The Development and Scale-Up of an Antibody Drug Conjugate Tubulysin Payload

Article abstract of DOI:10.1021/acs.oprd.7b00232

Significant development and scale-up work was completed on the synthesis of an antibody drug conjugate payload based on the tubulysin natural products. This work included the development of new routes to the tubuvaline and tubuphenylaniline portions of the molecules, as well as extensive optimization of the solid phase peptide synthesis used to assemble the molecule. The initial route (21 steps longest linear sequence, 0.01% overall yield) was improved to a new, more robust route (19 steps longest linear sequence, 2.4% overall yield) affording a 240-fold increase in overall yield and allowing delivery of over 86 g of the required molecule.

Full text of DOI:10.1021/acs.oprd.7b00232

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Full Paper
The Development and Scale-Up of an ADC Tubulysin Payload
Jeremy Stephen Parker, Marc McCormick, David W Anderson,
Beatrice A Maltman, Lakshmaiah Gingipalli, and Dorin Toader
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00232 • Publication Date (Web): 01 Aug 2017
Downloaded from http://pubs.acs.org on August 2, 2017
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The Development and Scale-Up of an ADC
Tubulysin Payload
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Jeremy S Parker†,*, Marc McCormick†, David W Anderson‡, Beatrice A Maltman‡, Lakshmaiah
Gingipalli§, and Dorin Toader§
†AstraZeneca, Pharmaceutical Sciences, Silk Road Business Park, Macclesfield, SK10 2NA, UK
‡Almac Sciences, 5 The Fleming Building, Edinburgh Technopole, Milton Bridge, Midlothian,
EH26 0BE, UK
§AstraZeneca, R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
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Key Words
Tubulysin
Antibody Drug Conjugate
ADC
Solid Phase Peptide Synthesis
SPPS
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Abstract
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Significant development and scale-up work was completed on the synthesis of an antibody drug
conjugate payload based on the tubulysin natural products. This work included development of
new routes to the tubuvaline and tubuphenylaniline portions of the molecules, as well as
extensive optimisation of the solid phase peptide synthesis used to assemble the molecule. The
initial route (21 steps longest linear sequence, 0.01% overall yield) was improved to a new, more
robust route (19 steps longest linear sequence, 2.4% overall yield) affording a 240-fold increase
in overall yield and allowing delivery of over 86g of the required molecule.
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Introduction
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Antibody Drug Conjugates (ADCs) represent an exciting new class of therapeutics, combining
a tumour targeting antibody with a cell-killing cytotoxic drug (Payload)1. Recent advances in
this field include the launches of brentuximab vedotin (Adcetris ®) for the treatment of relapsed
or refractory Hodgkin's lymphoma and relapsed or refractory systemic anaplastic large cell
lymphoma2, and trastuzumab emtansine (Kadcyla ®) for the treatment of advanced HER2
positive breast cancer3.
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One of the key challenges in developing new ADCs is the identification of suitable cytotoxic
payloads. To find molecules with the necessary potency, researchers have focussed on natural
product based cytotoxic molecules, for example the use of auristatins4 based on dolastatin 105
which have been used in Adcetris ®, and maytansinoids6 based on maytansine7 which have been
used in Kadcyla ®. The molecules that have been selected are typically highly complex natural
product based molecules, necessitating an important contribution to ADC projects from synthetic
organic chemistry.
As part of ongoing work to develop new ADCs, we identified the tubulysins as promising class
of molecules for use as payloads. The tubulysins were originally isolated from a culture broth of
strains of myxobacteria Archangium gephyra Ar 315 and Angiococcus disciformis An d488, with
further examples from Cystobacter sp. SBCb0049. The molecules were shown to be potent
inhibitors of tubulin polymerisation10, causing disintegration of the cytoskeleton and leading to
apoptosis. This activity has led to significant interest in the tubulysins as small molecule
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anticancer agents11 and more recently in conjugates with antibodies12, dendrimers13 and
nanoparticles14.
