Development of a safe process for manufacturing of the potent

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Development of a safe process for manufacturing of the potent anticancer agent melflufen hydrochloride Hanna Cotton, Birthe Bäckström, Ingela Fritzson, Fredrik Lehmann, Taraneh Monemi, Viveca Oltner, Ellen Sölver, Niklas Wahlström, and Johan Wennerberg Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00116 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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Organic Process Research & Development

Development of a safe process for manufacturing of the potent anticancer agent melflufen hydrochloride Hanna Cotton,‡ Birthe Bäckström,‡ Ingela Fritzson,‡ Fredrik Lehmann,† Taraneh Monemi,‡ Viveca Oltner,‡ Ellen Sölver,‡ Niklas Wahlström‡* and Johan Wennerberg‡* ‡Magle Chemoswed, Agneslundsvägen 27, SE 212 15 Malmö, Sweden †Recipharm OT Chemistry, Virdings Allé 32 B, 754 50 Uppsala, Sweden

ABSTRACT

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.

INTRODUCTION Melflufen (1)1,2 is an alkylating peptide, which belongs to a novel class of peptidase-enhanced compounds and targets the multiple myeloma (MM) tumor transformation process with a unique mechanism of action. Aminopeptidases are over-expressed in MM and are key to the transformational process of the tumor cells. Melflufen selectively targets cancer cells through

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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aminopeptidase-driven accumulation; in vitro experiments show a 50-fold enrichment of alkylating metabolites inside MM cells. The enrichment results in selective cytotoxicity (increased on-target potency and de-creased off-target toxicity). Additionally, resistance pathways of existing myeloma treatments (including alkylators) are overcome. Melflufen is currently undergoing clinical phase III studies. To continue the clinical program a method suitable for commercial manufacturing was de-sired. We present herein a novel process which is more efficient, more economical and much safer than the previous route used. Instead of handling toxic material throughout the synthetic sequence, the alkylating pharmacophore is introduced after formation of the core structure. A method for efficient introduction of the bis-chloroethyl groups was developed, which resulted in a scalable process for manufacture of melflufen of pharmaceutical quality.

RESULTS AND DISCUSSION Route development. In the original route, melflufen HCl (1) was synthesized starting from pharmaceutical grade melphalan (2) (Scheme 1).3 Boc-protection of (2) gave acid (3) which could be coupled with 4-fluoro-L-phenylalanine ethyl ester (4), to provide Boc-melflufen (5) after purification by chromatography. Deprotection gave the hydrochloride salt (1) in 32% overall yield. For early clinical trials some modifications were performed to adapt the synthesis to kilogram scale. By these actions it was possible to produce material for early clinical studies, but the route was still inadequate for safe manufacturing. Carrying the alkylating pharmacophore from the very beginning means that manufacturing must be performed with containment for all operations. Our strategy aimed at introducing the N,N-bis-chloroethylamine functionality at a later stage. The core structure, the aminopeptide (6) was prepared in two steps, via EDC mediated coupling of (4) with Boc-4-nitro-L-phenylalanine (7), providing the nitropeptide (8) (Scheme 2), which was


2

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hydrogenated to (6), employing palladium on charcoal as catalyst. Formation of the bis alcohol (9) could be efficiently performed using ethylene oxide or chloroethanol. Further manipulation (9) to the corresponding N,N-bis-chloroethylamine functionality has been reported for less functionalized precursors using either thionyl chloride,4 phosphorous oxychloride5 or conversion of the alcohols to mesylates with methanesulfonyl chloride followed by treatment with sodium chloride in DMF.6 Testing these reagents and reagent combinations did produce Boc-melflufen (5), but the yields, or the purities of obtained products were not satisfactory. We applied a protocol wherein the conversion from amine to N,N-bis-chloroethylamine was affected in one step using reductive alkylation with chloroacetic acid and borane-THF.7 In this method, by treating (6) with chloroacetic acid and borane-THF in excess, we obtained melflufen (1) as a chloroacetic acid salt in 53% yield with 63% purity. We considered the chloroacetic acid/borane-THF bis-alkylation reaction to have potential, despite the poor purity obtained, and set out to develop a process, which was suitable for production.

