Design, synthesis, and biological evaluations of tumor-targeting dual-warhead conjugates for a taxoid-camptothecin combination chemotherapy

Article abstract of DOI:10.1021/jm500631u

Novel tumor-targeting dual-warhead conjugates, 2 (DW-1) and 3 (DW-2), which consist of a next-generation taxoid, 1 (SB-T-1214), and camptothecin as two warheads, self-immolative disulfide linkers for drug release, biotin as the tumor-targeting moiety, and 1,3,5-triazine as the tripod splitter module, were designed and synthesized. The potency of 2 was evaluated against MX-1, MCF-7, ID8, L1210FR (BR+, biotin receptor overexpressed) and WI38 (BR-, normal) cell lines in the absence and presence of glutathione (GSH), which is an endogenous thiol that triggers drug release inside the cancer cells. With the GSH and resuspension protocol, 2 exhibited IC₅₀ values of 3.22-9.80 nM against all BR+ cancer cell lines, and 705 nM against WI38. Thus, there was a two orders of magnitude higher selectivity to cancer cells. Also, a clear cooperative effect was observed for the taxoid-camptothecin combination when two drugs were delivered to the cancer cells specifically in the form of a dual-warhead conjugate.

Full text of DOI:10.1021/jm500631u

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Article
Design, Synthesis and Biological Evaluations of Tumor-Targeting Dual-
Warhead Conjugates for a Taxoid-Camptothecin Combination Chemotherapy
Jacob G. Vineberg, Edison S Zuniga, Anushree Kamath, Ying-Jen Chen, Joshua D Seitz, and Iwao Ojima
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500631u • Publication Date (Web): 05 Jun 2014
Downloaded from http://pubs.acs.org on June 11, 2014
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Design, Synthesis and Biological Evaluations of
Tumor-Targeting Dual-Warhead Conjugates for a
Taxoid-Camptothecin Combination Chemotherapy
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Jacob G. Vineberg†, Edison S. Zuniga†, Anushree Kamath‡, Ying-Jen Chen†, Joshua D.
Seitz†, Iwao Ojima†,‡,*1
†Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400
‡Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794-
3400
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
*1 To whom correspondences should be addressed. Phone 631-632-1339; fax 631-632-7942;
e-mail, iwao.ojima@stonybrook.edu
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ABSTRACT:
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Novel tumor-targeting dual-warhead conjugates, 2 (DW-1) and 3 (DW-2), which consist of a next-
generation taxoid, 1 (SB-T-1214), and camptothecin as two warheads, self-immolative disulfide linkers
for drug release, biotin as the tumor-targeting moiety and 1,3,5-triazine as the tripod splitter module, were
designed and synthesized. The potency of 2 was evaluated against MX-1, MCF-7, ID8, L1210FR (BR+:
biotin receptor overexpressed) and WI38 (BR-: normal) cell lines in the absence and presence of
glutathione (GSH), which is an endogenous thiol that triggers drug release inside the cancer cells. With
the GSH and re-suspension protocol, 2 exhibited IC50 values of 3.22-9.80 nM against all BR+ cancer cell
lines, and 705 nM against WI38. Thus, there was a two-orders of magnitude higher selectivity to cancer
cells. Also, a clear cooperative effect was observed for the taxoid–camptothecin combination when two
drugs were delivered to the cancer cells specifically in the form of a dual-warhead conjugate.
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INTRODUCTION
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Over the past several decades, combination chemotherapy has been serving as a primary treatment option
for many types of cancers. Compared to the use of a single cytotoxic agent, the use of two or more
properly selected agents in combination can lead to a decrease in systemic toxicity and an increase in
efficacy due to synergistic or cooperative effects of the drugs on the tumor eradication.1 Furthermore, a
sequential treatment with chemotherapeutic agents bearing different mechanisms of action has been
shown to avert drug resistance and lead to synergistic enhancement of efficacy.1, 2
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Despite the profound advantages of combination chemotherapy, the lack of tumor-specificity
continues to be a serious issue for cancer treatment. In the last decade, significant advancement has been
made on the development of tumor-targeted drug delivery systems (TTDDSs), especially for monoclonal
antibody-drug conjugates (ADCs),3-9 and small molecule drug conjugates (SMDCs).7, 10-15 However, only
one such tumor-targeting dual-drug conjugate has been reported for the combination of mitomycin C and
desacetylvinblastine using folate as the tumor-targeting module.10, 16 Thus, there is an unmet need for the
development of efficacious TTDDSs amenable to anticancer drug combinations. Since a TTDDS delivers
anticancer drugs through receptor-mediated endocytosis (RME), it should be able to circumvent multidrug
resistance caused by ABC transporter efflux pumps such as P-glycoprotein, in the cancer cell membrane,17-
19 which is one of the beneficial features of TTDDSs.
