.HCl

Melphalan flufenamide hydrochloride

メルファランフルフェナミド塩酸塩;

L-Phenylalanine, 4-[bis(2-chloroethyl)amino]-L-phenylalanyl-4-fluoro-, ethyl ester, hydrochloride

FDA APPROVED PEPAXTO, 2021/2/26

• Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate
• J 1
• J 1 (prodrug)
• L-Melphalanyl-L-p-fluorophenylalanine ethyl ester
• Melflufen

• Melphalan flufenamide
• Pepaxto
• Prodrug J 1

Melflufen

• Molecular FormulaC₂₄H₃₀Cl₂FN₃O₃
• Average mass498.418 Da
• SP ROT +33.0 ° Conc: 1.3 g/100mL; chloroform ; 589.3 nm, Oncology Research 2003, V14(3), P113-132 

мелфалана флуфенамид [Russian] [INN]ميلفالان فلوفيناميد [Arabic] [INN]氟美法仑 [Chinese] [INN]380449-51-4[RN]
9493Ethyl 4-[bis(2-chloroethyl)amino]-L-phenylalanyl-4-fluoro-L-phenylalaninate
F70C5K4786L-Phenylalanine, 4-[bis(2-chloroethyl)amino]-L-phenylalanyl-4-fluoro-, ethyl ester

Melphalan flufenamide, sold under the brand name Pepaxto, is an anticancer medication used to treat multiple myeloma.^[3][4]

The most common adverse reactions include fatigue, nausea, diarrhea, pyrexia and respiratory tract infection.^[3]

Melphalan flufenamide is a peptidase enhanced cytotoxic (PEnC) that exerts a targeted delivery of melphalan in cells with high expression of aminopeptidases, such as aminopeptidase N, which has been described as over-expressed in human malignancies.Aminopeptidase N plays a functional role in malignant angiogenesis.

Melphalan flufenamide was approved for medical use in the United States in February 2021.^[4][5]

Medical uses

Melphalan flufenamide is indicated in combination with dexamethasone for the treatment of adults with relapsed or refractory multiple myeloma, with relapsed or refractory multiple myeloma who have received at least four prior lines of therapy and whose disease is refractory to at least one proteasome inhibitor, one immunomodulatory agent, and one CD-38 directed monoclonal antibody.^[3][4]

Metabolism

Melphalan flufenamide is metabolized by aminopeptidase hydrolysis and by spontaneous hydrolysis on N-mustard.^[6] Its biological half-life is 10 minutes in vitro.

Origin and development

Melphalan flufenamide is a peptidase enhanced cytotoxic (PEnC) with a targeted delivery within tumor cells of melphalan, a widely used classical chemotherapeutic belonging to a group of alkylating agents developed more than 50 years ago. Substantial clinical experience has been accumulated about melphalan since then. Numerous derivatives of melphalan, designed to increase the activity or selectivity, have been developed and investigated in vitro or in animal models.^[7] Melphalan flufenamide was synthesized, partly due to previous experience of an alkylating peptide cocktail named Peptichemio^[8] and its anti-tumor activity is being investigated.

Pharmacology

Compared to melphalan, melphalan flufenamide exhibits significantly higher in vitro and in vivo activity in several models of human cancer.^{[9][10][11][12][13][14][15][16]} A preclinical study, performed at Dana–Farber Cancer Institute, demonstrated that melphalan flufenamide induced apoptosis in multiple myeloma cell lines, even those resistant to conventional treatment (including melphalan).^[17] In vivo effects in xenografted animals were also observed, and the results confirmed by M Chesi and co-workers – in a unique genetically engineered mouse model of multiple myeloma – are believed to be predictive of clinical efficacy.^[18]

Structure

Chemically, the drug is best described as the ethyl ester of a dipeptide consisting of melphalan and the amino acid derivative para-fluoro-L-phenylalanine.

Pharmacokinetics

Pharmacokinetic analysis of plasma samples showed a rapid formation of melphalan; concentrations generally exceeded those of melphalan flufenamide during ongoing infusion. Melphalan flufenamide rapidly disappeared from plasma after infusion, while melphalan typically peaked a few minutes after the end of infusion. This suggests that melphalan flufenamide is rapidly and widely distributed to extravasal tissues, in which melphalan is formed and thereafter redistributed to plasma.^[19]

This rapid disappearance from plasma is likely due to hydrolytic enzymes.^[20] The Zn(2+) dependent ectopeptidase (also known as alanine aminopeptidase), degrades proteins and peptides with a N-terminal neutral amino acid. Aminopeptidase N is frequently overexpressed in tumors and has been associated with the growth of different human cancers suggesting it as a suitable target for anti-cancerous therapy.^[21]

Adverse effects

In a human Phase 1 trial, no dose-limiting toxicities (DLTs) were observed at lower doses. At doses above 50 mg, reversible neutropenias and thrombocytopenias were observed, and particularly evident in heavily pretreated patients.^[22] These side-effects are shared by most chemotherapies, including alkylating agents in general.

Drug interactions

No drug interaction studies have been reported. Several in vitro studies indicate that melphalan flufenamide may be successfully combined with standard chemotherapy or targeted agents.^[23][24]

Therapeutic efficacy

In a Phase 1/2 trial, in solid tumor patients refractory to standard therapy, response evaluation showed disease stabilization in a majority of patients.^[25][26] In relapsed and refractory multiple-myeloma (RRMM) patients, promising activity was seen in heavily pre-treated RRMM patients where conventional therapies had failed; the median Progression-Free Survival was 9.4 months and the Duration of Response was 9.6 months.^[27] An overall response rate of 41% and a clinical benefit rate of 56% were also shown, with similar results seen across patient populations regardless of their refractory status. Hematologic toxicity was common, but manageable with cycle prolongations, dose modifications and supportive therapy, and non-hematologic treatment-related adverse events were infrequent.