Scheme 1. Previous route to melflufen (1).


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F O Cl

NH2

N

O OH (Boc)2O, NEt3 THF 43%

Cl

Cl

F

F O

O Cl

N H O

HN

N

O

HCl (g), EtOH 90% Cl

O

O 5

Cl

4

O

O NMM, EDC, CH2Cl2 84%

O

3

2 Cl

OH O

HN

N

HCl H 2N

NH2 HCl

N

O

N H

O

1 Cl

Scheme 2. Route selection – synthesis of (6) and (9). F

F

HCl H 2N

O

O +

OH O

HN

O 2N

O 4

7

O

O N H O

HN

HN

O 2N

O

N H O

O

O F

H2, Pd/C EtOAc 97% H 2N

O EDC, HOBT, NMM EtOAc 82 %

Ethylene oxide, MeOH, THF, or ClCH2CH2OH, K2CO3

F

8 O

O O

quantitative yield

HO

N

HN

N H O

O O

O

O OH 6

9


4

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Amide coupling. An initial idea was to telescope the first two steps (Scheme 3), the coupling and hydrogenation, by performing the hydrogenation on the crude ethyl acetate solution, obtained after extractive work-up of the coupling product. Thus, coupling of the building blocks (4) and (7) under standard conditions with EDC (1.1 equiv.), HOBt (0.1 equiv.) and N-methylmorpholine (3 equiv.) in ethyl acetate gave nitropeptide (8) with 99.9% conversion and >98% purity in 5-10 grams scale (Scheme 3).

Increasing the scale showed the limitations with this approach as incrustations formed during the conversions reduced the conversion rate. An improvement was observed by changing to acetone as solvent for the reaction, which could now be performed with high conversion at room temperature without forming any adhesive incrustations. Telescoping the first two steps produced (6), but using this material in the next reaction, i.e. synthesis of (5), did not give a product with high enough purity required for the final step, since the nitro-peptide was difficult to purge downstream in the process. In order to achieve consistently higher conversion in the hydrogenation, higher purity of (8) was required. Crystallization of (8) was introduced and telescoping of step 1 and 2 was ruled out. Aqueous extractive work-up of (8) required addition of a second solvent, in this case 2methyltetrahydrofuran. The extractive work-up was less efficient with traces of acetone left, wherefore acetone was removed by distillation purging with 2-methyltetrahydrofuran at reduced pressure. The remaining (4) and NMM were removed by extractive work-up with 2 M sulfuric acid, whereas HOBt was removed by sodium bicarbonate washes. These extractions also effectively removed the urea by-product. Antisolvent crystallization with n-heptane produced (8) in good yield (89%) and excellent purity (99.9%). Other coupling conditions3 (PyBop or


5


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EDC/HOBt in several other solvents) used for coupling of compounds (3) with (4) producing (5) were also tested in the coupling of (7) and (4) with inferior results.

Scheme 3. New synthetic route for manufacture of melflufen (1). F O

O 2N

OH + HN O H 2N O HCl 7

O O

F EDC 1.1 eq. HOBT 0.1 eq. NMM 3.5 eq. acetone 89% O 2N

O HN

H 2N

N H O

HN

F O

THF, 20-25 °C

O

1 bar, 35-40 °C 83%

O

8

ClCH2COOH 26 eq. ClCH2COONa 10 eq. BH3Me2S 13 eq.

