Synthesis and biological evaluation of a library of hybrid derivatives as inhibitors of influenza virus PA-PB1 interaction

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Synthesis and biological evaluation of a library of hybrid derivatives

as inhibitors of Influenza Virus PA-PB1 interaction.

1,#

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1

Ilaria D’Agostino; Ilaria Giacchello; Giulio Nannetti; Anna Lucia Fallacara; Davide Deodato;

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Francesca Musumeci; Giancarlo Grossi; Giorgio Palù; Ylenia Cau; Iuni Margaret Trist; Arianna

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,*

2,*

1,4,5

Loregian; Silvia Schenone; Maurizio Botta.

1

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro, I-53100 Siena, Italy

Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132, Genova, Italy

Dipartimento di Medicina Molecolare, Università degli Studi di Padova, Via A. Gabelli 63, I-35121 Padova, Italy

Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, BioLife Science Building, Suite 333, 1900 N

2th Street, Philadelphia, Pennsylvania 19122, United States

Lead Discovery Siena s.r.l., Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy

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3

4

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5

*

Corresponding authors: arianna.loregian@unipd.it; schensil@unige.it.

#

These authors equally contributed to the work.

ABSTRACT. The limited treatment options against influenza virus along with the growing public health

concerns regarding the continuous emergence of drug-resistant viruses make essential the development of

new anti-flu agents with novel mechanisms of action. One of the most attractive targets is the interaction

between two subunits of the RNA-dependent RNA polymerase, PA and PB1. Herein we report the rational

design of hybrid compounds starting from a 3-cyano-4,6-diphenylpyridine scaffold recently identified as

disruptor of PA-PB1 interactions. Guided by the previously reported SAR data, a library of amino acid

derivatives was synthesized. The biological evaluation led to the identification of new PA-PB1 inhibitors,

that do not show appreciable toxicity. Molecular modeling shed further lights on the inhibition mechanism of

these compounds.

KEYWORDS: anti-influenza; PA-PB1; RdRp; amino acid coupling; SPPS; diphenyl-pyridine.

INTRODUCTION. Influenza, also known as flu, is an acute respiratory illness caused by the infection of

viruses which belong to the Orthomyxoviridae family. These pathogens are enveloped viruses containing a

single-stranded, negative-sense and segmented RNA genome and are classified in A, B, C and D,[1,2]

ABBREVIATIONS

AcOEt, Ethyl acetate; CC , Half maximal cytotoxic concentration; cRNA, complementar RNA; DCM, dichloromethane; DDQ, 2,3-

50

Dichloro-5,6-dicyano-1,4-benzoquinone; DIPEA, N,N-Diisopropylethylamine; DMEM, Dulbecco’s Modified Eagle’s Medium;

DMF, N,N-dimethylformamide; DMSO, N,N-dimethylsulfoxyde; EC , Half maximal effective concentration; EDC, 1-Ethyl-3-(3-

50

dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay; EtOH, ethanol; FluA, influenza A virus; FluB,

influenza B virus; FluC, influenza C virus; Fmoc, Fluorenilmetilossicarbonil; GAFF, General Amber Force Field; H O, water; HA,

2

hemagglutinin; HBTU, 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEK, human embryonic

kidney; HOBt, Hydroxybenzotriazole; IC , Half maximal inhibitory concentration; MD, molecular dynamics; MDCK,

50

Madin−Darby canine kidney; MeOH, methanol; mRNA, messenger RNA;MM-GBSA, Molecular Mechanics-Generalized Born

Surface Area; MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; MW, microwave oven; N , nitrogen; NA,

2

neuraminidase; NaN , sodium azide; NaOH, sodium hydroxide; OSV, oseltamivir carboxylate; PA, polymerase acidic protein;

3

PAINs, pan assay interference compounds; PB1, polymerase basic protein 1; PB1 , N-terminal of PB1; PB2, polymerase basic

N

protein 2; PDB, Protein Data Bank; PPI, protein−protein interaction; PRA, plaque reduction assay; RBV, ribavirin; RdRp, RNA-

dependent RNA polymerase; RNA, ribonucleic acid; SAR, structure−activity relationship; SD, standard deviation; SI, selectivity

index; SOCl , thionyl chloride; SPPS, solid phase peptide synthesis; tBu, tert-butyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran;

2

TIPS, triisopropylsilyl ether; vRNA, viral RNA.

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according to the antigenicity of their nucleoprotein (NP) and matrix protein 1 (M1).[3] Infections by

influenza A (FluA) and B (FluB) virus are the most relevant ad occur globally as epidemics (seasonal flu

almost every winter) or as more sporadic pandemics caused by always new and very different A viruses.

Seasonal flu accounts for a large number of cases worldwide each year (between 9.2 and 35.6 million; 5–

1

0% of adults and 20–30% of children)[4] and can cause severe illness especially in population at high risk

for influenza-related complications (e.g. immunocompromised, patients with chronic diseases and paediatric

and old people), resulting in hospitalization and death.[5]

Influenza viruses undergo continuous antigenic drifts and sporadic antigenic shifts that result in minor and

major antigenic changes of the viral surface glycoproteins and allow the viruses to evade the pre-existing

host immune response. For this reason, influenza vaccines must be constantly reformulated and fail to

provide protection against novel pandemic strains.[6–8]Only two classes of chemotherapeutics are currently

available: the M2-blockers, such as amantadine and rimantadine, that inhibit the replication by blocking the

M2 proton channel[9] and the neuraminidase inhibitors, such as zanamivir and oseltamivir, that prevent virus

release from infected cells. The increase of chemoresistance,[10–22] the serious side effects of the current

anti-flu drugs[23] and the difficulty of production of cross-protective vaccines, make current therapeutic

armamentarium limited and poorly effective,[24] molecules to fit.[29,55] After first attempts of PPI

inhibition through PB1-derived peptides,[44,51,53,56] several different libraries of small molecules were

investigated by in silico screenings, biochemical assays and rational design.[50,52,57–65]

In the last few years, our research group identified benzofurazan derivatives as inhibitors of PA–PB1

interaction through high throughput screening (HTS) and these compounds were characterized by an ELISA-

based assay followed by plaque reduction assay (PRA) on FluA/WSN/33 (H1N1)-infected MDCK cells.

This led us to identify the hit compound 1 (Figure1), showing micromolar antiviral activity (EC = 5 µM),

5

0

but cytotoxicity in MDCK cells (CC₅₀= 20 µM).[66] In order to explore the SARs and improve the

selectivity index (SI) and water solubility, a large library of benzofurazan derivatives was synthesized and

tested, giving a more potent compound 2 (IC = 35 µM, EC = 1 µM), but still cytotoxic (CC = 40 µM)

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50

(

Figure 1).[67] Despite the good anti-flu activity, this class of compounds was found to be unsuitable for

further development because of its poor SI. A model of the PA-PB1 complex was built based on PDB IDs

CM8[68] and 2ZNL[44]and it was used to perform a MD simulation to identify the hot-spot residues for the

3

inhibition. The following high throughput docking approach (a consensus docking protocol combining

Glide[69] and Gold[70] programs)[67] together highlighting the need for new targets discovery and

exploitation. Because of its critical role in FluA infection and its high amino acid conservation,[25–28] the

viral polymerase is widely recognized as one of the most promising anti-flu targets.[29–32]

The RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex composed of polymerase acidic

protein (PA), polymerase basic proteins 1 and 2 (PB1 and PB2, respectively) with a combined mass of

approximatively 250 KDa. [33,34] The enzymatic complex is associated to the NP-encapsulated RNA strand

to form the viral ribonucleoprotein complex (RNP).[33,35] It plays a key role in the viral life cycle, as it is

directly required for both transcription and replication.[25,26,33,36–41] The Flu RdRp cleaves the 5′-methyl

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cap from the host pre-mRNA and uses this capped structure as a primer for the synthesis of the viral mRNA

by a cap-snatching mechanism. The polymerase complex also catalyses the genome replication, generating a

positive-sense complementary RNA (cRNA), a replicative intermediate that serves as a template for the

synthesis of more copies of the viral genomic segments (vRNA).[33,34,42,43] The polymerase catalytic site

is in PB1; PB2 includes the capped-RNA recognition domain, required for generation of the primer for

transcription, while PA is involved in the complex assembly.[42,44–46] Only if correctly assembled RdRp

can work and the virus can replicate its genome.[41,44,47,48] Indeed, even if so far the search for new

antiviral targets has been mostly focused on the PA endonuclease active site, PB2 cap-binding domain and

PB1 subunit,[49] a very attractive strategy could be the disruption of RdRp complex assembly through

protein-protein interaction (PPI) inhibition. In fact, PPIs represent crucial events in many biological

processes under both physiological and pathological conditions. In particular, crystallization studies have

revealed that PA-PB1 interaction may be a very promising druggable target, [29,49–52] because of the high

degree of conservation of several interface residues among FluA, FluB and FluC,[27,53,54] that suggests

that its inhibitors may be active against many viral subtypes and less prone to drug-resistance, and because of

the little size of the specific interface that allows small

with ELISA evaluation revealed the 3-cyano-4,6-diphenyl-pyridine scaffold as a promising potential

inhibitor of the PA−PB1 complex by mimicking the hydrophilic N-terminal portion of PB1 (residues 1−4)

and the most important interactions with the C-terminal domain of PA. Compounds 3 and 4 (Figure 1),

showed the best activity profile.[63] The following validation of the reliability of the old homology model

was performed through comparison with the more complete X-ray structure of FluA H17N10 RdRp (PDB

ID: 4WSB)[41] and the superposition in the PB1-binding site region.[71] Chemical modifications aided by

molecular modeling studies allowed us to make preliminary SAR considerations and to identify some

compounds (5 and 6, Figure 1) as promising anti-flu agents, even if they suffer from low water solubility

that implied the design and validation of a new serum-free ELISA.[71]

Figure 1. Structures of compounds 1-6 from previous works.[63,66,67,71]

DISCUSSION. The biological evaluation of the compounds described above[71] allowed us to confirm this

novel class of diphenyl-pyridine derivatives as promising and non-toxic PA-PB1 disrupting RdRp inhibitors,

and to determine preliminary SARs. In order to increase the affinity to the PB1 binding site, a rational design

of new derivatives was performed.

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As already discussed, this library of derivatives contains a scaffold that directly competes and displaces the

N-terminal portion of PB1 (PB1 ). Indeed, the overlap of PB1 in the X-ray crystallography structure with

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the docking pose of compound 3 (Figure 2) revealed that the cyano-pyridine core fits very well with a

central region of PB1 , but the compound fails in establishing crucial interactions instead made with PA by

N

the three terminal amino acids (Met1-Asp2-Val3) of PB1 peptide.[63,71]

N

Figure 2. Superimposition of the crystallographic poses of PB1 and the docking pose of compound 3, a representative

N

molecule of the cyano-pyridine library, with the interaction protein region I, II and III. PB1 is showed as grey cartoon

N

and the last three amino acids (Met1-Asp2-Val3) are represented by sticks. The three amino acids of PA (Gln408,

Asn412 and Ile621) that interact with the C2 chain are represented by green sticks and labelled.

With the aim to enhance the ability of our compounds to completely displace PB1 and increase their affinity

toward the target protein PA, a hybrid derivative, combining the cyano-pyridine core linked to the PB1 last

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peptide side chain (Met-Asp-Val) has been designed and docked into the PB1 binding site on PA. The

rational design was performed choosing the cyano-pyridine ring of compound 3 as the nucleus to which

specific amino acids were added on the C2 side chain. Since the majority of the first virtual screening

hits[63] were characterized by a p-methyl group substituent on the C4 phenyl ring, this was maintained.