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Structural characterisation of the tubulysins15 showed the molecules to be tetrapeptides,
containing D-N-methyl-pipecolic acid (Mep), L-isoleucine (Ile), tubuvaline (Tuv) and either
tubutyrosine (Tut) or tubuphenylalanine (Tup) (Table 1).
Table 1. Structure of Tubulysins
Tubulysin R1
R2 R3
A
B
C
D
E
F
CH2OCOCH2CHMe2 Ac OH
CH2OCOCH2CH2CH3 Ac OH
CH2OCOCH2CH3
Ac OH
CH2OCOCH2CHMe2 Ac H
CH2OCOCH2CH2CH3 Ac H
CH2OCOCH2CH3
CH2OCOCH=CMe2
CH2OCOCH3
CH2OCOCH3
H
Ac H
G
H
I
Ac OH
Ac H
Ac OH
Ac H
U
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V
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CH2OCOCH2CH2CH3
OH
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X
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H
H
Ac OH
OH
H
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The variability in structure of the tubulysins is mostly observed in the Tuv portion of the
molecule. The first series (A-I) contains an unusual and synthetically challenging N,O-acetal
group (R1), as well as an acetylated hydroxyl group (R2). A second series (U-Z) is missing the
N,O-acetal group and/or the acetyl group, and is less active. On the basis of their complex
structure, limited availability and potential as anticancer agents, significant efforts have been
undertaken on the synthesis of tubulysins16, including total syntheses of Tubulysin B17, D18, U19
and V20. Work has also been completed on evaluating the key structural features21 that impart
the tubulysins with their activity, looking to deliver structurally simplified versions of these
molecules. Of particular interest in simplified analogues have been pretubulysins22 and N14-
Desacetoxytubulysin H23 (1), both of which replace the Tuv N,O-acetal group with a simple N-
methyl group without significant loss of activity.
Initial work24 to identify an effective ADC payload focussed on evaluation of structure activity
relationship (SAR) of the Mep and Tup residues of 1, with the new compounds being evaluated
using an Alamar blue assay against human prostate cancer cell line DU-145, human colon
carcinoma cell line HCT-116, and human breast cancer cell line MDA MB-23124 (Table 2). A
review of all the produced data led to the selection of 2, where the Mep residue is changed to a
(2R,4R)-1,4-dimethylpiperidine-2-carboxylic acid, as the starting point for further design work.
The next step was to identify a suitable attachment point for a linker which would allow
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conjugation to an antibody. The conversion of the phenolic group of Tut residues to an aniline
(3) had been exemplified previously25, although this modification was found to significantly
reduce potency compared to 1. This approach was evaluated for 2 affording 4, which had a
reduced potency compared to 2. Further structural modifications showed the replacement of the
N-methyl group of Tuv with an N-ethyl group was effective in restoring potency that matches 2,
and this molecule (5) was selected for use as the warhead for the new ADC payload. Design
work was completed by evaluating the inclusion of amino acids in the linker attached to 4-amino
Tup (to provide cleavable linkers), and looking at the length and type of spacer that could be
used to join the warhead to a conjugatable maleimide. Following a evaluation of the
performance of the different linker options, mc-Lys-MMETA (MethylMepN-
EthylTubulysinAniline) (6) was identified26 as an effective ADC payload27.
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Table 2. SAR of Tubulysin Analogues of N14-Descetoxytubulysin H
GI50 (nM)
Cpd
R1
R2
R3
DU-
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HCT- MDA
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MB-
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H
Me
Me
Me
H
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1.1
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Me
H
H
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0.9
3.4
NH2
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Me
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2.5
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With the successful identification and application of 6, further supplies of material were
required to support toxicology and clinical studies. Conducted on a similar scale to all previous
tubulysin syntheses, the initial work24,26 had only provided milligram quantities of material,
whereas multi-gram quantities of 6 were required to progress the development program,
requiring significant improvements to the synthesis of this molecule.
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In this article, we describe the development and scale-up of an efficient and robust synthesis of
6. The synthesis improvements were completed over four successive campaigns of development
and scale-up, during which the overall yield improved from 0.01% to 2.4% (a 240 fold increase),
ultimately affording over 86g of the required compound.
