A successful search for new, efficient, and silver-free manufacturing processes for key platinum(ii) intermediates applied in antibody-drug conjugate (ADC) production

Article abstract of DOI:10.1039/c9gc03130h

A silver-free amination procedure, here called "complexation", was developed to obtain a class of Pt(ii) complexes bearing a payload (such as a diagnostic or a therapeutic moiety). These complexes are crucial intermediates for the efficient development an

Full text of DOI:10.1039/c9gc03130h

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A successful search for new, efficient, and silver-
free manufacturing processes for key platinum(II)
intermediates applied in antibody–drug conjugate
Cite this: DOI: 10.1039/c9gc03130h
(
ADC) production†‡
a
a
a
a
Eugen Merkul, * Niels J. Sijbrandi, Ibrahim Aydin, Joey A. Muns,
b
b
a
Ruud J. R. W. Peters, Paul Laarhoven, Hendrik-Jan Houthoff and
Guus A. M. S. van Dongen
c
A silver-free amination procedure, here called “complexation”, was developed to obtain a class of Pt(II)
complexes bearing a payload (such as a diagnostic or a therapeutic moiety). These complexes are crucial
intermediates for the efficient development and production of antibody–drug conjugates (ADCs) based
on a novel Pt(II)-based linker technology. We termed this metal–organic linker, [ethylenediamineplatinum
2+
®
(
II)] , “Lx” . The present, newly developed procedure is a greener alternative for the classically applied
activation reaction of Pt-halido complexes with silver salts, followed by amination. The crucial finding is
that the leaving ligand of the classical process, chloride, can now be replaced by its higher homologue
iodide. This not only decisively improved the manufacturing process of the intermediate, but also was
found to be key to a more efficient conjugation procedure, i.e. a subsequent step in which this intermedi-
ate is coupled to an antibody. The new process allowed upscaling to be readily realized and the desired
intermediate was successfully manufactured on a multigram scale. The obtained Ag-free procedure can
be generalized and has a great potential to be applied for other Pt(II) complexes of high importance, such
as anti-cancer therapeutics.
Received 5th September 2019,
Accepted 20th December 2019
DOI: 10.1039/c9gc03130h
rsc.li/greenchem
process, as well as in the therapeutic efficacy and tolerability of
1
. Introduction
2
a–c
an ADC.
targeted to an improved ADC linker design by introducing
approach to treat cancer since they allow the delivery of toxic cationic metal–organic Pt(II)-based linker, [ethylenediamine-
Recently, we described a “plug-and-play” approach
Antibody–drug conjugates (ADCs) represent
a
a
2
+
® 3a–e
anti-cancer therapeutics selectively to the malignant cells, thus platinum(II)] , which we termed “Lx” .
The Lx concept
avoiding damage to healthy cells. Currently, six ADCs are consists of two key steps (Scheme 1).
®
1a
® 1b
®
approved by the FDA (Adcetris , Kadcyla , Mylotarg ,
In the first step (“complexation”), the commercially avail-
®
1c
2
Besponsa , the recent Polivy™, and the just approved able precursor complex LxCl bearing two chloride leaving
1
d
Padcev™) and >80 ADCs are under clinical evaluation. ADCs ligands can be coordinated to payloads bearing a suitable
comprise three components: a disease-selective monoclonal coordination group (payload-CG; CG typically is an
antibody (mAb), a small-molecule therapeutic payload, and a N-heterocycle such as piperidine (pip)), to provide storable
linker which connects mAb and payload to form a conjugate. intermediate products that we termed “semi-final” complexes.
Linkers are the most modifiable part of an ADC and can form In the second step (conjugation), the obtained “semi-final”
a critical factor in the efficiency and costs of the production complexes are conjugated to histidine residues of unmodified
mAbs. This novel ADC linker technology could become a game
changer in the field of next-generation ADCs.
a
We have also described earlier the distinguished in vitro
and preclinical in vivo targeting and therapeutic properties of
Lx-based ADCs. From the results that we obtained it can be
LinXis BV, De Boelelaan 1085c, Amsterdam, 1081 HV, The Netherlands.
E-mail: merkul@linxispharmaceuticals.com
b
3e
ChemConnection BV/Ardena Oss, Kloosterstraat 9, Oss, 5349 AB, The Netherlands
c
Department of Radiology and Nuclear Medicine, Amsterdam UMC, Location VU
concluded that the Lx-based ADCs are stable in serum and
Medical Center, Amsterdam, The Netherlands
3b
suitable for optimal tumor targeting. Results also revealed
†
‡
Dedicated to Lidia Aurelia Merkul on the occasion of her 5th birthday.
that Lx follows the biodistribution of an antibody and, unlike
Electronic supplementary information (ESI) available. See DOI: 10.1039/
C9GC03130H
platinum-based chemotherapeutics such as cisplatin, remains
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Scheme 3 The originally used classical Ag-mediated synthetic route
3
b
towards the crucial synthetic intermediate Cl-Lx-pip-AF (3d). pip =
piperidinyl.