At first, the hybrid Target Compound (see structure in Scheme S1 of Supplementary information) was

planned with the molecule bearing the peptide as N-terminal amine. Despite a lot of attempts (some of which

are reported in Supplementary information), the synthesis of this first designed Target Compound has

proven to be difficult to achieve; so we decided to prepare its “inverted amide”, inserting the peptide as C-

terminal. This modification was shown to be not relevant for the interaction with the most important residues

of PA, confirming a sufficiently similar docking pose.

In fact, from docking simulations, emerged that both the first designed Target Compound and the

compound with the inverted amide 11 (structure reported in Figure 3-4) were able to maintain the

characteristic interactions of the cyano-pyridine derivatives with Lys643 and Gln408 of PA also establishing

two additional hydrogen bonds with Val621 and Asn412 through the backbone of the terminal Asp and Val

amino acids (Figure 3A). The superimposition of the docking pose of compound 11 binding mode with the

PB1 fragment demonstrated a good overlap of the tripeptide region (Figure 3B).

N

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Figure 3. Docking pose of compound 11 in PA (Panel A) and superimposition of docking poses of compound 11 and

PB1 (Panel B). PA residues are represented by green sticks and PB1 is represented in grey.

N

The adducts with valine, valine-aspartate and valine-aspartate-methionine methyl esters (7a, 9 and 11

respectively, shown in Figure 4) were synthesized in order to explore the chemical space around the C2 side

chain. We decided to introduce amino acids as esters because they are more permeable through membranes

than the correspondent free acids.[72,73] Compound 11 turned out to be a weak inhibitor, while more

interesting activity profiles have been revealed for compounds 7a and 9. They showed a relevant discordance

between the low PA-PB1 interaction inhibition and the good intracellular activity, but minireplicon assays

confirmed the on-target activity (data reported in Table 1), suggesting a possible enzymatic hydrolysis in the

cell. To confirm this hypothesis, the free acid of Val and Val-Asp adducts were synthesized, through a

controlled hydrolysis in basic conditions, yielding 8 and 10 respectively. The biological data of these

derivatives seem to confirm that they are the actual responsible for the antiviral activity, as expected. The

good results of Val adducts 7a and 8 are in agreement with experimental data previously reported by

Wunderlich et al[74] that revealed Val3 on the PB1 peptide as one of the most important residues for the

interaction with PA, exhibiting the lowest binding free energy,[63] thus suggesting that its displacement

could inhibit the RdRp heterodimerization.

Encouraged by these findings, we decided to investigate the effects of other amino acids at the protein-

protein surface of the target through the addition of different L-amino acids as methyl esters (Leu, Ile, His,

Arg, Phe, Tyr, Glu: compounds 7b-h), whose structures are reported in Figure 4, through the validate

synthetic procedure of 7a (Scheme 2). Leucine (7b) and isoleucine (7c) have been chosen as hydrophobic

good replacement of valine, while the other residues have been selected as representatives of all the existing

classes of amino acids to investigate about the possible interactions of their different functions. Histidine

(

7d) and arginine (7e) bear a positive charge at physiological pH, phenylalanine (7f) and tyrosine (7g) are

characterized by aromatic lateral chains that can interact with the protein region I and glutamate (7h), if

hydrolysed, bears negative charges that could enhance the affinity of ligands towards the protein. Molecular

docking studies were then carried out to investigate the possible binding mode of these molecules within the

PB1 binding site of PA, while molecular dynamics (MD) simulations were performed to identify the

molecular determinants responsible for in vitro PA-PB1 PPI inhibition.

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Figure 4. Synthesized hybrid compounds 7-11.

CHEMISTRY. The first part of the synthetic work has been devoted to the set-up of a fast and high yielding

procedure for the preparation of the 4,6-diphenyl-3-cyanopyridinic nucleus. Starting materials were

acetophenone and p-tolualdehyde which were reacted via base catalysed Claisen-Schmidt condensation

reaction, affording the chalcone derivative 12.[75–77] This acts as Michael acceptor and the dihydropyridine

derivative 13 was obtained through a Hantzch-type cyclization of 12 with cyanothioacetamide using sodium

methoxide as a base.[78] Compound 13 was aromatized with 2,3-dichloro-5,6-dicyano-p-benzoquinone

(

DDQ) affording 2-mercapto derivative 14.[79] Then, a nucleophilic substitution of 2-bromoacetic acid was

carried out in DMF using potassium hydroxide as base affording the cyano-pyridinic core 15 (Scheme 1).

Scheme 1. Synthesis of cyano-pyridinic core. Reagents and conditions: (i) NaOH 4N, abs EtOH, N , r.t., 2 h; (ii)

2

0

Cyanothioacetamide, Na , dry MeOH, N , ref., 90 min; (iii) DDQ, DCM, r.t., 3 h; (vi) Bromoacetic acid, KOH 10%,

2

DMF, r.t., 4 h.

To synthesize the mono amino acid adducts 7a-h, we used the same amidation procedure with EDC (1-ethyl-

3

-(3-dimethylaminopropyl)carbodiimide) and HOBt (hydroxybenzotriazole) as coupling agents[78] and

racemization suppressors,[80] dry DIPEA (N, N- diisopropylethylamine), carboxylic acid 15 and the

appropriate amino acid methyl ester in dry DMF. Methyl ester 7a was hydrolysed in mild basic conditions

furnishing the free carboxylic derivative 8 (Scheme 2).

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Scheme 2. Synthesis of mono-amino acid adducts 7a-h and 8. Reagents and conditions: (i) 15, EDC, HOBt, dry

DIPEA, dry DMF, N , 0 °C to r.t., 3 h; (ii ) only for 7a: NaOH 1N, THF/EtOH/H O, r.t., 30 min. R is the lateral chain

2

a

2

of the appropriate amino acid.

The synthetic route to obtain di-amino acid adduct 9 is shown in Scheme 3. The Val-Asp dipeptide 16 was

synthesized in solution phase through the previous described amidation procedure with Fmoc-L-valine and

L-aspartic acid methyl ester. Dipeptide 16 was subjected to the cleavage of the Fmoc protecting group with

sodium azide (NaN ). The free amine 17 was then coupled with acid derivative 15 furnishing the di-amino

3

acid adduct 9. Eventually, the ester functions of 9 were hydrolysed by using a sodium hydroxide solution

affording the bicarboxylic acid derivative 10.

Scheme 3. Synthesis of di-amino acid adducts 9 and 10. Reagents and conditions: (i) EDC, HOBt, dry DIPEA, dry

DMF, N , 0 °C to r.t., 8 h; (ii) NaN , DMF, 50 °C, 5 h; (iii) EDC, HOBt, dry DIPEA, dry DMF, N 0°C to r.t., 4 h; (iv)

2

3

2,

NaOH 1N, THF/EtOH/H O, r.t., 30 min.

2

The synthesis of the tri-amino acid adduct 11 required the preparation of the tripeptide Val-Asp-Met, that

was performed in solid phase through the Fmoc strategy and is reported in Scheme 4. MW-SPPS

(

Microwave Solid Phase Peptide Synthesis) was conducted by using an automated peptide synthesizer, that

was charged with a resin (Wang resin) bearing the last amino acid of our tripeptide, L-methionine, covalently

linked. Prior to use, the resin was swallowed for 2 hours in DMF. In addition to resin and solvents (DMF and

DCM), the instrument was also charged with the other two protected amino acids (Fmoc-L-Asp-(OtBu) and

Fmoc-L-Val), the coupling activators (HOBt and HBTU), a base (DIPEA) and a deprotection solution (20%

piperidine in DMF). At the end of the deprotection and coupling cycles, we obtained the resin-bounded

tripeptide 18, which was manually cleaved to avoid the employment of corrosive trifluoroacetic acid (TFA)

inside the instrument. The cleavage of the resin was performed through a solution of TFA, H O and

2

triisopropylsilane (TIPS) as scavengers for the reactive species produced during resin cleavage, and

dithiotreithol (DTT) to avoid methionine oxidation. Under these acid conditions the tert-butyl ester in the

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aspartate lateral chain was hydrolysed furnishing the resin-free tripeptide 19 which was used in the next step

without further purification. Then the methyl esterification of dicarboxylic functions of 19, accomplished by

treating it with a methanolic solution of freshly distilled thionyl chloride (SOCl ), was necessary to avoid

2

side reactions in next steps. The reaction was carried out at room temperature and with controlled equivalents

of SOCl in order not to allow the acid-catalysed racemization of the methionine stereocenter. Dimethyl ester

2

tripeptide 20 was then deprotected from the Fmoc group furnishing the free amine 21 which was coupled

with carboxylic core 15 affording the final tri-amino acid adduct 11.

Scheme 4. Synthesis of tri-amino acid adduct (11). Reagents and conditions: (i) at Microwave Automated Peptide

Synthesizer; (ii) TFA/TIPS/H₂ O/DTT, N₂ , 3 h; (iii) SOCl dist., dry MeOH, N₂ , r.t., 6 h; (iv) NaN , DMF, 50 °C, 4

2

3

h; (v) 15, EDC, HOBt, dry DIPEA, dry DMF, 0 °C for 30 min., r.t., 12 h.

BIOLOGY. Amino acid derivatives 7-11, and the intermediates 15, 23 and 24 (the synthetic schemes of

these last two compounds are reported in Supplementary Information) were tested as inhibitors of the

PA−PB1 interaction. The in vitro biological evaluation of these compounds was conducted through enzyme-

linked immunosorbent assays (ELISA) and plaque reduction assays (PRA) in Madin−Darby canine kidney

(

MDCK) cells infected with FluA virus (A/PR/8/34 strain). The ELISA PA−PB1 interaction assay can

measure the physical inhibition between the two subunits, providing IC values. This assay was performed

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using the PB1(1−15)-Tat peptide[56] as a positive control for inhibition. The PRA evaluates the block of the

viral replication and includes oseltamivir carboxylic acid (OSV), the active form of oseltamivir (NA

inhibitor) and ribavirin (RBV) as positive controls. Cytotoxicity was assessed both in MDCK and in HEK

2

93T cell lines by MTT assays.

Moreover, to confirm the mechanism of action implied by the ELISA interaction assay, the ability of the

most interesting compounds (7a, 7c, 8, 9, 15, 24) to inhibit FluA RdRp enzymatic activity in a cellular

context was assessed by minireplicon assays in transfected HEK 293T cells, using ribavirin (RBV)[81] as a

reference drug, since it acts as RNA synthesis inhibitor thanks to its guanosine-like structure. In Table 1 all

the biological data are reported.

The tripeptide derivative (11) showed a weak inhibitory activity (IC = 68±14 µM) but such an activity is

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not retained in cells (EC > 100 µM in PRA).

50

The biological data for the methyl esters 9 and 7a, in particular the minireplicon assay results for 7a,

confirmed our hypothesis of a possible intracellular hydrolysis, so one could assume that their actual target is

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the PA-PB1 interaction, considering similar or lower EC values in minireplicon assays when compared to

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the ones observed in PRAs.

In fact, the free acid adduct 10 inhibited the interaction between PA and PB1 with a IC value of 77±9 µM,

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while it was less active in blocking viral replication in infected cells (EC = 100 µM in PRA), likely due to

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its inability to pass through the membranes. In contrast, the correspondent methyl ester 9 exhibited a good

activity in cell, according to PRA and minireplicon results (EC₅₀= 49±8 µM and EC₅₀= 59±18 µM,

respectively), but a low ability to disrupt the PA-PB1 interaction in ELISA (IC = 143±1 µM).