We set out to design a versatile TTDDS platform for two-drug combinations, and construct “dual-
warhead guided molecular missiles20” for tumor-targeted combination chemotherapy. Such a TTDDS
would have only single pharmacokinetics for the delivery of two different drugs to tumor since this
conjugate is a single molecule, which can get around potentially complicated treatment regimen to deal
with two different pharmacokinetic parameters for two drugs. This is a salient feature of the dual-warhead
TTDDS.
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For the selection of two drugs (warheads) for the novel “dual-warhead molecular missiles”, we
focused on the combination of a microtubule-stabilizing agent (taxanes) and a topoisomerase I inhibitor
(camptothecin and its analogs) in this study. One of the primary reasons for the selection of this
combination is the fact that these two classes of anticancer drugs possess clearly different and well-defined
molecular targets to investigate their synergistic or additive effects. Combinations of paclitaxel or
docetaxel (taxane) with topotecan or irinotecan (camptothecin analog) have been extensively studied for
preclinical and clinical drug development, exhibiting synergistic or additive effects depending on the
sequence of drug administration.21-24 For taxane anticancer agents, we have been developing highly potent
next-generation taxoids, which exhibit 2-3 orders of magnitude greater potency than paclitaxel and
docetaxel against multidrug resistant and paclitaxel-resistant cancer cell lines and tumors.25-30 Thus, we
have used one of these next-generation taxoids for combination with camptothecin in this study. We
report here the design, synthesis and biological evaluations of novel tumor-targeting dual-warhead
conjugates, bearing a next-generation taxoid, 1 (SB-T-1214),28 and camptothecin as warheads, and biotin
as the tumor-targeting module.
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Figure 1
We designed a versatile platform, consisting of 1,3,5-triazine as the key tripod splitter module,
self-immolative linkers with tetraethylene glycol diamine spacers to improve water-solubility, and a
propargylamine arm for the attachment of second warhead module. Then, novel dual-warhead conjugate,
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2 (DW-1), was synthesized based on this platform using “click” chemistry to attach the camptothecin
module with a self-immolative linker and a tetraethylene glycol diamine spacer (Figure 2). Another dual-
warhead conjugate, 3 (DW-2), bearing the camptothecin module with a simple ester linkage, was also
synthesized to secure stepwise drug release (Figure 2). In addition to these dual-warhead conjugates, we
also designed two more drug conjugates using the same platform, wherein one of the warheads was
replaced with a surrogate, i.e., phenol. Thus, a conjugate with taxoid 1 and phenol, 4 (SW-Tax) and another
with camptothecin and phenol, 5 (SW-CPT) were synthesized in order to properly compare the efficacy
of dual-warhead vs single-warhead in the same conjugate scaffold (Figure 3).
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2
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Figure 3. Chemical structures of conjugates 4 and 5
bearing a surrogate warhead.