2 3
vation of a sparingly soluble LxCl compound (2a) by AgNO in
DMF (e.g. a typical procedure can be found in ESI, 4.2.,‡ for
the synthesis of Cl-Lx-pip-AF (3d), and is represented in
3
b
Scheme 3). This activation step, in which ∼2 eq. of LxCl
2
(2a)
Scheme 1 The Pt(II)-based “Lx” approach to ADCs with its two crucial
steps: “complexation” and conjugation. mAb = monoclonal antibody,
CG = coordination group.
are activated with 1 eq. of AgNO – this to avoid a double acti-
3
vation of the precursor 2a leading to a double addition
product in which both chlorido ligands are substituted by the
payload –, was followed by the addition of a payload-CG com-
pound to achieve coordination of the modified payload moiety
to Pt(II).
an inert component of the ADC after in vivo administration
3
c
during the time frame of the investigation. This information
is important with respect to the tolerability and safety of Lx-
based ADCs. Currently we are developing a clinical candidate
This method has several drawbacks. First, Ag is a route-
5
dependent human toxicant added intentionally during the
3
b
manufacturing process, and therefore a risk assessment of
ADC trastuzumab-Lx-pip-AF
bearing the highly potent
+
4
a,b
such a process is necessary. Moreover, Ag has environmental
tubulin inhibitor auristatin F (AF).
Preclinical studies in
6
issues and needs to be removed from the waste water, as it is
tumor bearing mice showed that this anti-HER2 Lx-based ADC
7
®
highly toxic to a wide variety of organisms including bacteria.
outperformed the benchmark ADC Kadcyla , which is
Second, large amounts of DMF are used: these “complexation”
reactions are typically <0.01 M in substrate, and thus a large
amount of the high boiling point solvent DMF needs to be
removed under reduced pressure after completion of the reac-
tion in order to concentrate the reaction mixtures before the
purifications. This DMF removing step can be very tedious on
approved by the FDA for the treatment of patients with HER2-
positive metastatic breast cancer.
3
b
The crucial intermediates, which we call “semi-final” pro-
ducts to indicate that they are direct precursors of our “final”
+
Lx-ADC products, have a general formula [Cl-Lx-CG-payload]
(Scheme 2), where “payload-CG” typically stands for a diagno-
2
scale. Third, despite the use of an excess of LxCl (2a) for acti-
stic or a therapeutic payload modified with a coordination
group suitable for coordination to Pt(II).
vation with AgNO , some double activation does always occur
3
resulting in complexes bearing two payloads, and thus such
double addition products need to be separated from the
desired mono-addition products. And finally, the desired pro-
ducts need to be purified by preparative HPLC, known to be
very difficult in large scale production.
These storable “semi-final” complexes were initially pro-
duced on a laboratory scale using a classical approach via acti-
Since we aimed to manufacture our lead candidate trastuzu-
mab-Lx-pip-AF, which is obtained from the “semi-final”
complex Cl-Lx-pip-AF (3d), for toxicological and clinical trials,
it appeared necessary to improve the classical synthetic route
(
Scheme 3) to this crucial intermediate. We therefore aimed to
develop a scalable and greener route that would (1) avoid
usage of a Ag salt, (2) replace DMF or at least strongly reduce
Scheme 2 General structure of a “semi-final” product or complex.
CG = coordination group.
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its amount, and (3) avoid preparative HPLC for product purifi- program. Nevertheless,
a
high maximal conversion was
cation. Further, omission of a Ag salt would facilitate the achieved after 24 h at 80 °C (98.7%), while the product for-
product purification because Ag belongs to elements of class mation was 89.2%. Although this result already indicated that
2
B category according to the ICH Q3D guideline on elemental the process can be run without activation by Ag salts, the
−
1
impurities (parenteral intake limit of 10 µg day ) and would temperature at which the conversion became efficient (80 °C)
therefore require stringent removal. The analytical product was rather high considering our desire to develop a general
release would also be simpler since no Ag determination procedure that would also be applicable for more sensitive
would be required in case no Ag salt additive is used. Also for substrates. Interestingly, at 100 °C we observed an increase of
the sake of making the process as green as possible, an addi- the noreleagnine (1a) peak compared to 80 °C, indicating that
tive such as a Ag salt, if it is not absolutely necessary, has to be the product 3a apparently started to undergo a “de-complexa-
avoided. Therefore, a straightforward Ag-free and environmen- tion” process by which the chloride ligands that have been
tally benign production route appeared to be highly desirable.
released into the mixture during the reaction started to convert
the product back to noreleagnine 1a.
After this initial positive result, we reasoned that a replace-
ment of the chlorido leaving ligand in our key complex Cl-Lx-
CG-payload by its higher halido homologs, i.e. bromido or
iodido leaving ligands, might be the key to achieve our goal.