O

H2, Pd/C, 2-MeTHF

O

O

4

F O

N H O

Cl

N

O

N H O

HN

O O

O F

6

Cl 5

O HCl in EtOH 63 % over 2 steps Cl

N

NH2

N H

O O

HCl 1

Cl

Hydrogenation. During route selection the nitro-group reduction was tested in THF and ethyl acetate applying 1-4% wt of the active palladium catalyst (5% Pd on charcoal, 50% moist) and 13 bars of hydrogen pressure at 40 °C, but this gave rise to impurities, which did not form at all when switching to ethanol. The drawback with ethanol was the low solubility of both nitropeptide (8) and aminopeptide (6) requiring large volumes and high temperature for catalyst removal by filtration. When we employed a mixture of ethanol and ethyl acetate, the product was more soluble


6

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and none of the impurities were detected. Thus, nitropeptide (8) was hydrogenated in ethanol/ethyl acetate 70/30 with 1.5% wt of the active palladium catalyst (10% Pd on charcoal, 50% moist) at 7 bars of hydrogen and 40 °C, with full conversion after one hour. The purity was enhanced by crystallization, which afforded (6) in a yield of 75% and a purity of 99.2%. Later, solvent was changed to 2-methyltetrahydrofuran, with the primary benefit being increased solubility. Employing the same reaction conditions as above gave rise to rapid reactions, with full conversion after 45 minutes. An experimental design was set up with 2.25% wt active catalyst (5% Pd, 50% moist), 35 °C and 3 bars of hydrogen as the center point. This gave a reaction time of approximately 2 hours. The results of the experimental design showed that pressure had the least impact on the reaction performance, temperature was more influential, but that catalyst loading was the parameter of major importance. The limit for residues of palladium in melflufen hydrochloride was set to 10 ppm and to avoid re-processing late process steps, the same limit was assigned to (6). As we early on found out that palladium levels were higher than the set target, various options for reducing the levels were pursued. Activated charcoal treatment and metal scavengers were tested (see Supporting information). Among the scavengers, the silica-based trimercaptotriazine, Si-TMT, was most promising, and small quantities of the scavenger reduced the palladium content significantly. Using a loading of 3% of the Si-TMT scavenger decreased the palladium content from 21 to < 1 ppm. The hydrogenation was performed in 2-methyltetrahydrofuran, followed by treatment with 3% Si-TMT for 2 hours at 35 °C and antisolvent crystallization with n-heptane produced (6) in high yield (83%) and purity (99.7%). Reductive alkylation. Reductive alkylation of (6) was initially performed with chloroacetic acid in tetrahydrofuran using 1 M borane-tetrahydrofuran as the reducing agent. In this way crude


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melflufen was produced as the corresponding chloroacetic acid salt. This was at first considered an advantage, reducing the number of process steps, however, the α-amino group liberated in the deprotection was also accessible for reductive alkylation, which generated a too complex impurity profile. All attempts to purify the crude melflufen chloroacetic acid salt failed. Furthermore, to transform the material to the desired hydrochloride salt required an extra step and it was decided to research a step-wise process with initial isolation of Boc-melflufen (5). In order to suppress deprotection, we desired conditions which allowed reductive alkylation but prevented Boccleavage. Tests with basic additives such as N-methylmorpholine, sodium hydroxide or sodium chloroacetate showed that addition of the bases either did not give the desired reaction at all or caused incomplete reaction. Still, in the case involving a large excess of sodium chloroacetate (15 eq.) a clear effect in suppressing deprotection of the Boc-group during the alkylation reaction was evident. This minimized side reactions and a much purer product was obtained, compared to the original conditions without sodium chloroacetate. With a procedure suppressing deprotection at hand, the attention was turned to the borane reagent. A 1 M solution of borane tetrahydrofuran complex was applied initially. The large excess necessary for the reaction to run smoothly (15 eq.) and a dilute borane solution, would require large volumes at scale. To increase the capacity of the process, borane dimethyl sulfide complex, a 2 M solution in THF, and neat borane dimethyl sulfide complex (10 mol/L) were evaluated. Outlet gases were scrubbed with 10% aqueous sodium hypochlorite solution to oxidize the odorous dimethyl sulfide. Applying the concentrated borane dimethyl sulfide gave equal, if not better, results concerning both purity and conversion, compared to the 1 M solution in THF, and this reagent was thus henceforth used as hydride source. The order of addition of the reagents seemed to affect the outcome of the reductive alkylation. When (6) was


8

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added to a mixture of chloroacetic acid, sodium chloroacetate and borane reagent, 20% of a byproduct was formed (Figure 1).