Figure 2. Chemical structures of dual-warhead conjugates
2 and 3
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RESULTS AND DISCUSSION
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Overexpression of Biotin Receptor in Cancer Cells. All living cells depend on vitamins for
survival, but cancer cells require certain vitamins substantially more than most normal cells do in order to
sustain their rapid cell growth and enhanced proliferation. Thus, receptors for vitamins are overexpressed
on the cell surfaces of cancer cells to maintain sufficient vitamin uptake. Therefore, these cell surface
vitamin receptors serve as useful biomarkers for tumor-targeting drug delivery.12, 15, 31 The folate receptor
(FR) has been studied extensively for its overexpression in cancer cells and solid tumors, as well as the
relevant target for tumor-directed drug delivery,12 as exemplified by a highly promising SMDC,
“vintafolide”.10 In addition to the FR, recent studies have disclosed that the biotin receptor (BR) is
overexpressed even more than the FR in a good number of cancer cell lines.15 Thus, the BR has emerged
as a promising biomarker for tumor-targeted drug delivery, and we have successfully employed biotin as
a tumor-targeting module for small-molecule-based as well as single-walled carbon nanotube-based drug
conjugates.11, 20, 32, 33 In these previous studies, we designed, synthesized and used several fluorescent and
fluorogenic probes for the BR by employing fluorescein isothiocyanate (FITC), fluorescein and
coumarin.11, 20, 32-34 Among those fluorescent probes, biotin-NHNH-FITC was successfully used to verify
efficient receptor-mediated endocytosis (RME), targeting the BR.11 In the present study, we designed
fluorescent probe 6, which is more water-soluble than biotin-NHNH-FITC, for the assessment of the BR
overexpression level in several cancer cell lines to select appropriate cancer cell lines for evaluation of
biotin-drug conjugates, targeting the BR.
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Probe 6 was synthesized through coupling of FITC with N-biotinyl-NH-PEG3-(CH2)2NH2, which
was readily prepared from biotin and commercially available 11-azido-3,6,9-trioxaundecan-1-amine
through amide coupling, followed by Staudinger reduction of the resulting azide (Scheme 1).35, 36
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Scheme 1a
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aReagents and conditions: (i) DIPEA, DMF, dark, 25 oC, 48 h, 68%.
Assessment of the expression level of biotin receptor in cancer cell lines using fluorescent probe 7.
Although Russell-Jones et al. reported a dozen cell lines overexpressing the BR (BR+), most of those cell
lines are from murine cancers.15 Accordingly, we used probe 7 to assess the BR expression levels in
several human cancer cell lines by means of flow cytometry and confocal fluorescence microscopy
(CFM). Internalization of probe 7 into MX-1 (breast), MCF-7 (breast), ID8 (ovary) and WI38 (lung
fibroblast, normal) cell lines via RME was monitored at 0, 1 and 3 h periods. Previously studied15 murine
leukemia cell lines L1210FR (BR+) and L1210 (BR-) were also used for comparison. As Figure 4 clearly
indicates, the BR is highly expressed in MCF-7 and ID8 at the same level as known BR+++ cell line,
L1210FR, while the expression level is a little lower in MX-1 (BR++). As anticipated, the BR expression
level is negligible in human normal cell line, WI38, and known BR- cell line, L1210. The flow cytometry
histograms as well as the corresponding CFM images of probe 7 internalization into ID8 and MCF-7 are
shown in Figure 5 as typical examples.
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Figure 4. Internalization of probe 7 into various cell lines at 0, 1 and 3
h at 37 oC. Fluorescence intensity is a geometric mean of values
obtained from a flow cytometry histogram for each cell line, which is
provided in the Supporting Information.
Figure 5. Internalization of probe 7 monitered by flow cytometry and
CFM: (A) flow cytometry analysis of 7 in ID8 at 0 h (red, control), 1 h
(green), and 3 h (blue); (B) and (C) CFM images of 7 in ID8 at 1 h and
3 h, respectively; (D) flow cytometry analysis of 7 in MCF-7 at 0 h
(red, control), 1 h (green), 3 h (blue); (E) and (F) CFM images of 7 in
MCF-7 at 1 h and 3 h, respectively.
We also performed the assessment of the BR expression level in additional human breast cancer
cell lines and found that these cell lines also overexpress the receptor: BT-20 (BR++), LCC6-WT (BR++),
LCC6-MDR (BR+), MDA-MB 231 (BR++), and SkBr3 (BR++) (see Supporting Information). It was also
found that the BR and FR are equally overexpressed in BT-20, LCC6-WT, MDA-MB 231 and SkBr3 cell
lines, while the FR is more expressed than the BR in LCC6-MDR and MCF-7 cell lines (see Supporting
Information).