That would mean to perform the reaction of noreleagnine 1a
2
. Results and discussion
2
.1 Reaction of the model compound noreleagnine with the
dihalido Pt(II) complexes LxHal
2
Having a clear goal to eliminate a Ag salt auxiliary and to with the corresponding complexes LxBr2 (2b) and LxI2 (2c),
achieve direct complex formation with the platinum(II) which we expected to be more soluble in DMF or other organic
2
2
complex LxCl (2a), we first tried to react a model compound solvents then the classical hardly soluble complex LxCl (2a)
bearing the same coordination group as our lead candidate, (Scheme 5).
i.e. a piperidine (pip) moiety. For this purpose, we chose the
Indeed, since the halide ligand (Hal) will be lost anyway
commercially available indole natural product noreleagnine during the course of the subsequent conjugation reaction of
(
(
1a) and reacted it with an excess (1.5 eq.) of the complex LxCl
2a) under thermal conditions without AgNO (Scheme 4) in chloride can potentially be replaced by another leaving ligand,
2
the “semi-final” complex of compound class 3 to an antibody,
3
dry DMF (0.1 M in substrate 1a). It should be underlined that provided that the nature of such a new leaving ligand will not
no further additives or reagents were added to the reaction negatively affect the conjugation efficiency. Alternatively, since
mixture.
the literature suggested that those bromido and iodido con-
We started the reaction at 40 °C and increased the tempera- taining “semi-final” complexes could become less reactive
8
ture step-wise up to 100 °C with increments of 20 °C monitor- towards conjugation, we assumed that we could also achieve a
ing the conversion of the reaction and the product formation ligand exchange from the bromido or iodido leaving ligands
in the reaction mixture by HPLC (ESI, 3.1.1.‡). The product for- back to the chlorido leaving ligand after the completed “com-
mation is represented by the product peak area (a/a% value of plexation” reaction, simply by addition of an excess of a chlor-
the product 3a; a = area) on an HPLC chromatogram and the ide salt.
conversion is defined as product peak area divided by the sum
of starting material and product peak areas [a/a% excess of the complex LxBr2 (2b) (Scheme 6). Similar to the
product 3a)/a/a% (starting material 1a + product 3a)].
experiment with LxCl (2a), we started the reaction at 40 °C
We observed that the reaction did occur but was not par- and increased the temperature step-wise up to 80 °C with
ticularly clean. Because of the very low solubility of LxCl (2a), increments of 20 °C (ESI, 3.1.2.‡). The reaction time for each
To this end, as next we reacted noreleagnine 1a with an
(
2
2
the reaction mixture remained heterogeneous during the
whole course of the reaction until the end of the temperature
Scheme 4 The Ag-free “complexation” reaction of noreleagnine (1a)
Scheme 5 The Ag-free “complexation” reaction of noreleagnine (1a)
with LxHal complexes (2a–c).
with LxCl
2
(2a).
2
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complexation” reaction started to occur at a lower temperature
as well and was detectable already at 60 °C.
Because the reaction between noreleagnine (1a) and LxI
2c) was so efficient and clean, it was then repeated at 25 °C to
investigate its efficiency already at ambient temperature
Fig. 1).
We were pleased to find that the reaction proceeded
2
(
(
smoothly and was almost complete after 24–27 h. At 24 h reac-
tion time, 95.8% product 3c, 3.5% starting material 1a, and
0.7% intermediates or impurities were observed on HPLC.
After that, the conversion improved further giving 99.4%
product, 0.5% starting material, and only 0.1% intermediates
or impurities after 94 h reaction time. Interestingly, the total a/
a% of peaks that did not belong to the starting material 1a or
Scheme 6 The Ag-free “complexation” reaction of noreleagnine (1a)
with LxBr (2b).
2
temperature step was the same as in the previous experiment product 3c increased to ∼5% in the first 4–6 h but decreased
with LxCl (2a). Remarkably, due to a higher solubility of LxBr
later to only 0.1% after 94 h. That indicated that initially some
2b) compared to LxCl2 (2a), the reaction mixture became intermediates rather than impurities were formed, which were
2
2
(
homogeneous already at 40 °C after stirring for several hours. later transformed to the desired product 3c to give a very clean
Accordingly, we achieved a higher conversion (99.8%) and a HPLC chromatogram (Fig. 2).
better product formation (95.7%) at a lower temperature of
0 °C compared to the reaction with LxCl
(2a). The “de-com- without using preparative HPLC simply by diluting the reac-
plexation” reaction in this experiment started to occur at tion mixture with water, followed by filtration of the precipi-
0 °C.
tated unreacted/excessive LxI (2c) and applying the filtrate on
Finally, we performed the same experiment as performed the reverse phase silica gel RP-C18 (Merck LiChroprep
The isolation of the desired product 3c was possible
6
2
8
2
with LxCl2 (2a) and LxBr2 (2b) with the synthetically readily (15–25 μm)). A yield of 85% was obtained in total, with the
available (ESI, 2.2. and 5.2.‡), also on a large scale (ESI, 6.5.‡), purest fraction (53% yield) having the product purity of 98.5%
diiodido complex LxI
2
(2c) (Scheme 7). Since this complex has after addition of NaI (to improve the stability of the lyophili-
a very high solubility in DMF, the reaction mixture was homo- zate, vide infra) and lyophilization. This result indicated that
geneous already at room temperature just after mixing the manufacturing without the use of preparative HPLC for
components and adding the solvent.
product purification is possible.
