Harnessing the pyrroloquinoxaline scaffold for FAAH and MAGL interaction: Definition of the structural determinants for enzyme inhibition

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Marzo and S. Gemma, RSC Adv., 2016, DOI: 10.1039/C6RA12524G.

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Received 00th January

20xx,

Harnessing the pyrroloquinoxaline scaffold for FAAH and MAGL

interaction: definition of the structural determinants for enzyme

inhibition

Margherita Brindisi,^a,bSimone Brogi,^a,bSamuele Maramai,^a,bAlessandro Grillo,^a,bGiuseppe

Borrelli,^a,bStefania Butini,^a,b,*Ettore Novellino,^a,cMarco Allarà,^dAlessia Ligresti,^dGiuseppe

Campiani,^a,b,*Vincenzo Di Marzo,^dSandra Gemma^a,b

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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This paper describes the development of piperazine and 4-aminopiperidine carboxamides/carbamates supported on a

pharmacogenic pyrroloquinoxaline scaffold as inhibitors of the endocannabinoid catabolizing enzymes fatty acid amide

hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Structure–activity relationships and molecular modelling studies

allowed the definition of the structural requirements for dual FAAH/MAGL inhibition and led to the identification of a

small set of derivatives (compounds 5e,i,k,m) displaying a balanced inhibitory profile against both enzymes, with

compounds 5m being the frontrunner of the subset. Favorable calculated physico-chemical properties suggest further

investigation for specific analogues.

also on cancer and neuroinflammatory diseases.¹⁴

NO₂

Introduction

O

N

Endocannabinoids (ECs) N-arachidonoylethanolamine (AEA) and 2-

arachidonoylglycerol (2-AG) stimulate cannabinoid CB1 and CB2

receptors (CB1R and CB2R)¹thus modulating several physiological

responses, including nociception, anxiety, and depression.^2-4The

two key players involved in EC catabolism are the fatty acid amide

hydrolase (FAAH) and the monoacylglycerol lipase (MAGL)

enzymes.^5,6Additionally, the α/β-hydrolase domain containing

serine hydrolases, ABHD6 and ABHD12, may act as complementary

2-AG-degrading enzymes in the brain.⁷

O

N

NH

O

N

O

1, PF750

2

, JZL195

F

O

H

N

O

6

FAAH and MAGL are membrane-bound serine hydrolases,

which regulate the intracellular levels of AEA, 2-AG and other

ECs.^8,9FAAH utilizes a Ser-Ser-Lys catalytic triad as confirmed

by X-ray analysis,¹⁰and MAGL has a catalytic triad represented

by Ser-His-Asp.¹¹By elevating the endogenous concentration

of their substrates, FAAH and MAGL inhibition can promote

beneficial effects on several nervous system disorders,

including pain, inflammation, anxiety, and depression^4,12,13but

O

N

O

N

CONH₂

3, ST3913

4, NSD1819

O

-OBn

-NHBn

-NHPh

O

H

N

-OPh-pNO₂

N

R₂

7-F

7-Cl

7,8-diMe

R₁

1H-benzotriazolyl

1H-1,2,4-triazolyl

1H-1,2,3-triazolyl

1H-imidazolyl

X

N

Y

piperazine or

4-aminopiperidine

5a-n

6a-c

and

R₁, R₂, X and Y as defined in Table 1

Fig. 1. Reference FAAH, MAGL and dual inhibitors (1-4), and title compounds

5a-n and 6a-c.

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Accordingly, selective inhibition of FAAH and MAGL represents

an attractive approach for eliciting the desirable effects of CBR

activation with the remarkable benefit of avoiding the classical

CBR agonists side effects (hypomotility, hypothermia,

catalepsy, and psychotropic effects).¹⁵

A large number of FAAH inhibitors have been described over

the last years, such as

carbamates, and piperidine-/piperazine-based ureas.¹⁶To this

latter class belong the irreversible piperidine urea compound

α-keto heterocycles, lactams,

PF750 (1

, Fig. 1)¹⁷and the reversible piperazine urea JNJ-

1661010.¹⁸P750, however, may be classified as a weak FAAH

selective inhibitor.

Recently, Cravatt and co-workers described MAGL piperidine

carbamate selective inhibitors (e.g. JZL184¹⁹and KLM-29²⁰) and

piperazine carbamates dual FAAH/MAGL inhibitors, such as

JZL195²¹(2, Fig. 1). JZL195 binds both murine brain enzymes in the

nanomolar range; however, it is characterized by the hazardous p-

nitrophenylcarbamate functionality. Recently, Aaltonen and co-

workers reported the development of potent piperazine and

piperidine urea MAGL inhibitors.²²In the frame of our research

involving endocannabinoid metabolizing enzyme inhibitors, we

recently developed phenyl-1-pyrrole-based compounds (typified by

compound 3, Fig. 1) as ultrapotent and selective FAAH reversible

24

inhibitors^23,and the beta-lactam-based piperidintriazole urea 4

(Fig. 1) as an exceptionally potent and selective MAGL inhibitor.^{25, 26}

Increasing evidence highlights that a full spectrum of activities is not

observed upon inhibition of either FAAH or MAGL enzymes alone,^27-

29

therefore exploitation of compounds endowed with a dual

inhibition profile could represent a valuable therapeutic approach

in pathological states such as neuropathic pain³⁰and epilepsy.³¹

Remarkably, the enhancement of both the AEA and 2-AG signalling

pathways enables these two ECs to engage in extensive crosstalk.²¹

A critical balance between AEA and 2-AG levels, controlled by their

synthesizing and metabolizing enzymes, may thus be of pivotal

importance for normal physiological processes, as well as to face

pathological events.^32-34Accordingly, systemic administration of the

dual FAAH/MAGL inhibitor JZL195 (2) reduced allodynia in a chronic

constriction injury model of neuropathic pain with greater efficacy

with respect to selective FAAH or MAGL inhibitors. Notably, 2 also

displayed a better therapeutic window between anti-allodynia and

side effects than the CBR agonist WIN55212 and retained anti-

allodynia efficacy during repeated treatment.³⁰

Scheme 1. Synthetic procedures for the preparation of compounds 5a-n and

6a-c. Reagents and conditions: (a) Ac₂O, TEA, dry DCM, 0 °C to rt, 1 h; (b)

HNO₃, Ac₂O, AcOH, 0 °C to rt, 12 h; (c) 6N HCl, reflux, 4 h; (d) 2,5-

dimethoxytetrahydrofuran,

5 N HCl, 1,4-dioxane, 110 °C, 5 min; (e)

SnCl₂∙H₂O, EtOAc, reflux, 2 h; (f) AcCl, pyridine, dioxane, reflux, 4 h; (g)

POCl₃, toluene, reflux, 4 h; (h) SeO₂, 1,4-dioxane, reflux, 4 h; (i) benzyl

isocyanate for 14b, phenyl isocyanate for 14c or p-nitrophenyl

chloroformate for 14d, Et₃N, dry THF, 25 °C, 12 h; 1H-1,2,4-triazole for 14e,

1H-1,2,3-triazole for 14f or 1H-benzotriazole for 14h, phosgene (20 % in

toluene), DMAP, dry DCM, 25 °C, 12 h; CDI for 14g, dry DCM, 25 °C, 12 h; (j)

TFA, dry DCM, 25 °C, 2 h; (k) 1H-benzotriazole, phosgene (20 % in toluene),

Furthermore, the dual FAAH/MAGL inhibitor AM6701 resulted

more viable against excitotoxic brain injury with respect to the

FAAH selective analogue AM6702, showing protection against

cytoskeletal damage and synaptic decline in both a brain slice

model and in vivo, and seizure scores and behavioural deficits were DMAP, dry DCM, 25 °C, 12 h; (l) for 5a-c,e: NaCNBH₃, EtOH/AcOH 1%, 25 °C,

reduced in the kainic acid rat model.³¹

12 h; for 5d, 5f-n, 6a-c: Na(OAc)₃BH dry DCM, 25 °C, 12 h.

As a continuation of our efforts in this field, we herein describe the

development and structure-activity relationships of a series of novel

FAAH and MAGL inhibitors (5a-n and 6a-c, Fig. 1 and Table 1)

bearing the pyrroloquinoxaline pharmacogenic system as the key

substructure for development of drug-like compounds.^35-42In the

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specific frame of selective or dual acting FAAH/MAGL inhibitors, we incubation time of 10 and 60 minutes. In agreement with the

envisaged that the appropriate combination and outdistancing mechanism of action of “serine trap inhibitors”, the tested

between the pyrroloquinoxaline-based scaffold and a piperazine compounds displayed an increased enzyme inhibitory potency

carboxamide/carbamate⁴³portion could drive to compounds when pre-incubated for 10 min with the enzyme. This pre-

characterized by suitable size and geometrical shape for fitting both incubation time is satisfactory to reach the maximum activity, since

enzymes’ binding pockets. To this aim we synthesized compounds the same compounds, when assayed with 60 min pre-incubation,

5a-n (Fig.

1

and Table 1) in which the piperazine did not show further significant improvement of their inhibitory

carboxamide/carbamate moiety was connected to the C-4 of the potency (Table 1).

pyrroloquinoxaline skeleton by a methylene spacer, perceived as

The analysis of these biological data was performed by means of

flexibility element for multiple enzyme adaptation. Also compounds molecular docking studies which allowed us to better define the key

6a-c (Fig. 1 and Table 1) were synthesised, in which the piperazine elements for the inhibition of these ECs’ catabolizing enzymes.

moiety was replaced by a 4-aminopiperidine. Inhibition potency of

The first sub-series of compounds, namely 5a-h (Table 1),

the developed compounds was evaluated on FAAH (from rat brain) feature an unsubstituted pyrroloquinoxaline skeleton bridged at C-4

and MAGL (from COS cells and rat brain) enzymes. Classical by a methylene linker to a terminal piperazine carbamate/urea

medicinal chemistry approaches coupled with computational moiety, as the potential electrophilic centre to undergo nucleophilic

analysis and biological evaluation led to the definition of key attack by the target enzymes. In particular, the benzyl carbamate

molecular insights for both selective or dual FAAH/MAGL inhibition. (5a) and the benzylurea (5b) analogues showed poor inhibition

5e is the prototype of this new class of inhibitors, which led to 5m, potency on both enzymes being too long to correctly fit within the

an analogue with a potency comparable to JZL195 but without the FAAH and MAGL active sites (not shown). A terminal phenylurea

structural drawbacks mentioned above.

