Research

Introduction

Research Question

How does the presence of a PEG-linker change both the TLR7/8 activity and P-gp efflux in sulfonamide-conjugated imidazoquinolines?

A drug goes into a cancer cell. Next, an activated form of the drug is expelled from the cancer cell. Finally, this activated drug activates the immune system.

Figure 1. Overview of Bystander-Assisted Immunotherapy (BAIT)

MDR, Efflux, and TLR7/8 Agonism

Multi-drug-resistant (MDR) cancers have the ability to efflux a wide array of drug molecules out from its cancer cells. They use various efflux pumps like P-glycoprotein (P-gp) to achieve this(1) .

In the tumor microenvironment surrounding each of these cells, there are inert white blood cells, which can be activated through the NFκB pathway, which starts with the activation of Toll-like receptors (TLR), a family of receptor proteins(2) .

The Mancini Group is combining these two phenomena in a process they call bystander-assisted immunotherapy (BAIT). In this strategy, drugs are transformed in a cancer cell, then effluxed out to ultimately activate these nearby, inactive immune cells (Figure 1)(3).

The Mancini Group has previously synthesized various molecules that are substrates for both P-gp and TLR7/8. Using sulfonamides as the P-gp affinity fragment showed high efflux success(3).

PEG-Chain Linkers

Polyethylene glycol (PEG) is an organic polymer frequently used in biomolecule conjugation due to its low steric hindrance, its adjustable length, and its low price (4).

Adding a linker to TLR7/8 agonists has been shown to increase immune cell activation(5).

For our project, we are combining this principle with the high success of the sulfonamides by using a chain linker with a sulfonamide as the P-gp affinity fragment (Figure 2).

Currently, we are using a tosyl group as our R-group due to its low cost and poor TLR7/8 activation without a linker(3).

An imidazoquinoline derivative with a sulfonamide component on the left. On the right, there is a chain of poly ethylene glycol inserted between the imidazoquinoline and the sulfonamide fragment.

Figure 2. PEG Attachment Site(a) Mancini Group TLR7/8 Agonistst(3) (b) Mancini Group TLR7/7 Agonist with Linker

Methods

Methods 1 + 2

Mono-Tosylation and Di-Tosylation

Mono-Tosylation: Dry TEA (1.6 Eq) and the selected PEG (1 Eq) was stirred for 5 min. Then, TsCl (1.2 Eq, dissolved into dry DCM) was added into solution. This reaction mixture was then stirred for 2.5 hr at 0°C. The residue was chromatographed with DCM/MeOH(6).

Di-Tosylation: Same procedure as “For Mono-Tosylation”. However, the dry TEA was added at 2.2 Eq and the TsCl was added at 2 Eq Also, the reaction was instead stirred overnight at RT.

Methods 3 + 4

Azidation and Azide Reduction (Pathway For Amination)

Azidation: A solution of the tosylated product (1 Eq) and ACN was stirred for 5 min. NaN3 (1 Eq, dissolved in H2O) was added. The mixture was refluxed at 100° C for 36 hr. The residue was extracted with CHCl3. The organic layer was then dried (MgSO4), chromatographed, and eluted with DCM/EtOAc(6).

Azide Reduction: To a solution of the azidation product (1 eq), H2O (1.5 eq), and THF, P(Ph)3 (1 Eq) was added. The reaction was stirred for 4 hr. at RT. The residue was chromatographed with DCM/MeOH(6).

Method 5

Ammonium Bubbling (Alternative Pathway For Amination)

A reaction of NaOH (50 eq) and (NH4)2SO4 (25 Eq) generated NH3 (g), which flowed into a solution of the tosylated PEG in dry DCM (Figure 3). The reaction was stirred for 1 hr at RT. The residue was chromatographed with DCM/MeOH.

An erlenmeyer flask has a tube connected to a three-neck flask. The three-neck is covered in foil.