The reaction with the complex 2c impressed with its almost
To look into the “de-complexation” reaction more closely,
quantitative conversion (99.6%) and high purity (99.3% we performed a reaction of noreleagnine (1a) with LxI (2c) at
2
product 3c, along with 0.4% starting material 1a) that were 25 °C in the presence of different amounts of KI (0.2–5.0 eq.).
observed already at 40 °C (ESI, 3.1.3.‡). Its high efficiency and After 69 h reaction time, it was observed that the amount of KI
essentially lack of impurities (0.2%) suggested that the did not influence the conversion (ESI, 3.2.‡). Then, the temp-
product could be isolated without preparative HPLC, which is erature was increased step-wise to 40 °C, 60 °C, and 80 °C. It
a further improvement towards a greener alternative of the was observed that the conversion slightly decreased – also in
classical Ag-mediated “complexation” method. Thus, the the absence of KI due to the previously observed thermal “de-
complex 2c continued to follow the trend in the halide series, complexation” – but was again hardly influenced by the
giving the highest reaction efficiency among the homologous amount of KI (at 80 °C after 6 h reaction time, conversions
complexes 2a–c at even lower temperature (40 °C). The “de- were 87% and 82% for no KI added and 5.0 eq. KI added,
respectively).
Scheme 7 The Ag-free “complexation” reaction of noreleagnine (1a)
with LxI (2c).
Fig. 1 Reaction kinetics of noreleagnine (1a) with the diiodido complex
LxI (2c) at 25 °C.
2
2
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Table 1 Screening of solvents in the reaction of noreleagnine 1a with
LxI (2c). The best conversions are printed in bold
2
Conversion (%)
Entry
Solvent
24 h
82.1
80.7
76.9
77.3
1.3
48 h
91.1
89.9
87.9
88.7
2.6
158 h
a
1
2
3
4
5
6
7
8
9
DMF
DMF + 1 vol% H
DMF + 10 vol% H
DMA
MeOH
EtOH
MeCN
DMSO
Acetone
Ethyl acetate
NMP
DMI
DMPU
95.4
Fig. 2 The HPLC chromatogram of the reaction mixture of noreleag-
2
O
2
O
94.5
94.3a
95.2
—
—
—
81.3
—
—
—
—
nine (1a) with LxI
73 nm. t (1a) = 8.2 min (0.5 a/a%), t
.8 min (0.1 a/a%), t (3c) = 11.5 min (99.4 a/a%).
2
(2c) at 25 °C after 94 h reaction time, measured at
2
R
R
(unidentified intermediate) =
9
R
0.4
0.9
23.8
73.5
12.0
0.6
73.5
73.8
65.8
23.6
75.5
16.1
0.5
84.0
82.7
79.6
It should be noted that no undesired double addition
product could be identified by HPLC for the “complexation”
reactions using all three complexes LxHal
1
1
0
1
2a–c, in contrast to 12
2
13
—
the Ag-promoted procedure that is always plagued by the for-
mation of the double-substituted product, which then needs to
be separated from the desired mono-substituted product by
preparative HPLC. To further challenge the formation of only
the desired product 3c, we performed the reaction between 1.0
a
The reaction time was 136 h instead of 158 h.
not dissolve and the reaction mixtures remained hetero-
geneous until the end of the experiment. However, reactions in
NMP, DMI, and DMPU (entries 11–13) were found not clean.
Correspondingly, the best conversions and cleanest reaction
mixtures were observed in DMF, DMA, and DMSO (entries 1–4,
and 8); in almost all of the solvents that remained hetero-
geneous the reactions showed either no conversion at all (e.g.
2
eq. of LxI (2c) and 3.0 eq. of noreleagnine (1a) at 40 °C. HRMS
analysis indicated that no traces of the double addition
product were formed even after 50 h reaction time.
2
.2 Optimization of the “complexation” reaction between
noreleagnine and LxI
2
After these very encouraging results which indicated that the in MeOH (entry 5), EtOH (entry 6), and ethyl acetate (entry 10)
diiodido complex LxI
(2c) might become the new Pt(II)-precur- after 48 h reaction time) or a low conversion (16% in acetone
2
sor of choice for the synthesis of Lx “semi-final” complexes of (entry 9) and 24% in MeCN (entry 7) after 48 h reaction time).
compound class 3, we decided to optimize this direct “com- It should be noted that DMF that contained water performed
plexation” reaction using noreleagnine (1a) and LxI
(2c). similarly well compared with dry DMF (entries 1–3). To sum-
2
Thus, we screened the following parameters that we con- marize, fastest, cleanest, and highest conversions were
sidered to be of importance for the process upscaling: sol- achieved in DMF (91%; entry 1), closely followed by the reac-
vents, addition of an organic base, stoichiometry of the LxI
complex (2c), and reaction molarity (amount of DMF used).
