhibitory activity of CYP11B1

V79MZh cells expressing human CYP11B1 were incubated with [1,2-3H]-11-deoxycorticosterone

as substrate and the inhibitor at different concentrations. The assay was performed as previously

described38,39.

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CYP17 Preparation and Assay.

The inhibition of CYP17 was determined using the 50000g sediment of E. coli coexpressing human

CYP17and cytochrome P450 reductase40 with progesterone as substrate and NADPH as cofactor41.

Antiproliferative assays. Human T-leukemia (Jurkat), human B-cell leukemia RS4;11 and human

B-cell leukemia SEM, both with t(4;11) translocation were grown in RPMI-1640 medium, (Gibco,

Milano, Italy). Breast adenocarcinoma (MCF7), human cervix carcinoma (HeLa), human colon

adenocarcinoma (HT-29) human ovarian carcinoma cell lines IGROV-1 and OVCAR-3 were grown

in DMEM medium (Gibco, Milano, Italy), all supplemented with 115 units/mL of penicillin G

(Gibco, Milano, Italy), 115 µg/mL streptomycin (Invitrogen, Milano, Italy) and 10% fetal bovine

serum (Invitrogen, Milano, Italy). Human adrenocarcinoma cells, H295R were cultured in DMEM-

F12 1:1 additioned of 2% fetal bovine serum and supplemented with Insulin-transferrin-selenium

(ITSX Invitrogen, Milano, Italia). Stock solutions (10 mM) of the different compounds were

obtained by dissolving them in DMSO. Individual wells of a 96-well tissue culture microtiter plate

were inoculated with 100 µL of complete medium containing 8x103 cells. The plates were incubated

at 37 °C in a humidified 5% CO2 incubator for 18 h prior to the experiments. After medium

removal, 100 µL of fresh medium containing the test compound at different concentrations, were

added to each well and incubated at 37 °C for 72 h. The percentage of DMSO in the medium never

exceed 0.25% .Cell viability was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) test as previously described.22 The GI50 was defined as the compound

concentration required to inhibit cell proliferation by 50%.

Flow cytometric analysis of cell cycle distribution. For flow cytometric analysis of DNA content,

5x105 HeLa or MCF7 cells in exponential growth were treated with different concentrations of the

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test compounds for 24 h. After thisperiod, the cells were collected, centrifuged and fixed with ice-

cold ethanol (70%). The cells were then treated with lysis buffer containing RNAse A and 0.1%

Triton X-100 and then stained with PI. Samples were analyzed on a Cytomic FC500 flow cytometer

(Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow

Systems).

Molecular Modeling Studies. All pyrrolo[2,3-h] and pyrrolo[3,2-f]quinoline inhibitors were built

and their partial charges calculated after semi-empirical (AM1) energy minimization using the

Builder module of MOE201142. Chain A of the crystal structure of human CYP19 (placental

aromatase cytochrome P450 in complex with 4-androstene-3-17-dione; PDB code: 3EQM)35 was

retrieved from the Protein Databank (PDB, http://www.rcsb.org). Predocking procedure included

deletion of ligand and water molecules from the 3D protein structure and the addition of hydrogen

atoms to the protein. Protonate 3D, a module in MOE suite, was used to assign protons to donor

side chains. The final structure was energy optimized with the backbone atoms constraint with 1

kcal/mol using the LigX module of MOE2011.

All inhibitors were then docked using GOLD v5.0143 with the CYP dedicated scoring function

goldscore.p450_pdb.params. The active site was defined as all residues within 12 Å of the formerly

present androstenedione. Each ligand was docked 50 times. Default parameters were used

otherwise.

