Optimal linker length for small molecule PROTACs that selectively target p38α and p38β for degradation

1

Optimal linker length for small molecule PROTACs that selectively target p38α and

p38β for degradation

1

,†

1,†

Craig Donoghue , Monica Cubillos-Rojas , Nuria Gutierrez-Prat , Carolina Sanchez-

1

1,2

1,2,*

1,3,*

Zarzalejo , Xavier Verdaguer , Antoni Riera and Angel R. Nebreda

1

Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science

and Technology, Baldiri Reixac 10, 08028 Barcelona, Spain

2

Dept. Química Inorgànica i Orgànica, Universitat de Barcelona, Martí i Franquès 1, 08028

Barcelona, Spain.

3

ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain

†

These authors contributed equally

*

Correspondence: angel.nebreda@irbbarcelona.org, phone: +34-934031379,

antoni.riera@irbbarcelona.org, phone: +34-934037093

ABSTRACT

We report the design of hetero-bifunctional small molecules that selectively target p38α and

p38β for degradation. These proteolysis targeted chimeras (PROTACs) are based on an ATP

competitive inhibitor of p38α and p38β, which is linked to thalidomide analogues to recruit

the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a

copper catalyzed “click” reaction. We show that optimization of the linker length and

composition is crucial for the degradation-inducing activity of these PROTACs. We provide

evidence that these chemical compounds can induce degradation of p38α and p38β but no

other related kinases at nanomolar concentrations in several mammalian cell lines.

Accordingly, the PROTACs inhibit stress and cytokine-induced p38α signaling. Our

compounds contribute to understanding the development of PROTACs, and provide a useful

tool to investigate functions of the p38 MAPK pathway and its involvement in diseases.

KEYWORDS: Azide-alkyne click reaction, Cereblon, p38 MAPK, PROTAC linker

optimization, Protein degradation, Thalidomide derivative

2

1

. Introduction

The p38 mitogen-activated protein kinase (MAPK) family comprises four members, p38α,

p38β, p38γ and p38δ, with p38α being ubiquitously expressed and the most abundant family

member in almost all cell types. Many functions that are generally ascribed to p38 MAPKs

actually refer to p38α [1]. However, there is evidence that p38β may have redundant

functions with p38α, although it is usually expressed at lower levels [2]. In contrast, p38γ and

p38δ have more restricted expression patterns and perform different functions [1]. Extensive

evidence indicates that p38α is activated in response to stress and can regulate many cellular

processes by controlling the phosphorylation of a large variety of substrates [3].

Consequently, dysregulated p38α activity has been associated with several human pathologies

including inflammatory diseases and cancer. This has made p38α an attractive therapeutic

target and many small molecules of different structures have been developed by

pharmaceutical companies to inhibit the activity of this kinase [4]. It has also been reported

that p38α might regulate particular functions in a kinase-independent manner [5, 6],

supporting the need to develop novel therapeutic approaches.

The functions of p38α are highly dependent on both the cell type and the context. For

example, during tumorigenesis, p38α usually functions as a tumor suppressor in normal

epithelial cells, whereas in malignant cells p38α tends to support tumor development [7].

Considering that p38α can also play diverse roles in cells of the tumor microenvironment [8,

9

], it is clear that the contribution of p38α to tumor development is not straightforward.

Nevertheless, the available information suggests potential therapeutic opportunities in several

tumor types via intervention of the p38α pathway, especially when this is combined with

chemotherapeutic drugs [10]. However, currently available p38α inhibitors have not shown

the expected efficacy in clinical trials and there is a need to develop improved therapies that

target p38α signaling. A greater understanding of how p38α downregulation or inhibition

affects cellular functions would aid in therapeutically targeting this pathway.

Proteolysis targeting chimeras, referred to as PROTACs, are bifunctional compounds

that hijack ubiquitinating enzymes to induce the degradation of a target protein by the

ubiquitin proteasome pathway. Structurally, PROTACs consist of one end that binds to the

target protein covalently linked to an E3 ubiquitin ligase ligand. Several ligand-like small

molecules have been reported to engage E3 ligases and cause the poly-ubiquitination of target

proteins, including peptide recognition fragments and thalidomide analogues [11]. By

bringing the target protein and the E3 ligase in close proximity to each other, the target

3

protein can be poly-ubiquitinated and then degraded by the proteasome [12]. PROTAC-

induced protein degradation normally results in a superior level of selectivity, as

ubiquitination is dependent upon formation of a stable ternary complex and highly sensitive to

linker distance and geometries. Therefore, not all off-target interactions would result in

protein degradation [13]. To date, many targets have been reported using small-molecule,

polypeptide or hydrophobic tagging degraders to achieve promising pre-clinical results [14,

1

5].

Here, we present the design of chemical compounds that achieve the selective

degradation of p38α and p38β in mammalian cells at nanomolar concentrations. These

compounds will assist in uncovering further functions of the p38 MAPK pathway that are not

easily addressed by conventional inhibitors, and could aid in the development of improved

therapies.

2

. Results

2

.1. Generation of PROTACs that induce p38α degradation.

For the warhead, we selected the small molecular weight compound PH-797804, an ATP

competitor that potently inhibits p38α in various cellular models [10, 16-18]. PH-797804

possesses a methyl amide moiety amenable for linker attachment that resides in the solvent

exposed region, extending out from the ATP binding site when bound to p38α. Furthermore,

modifying this position was unlikely to interfere with the key interactions that determine the

inhibitor activity and selectivity [19] (Fig. 1a). We reasoned that this location would be best

for the attachment of the linker, directing the E3 ubiquitin ligase away from the p38α´s active

site and allowing heterodimerization of p38α and the E3 ligase. We used analogues of

thalidomide to recruit Cereblon (CRBN) E3 ligases and cause p38α ubiquitination [20].

Thalidomide analogues can be synthesized easily from commercially available starting

materials (Fig. 1b). A structurally unrelated PROTAC (SJFα) has been recently reported to

induce p38α degradation, featuring the c-Met kinase inhibitor foretinib as a warhead to bind

p38α, linked to a ligand to engage the von Hippel-Lindau E3 ligase [21]. We opted to

construct our PROTAC from basic and economic chemical starting materials and to utilize a

warhead that would selectively target p38α, in order to reduce the possibility of inducing the

degradation of other kinases [19].

4

Once the structural components at either side of the p38α PROTAC were decided, we

focused on the optimization of the linker length and composition, as well as the type of

attachment to the warhead and ligase ligand. An initial collection of compounds (NR-1a-c

and NR-2a-b) with varying linker lengths and attachment points was synthesized (Supporting

Information) and tested to monitor their ability to induce p38α degradation in mammalian

cells (Table 1). We chose the breast cancer cell lines BBL358 and T47D as p38α has been

shown to play a key role in breast tumors and we have previously reported the anti-

proliferative effect of p38α inhibition in breast cancer cells [10]. We found that compounds

with a linker length of 8 atomic units or less did not induce degradation of p38α in the cell

lines tested, presumably because the PROTACs were not long enough to allow sufficient

space for productive interaction between p38α and CRBN. However, PROTAC NR-1c with a

linker of 20 atoms efficiently induced p38α degradation in BBL358 cells and, to a lesser

extent, in T47D cells (Table 1 and Fig. S1a).

To further explore the influence of the linker length and composition on the

PROTAC’s activity, we developed a practical synthesis of PROTAC molecules aided by a

“click” reaction [22]. The PH-797804 derivatives containing a terminal alkyne 8a-c were

formed via peptide coupling of carboxylic acid intermediate 1 with corresponding alkyne

amines (Scheme 1). The thalidomide components containing an azide 9a-b and 15d were

formed from 4-fluorothalidomide (2) via S Ar followed by standard derivatization (Fig 2 and

N

Supplementary Information). Compound 12 was prepared from carboxylic acid 4. Finally, the

azide and alkyne components were “clicked” together via a copper catalyzed Huisgen

cycloaddition reaction to form 1,2,3-triazole containing PROTACs NR-3a-c and NR-4 (Fig

2

).

Our strategy introduced triazole and amide moieties that reduced linker flexibility and

also facilitated the synthesis of compounds with longer linkers. Another click platform has

been recently published for degradation of the bromodomain and extraterminal domain-4

(BRD4) [23]. Moreover, positive interaction of heteroatoms in the linker with amino acid

residues on the protein’s surfaces has been reported to enhance the protein-protein interaction

induced by PROTACs [24]. Therefore, we synthesized compounds NR-3-4 (Supporting

Information) and tested their ability to induce p38α degradation (Table 2). Of the NR-3

compounds with an amine linker at position 4’ of thalidomide, only NR-3c showed clear

activity in both BBL358 and T47D cells. It is worth noting that the connection to thalidomide

strongly influenced the PROTAC’s ability to induce p38α degradation, since PROTACs NR-

5

3

c and NR-4 featured the same linker with a different connection and showed different

activities (Table 2 and Supporting Information Fig. S1b). As expected, blocking the imide of

the piperidindione with an ortho-nitrobenzylgroup (NR-3d) strongly reduced the activity of

the compound.

To further test the influence of the attachment mode, we synthesized compounds NR-

5

a-b and NR-6a-b since it has been reported that the potency of PROTACs targeting BRD4

was improved by replacing the 4’ NH group of the thalidomide analogue with a 4’ methylene

group and removing one carbonyl from the indoline moiety [25, 26]. The synthesis of NR-5

and NR-6 started from thalidomide and deoxothalidomide derivatives 13a-d with an

aminoalkyl sidechain [26]. The azido group was incorporated as an amide with 4-

azidobutanoic acid (11). The click reaction of azides 14a-d with the acetylenic derivative 8a

afforded the desired PROTACs NR-5a-b and NR-6a-b (Fig 3) as expected. Thus, we

examined the effect of medium length methylene linkers on the ability of PROTACs to induce

p38α degradation with and without the pthalimide carbonyl. We found that NR-6a was very

potent at inducing p38α degradation in both BBL358 and T47D cells (Table 3 and Supporting

Information Fig. S1b).