2
tion in DMA (89%; entry 4) and DMSO (76%; entry 8) after
48 h reaction time. DMSO performed surprisingly well com-
For the initial solvent screening we made the following pared with DMF and DMA; however, at longer reaction times
selection (we used dry solvents to avoid possible hydrolysis of the reaction mixture became unclean. It is also known that
the product 3c leading to formation of the hydrolyzed complex DMSO, although being a very popular solvent for compounds
2
+
[H
2
O-Lx-noreleagnine] ): DMF, DMA, MeOH, EtOH, MeCN, to be tested in biological systems, is strongly discouraged to be
9
a,b
DMSO, acetone, ethyl acetate, 1-methyl-2-pyrrolidinone (NMP), used as a solvent for dissolution of cisplatin analogues.
In
1
3
,3-dimethyl-2-imidazolidinone (DMI), and 1,3-dimethyl- all three solvents the formation of the desired product was
,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU). This initial confirmed by HRMS analyses. Therefore, at the end of this
screening was performed at 25 °C, the concentration was experiment, we decided to isolate product 3c from the reaction
.1 M in noreleagnine (1a), and a strictly stoichiometric mixtures of the three best solvents DMF, DMA, and DMSO,
amount (i.e. 1.0 eq.) of LxI (2c) was used (Table 1, ESI, 3.3.‡).
and the isolated yields/purities of product 3c were 71%/97%,
0
2
Only in DMF, DMA, DMSO, NMP, DMI, and DMPU the mix- 71%/97%, and 47%/77% after preparative chromatographic
tures became homogeneous; in the other solvents LxI (2c) did purification and lyophilization, respectively.
2
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Next, we decided to screen the “complexation” reaction in idine, N-methyl morpholine (NMM), 1,8-diazabicyclo[5.4.0]
DMF analogues (other than DMA), i.e. solvents structurally undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), 2,6-
similar to DMF. Thus, the following solvents were screened: lutidine, and 2,4,6-collidine. The screening was performed at
N-formyl pyrrolidine, N-formyl piperidine, N-formyl morpho- 25 °C, the concentration was 0.1 M in noreleagnine (1a), and a
line, diethyl formamide, diisopropyl formamide, methyl for- strictly stoichiometric amount (i.e. 1.0 eq.) of LxI2 (2c) was
mamide, formamide, and tetramethyl urea. Similarly to the used (Table 3, ESI, 3.5.‡).
previous experiment, this screening was performed at 25 °C,
We found that the base had a pronounced effect on the
the concentration was 0.1 M in noreleagnine (1a), and a strictly “complexation” reaction with none of them improving the con-
stoichiometric amount (i.e. 1.0 eq.) of LxI
Table 2, ESI, 3.4.‡).
2
(2c) was used version. E.g. DBU completely prevented the formation of the
desired product (entry 6). Compared to the “complexation”
(
In none of the solvents tested was the reaction clearly better reaction without a base, only three bases showed a similar con-
than in DMF (that was used as a standard solvent in this version and no additional impurities: N-methyl morpholine,
experiment and showed a conversion of 95% after 136 h reac- 2,6-lutidine, and 2,4,6-collidine (entries 5, 8, and 9). TEA
tion time; entry 1), although five solvents showed conversions retarded the conversion (by ∼10% after 96 h reaction time,
comparable to DMF (95%, 94%, 95%, 91%, and 90% in entry 2); however, it is also a stronger base compared with the
N-formyl pyrrolidine, N-formyl piperidine, N-formyl morpho- ones above. So, for some salts this base will need to be used.
line, methyl formamide, and tetramethyl urea, respectively; DIPEA showed an even more pronounced retardation of the
entries 2–4, 7, and 9). In diethyl formamide the reaction was conversion (by ∼20% after 96 h reaction time, entry 3). Thus,
much less efficient when compared to DMF (conversion of 55% its use in the “complexation” reaction is not recommendable.
was observed after 24 h reaction time (entry 5), compared to
4% in DMF (entry 1)). In diisopropyl formamide and in forma- regarding LxI
mide the reactions showed low conversions (37% (entry 6) and ranging from 1.0 to 5.0 eq. The screening was performed at
9% (entry 8) after 24 h reaction time, respectively) and the reac- 25 °C and the concentration was 0.1 M in noreleagnine (1a)
tion mixtures remained heterogeneous until the end of the (Table 4, ESI, 3.6.‡).