History

Efficacy was evaluated in HORIZON (NCT02963493), a multicenter, single-arm trial.^[3] Eligible patients were required to have relapsed refractory multiple myeloma.^[3] Patients received melphalan flufenamide 40 mg intravenously on day 1 and dexamethasone 40 mg orally (20 mg for patients ≥75 years of age) on day 1, 8, 15 and 22 of each 28-day cycle until disease progression or unacceptable toxicity.^[3] Efficacy was evaluated in a subpopulation of 97 patients who received four or more prior lines of therapy and were refractory to at least one proteasome inhibitor, one immunomodulatory agent, and a CD38-directed antibody.^[3]

The application for melphalan flufenamide was granted priority review and orphan drug designations.^[3]

Society and culture

Names

Melphalan flufenamide is the International nonproprietary name (INN).^[28]

PAPER

 Organic Process Research & Development (2019), 23(6), 1191-1196.

https://pubs.acs.org/doi/pdf/10.1021/bk-2020-1369.ch005

Ethyl (2S)-2-[(2S)-2-amino-3-[bis-(2-chloroethyl)amino]phenyl]propaneamido]-3-(4-fluorophenyl)propanoate hydrochloride, (melphalan flufenamide or Melflufen), is an alkylating agent intended for the treatment of multiple myeloma. Initially only milligram quantities were synthesized, following a route starting from pharmaceutical-grade melphalan. Along with the pharmaceutical development, adjustments were made to the original medicinal chemistry route. This resulted in material for early clinical trials, but it became obvious that further development was necessary. Development resulted in a route in which two phenyl alanine derivatives were coupled to give a dipeptide. This intermediate was further manipulated to give an aniline which could be converted into the desired compound melflufen. The aniline derivative was converted to the corresponding N,N–bis-chloroethylaniline using chloroacetic acid and borane. Deprotection and conversion to the hydrochloride gave melflufen in good yield and excellent purity. Production was performed without chromatography at multi-kilogram scale to supply the API for Phase III studies and commercial validation batches.

PAPER

Antineoplastics

R.S. Vardanyan, V.J. Hruby, in Synthesis of Essential Drugs, 2006

Melphalan

Melphalan, l-3-[p-[bis-(2-chloroethyl)amino]phenyl]alanine (30.2.1.13), is a structural analog of chlorambucil in which the butyric acid fragment is replaced with an aminoacid fragment, alanine. This drug is synthesized from l-phenylalanine, the nitration of which with nitric acid gives 4-nitro-l-phenylalanine (30.2.1.8). Reacting this with an ethanol in the presence of hydrogen chloride gives the hydrochloride of 4-nitro-l-phenylalanine ethyl ester (30.2.1.9), the amino group of which is protected by changing it to phthalamide by a reaction with succinic anhydride to give 30.2.1.10. The nitro group in this molecule is reduced to an amino group using palladium on calcium carbonate as a catalyst. The resulting aromatic amine (30.2.1.11) is then reacted with ethylene oxide, which forms a bis-(2-hydroxyethyl)-amino derivative (30.2.1.12). The hydroxy groups in this molecule are replaced with chlorine atoms upon reaction with thionyl chloride, after which treatment with hydrochloric acid removes the phthalamide protection, giving melphalan (30.2.13) [47–50].

Melaphalan is used intravenously and orally to treat multiple myeloma and cancers of the breast, neck, and ovaries. A synonym of this drug is alkeran.

The racemic form of this drug, d,l-3-[p-[bis-(2-chloroethyl)amino]phenyl]alanine, is also widely used under the name sarcolysine or racemelfalan.
PATENT WO 2001096367PAPEROncology Research (2003), 14(3), 113-132PATENTWO 2016180740https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016180740

Alkylating agents, such as drugs derived from nitrogen mustard, that is bis(2-chloroethyl)amine derivatives, are used as chemotherapeutic drugs in the treatment of a wide variety of cancers. Melphalan, or p-bis-(2-chloroethyl)-amino-L-phenylalanine (compound (Id), CAS No. 148-82-3), is an alkylating agent which is a conjugate of nitrogen mustard and the amino acid phenylalanine (US 3,032,584). Melphalan is used clinically in the treatment of metastatic melanomas, but has limited efficacy, dose-limiting toxicities and resistance can develop.

Melphalan flufenamide ethyl ester (L-melphalanyl-L-p-fluorophenylalanine ethyl ester, melflufen, compound (Ib)) is a derivative of melphalan conjugated to the amino acid phenylalanine, creating a dipeptide (WO 01/96367):

The monohydrochloride salt of melflufen (L-melphalanyl-L-p-fluorophenylalanine ethyl ester monohydrochloride; hydrochloride salt of (Ib); CAS No. 380449-54-7) is referred to as melflufen hydrochloride.

When studied in cultures of human tumor cells representing approximately 20 different diagnoses of human cancers, including myeloma, melflufen showed 50- to 100-fold higher potency compared with that of melphalan (http://www.oncopeptides.se/products/melflufen/ accessed 26 March 2015). Data disclosed in Arghya, et al, abstract 2086 “A Novel Alkylating Agent Melphalan Flufenamide Ethyl Ester Induces an Irreversible DNA Damage in Multiple Myeloma Cells” (2014) 5^th ASH Annual Meeting and Exposition, suggest that melflufen triggers a rapid, robust and irreversible DNA damage, which may account for its ability to overcome melphalan-resistance in multiple myeloma cells. Melflufen is currently undergoing phase I/IIa clinical trials in multiple myeloma.