(5c) slightly improved potency against FAAH (IC₅₀= 960 nM, Table

1). In fact, docking studies indicated a better positioning within the

FAAH catalytic site of 5c, reaching the central region of the FAAH

active site with the phenylurea moiety projected towards F244.

Only limited interactions, such as π-π stacking with F192, were

observed and no other contacts with the key aromatic residues of

the binding site were detected (not shown). Although poorly

druggable, but in line with reference FAAH/MAGL inhibitors such as

compound 2, we explored the effect of a p-nitrophenylcarbamoyl

moiety on our pyrroloquinoxaline-based inhibitors. Derivative 5d

displayed submicromolar inhibition potency for both FAAH and

MAGL (FAAH IC₅₀= 562 nM; MAGL IC₅₀= 826 nM, Table 1) enzymes.

Introduction of a smaller 1,2,4-triazole urea, a moiety which already

demonstrated an optimum potential for MAGL inhibition, robustly

improved inhibition.²²Gratifyingly, compound 5e showed one of

the best inhibitory profile among the synthesized compounds with

inhibition potencies in the low nanomolar range against FAAH (IC₅₀

= 37 nM, rat brain;) and submicromolar for MAGL (IC₅₀= 44.66 nM,

611.37 nM, COS and rat brain, respectively; Table 1). The output of

our docking calculations performed by using Induced Fit Docking

(IFD) via Maestro GUI as previously described by us^45-47(Fig. 2)

confirmed the crucial role of the acyltriazole portion for the dual

inhibitory activity, coupled to the presence of the methylene linker

bridging the pyrroloquinoxaline core and the piperazine moiety. In

fact, 5e revealed a strong pattern of interaction with both target

enzymes. The compound spans the gorge of the FAAH enzyme

mainly interacting by hydrophobic contacts with the key residues as

well as with the catalytic triad (Fig. 2A). The triazole moiety is able

to establish a π-π stacking with F192, while the tricyclic portion of

5e establishes two π-π stacking with F192 and F432. Moreover a

series of hydrophobic contacts with I238, L380, F381, L401, L404

M436, and M495 was observed. Additionally, 5e also H-binds the

two catalytic serine residues (S217 and S241) (Fig. 2B). The

retrieved binding mode of 5e in complex with FAAH enzyme is

therefore in line with its strong enzyme inhibitory potency. Within

Results and discussion

Chemistry

Compounds 5a-n and 6a-c were synthesized as described in Scheme

1. The synthesis of the 4-fluoro-2-nitroaniline 8a was accomplished

starting from 4-fluoroaniline (7) which was first protected as

acetamide and then submitted to a classical nitration protocol.

Following acetamide hydrolisis led to the desired aniline.

Aniline 8a and the commercially available anilines 8b-d were

subjected to Clauson-Kaas reaction and to subsequent reduction of

the nitro group with tin(II) chloride, affording the o-pyrrolyl anilines

9a-d.³⁷These latter were converted into their corresponding

acetamide derivatives 10a-d and subsequently cyclized in the

presence of phosphorus oxychloride to provide the 4-

methylpyrroloquinoxaline derivatives 11a-d.⁴⁴The methyl group

was then oxydized to aldehyde by selenium(IV) oxide in 1,4-

dioxane.⁴⁵Intermediates 12a-d underwent reductive amination

with the piperazine derivatives 14a-h or the 4-aminopiperidine 16

leading to final compounds 5a-n and 6a-c, respectively.

Structure-activity relationships (SARs)

The inhibition potency of compounds 5a-n and 6a-c for FAAH

(rat brain membrane) and MAGL (COS cells) enzymes is presented

in Table 1; further details are provided in the Experimental Section).

Compounds displaying a significant affinity for COS MAGL were

further tested against rat brain MAGL (Table 1). Under these

conditions, we registered a general ~3-16-fold drop in inhibition

potency, with the only exception of 5m, which was nearly

equipotent on both preparations. These data are in line with typical

discrepancies previously observed among MAGL inhibitory activities

when using enzyme preparations from different animal species.

Moreover, compounds 5i, 5k and 5m the most potent inhibitors on

MAGL rat brain, were tested on the same enzyme applying a pre-

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the MAGL binding site, 5e mainly establishes a series of polar compounds 5f, 5g and 5h bearing a 1,2,3-triazole, an imidazole and

contacts. In fact, the 1,2,4-triazole moiety deeply projects into the benzotriazole ring, respectively. The 1,2,3-triazole-containing

a

enzyme, interacting with the catalytic site by two H-bonds with compound 5f led to a considerable drop of affinity for FAAH enzyme

Y194 and S122 (catalytic serine) and one with the backbone of A51 (FAAH IC₅₀= 175.50 nM; recombinant MAGL IC₅₀= 39.68 nM, Table

(oxyanion hole) (Fig. 2C). Furthermore, it establishes a double π-π 1), while compound 5h bearing the more hindered benzotriazole

stacking with the catalytic residue H269 and with H121. The moiety led to a dramatic loss of MAGL inhibitory activity (FAAH IC₅₀

pyrroloquinoxaline system gives a series of hydrophobic contacts = 23.59 nM; MAGL IC₅₀= 851.10 nM, Table 1). Therefore, for these

with M123, L148, L150, L151, L176, L205, L213, L214, V217 and two compounds the exploration of different terminal ureas led, to a

L241 (Fig. 2D). Notably, the carbonyl group, suitable for undergoing

different extent, to the loss of the dual inhibitory profile, giving

the nucleophilic attack from the catalytic serine residues, was molecular insights for selective inhibition. Compound 5g displayed a

found at a distance (< 2 Å in both enzymes) appropriate for loss of affinity for both enzymes.

generating a tetrahedral intermediate in both FAAH and MAGL.

A small set of derivatives was then conceived in order to explore

different terminal azole-containing carboxamides, namely

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Table 1. IC₅₀values on FAAH and MAGL enzymes for compounds 5a-n, 6a-c, and reference compounds 1 and 4.

IC₅₀FAAH (nM)^a,b

rat brain membrane

>10,000

IC₅₀MAGL (nM) ^a,b

COS cell cytosol

IC₅₀MAGL (nM) ^a

(rat brain cytosol)

Cpd

5a

X

Y

R₁

R₂

>10,000

N

-CH₂-

H

ND^c

ND

[3.73%]

>10,000

[13.80%]

[5.08%]

>10,000

[13.03%]

>10,000

[0%]

5b

5c

N

-CH₂-

H

960

5d

5e

N

-CH₂-

H

562

826

ND

37.0

44.7

611

5f

N

-CH₂-

H

175

39.7

632

ND

5g

2210

>10,000

5h

5i

N

-CH₂-

H

23.6

20.7

23.2

25.1

35.8

32.4

851

49.9

388

5000

329

4.3^d

2.1^e

7-F

5j

ND

7-F

284

4.0^d

3.8^e

5k

5l

7-Cl

95.9

1321

80.1

ND

7-Cl

133

4.7^d

4.2^e

5m

7,8-diMe

5n

6a

6b

N

-CH₂-

7,8-diMe

27.3

9.60

60.4

487

1600

1950

ND

-CH-

-CH₂NH-

ND

7-F

7-Cl

6c

1

-CH-

-

-CH₂NH-

-

7,8-diMe

-

25.1

3000

-

1830

-

20.7^d

6.4^e

4

^avalues are means of three experiments (n = 3) and all SD are within 10%; ^b% of enzyme inhibition at the maximum concentration tested is

reported in square brackets when IC₅₀is > 10 ꢀM; ^cND: not determined; ^dassay performed with 10 min of pre-incubation (n = 2); ^eassay

performed with 60 min of pre-incubation (n = 3).

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Fig. 2. (A,B) IFD pose and ligand interaction diagram of 5e (orange sticks) into FAAH enzyme (PDB ID: 3PPM in green cartoon). (C,D) IFD pose and ligand

interaction diagram of 5e (green sticks) into MAGL enzyme (PDB ID: 3HJU in orange cartoon). The catalytic triad of two enzymes is represented by sticks,

while the interacting residues are represented by lines. The H-bonds are represented by black dotted lines. The pictures were generated by PyMOL and

Maestro (Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012); HIP stands for protonated histidine.

Our SAR investigation also encompassed the exploration of a small pyrroloquinoxaline portion and the terminal benzotriazole urea led

set of structural decorations performed on the pyrroloquinoxaline to a dramatic loss of MAGL affinity while preserving an optimum

skeleton. To this aim 7-fluoro-, 7-chloro- and 7,8-dimethyl- FAAH inhibitory profile. The comparison of docking calculations of

substituted derivatives were prepared bearing either a terminal compound 5j with those obtained with compound 5e provides a

1,2,4-triazole or a benzotriazole moieties (5i-n, Table 1). In general, clear molecular rationalization for the above observations (Fig. S1 vs

the 1,2,4-triazole-containing derivatives (5i, 5k and 5m, Table 1) Fig. 2). Regarding compound 5j a remarkable activity against FAAH,

preserved an excellent dual FAAH/MAGL inhibitory profile, accompanied by a slight decrease of MAGL affinity was registered.

comparable to that displayed by the prototypical dual inhibitor 5e, As expected, docking studies suggest for 5j an accommodation into

with compound 5m showing the best balance of activities among FAAH (Fig. S1 panels A, B) very similar to that found for 5e (Fig. 2).

the enzymes taken under consideration. In line with our previous On the contrary, the analysis of the docking calculation of 5j into

observations the presence of the benzotriazole carboxamide moiety MAGL (Fig. S1 panels C, D) reveals a slight decrease of contacts into

(5j, 5l and 5n, Table 1) determined a loss of MAGL affinity.

the active site with respect to those found for 5e. In particular, 5j

This trend became even more marked with the last set of does not establish the key interactions with H121 and Y194 (Fig. S1

derivatives (6a-c, Table 1) in which the piperazine benzotriazole panels C,D) due to the higher steric hindrance of the benzotriazole

carboxamide was replaced by a 4-aminopiperidine benzotriazole moiety.

carboxamide moiety. Further distancing between the

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Fig. 3. (A,B) IFD pose and ligand interaction diagram of 6a (magenta sticks) into FAAH enzyme (PDB ID: 3PPM in green cartoon). (C,D) IFD pose and ligand

interaction diagram of 6a (magenta sticks) into MAGL enzyme (PDB ID: 3HJU in orange cartoon). The catalytic triad of two enzymes is represented by sticks,

while the interacting residues are represented by lines. The H-bonds are represented by black dotted lines. The pictures were generated by PyMOL and

Maestro (Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012); HIP stands for protonated histidine.