As next, we examined the stoichiometry of the reaction
8
2
(2c), using different stoichiometries of LxI (2c),
2
1
experiment. Thus, after this intensive solvent screening, DMF
2
Expectedly, increasing amounts of LxI (2c) increased and
remained the solvent of choice for the “complexation” reaction.
accelerated the conversions. We found that for long reaction
Since payloads bearing amines or functionalized with times, e.g. >72 h, already at 25 °C a conversion of >95% was
amines as coordination groups are sometimes used in the possible using all the stoichiometries examined, even 1.0 eq.
form of their salts (e.g. as a TFA salt) rather than as free bases, of LxI (2c). Using 1.5–2.0 eq. of LxI (2c), which is a prepara-
2
2
we decided to investigate the influence of organic bases on the tively reasonable amount also for working on large scale, con-
complexation” reaction. Thus, we tested conversions in the versions of >95% and of ∼100% were achieved after 24 h and
presence of an excess (5.0 eq.) of following bases: triethyl- 72 h reaction time, respectively (entries 4 and 5). It is note-
amine (TEA), diisopropylethylamine (DIPEA), N-methyl piper- worthy mentioning that the excess of LxI (2c) can be easily
“
2
Table 2 Screening of DMF analogues as solvents in the reaction of nor-
Table 3 Screening of organic bases in the reaction of noreleagnine 1a
eleagnine 1a with LxI
2
(2c). The best conversions are printed in bold
with LxI
2
(2c). The best conversions are printed in bold
Conversion (%)
Conversion (%)
Entry
DMF analogue
24 h
45 h
136 h Entry
Base
24 h
48 h
96 h
1
2
3
4
5
6
7
8
9
DMF (control)
83.6
78.5
82.5
87.0
55.3
29.8
76.5
14.2
70.1
87.7
85.3
87.7
90.6
66.7
37.4
79.3
18.9
80.3
95.3
94.6
93.6
95.2
—
—
91.3
—
1
2
3
4
5
6
7
8
9
No base (control)
TEA
DIPEA
N-Me piperidine
NMM
DBU
DABCO
2,6-Lutidine
2,4,6-Collidine
83.9
62.6
44.4
70.9
82.3
0.0
66.0
82.0
82.0
91.2
77.0
64.6
81.6
89.8
0.0
78.9
89.3
89.5
94.6
85.6
78.0
87.0
93.8
0.0
78.1
92.7
93.7
N-Formyl pyrrolidine
N-Formyl piperidine
N-Formyl morpholine
Diethyl formamide
Diisopropyl formamide
Methyl formamide
Formamide
Tetramethyl urea
89.8
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Table 4 Screening of the stoichiometry of the reaction of noreleagnine
Table 5 Screening of the molarity of the reaction of noreleagnine 1a
1
a with LxI
2
(2c)
2
with LxI (2c)
Conversion (%)
Conversion (%)
Entry
Eq. of LxI
2
(2c)
2 h
24 h
48 h
72 h
Entry
V (DMF)/concentration
24 h
48 h
144 h
1
2
3
4
5
6
7
8
1.0
1.1
1.2
1.5
2.0
3.0
4.0
5.0
27.5
29.9
32.6
40.5
51.3
66.9
76.7
82.5
84.1
86.3
89.6
95.1
98.4
99.7
99.8
99.8
92.4
93.9
96.1
98.9
99.7
99.9
99.9
100
95.1
96.5
98.1
99.7
99.8
99.9
99.9
100
1
2
3
4
5
6
7
8
9
50 µL/1 M
100 µL/0.5 M
95.1
95.8
92.8
89.5
87.6
84.6
81.3
79.0
76.8
74.0
71.8
97.1
97.1
95.3
93.3
93.1
91.2
89.0
87.8
86.4
84.7
83.4
97.3
97.8
97.1
96.2
97.0
96.2
94.9
94.5
94.3
93.3
92.8
200 µL/0.25 M
300 µL/0.167 M
400 µL/0.125 M
500 µL/0.1 M
600 µL/0.083 M
700 µL/0.071 M
800 µL/0.063 M
900 µL/0.056 M
1000 µL/0.05 M
1
1
0
1
removed and recovered by simply diluting the reaction mixture
with water and filtration of the thus formed precipitate (which
also can be reused if it is desirable, vide infra).
formed at short reaction times (5.5%, 0.7%, and 0.3% at 1 M,
.1 M, and 0.05 M, respectively, after 24 h), which disappeared
As an indicator for the robustness of manufacturing, we
0
2
also tested if there is a difference if the complex LxI (2c) is
again during the course of the reaction. Therefore, the stan-
dard concentration of 0.1 M or higher concentrations up to 1
M (entries 1–6) appear reasonable to be used on the laboratory
as well as on multigram scale, whereas working at concen-
trations lower than 0.1 M seems to be of no use (entries 7–11).
Finally, we performed an experiment in which the excess
added as one batch (2.0 eq.) or e.g. in two batches (2 × 1.0 eq.;
the second batch being added 24 h after the first batch). Two
reactions performed in parallel revealed no difference in the
final outcome, despite of course that the reaction with 2.0 eq.
of LxI (2c) was faster for shorter reaction times (after 24 h the
2
conversions were 98% (2.0 eq. LxI
2
(2c)) and 81% (1.0 eq. LxI
2
(
2
∼1.0 eq.) of the Pt-precursor complex LxI (2c) was recovered
(
2c))). Both reactions showed similar conversions (99.7% and
and reused in three subsequent runs. The runs were per-
formed at 25 °C (Table 6, ESI, 3.9.‡).