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As far as the valine adducts, the methyl ester derivative 7a seemed to have a similar biological profile than

the di-peptide adduct 9, showing a good antiviral activity (EC = 52±2 µM in PRA) and confirming the on-

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target activity in minireplicon assays (EC₅₀= 41±16 µM), while a high IC₅₀value of 167±20 µM was

detected. The free acid derivative 8 showed similar EC₅₀values in PRA and in minireplicon experiments

(

50±7 and 77±14 µM, respectively) and a more potent activity in ELISA (IC = 76±16 µM) when compared

50

to its methyl ester correspondent 7a.

Surprisingly, although the small difference in the side chain, leucine (7b) and isoleucine (7c) adducts were

found to have very different biological profiles: 7b appeared to have a low ability to disrupt the PA-PB1

interaction (IC =149±15 µM), but showed a weak inhibitory activity in infected cells (EC = 97±9 µM;

50

while 7c had a very promising antiviral activity in infected cells (EC = 39±2 µM in PRA) and likely acted

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by inhibiting the PA-PB1 PPI, as suggested by both the ELISA (IC = 36±3 µM) and the minireplicon

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assays (EC₅₀= 53±16µM). The histidine derivative (7d) resulted to have good activity in the ELISA

experiments (IC = 32±7 µM), but, unfortunately, a high cytotoxicity was detected (CC = 152±3 µM in

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50

MDCK cells). The arginine and the tyrosine adducts (7e and 7g respectively) were effective as disruptors of

the target interaction, with IC₅₀values of 48±8 and 18±5 µM, respectively, but did not retain the antiviral

activity in cells (EC > 100 µM). A particular case is the one of the phenylalanine derivative (7f) that seems

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not to have affinity with PA (IC > 200 µM), but in infected cells was active with an EC value of 68±10

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50

µM in PRA, perhaps after the ester hydrolysis or through an off-target mechanism of action. At the end, the

glutamate derivative (7h) is found to be inactive.

All tested compounds were non cytotoxic in MDCK and HEK 293T cells (CC > 200 µM) apart from 7d.

50

Comparing our best compound (7c) to the previous published compound 5, we can observe a similar ability

to disrupt the PA-PB1 interaction in ELISA. Moreover, even if compound 5 is a very potent antiviral agent

with an EC of 9.2±0.7 µM in PRA, its activity is probably due to other additional modes of action as

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revealed by minireplicon assays (EC = 86.6±15 µM).

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The intermediates 15, 23, 24 (the structures of the two last compounds are reported in Supplementary

Information) were also tested: while the amine 23 resulted inactive, the acid 15 and the amide 24 showed

interesting activity profiles, even if a low toxic behaviour in HEK 293T cell line was detected.

Table 1. Biological profiles of the synthesized compounds 7-11, 15, 23 and 24.

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ELISA

PRA in

MTT in

Minireplicon

Assay EC₅₀

MTT in

Compound

PA-PB1

MDCK cells

HEK 293T cells

a,e

b,e

c,e

d,e

c,e

IC [µM]

EC [µM]

CC [µM]

[µM]

CC [µM]

5

0

50

7

a

167 ± 20

149 ± 15

36 ± 3

52 ± 2

97 ± 9

210 ± 8

215 ± 28

>250

41 ± 16

> 250

b

-

7

c

39 ± 2

53 ± 16

229 ± 12

7

d

32 ± 7

80 ± 6

152 ± 13

>250

-

7

e

48 ± 8

>100

-

7

f

>200

68 ± 10

>100

240 ± 18

>250

-

7

g

18 ± 5

-

h

187 ± 14

76 ± 16

143 ± 17

77 ± 9

>100

>250

-

8

50 ± 7

> 250

>250

77 ± 14

250

9

49 ± 8

59 ± 18

> 250

-

1

0

1

5

3

4

100

-

1

2

68 ± 14

56 ± 5

>100

-

65 ± 11

> 100

43 ± 14

-

218 ± 21

-

>200

102 ± 18

35.2 ± 5.1

31.7 ± 10.8

-

41 ± 5

65 ± 14

86.6 ± 15.0

15.5 ± 2.6

-

218 ± 21

> 250

7

1

5

9.2 ± 0.7

49.7 ± 5.1

0.015 ± 0.006

12.8 ± 2.1

PB1₍₁₋₁₅₎-Tat peptide

OSV

> 250

RBV

-

23.8 ± 4.5

a

-

: not determined. Activity of the compounds in the ELISA PA−PB1 interaction assay. Values represent the compound

b

concentration (in µM) that reduces the interaction between PA and PB1 by 50% (IC ). Antiviral activity of the

50

compounds in plaque reduction assays against the Flu A/PR/8/34 strain. Values represent the compound concentration

in µM) that inhibits 50% of plaque formation (EC ). Compound concentrations higher than those reported could not be

(

50

c

tested in PRA due to cytotoxicity. Cytotoxicity of the compounds exhibited in MTT assays. Values represent the

compound concentration (in µM) that causes a 50% decrease in cell viability (CC50). The CC₅₀were assessed in

d

MDCK and HEK 293T cells. The EC values represent the compound concentration that reduces by 50% the activity

5

0

e

of FluA virus RNA polymerase in HEK 293T cells. All values represent the means ± SD of data derived from at least

three independent experiments in duplicate. Reference compounds are the previous described compound 5, PB1₍₁₋₁₅₎_-Tat

peptide, oseltamivir carboxylic acid (OSV) and ribavirin (RBV).

MOLECULAR MODELING. The ELISA PA−PB1 interaction assay provided a direct measure of the PA-

PB1 PPI inhibition by test compounds. Here, to add further insights into the inhibition mechanism, we

carried out molecular modeling investigations. First, the possible binding mode of compounds listed in

Table 1 to the PB1 binding site of PA was predicted by molecular docking, which was performed with the

GOLD program (GoldScore function)[82] according to previous works.[63,71] Notably, all compounds and

intermediates showed to fit the PB1 site of PA with a binding geometry that is highly comparable each other,

as well as to the binding mode already described above for compound 11 and the reference inhibitor PB1_N

ACCEPTED MANUSCRIPT

(

see Figure 2 and Figure 3). The binding mode of tested compounds is described in Supplementary

information. However, probably because of the narrow activity range observed in the ELISA assay (Table

), any attempt to correlate the ligands’ scores with experimental data was unsuccessful (data not shown).

1

Therefore, to identify the possible molecular determinants responsible for strong inhibition in the ELISA

assay, we carried out molecular dynamics (MD) investigations on the docking-based complexes between PA

and compounds 7g and 7f (Figure 4). Indeed, these two molecules differ each other only for the –OH phenol

group in 7g (L-Tyr residue) that is missing in 7f (L-Phe residue), but were notably different in the ELISA

experiment, as 7g proved the most potent PA-PB1 inhibitor of the tested set while 7f was inactive up to 200

µM concentration. Thus, they represent a suitable model system to challenge MD simulations. The

representative docking complex of 7g and 7f was solvated in a box of water molecules and was energy

minimized, heated and equilibrated in density as described previously,[83,84] before to run 250 ns of

unrestrained MD simulations. Cluster analysis performed on the whole MD production trajectories coupled

with visual inspection clearly indicated that 7g is stable in the PB1 binding site, with a conformation that is

highly consistent to that identified by molecular docking (Figure 5A). The nitrile group of 7g establishes a

H-bond interaction with the side chain of Lys643 (to allow comparison with results described in previous

works [63, 71] residue numbering follows the sequence of A/Wilson-Smith/1933 H1N1 strain). The two

phenyl rings of the 3-cyano-4,6-diphenylpyridine scaffold are included in a hydrophobic cleft bounded by

Pro625, Phe658, Leu666, Trp706, Phe707, and Phe710. The peptide moiety notably overlaps with the

crystallographic pose of the N-terminal end of PB1,[26] and establishes a network of H-bonds. First, the

amide bond contacts Glu623, and Gln408 (bridged by a water molecule, see Figure 5A) that was already

highlighted in prior works [63, 71] as a crucial residue for the binding of PA-PB1 inhibitors. The L-Tyr side

chain of 7g is accommodated in a hydrophobic sub-pocket of PA where it interacts with Asn412 (bridged by

a water molecule), Ser415, and Lys635. It is worth mentioning that these three H-bonds are established by

the –OH phenol group of 7g, which is missing in 7f. Different from the docking pose (Supplementary

information), the peptide moiety of 7f bends back towards the hydrophobic cleft of PA thus assuming a bad

placement as described previously (Figure 5B).[71] Moreover, in this conformation 7f is unable to establish

most of H-bond interactions with PA key residues with the only exception of a single H-bond to Lys635. The

different binding conformation and interaction network between these two compounds might explain their

opposite behaviour observed in the PA−PB1 interaction assay (ELISA). To further substantiate this

hypothesis, we estimated the ligand’s theoretical affinity by means of the MM-GBSA approach.[85] Delta

energy calculations clearly showed that the different binding conformation is associated to a significantly

different theoretical affinity, as reported in Table 2, which suggests that ligand affinity is mainly related to

enthalpy contributions. In summary, this molecular modeling investigation added further insights to the

inhibition mechanism of tested molecules and might drive the design of ligands with enhanced affinity for

PA.

ACCEPTED MANUSCRIPT

Table 2. Delta energy of binding of 7g and 7f to PA estimated by the MM-GBSA approach.

a

Compound

MM-GBSA (kcal/mol) ± SEM

-40.78 ± 1.12

7g

7f

-27.65 ± 0.78

a

Standard Error of the Mean

Figure 5. Binding pose of 7g (panel A) and 7f (panel B) extrapolated from the most representative cluster of MD

frames. The protein is shown as green cartoon, residues within 4 Å from the ligand are showed as lines, residues H-

bonded to the ligand are showed as sticks and are labelled. Non-polar H atoms are omitted. Bridged water molecules are

showed as red spheres, while the bulky solvent is omitted. 7g is shown as cyan sticks, 7f as white sticks. H-bond

interactions are highlighted by grey dashed lines.

CONCLUSIONS. In the frame of the limited armamentarium of drugs for the treatment of Flu infections

together with the chemo-resistant phenotypes increasing, the search for novel antivirals with innovative

mode of action became imperative. In recent years, the viral polymerase has proved to be an attractive target

for drug design, although the current efforts led to quite toxic compounds. In particular, some research

groups have been interested in the development of small molecules able to interfere with the correct

assembly of RdRp complex, through the inhibition of the interaction between PA and PB1, two of its

subunits. In this work, starting from previously published findings, a rational drug design led us to synthesize

hybrid compounds characterized by the diphenyl-pyridine nucleus, identified as promising inhibitor of the

above mentioned PPI, and the same last amino acids side chain of PB1 (Met-Asp-Val) in C2. Indeed, we

N

planned the synthesis of the tri-peptidic (11), di-peptidic (Val-Asp, 9) and the mono-amino acid (Val, 7a)

derivatives as methyl esters to ensure a higher passive membrane permeability. Biological data results

highlighted a major anti-flu activity (EC₅₀in PRA) of 9 and 7a, not correlated to the low enzymatic

inhibition (IC in ELISA). To better investigate this discrepancy, minireplicon experiments were performed,

50

allowing us to confirm the PA-PB1 interaction disruption as the main target of our compounds. Moreover,

ACCEPTED MANUSCRIPT

we hypothesize that these methyl ester derivatives could be hydrolysed in cell, giving the corresponding free

acids. In fact, the synthesized 10 and 8 (free acid dipeptide and mono-amino acid derivatives, respectively)

showed a better IC₅₀when compared to that of 9 and 7a. Notably, valine derivatives 7a and 8 emerged as

new hit compounds for further optimization. Aimed by the exploration of the chemical space around the C2

of the diphenyl-pyridine core, novel mono-amino acid derivatives (7b-h) were synthesized and tested.