A process for preparing melflufen in hydrochloride salt form is described in WO 01/96367, and is illustrated in Scheme 1, below. In that process N-tert-butoxycarbonyl-L-melphalan is reacted with p-fluorophenylalanine ethyl ester to give N-tert-butoxycarbonyl-L-melphalanyl-L-p-fluorophenylalanine ethyl ester. After purification by gradient column chromatography the yield of that step is 43%.

Scheme 1. Current route to melflufen (in hydrochloride salt form)

As shown in Scheme 1, the known process for preparing melflufen (in hydrochloride salt form) uses the cytotoxic agent melphalan as a starting material, and melflufen is synthesised in a multistep sequence. Melphalan is highly toxic, thus the staring materials and all of the intermediates, and also the waste stream generated, are extremely toxic. That is a major disadvantage in terms of safety, environmental impact and cost when using the process on a large scale. Therefore, an improved and safer method is highly desired, especially for production of melflufen on a large scale. Further, the purity of commercially available melphalan is poor due to its poor stability, the yield in each step of the process is poor, and purity of the final product made by the known process is not high.

A process for preparing melphalan is described in WO 2014/141294. In WO 2014/141294 the step to introduce the bis(2-chloroethyl) group into the molecule comprises conversion of a primary phenyl amine to a tertiary phenyl amine diol, by reaction with ethylene oxide gas. This gives a 52.6% yield. The amine diol is then converted to a bis(2-chloroethyl) phenylamine by reaction with phosphoryl chloride. Using ethylene oxide, or chloroethanol, to convert an aromatic amine to the corresponding bis-(2-hydroxy ethyl) amine, followed by

chlorination of that intermediate, is a common technique for producing aromatic bis-(2-chloroethyl) amines. It is also known to start from a chloroarene and let it undergo a SNAr-reaction with diethanolamine. The present inventors have applied those methods to produce melflufen (in its salt form), shown in Scheme 2 below.

Scheme 2. Alternative pathways to melflufen

The inventors have found that using ethylene oxide in THF (route (a) of Scheme 2), no alkylation occurs at 55 °C; increasing the temperature to 60 °C lead to the dialkylated intermediate being formed, but the reaction was very slow. To increase yield and reaction rate the reaction would require high temperatures, but this would cause increased pressure so that the reaction would need be performed in a pressure reactor. Such conditions are likely lead to formation of side products. Similar reaction conditions but using a 50:50 mixture of ethylene oxide and acetic acid (route (b) of Scheme 2) lead to faster reaction times but formation of side products. Using potassium carbonate and chloroethanol (route (c) of Scheme 2) also lead to formation of side product, possibly due to the chloroethanol undergoing partial trans-esterification with the ethyl ester.

The inventors also attempted chlorination of the di-alkylated compound. Chlorination of the bis-(2-hydroxyethyl) compound (4) of Scheme 2 using thionyl chloride in dichloromethane led to significant de-protected side product formation. Chlorination of the bis-(2-hydroxyethyl) compound (4) of Scheme 2 using POCl₃ required high temperature and long

reaction times. In addition, both thionyl chloride and POCl₃ are challenging to handle at large scale due to safety concerns. The inventors also converted the bis-(2-hydroxyethyl) compound (4) of Scheme 2 to the corresponding dimesylate by treatment with methanesulfonyl chloride and triethylamine. The dimesylate was treated then with sodium chloride in DMF at 120 °C. However, the crude product of this reaction contained significant side products making this route unsuitable to be used economically at scale.

In summary, none of these routes were found to be suitable for large scale production of high purity melflufen. They do not work well for the synthesis of melflufen, resulting in poor yields and are inefficient. Further, the routes shown in Scheme 2 require multiple steps to form the N, N-bis-chloroethyl amine and use toxic reagents.

Example 1 – Synthesis of compound (VIc)

To a reactor with overhead stirring, equipped with nitrogen inlet and reflux condenser, was charged Boc-nitrophenylalanine (compound (IVc)) (35.0 g, 112.8 mmol, 1 eq.), followed by acetone (420 mL), N-methylmorpholine (43.4 mL, 394.8 mmol, 3.5 eq.), fluoro-L-phenylalanine ethyl ester hydrochloride (compound (V)) (28.5 g, 115 mmol, 1.02 eq.), EDC (23.8 g, 124.1 mmol, 1.1 eq.) and HOBt·H₂O (1.7 g, 11.3 mmol, 0.1 eq.). The slurry was stirred at room temperature for 18.5 h which led to full consumption of compound (IVc) according to HPLC. Water (180 mL) and 2-MeTHF (965 mL) were charged. Approximately 640 g solvent was then removed by evaporation (T_J: 35 °C) from the clear two phase orange mixture. 360 mL 2-MeTHF was then added and evaporated off twice. The water phase was acidified to pH 3 via addition of 58 mL 2 M sulfuric acid. The organic layer was heated to 35-40 °C and was then sequentially washed with water (90 mL), twice with saturated aqueous NaHCO₃ solution (90 mL) and then brine (90 mL) and finally water (90 mL). To the 2-MeTHF dissolved product was added heptane (270 mL) drop wise at 35-40 °C before the mixture was allowed to reach room temperature overnight with stirring. Another 135 mL heptane was added drop wise before the beige slurry was cooled to 10 °C. The product was isolated and was rinsed with 100 mL cold 2-MeTHF/heptane 6/4. Product compound (VIc) was stored moist (82.5 g). A small sample of the product was analyzed by limit of detection (LOD) which revealed the solid to contain 43.8% solvent residues. Based on this, the purified product was obtained in a yield of 82 %. The purity was determined by HPLC to be: 99.4 area%.