Physicochemical and ADME Properties. The predicted ADME and physicochemical properties

have been calculated using StarDrop program.25

ASSOCIATED CONTENT

Supporting Information. 1H and 13C NMR spectra of all final compounds; HMBC and NOESY

spectra of compound 12 and HMBC spectrum of compound 21; antiproliferative activity of all

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compounds; cell cycle distribution analysis of compounds 8, 11, 13, 14, 21, and 22 and Letrozole;

combustion analysis data of all compounds; physicochemical properties; details of molecular

dynamics simulations are also summarised. This material is available free of charge via the Internet

at http://pubs.acs.org

CORRERSPONDING AUTHOR

Maria Grazia Ferlin

AUTHOR INFORMATIONS

Department oh Pharmaceutical and Pharmacological Sciences

Via Marzolo, 5

35131 Padova

Fax: (+39) 0498275366

Fhone: (+39) 0498271603

E-mail: [email protected]

ACKNOWLEDGMENT. The molecular modeling work coordinated by S.M. was carried out with

financial support from the University of Padova, Italy, and the Italian Ministry for University and

Research (MIUR), Rome, Italy. S.M. is also very grateful to Chemical Computing Group for the

scientific and technical partnership. Grant to VP from Associazione Italiana per la Ricerca sul

Cancro (AIRC) project n. IG10344

ABBREVIATIONS USED: AI, aromatase inhibitors; PPyQ, phenylpyrroloquinolinone; PyQ,

pyrroloquinoline; r.a., residual activity; SAR, structure-affinity relationship; RMSD, root mean

square deviation; MOE, Molecular Operating Environment; DMEM, Dulbecco’s Modified Eagle’s

Medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium

bromide.

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Legends, figures, tables and schemes

Figure 1. Structures of Aromatase inhibitors.

Figure 2. 2-Phenylpyrroloquinolin-4-ones and the designed molecules.

Figure 3. Structural similarities between the aromatase substrate androstenedione and the proposed

inhibitors 4-azolylmethyl-PQs.

Figure 4. HMBC (panel A, blue arrows) and NOE (panel B, red arrows) correlations for compound

12, taken as an example among [2,3-h] compounds.

Figure 5. The inhibition of aromatase activity in H295R cells by test compounds. H295R cells were

cultured for 24 h in DMEM-F12 (1:1) in the presence of the test compounds at the concentration of

0.1, 1.0 and 10 µM. Letrozole was also added as reference compound at the concentration of 5 µM.

After the treatment (24 h), the cells were further incubated for 2h with 0.5 µM of [1β-3H(N)]-

androst-4-ene-3,17-dione as substrate and then aromatase activity was assessed using a tritiated

water release assay. Results are expressed as percentage of [3H]H2O released respect to untreated

cells. Values represent means ± SEM of two independent experiments, each performed in triplicate..

Figure 6. Effect of compounds 8, 11 21 and 22 on cell cycle distribution of HeLa (right panels) and

MCF-7 cells (left panels). Cells were treated with different concentrations of the indicated

compounds, ranging from 6 to 50 µM for 24 h. Then the cells were fixed and stained with PI to

analyze DNA content by flow cytometry.

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Figure 7. Most energetically favoured docking poses obtained for compound 21 inside the catalytic

site of the placental aromatase cytochrome P450. To aid visualization, a zoom of the heme

prosthetic group region is partially performed and hydrogen atoms are not displayed. Side chains of

some amino acids important for ligand recognition and the iron-N(ligand) coordination are

highlighted.

Table 1. Inhibition of CYP19, CYP11B1 and CYP17 by compound 3-17 and 19-23

Table 2. Growth inhibition activity (GI50) of the new PyQs derivatives 11-15, 21 and 22.

Scheme 1. Synthesis of 4-substituted pyrrolo[2,3-h]quinoline derivatives 3-17.

Scheme 2. Synthesis of 9-substituted pyrrolo[3,2-f]quinoline derivatives 19-23.