Interestingly, the modulation in C2 of the starting chemical structures led to an increase in the on-target

activity. In particular, the best results were achieved with the isoleucine derivative (7c) that exhibited a good

biological profile with no cytotoxic behaviour. Molecular modeling simulations added further insights to the

inhibition mechanism of these compounds, showing that enthalpy contributions (i.e. H-bond interactions

with PA key residues) are crucial to achieve strong PA-PB1 PPI inhibition in vitro. In summary, a new small

library of hybrid derivatives endowed with anti-influenza activity was generated. Almost of all of these

compounds were found to be not cytotoxic at high concentrations in the two cell lines used for testing and to

inhibit actually the studied PPI. Moreover, we set up a quantitative four-step scaling-up procedure to

synthesize the diphenyl-pyridine scaffold that might be helpful to expand the chemical class and to improve

the inhibitory activity.

MATERIAL AND METHODS

1

. COMPUTATIONAL SECTION

The crystallographic structure of Influenza A virus PA (C-terminal region) in complex with PB1 coded by

N

PDB ID: 4WSB,[44] which was already validated as docking receptor in a previous work, [71] was used as

rigid receptor in molecular docking studies.[26] Tested compounds were designed by means of Maestro

program, and docked by using the GOLD docking program with the GoldScore function.[82] The receptor’s

site was centred on the carbon atom in position 2 of the indole ring of Trp706, and had a radius of 18 Å. The

search efficiency of the Genetic Algorithm was increased up to 200%, while all other parameters were kept

at their default values. MD simulations were carried out with Amber16, using the FF14SB force field for the

protein and GAFF force field for small molecules.[86] Ligands partial charges were assigned at the am1-bcc

level of theory. Each PA-ligand complex was solvated in a rectilinear box of TIP3P water molecules

+

buffering 8 Å from the protein surface. Na counter-ions were added to neutralize the total charge of the

system. First, the water solvent was energy minimized for 500 steps with the steepest descent algorithm (SD)

and 1500 steps with the conjugate gradient algorithm (CG); then, the whole system was energy minimized

for 1000 steps SD and 4000 steps CG before to be heated up to 300 K in 100 ps (time-step is 2 fs). The

heated system was equilibrated in density at constant pressure for 100 ps before to run an equilibration MD

at constant pressure for 50 ns. Subsequently, unrestrained MD production was run for 250 ns at constant

pressure. Analysis of MD trajectories, including cluster analysis, was carried out by cpptraj.[86] Graphics

representations were drawn for clusters being representative of more than 60% of MD frames.

ACCEPTED MANUSCRIPT

2

. CHEMISTRY SECTION

All commercially available chemicals and solvents were used as purchased (Merck-Sigma Aldrich, Alfa-

Aesar). Anhydrous reactions were run under a positive pressure of dry nitrogen. Anhydrous solvents were

prepared by distillation over the appropriate agent: DCM was dried over sodium hydride; THF was dried

over sodium and benzophenone; MeOH and EtOH were dried over iodide and magnesium. Anhydrous DMF

was used as purchased. Microwave reactions were conducted using a CEM Discover Synthesis Unit (CEM

Corp., Matthews, NC). Chromatographic separations were performed on columns packed with silica gel

(

230-400 mesh, for flash technique). TLC (thin layer chromatography) was carried out using Merck TLC

plates silica gel 60 F₂₅₄_._.Melting points (Mp) were determined with a Büchi B-540 apparatus and are

1

13

uncorrected. H NMR and C NMR spectra were recorded at 400 and 100 MHz respectively on a Bruker

AC200F spectrometer and are reported in parts per million (δ scale) and internally referenced to the CDCl₃,

CD OD or DMSO D signal, respectively at δ 7.26 ppm, 3.31 ppm and 2.50 ppm. Chemical shifts for carbon

3

6

are reported in parts per million (δ scale) and referenced to the carbon resonances of the solvent (CDCl at δ

3

7

7.16, CD OD at δ 49.00 ppm and DMSO D at 39.52 ppm). Data are shown as following: chemical shift,

3 6

multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet and/or multiplet resonances, b = broad),

coupling constant (J) in Hertz (Hz) and integration. Mass spectra (LC-MS) were acquired using an Agilent

1

100 LC-MSD VL system (G1946C) by direct injection with a 0.4 mL/min flow rate using a binary solvent

system of 95/5 CH OH/H O. UV detection was monitored at 254 nm. Mass spectra were acquired in positive

3

2

mode scanning over the mass range 105-1500 m/z, using a variable fragmentor voltage of 10-70 mV. IR

spectra were measured in KBr with a Perkin-Elmer 398 spectrophotometer. Elemental analysis for C, H, N

and S was determined using Thermo Scientific Flash 2000 and results were within ±0.4% of the theoretical

value. The purity of final products (7-11, 15, 23 and 24) was 90% or higher and it was assessed by HPLC-

MS, using a Kinetex EVO C18 column (00F-4633-EO: 150 x 4.6 mm, 5 µm particle size) at a flow rate of

0

.6 mL/min with an isocratic elution 60/50 v/v CH CN /H O (formic acid 0.5% v/v). UV detection was

3 2

monitored at 254 nm. Mass spectra were acquired in positive mode scanning over the mass range 105-1500

TM

m/z, using a fragmentor voltage of 0 mV. The MW-SPPS was performed using Fmoc strategy on a Liberty

TM

Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC), an additional module of Discover

(

CEM Corporation, Matthews, NC) on a 0.10 mmol scale. Wang resin served as the solid support. It was

preloaded with the first protected amino acid at a degree of functionalization of 0.60 mmol per gram of resin.

The coupling was performed in DMF and the peptide chain assembled under computer control of the

synthesizer. The synthesis cycles were composed of Fmoc-deprotection and coupling, both under microwave

irradiation. The deprotection was performed with 20% piperidine in DMF, the coupling mixture contained

Fmoc-protected amino acid the activating agents HOBt (1-Hydroxybenzotriazole) and HBTU (O-

(

benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate) 0.5 M in DMF and an excess of DIPEA

diisopropylethylamine) 34.8% v/v in DMF as the base. The final cleavage of the tripeptide from the solid

support and the ester deprotection of the side chains was performed with 88% trifluoroacetic acid (TFA), 5%

ACCEPTED MANUSCRIPT

water, 2% triisopropylsilane (TIS) and 5% DL-Dithiothreitol (DTT) to quench reactive intermediates under

microwave irradiation and to prevent the oxidation of the thioether side chain of methionine. The C-terminal

end and the carboxylic aspartate side chain of the peptide is obtained as free carboxylic acids. All parameters

(

t, p, T, Q) have been estabilished automatically by the instrument and are different in every cycle.

2

.1 Synthetic procedures for the acid core 15.

E)3-(4-Methylphenyl)-1-phenylpro-2-en-1-one (12)

(

To a stirred solution of acetophenone (3.8 mL; 33.0 mmol) in absolute EtOH (50 mL), a solution of p-

tolualdehyde (3.8 mL; 33.0 mmol) in dry EtOH (50 mL) and a 4 N aqueous solution of NaOH (20.6 mL)

were added. The mixture was stirred at room temperature for 6 h. The reaction was quenched with HCl 2 N

and filtered, then the solvent was partially evaporated under vacuum. The residue was dissolved in AcOEt,

washed with water, dried over Na SO , filtered and concentrated under reduced pressure. The solid obtained

2

4

was decanted with EtOH furnishing the pure chalcone as a pale yellow solid. TLC R (Hexane/AcOEt 1/6)

f

1

0

1

.70. Yield 99%. Mp: 189-190 °C. H-NMR (400 MHz CDCl ): δ (ppm) 7.97 (d, 2H, J = 7.6 Hz), 7.80 (d,

3

1

3

H, J = 15.6 Hz), 7.53 (m, 6H), 7.22 (d, 2H, J = 7.6 Hz), 2.38 (s, 3H). C-NMR (100 MHz CDCl ): δ (ppm)

3

90.27, 144.74, 140.96, 138.29, 132.60, 132.09, 129.65, 128.52, 128.46, 128.43, 120.94, 21.45. LRMS

-1

(

ESI) m/z = 222.9 [M+H]⁺ ; 244.9 [M+Na]⁺ . IR (KBr): cm 3022 (CH=CH), 1656 (CO). Anal. calcd. for

C H O: C 86.45, H 6.35; found: C 86.47, H 6.45.

16

14

4

‐ (4‐ Methylphenyl)‐ 6‐ phenyl‐ 2‐ sulfanyl‐ 1,4‐ dihydropyridine‐ 3‐ carbonitrile (13)

To a solution of sodium (186 mg, 8.10 mmol) in dry MeOH (22 mL) under N atmosphere, chalcone 12 (900

2

mg, 4.05 mmol) and thiocyanoacetamide (405 mg, 4.05 mmol) were added. The mixture was stirred at reflux

for 90 minutes. After cooling, the mixture was quenched with HCl 1 N and partially evaporated under

vacuum. The residue was diluted in DCM, washed with water, dried over Na SO , filtered and concentrated

2

4

under reduced pressure. The pure yellow solid obtained was used directly in the next step.TLC R_f

1

(

Hexane/AcOEt 4/1) 0.65. Yield 94%. Mp: 105-133 °C. H-NMR (400 MHz CDCl ): δ (ppm) 9.21 (s, 1H),

3

7

2

1

.46 (m, 5H), 7.19 (m, 4H), 5.92 (m, 0.4H), 5.78 (m, 0.6H), 4.33 (m, 0.4H), 4.09 (m, 0.6H), 2.35 (s, 3H),

1

3

.04 (s, 1H). C-NMR (100 MHz): δ (ppm) 137.38, 132.95, 129.91, 129.74, 129.21, 128.04, 127.52, 125.34,

+

16.26, 108.90, 108.64, 49.13, 48.38, 42.15, 21.04. LRMS (ESI) m/z = 304.9 [M+H] ; 326.8 [M+Na] . IR

-1

(

KBr) cm : 3203 (NH), 2196 (CN). Anal. calcd. for C H N S: C 74.97, H 5.30, N 9.20, S 10.53; found: C

19 16 2

7

4

4.86, H 5.68, N 8.84, S 10.73.

‐ (4‐ Methylphenyl)‐ 6‐ phenyl‐ 2‐ sulfanylpyridine‐ 3‐ carbonitrile (14)

To a stirred solution of 13 (679 mg, 2.23 mmol) in DCM (15 mL), DDQ (1014 mg, 4.46 mmol) was added.

The mixture was stirred at room temperature for 3 h. The mixture was filtered and evaporated under reduced

pressure. The dark solid obtained was decanted several times with MeOH, water and hexane to remove

DDHQ. The solid was then recrystallized from MeOH. TLC R (Hexane/AcOEt 4/1) 0.43 Yield: 84%. Mp:

f

1

2

04-207 °C. H-NMR (400 MHz CDCl ): δ (ppm) 7.96 (d, 2H, J = 8.0 Hz), 7.61 (s, 1H), 7.53 (d, 2H, J = 8.0

3

1

3

Hz), 7.37 (m, 5H), 2.46 (s, 3H), 1.56 (s, 1H). C-NMR (100 MHz CDCl ): δ (ppm) 160.57, 158.83, 154.86,

3

ACCEPTED MANUSCRIPT

1

42.72, 140.58, 136.74, 132.85, 130.64, 129.76, 128.82, 128.27, 127.32, 117.35, 115.48, 21.31. LRMS

+

-1

(

ESI) m/z = 302.9 [M+H] . IR (KBr) cm : 2215 (CN). Anal. calcd. for C H N S: C 75.47, H 4.67, N 9.26,

19 14 2

S 10.60; found: C 75.24, H 4.38, N 9.57, S 10.58.