Regarding compound 6a (Fig. 3), the computational analysis establishing H-bonds with the catalytic residues S217 and S241 (Fig.

clearly highlighted the negative effect of the 4-aminopiperidine 3A,B). On the contrary, the docking output of 6a into MAGL (Fig.

benzotriazole carboxamide for MAGL inhibition. Based on the 3C,D) reveals a limited number of interactions into the active site.

activity data reported in Table 1 the structural arrangement of 6a is The introduction of the 4-aminopiperidine benzotriazole

well-tolerated by FAAH being this compound one of the best FAAH carboxamide portion hampered the compound from completely

inhibitors of the series. The output of computational studies reaching the MAGL active site. In fact, the acyl-benzotriazole moiety

reported in Fig. 3A,B for FAAH supports the biological data and was not able to strongly interact with the catalytic residues and

clearly evidences the strong pattern of interaction of 6a within the only a cation-π stacking with H269 was observed. Compound 6a did

enzyme. Compound 6a is potentially able to engage a relevant not interact with the catalytic serine (S122) but only H-bound the

series of hydrophobic contacts (π-π stacking) with F192, F381 and backbone of the oxyanion hole residues (A51 and M123),

F432 by its tricyclic portion and the benzotriazole moiety can also precluding a strong interaction with the enzyme (lacking also some

establish a π-π stacking with F192 (face-to-edge). Moreover, the hydrophobic contacts at the boundary of the binding site).

carbonyl group is correctly located in front of the catalytic centre

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Next, we evaluated in silico ADME+T properties of five filtered and concentrated to afford title compound (quantitative

representative compounds (5d,e,i,j,m, Table S1) by means of yield) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 10.13 (br s, 1H),

QikProp program (QikProp, version 3.5; Schrödinger, LLC: New York, 8.74 (dd, J = 9.4, 5.2 Hz, 1H), 7.89 (dd, J = 8.5, 2.9 Hz, 1H), 7.41-7.31

2012) and TEST software. The output of the calculation indicated (m, 1H), 2.27 (s, 3H); ESI-MS m/z 199 [M+H]⁺. A mixture of N-(4-

satisfactory physico-chemical properties for the title compounds. fluoro-2-nitrophenyl)acetamide (6.0 g, 38.45 mmol) in 6 N HCl (40

Moreover, the favorable predicted LD₅₀value coupled to a potential mL) was refluxed for 4 h. After cooling to 25 °C, the mixture was

non-mutagenic profile support the drug-like propensity of the neutralized with aqueous NaHCO₃and extracted with EtOAc (3 x 20

developed compounds.

mL). The combined organic layers were washed with a saturated

solution of NaCl, dried over anhydrous Na₂SO₄, filtered and

concentrated. Silica gel column chromatography (PetEt/Et₂O 5:1)

afforded title compound as light yellow solid (70% yield). ¹H NMR

(300 MHz, CDCl₃) δ 7.82 (dd, J = 9.1, 3.0 Hz, 1H), 7.17 (dd, J = 10.0,

7.2 Hz, 1H), 6.80 (d, J = 9.2 Hz, 1H), 5.96 (br s, 2H); ESI-MS m/z 157

[M+H]⁺.

Experimental

Chemistry

Unless otherwise specified, materials were purchased from

commercial suppliers and used without further purification.

Reaction progress was monitored by TLC using silica gel 60 F254

(0.040-0.063 mm) with detection by UV. Silica gel 60 (0.040-0.063

mm) was used for column chromatography. ¹H NMR spectra were

recorded on a Varian 300 MHz spectrometer or a Bruker 400 MHz

spectrometer by using the residual signal of the deuterated solvent

as internal standard. Splitting patterns are described as singlet (s),

doublet (δ), triplet (t), quartet (q), and broad (br); the value of

chemical shifts (δ) are given in ppm and coupling constants (J) in

Hertz (Hz). ESI-MS spectra were performed by an Agilent 1100

Series LC/MSD spectrometer. Yields refer to purified products and

are not optimized. Elemental analyses (reported in Table S2) were

performed in a Perkin Elmer 240C elemental analyser, and the

results were within ± 0.4% of the theoretical values.

5-Fluoro-2-(1

1H-pyrrole.

H

-pyrrol-1-yl)aniline (9a). 1-(4-Fluoro-2-nitrophenyl)-

solution of 8a (2.5 g, 16.00 mmol) and 2,5-

A

dimethoxytetrahydrofuran (2.1 mL, 16.00 mmol) in 1,4-dioxane (40

mL) was heated to reflux. Then 5 N HCl (4 mL) was added and, after

5 min, the reaction mixture was diluted with cold water until a

white precipitate was formed. The aqueous phase was extracted

with CHCl₃(3 x 20 mL) and the combined organic layers were dried

over anhydrous Na₂SO₄, filtered and concentrated to obtain 1-(4-

fluoro-2-nitrophenyl)-1H-pyrrole (95% yield) as a pale yellow oil

1

that was used in the following step without further purification; H

NMR (300 MHz, CDCl₃) δ 7.61 (dd, J = 7.5, 2.8 Hz, 1H), 7.48 (dd, J =

8.8, 5.0 Hz, 1H), 7.42-7.33 (m, 1H), 6.75 (t, J = 2.1 Hz, 1H), 6.36 (t, J =

2.2 Hz, 2H); ESI-MS m/z 207 [M+H]⁺; To a solution of this latter (3.1

g, 15.20 mmol) in EtOAc (75 mL), SnCl₂∙2H₂O (20.6 g, 91.20 mmol)

was added and the solution was refluxed for 2 h. Then the reaction

mixture was quenched with saturated aqueous NaHCO₃and filtered

through Celite. The aqueous phase was extracted with EtOAc (3 x

25 mL), and the combined organic layers were dried over anhydrous

Na₂SO₄, filtered and concentrated. The crude residue was purified

by automated flash chromatography using a silica gel pre-packed

column (PetEt/Et₂O 15:1) to afford title compound (85% yield) as a

4-Fluoro-2-nitroaniline (8a). To a stirred solution of 4-fluoronaniline

(7, 10.0 g, 90.00 mmol) and triethylamine (18.6 mL, 134.00 mmol)

in DCM (50 mL), acetic anhydride (9.3 mL, 99.00 mmol) was added

dropwise at 0 °C and the resulting mixture was stirred for 1 h at 25

°C. 1 N HCl was added and the aqueous phase was extracted with

EtOAc (3 x 50 mL). The combined organic layers were washed with a

saturated solution of NaCl, dried over anhydrous Na₂SO₄, filtered

and concentrated to give pure N-(4-fluorophenyl)acetamide

(quantitative yield). ¹H NMR (300 MHz, CDCl₃) δ 7.70 (s, 1H), 7.45 (t,

J = 8.8 Hz, 2H), 6.98 (t, J = 8.7 Hz, 2H), 2.14 (s, 3H); ESI-MS m/z 154

1

colourless oil; H NMR (300 MHz, CDCl₃) δ 7.14-7.01 (m, 1H), 6.85-

6.58 (m, 2H), 6.55-6.41 (m, 2H), 6.34 (t, J = 2.0 Hz, 2H), 3.80 (br s,

[M+H]⁺.

N-(4-Fluoro-2-nitrophenyl)acetamide.

N-(4-

2H); ESI-MS m/z 177 [M+H]⁺

.

Fluorophenyl)acetamide (12.7 g, 83.25 mmol) was dissolved in

acetic acid (40 mL), then trifluoroacetic anhydride (40 mL) and

concentrated HNO₃(7.4 mL) were added dropwise at 0 °C. The

mixture was stirred for 12 h at 25 °C and a saturated solution of

NaHCO₃solution was added. The aqueous phase was extracted with

EtOAc (3 x 50 mL) and the combined organic layers were washed

with a saturated solution of NaCl, dried over anhydrous Na₂SO₄,

2-(1H-Pyrrol-1-yl)aniline (9b). Starting from 8b (4.0 g, 28.96 mmol)

and 2,5-dimethoxytetrahydrofuran (4.6 mL, 36.20 mmol), 1-(2-

nitrophenyl)-1H-pyrrole was obtained following the same procedure

reported for 9a. ¹H NMR (300 MHz, CDCl₃) δ 7.85 (dd, J = 8.4, 1.4 Hz,

1H), 7.65 (td, J = 7.9, 1.5 Hz, 1H), 7.51-7.45 (m, 2H), 6.83-6.76 (m,

2H), 6.40-6.32 (m, 2H); ESI-MS m/z 189 [M+H]⁺; Starting from this

latter, title compound was prepared according to the procedure

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previously described for 9a (68% yield, over 2 steps); ¹H NMR (300 obtained following the same procedure reported for 10a (65%

MHz, CDCl₃) δ 7.16 (d, J = 7.2 Hz, 2H), 6.93-6.76 (m, 4H), 6.40-6.29 yield), after purification by silica gel column chromatography

(m, 2H), 3.68 (br s, 2H); ESI-MS m/z [M+H]⁺159 (100).