¹ H-NMR (300 MHz, DMSO-D₆) δ 8.48 (broad d, 1H, J=7.5 Hz), 8.16 (2H, d, J=8.7 Hz), 7.55 (2H, d, J=9 Hz), 7.28 (2H, dd, J=8,7, 8.1 Hz), 7.12-7.02 (3H, m), 4.49 (1H, dd, J=14.4, 7.2 Hz), 4.32-4.24 (1 H, m), 4.04 (2H, dd, J=14.4, 7.2 Hz), 3.08-2.95 (3H,m), 2.84 (1H, dd, J=13.2, 10.8 Hz), 1.27 (s, 9H), 1.11 (3H, t, J=7.2Hz)

¹³C-NMR (75 MHz, DMSO-D₆) δ 171.4 (C=O), 171.2 (C=O), 161.2 (C-F, d, J=242.3 Hz), 155.2 (C=O), 146.6 (C), 146.2 (C), 133.1 (C), 131.1 (2 carbon, CH, d, J=8.3 Hz), 130.6 (2 carbon, CH), 123.1 (C), 114.9 (2 carbon, CH, J=20.4 Hz), 78.1 (C), 60.6 (CH₂), 55.1 (CH), 53.6 (CH), 37.3 (CH₂), 35.9 (CH₂), 28.0 (3 carbons, CH₃), 14.0 (CH₃)

Example 2 – Synthesis of compound (IIc)

To a hydrogenation autoclave was added wet solid product compound (VIc) (approximately 4.9 g dry weight, 9.7 mmol, 1 eq.), 2-MeTHF (75 mL) and 3 w/w% of a 5% Pd/C-catalyst (147 mg, 50% moist). The reaction mixture was degased with nitrogen and then 1 barg hydrogen gas was charged. Stirring was set to 600 rpm and T_J to 36 °C. The reaction was completed in four hours, The hydrogenation autoclave was rinsed with 10 mL 2-MeTHF and the rinsing portion was added to the reaction solution in the E-flask. Charcoal (250 mg, 5 wt%) was then added and the resulting mixture was stirred for 15 minutes at room temperature before it was filtered. The filter was rinsed with 10 mL 2-MeTHF and the rinsing portion was added to the filter. The light yellow/pink filtrate contained white precipitated product. The slurry was heated to approximately 40 °C to dissolve the solid before heptane (42 mL) was added drop wise during one hour. The heating was turned off and the mixture was allowed to reach room temperature with overnight stirring. Additional 21 mL heptane was the added before the mixture was cooled to approximately 7 °C (ice/water bath). The solid was isolated and was washed through with 10 mL cold 2-MeTHF/heptane 6/4. The moist solid (5.7 g) was vacuum dried at 35 °C overnight which gave a dry weight of

compound (IIc) of 4.2 g which corresponds to a yield of 91 %. The purity was determined by HPLC to be 99.1 area%.

¹H-NMR (300 MHz, DMSO-D₆) δ 8.26 (1H, d, J=7.5Hz), 7.26 (dd, 2H, J=8.1, 5.7 Hz), 7.09 (2H, t, J=8.7 Hz), 6.86 (2H, d, J=8.1 Hz), 6.71 (1H, d, J=8.7 Hz), 6.45 (1H, d, J=8.1 Hz), 4.87 (2H, s), 4.45 (1H, dd, J=14.4, 7.5 Hz), 4.07-4.00 (3H, m), 3.06-2.91 (2H, m), 2.71 (1H, dd, J=13.8, 3.9 Hz), 2.54-2.46 (1H, m), 1.31 (s, 9H), 1.11 (3H, t, J=6.9 Hz).

¹³C-NMR (75 MHz, DMSO-D₆) δ 171.4 (C=O), 171.2 (C=O), 161.2 (C-F, d, J=242.3 Hz), 155.1 (C=O), 146.9 (C), 133.2 (C, d, J=3.0 Hz), 131.1 (2 carbon, CH, d, J=8.3 Hz), 129.5 (2 carbon, CH), 124.8 (C), 114.8 (2 carbon, CH, J=21.1 Hz), 113.6 (2 carbon, CH), 77.9 (C), 60.5 (CH₂), 56.0 (CH), 53.5 (CH), 36.7 (CH₂), 35.9 (CH₂), 28.1 (3 carbons, CH₃), 13.9 (CH₃)

The present inventors have repeated Example 2 several times using crude compound (VIc) or recrystallised compound (VIc) (purity: 99.1 area%) as starting material and varying various reaction conditions, e.g. pressure of H₂, w/w% of Pd/C, solvent and temperature. The crude purity (97.2 area%) was a slightly higher when recrystallized compound (VIc) was used as starting material than when using crude compound (VIc), in which case the crude purity is generally 95-96 area%. Final yield and purity is also slightly higher than when starting from crude compound (VIc) (98-98.5 area%).

The present inventors have also repeated Example 2 several times varying the Pd/C w/w%, temperature, pressure of H₂ and concentration using 2-MeTHF as the solvent. A high conversion of Compound (VIc) (>99.5 area%) was achieved for Pd/C w/w% from 3 to 6 bar; temperature ranges from 30 to 40 °C, H₂ pressure from 1 to 6 barg, and for varying reaction concentrations. The resulting crude purity was similar in all attempts (95.3-96.2 area%), as was the purity of the isolated product after crystallization from 2-MeTHF/heptane (98.0-98.5 area%).