2

{[3 Cyano 4 (4 methylphenyl) 6 phenylpyridin 2 yl]sulfanyl}acetic acid (15)

To a stirred solution of 14 (418 mg, 1.38 mmol) in DMF (6 mL) a 10% aqueous solution of KOH (1.70 mL,

.04 mmol) and a solution of 2-bromoacetic acid (212 mg, 1.52 mmol) in DMF (1 mL) were added and the

3

mixture was stirred at room temperature for 3 h. The mixture became dark red. Then the reaction was treated

with HCl 3 N, becoming yellow, and extracted with AcOEt. The organic phase was washed with water, dried

over Na SO , filtered and concentrated under reduced pressure. The crude residue was purified through

2

4

recrystallization from MeOH to give compound 15 as yellow crystal. TLC R (AcOEt/MeOH 9/1) 0.43.

f

1

Yield: 75%. Mp: 218-220 °C. H-NMR (400 MHz DMSO D ): δ (ppm) 8.25 (m, 2H), 7.88 (s, 1H), 7.65 (d,

6

1

3

2

H, J = 8.0 Hz), 7.51 (m, 3H), 7.39 (d, 2H, J = 8.0 Hz), 4.15 (s, 2H), 2.40 (s, 3H). C-NMR (100 MHz

DMSO): δ (ppm) 170.29, 162.50, 158.40, 154.56, 140.47, 137.01, 133.11, 131.16, 129.84, 129.25, 128.97,

+

1

28.05, 116.44, 116.12, 102.81, 33.37, 21.27. LRMS (ESI): m/z = 360.9 [M+H] ; 382.8 [M+Na] ; 398.9

+

-1

[

M+K] . IR (KBr) cm : 3200-2400 (OH), 2219 (CN), 1710 (CO). Anal. calcd. for C H N O S: C 69.98, H

21 16 2 2

4

.47, N 7.77, S 8.90; found: C 69.73, H 4.78, N 8.13, S 8.51.

2

.2. Synthetic procedure for monoamino acid adducts (7a-h and 8)

General procedure for monoamino acid adducts (7a-h)

To a stirred solution of 15 (50 mg, 0.14 mmol) in dry DMF (0.5 mL) at 0 °C under N atmosphere, the

2

appropriate L-amino acid methyl ester (0.15 mmol), EDC (27 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol)

and dry DIPEA (0.03 mL, 0.20 mmol) were added. The mixture was stirred 30 min at 0 °C and then 5 h at

room temperature. The reaction was quenched with water and then extracted with AcOEt. The organic phase

was washed with water, dried over Na SO , filtered and concentrated under reduced pressure. The crude

2

4

residue was purified through flash chromatography on silica gel.

Methyl (2S) 2 (2 {[3 cyano 4 (4 methylphenyl) 6 phenylpyridin 2 yl]sulfanyl}acetamido)

3

methylbutanoate (L-Valine methyl ester adduct, 7a)

1

Eluted with Hexane/AcOEt 3/1. Yield: 20%. White solid. H -NMR (400 MHz CDCl ): δ (ppm) 8.05 (m,

3

2

H), 7.60 (s, 1H), 7.53 (m, 5H), 7.35 (d, 2H, J = 7.6 Hz), 7.17 (d, 1H, J = 6.4 Hz), 4.48 (m, 1H), 4.08 (q, 2H,

1

3

J = 16 Hz), 3.53 (s, 3H), 2.44 (s, 3H), 1.97 (m, 1H), 0.69 (d, 3H, J = 6.8 Hz), 0.62 (d, 3H, J = 6.8 Hz). C-

NMR (100 MHz CDCl ): δ (ppm) 167.92, 161.62, 158.97, 154.90, 140.65, 136.74, 132.83, 130.74, 129.75,

3

1

29.03, 128.22, 127.39, 116.52, 115.36, 103.79, 57.34, 51.84, 33.89, 30.87, 29.59, 21.29, 18.60, 17.43.

+

-1

LRMS (ESI) m/z = 474.7 [M+H]⁺ ; 496.8 [M+Na] . IR (KBr) cm : 3359 (NHCO), 2220 (CN), 1749 (CO

ester), 1646 (CO amide). Anal. calcd. for C H N 0 S: C 68.48, H 5.75, N 8.87, S 6.77; found: C 68.37, H

27

3 3

5

.59, N 8.77, S 6.64.

ACCEPTED MANUSCRIPT

Methyl (2S) 2 (2 {[3 cyano 4 (4 methylphenyl) 6 phenylpyridin 2 yl]sulfanyl}acetamido)

4

methylpentanoate (L-Leucine methyl ester adduct, 7b)

1

Eluted with AcOEt/MeOH 9/1. Yield: 31%. White solid. Mp: 150-151 °C. H -NMR (400 MHz CDCl ): δ

3

(

ppm) 8.06 (d, 2H, J = 7.6 Hz), 7.60 (s, 1H), 7.52 (m, 5H), 7.34 (d, 2H, J = 7.6 Hz), 7.13 (d, 1H, J = 8.0 Hz),

4

.56 (m, 1H), 4.05 (q, 2H, J = 15.6 Hz), 3.54 (s, 3H), 2.44 (s, 3H), 1.40 (m, 2H), 1.26 (m, 1H), 0.31 (d, 3H, J

1

3

=

11.4 Hz), 0.27 (d, 3H, J = 11.4 Hz). C-NMR (100 MHz CDCl3): δ (ppm) 172.73, 167.71, 161.64,

1

5

58.87, 154.90, 140.6,6, 136.65, 132.82, 130.81, 129.78, 129.07, 128.20, 127.37 116.43, 103.77, 52.02,

0.81, 41.12, 33.85, 24.53 22.50, 21.50, 21.31. LRMS (ESI) m/z = 407.7 [M+H]⁺ ; 509.8

+

-1

[

M+Na] ;525.5[M+K] . IR (KBr) cm : 3362 (NHCO), 2222 (CN), 1745 (CO ester), 1650 (CO amide).

Anal. calcd. for C H N O S: C 68.97, H 5.99, N 8.62, S 6.58; found: C 69.01, H 5.87, N 8.50, S 6.53.

2

8

29

3

Methyl (2S,3S)‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐

yl]sulfanyl}acetamido)‐ 3‐ methylpentanoate (L-Isoleucine methyl ester adduct, 7c)

1

Eluted with AcOEt/MeOH 9/1. Yield: 31%. White solid. Mp: 163-165 °C. H -NMR (400 MHz CDCl ): δ

3

(

ppm) 8.04 (m, 2H), 7.60 (s, 1H), 7.51 (m, 5H), 7.34 (d, 2H, J = 7.6 Hz), 7.18 (d, 1H, J = 8.4 Hz), 4.50 (m,

1

3

1

H), 4.06 (q, 2H, J = 15.6 Hz), 3.52 (s, 3H), 2.43 (s, 3H), 1.67 (m, 2H), 1.13 (m, 1H), 0.65 (m, 6H). C-

NMR (100 MHz CDCl3): δ (ppm) 171.66, 167.78, 161.64, 158.96, 154.89, 140.66, 136.73, 132.82, 130.78,

1

2

6

29.76, 129.04, 128.22, 127.40, 116.50, 115.37, 103.37, 56.68, 51.79, 37.51, 33.88, 24.83, 21.30, 15.07,

+

-1

1.23. LRMS (ESI) m/z = 488.0 [M+H]⁺ ; 510.0 [M+Na] ;526.0[M+K] . IR (KBr) cm : 3369 (NHCO),

215 (CN), 1732 (CO ester), 1657 (CO amide). Anal. calcd. for C H N O S: C 68.97, H 5.99, N 8.62, S

2

8

29

3

.58; found: C 68.75, H 5.97, N 8.72, S 6.43.

Methyl (2S)‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐ yl]sulfanyl}acetamido)‐

3

‐ (1H‐ imidazol‐ 4‐ yl) propanoate (L-Histidine methyl ester adduct, 7d)

1

Eluted with AcOEt/MeOH 9/1. Yield: 30%. White solid. Mp: 135-141 °C. H -NMR (400 MHz CDCl ): δ

3

(

ppm) 8.07 (m, 2H), 7.94 (d, 1H, J = 7.2 Hz), 7.57 (s, 1H), 7.52 (m, 5H), 7.39 (s, 1H), 7.34 (d, 2H, J = 8.0

Hz), 6.62 (s, 1H), 4.80 (m, 1H), 4.11 (s, 2H), 3.54 (s, 3H), 2.89-3.02 (2q, 2H, J = 15.0 Hz), 2.44 (s, 3H), 1.50

13

(

m, 1H). C-NMR (100 MHz CDCl ): δ (ppm) 171.42, 167.99, 161.54, 158.70, 154.80, 140.08, 140.04,

3

1

36.77, 133.10, 132.96, 130.74, 129.76, 129.02, 128.21, 127.42, 116.33, 115.62, 99.42, 90.97, 52.32, 52.21,

-1

4

5.20, 29.04, 21.28. LRMS (ESI) m/z = 512.0 [M+H]⁺ ; IR (KBr) cm : 3361 (NH), 2221 (CN), 1741 (CO

ester), 1654 (CO amide). Anal. calcd. for C H N O S: C 65.74, H 4.93, N 13.69, S 6.27; found: C 65.41,

28

25

5

3

H 4.70, N 14.07, S 6.44.

Methyl (2S)‐ 3‐ carbamoyl‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐

yl]sulfanyl}acetamido) propanoate (L-Arginine methyl ester adduct, 7e)

1

Eluted with DCM/MeOH 9/1. Yield: 27%. White solid. Mp: 161-171 °C. H -NMR (400 MHz CD OD): δ

3

(

ppm) 8.19 (m, 2H), 7.74 (s, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.50 (m, 3H), 7.37 (d, 2H, J = 8.0 Hz), 4.45 (m,

1

2

1

H), 4.22 (q, 2H, J = 16.0 Hz), 3.59 (s, 3H), 3.02 (m, 2H), 2.43 (s, 3H), 1.62 (m, 1H), 1.82 (m, 1H), 1.47 (m,

13

H). C-NMR (100 MHz CD OD): 171.90, 169.20, 161.91, 158.74, 154.86, 140.40, 136.90, 133.01, 132.02,

3

30.40, 129.30, 129.25, 128.71, 128.17, 127.49, 126.60, 115.13, 48.16, 47.94, 47.09, 46.87, 40.29, 29.01,

ACCEPTED MANUSCRIPT

+

-1

2

4.59. LRMS (ESI) m/z = 531.0 [M+H]⁺ ; 553.0 [M+Na] . IR (KBr) cm : 3364 (NH), 2225 (CN), 1744

(

CO ester), 1653 (CO amide). Anal. calcd. for C H N O S: C 63.38, H 5.70, N 15.84, S 6.04; found: C

28 30 6 3

6

3.44, H 5.44, N 15.75, S 6.42.

Methyl (2S)‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐ yl]sulfanyl}acetamido)‐

‐ phenylpropanoate (L-Phenylalanine methyl ester adduct, 7f)

3

1

Eluted with AcOEt/MeOH 9/1. Yield: 34%. White solid. Mp: 167-173 °C. H -NMR (400 MHz DMSO D ):

6

δ (ppm) 8.65 (d, 1H, J = 7.6 Hz), 8.25 (m, 2H), 7.87 (s, 1H), 7.64 (d, 2H, J = 7.6 Hz), 7.51 (m, 3H), 7.39 (d,

2

H, J = 7.6 Hz), 7.12 (m, 5H), 4.47 (m, 1H), 4.14 (s, 2H), 3.49 (s, 3H), 2.85-2.99 (2q, 2H, J = 14.0 Hz), 2.40

1

3

(

s, 3H). C-NMR (100 MHz DMSO D ): δ (ppm) 171.37, 167.67, 161.43, 158.77, 154.73, 140.71, 136.60,

6

1

5

35.43, 132.88, 130.80, 129.84, 129.06, 128.71, 128.52, 128.21, 127.35, 126.80, 116.37, 115.32, 103.54,

+

3.15, 52.12, 37.52, 33.38, 24.04. LRMS (ESI) m/z = 552.0 [M+H]⁺ ; 544.0 [M+Na] ; 560.0 [M+K] . IR

-1

(

KBr) cm : 3300 (NHCO), 2217 (CN), 1742 (CO ester), 1655 (CO amide). Anal. calcd. for C H N O S: C

31 27 3 3

7

1.38, H 5.22, N 8.06, S 6.15; found:C 71.21, H 5.30, N 8.11, S 5.78.