(PetEt/ Et₂O 10:1); ¹H NMR (300 MHz, CDCl₃) δ 7.47 (s, 1H), 7.40 (s,

5-Chloro-2-(1H-pyrrol-1-yl)aniline (9c). Starting from 8c (2.5 g, 1H), 7.25 (br s, 1H), 7.14-7.02 (m, 2H), 6.29-6.17 (m, 2H), 2.38 (s,

14.49 mmol) and 2,5-dimethoxytetrahydrofuran (1.9 mL, 14.49 6H), 2.24 (s, 3H); ESI-MS m/z 229 [M+H]⁺

.

mmol), 1-(4-chloro-2-nitrophenyl)-1H-pyrrole was obtained 7-Fluoro-4-methylpyrrolo[1,2-a]quinoxaline (11a). To a solution of

following the same procedure reported for 9a. ¹H NMR (300 MHz, 10a (50 mg, 0.25 mmol) in toluene (2 mL) phosphorous oxychloride

CDCl₃) δ 8.32 (d, J = 1.4 Hz, 1H), 7.78 (dd, J = 7.4, 1.5 Hz, 1H), 7.66 (115 µL, 1.25 mmol) was added dropwise and the reaction mixture

(d, J = 7.5 Hz, 1H), 7.21-7.09 (m, 2H), 6.33-6.19 (m, 2H); ESI-MS m/z was refluxed for 4 h. The yellow precipitate was filtered and the

223 [M+H]⁺. Starting from this latter, title compound was prepared resulting solution was washed with saturated aqueous NaHCO₃,

according to the procedure previously described for 9a (72% yield, dried over anhydrous Na₂SO₄, filtered and concentrated to obtain

over 2 steps). ¹H NMR (300 MHz, CDCl₃) δ 7.06 (d, J = 8.3 Hz, 1H), pure title compound (55% yield) as a yellow amorphous solid; ¹H

6.82-6.77 (m, 2H), 6.77-6.70 (m, 1H), 6.35 (t, J = 2.1 Hz, 2H), 3.78 (br NMR (300 MHz, CDCl₃) δ 7.88-7.84 (m, 1H), 7.76 (dd, J = 9.0, 5.1 Hz,

s, 2H). ESI-MS m/z 193 [M+H]⁺

1H), 7.57 (dd, J = 9.6, 2.8 Hz, 1H), 7.29-7.14 (m, 1H), 6.90 (dd, J =

.

4,5-Dimethyl-2-(1H-pyrrol-1-yl)aniline (9d). Starting from 8d (2.5 g, 4.0, 1.2 Hz, 1H), 6.84 (dd, J = 3.9, 2.7 Hz, 1H), 2.71 (s, 3H); ESI-MS

15.04 mmol) and 2,5-dimethoxytetrahydrofuran (2.0 mL, 15.04 m/z 201 [M+H]⁺.

mmol), 1-(4,5-dimethyl-2-nitrophenyl)-1H-pyrrole was obtained 4-Methylpyrrolo[1,2-a]quinoxaline (11b). Starting from 10b (47

following the same procedure reported for 9a. ¹H NMR (300 MHz, mg, 0.23 mmol), the title compound was obtained following the

CDCl₃) δ 7.68 (s, 1H), 7.22 (s, 1H), 6.81-6.69 (m, 2H), 6.38-6.18 (m, same procedure reported for 11a (59% yield); ¹H NMR (300 MHz,

2H), 2.36 (s, 6H); ESI-MS m/z 217 [M+H]⁺Starting from this latter, CDCl₃) δ 8.02-7.90 (m, 2H), 7.84 (d, J = 8.1 Hz, 1H), 7.55-7.36 (m,

.

title compound was prepared according to the procedure previously 2H), 6.95 (d, J = 2.9 Hz, 1H), 6.89 (d, J = 2.6 Hz, 1H), 2.78 (s, 3H); ESI-

described for 9a (76% yield, over 2 steps); ¹H NMR (300 MHz, CDCl₃) MS m/z 183 [M+H]⁺.

δ 6.94 (s, 1H), 6.82 (t, J = 2.1 Hz, 2H), 6.63 (s, 1H), 6.33 (t, J = 2.1 Hz, 7-Chloro-4-methylpyrrolo[1,2-a]quinoxaline (11c). Starting from

2H), 3.53 (br s, 2H), 2.23 (s, 3H), 2.18 (s, 3H); ESI-MS m/z 187 10c (50 mg, 0.21 mmol), the title compound was obtained following

[M+H]⁺

the same procedure reported for 11a (52% yield); ¹H NMR (300

MHz, CDCl₃) δ 7.88 (dd, J = 7.0, 1.9 Hz, 2H), 7.75 (d, J = 8.7 Hz, 1H),

.

N-(5-Fluoro-2-(1H-pyrrol-1-yl)phenyl)acetamide (10a). To

a

solution of 9a (110 mg, 0.63 mmol) in 1,4-dioxane (2 mL), pyridine 7.43 (dd, J = 8.7, 2.3 Hz, 1H), 6.87 (dt, J = 32.2, 14.7 Hz, 2H), 2.72 (s,

(56 µL, 0.69 mmol) and acetyl chloride (45 µL, 0.63 mmol) were 3H); ESI-MS m/z 217 [M+H]⁺

.

added. The solution was refluxed for 4 h then the solvent was 4,7,8-Trimethylpyrrolo[1,2-a]quinoxaline (11d). Starting from 10d

removed. The residue was taken up with water and extracted with (50 mg, 0.22 mmol), the title compound was obtained following the

1

EtOAc (3 x 10 mL). The combined organic phases were dried over same procedure reported for 11a (50% yield); H NMR (300 MHz,

anhydrous Na₂SO₄, filtered and concentrated. The crude residue CDCl₃) δ 7.84 (s, 1H), 7.66 (s, 1H), 7.59 (s, 1H), 6.83 (dt, J = 6.5, 2.4

was purified by silica gel column chromatography (PetEt/ EtOAc 1:1) Hz, 2H), 2.70 (s, 3H), 2.43 (s, 3H), 2.39 (s, 3H); ESI-MS m/z 211

to provide title compound (68% yield) as a white amorphous solid; [M+H]⁺

.

¹H NMR (300 MHz, CDCl₃) δ 8.25 (dd, J = 10.8, 2.4 Hz, 1H), 7.28-7.18 7-Fluoropyrrolo[1,2-a]quinoxaline-4-carbaldehyde (12a). To

a

(m, 1H), 6.96 (br s, 1H), 6.89-6.79 (m, 1H), 6.75 (t, J = 2.1 Hz, 2H), solution of 11a (100 mg, 0.50 mmol) in 1,4-dioxane (2 mL), selenium

6.40 (t, J = 2.1 Hz, 2H), 2.03 (s, 3H); ESI-MS m/z 219 [M+H]⁺

dioxide (83 mg, 0.75 mmol) was added. The resulting suspension

.

N-(2-(1H-Pyrrol-1-yl)phenyl)acetamide (10b). Starting from 9b (100 was refluxed for 4 h and then cooled to 25 °C, filtered and dried by

mg, 0.63 mmol), the title compound was obtained following the rotary evaporation. The residue was purified by silica gel column

same procedure reported for 10a (63% yield), after purification by chromatography (PetEt/EtOAc 5:1) to obtain title compound (56%

1

silica gel column chromatography (PetEt/ EtOAc 1:1); H NMR (400 yield) as a yellowish solid; ); ¹H NMR (300 MHz, CDCl₃) δ 10.10 (s,

1H), 8.01 (s, 1H), 7.93-7.88 (m, 1H), 7.80 (d, J = 9, 1H), 7.7-7.68 (m,

MHz, CDCl₃) δ 7.78 (d, J = 7.8 Hz, 1H), 7.29-7.33 (m, 1H), 7.20 (d, J =

7.0 Hz, 1H), 7.07-7.11 (m, 1H), 6.95 (br s, 1H), 6.72 (s, 2H), 6.33 (s,

2H), 1.97 (s, 1H); ESI-MS m/z [M+H]⁺201 (100), [M+Na]⁺223.

1H), 7.46-7.39 (m, 1H), 7.04-7.01 (m, 1H); ESI-MS m/z 215 [M+H]⁺

.

Pyrrolo[1,2-a]quinoxaline-4-carbaldehyde (12b). Starting from 11b

(100 mg, 0.55 mmol), the title compound was obtained following

the same procedure reported for 12a (53% yield), after purification

by silica gel column chromatography (PetEt/EtOAc 5:1); ¹H NMR

(300 MHz, CDCl₃) δ 10.13 (s, 1H), 8.13 (d, J = 8.1 Hz, 1H), 8.04 (d, J =

1.5 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.74-7.61 (m, 2H), 7.54 (t, J = 7.7

Hz, 1H), 7.06-6.98 (m, 1H).

N-(5-Chloro-2-(1H-pyrrol-1-yl)phenyl)acetamide (10c). Starting

from 9c (100 mg, 0.52 mmol), the title compound was prepared

according to the procedure previously described for 10a (70%

yield), after purification by silica gel column chromatography

1

(PetEt/ Et₂O 10:1); H NMR (300 MHz, CDCl₃) δ 7.60 (d, J = 1.4 Hz,

1H), 7.53 (d, J = 7.5 Hz, 1H), 7.30 (dd, J = 7.4, 1.5 Hz, 1H), 7.25 (br s,

7-Chloropyrrolo[1,2-a]quinoxaline-4-carbaldehyde (12c). Starting

from 11c (100 mg, 0.46 mmol), the title compound was obtained

following the same procedure reported for 12a (50% yield) after

purification by silica gel column chromatography (PetEt/EtOAc 5:1);

1H), 7.13-7.06 (m, 2H), 6.28-6.21 (m, 2H), 2.24 (s, 3H); ESI-MS m/z

219 [M+H]⁺

.

N-(4,5-Dimethyl-2-(1H-pyrrol-1-yl)phenyl)acetamide

(10d).