We could demonstrate that just by diluting the “complexa-
tion” reaction mixture with water (DMF : water = 1 : 1) after
9
9.3%, respectively) and purity of the reaction mixtures
(
∼0.3–0.7% starting material, ∼99.3–99.7% product, and
0.0–0.1% intermediate) after 48 h reaction time (ESI, 3.7.‡).
This result shows that if the conversion is not satisfactory,
simply more LxI (2c) can be added; this observation can be of
∼
2
relevance for a large-scale manufacturing.
As next, we studied the influence of the concentration of
2
Table 6 LxI (2c) recovery runs
noreleagnine (1a) and LxI
2
(2c), i.e. the influence of the
amount of solvent DMF (the standard concentration used in
all the previous experiments was 0.1 M in noreleagnine 1a), on
the reaction conversion. To this end, we used 11 different con-
centrations between 0.05 M and 1 M. The screening was per-
formed at 25 °C and a strictly stoichiometric amount (i.e. 1.0
eq.) of LxI (2c) was used (Table 5, ESI, 3.8.‡).
2
We found that, as expected, the reaction was considerably
faster at higher concentrations (95%, 85%, and 72% at 1 M,
0
.1 M, and 0.05 M, respectively, after 24 h; entries 1, 6, and 11)
Scale (2c)/ Conversion to
Product purity
of 3c/(%)
Recovery of
2c/(%)
but the conversions became comparable after a prolonged
reaction time (97%, 96%, and 93% at 1 M, 0.1 M, and 0.05 M,
respectively, after 144 h). It was comparably clean at all con-
centrations tested. However, at higher concentrations also a
higher amount of an unidentified reactive intermediate was
Run [mg]
3c/(%)
1
2
3
4
509
202
83
99.3
98.8
99.3
99.3
99.0
98.8
99.3
99.3
80
83
73
53
30
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4 h reaction time and filtration of the thus formed precipi- 2.4 Results of the conjugation of the next-generation “semi-
tate, followed by washing and drying, ∼80% of LxI (2c) could final” complex I-Lx-pip-AF with trastuzumab
2
be easily recovered (except for the last run 4, in which ∼53% 2c
With the lyophilizate 3e in hands, we successfully synthesized
were recovered probably due to small amount of remaining
material and thus a relatively high loss of material during the
filtration step). The recovery can be further improved by apply-
ing a larger amount of an aqueous solution, as was demon-
strated later during the manufacturing campaign: the complex
the corresponding ADC 4 (Scheme 9) with unchanged pro-
perties compared with an ADC 4 synthesized from the chlorido
“semi-final” complex 3d.
This result means that the kind of the leaving group does
not affect the properties of the obtained Lx-ADCs of compound
class 4. In fact, we found that conjugation of I-Lx-pip-AF (3e) to
mAbs is much more efficient than the conjugation of Cl-Lx-
pip-AF (3d); this unexpected improvement was found to be
2
LxI (2c) could be recovered quantitatively (ESI, 6.6.‡). The
model experiment described above showed that the recovered
LxI2 (2c) possessed a similar quality compared to the fresh
material (used in run 1), as demonstrated by similar conver-
sions (∼99%) and product purities (∼99%) obtained in these
runs after 24 h at 25 °C. No Pt black was observed, even after
10
general and forms a counterintuitive separate finding that
will be described in details elsewhere (manuscript in prepa-
ration). Thus, the whole Lx approach to ADCs improved
greatly, with respect to the economics of manufacturing as
well as to environmental and safety aspects, from this see-
mingly subtle change of the leaving ligand from chloride to
iodide (Scheme 10).
2
the last run 4. The recovered material LxI (2c) only contained
a small amount of the product 3c, as detected by analytical
HPLC. Thus, our procedure allows to recover the excessive Pt-
precursor 2c quantitatively from the reaction mixture, giving
an opportunity to recycle or reuse the complex easily.
