DAZDOTUFTIDE

• TRS-01
• CAS 2522933-44-2
• 4-((E)-(5-(2-(2-((S)-2-((S)-1-(L-Threonyl-L-lysyl)pyrrolidine-2-carboxamido)-5-guanidinopentanamido)acetamido)-2-carboxyethyl)-2-hydroxyphenyl)diazenyl)phenyl (2-(trimethylammonio)ethyl) phosphate
• L-Tyrosine, L-threonyl-L-lysyl-L-prolyl-L-arginylglycyl-3-((1E)-2-(4-((hydroxy(2-(trimethylammonio)ethoxy)phosphinyl)oxy)phenyl)diazenyl)-, inner salt
• [4-[[5-[(2S)-2-[[2-[[(2S)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-2-carboxyethyl]-2-hydroxyphenyl]diazenyl]phenyl] 2-(trimethylazaniumyl)ethyl phosphate

C₄₃H₆₈N₁₃O₁₃P

1006.1 g/mol

L-Tyrosine, L-threonyl-L-lysyl-L-prolyl-L-arginylglycyl-3-[(1E)-2-[4-[[hydroxy[2-(trimethylammonio)ethoxy]phosphinyl]oxy]phenyl]diazenyl]-, inner salt

L-threonyl-L-lysyl-L-prolyl-L-arginylglycyl-3-{(E)-[4- ({oxido[2-(trimethylazaniumyl)ethoxy] phosphoryl}oxy)phenyl]diazenyl}-L-tyrosine

SQ

1

TKPRGY

Protein/Peptide Sequence, Sequence Length: 6

modified (modifications unspecified)

• OriginatorTarsius Pharma
• DeveloperTarsier Pharma
• ClassAnti-inflammatories; Eye disorder therapies; Small molecules
• Mechanism of ActionImmunomodulators
• Orphan Drug StatusYes – Uveitis

• Phase IIIUveitis
• Phase I/IIOcular inflammation
• PreclinicalDiabetic macular oedema; Diabetic retinopathy; Dry age-related macular degeneration

• 16 Jan 2024Tarsier Pharma receives an agreement from the US FDA under Special Protocol Assessment for Tarsier-04 phase III trial for TR S01 eye drops for Uveitis
• 13 Nov 2023Tarsier Pharma announces successful outcome of a Type C meeting with the US FDA supporting the advancement of TRS 01 eye drop for Uveitis
• 13 Nov 2023Tarsier Pharma plans a Tarsier-04 phase III registrational trial of TR S01 for Uveitis in USA

Molecular Formula	C43H68N13O13P.C2HF3O2
Molecular Weight	1120.0764

Ocular inflammation, an inflammation of any part of the eye, is one of the most common ocular diseases. Ocular inflammation refers to a wide range of inflammatory disease of the eye, one of them is uveitis. These diseases are prevalent in all age groups and may be associated with systemic diseases such as Crohn’s disease, Behcet disease, Juvenile idiopathic arthritis and others. The inflammation can also be associated with other common eye symptoms such as dry eye and dry macular degeneration. Several drugs have the known side effect of causing uveitis and/or dry eye. The most common treatment for ocular inflammation, is steroids and specifically corticosteroids. However, these treatments have several known and sometimes severe side effects.

Phosphorylcholine (PC) is a small zwitterionic molecule secreted by helminths which permits helminths to survive in the host inducing a situation of immune tolerance as well as on the surface of some bacteria and apoptotic cells. Tuftsin-PhosphorylCholine (TRS) is bi-specific small molecule with immunomodulatory activities. TRS (Thr-Lys-Pro-Arg-Gly-Tyr-PC) is an immunomodulating peptide derivative.

Currently, TRS has been synthesized by post-synthesis modification of Thr-Lys-Pro-Arg-Gly-Tyr, so as to couple the PC moiety to the phenol ring of tyrosine. However, this synthetic approach results in very low yield, thus making the synthesis of TRS ineffective and costly. New simple and efficient methods of synthesizing TRS are highly required.

SCHEME

PATENT

WO2022224259

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2022224259&_cid=P11-MAOYY3-78105-1

EXAMPLES

EXAMPLE 1

CONJUGATION OF PHOSPHORYLCHOLINE TO BOC-TYR

[0151] 1) Preparation of diazonium salt

[0152] 4-Aminophenyl (2-(trimethylammonio)ethyl) phosphate (50 mg, 0.18 mmol)) was dissolved in 1M aqueous HC1 (1 mL), cooled in an ice-water bath and sodium nitrite (12.6 mg, 0.18 mmol) was added in a single batch. The resulting solution was stirred at 0°C for 30 min.