Starting from 9d (100 mg, 0.54 mmol), the title compound was

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¹H NMR (300 MHz, CDCl₃) δ 10.09 (s, 1H), 8.12 (d, J = 2.2 Hz, 1H), quantitative yield and carried on without further purification; ¹H

8.01 (s, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.72-7.57 (m, 2H), 7.07-6.98 (m, NMR (300 MHz, CD₃OD) δ 9.02 (s, 1H), 8.15 (s, 1H), 4.22-3.98 (m,

1H); ESI-MS m/z 231 [M+H]⁺

4H), 3.45-3.34 (m, 4H). ESI-MS m/z [M+H]⁺182.

.

7,8-Dimethylpyrrolo[1,2-a]quinoxaline-4-carbaldehyde

(12d). Piperazin-1-yl(1H-1,2,3-triazol-1-yl)methanone (14f). Starting from

Starting from 11d (100 mg, 0.48 mmol), the title compound was 1H-1,2,3-triazole (100 mg, 1,45 mmol), phosgene (20% solution in

obtained following the same procedure reported for 12a (40% toluene, 715 µL, 1.45 mmol) and DMAP (354 mg, 2.90 mmol), tert-

yield) after purification by silica gel column chromatography butyl 4-(1H-1,2,3-triazole-1-carbonyl)piperazine-1-carboxylate was

(PetEt/EtOAc 5:1); ¹H NMR (300 MHz, CDCl₃) δ 10.09 (s, 1H), 7.95 (s, obtained following the same procedure reported for 14e (50%

1H), 7.87 (s, 1H), 7.68-7.63 (m, 2H), 6.99-6.95 (m, 1H), 2.50 (s, 3H), yield), after purification by silica gel column chromatography

1

2.43 (s, 3H); ESI-MS m/z 225 [M+H]⁺.

(DCM/MeOH 20:1). H NMR (300MHz, CDCl₃) δ 8.98 (d, J = 7.5 Hz,

N-Benzylpiperazine-1-carboxamide (14b). To

a

solution of 1H), 8.38 (d, J = 7.5 Hz, 1H), 3.84-3.67 (m, 4H), 3.51-3.29 (m, 4H),

piperazine (13a, 100 mg, 1.16 mmol) in dry THF (5 mL), benzyl 1.50 (s, 9H); ESI-MS m/z 282 [M+H]⁺. Starting from this latter, title

isocyanate (154 mg, 1.16 mmol) and triethylamine (645 μL, 4.04 compound was prepared according to the procedure previously

1

mmol) were sequentially added dropwise. The reaction mixture was described for 14e (quantitative yield); H NMR (300 MHz, CD₃OD) δ

stirred at 25 °C for 12 h, then volatiles were removed by rotary 8.02 (s, 2H), 4.15-4.00 (m, 4H), 3.50-3.37 (m, 4H); ESI-MS m/z 182

evaporation. Silica gel column chromatography (DCM/MeOH 5:1) [M+H]⁺.

afforded title compound (53% yield) as a colourless oil; ¹H NMR (1H-Imidazol-1-yl)(piperazin-1-yl)methanone (14g). tert-butyl-4-

(300 MHz, CD₃OD) δ 7.40-7.11 (m, 5H), 4.35 (s, 2H), 3.80-3.56 (m, (1H-imidazole-1-carbonyl)piperazine-1-carboxylate. To a solution of

4H), 3.25-3.09 (m, 4H).

4-Boc-piperazine (13b, 100 mg, 0.54 mmol) in dry DCM (10 mL), 1,1-

N-Phenylpiperazine-1-carboxamide (14c). Starting from piperazine carbonyldiimidazole (96 mg, 0.59 mmol) was added and the

(13a, 100 mg, 1.16 mmol), phenyl isocyanate (127 µL, 1.16 mmol) reaction was stirred at 25 °C for 12 h. Volatiles were removed under

and triethylamine (645 µL, 4.04 mmol), the title compound was reduced pressure. The crude was purified by silica gel column

prepared according to the procedure previously described for 14b. chromatography (PetEt/EtOAc 1:1) to afford intermediate tert-butyl

The residue was purified by silica gel column chromatography 4-(1H-imidazole-1-carbonyl)piperazine-1-carboxylate (51% yield) as

(DCM/MeOH 5:1) to obtain pure compound (65% yield) as a white a transparent oil. ¹H NMR (300 MHz, CDCl₃) δ 7.98 (d, J = 7.5 Hz,

amorphous solid; ¹H NMR (300 MHz, CD₃OD) δ 7.45-7.33 (m, 2H), 1H), 7.82 (s, 1H), 7.07 (d, J = 7.5 Hz, 1H), 3.69 (dt, J = 20.2, 5.1 Hz,

7.27 (t, J = 7.7 Hz, 2H), 7.03 (t, J = 7.4 Hz, 1H), 3.84-3.73 (m, 4H), 4H), 3.37 (dt, J = 15.4, 5.0 Hz, 4H), 1.50 (s, 9H); ESI-MS m/z 281

3.29-3.22 (m, 4H).

[M+H]⁺Starting from this latter, title compound was prepared

.

4-Nitrophenyl piperazine-1-carboxylate (14d). Starting from according to the procedure previously described for 14e

1

piperazine (13a, 130 mg, 1.50 mmol), 4-nitrophenylchloroformate (quantitative yield). H NMR (300 MHz, CD₃OD) δ 9.38 (s, 1H), 7.95

(272 mg, 1.35 mmol) and triethylamine (630 μL, 4.50 mmol), title (s, 1H), 7.69 (s, 1H), 3.98-3.81 (m, 4H), 3.51-3.35 (m, 4H); ESI-MS

compound was prepared according to the procedure previously m/z 181 [M+H]⁺.

described for 14b. Silica gel column chromatography (DCM/MeOH (1H-Benzo[d][1,2,3]triazol-1-yl)(piperazin-1-yl)methanone (14h).

10:1) afforded pure compound (45% yield) as a yellow amorphous Starting from 1H-benzotriazole (100 mg, 0.84 mmol), phosgene

solid; ¹H NMR (300 MHz, CDCl₃) δ 8.25 (d, J = 9.0 Hz, 2H), 7.30 (d, J = (20% solution in toluene, 415 µL, 0.84 mmol) and DMAP (205 mg,

9.0 Hz, 2H), 3.73-3.51 (m, 4H), 3.01-2.89 (m, 4H).

Piperazin-1-yl(1H-1,2,4-triazol-1-yl)methanone (14e). tert-Butyl-4- carbonyl)piperazine-1-carboxylate was obtained following the same

(1H-1,2,4-triazole-1-carbonyl)piperazine-1-carboxylate. 1H-1,2,4- procedure reported for 14e (56% yield), after purification by silica

2.90

mmol),

tert-butyl

4-(1H-benzo[d][1,2,3]triazole-1-

triazole (100 mg, 1.45 mmol), phosgene (20% solution in toluene, gel column chromatography (PetEt/EtOAc 2:1). ¹H NMR (300 MHz,

715 μL, 1.45 mmol) and dimethylaminopiridine (354 mg, 2.90 CDCl₃) δ 7.96 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.48 (t, J =

mmol) were sequentially dissolved in dry DCM (5 mL) and stirred at 7.7 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 3.79 (m, 4H), 3.65-3.41 (m, 4H),

25 °C for 30 min. Then 4-Boc-piperazine (13b, 135 mg, 0.73 mmol) 1.39 (s, 9H); ESI-MS m/z 332 [M+H]⁺. Starting from this latter, title

was added and the reaction mixture was stirred at 25 °C for 12 h. compound was prepared according to the procedure previously

Volatiles were then removed by rotary evaporation. Silica gel described for 14e (quantitative yield). ¹H NMR (300 MHz, CD₃OD) δ

column chromatography (DCM/MeOH 20:1) afforded tert-butyl-4- 8.10 (d, J = 8.3 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.69 (t, J = 7.7 Hz,

(1H-1,2,4-triazole-1-carbonyl)piperazine-1-carboxylate (42% yield) 1H), 7.54 (t, J = 7.7 Hz, 1H), 4.32-4.05 (m, 4H), 3.61-3.41 (m, 4H);

as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 8.74 (s, 1H), 7.93 (s, ESI-MS m/z 232 [M+H]⁺.

1H), 3.85-3.65 (m, 4H), 3.49 (m, 4H), 1.40 (s, 9H). ESI-MS m/z (4-Aminopiperidin-1-yl)(1H-benzo[d][1,2,3]triazol-1-yl)methanone

[M+H]⁺282. This latter was then dissolved in dry DCM and (16). Starting from 4-(Boc-amino)piperidine (15, 100 mg, 0.50

trifluoroacetic acid (700 μL) was added. The reaction mixture was mmol) and 1H-benzotriazole (120 mg, 1.00 mmol), tert-butyl-(1-

stirred at 25 °C for

2 h. Solvents were removed by rotary (1H-benzo[d][1,2,3]triazole-1-carbonyl)piperidin-4-yl)carbamate

evaporation and the residue was dissolved in EtOAc, treated with a was obtained following the same procedure reported for 14e (56%

saturated solution of NaHCO₃, dried over anhydrous Na₂SO₄, yield), after purification by silica gel column chromatography

filtered and concentrated. Title compound was obtained in (PetEt/EtOAc 2:1). ¹H NMR (300 MHz, CDCl₃) δ 8.09 (d, J = 8.3 Hz,

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1H), 7.96 (d, J = 8.3 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.45 (t, J = 7.7 129.1, 127.9, 127.7, 126.0, 125.4, 123.3, 120.2, 114.4, 113.9, 113.8,

Hz, 1H), 4.71-4.36 (m, 3H), 3.93-3.65 (m, 1H), 3.29 (t, J = 12.0 Hz, 107.5, 63.0, 53.4, 44.3; ESI-MS m/z [M+H]⁺386.