Methyl (2S)‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐ yl]sulfanyl}acetamido)‐

3

‐ (4‐ methoxyphenyl) propanoate (L-Tyrosine methyl ester adduct, 7g)

1

Eluted with Hexane/AcOEt 6/4. Yield: 21%. White solid. Mp: 215-217 °C. H -NMR (400 MHz CDCl ): δ

3

(

ppm) 8.03 (m, 2H), 7.58 (d, 2H, J = 8 Hz), 7.50 (m, 3H), 7.36 (d, 2H, J = 7.6 Hz), 7.25 (s, 1H), 7.10 (d, 1H,

J = 8.0 Hz), 6.68 (d, 2H, J = 8.2 Hz), 6.45 (d, 2H, J = 8.2 Hz), 5.12 (bs, 1H), 4.77 (m, 1H), 3.99 (q, 2H, J =

1

3

1

=

5.6 Hz), 3.57 (s, 3H), 2.75-2.94 (2q, 2H, J = 14.0 Hz), 2.45 (s, 3H). C-NMR (400 MHz CDCl ): δ (ppm)

3

71.47, 167.80, 161.42, 158.90, 154.81, 144.96, 140.69, 136.59, 132.84, 130.94, 129.79, 129.12, 129.02,

28.39, 127.57, 127.15, 116.56, 116.10, 115.40, 103.46, 53.17, 49.84, 36.58, 31.31, 21.29. LRMS (ESI) m/z

+

-1

538.0 [M+H]⁺ ; 540.6 [M+Na] . IR (KBr) cm : 3400-3200 (OH), 3306 (NH), 2218 (CN), 1730 (CO

ester), 1656 (CO amide). Anal. calcd. for C H N O S: C 69.25, H 5.06, N 7.82, S 5.96; found: C 69.61, H

3

1

27

3

4

5

.32, N 7.99, S 5.83.

,5‐ Dimethyl (2S)‐ 2‐ (2‐ {[3‐ cyano‐ 4‐ (4‐ methylphenyl)‐ 6‐ phenylpyridin‐ 2‐

1

yl]sulfanyl}acetamido) pentanedioate (L-Glutammate methyl diester adduct, 7h)

1

Eluted with Hexane/AcOEt 1/1. Yield: 27%. White solid. Mp: 179-193 °C. H -NMR (400 MHz CDCl ): δ

3

(

ppm) 8.06 (m, 2H), 7.61 (s, 1H), 7.54 (d, 2H, J = 7.6 Hz), 7.50 (m, 3H), 7.35 (d, 2H, J = 7.6 Hz), 7.25 (s,

1

2

1

3

H), 4.58 (m, 1H), 4.05 (q, 2H, J = 15.8 Hz), 3.55 (s, 3H), 3.54 (s, 3H), 2.44 (s, 3H), 2.09 (m, 2H), 1.78 (m

13

H). C-NMR (400 MHz CDCl ): δ (ppm) 171.30, 168.01, 162.70, 161.80, 158.97, 155.00, 140.66, 136.66,

3

32.85, 130.80, 129.78, 129.11, 128.29, 127.47, 116.61, 115.37, 103.86, 59.91, 59.12, 53.05, 52.20, 41.80,

+

-1

7.02, 20.50. LRMS (ESI) m/z = 518.0 [M+H]⁺ ; 540.0 [M+Na] ; 556.0 [M+K]⁺ . IR (KBr) cm : 3312

(

NHCO), 2215 (CN), 1732 (CO ester), 1653 (CO amide). Anal. calcd. for C H N O S: C 64.97, H 5.26, N

28 27 3 5

8

.12, S 6.20; found: C 65.01, H 5.43, N 8.49, S 6.06.

ACCEPTED MANUSCRIPT

(

2S) 2 (2 {[3 Cyano 4 (4 methylphenyl) 6 phenylpyridin 2 yl]sulfanyl}acetamido) 3

methylbutanoic acid (L-Valine free acid adduct, 8)

To a stirred solution of 7a (24 mg, 0.05 mmol) in a 3/1/0.5 mixture of THF/EtOH/H O (2 mL), NaOH 1 N

2

(

0.22 mL, 0.22 mmol) was added. The mixture was stirred at room temperature for 15 min until the solution

becomes yellow. The reaction was quenched with HCl 2 N and extracted with AcOEt. The organic phase

was washed with water, dried over Na SO , filtered and concentrated under reduced pressure. The crude

2

4

residue was purified through flash chromatography on silica gel, eluting with AcOEt/MeOH/AcOH 9/1/0.5

1

to give pure 8. TLC R (DCM/MeOH 9/1) 0.61. Yield: 40%. H-NMR (400 MHz CD OD): δ (ppm) 8.19 (d,

f

3

2

H, J = 8.0 Hz), 7.76 (s, 1H), 7.58 (d, 2H, J = 8.0 Hz), 7.50 (m, 3H), 7.38 (d, 2H, J = 8.0 Hz), 4.24 (d, 1H, J

=

16.0 Hz), 4.17 (q, 2H, J = 16.0 Hz), 2.44 (s, 3H), 2.06 (m, 1H), 0.74 (d, 3H, J = 16.0 Hz), 0.73 (d, 3H, J =

13

1

6.0 Hz). C-NMR (100 MHz CD OD): δ (ppm) 174.15, 171.31, 162.55, 158.39, 154.47, 140.11, 137.09,

3

1

33.22, 131.31, 129.74, 129.31, 128.80, 128.15, 116.41, 116.21, 102.78, 31.10, 30.40, 23.40, 21.30, 18.99.

+

-1

LRMS (ESI) m/z = 459.8 [M+H] ; 481.8 [M+Na] . IR (KBr) cm : 3450-3100 (OH), 3362 (NHCO), 2221

(

CN), 1690 (CO acid), 1646 (CO amide). Anal. calcd. for C H N 0 S: C 67.95, H 5.48, N 9.14, S 6.98;

26 25 3 3

found: C 68.02, H 5.33, N 8.96, S 6.79.

2

1

.3. Synthetic procedure for diamino acid adducts (9 and 10)

,4 Dimethyl(2S) 2 [(2S) 2 ({[(9H fluoren 9 yl)methoxy]carbonyl}amino) 3

methylbutanamido] butanedioate (Fmoc-Val-Asp-OMe(OMe), 16)

Same amidation protocol used for 7a-h: To a stirring solution of Fmoc-Valine (500 mg, 1.47 mmol) in dry

DMF (5 mL) a solution of L-aspartate dimethyl ester (hydrochloride salt; 291 mg, 1.47 mmol) in dry DMF

(

53 mL) was added dropwise at 0 °C under N atmosphere. Then EDC (282 mg, 1.47 mmol), HOBt (199 mg,

2

1

.47 mmol) and dry DIPEA (0.31 mL, 1.77 mmol) were added. The mixture was stirred 30 min at 0 °C and

then 4 h at room temperature. The reaction was quenched with water and then it was extracted with AcOEt.

The organic phase was washed with water, dried over Na SO , filtered and concentrated under reduced

2

4

pressure. The crude residue was purified through flash chromatography on silica gel. Eluted with

1

Hexane/AcOEt 1/1. TLC R (Hexane/AcOEt 1/1) 0.55. Yield: 79%. H-NMR (400 MHz CDCl ): δ (ppm)

f

3

7

1

3

0

1

3

.74 (d, 2H, J = 8.0 Hz), 7.58 (d, 2H, J = 6.4 Hz), 7.38 (t, 2H, J = 7.2 Hz), 7.29 (t, 2H, J = 7.2 Hz), 6.92 (d,

H, J = 7.6 Hz), 5.56 (d, 1H, J = 8.4), 4.89 (m, 1H), 4.37 (m, 2H), 4.21 (t, 1H, J = 6.8 Hz), 4.10 (m, 1H),

.73 (s, 3H), 3.66 (s, 3H), 3.04 (dd, 1H, J = 13.6 Hz), 2.83 (dd, 1H, J = 4.4 Hz, J = 17.2 Hz), 2.13 (m, 1H),

1

3

.99 (d, 3H, J = 6.0 Hz), 0.96 (d, 3H, J = 6.0 Hz). C-NMR (100 MHz CDCl ) δ (ppm) 171.08, 170.70,

3

56.23, 143.71, 141.21, 127.62, 126.99, 125.08, 119.79, 67.10, 60.06, 52.68, 52.04, 48.32, 47.06, 35.80,

+

-1

1.44, 18.88, 17.62 LRMS (ESI) m/z = 483.0 [M+H] ; 505.2 [M+Na] . IR (KBr) cm : 3362 (NHCO), 1745

(

CO ester), 1649 (CO amide). Anal. calcd. for C H N 0 : C 64.72, H 6.27, N 5.81; found: C 64.51, H 6.25,

2

6

30

2 7

N 6.09.

ACCEPTED MANUSCRIPT

1

,4 Dimethyl

(2S) 2 [(2S) 2 amino 3 methylbutanamido]butanedioate

(NH -Val-Asp-

2

OMe(OMe), 17)

To a stirred solution of 16 (271 mg, 0.56 mmol) in DMF (7 mL), NaN (73 mg, 1.12 mmol) was added. The

3

mixture was stirred at 50 °C for 12 h. The reaction was quenched with water and extracted with AcOEt. The

organic phase was washed with water, dried over Na SO , filtered and concentrated under reduced pressure.

2

4

The crude residue was used in the next step without further purification. TLC R (Hexane/AcOEt 3/1) 0.57.

f

+

LRMS (ESI) m/z = 261.8 [M+H] .

1

,4 Dimethyl (2S) 2 [(2S) 2 (2 {[3 cyano 4 (4 methylphenyl) 6 phenylpyridin 2

yl]sulfanyl} acetamido) 3 methylbutanamido]butanedioate (Diamino acid methyl diester adduct, 9)

Same amidation protocol used for 7a-h: To the crude of 17a, dry DMF (3 mL) was added under N₂

atmosphere. Then a solution of 15 (112 mg, 0.31 mmol) in dry DMF (3 mL) was added dropwise at 0 °C.

EDC (42 mg, 0.31 mmol), HOBt (59 mg, 0.31 mmol) and dry DIPEA (0.06 mL, 0.37 mmol) were added.