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Synthesis of the key components of tumor-targeting drug conjugates with “smart” linkers.
We have developed novel mechanism-based “smart” disulfide linkers, which can be conjugated to a
cytotoxic warhead on one end and a tumor-targeting module on the other end. These “smart” linkers were
designed to be stable during circulation in blood stream, but readily cleavable inside the tumor
microenvironment.20 These “smart” linkers consist of a 2-sulfhydrylphenylacetic acid component and a
sulfanylalkanoic acid component, and the former is connected to a cytotoxic agent and the latter to a
tumor-targeting module or a splitter unit linked to a tumor-targeting module (see Figure 2). In this study,
4-sulfanylpentanoic acid was used as the sulfanylalkanoic acid component, since it has been shown37 that
linkers bearing 4-disulfanylpentanoic acid moiety are more stable than disulfanylpropanoic acid moiety,
which was used in our previous studies.11, 20, 32 This “smart” disulfide linker was designed to be self-
immolative for releasing a cytotoxic warhead through disulfide-thiol exchange reaction with endogenous
thiols, represented by glutathione (GSH), followed by facile benzothiolactonization.11, 20, 38 The phenyl
moiety was placed to electronically direct the cleavage of the disulfide linkage by intracellular thiol and
entropically drive thiolactionization.11, 20 Thus, when the tumor-targeting module navigates the drug-
conjugate to the target biotin receptors on the tumor surface, the whole conjugate is internalized via
receptor-mediated endocytosis (RME).11, 20 Then, an intracellular thiol-triggered cascade drug-release
takes place through thiolactonization. Since it has been shown that the glutathione level in tumor tissues
(2-8 mM) is more than 1,000 times higher than that in blood stream (1-2 μM),39, 40 glutathione and other
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endogenous intracellular thiols would act as excellent tumor-specific agents to trigger the drug release.11,
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The preparation of bifunctional disulfide linker unit 11 and its use for the synthesis of coupling-
ready (14T, 14P) or “click”-ready constructs (15C, 15P) of drug-linker conjugates with taxoid 1,
camptothecin (CPT) and phenol are illustrated in Scheme 2. First, 4-sulfanylpentanoic acid (9) was
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prepared by ring-opening substitution of γ-valerolactone (8) with hydrobromic acid and thiourea, followed
by basic hydrolysis. The thiol-disulfide exchange reaction of 9 with 4,4’-dipyridinedisulfide, followed by
TIPS protection of the carboxylic acid moiety gave TIPS pyridinyldisulfanylpentanoate 10 in 92% yield
for two steps. Another thiol-disulfide exchange reaction of 10 with 2-sulfanylphenylacetic acid afforded
11 in 68% yield. The coupling reactions of 11 with 1, CPT and phenol in the presence of DIC and DMAP
gave the corresponding drug-disulfide linker constructs 12T, 12C and 12P, respectively, in 57-79% yields.
TIPS groups of 12T, 12C and 12P were removed with HF-pyridine to afford 13T, 13C and 13P,
respectively.
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Scheme 2a
aReagents and conditions: (i) thiourea, 48% HBr, 100 oC, 24 h; (ii) KOH, H2O, 100 oC, 24 h, 68%; (iii) 4,4’-
dipyridinedisulfide, CH3CH2OH, 80 oC, 2 h; (iv) i-Pr3SiCl, Et3N, CH2Cl2, 0→25 oC, 24 h, 92%; (v) (2-sulfanylphenyl)acetic
acid, THF, -10 oC, 3 h, 68%; (vi) 1 or CPT or phenol, DIC, DMAP, CH2Cl2, 25 oC, 24 h, 57-79%; (vii) 70% HF in pyridine,
CH3CN/pyridine (1:1), 0→25 oC, 16 h, 85-91%; (viii) HOSu, EDC, THF/pyridine, 25 oC, 36 h, 70-84%.; (ix) H2N-PEG3-
(CH2)2N3, DIC, CH2Cl2, 25 oC, 10 h, 56-71%.