Figure 1. Chloroacetamide impurity. F O O Cl

N H

HN

N H O

O O

O

The following order of addition protected (6) from by-product formation; dissolution of (6) in tetrahydrofuran, addition of chloroacetic acid for protonation, followed by the addition of sodium chloroacetate and slow addition of borane dimethyl sulfide complex. It was understood that the pH must be acidic for the reaction to work, since too high a pH resulted in incomplete reaction or decreased purity. By applying a set ratio for the three reagents chloroacetic acid, sodium chloroacetate and borane dimethyl sulfide complex of 30:10:15, Boc-deprotection was minimized during the reaction and by charging the borane over prolonged time at 20 to 35 °C and continuing the reaction at 40 °C gave a fast reaction, with practically no side reactions. The ratio for the reagents could be somewhat further reduced to 26:10:13 of chloroacetic acid, sodium chloroacetate and borane dimethyl sulfide complex. It was also decided that for large scale application the reaction temperature was further decreased and kept at 10 to 15 °C during the borane addition and increased to 25 °C for completion of the reaction for safety reasons. After completed reaction,


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excess borane was quenched by the addition of ethanol, followed by water to precipitate Bocmelflufen (5), which could be isolated in high yield (94%) and purity (99.6%) (Scheme 3).

Deprotection and salt formation. An advantage with stepwise reductive alkylation and deprotection was that the deprotection protocol used for early clinical batches could be applied, securing melflufen hydrochloride with predictable solid-state properties and impurity profile. The development work revealed that a high number of equivalents of hydrogen chloride was important to obtain fast reaction with low levels of by-products. On the other hand, the subsequent crystallization was inhibited by large excess of hydrogen chloride. A compromise was found in treating Boc-melflufen (5) with seven equivalents of 2.7 M HCl in ethanol overnight at ambient temperature, followed by repeated evaporations and additions of fresh ethanol. After crystallization had been induced, methyl-tert-butyl ether was added to increase the yield. Isolation gave melflufen hydrochloride (1) in 63% yield over two steps and in high purity (99.6%). Albeit hydrogen chloride could be obtained in ethanol of USP quality, the stability of the solution was a concern. It is known that hydrogen chloride in alcohols degrades the alcohol and gives rise to the corresponding alkyl chlorides, which are considered genotoxic impurities and should be controlled.8 We found indeed substantial amounts of chloroethane in the HCl/ethanol solution but showed that up to 2 % was efficiently purged in the crystallization and drying of melflufen hydrochloride (1). Degradation was quicker than we had anticipated but could nevertheless be suppressed by lowering the temperature. Storage at 5 °C was necessary (see Supporting information for a figure of the formation of chloroethane over time). Whereas the presence of chloroethane per se was not a potential threat to the quality of (1), the decrease in concentration of


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hydrogen chloride over time, with the potential consequence of charging too little hydrogen chloride in the reaction, was a potential challenge to the process.

CONCLUSION A four-step process for manufacture of melflufen HCl has been designed, involving late introduction of the alkylating pharmacophore, returning the same high-quality product as the previous method. The overall yield was 47%. This was accomplished by purifications in each step and careful control of reaction conditions and impurities. Furthermore, a novel method for bischloroalkylation in the presence of the N-Boc functionality has been developed, including chloroacetic acid, sodium chloroacetate and borane dimethyl sulfide complex. EXPERIMENTAL SECTION General. HPLC analyses were performed using a Waters, Atlantis T3, 4.6 X 150 mm, 3 μm, column. Gradient elution was performed with 0.05% phosphoric acid in acetonitrile:water 10:90 and 0.05% phosphoric acid in acetonitrile:water 90:10. The flow rate was 1.5 mL/min and UV detection were measured at 262 nm. NMR spectra were recorded on a Bruker 300 Mhz spectrometer operating at 300 MHz and 75 MHz for 1H and 13C respectively. Solvents and reagents were obtained from commercial sources and were used as such without any further purification. All reactors used are standard multipurpose equipment, either glass-lined or stainless steel. All reactions in production are for safety reasons routinely carried out under an atmosphere of nitrogen. L-Phenylalanine, N-[(1,1-dimethylethoxy)carbonyl]-4-nitro-L-phenylalanyl-4-fluoro-, ethyl ester (8). To a 600 L glass-lined reactor charged with acetone (70 L) was added L-Boc-4-