2H), 2.13 (d, J = 11.5 Hz, 2H), 1.63 (q, J = 15.3 Hz, 2H), 1.45 (s, 9H); 4-Nitrophenyl-4-(pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazine-

ESI-MS m/z 346 [M+H]⁺. Starting from this latter, title compound 1-carboxylate (5d). Starting from 12b (15 mg, 0.08 mmol) and 14d

was prepared according to the procedure previously described for (20 mg, 0.08 mmol), title compound was prepared according to the

14e (quantitative yield). ¹H NMR (300 MHz, CD₃OD) δ 8.10 (d, J = 8.3 procedure previously described for 5a. Column chromatography on

Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.53 (t, J = Al₂O₃(n-Hex/EtOAc 3:1) afforded pure compound (55% yield) as a

1

7.7 Hz, 1H), 4.68-4.46 (m, 2H), 3.66-3.45 (m, 1H), 3.43-3.30 (m, 2H), pale yellow oil. H NMR (300 MHz, CDCl₃) δ 8.24 (d, J = 9.2 Hz, 2H),

2.29-2.10 (m, 2H), 1.83 (qd, J = 12.4, 4.5 Hz, 2H); ESI-MS m/z 246 8.01 (d, J = 7.6 Hz, 1H), 7.98-7.94 (m, 1H), 7.88 (dd, J = 8.1, 1.3 Hz,

[M+H]⁺.

1H), 7.59-7.42 (m, 2H), 7.29 (d, J = 9.2 Hz, 2H), 7.19 (d, J = 3.0 Hz,

Benzyl-4-(pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazine-1-

1H), 6.94-6.87 (m, 1H), 4.01 (s, 2H), 3.83-3.56 (m, 4H), 2.90-2.61 (m,

carboxylate (5a). Compound 12b (57 mg, 0.29 mmol) was dissolved 4H); ESI-MS m/z [M+H]⁺432 (100), [M+Na]⁺454.

in a 99:1 ethanol/acetic acid mixture (3 mL), then commercially (4-(Pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazin-1-yl)(1H-1,2,4-

available benzyl piperazine-1-carboxylate (14a, 64 mg, 0.29 mmol) triazol-1-yl)methanone (5e). Starting from 12b (15 mg, 0.08 mmol)

was added and the resulting solution was stirred at 25 °C for 1 h. and 14e (14 mg, 0.08 mmol), title compound was prepared

NaCNBH₃was added and the mixture was stirred at 25 °C for 12 h. A according to the procedure previously described for 5a. Silica gel

saturated solution of NaHCO₃was added and ethanol was removed column chromatography (CHCl₃/MeOH 100:1 to 70:1) provided

by rotary evaporation. The aqueous phase was extracted with CHCl₃pure compound (57% yield) as a pale yellow solid. ¹H NMR (300

(3 x 10 mL) and the combined organic layers were dried over MHz, CDCl₃) δ 8.78 (s, 1H), 8.03-7.92 (m, 3H), 7.86 (dd, J = 8.1, 1.4

anhydrous Na₂SO₄, filtered and concentrated. The crude residue Hz, 1H), 7.57-7.39 (m, 2H), 7.15 (dd, J = 4.0, 1.2 Hz, 1H), 6.88 (dd, J =

was purified by silica gel column chromatography (CHCl₃/MeOH 4.0, 2.7 Hz, 1H), 4.08-3.76 (m, 6H), 2.88-2.70 (m, 4H); ¹³C NMR (75

100:1) to obtain title compound (57% yield) as a white amorphous

solid. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 8.0 Hz, 1H), 7.93- 7.89

(m, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.42 (t, J =

7.6 Hz, 1H), 7.38-7.26 (m, 5H), 7.15 (d, J = 3.9 Hz, 1H), 6.87-6.83 (m,

1H), 5.11 (s, 2H), 3.93 (s, 2H), 3.64-3.47 (m, 4H), 2.77-2.50 (m, 4H);

MHz, CDCl₃) δ 152.6, 152.2, 148.7, 146.9, 135.7, 130.2, 128.0, 127.7,

126.0, 125.5, 114.6, 114.0, 113.9, 107.4, 62.5, 53.6, 53.4; ESI-MS

m/z [M+H]⁺362 (100), [M+Na]⁺384.

(4-(Pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazin-1-yl)(1H-1,2,3-

triazol-1-yl)methanone (5f). Starting from 12b (15 mg, 0.08 mmol)

¹³C NMR (100 MHz, CDCl₃) δ 155.5, 153.1, 137.0, 135.7. 130.2, and 14f (14 mg, 0.08 mmol), title compound was prepared

128.7, 128.2, 128.1, 127.8, 127.7, 126.1, 125.3, 114.4, 113.9, 113.8, according to the procedure previously described for 5a. Silica gel

107.5, 67.3, 53.4, 53.1, 44.0. ESI-MS m/z [M+H]⁺401 (100), [M+Na]⁺column chromatography (EtOAc) provided pure compound (30%

423.

yield) as a pale yellow solid; ¹H NMR (300 MHz, CDCl₃) δ 8.16 (d, J =

N-Benzyl-4-(pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazine-1-

1.2 Hz, 1H), 8.02-7.92 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H), 7.71 (d, J =

carboxamide (5b). Starting from 12b (18 mg, 0.09 mmol) and 14b 1.2 Hz, 1H), 7.55-7.42 (m, 2H), 7.15 (d, J = 3.9 Hz, 1H), 6.92-6.84 (m,

(20 mg, 0.09 mmol), title compound was prepared according to the 1H), 3.99 (s, 2H), 3.88 (m, 4H), 2.81 (m, 4H); ¹³C NMR (75 MHz,

procedure previously described for 5a. Silica gel column CDCl₃) δ 152.6, 148,7, 136.2, 133.1, 130.2, 127.9, 127.7, 126.0,

chromatography (CHCl₃/MeOH 100:1) gave pure compound (71% 125.6, 125.4, 114.6, 113.9, 113.8, 107.4, 62.35, 53.1, 52.9; ESI-MS

yield) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.95 (dd, J = m/z 362 [M+H]⁺.

7.9, 1.1 Hz, 1H), 7.90 (d, J = 1.4 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.52- (1H-Imidazol-1-yl)(4-(pyrrolo[1,2-a]quinoxalin-4-

7.45 (m, 1H), 7.45-7.38 (m, 1H), 7.33-7.19 (m, 5H), 7.14 (dd, J = 3.8, ylmethyl)piperazin-1-yl)methanone (5g). Starting from 12b (15 mg,

0.8 Hz, 1H), 6.84 (t, J = 3.7 Hz, 1H), 4.76 (t, J = 5.1 Hz, 1H), 4.40 (d, J 0.08 mmol) and 14g (14 mg, 0.08 mmol), title compound was

= 5.4 Hz, 2H), 3.88 (s, 2H), 3.46-3.37 (m, 4H), 2.70-2.55 (m, 4H); ¹³C prepared according to the procedure previously described for 5a.

NMR (75 MHz, CDCl₃) δ 157.89, 153.1, 139.7, 135.7, 130.2, 128.8, Silica gel column chromatography (EtOAc) provided pure compound

128.0, 127.8, 127.7, 127.5, 126.0, 125.3, 114.4, 113.9, 113.8, 107.6, (35% yield) as a colourless oil; ¹H NMR (300 MHz, CDCl₃) δ 8.01-7.92

63.1, 53.4, 45.2, 44.0; ESI-MS m/z [M+H]⁺400.

(m, 2H), 7.90-7.82 (m, 2H), 7.57-7.40 (m, 2H), 7.21-7.15 (m, 1H),

N-Phenyl-4-(pyrrolo[1,2-a]quinoxalin-4-ylmethyl)piperazine-1-

7.12 (dd, J = 4.0, 1.2 Hz, 1H), 7.10-7.06 (m, 1H), 6.88 (dd, J = 3.9, 2.8

carboxamide (5c). Starting from 12b (51 mg, 0.26 mmol) and 14c Hz, 1H), 3.96 (s, 2H), 3.73-3.60 (m, 4H), 2.81-2.66 (m, 4H); ¹³C NMR

(53 mg, 0.26 mmol), title compound was prepared according to the (75 MHz, CDCl₃) δ 152.4, 150.9, 137.1, 135.7, 130.2, 129.9, 128.0,

procedure previously described for 5a. Silica gel column 127.7, 125.9, 125.5, 118.1, 114.6, 114.0, 113.9, 107.3, 62.7, 53.3,

chromatography (CHCl₃/MeOH 100:1) provided pure compound 46.7; ESI-MS m/z 361 [M+H]⁺.

(83% yield) as a white amorphous solid. ¹H NMR (300 MHz, CDCl₃) δ (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(pyrrolo[1,2-a]quinoxalin-4-

7.97 (dd, J = 7.9, 1.5 Hz, 1H), 7.94-7.89 (m, 1H), 7.83 (dd, J = 8.1, 1.3 ylmethyl)piperazin-1-yl)methanone (5h). Starting from 12b (15 mg,

Hz, 1H), 7.55-7.38 (m, 2H), 7.38-7.29 (m, 2H), 7.29-7.18 (m, 2H), 0.08 mmol) and 14h (19 mg, 0.08 mmol), title compound was

7.15 (dd, J = 4.0, 1.3 Hz, 1H), 7.04-6.93 (m, 1H), 6.86 (dd, J = 4.0, 2.7 prepared according to the procedure previously described for 5a.

Hz, 1H), 6.61 (br s, 1H), 3.89 (s, 2H), 3.56-3.46 (m, 4H), 2.67-2.57 (m, Silica gel column chromatography (PetEt/EtOAc 2:1) provided pure

4H); ¹³C NMR (75 MHz, CDCl₃) δ 155.3, 153.1, 139.2, 135.7, 130.2, compound (32% yield) as a colourless oil; ¹H NMR (300 MHz, CDCl₃)

J. Name., 2013, 00, 1-3 | 11

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δ 8.08 (d, J = 8.3 Hz, 1H), 8.04-7.93 (m, 3H), 7.87 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.46 (dt,

7.64-7.38 (m, 4H), 7.18 (d, J = 3.9 Hz, 1H), 6.91-6.80 (m, 1H), 4.00 J = 15.4, 5.4 Hz, 2H), 7.19 (d, J = 4.0 Hz, 1H), 6.94-6.86 (m, 1H), 3.99

(m, 6H), 2.85 (m, 4H); ¹³C NMR (75 MHz, CDCl₃) δ 152.7, 149.6, (m, 6H), 2.85 (m, 4H); ¹³C NMR (75 MHz, CDCl₃) δ 153.9, 149.6,

145.6, 135.7, 133.4, 130.2, 129.6, 127.9, 127.7, 126.0, 125.4 (2C), 145.6, 136.7, 133.4, 130.5, 129.7, 129.6, 127.9, 126.3, 125.9, 125.5,

120.1, 114.5, 114.0, 113.9, 113.8, 107.4, 62.7, 53.5, 45.3; ESI-MS 120.1, 115.0, 114.9, 114.4, 113.8, 107.9, 62.4, 53.5, 53.4; ESI-MS

m/z 412 [M+H]⁺.

m/z: 446 [M+H]⁺

.