We, then synthesized a small series of N-linked PROTACs NR-7a-h starting from

amines 3a-c which were prepared from 4-fluorothalidomide (2) by SNAr. The azido

functionality was introduced again by amide formation with 4-azidobutanoic acid (11). This

family of compounds was generated making minor adjustments to the length at either side of

the triazole and amide moieties using alkyl fragments of 2-4 carbons in length. These

PROTACs were then tested in cancer cell lines (Table 3). We found that a linker length of 15-

1

7 atoms was optimum for the degradation of p38α in both BBL358 cells and T47D cells,

whereas PROTACs bearing linkers shorter than 15 atoms poorly induced p38α degradation

(Table 3 and Supporting Information Fig. S1c).

Out of the 24 compounds that were synthesized following the design mentioned

above, we selected five compounds (NR-6a, NR-7f-i) that at 10 µM efficiently induced

p38α downregulation in both BBL358 and T47D cancer cells (Supporting Information Fig.

S1). To compare the efficacy of p38α downregulation induced by these five PROTACs, we

treated T47D cells with different concentrations of the compounds (Supporting Information

Fig. S2). Based on these results, we selected the PROTACs NR-6a and NR-7h (Fig. 4a) for

further characterization, as both of them efficiently induced p38α downregulation at

nanomolar concentrations. We calculated the concentrations of NR-6a and NR-7h required to

6

induce 50% of p38α degradation (DC ) in two different cancer cell lines, and obtained

5

0

values of 2.9 nM in T47D cells and 18.4 nM in MB-MDA-231 cells for NR-6a, and 24 nM

in T47D cells and 27.2 nM in MB-MDA-231 cells for NR-7h (Fig. 4b).

In summary, we synthesized and tested 24 compounds. We have not investigated other

p38α inhibitors as warheads, as PH-79780 gave superb potencies in the nanomolar range.

Neither have we tested other E3 ligases, e.g. VHL, which has been thoroughly investigated by

others [21]. Therefore, our efforts concentrated on linker composition and length. From our

small structure-activity study (SAR) we conclude that a minimum linker length of 15 atoms is

necessary to have good activity. The linker can be as long as 20 atoms but the optimum size is

1

6-17. The connection to the indoline moiety is a crucial aspect, with the alkyl and amino

alkyl substituent at the 4’-position giving the best activities. Compounds derived from

carboxylic acid 4 did not lead to degradation of p38α.

Next, we synthesized the compounds NR-7h* and NR-6a*, featuring an additional

methyl group to block the NH functionality of the thalidomide group (Supporting

Information Fig. S3), which has been shown to prevent binding to CRBN [25] and therefore

prevent ubiquitination of the target protein. These modifications impaired the ability of the

PROTACs to induce the degradation of p38α in two different cells lines (Fig. 5),

supporting the implication of CRBN-mediated ubiquitination.

To try to improve the aqueous solubility of NR-7h, compounds NR-7i and NR-7j

were synthesized, featuring an additional branched functionality on the linker, a tert-butyl

carbamate or a hydrochloride amine salt respectively (Fig. 6). Whereas the amine salt group

of NR-7j improved aqueous solubility, it did not induce degradation of p38α in cells,

potentially affecting the interaction between p38α and the E3 ligase or diminishing cell

permeability. However, NR-7i induced degradation of p38α to a similar level as the parental

NR-7h, which although did not improve aqueous solubility, demonstrated the potential for

this position to serve as a derivatization point to improve the pharmacokinetic properties or

for conjugation for directed delivery.

2

.2 PROTACs target p38α and p38β for degradation.

It has been reported that PH-797804 potently inhibits p38α but it can also inhibit p38β at

higher concentrations, whereas it has no inhibitory effect on other MAPKs, including the

structurally related p38γ and p38δ, as well as JNK and ERK family members [27].

Therefore, PROTACs based on PH-797804 might also be able to induce p38β degradation in

7

cells. We found that both compounds NR-7h and NR-6a induced the degradation of

p38β in several cancer cell lines (Fig. 6a). However, NR-7h and NR-6a treatment affected

neither the expression levels of p38γ and p38δ nor those of other related MAPK family

members such as JNK1/2 and ERK1/2, in any of the cell lines analyzed (Fig. 7a). We

estimated the DC values for p38β in two different cancer cell lines, and obtained values of

5

0

3

5.19 nM in T47D cells and 19.10 nM in MB-MDA-231 cells for NR-6a, and 48.47 nM in

T47D cells and 48.90 nM in MB-MDA-231 cells for NR-7h (Supporting Information Fig.

S4). Interestingly, we extended our observations to non-malignant cell lines, and confirmed

that NR-7h and NR-6a induced the degradation of p38α and p38β in retina epithelial cells,

cardiomyocytes and bone marrow derived macrophages (BMDMs) (Fig. 7b). These results

indicate that PROTACs NR-6a and NR-7h are able to target specifically p38α and p38β

but not other related MAPK family members in a variety of cell types.

Next, we confirmed that treatment of cells with NR-7h or NR-6a did not change the

p38α and p38β mRNA expression levels (Supporting Information Fig. S5a), suggesting that

the reduced expression levels of p38α and p38β induced by these compounds was due to

protein degradation. Furthermore, the ability of NR-7h and NR-6a to induce p38α and

p38β downregulation was blocked when cells were pre-treated with the proteasome

inhibitor bortezomib or with the NEDD8-Activating Enzyme (NAE) inhibitor, which

prevents activation of cullin-RING ubiquitin ligases like CRBN (Supporting Information

Fig. S5b). The data indicate that indeed these PROTACs target p38α and p38β proteins for

degradation by the proteasome pathway in a CRBN ubiquitin ligase dependent manner.

To address the specificity of the PROTACs, we performed a proteomic study.

MDA-MB-231 cells were subjected to a protocol of stable isotope labeling by amino acids

in cell culture (SILAC) until they reached 95% or higher labeling, and then were incubated

for 16 h with 1 µM of NR-7h or with DMSO. Whole protein lysates were prepared and

digested, and the peptides were analyzed by mass spectrometry (MS). We identified 17423

unique peptides that corresponded to 2812 protein groups, of which p38α was the most

downregulated protein in NR-7h-treated cells (Fig. 8).

2

.3 PROTACs impair p38α pathway activation in cells.

p38α is activated by a broad range of cellular stimuli including many stresses and pro-

inflammatory cytokines. We analyzed the ability of the PROTACs to inhibit the p38α

pathway activity in cells. Using MDA-MB-231 cancer cells, we detected a robust

8

upregulation of MK2 phosphorylation in response to ultraviolet light (UV), which was

strongly reduced upon incubation with PH-797804 or the PROTACs (Fig. 9a). Since UV-

induced phosphorylation of MK2 is known to depend mostly on p38α activity, the results

indicate that PROTAC treatment efficiently downregulates this signaling pathway in cells.

We also confirmed that the N-methyl inactive controls NR-7h* and NR-6a* did not induce

p38α protein degradation, but surprisingly compound NR-7h* was able to inhibit p38α

signaling, probably because the PH-797804 component of the PROTAC remains active. On

the other hand, NR-6a* does not inhibit p38α signaling despite possessing the same

warhead, differing only in the linker and E3 ligase exit vector. Moreover, the inhibition of

p38α was observed for at least two days after addition of the PROTACs to the cell culture

media (Fig. 9b). To further validate the effect of PROTACs on p38α signaling beyond

cancer cells, we used BMDMs, which are known to activate the p38α pathway in response

to lipopolysaccharide (LPS) and interferon (IFN)-γ stimulation. Accordingly, we detected a

robust phosphorylation of MK2 in cells treated with LPS and IFN-γ, which was strongly

reduced upon incubation with PH-797804 or the PROTACs (Fig. 9c). These results indicate

that PROTAC treatment can efficiently downregulate the p38α signaling pathway in primary

cells.

3

. Discussion

In this manuscript, we report the design, synthesis and characterization of small molecules

that can trigger p38α downregulation in mammalian cells. These PROTACs were based on an

ATP competitive inhibitor of p38α and p38β (PH-797804) that was linked to thalidomide

analogues to recruit CRBN E3 ligases. Our work confirms the importance of the length and

composition of the linker to bring together the target protein and the E3 ligase in the correct

spatial orientation so that a productive protein-protein interaction is formed that ultimately

allows ubiquitination of the target protein [12]. Accordingly, we found that an appropriate

linker length is critical for the activity of PROTACs that target p38α, with linkers having 15-

1

7 atoms showing optimal performance, which is longer than most PROTACs for other

targets [26, 28]. Moreover, we observed that compounds with the same linker but that differ

only in their connection to thalidomide (i.e., NR-3c and NR-4) showed different abilities to

induce p38α degradation, emphasizing the importance of the exit vector of the thalidomide

fragment in engaging productive CRBN binding.

9

Our work allowed us to identify several PROTACs that induce the degradation of both

p38α and p38β, without affecting other MAPKs, when administered at nanomolar

concentrations to the culture medium of several cell lines. These PROTACs show a distinct

selectivity profile over the recently reported p38α degrader SJFα, which also degrades p38δ

at nanomolar concentrations, albeit higher than for p38α, and presumably degrades c-Met

kinase too [13, 21]. Moreover, we provide evidence that these PROTACs can function in

several cell types, including epithelial cells, cardiomyocytes and macrophages.

Protein degradation is thought to have several benefits over traditional inhibition of the

enzymatic activity. For example, it does not depend on occupation of the active site of the

target protein, requiring only a transient binding effect in order to initiate degradation,

therefore enabling sub-stoichiometric quantities of PROTAC to be used [11]. However, our

initial characterization did not show a marked improvement in the extent of p38α pathway

inhibition in cells, as determined by the phosphorylation of the p38α substrate MK2, for NR-

6

a or NR-7h compared to PH-797804. This might be due to the potent inhibitory capacity of

PH-797804 being difficult to improve upon. Further work will be needed to determine if

particular p38α functions could be differentially affected by PROTACs and chemical

inhibitors, but our observations highlight the need for further understanding the advantages

and mechanisms of PROTAC-induced degradation for each given target.