The mixture was stirred 30 min at 0 °C and then 12 h at room temperature. The reaction was quenched with

water and then it was extracted with AcOEt. The organic phase was washed with water, dried over Na SO ,

2

4

filtered and concentrated under reduced pressure. The crude residue was purified through flash

1

chromatography on silica gel. Eluted with AcOEt. TLC R (AcOEt) 0.56. Yield: (over two steps): 21%. H

f

NMR (400 MHz CDCl ): δ (ppm) 8.04 (d, 2H, J = 6.0 Hz), 7.58 (s, 1H), 7.53 (m, 2H), 7.48 (m, 3H), 7.30

3

(

m, 2H), 6.79 (d, 1H, J = 8.0 Hz), 4.73 (m, 1H), 4.25 (t, 1H, J = 7.2 Hz), 4.16 (d, 1H, J = 15.6 Hz), 4.00 (d,

H, J = 15.6 Hz), 3.69 (s, 3H), 3.64 (s, 3H), 2.91 (dd, 1H, J = 17.2 Hz), 2.62 (dd, 1H, J = 4.4 Hz, J = 17.2

2

1

3

Hz), 2.43 (s, 3H), 1.93 (m, 1H), 0.75 (d, 3H, J = 6.8 Hz), 0.67 (d, 3H, J = 6.8 Hz). C-NMR (100 MHz

CDCl ): δ (ppm) 171.08, 170.70, 156.23, 143.85, 130.73, 129.97, 129.76, 129.04, 128.23, 127.40, 116.58,

3

1

15.37, 79.41, 58.60, 52.67, 52.02, 48.15, 35.65, 31.82, 29.59, 29.24, 26.30, 22.58. 21.28, 18.66, 17.54,

+

-1

1

4.01. LRMS (ESI) m/z = 602.8 [M+H] ; 624.8 [M+Na] . IR (KBr) cm : 3361 (NHCO), 2225 (CN), 1742

(

CO ester), 1651 (CO amide). Anal. calcd. for C H N 0 S: C 63.77, H 5.69, N 9.30, S 5.32; found: C

32 34 4 6

6

3.81, H 5.75, N 9.09, S 5.64

(

2S) 2 [(2S) 2 (2 {[3 Cyano 4 (4 methylphenyl) 6 phenylpyridin 2

yl]sulfanyl}acetamido) 3 methylbutanamido]butanedioic acid (Diamino acid acid free adduct, 10)

To a stirred solution of 9 (34 mg, 0.05 mmol) in a 3/1/0.5 mixture of THF/EtOH/H O (2 mL), NaOH 1 N

2

(

0.22 mL, 0.22 mmol) was added. The mixture was stirred at room temperature for 15 min until the solution

becomes yellow. The reaction was quenched with HCl 2 N and extracted with AcOEt. The organic phase

was washed with water, dried over Na SO , filtered and concentrated under reduced pressure. The crude

2

4

residue was purified through flash chromatography on silica gel, eluting with AcOEt/MeOH/AcOH 9/1/0.5

1

to give pure 10. TLC R (AcOEt) 0.41. Yield: 61%. H-NMR (400 MHz CD OD): δ (ppm) 8.04 (d, 2H, J =

f

3

6

.8Hz), 7.56 (s, 1H), 7.54 (m, 3H), 7.40 (m, 4H), 4.65 (t, 1H, J = 5.6 Hz), 4.41 (m, 1H), 3.83 (m, 1H), 3.16

1

3

(

m, 1H), 2.20 (m, 2H), 1.02 (d, 3H, J = 7.2 Hz), 1.01 (d, 3H, J = 7.2 Hz). C-NMR (100 MHz CD OD): δ

3

ppm) 173.08, 171.90, 165.58; 160.06, 156.68, 148.15, 145.94, 139.17, 137.92; 129.29; 128.42; 128.23;

1

27.72; 126.88; 121.38; 118.27, 98.30, 63.48, 58.54, 49.71, 36.71, 30.82, 29.25, 19.87, 18.39, 17.39. LRMS

ACCEPTED MANUSCRIPT

+

-1

(

ESI) m/z = 574.9 [M+H] ; 573.0 [M-H]. IR (KBr) cm : 3420-3090 (OH), 3361 (NHCO), 2225 (CN), 1688

CO acid), 1640 (CO amide). Anal. calcd. for C H N 0 S: C 62.70, H 5.26, N 9.75, S 5.58; found: C

(

3

0

30

4 6

6

2.80, H 5.35, N 9.89, S 5.62.

2

.4. Synthetic procedure for triamino acid adduct (11)

(

2S) 2 [(2S) 3 Carboxy 2 [(2S) 2 ({[(9H fluoren 9 yl)methoxy]carbonyl}amino) 3

methylbutanamido] propanamido] 4 (methylsulfanyl)butanoic acid (Resin-free Fmoc-tripeptide, 19)

Resin-bounded compound 18 was charged in a gooch filter and treated with a 88/2/5/5 cleavage solution of

TFA/TIPS/H O/DTT (20 mL) under an upward flux of N . After 1 h the resin was filtered and the filtrate

2

was recovered and evaporated under reduced pressure. The white solid obtained was washed with hexane

and toluene and used directly in the next step.

Resin-bounded tripeptide (18)

Compound 18 has been obtained by MW-SPPS (microwave solid phase peptide synthesis) using an MW

automated CEM Peptide Synthesizer. The instrument has been charged with Fmoc-methionine on Wang

resin (167 mg) previously swallowed in DMF through a fritted buchner funnel under N flux for 2 h. The

2

instrument was also charged with: Fmoc-Val-OH (2N, 408 mg in 6 mL of DMF); Fmoc- Asp(OtBu)-OH

(

2N, 492 mg in 6 mL of DMF); a HOBt and HBTU (270 mg and 758 mg respectively in 4 mL of DMF);

DIPEA (2 mL of 34.8 % v/v DMF); piperidine (100 mL of 20% v/v DMF); DCM (2 L); DMF (2 L). Then

the instrument was set and the synthetic procedure lasted for 3 h and 37 min. The obtained resin was

removed from the instrument and washed several times with DMF and DCM.

Methyl (2S) 2 [(2S) 2 [(2S) 2 ({[(9H fluoren 9 yl)methoxy]carbonyl}amino) 3

methylbutanamido] 4 methoxy 4 oxobutanamido] 4 (methylsulfanyl)butanoate (methyl diester

Fmoc-tripeptide, 20)

To a stirred solution of 19 (170 mg, 0.29 mmol) in dry MeOH (10 mL), freshly distilled SOCl (0.22 mL,

2

.90 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 30 min and then at room temperature for

6

h. Solvent was removed under reduced pressure. The crude residue was purified through flash

1

chromatography on silica gel, eluting with AcOEt to give 20. TLC R (AcOEt) 0.69. Yield: 28%. H-NMR

f

(

400 MHz CDCl ): δ (ppm) 7.75 (d, 2H, J = 7.6 Hz), 7.58 (d, 2H, J = 7.2 Hz), 7.39 (t, 2H, J = 7.6 Hz), 7.31

3

t, 2H, J = 7.6 Hz), 5.40 (m, 1H), 4.82 (m, 1H), 4.62 (m, 1H), 4.44 (m, 2H), 4.23 (t, 1H, J = 6.8 Hz), 4.00 (m,

1

3

1

5

6

H), 3.72 (s, 3H), 3.68 (s, 3H), 3.04 (m, 1H), 2.99 (m, 1H), 2.67 (t, 2H, J = 6.4 Hz), 2.53 (m, 2H), 2.16 (s,

1

3

H), 1.99 (m, 1H), 0.98 (d, 3H), 0.95 (d, 3H). C-NMR (100 MHz DMSO): δ (ppm) 172.91, 170.70,

70.31, 156.23, 143.71, 141.21, 127.62, 126.99, 125.08, 119.98, 119.79, 67.10, 65.69, 60.06, 55.11, 52.04,

+

1.89, 48.32, 47.06, 35.80, 31.44, 31.00, 27.91, 18.88, 17.62, 15.47. LRMS (ESI) m/z = 613.7 [M+H] ;

+

-1

36.7 [M+ Na] ; 652.0 [M+ K] . IR (KBr) cm : 3361 (NHCO), 1742 (CO ester), 1651 (CO amide). Anal.

calcd. for C H N 0 S: C 60.67, H 6.41, N 6.85, S 5.22; found: C 60.42, H 6.35, N 7.09, S 5.44.

3

1

39

3 8

ACCEPTED MANUSCRIPT

Methyl (2S) 2 [(2S) 2 [(2S) 2 amino 3 methylbutanamido] 4 methoxy 4

oxobutanamido] 4 (methylsulfanyl) butanoate (Free amine tripeptide, 21)

Same deprotection protocol used for 17. To a stirred solution of 20 (48 mg, 0.79 mmol) in DMF (a mL),

NaN (13 mg, 0.20 mmol) was added. The mixture was stirred at 50 °C for 4 h. The reaction was quenched

3

with water and extracted with AcOEt. The organic phase was washed with water, dried over Na SO , filtered

2

4

and concentrated under reduced pressure. The crude residue was purified through flash chromatography on

silica gel, eluting with AcOEt/MeOH 9/1 to give 21. TLC R (AcOEt) 0.47. Yield: 62%. LRMS (ESI) m/z

f

+

-1

=

391.8 [M+H] ; 414.0 [M+ Na] ; 430.1 [M+ K] . IR (KBr) cm : 3401 (NH), 3361 (NHCO), 1742 (CO

ester), 1651 (CO amide). Anal. calcd. for C H N 0 S: C 49.09, H 7.47, N 10.73, S 8.19; found: C 49.24, H

1

6

29

3 6

7

.38, N 10.89, S 8.44.

Methyl (2S) 2 [(2S) 2 [(2S) 2 (2 {[3 cyano 4 (4 methylphenyl) 6 phenylpyridin 2

yl]sulfanyl} acetamido) 3 methylbutanamido] 4 methoxy 4 oxobutanamido] 4

(

methylsulfanyl)butanoate (Tripeptide methyl diester adduct, 11)

Same amidation protocol used for 7a-h. To a stirring solution of 21 (7 mg, 0.02 mmol) in dry DMF (1 mL) a

solution of 15 (6 mg, 0.02 mmol) in dry DMF (0.5 mL) was added dropwise at 0 °C under N atmosphere.

2

Then EDC (3 mg, 0.02 mmol), HOBt (2 mg, 0.02 mmol) and dry DIPEA (0.01 mL, 0.02 mmol) were added.

The mixture was stirred 30 min at 0 °C and then 12 h at room temperature. The reaction was quenched with

water and then extracted with AcOEt. The organic phase was washed with water, dried over Na SO , filtered

2

4

and concentrated under reduced pressure. The crude residue was purified through flash chromatography on

1

silica gel, eluting with AcOEt to give 11. Yield: 41%. H-NMR (400 MHz CD OD): δ (ppm) 8.19 (d, 2H, J

3

=

2.4 Hz), 7.77 (s, 1H), 7.60 (d, 2H, J = 8.0 Hz), 7.51 (m, 3H), 7.38 (d, 2H, J = 8.0 Hz), 4.94 (m, 1H), 4.52

m, 1H), 4.43 (m, 1H), 3.70 (m, 2H), 3.30 (s, 6H), 2.94 (d, 1H, J = 8.0 Hz), 2.90 (d, 1H, J = 8.0 Hz), 2.54 (m,

H), 2.53 (m, 1H), 2.44 (s, 3H), 2.29 (m, 2H), 2.00 (s, 3H), 0.81 (d, 3H, J = 6.8 Hz), 0.76 (d, 3H, J = 76.8

(

2

13

Hz). C-NMR (100 MHz DMSO): δ (ppm) 172.09, 171.65,156.25, 143.70, 130.69, 129.97, 129.76,129.04,

1

28.25, 127.50, 116.45, 115.37, 79.41, 58.60, 55.10, 52.00, 48.15, 35.65, 31.82, 31.09, 29.59,29.31, 29.24,

-1

2

6.30, 23.33, 22.49. 21.28, 18.66, 17.54, 15.13, 14.01. IR (KBr) cm : 3369 (NHCO), 2218 (CN), 1749 (CO

ester), 1647 (CO amide). Anal. calcd. for C H N 0 S : C 60.55, H 5.91, N 9.54, S 8.74; found: C 60.41, H

3

7

43

5 7 2

6

.15, N 9.59, S 8.84.

