Referentes: Experiencias exitosas en la gestión comunitaria a través de la

10.2.1 Rationale

The crystal structure of ATAD2 in complex with fragment 158 revealed that the amide group mimics the acetyllysine binding mode (Figure 10.2). The oxygen atom of the amide group forms H-bond with Asn1064 and a water mediated H-bond with Tyr1021.

Figure 10.2: Crystal structure of fragment 158 (carbons: grey) bound to ATAD2. The binding surface is coloured based on the charge, positive: blue and negative: red. The ZA-shelf is shown by dark pink coloured circle. The pink arrow denotes the area of potential SAR investigation.

A shelf around the ZA loop (hereafter referred to as ZA-shelf) was identified from the crystal structure (Figure 10.2). Small alkyl substituents at the 3-position of fragment 158 were selected to exploit the ZA-shelf (Figure 10.3).

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Similarly, the crystal structure of fragment 160 bound to ATAD2 showed that the oxygen of amide forms H-bond with Asn1064, and a water mediated H-bond with Tyr1021 (Figure 10.4).

Figure 10.4: Crystal structure of fragment 160 (carbons: grey) bound to ATAD2. The binding surface is coloured based on the charge, positive: blue and negative: red. The ZA-shelf is shown by pink coloured circle. The pink arrow denotes the area of potential SAR investigation.

It was postulated that substitution at the 2-position of the phenyl ring could access the ZA-shelf. Alkyl and alkoxy substituents were selected for preliminary investigation (Figure 10.5).

Figure 10.5: 2-Substituted phenylacetamides to be synthesised.

Crystal structure of triazole fragment 157 bound to ATAD2 showed that the compound adopted a different binding mode compared to other fragments (Figure 10.6). The H-bond with Asn1064 and water mediated H-bond with Tyr1021 was formed with two different nitrogen atoms in the

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triazole ring, whereas, the two H-bonds were formed with one oxygen atom in fragments 158 and 160.

Figure 10.6: Crystal structure of fragment 157 (carbons: grey) bound to ATAD2. The binding surface is coloured based on the charge, positive: blue and negative: red. The ZA-shelf is shown by pink coloured circle. The pink arrow denotes the area of potential SAR investigation.

Interestingly, fragment 157 has a bromine atom on the pyridyl ring that could be utilised as a vector to exploit the ZA-shelf. Therefore, small alkyl substituents were selected for initial investigation (Figure 10.7).

Figure 10.7: 5-substituted triazolopyridines to be synthesised.

10.2.2 Synthesis

Synthesis of compound 172 began with formylation of the commercially available phenol 167 using magnesium chloride and paraformaldehyde (Scheme 10.1).178 _{Subsequent Pinnick}

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oxidation of the aldehyde 168 gave carboxylic acid 169, which was bis-methylated to obtain compound 170. Hydrolysis of ester 170 followed by amide coupling with methylamine provided the target 172 in good yield.

Scheme 10.1: Reagents and conditions: (a) paraformaldehyde, NEt3, MgCl2, MeCN, reflux, 3

h, 93%; (b) NaClO2, sulfamic acid, H2O, MeCN, r.t., 8 h, 88%; (c) MeI, Cs2CO3, DMF, r.t., 18

h, 58%; (d) NaOH, H2O, MeOH, 60 ˚C, 18 h, 95%; (e) MeNH2, DIC, HATU, DMAP, THF,

r.t., 18 h, 71%.

Compounds 175 and 178 were prepared following a two-step reaction sequence (Scheme 10.2). Reduction of commercially available nitrophenols 173 and 176 provided the corresponding amines, subsequent acetylation afforded targets 175 and 178.

Scheme 10.2: Reagents and conditions: (a) H2, 10% Pd/C, MeOH, r.t., 24 h; (b) Ac2O, AcOH,

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To access triazolo[4,3-a]pyridine analogues, two different routes were employed. The first route involved SNAr reaction on commercially available compound 179. 2-Fluoro-5-

methylpyridine 179 was resistant to SNAr reaction with acethydrazide 180 at various conditions

(Table 10.1).