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Results and Discussion
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The initial work on the project had established solid phase peptide synthesis (SPPS) using 2-
chlorotrityl resin (CTR) as an effective method of assembling tubulysins, and this approach was
used as the start point for the development and scale-up. This approach required the separate
synthesis of each of the components of 6, with the use of N-Fmoc protecting groups to facilitate
the SPPS (Figure 1).
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Figure 1. Retrosynthetic analysis of mc-Lys-MMETA (6)
Tup Synthesis and Coupling. Tup derivatives were initially prepared using the methodology
reported by Wipf28, but for larger amounts, with a focus on preparing N-Fmoc 4-nitro-Tup (9a),
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key elements of the approach reported by Zanda19b were adopted24,26. This methodology was
effective at supplying material for early development campaigns and was scaled-up directly
affording 22g of 9a. The overall yield for the sequence (8 steps) was 2%, thus for later
development and scale-up it was decided to review the route, looking to improve both the yield
and the efficiency of the synthesis. In the previous sequence, the majority of the structure had
been derived from 4-nitro-L-phenylalanine, with the remaining three carbons being introduced
from Meldrum’s Acid (2 carbons) and methyl iodide (1 carbon). Thus to improve the efficiency
of the synthesis, it was decided to investigate the introduction of all three carbon atoms in a
single step using a 2-methylmalonate derivative.
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To introduce the 2-methylmalonate derivative, it was decided to use cyclic sulfamidate
chemistry29. N-Boc 4-Nitro-L-phenylalanine was converted to the corresponding alaninol (10)
by preparing a functionalised acid and reducing. Initial work used CDI to prepare the
imidazolide, followed by reduction with sodium borohydride, but this was found to cause
racemisation. Similar results were obtained with isobutylchloroformate and sodium borohydride,
but conversion to the methyl ester followed by reduction with sodium borohydride gave high
conversion and good quality product without racemisation. The alaninol (10) was then cyclised
to the sulfamidite (11) with thionyl chloride and oxidised to the sulfamidate (12) using standard
procedures. This was then reacted with the anion of diethyl methylmalonate to successfully
afford the diester (13). Ensuring good purity of input materials and low water content ensured
good conversion.
The product of the reaction was directly telescoped into the
decarboxylation/saponification step affording racemic 14 which was Fmoc protected using
Fmoc-OSu to afford racemic 9a. Development work showed that this was the optimum point to
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separate the diastereomers, which was achieved using Supercritical Fluid Chromatography (SFC)
allowing the required diastereomer (9a) to be recovered efficiently from the separation. The
improved overall yield for the 7 step sequence was 19% (Scheme 1).
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Scheme 1. Improved Synthesis of N-Fmoc 4-nitro-Tup (9a)a
aReagents and Conditions: (a) Me2SO4, Na2CO3, THF then NaBH4, MeOH/THF, 87%; (b)
SOCl2, DCM; (c) RuCl3.3H2O, NaIO4, MeCN/H2O, 74% (2 steps); (d) Diethyl methylmalonate,
NaH, THF; (e) HCl; (f) Fmoc-OSu, NaHCO3, acetone/water, 72% (3 steps); (g) SFC, 40%.
Reviewing the use of 9a in the SPPS identified the tin mediated reduction of the nitro group as
a major issue, with loss of material from the solid phase as well as significant deacetylation.
Thus during the first development and scale-up campaign it was decided to change the timing of
the reduction of the nitro group, completing this on the Tup prior to the peptide assembly. Thus
9a was reduced to the aniline (15) using a Pd/C mediated hydrogenation. Some care was
required with the reaction, as hydrogenolysis of Fmoc protecting group could occur with
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extended reaction times30. However, this issue could be overcome by careful dosing of the
hydrogen, or by the use of Pt/C catalysts. The resultant 15 was then protected with an Alloc
protecting group affording 9b (Scheme 2). The Alloc protecting group was initially introduced
using Alloc-Cl/citrate buffer, but this was later changed to Alloc-Cl/sodium hydrogen carbonate
in aqueous THF, which gave an improved reaction profile and crude product purity.