Tert-butyl (hydroxymethyl)carbamate (22)

t-butoxy carbamide (300 mg, 2.56 mmol), water (2 mL), formaldehyde (37% in H O, 0.52 mL, 6.41 mmol)

2

and K CO (7 mg, 0.05 mmol) were charged into a round bottomed flask with a magnetic stirrer. The

2

3

mixture was heated to 65 °C and stirred for 1 h. After cooling the mixture was extracted with AcOEt. The

organic phase was washed with water, dried over Na SO , filtered and concentrated under reduced pressure.

2

4

The crude residue was purified through flash chromatography on silica gel, eluting with Hexane/AcOEt 3/2

1

to give compound 23b as white solid. Yield: 42%. H-NMR (400 MHz CDCl ): δ (ppm) 5.93 (s, 1H), 4.59

3

1

3

(

s, 2H), 4.39 (s, 1H), 1.39 (s, 9H). C-NMR (100 MHz CDCl ): δ (ppm) 156.07, 80.02, 65.41, 28.19.

3

ACCEPTED MANUSCRIPT

+

-1

LRMS (ESI) m/z = 169.9 [M+Na] . IR (KBr) cm : 3402-3195 (OH), 3360 (NHCO), 1755 (CO ester), 1640

(

CO amide). Anal. calcd. for C H NO : C 48.97, H 8.90, N 9.52; found: C 48.84, H 8.85, N 9.44.

6 13 3

Tert-butyl (((3-cyano-6-phenyl-4-(p-tolyl)pyridin-2-yl)thio)methyl)carbamate (23)

To a stirred solution of compound 23 (80 mg, 0.54 mmol) in DCM (1 mL), triphenylphosphine (PPh ) (214

3

mg, 0.81 mmol) was added. The mixture was cooled in an ice-bath and CBr (334 mg, 1.09 mmol) was

4

added. The mixture was irradiated with MW for 15 min at 40 °C. The solution obtained was added dropwise

to 14 (82 mg, 0.27 mmol) and trimethylamine (0.07 mL, 0.54 mmol) in DCM (2 mL). The mixture was

stirred at room temperature for 12 h. The reaction was quenched with water and extracted with DCM. The

organic phase was washed with water, dried over Na SO , filtered and concentrated under reduced pressure.

2

4

The crude residue was purified through flash chromatography on silica gel, eluting with Hexane/AcOEt 9/1

1

give compound 23. Yield: 49%. H-NMR (400 MHz CDCl ): δ (ppm) 8.03 (m, 2H), 7.55 (s, 1H), 7.52 (m,

3

5

H), 7.39 (d, 2H, J = 8.0 Hz), 5.77 (s, 1H), 5.11 (d, 2H, J = 4.0 Hz), 2.43 (s, 3H), 1.49 (s, 9H). LRMS (ESI)

+

-1

m/z = 432.8 [M+H] . IR (KBr) cm : 3360 (NHCO), 2218 (CN), 1755 (CO ester), 1640 (CO amide). Anal.

calcd. for C H N 0 S: C 69.58, H 5.84, N 9.74, S 7.43; found: C 69.60, H 5.95, N 9.81, S 7.74.

2

5

25

3 2

2

-((3-cyano-6-phenyl-4-(p-tolyl)pyridin-2-yl)thio)acetamide (24)

To a stirred solution of 2-mercapto-pyridine 14 (120 mg, 0.40 mmol) in DMF (2 mL) a 10% aqueous

solution of KOH (0.15 mL, 0.27 mmol) was added and the mixture was stirred at room temperature for 30

min. The mixture becomes red and then a solution of 2-Bromoacetamide (60 mg, 0.40 mmol) in DMF (0.5

mL) was added dropwise. The mixture was stirred for further 3h at room temperature. The reaction was

treated with HCl 1 N and extracted with AcOEt. The organic phase was washed with water, dried over

Na SO , filtered and concentrated under reduced pressure. The crude residue was purified through flash

2

4

chromatography on silica gel, eluting with Hexane/AcOEt 1/1 to give compound 24 as pale yellow solid.

1

Yield: 50%. H-NMR (400 MHz CDCl ): δ (ppm) 8.08 (d, 2H, J = 8.0 Hz), 7.50 (s, 1H), 7.48-7.44 (m, 3H),

3

1

3

7

1

2

6

.38-7.31 (m, 4H), 5.96 (bs, 2H), 5.70 (s, 2H), 2.45 (s, 3H). C-NMR (100 MHz CDCl ): δ (ppm) 170.54,

3

62.50, 158.40, 154.56, 140.47, 137.01, 133.11, 131.16, 129.66, 129.25, 128.97, 128.05, 116.44, 116.12,

+

-1

02.42, 33.37, 21.27. LRMS (ESI) m/z = 359.8 [M+H] ; 381.8 [M+Na] . IR (KBr) cm : 3300 (NHCO),

218 (CN) 1644 (CO amide). Anal. calcd. for C H N 0S: C 70.17, H 4.77, N 11.69, S 8.92; found: C

2

1

17

3

9.98, H 4.95, N 11.77, S 8.74.

3

. BIOLOGY SECTION

Cells and Virus. Human embryonic kidney 293T (HEK 293T) and Madin−Darby canine kidney (MDCK)

cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented

with 10% (v/v) fetal bovine serum (FBS, Life Technologies) and antibiotics (100 U/mL penicillin and 100

µg/mL streptomycin, Life Technologies). The cells were maintained at 37 °C in a 5% CO humidified

2

atmosphere. Influenza A/PR/8/34 virus (PR8 strain, H1N1, Cambridge lineage) was obtained from P. Digard

(

Roslin Institute, University of Edinburgh, United Kingdom).

ACCEPTED MANUSCRIPT

Compounds and Peptide. Ribavirin (RBV; 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and

oseltamivir carboxylic acid (OSV), the active form of oseltamivir [(3R,4R,5S)-4-acetamido-5-amino-3-(1-

ethylpropoxy)-1-cyclohexene-1-carboxylic acid] were obtained from Roche. The PB1₁_–₁₅–Tat peptide was

synthesized and purified by the Peptide Facility of CRIBI Biotechnology Center (University of Padua,

Padua, Italy). The PB1₁_–₁₅–Tat peptide possesses a C-terminal sequence from the HIV Tat protein (amino

acids 47–59) to mediate cell entry.

PA−PB1 Interaction Enzyme-Linked Immunosorbent Assay (ELISA). The PA−PB1 interaction was

detected as described by Trist et al.[71] Briefly, microtiter plates (Nuova Aptca) were incubated with 400 ng

of 6His-PA₍₂₃₉₋₇₁₆₎for 3 h at 37 °C and then blocked with 2% BSA (Sigma) in PBS for 1 h at 37 °C. After

washing, 200 ng of GST-PB1₍₁₋₂₅₎or GST as a control, dissolved in serum-free DMEM, in the presence of

test compounds at various concentrations or DMSO, were added and incubated overnight at room

temperature. E. coli-expressed, purified 6His-PA₍₂₃₉₋₇₁₆₎, GST, and GST-PB1₍₁₋₂₅₎proteins were obtained as

previously described.[50,88] After washing, the interaction between 6His−PA₍₂₃₉₋₇₁₆₎and GST−PB1₍₁₋₂₅₎was

detected with an anti-GST monoclonal antibody conjugated to horseradish peroxidase (HRP; GenScript).

After the final wash step, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, KPL) was added, and the

optical density was determined at 450 nm by an ELISA plate reader (Tecan Sunrise). Values obtained from

the samples treated with only DMSO were used to set as 100% of PA−PB1 interaction. The PB1₍₁₋₁₅₎_-Tat

peptide was included in all experiments as positive control for inhibition.

Plaque Reduction Assay (PRA). Antiviral activity of compounds was investigated using PRAs, as

described elsewhere.[89] Briefly, a confluent monolayer of MDCK cells was seeded in 12-well plates. Cells

were infected with FluA virus (PR8 strain) at 40 PFU/well in DMEM supplemented with 1 µg/mL of TPCK-

treated trypsin (Worthington Biochemical Corporation) and 0.14% BSA in the presence of various

concentrations of test compounds for 1 h at 37 °C. Medium containing 1 µg/mL of TPCK-treated trypsin,

0

.14% BSA, 1.2% Avicel, and test compounds at the same concentrations was then added. After 2 days of

incubation, cell monolayers were fixed with 4% formaldehyde and stained with 0.1% toluidine blue, and

viral plaques were counted. Ribavirin and oseltamivir carboxylic acid were included in all experiments as

reference compounds. The mean plaque number obtained from the samples treated with DMSO was set at

1

00%.

Cytotoxicity Assay. Cytotoxicity of test compounds was assessed in MDCK and HEK 293T cells by the 3-

4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, as previously reported.[90,91]

(

3

4

Briefly, MDCK cells (5 × 10 cells/well on 96-well plates) and HEK 293T (2 × 10 cells/well on 96-well

plates) cells were incubated with test compounds at 2-fold serial dilutions from 250 µM or DMSO. After 24

or 48 h for HEK 293T and MDCK cells, respectively, MTT solution (5 mg/mL in PBS) was added to each

well, and plates were incubated for 4 h at 37 °C. Then, a solubilization solution was added to lyse cells. After

3

h of further incubation at 37 °C, absorbance was read at 620 nm using an ELISA plate reader (Tecan

Sunrise). Values obtained from the wells treated with only DMSO were used to set as 100% of viable cells.

ACCEPTED MANUSCRIPT

5

Minireplicon Assay. HEK 293T cells were seeded at a density of 2 × 10 per well into 24-well plates. After

4 h, cells were transfected with pcDNA-PB1, pcDNA-PB2, pcDNA-PA, and pcDNA-NP plasmids (100

2

ng/well of each), expressing the Flu A/PR/8/34 virus PB1, PB2, PA, and NP proteins, respectively, along

with pPolI-Flu-ffLuc plasmid (50 ng/well), containing an influenza virus-based luciferase minireplicon

vRNA under the control of the human RNA polymerase I promoter. Plasmids pcDNA-PB1, pcDNA-PB2,

pcDNA-PA, and pcDNA-NP were generated as described elsewhere[92] and were kindly provided by P.

Digard (Roslin Institute, University of Edinburgh, United Kingdom), whereas the plasmid pPolIFlu-ffLuc

was provided by L. Tiley (University of Cambridge, United Kingdom). The transfection mixture also

contained pRL-SV40 plasmid (50 ng/well. Promega), which constitutively expresses the Renilla luciferase,

to normalize variations in transfection efficiency. Transfections were performed using calcium phosphate

protocol in the presence of the test compounds or DMSO. Cell medium was replaced 4 h post-transfection

with DMEM containing compounds or DMSO. At 24 h post-transfection, cells were harvested and both

firefly luciferase and Renilla luciferase expression were determined using the Dual Luciferase Assay Kit

from Promega. Ribavirin was included in all experiments as the reference compound.

Interference compound assessment. The behaviour of all the tested compounds (7-11, 15, 23 and 24) as

PAINS (Pan-Assay Interference Compounds) [93] was examined through prediction by the online

FAFDrugs4[94–96] (Free ADME-Tox Filtering Tool) program. Through its tool Bank-Formatter the

compound library was prepared and then screened with the three different available filters A, B and C. All

the analysed compounds resulted “accepted” by the software.[97]

ACKNOWLEDGMENTS

We thank P. Digard for providing plasmids and the Flu A A/PR/8/34 strain and L. Tiley for providing the

plasmid pPolIFlu-ffLuc. This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC,

grant n. IG18855) and by the University of Padua (Progetto di Ricerca di Ateneo 2014, grant no.

PDA141311) to A. Loregian.

Notes. The authors declare no competing financial interest.

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Article Doi

Synthesis and biological evaluation of a library of hybrid derivatives as inhibitors of influenza virus PA-PB1 interaction

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