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nitrophenylalanine 7 (5.80 kg, 18.7 mol), 4-methylmorpholine (6.62 kg, 65.5 mol), L-4-fluorophenylalanine ethyl ester HCl 4 (4.84 kg, 19.1 mol), HOBt·H2O (286 g, 1.85 mol) and EDC·HCl (3.94 kg, 20.6 mol). The resulting slurry was stirred for 21 h at 20 °C. Acetone (34 L) was removed under reduced pressure and replaced with 2-methyltetrahydrofuran (100 L) and water (40 L). An additional 100 L of solvents were distilled off under reduced pressure. Again 2methyltetrahydrofuran (100 L) was added and solvents (100 L) were removed as above. A final addition of 2-methyltetrahydrofuran (120 L) was made and then the phases were allowed to separate at 35 °C. Still at 35 °C, the organic phase was washed with sodium hydrogen carbonate (5% aq., 2 × 30 L), sodium chloride (2% aq., 43 L) and 2 M sulfuric acid (3 L), sodium chloride (20%, aq., 29 L), and finally water (30 L). After distillation of solvents (66 L) under reduced pressure, n-heptane (81 L) was added at 40 °C. After cooling to 15 °C and stirring for 2 h, the product was isolated with centrifugation. The product was washed with a mixture of n-heptane (8 L) and 2-methyltetrahydrofuran (10 L). The moist material was dried at 35 °C under reduced pressure to give 8.4 kg (89%) of the title compound as an off-white solid. Purity was 99.9% according to HPLC: mp 160 °C (dec.); IR (neat): 3333 (br), 2983, 1741, 1722, 1660, 1511, 1519, 1298, 1158 cm-1; 1H NMR (DMSO-d6) δ 8.49 (broad d, J = 7.5 Hz, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8,4 Hz, 2H), 7.28 (dd, J = 8.1, 7.8 Hz, 2H), 7.12-7.02 (m, 3H), 4.49 (dd, J = 14.4, 7.2 Hz, 1H), 4.32-4.24 (m, 1H), 4.04 (dd, J = 14.2, 6.9 Hz, 2H), 3.08-2.95 (m, 3H), 2.84 (dd, J = 13.2, 10.8 Hz, 1H), 1.26 (s, 9H), 1.11 (t, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 171.4, 171.2, 161.2 (d, J = 242.3 Hz), 155.2, 146.6, 146.2, 133.1, 131.1 (d, J = 8.3 Hz), 130.6, 123.1, 114.9 (d, J = 20.4 Hz), 78.1, 60.6, 55.0, 53.6, 37.3, 35.8, 28.0, 13.9. MS (M+TFA-H)- ion of 616.2 Da which corresponds to M =503.2 Da. Data in agreement with published procedure.9