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Figure 1

First generationNon-steroidal competitive inhibitors

NO O

C2H5

NH2

H

aminoglutethimide

NO O

C2H5

N

H

rogletimide (pyridoglutethimide)

Second generationSteroidal, mechanism based inhibitors

O

OCH3

CH3

OH

O

OCH3

CH3

CH2

Non-steroidal competitive inhibitors

N

N

CN fadrozole

exemestanelentanor

Third generationNon-steroidal competitive inhibitors

N

NNNC

CN

N

NN

CN

H3C CH3

NC

CH3H3CN

N

NCH

N

N

N

Cl

CH3

letrozole arimidex vorozole

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51

Figure 2

NN

NX

N

R1NH

O

N

R2

R1

X = CH, N R2

2-phenyl-pyrroloquinolinones 4-azolylmethyl-pyrroloquinolines

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Figure 3

NHN

N

N

X

A

O

A

BC

DO

BC

D

Androstenedione 4-methylazolyl-pyrroloquinolines

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Figure 4.

A

NHN

NN

O

HMBC correlations for compound 12.

B

NHN

NN

O

NOE correlations for compound 12.

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Figure 5

0

25

50

75

100

125

Alcohol

Aro

mata

se a

ctivity

(% o

f contr

ol)

0.1 µM

1.0 µM

10 µM

Ctr 3 4 5 6 19 7 8 9 10 20 Letr

Ester

0

25

50

75

100

125

150

175

Aro

ma

tase

activity

(% o

f co

ntr

ol)

0.1 µM

1.0 µM

10 µM

Ctr 11 12 13 14 21 15 22 16 17 23 Letr

Imidazol Triazol Acid

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Figure 6

0 10 20 30 40 500

20

40

60

80

100

Ce

lls (

%)

Concentration (µM)

G1

G2/M

S

8

0 10 20 30 40 500

20

40

60

80

100

Ce

lls (

%)

Concentration (µM)

G1

G2/M

S

8

0 10 20 30 40 500

20

40

60

80

100

Ce

lls (

%)

Concentration (µM)

G1

G2/M

S

11

0 10 20 30 40 500

20

40

60

80

100

Cells

(%

)

Concentration (µM)

G1

G2/M

S

11

0 10 20 30 40 500

20

40

60

80

100

Ce

lls (

%)

Concentration (µM)

G1

G2/M

S

21

0 10 20 30 40 500

20

40

60

80

100

Cells

(%

)

Concentration (µM)

G1

G2/M

S

21

0 10 20 30 40 500

20

40

60

80

100

Cells

(%

)

Concentration (µM)

G1

G2/M

S

22

0 10 20 30 40 500

20

40

60

80

100

Cells

(%

)

Concentration (µM)

G1

G2/M

S

22

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Figure 7

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TABLE 1. Inhibition of CYP19, CYP11B1 and CYP17 by compound 3-17 and 19-23

aIC50= compound concentration required to inhibit enzimatic activity by 50%. The inhibition of

CYP19, CYP11B1 and CYP17 was carried out as described in the Experimental Section. Data

are presented as the mean ± SEM from three independent experiments performed in duplicate.

n.d. Not determined. b Data taken from REF. 14

COMPD CYP19

IC50 (nM)a CYP11B1

IC50 (nM)a CYP17

IC50 (nM)a

ester

3 >10000 n.d. n.d. 4 >10000 n.d. n.d. 5 >10000 n.d. n.d. 6 >10000 n.d. n.d. 19 >10000 n.d. n.d.

alcohol

7 2112±187 n.d. n.d. 8 922±85 n.d. n.d. 9 >10000 n.d. n.d. 10 4152±212 n.d. n.d. 20 >10000 n.d. n.d.

imidazole

11 11.4±1.5 230±18 910±52 12 454±36 960±87 550±67 13 5.3±0.6 250±34 480±71 14 6.0±0.7 75±16 >5000 21 3.1±0.3 1010±98 2160±180

triazole 15 >10000 >5000 >5000 22 13.3±1.5 1480±118 >5000

acid

16 >10000 n.d. n.d. 17 6195±256 n.d. n.d. 23 1472±53 n.d. n.d.

