therapeutic index and improving treatment efficacy while limiting off-target effects.
With an LD50 of 50 – 100 µ g / kg, α-amanitin is among the most lethal naturally occurring toxins. It acts as a selective allosteric inhibitor of RNA polymerase II, with its toxicity attributed in part to two oxidised amino acids( AA6 and AA7), and a unique cross-link— 6-hydroxytryptathionine-( R)-sulfoxide— between AA1 and AA5( Figure 1).
Interest in this compound class was big both at industry and academia5. In fact, Heidelberg Pharma investigated the synthetic routes to focus on advancing amanitin-based ADCs as a novel mode of action in oncology, leveraging RNA polymerase II inhibition as a therapeutic strategy6 prior to Perrin’ s publication.
From a retrosynthetic perspective, disconnection via macrolactamisation between the dihydroxy leucine( AA6) and the masked tyrosine( AA5) derivatives, followed by a Savige – Fontana cyclisation, enables access to the key octapeptide intermediate( Figure 1). 7
This building block can be further traced back to a diastereomeric pair of hydroxypyrroloindolines( syn-cis / anti-cis) and ultimately to commercially available L-aspartic acid β-methyl ester hydrochloride and five protected amino acids: glycine,
L-isoleucine, L-cysteine, L-asparagine and trans-4-hydroxy-L-proline.
Cross-functional work
Following a detailed knowledge transfer from Heidelberg Pharma, initial small-scale and research activities were conducted at the Neuland site in Switzerland. Drawing on cross-functional expertise in chemistry, research and manufacturing, the team advanced the industrial synthesis of this highly potent, structurally complex natural product.
According to Carbogen Amcis’ internal categorisation system for containment( ranging from category 0 to 5), the final synthetic steps were classified as category 5— requiring the highest containment level for highly potent compounds— while earlier stages were classified as category 2.
This classification influenced the distribution of manufacturing activities: early-stage synthesis was carried out at Aarau, Switzerland, late-stage steps at Bubendorf and early building block preparation at Manchester, UK. The project required meticulous planning and synchronisation across three countries and also across multiple departments, including development, CS, QC & A, manufacturing, QA and ADC operations.
Synthesis of compound 6
This article focuses primarily on compound 6( Figure 3). The development and synthesis of its key intermediate— compound 2, a diastereomeric pair of hydroxypyrroloindolines( syn-cis / anti-cis)— was carried out at the Manchester site.
The development and manufacturing of the dihydroxy leucine derivative( compound 3) was performed at the Manchester site.
Synthesis of compound 4
The synthesis of the monocyclic intermediate( compound 4) was carried out in Switzerland according to the sequence shown in Figure 2. The strategy involves a protected proline derivative, as seen in Perrin’ s earlier work, but follows a distinct reaction order. 8
In the Heidelberg Pharma route, the proline derivative is first anchored to the solid-phase resin, followed by peptide couplings at the carboxyl and amino positions. Manufacturing was performed, affording compound 4 after purification by preparative HPLC.
Progression beyond compound 4 involved a shift in containment classification— from category 2 to category 5— which necessitated the transfer of activities to Bubendorf( Figure 3). In this
|
|
|
O |
|
O OH O
N
O
HO
Fmoc-hydroxyproline
|
O |
OH |
|
|
|
|
O |
|
Steps 1-3 |
PS |
O |
N |
O |
Step 4 |
O |
O |
|
HO
AcO
|
OAc
NH
O
N AcO
O
O
O
H N
OH
H 2 N
O
N H
O
N H
S O
O
N H
HN
O
NH
Compound 4 Monocyclic intermediate
|
|
O O
O O
O NH 3
O Cl
|
Fmoc-Asp( OAll) OH Fmoc-Cys( Trt) OH Fmoc-GlyOH Fmoc-IleOH Fmoc-GlyOH |
AcO |
HO
N H
|
O
OH
N O
H O
|
Figure 2 – Manufacture of monocyclic intermediate( compound 4) |
20 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981