An important consideration for PROTAC use is that the degraded protein’s function is

subdued until new protein synthesis has taken place. This generally lasts longer than

inhibition of the target protein, which requires occupation of the active site and is therefore

limited by the off-rate of the inhibitor. Indeed, the rate of degradation and subsequent re-

synthesis is vital for selecting an appropriate target for development of novel PROTAC

therapies. It should be noted that p38α expression is maintained to minimal levels for several

days, suggesting that protein re-synthesis is not a limiting factor for the use of p38α-targeted

PROTACs.

Further work will be needed to explore the therapeutic potential of using PROTACs to

target p38α for degradation, and whether this approach provides any advantage over the use

of traditional inhibitors that target p38α kinase activity. These tool compounds should also

allow further investigation of the poorly understood pseudo-kinase activity of p38α and

determination of its role in diseases.

1

0

4

. Experimental section

4

.1. Chemical synthesis.

General.

All compounds were chemically synthesized, purified by chromatography and characterized

1

13

by H NMR, C NMR, IR and HRMS. Compounds were of a purity ≥95% as determined by

1

3

HRMS and C NMR spectroscopy, or by HPLC/ms. NMR spectra were recorded at 23°C on

1

13

a Varian Mercury 400 or Varian 500 apparatus. H NMR and C NMR spectra were

referenced either to relative internal TMS or to residual solvent peaks. Signal multiplicities in

1

3

the C NMR spectra were assigned by HSQC or DEPT experiments. Melting points were

determined using a Büchi M-540 apparatus or by DSC without recrystallization of the final

solids. IR spectra were recorded in a Thermo Nicolet Nexus FT-IR apparatus by depositing a

film of the product on a sodium chloride disk. HRMS were recorded in a LTQ-FT Ultra

(

Thermo Scientific) using Nanoelectrospray technique. HPLC chromatography was

performed on Hewlett-Packard 1050 equipment with UV detection using a Kinetix EVO C18

0x 4.6 mm, 2.6 μm column (Standard gradient: 10 mM NH CO / MeCN (95:5) – (0:100)).

5

4

3

PH-797804 was prepared according to literature precedents [16].

The synthesis of compounds NR-1-5 and NR-7i-j and their intermediates is described

in the Supporting Information.

Amide formation (General procedure D).

The carboxylic acid (1 equiv) was dissolved in anhydrous DMF, cooled to 0°C, then added

Oxyma (ethyl cyanohydroxyiminoacetate) (1.66 equiv) and DIC (N,N′-

Diisopropylcarbodiimide) (1.66 equiv). After 15 min the solution turned yellow, then NEt₃

(1.66 mL) was added, followed by the corresponding amine starting material (1.1 equiv). The

reaction was left 18 – 24 h before quenching with water. The mixture was separated between

EtOAc and water, then the aqueous layer re-extracted using EtOAc. The combined organic

layers were washed with brine, dried over MgSO , and then concentrated by rotary evaporator

4

under reduced pressure. The corresponding crude amide was then purified by flash column

chromatography.

Click reaction (General procedure E).

-1

To a solution of the azide component (1 equiv) in t-BuOH (1.0 mmol mL ) and water (1.0

-1

mmol mL ) at 30°C was added CuSO (0.3 equiv), sodium ascorbate (0.1 equiv) and the

4

alkyne component (1 equiv). The reaction mixture was then degassed using N for 20 min,

2

1

then left to stir at this temperature for 20 h. The reaction mixture was then separated between

EtOAc and water, the aqueous layer re-extracted using EtOAc (x3). The combined organic

layers were then washed with water and brine, dried over MgSO and concentrated under

4

reduced pressure to afford a fluorescent yellow film. The crude was then purified by flash

column chromatography, to afford the desired product.

3

4

-(5-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(hex-5-yn-1-yl)-

-methylbenzamide (8a)

Following general procedure D starting from 1 and hex-5-yn-1-aminium chloride (7a),

compound 8a was obtained in 77% yield. The product eluted from the silica column at 70%

EtOAc/hexanes, as a light beige, foamy solid. TLC: R = 0.36 (50% EtOAc/hexanes). Mp:

f

1

9

42–143°C. H NMR (400 MHz, CDCl ) δ 7.79 (dd, J = 8, 2 Hz, 1H), 7.61 (td, J = 9, 6 Hz,

3

H), 7.50 (d, J = 2 Hz, 1H), 7.36 (dd, J = 8, 1 Hz, 1H), 7.01 – 6.92 (m, 1H), 6.84 (ddd, J = 10,

, 3 Hz, 1H), 6.74 (t, J = 6 Hz, 1H), 6.14 (d, J = 1 Hz, 1H), 5.22 (d, J = 1 Hz, 2H), 3.39 – 3.21

(m, 2H), 2.21 (td, J = 7, 3 Hz, 2H), 2.08 (s, 3H), 1.94 (t, J = 3 Hz, 1H), 1.92 (d, J = 2 Hz, 3H),

13

1

.66 – 1.52 (m, 4H) ppm. C NMR (101 MHz, CDCl ) δ 165.9 (C), 164.2 (d, J = 12 Hz,

3 F

C), 163.2 (C), 161.4 (dd, J = 57, 12 Hz, C), 160.4 (C), 158.7 (d, J = 12 Hz, C), 146.4 (C),

F

1

38.4 (C), 137.3 (C), 134.4 (C), 131.4 (CH), 130.0 (dd, J = 10, 5 Hz, CH), 128.5 (CH), 126.4

F

(

CH), 118.5 (dd, J = 14, 4 Hz, C), 111.9 (dd, J = 21, 4 Hz, CH), 103.9 (t, J = 25 Hz, CH),

F F F

9

2

1

6.3 (CH), 84.2 (C), 68.6 (CH), 64.4 (d, J = 4 Hz, CH ), 39.6 (CH ), 28.4 (CH ), 25.8 (CH ),

F 2 2 2 2

1.6 (CH ), 18.1 (CH ), 17.3 (CH ) ppm. IR (NaCl) ʋ_m_a_x: 3304, 2925, 1640, 1527, 1340,

3

2

3

-1

+

101 cm . HRMS (ESI): calc. for [C H O N BrF ] : 543.10894 found 543.10700.

2

7

26

3

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-4-methyl-N-(pent-4-

yn-1-yl)benzamide (8b)

Following general procedure D starting from 1 and pent-4-yn-1-aminium chloride, compound

7

b was obtained in 73% yield. The product eluted from the silica column at 70%

EtOAc/hexanes, as a white solid. TLC: R = 0.43 (80% EtOAc/hexanes). Mp: 144–145°C.

f

1

H NMR (400 MHz, CDCl ) δ 7.82 (dd, J = 8, 2 Hz, 1H), 7.61 (td, J = 9, 6 Hz, 1H), 7.54 (d,

3

J = 2 Hz, 1H), 7.37 (dd, J = 8, 1 Hz, 1H), 7.00 – 6.93 (m, 1H), 6.91 (t, J = 6 Hz, 1H), 6.83

(ddd, J = 10, 9, 3 Hz, 1H), 6.14 (d, J = 1 Hz, 1H), 5.20 (s, 2H), 3.45 – 3.25 (m, 2H), 2.22 (td,

J = 7, 3 Hz, 2H), 2.08 (s, 3H), 1.96 (t, J = 3 Hz, 1H), 1.92 (d, J = 1 Hz, 3H), 1.71 (quint, J = 7

13

Hz, 2H) ppm. C NMR (101 MHz, CDCl ) δ 165.8 (C), 164.2 (d, J = 12 Hz, C), 163.2 (C),

3

F

1

61.4 (dd, J = 60, 12 Hz, C), 160.3 (C), 158.6 (d, J = 12 Hz, C), 146.5 (C), 138.4 (C), 137.3

F F

1

2

(

C), 134.2 (C), 131.4 (CH), 129.8 (dd, J = 10, 5 Hz, CH), 128.7 (CH), 126.4 (CH), 118.5

F

(

dd, J = 14, 4 Hz, C), 111.9 (dd, J = 21, 4 Hz, CH), 103.9 (t, J = 25 Hz, CH), 96.4 (CH),

F

8

3.9 (C), 68.9 (CH), 64.4 (d, J = 4 Hz, CH ), 39.3 (CH ), 27.9 (CH ), 21.6 (CH ), 17.3

F 2 2 2 3

-1

(

CH ), 16.2 (CH ) ppm. IR (NaCl) ʋ : 3299, 2929, 1644, 1527, 1340, 1088 cm . HRMS

3 2 max

+

(

ESI): calc. for [C H O N BrF ] : 529.09329, found 529.09285.