Example 3 – Preparation of compound (IIIc)

(i) carried out using BH₃SMe₂ in the presence of chloroacetic acid salt

In a 0.5 L dried reactor with overhead stirrer, compound (IIc) (6.99 g, 14.76 mmol) was added, followed by anhydrous tetrahydrofuran (46 mL), chloroacetic acid (36.3 g, 383.8 mmol), chloroacetic acid sodium salt (17.2 g, 147.6 mmol) at T_I=5-13°C. A solution of

BH₃SMe₂ (14.6 g, 191.9 mmol, 18.2 mL) was then added over 45 minutes. After the addition, the reaction temperature was adjusted to T_I=25-30°C and kept for 2 hr after reaching this temperature. The reaction was slowly quenched with ethanol (17.7 g, 383.8 mmol, 22.4 mL) and was stirred overnight at T_J=5°C and then slowly diluted with distilled water (138 mL) to precipitate the product, compound (IIIc). The temperature was adjusted to T_I=15°C and the stirring rate was increased before addition of a solution of aqueous K₂CO₃ (8.0 M, 27 mL) to pH = 7.0-7.5. The reaction slurry was collected on a filter and reaction vessel and filter-cake were washed with water (2×40 mL). The filter-cake was re-slurred in water (200 mL) for 1 hr at T_J=20°C and then filtered again. Washing with water (50 mL), followed by drying at T_J=35°C under high vacuum, produced the crude white product, compound (IIIc), in 7.85 g (88.8%) uncorrected yield. HPLC purity 97.5 area %.

Crude compound (IIIc) (7.5 gram) prepared according to the described procedure was charged to a reactor and washed down with 2-MeTHF (80 mL). Heating at T_J=50°C dissolved the substance. Heptane (80 mL) was added with stirring at T_I=45-50°C and then stirred before adjusting the temperature to T_J=10°C. The precipitated solid was collected by filtration and dried at T_J=35°C under high vacuum which produced white product, compound (IIIc), in 6.86 g (91.5%). HPLC purity 99.1 area %.

¹H-NMR (300 MHz, DMSO-D₆) δ 8.30 (1H, d, J=7.8 Hz), 7.26 (2H, dd, J=8.1, 6 Hz), 7.09-7.05 (3H, m), 6.79 (1H, d, J=8.9 Hz), 6.63 (2H, d, J=8.4 Hz), 4.49-4.42 (1H, dd, J=14.7, 7.5 Hz), 4.07-3.99 (3H, m), 3.68 (8H, s), 3.06-2.91 (2H, m), 2.76 (1H, dd, J=13.8, 4.2 Hz), 2.56 (1H, m), 1.29 (9H, s), 1.1 (3H, t, J=6.6 Hz)

¹³C-NMR (75 MHz, DMSO-D₆) δ 172.1 (C=O), 171.3 (C=O), 161.2 (C-F, d, J=242.3 Hz), 155.2 (C=O), 144.7 (C), 133.2 (C, d, J=3.0 Hz), 131.1 (2 carbon, CH, d, J=7.5 Hz), 130.2 (2 carbon, CH), 126.1 (C), 114.9 (2 carbon, CH, J=21.1 Hz), 111.6 (2 carbon, CH), 78.0 (C), 60.6 (CH₂), 55.9 (CH), 53.5 (CH), 52.2 (CH₂), 41.2 (CH₂), 36.4 (CH₂), 35.9 (CH₂), 28.1 (3 carbons, CH₃), 14.0 (CH₃)

(ii) Carried out using BH₃SMe₂ in the presence of chloroacetic acid salt

In a 0.5 L dried reactor with overhead stirrer, compound (IIe) (7.5 g, 15.84 mmol) was added, followed by 2-MeTHF (150 mL). The mixture was heated to 45 °C to form a clear solution. The solution was cooled to 4 °C and chloroacetic acid (38.9 g, 411.8 mmol), followed by chloroacetic acid sodium salt (18.4 g, 158.4 mmol) was added at T_I=5-13°C. A solution of BH₃SMe₂ (15.6 g, 205.9 mmol, 19.5 mL) was then added over 90 minutes. After the addition, the reaction temperature was adjusted to T_I=20-25°C and kept for 5 hr after reaching this temperature. The reaction was slowly quenched with water at T_I=15-25 °C (150 g, 8333 mmol, 150 mL), pH=3.5 in water phase, and left overnight without stirring at T_I=6 °C.

Product, compound (IIIc), had precipitated out in the organic phase and the temperature was adjusted to T_I=35 °C while stirring, and two clear phases formed. The phases were allowed to separate and the water phase was removed. The organic phase was washed three times with 20% NaCl(aq). pH in the three water phases were: 1.7, 1.1, and 1.1. After the removal of the third water phase, the organic phase was transferred to a round bottom flask and concentrated to half its volume on an evaporator. Product, compound (IIIc), started to precipitate out and the product slurry was allowed to mature at 6 °C for 19 hr. The slurry was collected on a filter and round bottom flask and filter-cake were washed with 2-MeTHF:n-heptane (2×40 mL), followed by drying at T_J=35 °C under high vacuum, to produce the crude white product, compound (IIIc), in 8.3 g (87.6%) uncorrected yield. HPLC purity 99.4 area % .

(iii) Carried out using borane-tetrahydrofuran in the presence of chloroacetic acid salt

In a 100 mL dried round bottom flask with magnet stirrer bar, compound (IIc) (0.75 g, 1.58 mmol) was added under a slow nitrogen flow followed by anhydrous tetrahydrofuran (6 mL), chloroacetic acid (3.89 g, 41.2 mmol), and chloroacetic acid sodium salt (1.84 g, 15.8 mmol). At T_I=5-13°C °C a 1 M solution of BH₃THF (20.6 mmol, 20.6 mL) was added over 30

_minutes. After the addition the reaction temperature was adjusted between T_I=23-28 °C and kept for 2 hr after reaching this temperature. In process control sample (HPLC) indicated in-complete reaction and the jacket temperature was set to T_J=40°C and when the internal temperature reached T_I=40°C the reaction was kept at this temperature for 2 hr when in-process sample (HPLC) showed 6.7 area% starting material, 7.1% acylation adduct

(impurity) and 84.1% compound (IIIc). The reaction was progressed at T_I=23°C and left for 4 days before slowly quenched with ethanol (2.4 g, 3 mL). Water (100 mL) was added and the pH adjusted with 1 M aqueous K₂CO₃ to pH 7. The reaction slurry was collected on a filter and reaction vessel and filter-cake were washed with water (2×20 mL) followed by drying at T_J=35°C under high vacuum produced the crude colorless product in 0.85 g (89.6%) uncorrected yield. HPLC purity was 94.3 area %, with one major impurity attributed to a chloroacylation adduct of the starting material in 3.8 area %.