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L-Phenylalanine, N-[(1,1-dimethylethoxy)carbonyl]-4-amino-L-phenylalanyl-4-fluoro-, ethyl ester (6). To a stainless steel 250 L pressure reactor charged with 2-methyltetrahydrofuran (112 L) was added (8) (7.00 kg, 13.9 mol). Palladium on charcoal (320 g, 5% Pd, 50% moist) was added and the temperature was adjusted to 35 °C. Hydrogenation was performed for 4 h at 2 bars. After the reaction was completed the reaction mixture was filtered. The catalyst was washed with 2methyltetrahydrofuran (14 L) and scavenger Si-TMT (0.21 kg) was added. The mixture was stirred for 2 hours at 35 °C. After filtration and wash with 2-methyltetrahydrofuran (14 kg), the temperature was adjusted to 40 °C and n-heptane (114 L) was added. The temperature was decreased to 15 °C and the suspension was stirred at this temperature for 15 h. After isolation via centrifugation the product was washed with a mixture of n-heptane (7.6 L) and 2methyltetrahydrofuran (9.2 L). Drying at 35 °C under reduced pressure gave 5.5 kg (83%) of the title compound as an off-white solid. Purity was 99.7% according to HPLC: mp 142 °C (dec.); IR (neat): 3472 (br), 3378, 3328, 2979, 2924, 1727, 1685, 1651, 1509, 1288, 1170 cm-1; 1H NMR (DMSO-d6) δ 8.26 (d, J = 7.5 Hz, 1H), 7.26 (dd, J = 8.1, 5.7 Hz, 2H), 7.09 (t, J = 8.7 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 6.71 (d, J = 8.7 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.87 (s, 2H), 4.45 (dd, J = 14.4, 7.5 Hz, 1H), 4.07-4.00 (m, 3H), 3.06-2.91 (m, 2H), 2.71 (dd, J = 13.8, 3.9 Hz, 1H), 2.542.46 (m, 1H), 1.31 (s, 9H), 1.11 (t, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 172.1, 171.2, 161.1 (d, J = 241.5 Hz), 155.1, 146.9, 133.2 (d, J = 3.0 Hz), 131.1 (d, J = 8.3 Hz), 129.5, 124.8, 114.9 (d, J = 21.1 Hz), 113.6, 77.9, 60.5, 56.0, 53.5, 36.7, 35.9, 28.1, 13.9; MS; Positive ionisation mode shows a major peak at 418.2 Da, corresponding to a M = 473.2 Da (M-55). Data in agreement with published procedure.9


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L-Phenylalanine,

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4-[bis(2-chloroethyl)amino]-N-[(1,1-dimethylethoxy)carbonyl]-L-

phenylalanyl-4-fluoro-, ethyl ester (5). A glass-lined 250 L reactor was connected to a scrubber containing a sodium hypochlorite solution (117 L, 190 mol) and water (120 L). To the reactor was added THF (31 L) and (8) (4.5 kg, 9.5 mol) at 35 °C followed by stirring. The temperature was decreased to 5 °C, then chloroacetic acid (23.3 kg, 247 mol) and sodium chloroacetate (11.1 kg, 95 mol) were added. Borane-dimethyl sulfide (9.4 kg, 123.5 mol) complex was added over 3 h so that temperature was kept between 5 and 10 °C. The temperature was adjusted to 20-25 °C, followed by stirring for 2 h. The temperature was adjusted to 5 °C and ethanol (22 L) and 1 M potassium carbonate (105 kg) were added at a rate where the temperature did not exceed 15 °C. After an adjustment to 10 °C and stirring for 1 h, the reaction mixture was transferred to a filter drier and filtered. The filter cake was washed with water (19 L). Water (72 L) was charged to the filter drier and the material was stirred for 1 h, thereafter the solvent was filtered off. The filter cake was washed with water (27 L). The product was dried at 30 °C under reduced pressure to give the product, which was recrystallized by addition of 2-methyltetrahydrofuran (68 L) and nheptane (68 L) at 50 °C. The suspension was cooled to 10 °C and left with stirring for 2 h. The material was filtered, washed with 2-methyltetrahydrofuran:n-heptane 1:1 (18 L) and dried at 35 °C under reduced pressure to obtain off-white product, which was kept in the filter drier and used in the next step. Purity was 99.6% according to HPLC.: mp 132 °C (dec.); IR (neat): 3334 (br), 2985, 1749, 1737, 1662, 1510 cm-1; 1H NMR (CDCl3) δ 7.07 (d, J = 8.1 Hz, 2H), 7.01-6.90 (m, 4H), 6,60 (d, J = 8.1 Hz, 2H), 6.34 (d, J = 6,6 Hz, 1H), 4.95 (d, J = 6,6 Hz, 1H), 4.75 (m, 1 H), 4,27 (d, J = 6,0 Hz, 1H), 4.16-4.09 (m, 2H), 3.70 (app dd, J =7.2, 6.3 Hz, 4H), 3.61 (app dd, J = 6.9, 6.6 Hz, 4H), 3.05 (d, J = 5.1 Hz, 2H), 2,94 (d, J = 5.7 Hz, 2H), 1.42 (s, 9H), 1.22 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 171.1, 171.0, 162.2 (d, J = 245.3 Hz), 155.5, 145.3, 131.7 (d, J =