The change of the Pt-precursor complex from LxCl (2a) to
2
2
LxI (2c) not only gave an improvement of the overall Lx
technology and allowed to omit Ag additives, as outlined
above, but also allowed to reduce the costs of the Pt-precursor,
2
.3 Application of the improved “complexation” reaction for
the large scale synthesis of I-Lx-pip-AF
The described Ag-free amination procedure was successfully thus making the technology even greener. Although LxCl
applied in our laboratory on a milligram scale (ESI, 5.‡) and is commercially available, its price is rather high: 367 € per 1 g
8.0 mg of the “semi-final” complex I-Lx-pip-AF (3e) × 2.0 TFA (Aldrich, product code 244929). On the other hand, LxI (2c),
2
(2a)
1
2
in 98.9% purity were obtained. Then, it was applied without although not commercially available, can be made very easily,
issues on a multigram scale (Scheme 8, ESI, 6.‡) to produce in high yield (97.5%) and purity (the complex batches are con-
1
6.0 g of I-Lx-pip-AF (3e), formulated as a lyophilizate (ESI, sistently pure by elemental analysis), from the commercially
6
.7.‡) with a composition I-Lx-pip-AF (3e) × 2.0 TFA × 2.5 NaI available K
2
PtCl
4
, which price is much lower than that of LxCl
2
(
in total, 22.6 g of this lyophilizate were obtained). It should be (2a): 392 £ per 10 g (Fluorochem, product code 053555). LxI
2
noted that NaI was added as an excipient in order to increase (2c) could be easily prepared on a 12 g scale during the first
the product purity (the HPLC purity increased from 98.2%, manufacturing campaign. Moreover, preparation of the diio-
which was the average purity of five combined batches, to dido complexes introduced modularity in respect to the Lx var-
9
9.5%) and to ensure product stability during the solvent evap- iants: the bidentate ligand can now be varied easily, thus
oration, presumably by preventing hydrolysis and/or convert-
ing the already hydrolysed product H O-Lx-pip-AF back into
2
the desired product 3e.
Scheme 8 The new large scale manufacturing route towards the lyo-
philizate I-Lx-pip-AF (3e) × 2.0 TFA × 2.5 NaI. pip = piperidinyl, TFA =
trifluoroacetic acid.
Scheme 9 Synthesis of the Lx-ADC trastuzumab-Lx-pip-AF 4 from the
lyophilizate I-Lx-pip-AF (3e) × 2.0 TFA × 2.5 NaI. pip = piperidinyl, TFA =
trifluoroacetic acid.
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(
>95%), isolated yield, and product purity; (3) drastical
reduction of solvent volume; (4) recovery and recyclability of
the precursor LxI complex; and (5) formation of only the
2
desired mono-substituted product with no double substi-
tution, as confirmed by HPLC and HRMS. The product
obtained during the described manufacturing campaign is
being used successfully for the production of an anti-cancer
antibody–drug conjugate (ADC) which will undergo toxicologi-
cal and clinical studies in the near future. It turned out that
changing the leaving ligand from chloride to iodide not only
was a successful strategy in the “complexation” reaction but
1
1
was also found to be a crucial invention for an improved con-
1
0
jugation efficiency. It should be mentioned that the Lx conju-
gation using the new generation of the iodido “semi-final”
complexes described herein works generally and reliably with
various payloads and mAbs and is not restricted to the herein
presented noreleagnine and AF based “semi-final” complexes
and trastuzumab as a mAb. This conjugation work will be
described later elsewhere in a great detail. Finally, the
obtained Ag-free procedure can be potentially applied in the
synthesis of other Pt(II) or other transition metal complexes of
1
2a–i
high importance as anti-cancer therapeutics.
Scheme 10 The improved Lx approach to ADCs operating with LxI
2
Conflicts of interest
(
2
2c) instead of LxCl (2a). mAb = monoclonal antibody, CG = coordi-
nation group.
E. Merkul, N. J. Sijbrandi, J. A. Muns, and I. Aydin are
employed by LinXis B. V., H.-J. Houthoff is Scientific Director
of LinXis B. V. and has ownership (including patents) in
LinXis B. V., G. A. M. S. van Dongen is a non-profit scientific
advisory board member of LinXis B. V. R. J. R. W. Peters and
P. Laarhoven are employed by Ardena/ChemConnection B. V.,
which is a CDMO contracted by LinXis B. V. for the large-scale
manufacturing.
giving access to a family of diiodido complexes, and is not
limited to ethylenediamine as it is the case with the commer-
cial LxCl (2a). These new Lx family members are currently
2
under investigation and the results will be published
elsewhere.
We are now planning to apply the described “complexation”
method in the GMP manufacturing of the “semi-final” product
3
e, followed by a GMP manufacturing of the corresponding
Acknowledgements
Lx-ADC 4 for our ongoing clinical trials.
The authors thank Prof. Dr Jan Reedijk (Leiden University) for
critical reading of the manuscript.
3
. Conclusions
A greener (environmentally less demanding) and more practi-
cal route towards a crucial Pt(II) intermediate for ADC pro-
duction bearing the cytotoxic payload auristatin F and an
iodido leaving ligand, has been developed. This synthesis was
successfully implemented on a multigram scale and the thus
obtained important synthetic intermediate was successfully
used in a subsequent bioconjugation reaction to obtain the
Lx-based ADC trastuzumab-Lx-pip-AF. Additionally, we demon-
strated that the excess of the Pt-diiodido complex that was
used to achieve good conversions could be completely recov-
ered, thus avoiding waste. The process demonstrated the
following noteworthy improvements over the classical
Ag-mediated route: (1) Ag-free direct and operationally simple
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