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Scheme 2. Synthesis of N-Fmoc N’-Alloc Tup (9b)a
aReagents and Conditions: (a) H2, Pd/C, MeOH; (b) AllocCl, NaHCO3, THF/H2O, 56% (2
steps).
Overall the new route (Schemes 1 and 2) was used to supply 527g of 9b (9 steps, 10% overall
yield) for the final development and scale-up campaign.
With the successful preparation of 9b, SPPS was started using 2-chlorotrityl resin affording
resin-bound Tup (16). In early development and scale-up campaigns, loading was allowed to
vary between 0.5mmol/g and 0.75mmol/g, but a more detailed study demonstrated that the
maximum reliable capacity of the resin was 0.50mmol/g and this value was used to provide
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consistency as well as ensuring efficient use of 9b. The Fmoc protecting group was then
removed using piperidine affording 17, ready for Tuv coupling.
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Tuv Synthesis and Coupling. N-Alkyl Tuv derivatives were initially prepared starting from
protected valine as demonstrated by Wipf23a, but to circumvent the use of diazomethane, a
simple synthesis of Weinreb amides analogous to those used by Fecik31 was developed from L-
β-leucine. This synthesis afforded N-Boc N-Et Tuv (8c) whch was deprotected and the resultant
amino acid (8a) N-Fmoc protected to afford N-Fmoc N-Et Tuv (8b)26. This sequence was
directly scaled-up, supplying all four campaigns of development and scale-up, with the largest
scale preparation affording 760g of material (13 steps, 1% overall yield).
Although effective at supplying material, the key issue with the route was the low overall
yield. Particularly of note is the low yielding final N-Fmoc protection (32% yield), mostly likely
due to the steric hindrance around the secondary amine centre (presence of N-ethyl and α-
isopropyl groups). The route represents poor efficiency in terms of Redox Economy32, with an
initial reduction of a carboxylic acid group followed by a final oxidation, as well as an inefficient
non-chiral reduction of a ketone to a secondary alcohol (even with a recycle demonstrated as part
of the 760g campaign). These issues were tackled by the development of a new, more efficient
route to 18. Thus carboxylic acid (19) was protected as the tert-butyl ester and this molecule
(20) could be metallated and reacted with the previously prepared Weinreb Amide affording 21.
An asymmetric hydrogenation screen was completed which identified (Mes)RuCl(R,R-
MsDPEN)33 as a suitable catalyst, affording the chiral alcohol (22) in >99% chiral purity. The
alcohol could be acetylated using the previously demonstrated conditions affording 18. This
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molecule can be converted to 8a by removing both the N-Boc and O-tert-butyl ester protecting
groups in a single step, improving the overall efficiency of the protecting group strategy. The
route (Scheme 3) was successfully scaled-up to afford 2.00kg of 18 (7 steps, 20% overall yield)
for use in later campaigns.
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Scheme 3. Improved Synthesis of Protected N-Et Tuv (18)a
aReagents and Conditions: (a) CDI, DBU, tBuOH, DMF, 85%; (b) (R)-tert-butyl ethyl(1-
(methoxy(methyl)amino)-4-methyl-1-oxopentan-3-yl)carbamate, iPrMgBr, 2MeTHF, 35%; (c)
(Mes)RuCl(R,R-MsDPEN), HCOOH:TEA (5:2), THF/water; (d) AcCl, py, 82% (2 steps).
Following the procedures developed in the initial work24,26, N-Fmoc N-Et Tuv (8b) was used in
the SPPS, coupling the material with 17 using HATU/TMP, followed by Fmoc deprotection
using piperidine to afford 23. However, significant issues were observed when the subsequent
coupling of Fmoc-Ile-F was undertaken, with a significant new impurity being observed at 50%
HPLC area. Investigations showed that under a range of conditions, the impurity could be
converted back to 23, and that the impurity had a m/z of 982, 44 units higher than the mass of the
expected product (24). On the basis of this information, a tentative structural assignment of a
mixed anhydride (25) was made, with a carbon dioxide group inserted between the secondary
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amine of 23 and the Fmoc-Ile. The mixed anhydride is proposed to form from a carbamic acid
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The reason and mechanism for the formation of the impurity are not fully understood, but
experimental work found that treating the batch containing the mixture of 24 and 25 with a range
of acids, bases and nucleophiles was effective at converting the impurity to 23, and the use of 3%
HOBT in DMF was selected for convenience. The resulting mixture of 23 and 24 could all be
converted to 24 using Fmoc-Ile-F.