(iv) Carried out using BH₃SMe₂ without addition of chloroacetic acid salt

In a 100 mL dried round bottom flask with magnet stirrer bar, compound (IIc) (0.75 gram, 1.58 mmol) was added under a slow nitrogen flow followed by anhydrous tetrahydrofuran (6 mL) and chloroacetic acid (3.89 g, 41.2 mmol). At T_I=5-16°C a solution of BH₃SMe₂ (1.56 g, 20.6 mmol, 2.0 mL) was added over 30. After the addition the reaction temperature was adjusted between T_I=25°C and kept for 2.5 h after reaching this temperature. A process control sample (HPLC) indicated melflufen (Compound (Ib)), the Boc-deprotected form of Compound (IIIc), in 66 area %. The reaction was slowly quenched with ethanol (2.9 g, 3.7 mL). The pH of the reaction was adjusted with 1 M aqueous K₂CO₃ solution to pH=8, followed by addition of EtOAc (40 mL). Layers were separated and the aqueous layer re-extracted with EtOAc (50 mL). The organic layers were combined and reduced at <30 mbar / 35°C to an oil. The oil was re-distilled from EtOAc (30 mL) twice and the residue was dried at T_J=23°C / 5 mbar to leave 1.6 g brownish oil. HPLC purity of Compound (Ib) was 66.1 area %.

Example 4 – Preparation of compound (Ib) as hydrochloride salt

Boc-melflufen (compound (IIIc)) (5.0 g, 8.3 mmol) was charged to a round bottomed flask, equipped with magnet stirrer bar, and nitrogen inlet. 1.3 M HCl (anhydrous) in ethanol (64 mL, 83.5 mmol, 10 eq.) was added. After 19 h the conversion was 99.4%. The solvents were partially distilled at T_J=33°C on a rotary evaporator, followed by the addition of ethanol (18 mL). This was repeated twice. Seed crystals were added and after 30 minutes product had precipitated. The slurry was stirred for 21 h and was then concentrated. Methyl tert-butyl ether (MTBE) (108 mL) was added at room temperature with an even rate over 30 minutes. After 100 minutes of stirring at room temperature the precipitate was collected by vacuum filtration and washed with 2×25 mL ethanol: MTBE (1:6). Drying was performed overnight at T_J=35°C / 5 mbar in vacuum oven. Yield of compound (Ib) in the form of its hydrochloride salt, 4.0 g (90%). HPLC-purity 98.7 area%.

¹H-NMR (300 MHz, MeOH-D₄) δ 7.26 (2H, dd, J=8.4, 8.1 Hz), 7.17 (2H, d, J=8.4 Hz), 7.02 (2H, dd, J=9, 8.4 Hz), 6.74 (2H, d, J=8.4 Hz), 4.69 (1H, dd, J=7.8, 6.3 Hz), 4.15 (2H, dd, J=14.1, 7.2 Hz), 4.04 (1H, dd, J=8.4, 5.4 Hz), 3.76 (4H, dd, J=6.3, 6 Hz), 3.67 (4H, dd, 6.6, 5.7 Hz), 3.17 (2H, dd, J=14.4, 6 Hz), 3.06-2.88 (2H, m), 1.22 (3H, t, J=7.2 Hz)

¹³C-NMR (75 MHz, MeOH-D₄) δ 172.2 (C=O), 169.8 (C=O), 163.4 (C-F, d, J=244.5 Hz), 147.4 (C), 133.9 (C, d, J=3 Hz), 132.1 (2 carbon, CH, d, J=7.5 Hz), 131.8 (2 carbon, CH), 123.4 (C), 116.2 (2 carbon, CH, d, J=21.9 Hz), 113.7 (2 carbon, CH), 62.6 (CH₂), 55.6 (CH), 55.5 (CH), 54.3 (CH₂), 41.6 (CH₂), 37.6 (CH₂), 37.6 (CH₂), 14.5 (CH₃)

Example 4 was repeated successfully in the presence ethyl acetate and with varying concentrations of HCl from 1.3 M to 2.5 M and at varying temperatures from 6 °C to room temperature.PAPERhttps://pubs.acs.org/doi/10.1021/acs.oprd.9b00116 Organic Process Research & Development (2019), 23(6), 1191-1196.Melflufen is a novel cytostatic currently in phase III clinical trials for treatment of multiple myeloma. Development of a process suitable for production is described. The two key features of the novel method are late introduction of the alkylating pharmacophore and an improved method for formation of the bis-chloroethyl group.

1H NMR spectrum of L-Phenylalanine, 4-[bis(2-chloroethyl)amino]-L-phenylalanyl-4-fluoro-, ethyl ester, hydrochloride (1) (in D4–MeOH).

13C NMR spectrum of L-Phenylalanine, 4-[bis(2-chloroethyl)amino]-L-phenylalanyl-4-fluoro-, ethyl ester, hydrochloride (1) (in D4–MeOH).