26 24 3 2 2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(but-3-yn-1-yl)-

4

-methylbenzamide (8c)

Following general procedure D starting from 1 and but-3-yn-1-aminium chloride (7c),

compound 8c was obtained in 50% yield. The product eluted from the silica column at 70%

EtOAc/hexanes, as a white solid. TLC: R = 0.63 (80% EtOAc/hexanes). Mp: 148–149°C.

f

1

H NMR (400 MHz, CDCl ) δ 7.73 (dd, J = 8, 2 Hz, 1H), 7.54 (td, J = 8, 6 Hz, 1H), 7.45 (d,

3

J = 2 Hz, 1H), 7.31 (dd, J = 8, 1 Hz, 1H), 6.90 (tdd, J = 9, 3, 1 Hz, 1H), 6.85 (t, J = 6 Hz, 1H),

6

–

.78 (ddd, J = 10, 9, 3 Hz, 1H), 6.08 (d, J = 1 Hz, 1H), 5.16 (s, 2H), 3.50 – 3.36 (m, 1H), 3.36

3.26 (m, 1H), 2.34 (td, J = 7, 3 Hz, 2H), 2.03 (s, 3H), 1.92 (t, J = 3 Hz, 1H), 1.85 (s, 3H)

1

3

ppm. C NMR (101 MHz, CDCl ) δ 165.8 (C), 164.2 (d, J = 12 Hz, C), 163.2 (C), 161.5

3

F

(

dd, J = 56, 12 Hz, C), 160.4 (C), 158.7 (d, J = 12 Hz, C), 146.3 (C), 138.7 (C), 137.5 (C),

F

1

34.1 (C), 131.4 (CH), 130.0 (dd, J = 10, 5 Hz, CH), 128.4 (CH), 126.5 (CH), 118.5 (dd, J_F

F

=

14, 4 Hz, C), 111.9 (dd, J = 21, 4 Hz, CH), 104.3 – 103.5 (m, CH), 96.3 (CH), 81.6 (C),

F

6

9.8 (CH), 64.4 (d, J = 4 Hz, CH ), 38.8 (CH ), 21.6 (CH ), 19.2 (CH ), 17.3 (CH ) ppm. IR

F 2 2 3 2 3

-1

(

NaCl) ʋ_m_a_x: 3304, 2933, 1649, 1527, 1349, 1088 cm . HRMS (ESI): calc. for

+

[

C H O N BrF ] : 515.07764, found 515.07740.

2

5

22

3

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(4-(1-(4-((6-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)hexyl)amino)-4-oxobutyl)-1H-1,2,3-triazol-4-

yl)butyl)-4-methylbenzamide (NR-6a)

Following general procedure E starting from 8a and 14c, compound NR-6a was obtained as a

1

white solid in 56% yield. Mp: 148ºC. H NMR (400 MHz, CDCl ) δ 9.08 (s, HN), 9.03 (s,

3

HN), 7.83 (d, J = 8 Hz, 1H), 7.69 (d, J = 7 Hz, 1H), 7.65 – 7.54 (m, 2H), 7.45 – 7.31 (m, 5H),

7.02 – 6.92 (m, 1H), 6.90 – 6.82 (m, 1H), 6.60 (s, 1H), 6.17 (s, 1H), 5.27 – 5.16 (m, 3H), 4.46

– 4.23 (m, 4H), 3.37 – 3.22 (m, 2H), 3.22 – 3.04 (m, 2H), 2.89 – 2.75 (m, 2H), 2.72 – 2.64 (m,

2H), 2.60 (t, J = 8 Hz, 2H), 2.48 – 2.35 (m, 1H), 2.24 – 2.01 (m, 8H), 1.92 (s, 3H), 1.73 – 1.49

13

(

m, 6H), 1.48 – 1.38 (m, 2H), 1.37 – 1.29 (m, 4H) ppm. C NMR (101 MHz, CD OD) δ

3

1

71.8 (C), 170.2 (C), 169.8 (C), 166.4 (C), 164.4 (d, J = 12 Hz, C)163.5 (C), 161.8 (d, J =

F F

1

3

1

4 Hz, C), 160.6 (C), 159.0 (d, J = 12 Hz, C), 147.8 (C), 146.7 (C), 140.1 (C), 138.7 (C),

F

37.7 (C), 137.6 (CH), 134.4 (d, J = 5 Hz, C), 132.0 (C), 131.6 (CH), 131.4 (CH), 130.5 –

F

29.9 (m, CH), 128.7 (CH), 128.6 (CH), 126.7 (d, J = 2 Hz, CH), 121.9 – 121.4 (m, CH),

F

18.7 (dd, J = 14, 4 Hz, C), 112.1 (dd, J = 21, 4 Hz, CH), 104.3 (t, J = 25 Hz, CH), 96.7

F

(

CH), 96.5 (C), 64.6 (d, J = 4 Hz, CH ), 51.9 (CH ), 49.3 (CH), 46.5 (CH ), 40.0 (CH ), 39.5

F 2 2 2 2

(

CH ), 32.7 (CH ), 32.1 (CH ), 30.6 (CH ), 29.8 (CH ), 29.4 (CH ), 29.0 (CH ), 28.4 (CH ),

2 2 2 2 2 2 2 2

2

6.7 (CH ), 26.6 (CH ), 26.2 (CH ), 25.1 (CH ), 23.5 (CH ), 21.7 (CH ), 17.4 (CH ) ppm. IR

2 2 2 2 2 3 3

-1

+

(

NaCl) ʋ_m_a_x: 3408, 2931, 1644, 1349, 1101 cm . HRMS (ESI): calc. for [C H O N BrF ] :

50 56 7 8 2

9

97.3418; found: 997.3426.

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(4-(1-(4-((5-(2-

(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pentyl)amino)-4-oxobutyl)-1H-1,2,3-triazol-4-

yl)butyl)-4-methylbenzamide (NR-6b)

Following the general procedure E starting from 8a and 14d, compound NR-6b was obtained

1

as a white solid in 26% yield. Mp: 136ºC. H NMR (400 MHz, CDCl ) δ 8.99 (s, 1H), 7.79

3

(

ddd, J = 8, 3, 2 Hz, 1H), 7.69 (dt, J = 7, 2 Hz, 1H), 7.60 (td, J = 9, 6 Hz, 1H), 7.54 (s, 1H),

.40 (t, J = 8 Hz, 1H), 7.37 – 7.31 (m, 3H), 7.26 – 7.16 (m, 1H), 7.01 – 6.93 (m, 1H), 6.86

ddd, J = 10, 9, 3 Hz, 1H), 6.46 (t, J = 6 Hz, 1H), 6.15 (dd, J = 2, 1 Hz, 1H), 5.25 – 5.17 (m,

7

(

3

2

H), 4.42 (dd, J = 16, 5 Hz, 1H), 4.35 – 4.26 (m, 3H), 3.37 – 3.25 (m, 2H), 3.23 – 3.05 (m,

H), 2.88 – 2.79 (m, 2H), 2.69 (t, J = 7 Hz, 2H), 2.65 – 2.55 (m, 2H), 2.49 – 2.36 (m, 1H),

.25 – 2.01 (m, 8H), 1.91 (s, 3H), 1.81 – 1.52 (m, 6H), 1.53 – 1.38 (m, 2H), 1.37 – 1.26 (m,

1

3

H) ppm. C NMR (101 MHz, CD OD) δ 171.8 (C), 170.2 (C), 169.8 (C), 167.7 (C), 166.4

3

(C), 163.5 (C), 162.0 (C), 160.6 (C), 159.1 (C), 148.7 – 147.7 (m, C), 146.7 (C), 143.1 (C),

1

40.3 (d, J = 2 Hz, C), 138.7 (C), 137.6 (C), 137.4 (CH), 134.4 (m, C), 132.0 (C), 131.6

F

(

CH), 131.4 (CH), 130.4 (m, CH), 128.7 (CH), 126.7 (CH), 121.8 (CH), 118.6 (d, J = 4 Hz,

F

C), 118.1 – 117.2 (m, CH), 112.08 (dd, J = 21, 4 Hz, CH), 104.6 – 103.3 (m, CH), 96.7 (C),

F

9

6.5 (CH), 64.7 (d, J = 3 Hz, CH ), 52.0 (CH ), 49.3 (CH), 46.5 (CH ), 40.0 (CH ), 39.4

F 2 2 2 2

(

CH ), 32.7 (CH ), 31.9 (CH ), 31.8 (CH ), 29.8 (CH ), 29.6 (CH ), 29.3 (CH ), 28.4 (CH ),

2 2 2 2 2 2 2 2

2

6.5 (CH ), 26.2 (CH ), 25.1 (CH ), 23.5 (CH ), 21.7 (CH ), 17.4 (CH ) ppm. IR (NaCl)

2 2 2 2 3 3

-1

+

ʋ_m_a_x: 3385, 2929, 1642, 1528, 1349, 1101 cm . HRMS (ESI): calc. for [C H O N BrF ] :

49

54

7

8

2

9

83.3261; found 983.3238.

1

4

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(2-(1-(4-((2-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-4-oxobutyl)-1H-1,2,3-

triazol-4-yl)ethyl)-4-methylbenzamide (NR-7a)

Following the general procedure E starting from 8c and 15c, compound NR-7a was obtained

1

as a bright yellow solid in 96% yield. H NMR (400 MHz, CDCl ) δ 9.16 (s, HN), 9.10 (s,

3

HN), 7.72 (dt, J = 8, 2 Hz, 1H), 7.57 – 7.49 (m, 1H), 7.45 (s, 1H), 7.42 – 7.35 (m, 2H), 7.31

(

s, 1H), 7.25 – 7.20 (m, 2H), 6.98 (dd, J = 7, 2 Hz, 1H), 6.94 – 6.84 (m, 2H), 6.80 (ddd, J =

0, 9, 3 Hz, 1H), 6.36 (s, 1H), 6.09 (d, J = 3 Hz, 1H), 5.16 (s, 2H), 4.87 – 4.80 (m, 1H), 4.21

s, 2H), 3.61 – 3.44 (m, 2H), 3.37 – 3.37 (m, 4H), 2.83 – 2.61 (m, 5H), 2.04 – 1.94 (m, 6H),

1

(

1

3

1

.89 – 1.80 (m, 5H) ppm. C NMR (101 MHz, CDCl ) δ 172.6 (C), 171.9 (C), 171.7 (C),

3

69.2 (C), 169.1 (C), 167.6 (C), 166.2 (d, J = 8 Hz, C), 164.5 – 164.1 (m, C), 163.3 (C),

F

62.0 (C), 161.7 (C), 160.5 (d, J = 2 Hz, C), 158.9 (d, J = 12 Hz, C), 146.7 (C), 146.4 (C),

F

38.8 (C), 137.5 (C), 136.2 (CH), 134.0 (d, J = 6.3 Hz, C), 132.4 (C), 131.4 (d, J = 5 Hz,

F

CH), 130.3 (dd, J = 10, 6 Hz, CH), 128.3 (CH), 126.7 (CH), 118.5 (d, J = 3 Hz, CH), 116.8

F

(

CH), 111.9 (dd, J = 21, 3 Hz, CH), 111.6 (CH), 110.1 (C) , 104.4 – 103.5 (m, CH), 96.4

F

(

CH), 64.5 (d, J = 4 Hz, CH ), 49.1 (CH ), 48.9 (CH), 42.2 (CH ), 39.2 (CH ), 38.6 (CH ),

F

2

3

1.9 (CH ), 31.4 (CH ), 25.7 (CH ), 25.5 (CH ), 22.7 (CH ), 21.5 (CH ), 17.3 (CH ) ppm. IR

2 2 2 2 2 3 3

-1

(

NaCl) ʋ_m_a_x: 3338, 2938, 1702, 1641, 1501, 1340, 1040 cm . HRMS (ESI): calc. for

+

[

C H O N BrF ] : 942.23805, found 942.23882.