With both the issue of the formation of the new impurity (25) the low yielding introduction of
the Fmoc protecting group (8a to 8b), it was decided to investigate whether unprotected N-Et
Tuv (8a) could be used in the SPPS, with the low reactivity of secondary amine centre limiting
the amount of double inclusion product that could potentially be generated. A laboratory
investigation demonstrated that 8a could be successfully coupled to 17 with only 4% HPLC area
of double inclusion product (26) observed (Scheme 4). The double inclusion products have
significantly different retention times on HPLC and the final impurity that this material produces
can be removed in the final purification.
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Scheme 4. Coupling of Unprotected Tuv (8a)a
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aReagents and Conditions: (a) 8a, HATU, DIPEA, DMF.
With the SPPS now requiring N-Et Tuv (8a), the deprotection of 8c was examined in more
detail. Analysis showed that the deprotection conditions used previously caused significant
deacetylation (30-40%) during processing. A simpler process using a controlled amount of TFA
and DCM was found to be effective at removing the Boc group without loss of the acetyl group.
Following solvent removal the product could be dissolved in diethyl ether and precipitated by the
addition of diisopropylethylamine. The resultant solid, containing both 8a.DIPEA and
DIPEA.TFA could be used directly in the SPPS after the w/w assay of 8a had been accurately
determined. The coupling of 8a onto 17 was then investigated. It was found that the use of
HATU/DIEA as the coupling agents caused both deacetylation of the product (23) and
trifluoroacetylation of 17. Changing the coupling reagents to OxymaPure/DIC34 eliminated the
trifluoroacetylation and significantly reduced the amount of deacetylation. This procedure was
investigated on an intermediate scale and although the coupling was shown to be effective,
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significant deacetylated product was also present. The formation of this was traced to the use of
a methanol wash of the resin, and removing this (from this and subsequent stages) ensured
excellent conversion to 23 without loss of the acetyl group.
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Ile Synthesis and Coupling. The Ile residue was introduced using Fmoc-Ile-F35 prepared by
reacting DAST with Fmoc-Ile-OH. The chemistry was directly scaled-up with the largest scale
preparation affording 6.45kg of material.
The initial SPPS issues encountered with the formation of the mixed anhydride impurity (25)
were resolved by introducing unprotected N-Et Tuv (8a) and further development started from
23, using the previous conditions of 3 cycles of 8eq Fmoc-Ile-F. A more detailed study was
completed looking at the efficiency of the three cycles of Fmoc-Ile-F coupling. This showed that
after the first coupling cycle a conversion of 70% 23 to 24 had been achieved; after the second
coupling cycle 90% and after the third coupling cycle 95%. As noted previously, the coupling
onto the secondary amine centre of Tuv is challenging due to steric hindrance and this study
clearly demonstrated the need for three cycles of Fmoc-Ile-F coupling. As this stage generates
an acid byproduct (HF), there were concerns about this resulting in loss of the peptide from the
resin, thus bis(trimethylsilyl)acetamide was introduced as a fluoride scavenger36 following which
a range of coupling conditions showed no significant loss of material. The Fmoc group was
removed from 24 using 2 cycles of piperidine/DMF affording 27.
Mep Synthesis and Coupling. (2R,4R)-1,4-dimethylpiperidine-2-carboxylic acid (7) was
prepared by a reductive amination of (2R,4R)-4-methylpiperidine-2-carboxylic acid (Scheme 5).
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Scheme 5. Synthesis of (2R,4R)-1,4-dimethylpiperidine-2-carboxylic acid (7)a
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aReagents and Conditions: (a) CH2O(n), H2, Pd/C, MeOH/H2O, 85%.