4-((7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)methyl)piperazin-1-

(4-((7,8-Dimethylpyrrolo[1,2-a]quinoxalin-4-yl)methyl)piperazin-1-

yl)(1H-1,2,4-triazol-1-yl)methanone (5i). Starting from 12a (15 mg, yl)(1H-1,2,4-triazol-1-yl)methanone (5m). Starting from 12d (75

0.08 mmol) and 14e (14 mg, 0.08 mmol), title compound was mg, 0.33 mmol) and 14e (60 mg, 0.33 mmol), title compound was

prepared according to the procedure previously described for 5a. prepared according to the procedure previously described for 5a.

Silica gel column chromatography (EtOAc) provided pure compound Silica gel column chromatography (EtOAc) provided pure compound

1

(25% yield) as a colourless oil; H NMR (300 MHz, CDCl₃) δ 8.78 (s, (35% yield) as a colourless oil; H NMR (300 MHz, CDCl₃) δ 8.78 (s,

1H), 7.97 (s, 1H), 7.90 (s, 1H), 7.81 (dd, J = 9.0, 5.0 Hz, 1H), 7.65 (dd, 1H), 7.97 (s, 1H), 7.88 (d, J = 1.3 Hz, 1H), 7.75 (s, 1H), 7.62 (s, 1H),

J = 9.5, 2.6 Hz, 1H), 7.32-7.18 (m, 1H), 7.15 (d, J = 3.9 Hz, 1H), 6.91- 7.10 (d, J = 4.0 Hz, 1H), 6.88-6.76 (m, 1H), 3.94 (m, 6H), 2.76 (m,

6.82 (m, 1H), 3.94 (m, 6H), 2.76 (m, 4H); ¹³C NMR (75 MHz, CDCl₃) δ 4H), 2.45 (s, 3H), 2.39 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 152.2,

160.0 (d, J_C-F= 243.8 Hz), 153.9, 152.2, 148.7, 146.9, 136.9 (d, J_C-F

=

151.5, 148.7, 146.8, 137.5, 134.3, 133.7, 130.2, 126.1, 125.6, 114.3,

11.4 Hz), 125.8, 124.4 (d, J_C-F= 1.9 Hz), 115.7 (d, J_C-F= 10.2 Hz), 114.0, 113.6, 106.8, 62.6, 53.4, 53.2, 20.5, 19.8; ESI-MS m/z: 390

115.4 (d, J_C-F= 8.2 Hz), 115.0 (d, J_C-F= 9.2 Hz), 114.7, 114.1, 107.7, [M+H]⁺

.

62.5, 53.4, 47.1; ¹⁹F NMR (282 MHz, CDCl₃) δ -116.9; ESI-MS m/z (1H-Benzo[d][1,2,3]triazol-1-yl)(4-((7,8-dimethylpyrrolo[1,2-

380 [M+H]⁺.

a]quinoxalin-4-yl)methyl) piperazin-1-yl)methanone (5n). Starting

from 12d (48 mg, 0.21 mmol) and 14h (50 mg, 0.21 mmol), title

(1H-Benzo[d][1,2,3]triazol-1-yl)(4-((7-fluoropyrrolo[1,2-

a]quinoxalin-4-yl)methyl)piperazin-1-yl)methanone (5j). Starting compound was prepared according to the procedure previously

from 12a (40.0 mg, 0.18 mmol) and 14h (42 mg, 0.18 mmol), title described for 5a. Silica gel column chromatography (PetEt/EtOAc

compound was prepared according to the procedure previously 2:1) provided pure compound (26% yield) as a yellow solid; ¹H NMR

described for 5a. Silica gel column chromatography (PetEt/EtOAc (300 MHz, CDCl₃) δ 8.08 (d, J = 8.3 Hz, 1H), 8.02-7.95 (m, 1H), 7.90-

2:1) provided pure compound (28% yield) as a colourless oil; ¹H 7.86 (m, 1H), 7.75 (s, 1H), 7.62 (s, 1H), 7.60-7.56 (m, 1H), 7.49-7.39

NMR (300 MHz, CDCl₃) δ 8.08 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.3 Hz, (m, 1H), 7.12 (d, J = 4.0 Hz, 1H), 6.88-6.82 (m, 1H), 3.97 (m, 6H),

1H), 7.91 (s, 1H), 7.82 (dd, J = 9.0, 5.1 Hz, 1H), 7.71-7.54 (m, 2H), 2.83 (m, 4H), 2.45 (s, 3H), 2.39 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ

7.50-7.39 (m, 1H), 7.30-7.22 (m, 1H), 7.18 (d, J = 3.9 Hz, 1H), 6.92- 151.6, 149.6, 145.5, 137.4, 134.2, 133.9, 133.4, 130.3, 129.5, 126.1,

6.85 (m, 1H), 3.99 (m, 6H), 2.85 (m, 4H); ¹³C NMR (75 MHz, CDCl₃) δ 125.7, 125.4, 120.1, 114.3, 114.0, 113.8, 113.6, 106.8, 62.7, 53.5,

158.1 (d, J_C-F= 244.1 Hz), 147.7, 146.6, 143.6, 135.0 (d, J_C-F= 11.6 53.4, 20.5, 19.8; ESI-MS m/z 440 [M+H]⁺.

Hz), 131.5, 127.7, 123.9, 123.5, 122.5 (d, J_C-F= 2.1 Hz), 118.2, 113.9 (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(((7-fluoropyrrolo[1,2-

(d, J_C-F= 8.2 Hz), 113.5 (d, J_C-F= 6.3 Hz), 113.1 (d, J_C-F= 9.4 Hz), a]quinoxalin-4-yl)methyl)amino) piperidin-1-yl)methanone (6a).

112.8, 112.2, 111.9, 105.9, 60.6, 58.7, 51.6; ¹⁹F NMR (282 MHz, Starting from 12a (40 mg, 0.18 mmol) and 16 (45 mg, 0.18 mmol),

CDCl₃) δ -117.0; ESI-MS m/z 430 [M+H]⁺

title compound was prepared according to the procedure previously

.

(4-((7-Chloropyrrolo[1,2-a]quinoxalin-4-yl)methyl)piperazin-1-

described for 5a. Silica gel column chromatography (EtOAc)

yl)(1H-1,2,4-triazol-1-yl)methanone (5k). Starting from 12c (25 mg, provided pure compound (30% yield) as a yellow solid; ¹H NMR (300

0.11 mmol) and 14e (20 mg, 0.11 mmol), title compound was MHz, CDCl₃) δ 8.09 (d, J = 7.0 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.93-

prepared according to the procedure previously described for 5a. 7.89 (m, 1H), 7.82 (dd, J = 9.0, 5.0 Hz, 1H), 7.68-7.55 (m, 2H), 7.45 (t,

Silica gel column chromatography (DCM/Acetone 9:1) provided J = 7.7 Hz, 1H), 7.25 (dd, J = 17.0, 2.8 Hz, 1H), 6.95 (d, J = 2.8 Hz, 1H),

pure compound (30% yield) as a colourless oil; ¹H NMR (300 MHz, 6.90-6.85 (m, 1H), 4.69 (br s, 1H), 4.46 (d, J = 13.4 Hz, 2H), 4.26 (s,

CDCl₃) δ 8.79 (s, 1H), 7.97 (d, J = 3.9 Hz, 2H), 7.91 (d, J = 1.4 Hz, 1H), 2H), 3.40 (t, J = 12.1 Hz, 2H), 3.16-3.00 (m, 1H), 2.29-2.10 (m, 2H),

7.79 (d, J = 8.8 Hz, 1H), 7.47 (dd, J = 8.8, 2.2 Hz, 1H), 7.16 (d, J = 4.0 1.90-1.69 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 158.1 (d, J_C-F= 244.1

Hz, 1H), 6.93-6.85 (m, 1H), 3.95 (m, 6H), 2.78 (m, 4H); ¹³C NMR (75 Hz), 152.0, 147.7, 143.7, 135.0 (d, J_C-F= 11.6 Hz), 131.5, 127.7,

MHz, CDCl₃) δ 155.2, 149.6, 145.5, 136.6, 133.3, 129.3, 127.6, 126.4, 123.9, 123.5, 122.5 (d, J_C-F= 2.1 Hz), 118.2, 113.9 (d, J_C-F= 8.2 Hz),

125.5, 125.0, 115.1, 114.4, 113.7, 106.4, 60.21, 54.47, 53.40; ESI-MS 113.5 (d, J_C-F= 6.3 Hz), 113.1 (d, J_C-F= 9.4 Hz), 112.8, 112.2, 111.9,

m/z: 396 [M+H]⁺

105.8, 60.6, 58.7, 51.7, 51.6; ¹⁹F NMR (282 MHz, CDCl₃) δ -117.0;

.