4

43

8

9

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(2-(1-(4-((3-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-4-oxobutyl)-1H-

1

,2,3-triazol-4-yl)ethyl)-4-methylbenzamide (NR-7b)

Following the general procedure E starting from 8c and 15b, compound NR-7b was obtained

1

as a bright yellow solid in 78% yield. H NMR (400 MHz, CDCl ) δ 8.74 (s, HN), 8.67 (s,

3

HN), 7.69 (d, J = 8 Hz, 1H), 7.53 (q, J = 8 Hz, 1H), 7.42 (s, 1H), 7.38 (t, J = 8 Hz, 1H), 7.34

(

s, 1H), 7.26 (dd, J = 8, 4 Hz, 1H), 7.21 (s, 1H), 6.98 (d, J = 7 Hz, 1H), 6.91 (t, J = 8 Hz, 1H),

.85 – 6.77 (m, 3H), 6.32 (s, 1H), 6.09 (s, 1H), 5.17 (s, 2H), 4.85 (dd, J = 12, 6 Hz, 1H), 4.28

s, 2H), 3.65 – 3.56 (m, 2H), 3.26 – 3.16 (m, 4H), 2.90 – 2.61 (m, 5H), 2.08 – 1.94 (m, 6H),

6

(

1

3

1

.90 – 1.80 (m, 5H), 1.75 – 1.63 (m, 2H) ppm. C NMR (101 MHz, CDCl ) δ 172.1 (2 x C),

3

71.4 (C), 169.3 (C), 168.7 (C), 167.6 (C), 166.2 (d, J = 6 Hz, C), 164.3 (dd, J = 13, 3 Hz,

F

C), 163.3 (C), 162.0 (C), 160.7 (C), 160.5 (C), 157.8 (C), 146.7 (C), 146.3 (C), 138.9 (C),

37.5 (C), 136.1 (CH), 134.0 (C), 132.5 (C), 131.5 (CH), 130.4 (CH), 128.2 (CH), 126.5

CH), 122.5 – 122.0 (m, CH), 118.5 (m, C), 116.7 (CH), 112.0 (dd, J = 21, 3 Hz, CH), 111.4

1

(

F

1

5

(

CH), 110.0 (C), 104.4 – 103.6 (m, CH), 96.4 (CH), 64.4 (d, J = 4 Hz, CH ), 49.3 (CH ),

F 2 2

4

8.9 (CH), 40.2 (CH ), 39.0 (CH ), 37.0 (CH ), 31.9 (CH ), 31.4 (CH ), 29.0 (CH ), 25.8

2 2 2 2 2 2

(

CH ), 25.5 (CH ), 22.8 (CH ), 21.5 (CH ), 17.3 (CH ) ppm. IR (NaCl) ʋ_m_a_x: 3303, 2925,

2 2 2 3 3

-1

+

1

697, 1641, 1501, 1358, 1192 cm . HRMS (ESI): calc. for [C H O N BrF ] : 956.25370,

45 45 8 9 2

found 956.25392.

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(3-(1-(4-((3-((2-

2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-4-oxobutyl)-1H-

,2,3-triazol-4-yl)propyl)-4-methylbenzamide (NR-7c)

(

1

Following the general procedure E but starting from 8b and 15b, compound NR-7c was

1

obtained as a bright yellow solid in 52% yield. H NMR (400 MHz, CDCl ) δ 8.91 (s, HN),

3

8

.89 (s, HN), 7.74 – 7.69 (m, 1H), 7.63 – 7.59 (m, 2H), 7.46 – 7.38 (m, 3H), 7.32 (dd, J = 8, 2

Hz, 1H), 7.18 (q, J = 5 Hz, 1H), 7.03 (dd, J = 7, 2 Hz, 1H), 7.00 – 6.93 (m, 1H), 6.89 – 6.82

(

m, 2H), 6.35 (t, J = 6 Hz, 1H), 6.18 (t, J = 1 Hz, 1H), 5.22 (s, 2H), 4.94 – 4.87 (m, 1H), 4.31

h, J = 8 Hz, 2H), 3.49 – 3.35 (m, 1H), 3.33 – 3.15 (m, 5H), 2.87 – 2.65 (m, 5H), 2.15 – 1.98

13

m, 8H), 1.97 – 1.81 (m, 5H), 1.81 – 1.68 (m, 2H) ppm. C NMR (101 MHz, CDCl ) δ

3

1

72.3 (2 x C), 171.5 (2 x C), 169.3 (C), 168.8 (2 x C), 167.6 (C), 165.7 (C), 165.6 (C), 163.4

(

C), 161.6 (dd, J = 47, 12 Hz, C), 160.5 (C), 146.7 (C), 146.6 (C), 138.7 (C), 137.5 (C),

F

1

36.1 (CH), 133.9 (d, J = 8 Hz, C), 132.5 (C), 131.4 (CH), 130.3 (CH), 128.2 (CH), 126.6

F

(

CH), 122.0 (CH), 118.5 (dd, J = 15, 5 Hz, C), 116.7 (CH), 111.9 (d, J = 25 Hz, CH), 111.3

F

(

CH), 110.0 (d, J = 9 Hz, C), 104.0 (t, J = 25 Hz, CH), 96.5 (CH), 64.5 (d, J = 4 Hz, CH ),

F F F 2

4

9.3 (CH ), 48.9 (CH), 40.1 (CH ), 39.6 (CH ), 36.8 (CH ), 32.0 (CH ), 31.4 (CH ), 29.0

2 2 2 2 2 2

(

[

CH ), 28.2 (CH ), 25.9 (CH ), 23.3 (CH ), 22.8 (CH ), 21.6 (CH ), 17.3 (CH ) ppm. IR

2

3

-1

NaCl) ʋ_m_a_x: 3308, 3082, 2942, 1693, 1641, 1358, 1192, 726 cm . HRMS (ESI): calc. for

+

C H O N BrF ] : 970.26935, found 970.26913.

4

6

47

8

9

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(2-(1-(4-((4-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-4-oxobutyl)-1H-1,2,3-

triazol-4-yl)ethyl)-4-methylbenzamide (NR-7d)

Following the general procedure E starting from 8c and 15a, compound NR-7d was obtained

1

as a bright yellow solid in 99% yield. H NMR (400 MHz, CDCl ) δ 8.96 (s, HN), 8.90 (s,

3

HN), 7.76 (d, J = 8 Hz, 1H), 7.60 (q, J = 8 Hz, 1H), 7.51 (s, 1H), 7.48 – 7.31 (m, 4H), 7.06 –

7

1

.02 (m, 1H), 7.01 – 6.93 (m, 1H), 6.90 – 6.81 (m, 2H), 6.78 (s, 1H), 6.21 (s, 1H), 6.16 (s,

H), 5.23 (s, 2H), 4.91 (dd, J = 12, 6 Hz, 1H), 4.33 (s, 2H), 3.72 – 3.52 (m, 2H), 3.29 – 3.13

1

6

(m, 4H), 2.95 – 2.68 (m, 5H), 2.15 – 2.02 (m, 6H), , 1.97 – 1.87 (m, 5H), 1.69 – 1.53 (m, 4H)

13

ppm. C NMR (101 MHz, CDCl ) δ 171.8 (C), 171.4 (2 x C), 169.4 (2 x C), 168.8 (C),

3

1

67.6 (C), 166.2 (d, J = 16 Hz, C), 164.3 (C), 163.3 (C), 161.5 (dd, J = 48, 12 Hz, C), 160.4

F F

(

d, J = 5 Hz, C), 158.8 (d, J = 12 Hz, C), 146.8 (C), 146.4 (C), 138.9 (d, J = 2 Hz, C),

F F F

1

37.5 (d, J = 2 Hz, C), 136.1 (CH), 134.0 (d, J = 9 Hz, C), 132.4 (C), 131.6 – 131.3 (m,

F F

CH), 130.4 – 130.0 (m, CH), 128.5 – 128.2 (m, CH), 126.6 (CH), 122.3 (CH), 118.5 (dd, J =

F

1

4, 4 Hz, C), 116.7 (CH), 112.1 – 111.7 (m, CH), 111.4 (CH), 109.8 (d, J = 3 Hz, C), 104.0

F

(

t, J = 25 Hz, CH), 96.7 – 96.3 (m, CH), 64.5 (d, J = 4 Hz, CH ), 49.2 (CH ), 48.9 (CH), 42.2

F 2 2

(

CH ), 39.1 (CH ), 38.9 (CH ), 32.0 (d, J = 8 Hz, CH ), 31.4 (CH ), 26.8 (CH ), 26.5 (CH ),

2 2 2 F 2 2 2 2

2

9

5.9 (CH ), 25.5 (CH ), 22.8 (CH ), 21.5 (CH ), 17.3 (CH ) ppm. IR (NaCl) ʋ_m_a_x: 3299,

2 2 2 3 3

-1

+

929, 1702, 1641, 1349, 1349, 1044 cm . HRMS (ESI): calc. for [C H O N BrF ] :

46

47

8

9

2

70.26935, found 970.26984.

