D. Zhou et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
i
Scheme 8. aReagents and conditions: (a) propargylamine, HATU, Pr2EtN; (b) 2-azidoethan-1-ol (neat), NaHCO3; (c) 18, CuSO4⋅5H2O, sodium ascorbate, DMF.
Table 1
Cu(I)-catalyzed [3 + 2] cycloaddition with acetylene 18 to afford
neolymphostin-based linker-payload 20.
Characterization of ADCs.
The neolymphostin-based ADC precursors, linker-payloads 9, 11, 16
and 20, were then conjugated to site-specific cysteine mutant trastu-
zumab-A114C by 1,4-addition across the maleimide. The crude ADCs
were purified by size exclusion chromatography (SEC) using GE AKTA
Explorer system with GE Superdex 200 (10/300 GL) column and DPBS
(pH7.4) eluent. A mean drug-antibody ratio (DAR) of ADCs was de-
termined by analytical LC-MS method. The results were listed in
ADC Linker-
DAR
Theoretical Observed Yield after
payload
(drug/
antibody)
Δ mass
Δ mass
purification
21
22
23
24
9
1.6
1.6
1.9
17
993
678
693
764
994
678
695
765
38%
54%
66%
52%
11
16
20
assays were conducted as reported previously.10 Briefly, HER2 antigen
expressing cells (BT474 breast cancer, N87 gastric cancer, MDA-MB-
361-DYT2 breast cancer) or non-expressing cells (MDA-MB-468 breast
cancer) were seeded in 96-well cell culture plates for 24 h before
treatment. Cells were treated with 3-fold serially diluted antibody-drug
conjugates or free compounds (i.e., no antibody conjugated to the drug)
in duplicate at 10 concentrations. Cell viability was determined by
CellTiter 96® AQueous One Solution Cell Proliferation MTS Assay
(Promega, Madison, WI) 96 h after treatment. Relative cell viability was
determined as percentage of untreated control. IC50 values were cal-
culated using four parameter logistic model 203 with XLfit v4.2.
As shown in Table 2, the ADCs 21 and 22 displayed moderate cy-
totoxic potency against high HER2 (BT-474 and N87) expressing cell
lines in the range of 77 to 423 nM. Moreover, both ADCs showed > 6-
fold selectivity vs non-HER2 expressing cells (MDA-MB-468) compared
with the neolymphostin (1) payload alone. ADC 23 was approximately
as potent against BT-474 and N87 cell lines (IC50 = 286 nM and
274 nM, respectively) when compared to neolymphostin (1) alone
(IC50 = 97 nM and 210 nM, respectively). ADC 24 demonstrated cyto-
toxic activity against BT474 and N87 cell lines with IC50 of 195 and
202, respectively. Selectivity of > 4-fold was observed with trastu-
zumab ADCs 23 and 24 vs non-HER2-expressing MDA-MB-468 cells
compared with payload 1 alone. Furthermore, ADCs 23 and 24 de-
monstrated comparable in vitro cytotoxic activity against medium HER2
expressing MDA-MB-261-DYT2 cell line (IC50 of 162 and 216, respec-
tively) compared with high HER2 (BT-474 and N87) expressing cell
lines, providing additional evidence of the attractiveness of ADC-based
In searching for a practical method to conjugate neolymphostin (1)
with a linker, we became interested in utilizing the copper (I)-catalyzed
Huisgen cycloaddition of azides and terminal alkynes to form 1,2,3-
triazoles9 as the key transformation to construct a linker-payload. We
envisioned that the generality and mild reaction conditions of this ap-
proach would be suitable for such a labile payload 1.
To this end, we examined the feasibility of preparing neolymphostin
alkyne 16 and using it in the Cu(I)-catalyzed reaction with PEGylated
maleimide azide 15. As shown in Scheme 7, the alkyne bearing neo-
in neat propynyl alcohol. However, the azide component 15, prepared
by reacting 11-azido-3,6,9-trioxaundecan-1-amine with N-(methyoxy-
carbonyl)maleimide, was unstable at room temperature. Azide-acet-
ylene cycloaddition reaction was nonetheless carried out using copper
sulfate and sodium ascorbate under ambient temperature within
15 min, but the desired linker payload 16 was only isolated in less than
10% yield (presumably due to the instability of 15).
The poor reaction yield and unstable maleimido-azide partner led us
to consider an alternative route wherein a maleimide-containing alkyne
would be employed as a acetylenic counterpart in combination with
suitable neolymphostin-azide for the Cu(I)-mediated “click” cycload-
The requisite PEGylated maleimido-alkyne 18 was prepared by re-
action of propargyl amine with acid 17 in the presence of HATU.
Although the reaction of neolymphostin (1) with neat 2-azidoethanol in
the presence of sodium bicarbonate was sluggish, the resulting neo-
lymphostin azide 19 was isolated in good yield and readily underwent
Table 2
In vitro cytotoxicity of neolymphostin ADCs.a
ADC
Linker-payload
DAR
IC50 (nM)
(drug/antibody)
HER2+++
BT474
HER2+++
N87
HER2++
HER2-negative
MDA-MB-468
MDA-MB-361-DYT2
–
1
–
97.5(8)
77.3(4)
423(3)
286(3)
195(4)
210(10)
225(5)
251(3)
274(3)
202(1)
92.9(3)
ND
139(6)
21
22
23
24
9
1.6
1.6
1.9
1.7
> 1000(6)
> 1000(1)
> 1000(2)
> 750(2)
11
16
20
> 1000(1)
162(1)
216(2)
a
Relative antigen expression is classified as high (+++), medium (++), and low (−). Reported IC50 is the mean of the indicated number of independent
determinations (parentheses).
4