(1H-Benzo[d][1,2,3]triazol-1-yl)(4-((7-chloropyrrolo[1,2-

ESI-MS m/z 444 [M+H]⁺

.

a]quinoxalin-4-yl)methyl)piperazin-1-yl)methanone (5l). Starting (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(((7-chloropyrrolo[1,2-

from 12c (30 mg, 0.13 mmol) and 14h (30 mg, 0.13 mmol), title a]quinoxalin-4-yl)methyl)amino) piperidin-1-yl)methanone (6b).

compound was prepared according to the procedure previously Starting from 12c (30 mg, 0.13 mmol) and 16 (32 mg, 0.13 mmol),

described for 5a. Silica gel column chromatography (PetEt/EtOAc title compound was prepared according to the procedure previously

9:1) provided pure compound (32% yield) as a colourless oil; ¹H described for 5a. Silica gel column chromatography (PetEt/EtOAc

NMR (300 MHz, CDCl₃) δ 8.09 (d, J = 7.6 Hz, 1H), 8.02-7.95 (m, 2H), 1:1) provided pure compound (33% yield) as a yellow solid; ¹H NMR

12 | J. Name., 2012, 00, 1-3

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(300 MHz, CDCl₃) δ 8.09 (d, J = 8.3 Hz, 1H), 8.01-7.87 (m, 3H), 7.78 steps to (1) add hydrogens, (2) optimize the orientation of hydroxyl

(d, J = 8.8 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.46 (dd, J = 12.9, 4.7 Hz, groups, Asn, and Gln, and the protonation state of His, and (3)

2H), 6.96 (d, J = 4.0 Hz, 1H), 6.92-6.86 (m, 1H), 4.45 (d, J = 13.2 Hz, perform a constrained refinement with the impref utility, setting

2H), 4.24 (s, 2H), 3.46-3.34 (m, 2H), 3.08-3.01 (m, 1H), 2.17 (m, 3H), the max RMSD of 0.30. The impref utility consists of a cycles of

1.86-1.74 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 155.2, 149.6, 145.6, energy minimization based on the impact molecular mechanics

136.6, 133.7, 133.4, 130.5, 129.5, 129.4, 127.7, 126.3, 125.4, 125.1, engine and on the OPLS_2005 force field.⁴⁸

120.0, 115.2, 115.1, 114.4, 113.7, 106.4, 60.6, 54.5, 53.6, 49.2; ESI- c) Molecular Docking

MS m/z 460 [M+H]⁺

Molecular docking was carried out using the Schrödinger suite 2011

by applying the IFD protocol (Schrödinger Suite 2012 Induced Fit

(6c). Docking protocol; Glide version 5.8, Schrödinger, LLC, New York, NY,

.

(1H-Benzo[d][1,2,3]triazol-1-yl)(4-(((7,8-dimethylpyrrolo[1,2-

a]quinoxalin-4-yl)methyl)amino)piperidin-1-yl)methanone

Starting from 12d (55 mg, 0.24 mmol) and 16 (60 mg, 0.24 mmol), 2012; Prime version 3.1, Schrödinger, LLC, New York, NY, 2012). This

title compound was prepared according to the procedure previously procedure induces conformational changes in the binding site to

described for 5a. Silica gel column chromatography (EtOAc) accommodate the ligand and exhaustively identify possible binding

provided pure compound (28% yield) as a yellow solid; ¹H NMR (300 modes and associated conformational changes by side-chain

MHz, CDCl₃) δ 8.09 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.88 sampling and backbone minimization. The proteins and the ligands

(d, J = 1.4 Hz, 1H), 7.71 (s, 1H), 7.65-7.55 (m, 2H), 7.45 (t, J = 7.7 Hz, used were prepared as reported in the previous paragraphs. The

1H), 6.86 (dt, J = 15.5, 3.4 Hz, 2H), 4.44 (d, J = 13.4 Hz, 2H), 4.22 (s, boxes for docking calculation were built taking into account the

2H), 3.49 - 3.46 (m, 2H), 3.15-2.94 (m, 1H), 2.45 (s, 3H), 2.40 (s, 3H), centroid of the co-crystallized ligand for FAAH enzyme, while for

2.16-2.06 (m, 3H), 1-83-1.70 (m, 2H); ESI-MS m/z 454 [M+H]⁺

MAGL enzyme the catalytic serine 122, employing default setting.

IFD includes protein side-chain flexibility in a radius of 5.0 Å around

the poses found during the initial docking stage of the IFD protocol.

Complexes within 30.0 kcal/mol of minimum energy structure were

taken forward for redocking. The Glide redocking stage was

.

Molecular Modelling studies

Molecular Docking

a) Ligand preparation

Three-dimensional structures of all compounds in this study were performed by XP (Extra Precision) methods. No hydrogen bonding

built by means of Maestro (Maestro, version 9.3, Schrödinger, LLC, or other constraints were used. Hydrophobic contacts for the

New York, NY, 2012). Molecular energy minimizations were described ligand/protein complexes were visualized by using the

performed by means of MacroModel (MacroModel, version 9.9, python script "display_hydrophobic_interactions.py" implemented

Schrödinger, LLC, New York, NY, 2012) using the Optimized in Maestro suite.

Potentials for Liquid Simulations-all atom (OPLS-AA) force field

2005.⁴⁸The solvent effects are simulated using the analytical Enzyme assays

Generalized-Born/Surface-Area (GB/SA) model,⁴⁹and no cutoff for MAGL and FAAH activities were detected in COS cells and rat brain,

nonbonded interactions was selected. Polak-Ribiere conjugate respectively. In particular, 2-AG hydrolysis was measured by

gradient (PRCG) method with 1000 maximum iterations and 0.001 incubating the 10,000xg cytosolic fraction of either COS cells or rat

gradient convergence threshold was employed. All compounds

reported in this paper were treated by LigPrep application (LigPrep,

version 2.5, Schrödinger, LLC, New York, NY, 2012), implemented in

Maestro suite 2011, generating the most probable ionization state

of any possible enantiomers and tautomers at cellular pH value (7 ±

0.5). QikProp (QikProp, version 3.5; Schrödinger, LLC: New York,

2012) was used to assess the physico-chemical properties of

selected compounds. The output is reported in Supplementary

Information in Table S1.

brain homogenate (100 ꢀg/sample) in Tris–HCl 50 mM, at pH 7.4 at

37 °C for 20 min, with synthetic 2-arachidonoyl-[³H]-glycerol (40

Ci/mmol, ARC St. Louis, MO, USA) properly diluted with 2-AG

(Cayman Chemicals, Ann Arbor, MI, USA). After incubation, the

amount of [³H]-glycerol produced was measured by scintillation

counting of the aqueous phase after extraction of the incubation

mixture with 2 volumes of CHCl₃/MeOH 1:1 (by vol.). For time-

course experiments, the effect of compounds on MGL activity was

measured after 10 min and 60 min of pre-incubation with the

enzyme followed by a 20 min of incubation with the specific

substrate. AEA hydrolysis was measured by incubating the 10,000xg

membrane fraction of rat brain (70 ꢀg/sample) in Tris–HCl 50 mM,

at pH 9.5 at 37 °C for 30 min, with synthetic N-arachidonoyl-[¹⁴C]-

ethanolamine (110 mCi/mmol, ARC St. Louis, MO, USA) properly

diluted with AEA (Tocris Bioscience, Avonmouth, Bristol, UK). After

b) Protein preparation

The three-dimensional structures of FAAH (PDB ID: 3PPM⁵⁰) and

MAGL enzymes (PDB ID: 3HJU¹¹

) were taken from PDB and

imported into Schrödinger Maestro molecular modeling

environment. Water molecules, compounds used for the

crystallization and the co-crystallized ligand were removed from the

available experimental structures. The obtained enzymes were

submitted to protein preparation wizard implemented in Maestro

suite 2012 as described by us.^51-53This protocol through a series of

computational steps, allowed us to obtain a reasonable starting

structure of the proteins for molecular docking calculations by a

series of computational steps. In particular, we performed three

incubation, the amount of

[

¹⁴C]-ethanolamine produced was

measured by scintillation counting of the aqueous phase after

extraction of the incubation mixture with 2 volumes of CHCl₃/MeOH

1:1 (by vol.).

J. Name., 2013, 00, 1-3 | 13

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Journal Name

12. S. G. Kinsey, J. Z. Long, B. F. Cravatt and A. H. Lichtman, J Pain,

2010, 11, 1420-1428.

Conclusions

In conclusion, we described herein the identification of novel

FAAH and MAGL inhibitors characterized by a pyrroloquinoxaline-

based structure. Among the newly synthesized chemical entities,

compounds 5e, 5i, 5k and 5m showed relatively high potency on

both target enzymes and favorable predicted drug-like properties,

thus fostering further investigation and optimization for this novel

class of dual endocannabinoid metabolizing enzyme inhibitors.

13. M. N. Hill, C. J. Hillard, F. R. Bambico, S. Patel, B. B. Gorzalka and

G. Gobbi, Trends Pharmacol Sci, 2009, 30, 484-493.

14. V. Di Marzo, N. Stella and A. Zimmer, Nat Rev Neurosci, 2015,

16, 30-42.

Moreover we gained insights into the molecular determinants for 15. S. M. Saario, J. R. Savinainen, J. T. Laitinen, T. Jarvinen and R.

Niemi, Biochem Pharmacol, 2004, 67, 1381-1387.

specific enzyme inhibition.

16. K. Otrubova, C. Ezzili and D. L. Boger, Bioorg Med Chem Lett,

2011, 21, 4674-4685.

Acknowledgements

PRIN-Miur is kindly acknowledged.

17. K. Ahn, D. S. Johnson, L. R. Fitzgerald, M. Liimatta, A. Arendse, T.

Stevenson, E. T. Lund, R. A. Nugent, T. K. Nomanbhoy, J. P.

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Harnessing the pyrroloquinoxaline scaffold for FAAH and MAGL interaction:

definition of the structural determinants for enzyme inhibition

Margherita Brindisi, Simone Brogi, Samuele Maramai, Alessandro Grillo, Giuseppe Borrelli, Stefania Butini,^*Ettore

Novellino, Marco Allarà, Alessia Ligresti, Giuseppe Campiani,^*Vincenzo Di Marzo, Sandra Gemma

Article Doi

Harnessing the pyrroloquinoxaline scaffold for FAAH and MAGL interaction: Definition of the structural determinants for enzyme inhibition

DOI: 10.1039/c6ra12524g

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c6ra12524g

Full text of DOI:10.1039/c6ra12524g

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