References

1. ^ Berglund, Åke; Ullén, Anders; Lisyanskaya, Alla; Orlov, Sergey; Hagberg, Hans; Tholander, Bengt; Lewensohn, Rolf; Nygren, Peter; Spira, Jack; Harmenberg, Johan; Jerling, Markus; Alvfors, Carina; Ringbom, Magnus; Nordström, Eva; Söderlind, Karin; Gullbo, Joachim (2015). “First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies”. Investigational New Drugs. 33 (6): 1232–41. doi:10.1007/s10637-015-0299-2. PMID 26553306. S2CID 8207569.
2. ^ Strese, Sara; Wickström, Malin; Fuchs, Peder Fredlund; Fryknäs, Mårten; Gerwins, Pär; Dale, Tim; Larsson, Rolf; Gullbo, Joachim (2013). “The novel alkylating prodrug melflufen (J1) inhibits angiogenesis in vitro and in vivo”. Biochemical Pharmacology. 86(7): 888–95. doi:10.1016/j.bcp.2013.07.026. PMID 23933387.
3. ^ Jump up to:^{a b c d e f g h i} “FDA grants accelerated approval to melphalan flufenamide for relapsed”. U.S. Food and Drug Administration(FDA). 26 February 2021. Retrieved 1 March 2021.  This article incorporates text from this source, which is in the public domain.
4. ^ Jump up to:^a b c “FDA Approves Oncopeptides’ Pepaxto (melphalan flufenamide) for Patients with Triple-Class Refractory Multiple Myeloma” (Press release). Oncopeptides AB. 1 March 2021. Retrieved 1 March 2021 – via PR Newswire.
5. ^ “Pepaxto: FDA-Approved Drugs”. U.S. Food and Drug Administration (FDA). Retrieved 1 March 2021.
6. ^ Gullbo, J; Tullberg, M; Våbenø, J; Ehrsson, H; Lewensohn, R; Nygren, P; Larsson, R; Luthman, K (2003). “Structure-activity relationship for alkylating dipeptide nitrogen mustard derivatives”. Oncology Research. 14 (3): 113–32. doi:10.3727/000000003771013071. PMID 14760861.
7. ^ Wickstrom, M.; Lovborg, H.; Gullbo, J. (2006). “Future Prospects for Old Chemotherapeutic Drugs in the Target-Specific Era; Pharmaceutics, Combinations, Co-Drugs and Prodrugs with Melphalan as an Example”. Letters in Drug Design & Discovery. 3(10): 695. doi:10.2174/157018006778631893.
8. ^ Gullbo, J; Dhar, S; Luthman, K; Ehrsson, H; Lewensohn, R; Nygren, P; Larsson, R (2003). “Antitumor activity of the alkylating oligopeptides J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester) and P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester): Comparison with melphalan”. Anti-Cancer Drugs. 14 (8): 617–24. doi:10.1097/00001813-200309000-00006. PMID 14501383. S2CID 10282399.
9. ^ Berglund, Åke; Ullén, Anders; Lisyanskaya, Alla; Orlov, Sergey; Hagberg, Hans; Tholander, Bengt; Lewensohn, Rolf; Nygren, Peter; Spira, Jack; Harmenberg, Johan; Jerling, Markus; Alvfors, Carina; Ringbom, Magnus; Nordström, Eva; Söderlind, Karin; Gullbo, Joachim (2015). “First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies”. Investigational New Drugs. 33 (6): 1232–41. doi:10.1007/s10637-015-0299-2. PMID 26553306. S2CID 8207569.
10. ^ Strese, Sara; Wickström, Malin; Fuchs, Peder Fredlund; Fryknäs, Mårten; Gerwins, Pär; Dale, Tim; Larsson, Rolf; Gullbo, Joachim (2013). “The novel alkylating prodrug melflufen (J1) inhibits angiogenesis in vitro and in vivo”. Biochemical Pharmacology. 86(7): 888–95. doi:10.1016/j.bcp.2013.07.026. PMID 23933387.
11. ^ Wickström, M; Johnsen, J. I.; Ponthan, F; Segerström, L; Sveinbjörnsson, B; Lindskog, M; Lövborg, H; Viktorsson, K; Lewensohn, R; Kogner, P; Larsson, R; Gullbo, J (2007). “The novel melphalan prodrug J1 inhibits neuroblastoma growth in vitro and in vivo”. Molecular Cancer Therapeutics. 6 (9): 2409–17. doi:10.1158/1535-7163.MCT-07-0156. PMID 17876040.
12. ^ Gullbo, J; Lindhagen, E; Bashir-Hassan, S; Tullberg, M; Ehrsson, H; Lewensohn, R; Nygren, P; de la Torre, M; Luthman, K; Larsson, R (2004). “Antitumor efficacy and acute toxicity of the novel dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) in vivo”. Investigational New Drugs. 22 (4): 411–20. doi:10.1023/B:DRUG.0000036683.10945.bb. PMID 15292711. S2CID 31613292.
13. ^ Gullbo, J; Wickström, M; Tullberg, M; Ehrsson, H; Lewensohn, R; Nygren, P; Luthman, K; Larsson, R (2003). “Activity of hydrolytic enzymes in tumour cells is a determinant for anti-tumour efficacy of the melphalan containing prodrug J1”. Journal of Drug Targeting. 11(6): 355–63. doi:10.1080/10611860310001647140. PMID 14668056. S2CID 25203458.
14. ^ Gullbo, J; Dhar, S; Luthman, K; Ehrsson, H; Lewensohn, R; Nygren, P; Larsson, R (2003). “Antitumor activity of the alkylating oligopeptides J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester) and P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester): Comparison with melphalan”. Anti-Cancer Drugs. 14 (8): 617–24. doi:10.1097/00001813-200309000-00006. PMID 14501383. S2CID 10282399.