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3.0 Hz), 131. 01 (d, J = 8.3 Hz), 130.8, 125.4, 115.5 (d, J = 21.1 Hz), 112.4, 80.4, 61.8, 56.0, 53.6, 53.4, 40.6, 37.4, 37.3, 28.4, 14.3; MS (M+TFA-H)- = 710.2 Da corresponds to Boc-melflufen (5). Data in agreement with published procedure.3

Ethyl

(2S)-2-[(2S)-2-amino-3[4-[bis(2-chloroethyl)amino]phenyl]propanamido]-3-(4-

fluorophenyl)propanoate hydro chloride

or L-Phenylalanine, 4-[bis(2-chloroethyl)amino]-L-

phenylalanyl-4-fluoro-, ethyl ester, hydrochloride (1). To (5), still in the filter drier from the previous step, was added ethyl acetate (95 L). After stirring at 50 °C for 1 h the solution was transferred to a glass-lined 250 L reactor. The filter drier was washed with ethyl acetate (10 L). The temperature was adjusted to 25 °C and hydrogen chloride in ethanol (20.8 L, 2.7 M, 56.5 mol) was added. The mixture was stirred at 25 °C for 24 h, after which 62 L of solvent was distilled off under reduced pressure. Ethanol (96 L) was added and distillation was repeated until 60 L were distilled off, whereby the material precipitated. Methyl-tert-butyl ether (144 L) was added at 20 °C over 1 hour. The mixture was stirred for 2 hours then the temperature was adjusted to 10 °C followed by stirring for 19 h. The product was isolated through filtration and the filter cake was washed with ethanol and methyl-tert-butyl ether (30 L, 1:3). After drying at 35 °C and reduced pressure the product was obtained as a white solid with a yield over 2 steps of 3.18 kg, 63%. Purity was 99.6% according to HPLC; Mp 199 °C (Dec.); IR (neat): 3340 (br), 2797, 1723, 1666, 1520 cm-1; 1H NMR (MeOH-d4) δ 7.26 (dd, J = 8.4, 8.1 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.02 (dd, J = 9.0, 8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 4.69 (dd, J = 7.8, 6.3 Hz, 1H), 4.15 (dd, J = 14.1, 7.2 Hz, 2H), 4.04 (dd, J = 8.4, 5.4 Hz, 1H), 3.76 (dd, J = 6.3, 6 Hz, 4H), 3.67 (dd, J = 6.6, 5.7 Hz, 4H), 3.17 (dd, J = 14.4, 6 Hz, 2H), 3.06-2.88 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H); 13C NMR (MeOH-d4) δ 172.2, 169.8, 163.4 (d, J = 243.8 Hz), 147.4, 133.9 (d, J = 3 Hz), 132.1 (d, J = 8.3 Hz), 131.8,


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123.4, 116.2 (d, J = 21.9 Hz), 113.7, 62.6, 55.6, 55.5, 54.3, 41.6, 37.6, 37.6, 14.5; MS (M+H)+ 498.2 Da (Melflufen). Data in agreement with published procedure.9


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ASSOCIATED CONTENT The supporting information is available free of charge at the ACS Publications webpage at DOI: Table of scavengers tested for removal of residual palladium. A picture of ethyl chloride formation over time at 5 and 25 °C. 1H and