Table 10.1: Reagents and conditions used in step a

Solvent Base Temp ˚C

Time

h Result

a

Ethanol None 100 3 No conversion

Ethanol None 100 under µW 3 No conversion

Ethanol NEt3 100 18 No conversion

Ethanol DIPEA 100 18 No conversion

a _{Observations from TLC and LCMS analyses}

Failure of the SNAr reaction might be due to the decrease in nucleophlicity of hydrazine in

acethydrazide 180, caused by the electron withdrawing acetyl group. Therefore, the SNAr

reaction was performed on commercially available compound 179 with unsubstituted hydrazine to get compound 183 (Scheme 10.3). Subsequent ring closure was performed in the presence of acetic acid and acetic anhydride, and gave target 182 in poor isolated yield.

Scheme 10.3: Reagents and conditions: (a) hydrazine, EtOH, reflux, 72 h, 43%; (b) Ac2O,

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The second route to access triazolo[4,3-a]pyridine analogues involved Suzuki-Miyaura coupling of compound 157 with alkyl boronic acids. Unfortunately, the coupling reaction with ethyl boronic acid was not successful. Further cross-coupling attempts were therefore performed with vinylboronic acid pinacol ester, followed by reduction (Scheme 10.4). However, the intermediate was found to be unstable with a noticeable colour change from beige to dark brown upon standing. A plausible explanation for the observed degradation is that the ring is very electron rich, therefore, susceptible to oxidation in presence of air. Literature searches of compound 186 and 187 were undertaken using Reaxys® and SciFinder® but with no success as the compounds had not been reported yet. Degradation of compounds with the triazolo[4,3-a]pyridine scaffold was also observed by our bioscientist colleague Dr Mathew Martin whilst performing the HTRF assay. Therefore, the series was not explored further due to the observed degradation of the intermediates 184 and 185.

Scheme 10.4: Reagents and conditions: (a) vinylboronic acid pinacol ester, or isopropenylboronic acid pinacol ester, NaOH, N,N-dicyclohexylmethylamine,

Pd(dppf)Cl2.DCM, THF, 95 ˚C, 2.5 h, (184, 51%; 185, 62%, over 2 steps); (b) H2, 10% Pd/C,

MeOH.

10.2.3 Biological Evaluation

A homogenous time resolved FRET assay (HTRF) was used to measure the ATAD2 inhibitory activity. GST-tagged ATAD2 was mixed with biotinylated acetylated ligand (Figure 10.8). Streptavidin labelled phycobiliprotein pigment purified from red algae (SA-XL665),111 which binds to the biotinylated acetylated ligand, and a terbium-labelled anti-GST antibody that binds to GST-tagged ATAD2 was added to the mixture. The terbium label acts as a donor of FRET signal, and SA-XL665 acts as an acceptor of the FRET signal. Upon excitation, there is a

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transfer of FRET from donor to acceptor which can be measured. In the absence of an inhibitor, the donor (terbium) and the acceptor (SA-XL665) are in close proximity. The donor terbium absorbs energy and transfers it to SA-XL665. The transfer of energy is measured as a FRET signal. In the presence of an inhibitor, the donor and acceptor are separated which results in a decrease in the amount of FRET. The experimental details of the HTRF assay are discussed in 16.4.1.

Figure 10.8: Schematic representation of ATAD2-HTRF assay.

Compounds 172, 175, 178 and 182 did not show measurable inhibition by Homogenous Time Resolved FRET assay (HTRF) at 4 mM concentration. In addition, co-crystallisation experiments of ATAD2 with the compounds had failed. Meanwhile, Dr Duncan Miller had demonstrated that subtle modifications in fragment 5 improved ATAD2 inhibition and the compounds were successfully co-crystallised with ATAD2. Besides, fragment 5 was a novel scaffold which had not been reported to date for bromodomain inhibition. Therefore, most of the medicinal chemistry effort was focussed in SAR studies around fragment 5 which will be discussed in detail in following chapters.

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