Angewandte
Communications
Chemie
Table 1: MAC analogue structures and hydrolysis under physiological
conditions.
MS, presumably because of rapid decomposition and release
of the free alcohol.
We next turned our attention toward the utility of the
MAC unit for ADCs using the potent microtubule-disrupting
[
5]
agent AE (2). The dimethylaminoethyl-containing construct
was selected, based on the stability characteristics from the
model compounds 10e and 10 f. Reaction of the chloromethyl
adduct 6b with 2 gave the AE-alkoxy carbamate 8h
1
2
3
[a]
Compound
R
R
R
Hydrolysis (%)
/14 days
7
(Scheme 1). Nitro group reduction afforded 9h, which was
coupled to Fmoc b-alanine (17) using EEDQ, to give 18.
Saponification with concomitant removal of the Fmoc
protecting group using aqueous lithium hydroxide, followed
by maleimide installation using the N-hydroxysuccinimide
ester 19, gave the fully elaborated MAC b-glucuronide AE
drug linker 1. The compound 1 was incubated in phosphate-
buffered saline at 378C, and like the model MAC constructs
possessing the dimethylaminoethyl residue (10e and 10 f), no
hydrolysis of the MAC-AE unit was observed over the 14 day
incubation period (Table 1; see Figure S1 in the Supporting
Information). As expected, b-glucuronidase treatment
resulted in clean and facile formation of AE (2; see Fig-
ure S2).
1
1
1
0a
0b
0c
Et
Et
Et
Ac
Ac
Ac
44/n.d.
0/0
61/n.d.
10d
Et
Ac
0/0
1
1
1
1
0e
0f
0g
1
Ac
Ac
Ac
Ac
MP
0/0
0/0
0/0
The method for conjugating 1 was as previously de-
H
100/n.d.
0/0
[
12]
[1]
scribed. Briefly, the anti-CD30 mAb cAC10 was partially
reduced with tris(2-carboxyethyl)phosphine and then purified
by gel filtration, thus affording approximately four free thiol
residues/mAb. Addition of a slight excess of 1 to the reduced
mAb resulted in conjugation with an average of 4 drugs/mAb.
The cAC10-1 conjugate was evaluated against CD30-
positive and -negative cell lines (Table 2). As a positive
[
b]
1
AE
[
a] In Gibco pH 7.4 (10X) PBS at 378C, as determined by LC-MS. [b] The
compound 1 undergoes maleimide hydrolysis to the ring-open form
during the course of the stability study. n.d.=not determined because of
instability at the 7 day time point.
saline and incubated at 378C for up to 14 days. Changes were
monitored by liquid chromatography/mass spectroscopy (LC/
MS) and integration of the parent peak of each model
compound was used to determine the degree of hydrolysis.
The results for the substituted carbamate model compounds
are shown in Table 1. The compounds 10a–d, with N-ethyl
carbamate residues derived from primary, secondary, tertiary,
and 1-napthyl alcohols (7a–d), were differentially stable. The
compounds 10b and 10d were completely stable up to 14
days, while the primary and tertiary alcohol constructs 10a
and 10c showed significant hydrolysis at 7 days.
[
a]
Table 2: In vitro activity and immunologic specificity.
ADC
Karpas 299
CD30 290K
ALCL
L540cy
CD30 433K
HL
Ramos
CD30 (À)
Burkitt’s
cAC10-1
hIgG-1
brentuximab vedotin
0.5
>1000
0.5
2
>1000
4
>1000
>1000
>1000
À1
[
a] Activity reported as IC50 value in ngmL of ADC. Cells were treated
with ADC for 96 h and viability was assessed using Cell TiterGlo.
Anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL).
To stabilize the MAC linkage, we introduced both basic
and electron-withdrawing groups proximal to the aminal
linkage. Replacement of the R ethyl group of 10a and 10c,
control for the assay, the anti-CD30 ADC brentuximab
vedotin, employing monomethyl auristatin E (MMAE) at-
tached by a valine-citrulline para-aminobenzyl carbamate
1
with a dimethylaminoethyl group, resulted in the compounds
[1]
1
0e and 10 f, respectively, which were stable for the 14 day
linker, was included (Figure 1). The ADC cAC10-1 was
comparable to brentuximab vedotin in activity on both the
antigen positive cells, and on the antigen negative cell line, in
which both constructs were relatively inactive. Further
evidence for antigen-dependent activity was obtained with
the nonbinding hIgG-1 conjugate, which was inactive (IC >
incubation period. Likewise, incorporation of the methane-
sulfonylethyl group in 10g also improved stability relative to
the N-ethyl compound 10a (and 10c), presumably by
reducing electron density at the carbamate nitrogen atom.
High electron density at the carbamate nitrogen atom may
explain why the secondary carbamate, 11, was not stable and
showed complete hydrolysis at 7 days.
Each model construct released the corresponding alcohol
compound when treated with bovine liver b-glucuronidase.
As anticipated, the intermediate aminal released from the
b-glucuronide MAC construct could not be detected by LC/
5
0
À1
1000 ngmL ) on all three cell lines tested. These results are
consistent with the high degree of stability observed with 1 in
aqueous buffer (Table 1). A stability assessment under
physiological-type conditions was also obtained by incubating
cAC10-1 in mouse plasma at 378C. An LC/MS assay detected
less than 1% AE (2) liberated after a 7 day incubation (see
Figure S3).
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3
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