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(4-(1-(4-((2-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-4-oxobutyl)-1H-1,2,3-

triazol-4-yl)butyl)-4-methylbenzamide (NR-7e)

Following the general procedure E starting from 8a and 15c, compound NR-7e was obtained

1

as a bright yellow solid in 96% yield. H NMR (400 MHz, CDCl ) δ 9.13 (s, HN), 9.00 (s,

3

HN), 7.81 – 7.75 (m, 1H), 7.60 (td, J = 8, 6 Hz, 1H), 7.51 (dd, J = 4, 2 Hz, 1H), 7.45 (dd, J =

9

, 7 Hz, 1H), 7.39 – 7.27 (m, 4H), 7.04 (dd, J = 7, 3 Hz, 1H), 7.00 – 6.90 (m, 2H), 6.86 (ddd,

J = 11, 9, 3 Hz, 1H), 6.40 (t, J = 5 Hz, 1H), 6.17 (dd, J = 2, 1 Hz, 1H), 5.22 (s, 2H), 4.92 –

4

.84 (m, 1H), 4.36 – 4.26 (m, 2H), 3.52 – 3.41 (m, 1H), 3.41 – 3.29 (m, 3H), 3.16 (ddd, J =

3

0, 14, 7 Hz, 2H), 2.83 – 2.59 (m, 5H), 2.18 – 2.01 (m, 8H), 1.93 – 1.88 (m, 3H), 1.67 – 1.54

1

3

(

m, 2H), 1.52 – 1.42 (m, 2H) ppm. C NMR (101 MHz, CDCl ) δ 172.6 (C), 171.6 (2 x C),

3

1

69.2 (d, J = 5 Hz, C), 169.0 (C), 167.5 (2 x C), 166.5 (C), 166.2 (C), 163.3 (C), 161.7 (C),

F

1

60.5 (d, J = 7 Hz, C), 146.7 (C), 146.5 (d, J = 3 Hz, C), 138.6 (d, J = 5 Hz, C), 137.4 (d,

F

J = 2 Hz, C), 136.1 (d, J = 2 Hz, CH), 134.2 (d, J = 20 Hz, C), 132.4 (C), 131.4 (CH),

F

1

2

30.3 (CH), 128.5 (CH), 126.6 (d, J = 10 Hz, CH), 121.7 (CH), 118.5 (d, J = 14 Hz, C),

F

16.8 (CH), 111.9 (dd, J = 22, 4 Hz, CH), 111.6 (CH), 110.0 (d, J = 4 Hz, C), 104.0 (t, J =

F

5 Hz, CH), 96.6 (d, J = 5 Hz, C), 96.4 (CH), 64.5 (d, J = 4 Hz, CH ), 49.2 (d, J = 8 Hz,

F

2

CH ), 48.8 (d, J = 3 Hz, CH), 42.20 (d, J = 10 Hz, CH ), 39.8 (CH ), 38.8 (d, J = 6 Hz, CH ),

2

3

2.2 (d, J = 20 Hz, CH ), 31.4 (CH ), 28.1 (CH ), 26.3 (CH ), 25.7 (d, J = 11 Hz, CH ), 24.8

2 2 2 2 2

(

CH ), 22.7 (CH ), 21.5 (CH ), 17.3 (CH ) ppm. IR (NaCl) ʋ_m_a_x: 3307, 2929, 1693, 1641,

2 2 3 3

1

7

-1

+

1

353, 1196, 731 cm . HRMS (ESI): calc. for [C H O N BrF ] : 970.26935, found

46 47 8 9 2

9

70.26872.

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(3-(1-(4-((4-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-4-oxobutyl)-1H-1,2,3-

triazol-4-yl)propyl)-4-methylbenzamide (NR-7f)

Following the general procedure E starting from 8b and 15a, compound NR-7f was obtained

1

as a bright yellow solid in 53% yield. H NMR (400 MHz, CDCl ) δ 8.79 (s, HN), 8.76 (s,

3

HN), 7.71 (d, J = 8 Hz, 1H), 7.63 – 7.53 (m, 2H), 7.44 (ddd, J = 9, 7, 5 Hz, 1H), 7.40 – 7.31

(

m, 3H), 7.07 – 6.93 (m, 3H), 6.90 – 6.82 (m, 2H), 6.17 (s, 2H), 5.22 (s, 2H), 4.90 (dd, J = 12,

Hz, 1H), 4.33 (dt, J = 11, 7 Hz, 2H), 3.42 (dd, J = 13, 6 Hz, 1H), 3.34 – 3.17 (m, 4H), 3.09

td, J = 14, 13, 6 Hz, 1H), 2.88 – 2.66 (m, 6H), 2.09 (d, J = 7 Hz, 3H), 2.06 (d, J = 2 Hz, 3H),

6

(

1

3

1

4

.95 – 1.85 (m, 6H), 1.63 – 1.47 (m, 5H) ppm. C NMR (101 MHz, CDCl ) δ 172.0 (d, J =

3

Hz, C), 171.3 (2 x C), 169.4 (C), 168.8 (d, J = 4 Hz, C), 167.6 (2 x C), 165.8 (2 x C), 163.4

(

d, J = 3 Hz, C), 160.5 (d, J = 9 Hz, C), 146.8 (C), 146.5 (d, J = 5 Hz, C), 138.7 (d, J = 6 Hz,

C), 137.6 (C), 136.1 (CH), 134.0 (C), 132.4 (C), 131.4 (CH), 130.5 – 130.0 (m, CH), 128.1

CH), 126.7 (CH), 122.0 (d, J = 6 Hz, CH), 118.4 (C), 116.7 (CH), 112.2 – 111.6 (m, CH),

11.3 (d, J = 3 Hz, CH), 109.7 (C), 104.46 – 103.41 (m, CH), 96.6 (CH), 96.4 (C), 64.5 (d, J_F

4 Hz, CH ), 49.3 (d, J = 9 Hz, CH ), 48.9 (d, J = 2 Hz CH), 42.1 (CH ), 39.7 (CH ), 39.5

(

1

=

2

(

CH ), 38.8 (CH ), 31.9 (d, J = 10 Hz, CH ), 31.4 (CH ), 28.3 (CH ), 26.7 (d, J = 30 Hz,

2 2 2 2 2

CH ), 25.9 (CH ), 23.2 (CH ), 22.8 (CH ), 21.5 (CH ), 17.3 (CH ) ppm. (1 C not observed in

2

3

-1

spectrum) IR (NaCl) ʋ_m_a_x: 3308, 3090, 2920, 1697, 1640, 1353, 726 cm . HRMS (ESI):

+

calc. for [C H O N BrF ] : 984.28500, found 984.28549.

47

49

8

9

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(4-(1-(4-((3-((2-

2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-4-oxobutyl)-1H-

,2,3-triazol-4-yl)butyl)-4-methylbenzamide (NR-7g)

(

1

Following the general procedure E starting from 8a and 15b, compound NR-7g was obtained

1

as a bright yellow waxy solid in 44% yield. H NMR (400 MHz, CDCl ) δ 8.95 (s, HN), 8.92

3

(

s, HN), 7.71 (d, J = 8 Hz, 1H), 7.53 (td, J = 9, 6 Hz, 1H), 7.48 (s, 1H), 7.41 – 7.19 (m, 4H),

.00 – 6.86 (m, 3H), 6.82 – 6.75 (m, 2H), 6.30 (s, 1H), 6.10 (s, 1H), 5.15 (s, 2H), 4.89 – 4.81

m, 1H), 4.26 (s, 2H), 3.28 – 3.10 (m, 6H), 2.78 – 2.51 (m, 5H), 2.13 – 1.99 (m, 5H), 1.97 (s,

7

(

1

3

H), 1.83 (s, 3H), 1.69 (t, J = 6 Hz, 2H), 1.69 – 1.37 (m, 4H) ppm. C NMR (101 MHz,

CDCl ) δ 172.2 (C), 171.6 (d, J = 4 Hz, C), 169.3 (C), 168.9 (d, J = 4 Hz, C), 167.6 (C),

3

1

8

1

66.2 (d, J = 8 Hz, C), 164.2 (C), 163.3 (C), 161.8 (d, J = 12 Hz, C), 161.3 (d, J = 12 Hz, C),

60.5 (C), 158.8 (d, J = 12 Hz, C), 146.6 (d, J = 7 Hz, C), 138.5 (C), 137.4 (C), 136.1 (CH),

34.2 (d, J = 7 Hz, C), 132.5 (C), 131.4 (CH), 130.2 (dd, J = 10, 5 Hz, CH), 128.5 (CH),

26.5 (CH), 118.5 (dd, J = 14, 4 Hz, C), 116.7 (CH), 111.9 (dd, J = 21, 4 Hz, CH), 111.3

(CH), 109.9 (C), 103.9 (t, J = 25 Hz, CH), 96.5 (d, J = 9 Hz, CH), 64.5 (d, J = 4 Hz, CH₂),

4

9.3 (CH ), 48.9 (CH), 40.1 (CH ), 39.8 (CH ), 36.9 (CH ), 32.4 (CH ), 31.4 (CH ), 29.0

2

(

CH ), 28.2 (CH ), 26.3 (CH ), 25.8 (CH ), 24.9 (CH ), 22.8 (CH ), 21.6 (CH ), 17.3 (CH )

2 2 2 2 2 2 3 3

-1

ppm. IR (NaCl) ʋ_m_a_x: 3282, 2929, 1702, 1644, 1349, 1192, 1031 cm . HRMS (ESI): calc.

+

for [C H O N BrF ] : 984.28531, found 984.28505.