2-PPyQ 590±29 n.d. n.d. Letrozole 3.4±0.5 2620b 7b

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Table 2. Growth inhibition activity (GI50) of the pyrroloquinoline derivatives 11-15, 21 and 22, 2-PPyQ and Letrozole

COMPD GI50 (M)

a

RS 4;11 SEM Jurkat HeLa HT-29 MCF-7 IGROV-1 OVCAR-3 H295R

imidazole

11

12

13

14

21

29.6±1.9

23.3±1.9

20.7±6.7

34.7±0.5

9.0±2.5

17.0±2.7

25.5±5.8

31.2±6.7

16.8±1.7

4.2±0.1

27.0±2.1

73.8±10.9

16.7±2.6

23.8±5.9

2.2±0.4

21.2±1.2

67.9±9.7

19.5±2.2

>100

26.2±6.0

13.5±1.7

>100

6.5±0.62

89.3±9.0

15.0±0.7

23.3±2.9

>100

3.8±0.2

54.6±1.7

12.5±4.0

23.6±1.6

>100

18.9±2.1

>100

19.4±0.9

16.9±0.23

>100

17.5±2.1

40.8±11.8

21.5±3.9

36.5±5.7

46.2±6.4

19.0±7.2

51.7±7.4

3.8±0.77

triazole

15

22

43.8±8.9

2.7±0.3

28.4±1.8

5.5±1.6

39.5±6.5

65.8±3.8

>100

59.8±6.4

>100

29.5±1.5

>100

23.0±1.5

82.0±4.7

54.3±4.3

50.6±10.1

13.6±3.8

>100

75.3±3.4

2-PPyQ 25.0±1.5 35. ±0.3 21.2±1.9 38.4±5.9 17.7±5.9 0.81±0.15 0.92±0.14 0.39±0.1 0.7±0.2

Letrozole >100 >100 >100 >100 >100 >100 >100 >100 >100 aGI50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean ± SE from the dose-response curves of at least three independent experiments.

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SCHEME 1

N

NH2

R1

N R2

COOC2H5

NR1

N

CH2OH

NR1N R2N

NN

R1

N R2

COOH

NR1

N R2N

NN

N

R1

a

b

c d

e

Reagents e conditiones:

a) benzaldehyde, p-methoxy-benzaldehyde, acetaldehyde,

ethyl pyruvate, absolute ethanol, HCl 36%, 100°C, 3 h;

b) LiAlH4, THF anhydrous, rt, 2 h;

c) CDI, NMP, 170°C, 2 h;

d) CDT, NMP, 170°C, 8 h;

e) MeOH, NaOH 20%, reflux, 2 h.

3-6

7-1011-14

R2

15

16, 171, 2

R1R2

1, 3, 7, 11 H phenyl 4, 8, 12, 15, 16 H p-OCH3-phenyl2, 5, 9, 13 C2H5 phenyl 6, 10, 14, 17 H CH3

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SCHEME 2

N

C2H5

N

COOC2H5

N

NN

N

COOH

N

NN

N

a

b

c d

eH2N

NC2H5

N

CH2OH

NC2H5N

N

NC2H5 C2H5

C2H5

18 19

2021 22

23

Reagents e conditiones: a) benzaldehyde, ethyl pyruvate, absolute ethanol, HCl 36%, 100°C, 3 h;

b) LiAlH4, THF anhydrous, rt, 2 h. c) CDI, NMP, 170°C, 2 h; d) CDT, NMP, 170°C, 8 h;

e) MeOH, NaOH 20%, reflux, 2 h.

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Table of Contents Graphic

NR

N

N

XN

N

R1

R1

f

h

X = C, NR = phenyl, CH3R1 = H, C2H5

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Documents

Design, Synthesis, and Structure–Activity Relationships of Azolylmethylpyrroloquinolines as Nonsteroidal Aromatase Inhibitors