This compound had initially been coupled in the SPPS using HATU/TMP, but following an
evaluation of the reaction the coupling was changed to the preferred conditions of OxymaPure
and DIC to afford 28.
Related to this transformation, at the end of the SPPS an impurity with similar retention time
(RRT 0.97) to 6 was observed, meaning that it was not possible to purify the product to the
required purity of >95%. The impurity was identified as a dimaleimide (29), causing significant
concern as the molecule contained two maleimide groups and thus had the potential to cross-link
two antibodies. After a careful review of the synthesis, it was concluded that the formation of 29
could be traced back to the low solubility of 7. This low solubility had resulted in incomplete
dissolution of 7 and when the resultant solution was charged to the SPPS, less than the required
1.60eq was present leaving a portion of 27 present. Following Alloc deprotection, Fmoc-
Lys(Boc)-OH and the maleimide linker could be added to both ends of this molecule resulting in
29. To circumvent this problem a more detailed check was introduced after the coupling of 7 to
ensure complete coupling, with the option for an additional coupling cycle to be used if required.
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Alloc Deprotection. Deprotection of the Alloc protecting group on 28 was initially achieved
using using 3 cycles of tetrakis(triphenylphosphine) palladium (0) with triphenylsilane as the
allyl-scavenger37 affording 30. More detailed analysis of the process showed the deprotection to
have proceeded to 40%, 80% and 90% after first, second and third cycles respectively
confirming the requirement for 3 cycles. As part of the ongoing analysis at the end of the SPPS,
another impurity with similar retention time (RRT 0.97) to 6 was observed which was identified
as N-Allyl MMETA (31). To avoid the issues with the formation and removal of this impurity
the scavenging agent for the deprotection was changed from triphenylsilane to dimethylamine
borane complex38, which completely eliminated the formation of 31.
Fmoc-Lys(Boc)-OH and NHS-(CH2)5-Mal Coupling. Following optimisation studies,
coupling of Fmoc-Lys(Boc)-OH was changed from the inital conditions of HATU/TMP to
OxymaPure/DIC affording 32, and the 2 cycles of piperidine/DMF were retained to deprotect the
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Fmoc group affording 33. Finally the NHS-(CH2)5-Mal was coupled using DIPEA in DMF
affording the resin-bound Boc-protected product (34).
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Detachment from Resin and Purification. Detachment of the product and deprotection was
initially achieved using trifluoroacetic acid affording crude 6, which could be purified by
Reverse Phase HPLC24,26. Initial deliveries were hindered by the low purity of the crude material
(HPLC purity of ca. 30% area), which could only be purified to an HPLC purity of 88% area
even with 2 sequential Reverse Phase HPLC runs.
To resolve this issue, significant process development work was carried out on all aspects of
the detachment, purification and isolation. This work was aided by the development of the SPPS
(described previously), which improved the HPLC purity of the crude material to 60-70% area.
Detachment and deprotection of 34 was found to be reliably achieved using 25% trifluoroacetic
acid in dichloromethane affording a solution of crude 6. Stability data was generated on the
various solutions of 6 post-cleavage and during purification. This work showed that solutions
held for extended periods should be stored at -20°C to avoid the formation of deacetylated mc-
Lys-MMETA (35).
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An investigation into whether precipitation of this material would improve purity was initiated,
and isolated crude material was azeotroped with toluene, DCM and diethyl ether affording a
gummy solid. Isolation of the product by precipitation from diethyl ether was investigated, but a
filterable solid could not be obtained due to the extremely hygroscopic nature of the product.
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Thus purification was undertaken using Reverse Phase HPLC followed by a further Reverse
Phase HPLC concentration step, with a focus on improving the system resolution to enable
separation of the product from impurities, as well as improving product loading, recovery and
stability.