47

49

8

9

2

3

-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N-(4-(1-(4-((4-((2-

(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-4-oxobutyl)-1H-1,2,3-

triazol-4-yl)butyl)-4-methylbenzamide (NR-7h)

Following the general procedure E starting from 8a and 15a, compound NR-7h was obtained

1

as a bright yellow solid in 78% yield. DSC = 231.6°C (peak), 188.7°C (onset). H NMR (400

MHz, CDCl ) δ 8.86, 8.82 (2s, 1H), 7.78 (d, J = 8 Hz, 1H), 7.60 (td, J = 9, 6 Hz, 1H), 7.53

3

(

bs, 1H), 7.45 (dd, J = 9, 7 Hz, 1H), 7.34 (d, J = 8 Hz, 2H), 7.21 (s, 1H), 7.04 (dd, J = 7, 1 Hz,

H), 7.00 – 6.92 (m, 1H), 6.88 – 6.72 (m, 3H), 6.20 (dd, J = 9, 5 Hz, 1H), 6.16 (s, 1H), 5.22

s, 2H), 4.95 – 4.85 (m, 1H), 4.32 (t, J = 5, 2H), 3.34 – 3.08 (m, 6H), 2.91 – 2.59 (m, 5H),

1

(

1

3

2

1

.19 – 2.02 (m, 8H), 1.90 (s, 3H), 1.73 – 1.47 (m, 8H) ppm. C NMR (101 MHz, CDCl ) δ

3

72.0 (C), 171.8 (C), 171.7 (C), 169.6 (C), 169.1 (C), 167.7 (2 x C), 166.1 (d, J = 7 Hz, C),

F

64.4 (C), 163.5 (C), 161.9 (C), 161.8 (C), 160.6 (d, J = 3 Hz, C), 146.9 (C), 146.8 (C),

F

38.6 (d, J = 2 Hz, C), 137.5 (C), 136.3 (CH), 134.3 (d, J = 4 Hz, C), 132.5 (C), 131.5 (CH),

F

30.3 (dd, J = 10, 4 Hz, CH), 128.7 (CH), 126.7 (CH), 121.9 – 121.2 (m, CH), 118.9 – 118.3

(

m, C), 116.9 (CH), 112.0 (dd, J = 22, 4 Hz, CH), 111.4 (CH), 109.9 (d, J = 3 Hz, C), 104.1

F

(

t, J = 25 Hz, CH), 96.6 (CH), 64.7 (d, J = 4 Hz, CH ), 49.4 (CH ), 49.0 (CH), 42.3 (CH ),

F 2 2 2

3

9.9 (CH ), 39.0 (CH ), 32.6 (CH ), 32.5 (CH ), 31.6 (CH ), 27.0 (CH ), 26.7 (CH ), 26.6

2 2 2 2 2 2 2

(

CH ), 26.1 (CH ), 25.1 (CH ), 22.9 (CH ), 21.7 (CH ), 17.4 (CH ) ppm. IR (NaCl) ʋ_m_a_x:

2 2 2 2 3 3

-1

+

3

9

388, 2925, 1697, 1641, 1354, 1198, 1101 cm . HRMS (ESI): calc. for [C H O N BrF ] :

48 51 8 9 2

98.3007, found 998.2978.

4

.2. Cell culture and treatments.

Experiments were done using the cancer cell lines BBL358, MB-MDA-231, T47D, and

MCF7, HT29, and HeLa, as well as RPE cells, H9c2 cells and BMDMs. MB-MDA-231,

1

9

T47D, MCF7, HeLa, HT29, RPE and H9c2 cells were purchased from ATCC. BBL358 were

generated as described [10] and BMDMs were obtained from bone marrow precursor cells

isolated from femurs and tibias of mice. Once isolated BMDMs were incubated in

conditioned media from the mouse fibroblast cell line L929 (L-cell) for 6 days, and then were

cultured in complete DMEM as described [8]. All cells were cultured in DMEM or DMEM-

F12 according to ATCC recommendations, supplemented with 10% FBS, 1 % L-Glutamine

and 1% penicillin-streptomycin at 37ºC and 5% CO . For passaging, cells were washed in

2

PBS and incubated in 1 ml trypsin at 37ºC until detached. Then, complete media was added,

and cells were diluted as desired depending on the confluence and re-plated in a new culture

dish.

Cells were treated with the indicated concentrations of PROTACs or with 2 µM of

2

PH797804 (Selleckchem #S2726). p38α pathway activation was induced with UV (30 J/m )

or 100 ng/ml LPS (Sigma #L4005) and 50 ng/ml IFNγ (Peprotech #315-05). The proteasome

was inhibited with 100 nM bortezomib (Selleckchem #PS-341). The NEDD8-Activating

Enzyme (NAE) inhibitor MLN4924 (Calbiochem #505477) was used at 1 µM.

4

.3. Protein extraction and immunoblotting.

Cells were lysed in RIPA buffer containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS,

0mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM sodium fluoride, 1 mM

5

PMSF, 1 mM sodium orthovanadate, 2.5 mM benzamidine, 10 µg/ml pepstatin A, 1 µM

mycrocystin, 10 µg/ml leupeptin and 10 µg/ml aprotinin. Cell lysates were maintained for 15

min on ice and then centrifuged at 13000xg at 4°C for 15 min. Supernatants were collected,

separated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting. The

following antibodies were used: p38α (Santa cruz #sc-81621; 1:500), human p38β (Cell

signaling #2339; 1:500), mouse p38β (Thermo Fisher 33-8700, 1:500), p38δ (Cell signaling

#2308; 1:1000), p38γ (Cell signaling #2307; 1:1000), phospho-p38 (Cell signaling #9211,

1:1000); Erk1/2 (Cell signaling #9102; 1:1000), phospho-MK2 ( Cell signaling #3007;

1:500), α-tubulin (Sigma #T9026; 1:10000), and Jnk (Santa cruz #sc-571; 1:2000). Band

intensities were determined using the Odyssey Infrared Imaging System (Li-Cor, Biosciences)

and quantified by ImageJ.

4

.4. SILAC experiment.

2

0

MDA-MB-231 cells were cultured with SILAC medium DMEM F-12 (Invitrogen, USA)

without arginine or lysine, and supplemented with 2% dialyzed fetal bovine serum (dFBS)

(

Invitrogen), 2 g/l glucose (Invitrogen), 0.29 g/l L-glutamine (Invitrogen), 5 mg/l Phenol Red

Invitrogen) and 200 mg/l L-proline (Sigma, USA). “Light” growth medium was additionally

supplemented with 100 mg/l L-lysine (SIGMA), 100 mg/l L-arginine (SIGMA), whereas

heavy” growth medium was supplemented with 100 mg/l heavy lysine ([13C6, 15N2]-L-

lysine (SILANTES, Germany)) and 100 mg/l heavy arginine ([13C6, 15N4]-L-arginine

SILANTES, Germany)). Cells labelled with “light” and “heavy” amino acids were treated

“

(

with DMSO or NR-7h (1 µM), respectively, for 16 h, and then were lysed in 0.1 M Tris-HCl,

pH 7.6, 4% SDS, 0.1 M DTT at RT and briefly sonicated. Samples were digested in FASP

with trypsin overnight at 37ºC following FASP protocol [29]. Digested peptides were diluted

in 3% ACN/1% FA. Sample was loaded to a 300 µm × 5 mm PepMap100, 5 µm, 100 Å, C18

µ-precolumn (Thermo Scientific) at a flow rate of 15 µl/min using a Thermo Scientific

Dionex Ultimate 3000 chromatographic system (Thermo Scientific). Peptides were separated

using a C18 analytical column (nanoEase M/Z HSS C18 T3 100Å, 1.8 μm, 75 µm x 250 mm,

Waters) with a 300 min run with a linear gradient from 3 to 35% B in 267 min (A= 0.1% FA

in water, B= 0.1% FA in CH3CN). Survey MS scans were acquired in the Orbitrap with the

resolution (defined at 200 m/z) set to 120,000. The lock mass was user-defined at 445.12 m/z

in each Orbitrap scan. The top speed (most intense) ions per scan were fragmented by CID

and detected in the Iontrap. An additional event for targeted mass difference was included

(only one of the precursor triplet is fragmented). The ion count target value was 400,000 and

4

000 for the survey scan and for the MS/MS scan respectively. Target ions already selected

for MS/MS were dynamically excluded for 15s. Spray voltage in the NanoMate source was

set to 1.60 kV. RF Lens were tuned to 30%. Minimal signal required to trigger MS to MS/MS

switch was set to 5,000.

MS/MS spectra were searched against the SwissProt (human, release 2019_05) and

contaminants database using MaxQuant v1.6.6.0 with andromeda search engine [30]. Peptide

and fragment mass tolerances were 20 ppm and 0.5 Da respectively. Peptides and proteins

were filtered at a false discovery rate (FDR) of 1 % based on the number of hits against the

reversed sequence database.

Protein intensities were used for the statistical analysis. Within each SILAC

experiment, protein quantitation was normalized to the total intensity for channel and

experiment. Data was first transformed to log scale to apply a linear model. For each

2

1

treatment, 6 samples were analyzed corresponding to three biological replicates with two

technical replicates of each biological sample. We filtered the data to retain only proteins with

valid quantification values in at least 4 out of 6 samples in at least one group. Missing values

were imputed with normally distributed random numbers (centered at -1.8 standard deviations

units and spread 0.3 standard deviations units with respect to non-missing values). To adjust

for batch effect a linear model was used with SILAC batch as fixed effect. Model fitting was

accomplished with the lmFit function of the limma package [31] of R statistical software

(http://www.Rproject.org). Comparison between the two groups (H and L) was done and p-

values were adjusted using the Benjamini & Hochberg correction. Proteins with an adjusted

p-value lower than 0.05 and fold change higher than 2 were considered statistically significant

between groups. Clustering analysis was performed using the Perseus software vs 1.6.3 [32].