13C

NMR spectra of compounds 1, 5, 6 and 8 are

available. AUTHOR INFORMATION Corresponding Author * E-mail [email protected]. ACKNOWLEDGMENT The authors would like to thank Hans Uvelius, Lars-Åke Scherman and Peter Jönsson for production of melflufen HCl, Mats Frohm for Pd-analyses and Carl-Magnus Andersson for valuable comments on the manuscript. REFERENCES 1. Berglund, Å.,; 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. (1) 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. Invest. New Drugs, 2015, 33, 1232– 1241.


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2. Strese, S.; Wickström, M.; Fuchs Fredlund, P.; Fryknäs, M.; Gerwins, P.; Dale, T.; Larsson, R.; Gullbo, J. The novel alkylating prodrug melflufen (J1) inhibits angiogenesis in vitro and in vivo. Biochem. Pharmacol. 2013, 86, 888–895. 3. Gullbo, J.; Tullberg, M.; Våbenø, J.; Ehrsson, H.; Lewensohn, R.; Nygren, P.; Larsson, R.; Luthman K. Structure-activity relationship for alkylating dipeptide nitrogen mustard derivatives. Oncol Res., 2003, 14, 113–132. 4. (a) Cammak, T.; Reeves, P. C. Synthesis of Ketobemidone Precursors via Phase-Transfer Catalysis. J. Heterocyclic Chem., 1986, 23, 73–75. (b) Fang, K.; Dong, G.; Wang, H.; He, S.; Wu, S.; Wang, W.; Sheng C. Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA. ChemMedChem, 2018, 13, 30–36. 5. (a) Elderfield, R.C.; Covey, I. S.; Geiduschek, J. B.; Meyer, W. L.; Ross, A. B.; Ross, J. H. Synthesis of Potential Anticancer Agents. I. Nitrogen Mustards Derived from p-[N,NBis(2-chloroethyl)amino]benzaldehyde. J. Org. Chem., 1958, 23, 1749–1753. (b) Keskin, S. G.; Stanley, J. M.; Cowley, A. H. Synthesis, characterization and theoretical investigations

of

molybdenum

carbonyl

complexes

with

phosphorus/nitrogen/phosphorus ligand as bidentate and tridentate modes. Polyhedron, 2017, 138, 206–217. 6. (a) Gourdie, T. A.; Valu, K. K.; Gravatt, G. L.; Boritzki, T. J.; Baguley, B. C.; Wakelin, L. P. G.; Wilson, W. R.; Woodgate, P. D.; Denny, W. A. DNA-Directed Alkylating Agents. 1. Structure–Activity Relationships for Acridine-Linked Aniline Mustards: Consequences of Varying the Reactivity of the Mustard. J. Med. Chem., 1990, 33, 1177– 1186. (b) Kamal, A.; Kumar, B. A.; Suresh, P.; Agrawal, S. K.; Chashoo, G.; Singh, S.


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K.; Saxena, A. K. Synthesis of 4β-N-polyaromatic substituted podophyllotoxins: DNA topoisomerase inhibition, anticancer and apoptosis-inducing activities. Bioorg. Med. Chem., 2010, 18, 8493–8500. 7. Chen, J.; Przyuski, K.; Roemmele, R.; Bakale, R. P. Discovery of a Novel, Efficient, and Scalable Route to Bendamustine Hydrochloride: The API in Treanda. Org. Process Res. Dev. 2011, 15, 1063–1072. 8. (a) Guideline on the limits of genotoxic impurities (EMEA, 2006). (b) Guidance for Industry – Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches (FDA, Draft 2008). (c) ICH S9 Nonclinical evaluation for anticancer pharmaceuticals (2009). (d) ICH M7 Assessment and Control of DNA Reactive (Mutagentic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (2014). 9. PCT Int. Appl. (2016), WO2016180740 A1


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Development of a safe process for manufacturing of the potent

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