Figure 3. Ammonium Bubbling Setup

Results

Reaction Optimization

Method 1 provided acceptable yield and easy separation, but it was a significant challenge to prevent ditosylation of the PEG. The reaction scheme was subsequently altered to use the ditosylated product in later steps, which resulted in Method 2.

Method 3 proved to be difficult when attempting to aminate the monotosylated product or only one side of the ditosylated product. The ammonium gas was produced too quickly when performing the reaction. The yield was extremely low and prevented further reactions with the isolated product.

Method 4 had acceptable yield but Method 5 had a diminished yield and proved difficult when separating the products which led to our current reaction scheme (Figure 5).

Combining the di-tosylated product of Method 2 with para-toluene sulfonamide will avoid unnecessary and difficult steps and achieve the same end goal.

Figure 4. Ditosylated product of Method 2 performed on Diethylene glycol.

A synthetic reaction scheme with 3 reaction steps. It starts with a diethylene glycol molecule. Both hydroxyls get tosylated. Next, it is reacted with a sulfonamine. This product is reacted with an imidazoquinoline derivative to create the final product.

Figure 5. Current Synthetic Scheme

Step (i) TsCl, TEA, dry DCM (ii) TEA, dry DCM (iii) DMAP, TEA, dry DCM, Ar (g)(3)

Attempted Compounds (Before Optimization)

Method 1:

Outcome: Low yield, difficult to prevent di-tosylation

A polyethylene glycol molecule with a tosyl group in the place of one of the original hydroxyl groups.

Figure 6. Mono-tosylated Product. n = 1, 2, 3

Method 2:

Outcome: Acceptable yield, inexpensive reagents, good atom economy, easy separation

A polyethylene glycol molecule with tosyl groups in the place of both of the original hydroxyl groups.

Figure 7. Di-tosylated Product. n = 1, 2

Method 3:

Outcome: Difficult to aminate one side of Method 2 product, gas production occurs in 10 - 30 min, low yield for further reactions

A diethylene glycol molecule with an amine in the place of one of its original hydroxyl groups.

Figure 8. Amination of mono-tosylated Product.

A diethylene glycol molecule with an amine in the place of one of its original hydroxyl groups and a tosyl group in the place of the other original hydroxyl group.

Figure 9. Amination of di-tosylated Product.

Method 4:

Outcome: Acceptable yield, long reflux time. Difficult to dissolve NaN3

A diethylene glycol molecule with an azide in the place of one of its original hydroxyl groups and a tosyl group in the place of the other original hydroxyl group.

Figure 10. Azidation of Figure 9 product

Method 5:

Outcome: Very low yield, difficult to separate products, resulted in unintended products

Figure 11. Reduction of Figure 10 product

Current Scheme Progress

DiDEG-1 (Figure 12)

Di-tosylated diethylene glycol (Method 2)
Produces a fine, white powder (Figure 4)
Structure confirmed by 1H (Figure 13), 13 C (Figure 14), and COSEY

A diethylene glycol molecule with tosyl groups in the place of both of the original hydroxyl groups.

Figure 12. Structure of DiDEG-1, a di-tosylated diethylene glycol

A Hydrogen-1 NMR Spectra of DiDEG-1. An aliphatic singlet, two aliphatic triplets, two aromatic doublets.

Figure 13. 1H NMR of DiDEG-1δ 2.47 (s), 3.62 (t), 4.10 (t), 7.37 (d), 7.80 (d)

A Carbon-13 NMR Spectra of DiDEG-1. 3 aliphatic peaks and 4 aromatic peaks.

Figure 14. 13C NMR of DiDEG-1δ 21.7, 68.8, 68.9, 127.9, 129.9, 132.9, 144.9

Conclusions

We had hypothesized that both the TLR7/8 activity and the P-gp efflux would increase with an increasing PEG chain length. However, we are unable to accept or reject it, since we are currently optimizing the synthetic steps in achieving our final product.

In optimizing the synthetic steps toward our final product, we have settled on a more efficient and productive reaction scheme.

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