[0153] 2) Azo coupling

[0154] A new mixture was prepared with BOC-L-tyrosine (107 mg, 0.38 mmol) in NaHC03(lM)+NaOH buffer (pH 10) (3.3 mL) + acetonitrile (1.2 mL). The mixture was cooled in an ice-water bath. The diazonium salt mixture was added drop-wise. A red solution was formed. Stirring of this was continued at 0 °C for 6 minutes. The reaction mixture was acidified with IN aqueous HC1 to pH=~3.

[0155] The obtained solution was lyophilized overnight, and subsequently purified (e.g. by preparative MPLC), to obtain the compound:

, wherein R is Boc.

EXAMPLE 2

PREPARATION OF AN EXEMPLARY COMPOUND OF THE INVENITON

Preparation of diazonium salt:

Fmoc-Tyr-PPC

(compound 10)

[0156] 4-Aminophenyl (2-(trimethylammonio)ethyl) phosphate (250 mg, 0.912 mmol)) was dissolved in 1M aqueous HC1 (5 mL), cooled in an ice-water bath and sodium nitrite (62.9 mg, 0.912 mmol) was added in a single batch. The resulting solution was stirred at 0°C for 30 min. Azo coupling, a new mixture was prepared with Fmoc-Tyr-OH (739 mg, 1.832 mmol) in saturated NaHC03 (17 mL) + acetonitrile (12.5 mL). The resulting suspension/solution was cooled in an ice-water bath. The diazonium salt mixture was added drop-wise. Stirred at 0°C. The reaction mixture slowly turned yellow. After 5.5 h LCMS showed complete conversion. The reaction mixture was acidified with IN HC1 to pH~6, the yellowish suspension turned into a clear orange solution, which was lyophilized. This afforded 2.10 g. Dissolved in a mixture of DMSO/H20/MeCN (-1:1:1) and purified in 5 runs by acidic preparative MPLC. The fractions were combined and lyophilized overnight, to obtain the desired product (compound 10).

EXAMPLE 3

SPPS SYNTHESIS OF TRS

[0157] While facing difficulties with protection of the hydroxy group of compound 10, the inventors explored a novel strategy for SPPS synthesis of TRS :

[0158] The inventors initiated the SPPS synthesis by implementing the N-protected (Fmoc) phosphorylcholine modified tyrosine (e.g. compound 10) 200 mg of compound 10 were loaded onto the CTC resin. In brief, 2-Chlorotrityl chloride resin (1.0 – 1.2 mmol/g, 200 – 400 mesh) (450 mg, 1.441 mmol) was allowed to swell in dichloromethane (12 mL) by rocking for 30 min. The solvent was removed and a solution of (S,E)-4-((5-(2-((((9//-f1uoren-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)-2-hydroxyphenyl)diazenyl)phenyl(2-(trimethylammonio)-ethyl) phosphate (200 mg, 0.290 mmol) in dichloromethane (12 mL) containing DIPEA (0.177 mL, 1.016 mmol) (substrate did not dissolve in DCM, after addition of DIPEA a solution was obtained) was added.

[0159] After 17 h the solvent was removed and the resin was washed with dichloromethane (3×10 mL, each washing step > 2 minutes). The capping solution (CH2C12:MeOH: DIPEA 9: 1:0.5) was added (10.5 mL) and the resin was rocked for 1 hour. Then the resin was washed with dichloromethane (3×10 mL) and dried in vacuo.

[0160] This resin was then split into equal portions in order to investigate a number of conditions for the subsequent chemistry in parallel, aimed at preventing the formation of the previously found tyrosine O-acylation, as witnessed by the isolation of compound 13 (see Scheme 2). The different reaction conditions were outlined in Table 1 (see below).

Scheme 2: Solid phase peptide synthesis

Table 1: exemplary coupling conditions tested

[0161] As shown in Table 1, various coupling conditions have been tested. Entries a-c resulted in the formation of a substantial amount of the byproduct (13). An improvement was obtained by using Fmoc-Gly-OSu in DMF (entry d). In this case the formation of byproduct (13) was reduced to only 3% relative to the desired compound 12. Nonetheless, neither of these methods was capable of suppressing the formation of 13 completely, therewith still posing a risk for further peptide synthesis, as this may lead to the accumulation of byproducts (compound 13).

[0162] Surprisingly, the inventors found that the byproduct (or phenolic ester byproduct, represented by compound 13 in Scheme 3) can be cleaved under standard Fmoc deprotection conditions with piperidine or with DBU in DMF, affording compound 15 cleanly, as illustrated below:

/////////DAZDOTUFTIDE, PHASE 3, TRS-01, TRS 01