Control of pH was found to be critical during the purification. Following purification, the pH
of product fractions was increased from pH 2. However, adjustment to pH 6 resulted in the
formation of a significant impurity, with levels up to 50% in the product fractions. The new
impurity was identified as the product where the amino group of the Lysine had cyclised onto the
maleimide group (36). Further studies confirmed that the formation of this impurity occurs at pH
6, but not at lower pH (4-5). It was not possible to reverse the Michael addition of the Lysine to
the maleimide, and the impurity ran very close to 6 in the Reverse Phase HPLC method, making
it impossible to purify the remaining product present to the required purity of >95%. As result of
this work, careful control of both mixing and pH control of all product fractions was introduced.
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Following the Reverse Phase HPLC purification and concentration, 6 was isolated by
lyophilisation, affording the product as a white solid with an assay of ca 73%w/w (corresponding
to the tri-TFA salt) and an HPLC purity of >95% area.
Synthesis Scale-Up. The improvements described above were introduced over a number of
campaigns of development, during which material was provided for toxicology and early clinic
studies. The largest delivery was completed using 2 SPPS runs (Scheme 6), using 300g of 2-
chlorotrityl chloride resin for each run. The two runs were completed with a synthesis scale of
170mmol and 158mmol and the purity of the crude 6 following cleavage from the resin was 64
and 70% respectively. Following the 2 Reverse Phase HPLC procedures, 122g of product was
obtained with a HPLC purity of 95.8%. The assay of the material was 71%w/w thus 86.4g of 6
was present (24% yield for SPPS and purification).
Scheme 6. Improved Synthesis of mc-Lys-MMETA (6)a
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aReagents and Conditions: (a) Chlorotrityl Resin, Et3N, DMF/DCM; (b) Piperidine, DMF; (c)
8a, OxymaPure, DIC, DMF; (d) Fmoc-Ile-F, BSA, DIPEA, DMF; (e) Piperidine, DMF; (f) 7,
OxymaPure, DIC, DMF; (g) Pd(PPh3)4, BH3.HNMe2, DCM; (h) Fmoc-Lys(Boc)-OH,
OxymaPure, DIC, DMF; (i) Piperidine, DMF; (j) NHS-(CH2)5-Mal, DIPEA, DMF; (k) TFA,
DCM, 24% (11 steps).
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Conclusions
An efficient and robust synthesis of an ADC payload based on the Tubulysin natural products
has been successfully developed and scaled-up. This work has included the development of new,
more efficient routes to tubuphenylalanine and tubuvaline portions of the molecule, as well as
improvements in the robustness of the SPPS assembly. A first scale-up of the chemistry had
afforded a yield of 1% for the synthesis of the tubuphenylaniline portion (9a) and a yield of 1%
for the SPPS, giving an overall yield of 0.01% for the longest linear sequence of the synthesis.
Following the development work, 9a was synthesised in a 10% yield, and with a 24% yield for
the SPPS, an overall yield of 2.4% for the longest linear sequence was achieved (a 240-fold
increase) delivering over 86g of 6. This work has supported toxicology and early clinic studies,
and further development is planned in anticipation of the progression of the molecule into later
clinical trials.
Associated Content
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at
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XXX.
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Full description of experimental procedures including large scale preparation of 8c using
original procedures.
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Author Information
Corresponding Author
*E-mail: jeremy.parker@astrazeneca.com.
Present address
Dorin Toader, Mersana Therapeutics Inc. 840 Memorial Drive, Cambridge, MA 02139, USA
Notes
The authors declare no competing financial interest.
Acknowledgements
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Will Goundry, Lorraine Graham, Matt Welham (project leadership); Kevin Leslie (asymmetric
hydrogenation screening); Ian Jones, Andrew Turner (NMR characterisation); Daniel Ma,
Kunshan Xie, Weifang Yuan, Yitian Zhang (large scale preparation of Tuv and Tub using
original procedures); Jianhui Sun, Youchu Wang, Jianqian Zhang (improved synthesis of Tup);
Heng Song, Jianbo Yang (improved synthesis of Tuv); Gregoire Bonvin, Laurent Ducry, Marcel
Suhartono, Andreas Peer (early SPPS development and manufacturing); Alistair Hay, Steve
McIntyre, Gareth McConville, Lynn O’Neill (late SPPS development and manufacturing).
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