15. ^ Chauhan, D.; Ray, A.; Viktorsson, K.; Spira, J.; Paba-Prada, C.; Munshi, N.; Richardson, P.; Lewensohn, R.; Anderson, K. C. (2013). “In Vitro and in Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells”. Clinical Cancer Research. 19 (11): 3019–31. doi:10.1158/1078-0432.CCR-12-3752. PMC 4098702. PMID 23584492.
16. ^ Viktorsson, K; Shah, C. H.; Juntti, T; Hååg, P; Zielinska-Chomej, K; Sierakowiak, A; Holmsten, K; Tu, J; Spira, J; Kanter, L; Lewensohn, R; Ullén, A (2016). “Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma”. Molecular Oncology. 10 (5): 719–34. doi:10.1016/j.molonc.2015.12.013. PMC 5423156. PMID 26827254.
17. ^ Chauhan, D; Ray, A; Viktorsson, K; Spira, J; Paba-Prada, C; Munshi, N; Richardson, P; Lewensohn, R; Anderson, K. C. (2013). “In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells”. Clinical Cancer Research. 19 (11): 3019–31. doi:10.1158/1078-0432.CCR-12-3752. PMC 4098702. PMID 23584492.
18. ^ Chesi, M; Matthews, G. M.; Garbitt, V. M.; Palmer, S. E.; Shortt, J; Lefebure, M; Stewart, A. K.; Johnstone, R. W.; Bergsagel, P. L. (2012). “Drug response in a genetically engineered mouse model of multiple myeloma is predictive of clinical efficacy”. Blood. 120 (2): 376–85. doi:10.1182/blood-2012-02-412783. PMC 3398763. PMID 22451422.
19. ^ Berglund, Åke; Ullén, A; Lisyanskaya, A; Orlov, S; Hagberg, H; Tholander, B; Lewensohn, R; Nygren, P; Spira, J; Harmenberg, J; Jerling, M; Alvfors, C; Ringbom, M; Nordström, E; Söderlind, K; Gullbo, J (2015). “First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies”. Investigational New Drugs. 33 (6): 1232–41. doi:10.1007/s10637-015-0299-2. PMID 26553306. S2CID 8207569.
20. ^ Wickström, M; Viktorsson, K; Lundholm, L; Aesoy, R; Nygren, H; Sooman, L; Fryknäs, M; Vogel, L. K.; Lewensohn, R; Larsson, R; Gullbo, J (2010). “The alkylating prodrug J1 can be activated by aminopeptidase N, leading to a possible target directed release of melphalan”. Biochemical Pharmacology. 79 (9): 1281–90. doi:10.1016/j.bcp.2009.12.022. PMID 20067771.
21. ^ Wickström, M; Larsson, R; Nygren, P; Gullbo, J (2011). “Aminopeptidase N (CD13) as a target for cancer chemotherapy”. Cancer Science. 102 (3): 501–8. doi:10.1111/j.1349-7006.2010.01826.x. PMC 7188354. PMID 21205077.
22. ^ Berglund, Åke; Ullén, A; Lisyanskaya, A; Orlov, S; Hagberg, H; Tholander, B; Lewensohn, R; Nygren, P; Spira, J; Harmenberg, J; Jerling, M; Alvfors, C; Ringbom, M; Nordström, E; Söderlind, K; Gullbo, J (2015). “First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies”. Investigational New Drugs. 33 (6): 1232–41. doi:10.1007/s10637-015-0299-2. PMID 26553306. S2CID 8207569.
23. ^ Wickström, M; Haglund, C; Lindman, H; Nygren, P; Larsson, R; Gullbo, J (2008). “The novel alkylating prodrug J1: Diagnosis directed activity profile ex vivo and combination analyses in vitro”. Investigational New Drugs. 26 (3): 195–204. doi:10.1007/s10637-007-9092-1. PMID 17922077. S2CID 19915448.
24. ^ Chauhan, D; Ray, A; Viktorsson, K; Spira, J; Paba-Prada, C; Munshi, N; Richardson, P; Lewensohn, R; Anderson, K. C. (2013). “In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells”. Clinical Cancer Research. 19 (11): 3019–31. doi:10.1158/1078-0432.CCR-12-3752. PMC 4098702. PMID 23584492.
25. ^ Berglund, Åke; Ullén, A; Lisyanskaya, A; Orlov, S; Hagberg, H; Tholander, B; Lewensohn, R; Nygren, P; Spira, J; Harmenberg, J; Jerling, M; Alvfors, C; Ringbom, M; Nordström, E; Söderlind, K; Gullbo, J (2015). “First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies”. Investigational New Drugs. 33 (6): 1232–41. doi:10.1007/s10637-015-0299-2. PMID 26553306. S2CID 8207569.
26. ^ Viktorsson, K; Shah, C. H.; Juntti, T; Hååg, P; Zielinska-Chomej, K; Sierakowiak, A; Holmsten, K; Tu, J; Spira, J; Kanter, L; Lewensohn, R; Ullén, A (2016). “Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma”. Molecular Oncology. 10 (5): 719–34. doi:10.1016/j.molonc.2015.12.013. PMC 5423156. PMID 26827254.
27. ^ https://ash.confex.com/ash/2015/webprogram/Paper85666.html
28. ^ World Health Organization (2012). “International nonproprietary names for pharmaceutical substances (INN): recommended INN: list 67”. WHO Drug Information. 26 (1): 72. hdl:10665/109416.

External links

• “Melphalan flufenamide”. Drug Information Portal. U.S. National Library of Medicine.
• Clinical trial number NCT02963493 for “A Study of Melphalan Flufenamide (Melflufen) in Combination With Dexamethasone in Relapsed Refractory Multiple Myeloma Patients (HORIZON)” at ClinicalTrials.gov

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