The adjusted intensities after removing batch effect were filtered with those proteins

significant in at least one comparison and z-score normalized. Clustering was done by using

multiple iterations of hierarchical clustering.

4

.5. RNA extraction and gene expression analysis.

Total RNA was extracted using PureLink RNA mini kit (Invitrogen) following the

manufacturer’s instructions. Reverse transcription reactions were performed using 500 ng of

total RNA using Superscript IV reverse transcriptase (Invitrogen) following manufacturer’s

instructions for cDNA synthesis. qRT-PCR reactions were performed in triplicates using 5 ng

of cDNA and SYBR Green reagent (Life Technologies #4472954) in a Quant-Studio 6 Flex

Instrument (Life Technologies). The following primers were used:

ACTCAGATGCCGAAGATGGAAC and GTGCTCAGGACTCCATCTCT for p38α,

CCCGGACATATATCCAGTCC and TCACTGCTCAATCTCCAGG for p38β,

GGATTTGGTCGTATTGGG and GGAAGATGGTGATGGGATT for GAPDH. GAPDH

mRNA levels were used for normalization.

Declaration of competing interest

The authors declare no conflicts of interest.

Author Contributions

C. D. designed and synthesized chemical compounds, analyzed data and wrote the

manuscript. C.S.-Z. and X.V. contributed to the design and synthesis of chemical compounds.

2

M.C.-R. and N.G.-P. designed and performed experiments in cells to characterize the activity

of the compounds, analyzed data and wrote the manuscript. A.R. and A.R.N. conceived the

project, provided funding, supervised the overall study, designed experiments, analyzed data,

and wrote the manuscript.

Acknowledgements

We are grateful to Marina Gay, Gianluca Arauz, and Marta Vilaseca for help in the design of

the SILAC experiment and for analysing of the samples by Mass Spectrrometry, and Oskar

Fernandez-Capetillo (CNIO) for inspiring discussions. This work was supported by grants

from FEDER/Ministerio de Ciencia, Innovación y Universidades (MICINN) - Agencia Estatal

de Investigación (SAF2016-81043-R and CTQ2017-87840-P), Agència de Gestió d'Ajuts

Universitaris i de Recerca (2017 SRG-557), and “La Caixa” Foundation (Young BioMedTec).

C.D. thanks “La Caixa” Foundation for a predoctoral fellowship. IRB Barcelona

is the recipient of institutional funding from MICINN through the Centres of Excellence

Severo Ochoa award and from the CERCA Program of the Catalan Government.

Appendix A. Supplementary data

Experimental procedures for the synthesis of PROTACs NR-1-5, NR-7i-j, NR-7h* and NR-

1

6

a*, and for the synthesis of all intermediates and assignations of the H NMR spectra.

Additional figures illustrating the structures and activities of different PROTACs, as well as

1

13

examples of H and C NMR spectra.

Abbreviations used

BMDM, bone-marrow-derived macrophages; DC , 50% of protein degradation; DIC, N,N′-

5

0

diisopropylcarbodiimide, DMEM, dulbecco’s modified eagle medium; DMF,

dimethylformamide, FBS, fetal bovine serum; LPS, lipopolysaccharide; MAPK, mitogen-

activated protein kinase; MS, mass spectrometry; NEt , trimethylamine; Oxyma, ethyl

3

cyanohydroxyiminoacetate, PROTAC, proteolysis targeted chimera; SDS, sodium dodecyl

sulfate; UV, ultraviolet light.

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2

5

Table 1. Structures of the initial PROTACs NR-1a-c and NR-2a-b and their abilities to

induce p38α degradation.

b

Compound

X

Linker Linker

p38α degradation

a

length

connection

BBL358

8

T47D

NR-1a

NR-2a

NR-1b

NR-2b

NR-1c

-(CH

2

)

4

-

4

4’ -NH-

0

-(CH

)

5’ -NHC(O)-

4’ -NH-

40±27

60±15

55±23

82±2

-CH

-(CH

-O-CH

)

2

)

2

)

6

-CH

-

8

0

2

8

5’ -NHC(O)-

4’ -NH-

0

20

a

b

Number of atoms of the linker. Percentage of p38α degradation after treatment of BBL358

cells or T47D cells with the indicated PROTACs at 10 µM for 24 h. Immunoblotting band

intensities were analyzed using Image J and the p38α/Tubulin ratio was calculated and

normalized to non-treated cells. Data are mean ±SEM, n=2 in BBL358 cells, and n=1 in T47D

cells.

2

6

Table 2. Structures of PROTACs NR-3a-d and NR-4 and their abilities to induce p38α

degradation

b

Compound

X

Linker

length

Linker

connection

p38α degradation

a

BBL358

78 ± 4

T47D

NR-3a

NR-3b

NR-3c

NR-3d

NR-4

-CH

2

-(CH

2

-O-CH

2

)

3

-CH

2

-

18

21

20

4’-NH-

5’-NHCO-

26 ± 9

41 ± 16

85 ± 3

20 ± 20

76

92 ± 2

2

4

2

79 ± 1

3 ± 3

0.5

2

a

b

Number of atoms of the linker. Percentage of p38α degradation after treatment of BBL358

cells or T47D cells with the indicated PROTACs at 10 µM for 24 h. Immunoblotting band

intensities were analyzed using Image J and the p38α/Tubulin ratio was calculated and

normalized to non-treated cells. Data are mean ±SEM, n ≥ 2.

2

7

Table 3. Structures of PROTACs NR-5a-b and NR-7a-j and their abilities to induce p38α

degradation

O

N

H

F

X

N

HN

F

X

HN

NH

O

N

HN

N

n'

N

O

n

O

Y

n

O

N

O

N

O

NH

O

Br

NH

O

Br

O

NR-5a-b, Y = CO

NR-6a-b, Y = CH₂

NR-7a-j

b

Compound

X

n

n’

Linker

length

Linker

connection

p38α degradation

a

BBL358

67 ± 17

77 ± 3

93 ± 5

74 ± 3

0

T47D

NR-5a

NR-5b

NR-6a

NR-6b

NR-7a

NR-7b

NR-7c

NR-7d

NR-7e

NR-7f

NR-7g

NR-7h

NR-7i

NR-7j

-(CH

2

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

3

)

2

)

2

-

4

2

3

2

4

3

4

6

5

6

5

2

3

4

2

4

3

4

17

16

17

16

12

13

14

15

16

-CH

2

-

47 ± 32

82 ± 10

87 ± 3

16 ± 13

7 ± 8

4’-NH-

36 ± 20

60 ± 12

40 ± 24

12 ± 10

84 ± 6

74 ± 10

84 ± 2

81 ± 1

0

42 ± 9

68

25 ± 17

40 ± 8

91

87

89 ± 2

93 ± 2

4 ± 3

-CH(NHBoc)-

-CH(NH Cl)-

3

a

b

Number of atoms of the linker. Percentage of p38α degradation after treatment of BBL358

cells or T47D cells with the indicated PROTACs at 10 µM for 24 h. Immunoblotting band

intensities were analyzed by Image J software and the p38α/Tubulin ratio was calculated and

normalized to non-treated cells. Data are mean ±SEM, n ≥ 2.

2

8

Figure Legends

Fig. 1. Molecules used to generate the PROTACs. (a) Diagrammatic representation of the key

interactions of PH-797804 within the ATP binding site of p38α, as reported by Xing et al.

[

19]. (b) Formulas of thalidomide, marketed drugs Pomalidomide and Lenalidomide, and a

carboxylic acid analogue.

Fig. 2. Synthesis of PROTACs of the general structure NR-3 and NR-4. Reaction conditions.

General procedure D (GP-D): R-NH , Oxyma (ethyl cyanohydroxyiminoacetate), DIC (N,N′-

2

Diisopropylcarbodiimide), NEt , DMF. General procedure E (GP-E): CuSO , Sodium

3

4

ascorbate, t-BuOH/H O 35ºC.

2

Fig. 3. Synthesis of PROTACs of the general structure NR-5, NR-6 and NR-7. Reaction

conditions. General procedure D (GP-D): 4-azidobutanoic acid (11), Oxyma (ethyl

cyanohydroxyiminoacetate), DIC (N,N′-Diisopropylcarbodiimide), NEt , DMF. General

3

procedure E (GP-E): CuSO , Sodium ascorbate, t-BuOH/H O 35ºC.

4

2

Fig. 4. Determination of the DC for p38α degradation of PROTACs NR-6a and NR-7h. (a)

5

0

Chemical structures of the most active PROTACs NR-6a and NR-7h. (b) T47D cells and

MB-MDA-231 cells were treated with the indicated concentrations of the PROTACs NR-6a

or NR-7h for 24 h, and cell lysates were analyzed by immunoblotting. Band intensities were

quantified using Image J and the p38α/Tubulin ratio was calculated and normalized to non-

treated cells. DC values were determined in biological duplicates using the GraphPad Prism

5

0

software 6.01. The DC values for NR-7h were 24 + 1.3 nM in T47D cells and 27.2 + 2.7

5

0

nM in MB-MDA-231 cells, while for NR-6a were 2.9 + 1.3 nM in T47D cells and 18.4 +

.6 nM in MB-MDA-231 cells.

5

Fig. 5. N-methylation of the imide group impairs the PROTAC’s activity. MB-MDA-231 and

T47D cells were treated for 24 h with 1 µM of the PROTACs NR-7h or NR-6a or inactive

derivatives with the imide group modified by N-methylation (NR-7h* and NR-6a*), and then

p38α protein levels were analyzed by immunoblotting.

Fig. 6. Structures of compounds NR-7i designed to have a protected functionality as

derivatization point and NR-7j designed to improve solubility.

Article Doi

Optimal linker length for small molecule PROTACs that selectively target p38α and p38β for degradation

DOI: 10.1016/j.ejmech.2020.112451

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2020.112451

Full text of DOI:10.1016/j.ejmech.2020.112451

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