Then, the esterification of 13T and 13P with N-hydroxysuccinimide (HOSu) gave the corresponding
coupling-ready taxoid-linker construct 14T and phenol-linker construct 14P, respectively, as activated
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esters in high yields. Finally, the DIC coupling of 13C and 13P with 1-amino-11-azido-3,6,9-
trioxaundecane afforded the corresponding “click”-ready drug-linker constructs, 15C and 15P,
respectively, in good yields.
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Scheme 3a
aReagents and conditions: (i) H2N-PEG3-(CH2)2N3, Et3N,
CH2Cl2, 25 oC, 16 h, 67%; (ii) CPT, DIC, DMAP, CH2Cl2,
25 oC, 36 h, dark, 72%.
Design and synthesis of tumor-targeting dual-warhead conjugates
Design and synthesis of dual-warhead conjugates and their single-warhead surrogates.
Among possible tripod splitter modules, we chose 1,3,5-triazine for novel dual-warhead conjugates with
three arms. Also, we decided to implement water-soluble linker components by incorporating
polyethylene glycol oligomers, i.e., PEG3, as well as the use of “click chemistry” to introduce the third
arm at the end of the synthesis. Thus, dual-warhead conjugates, 2 and 3, as well as their single-warhead
surrogates, i.e., 4 and 5, were designed for construction by incorporating those components and strategies
(see Figure 2).
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The syntheses of conjugates 2, 3 and 4 are illustrated in Scheme 4. First, cyanuric acid (19) was
reacted with N-Boc-ethylenediamine (1.0 equiv.) in the presence of N,N-diisopropylethylamine (DIPEA)
at 0 ºC, followed by the subsequent substitution reaction with biotinyl-NH-PEG3-NH2 (6) (1.0 equiv.) in
the presence of DIPEA at 40 ºC to give 20 in moderate yield for 2 steps. The reaction of 20 with excess
propargylamine in the presence of DIPEA at 50 ºC afforded fully functionalized triazine 21 in 69% yield.
The deprotection of 21 with TFA gave 22, which was coupled with taxoid-linker-OSu 14T in the presence
of pyridine at 25 ºC to afford 23 in 73% yield. Then, 23 was subjected to three “click” reactions with
azides 15C, 18 and 15P in the presence of cupric sulfate and ascorbic acid at 25 ºC to give the
corresponding conjugates, 2, 3 and 4, respectively, in 73-92% yields.
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Scheme 4a
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aReagents and conditions: (i) N-Boc-ethylenediamine, DIPEA, 0 C; 3 h (ii) DIPEA, 40 C, 16 h, 43% for two steps; (iii)
propargylamine, DIPEA, THF, 50 oC, 12 h, 69%; (iv) TFA, CH2Cl2, 25 oC, 24 h, 99%; (v) CH2Cl2/pyridine, 25 oC, 36 h, 73%;
(vi) CuSO4•5H2O, ascorbic acid, THF/H2O/CH2Cl2, 25 oC, 14 h, dark, 92%; (vii) CuSO4•5H2O, ascorbic acid, THF/H2O/CH2Cl2,
25 oC, 10 h, dark, 54%; (viii) CuSO4•5H2O, ascorbic acid, THF/H2O, 25 oC, 15 h, 79%.
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For the synthesis of the single-warhead surrogate 5 bearing camptothecin, triazine 22 was reacted
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with phenol-linker-OSu 14P in the presence of pyridine at 25 ºC to afford 24 in 52% yield. Then, the
“click” reaction of 24 with azide 15C under the standard conditions gave 5 in 54% yield (Scheme 5).
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Scheme 5
aReagents and conditions: (i) CH2Cl2/pyridine, 25 oC, 10 h, 52%; (ii) CuSO4•5H2O, ascorbic acid, THF/H2O/CH3OH, 25 oC,
4 h, dark, 54%.
Effect of equimolar combinations of taxoid 1 and camptothecin on cytotoxicity against cancer cell
lines.
The effect of the combinations of taxoid 1 and camptothecin (1:1) on cytotoxicity was examined
using three different timings for drug mixing against human breast carcinoma (MX-1 and MCF-7), murine
ovarian (ID8), and murine leukemia (L1210FR) cell lines. Taxoid 1 and camptothecin were also used as
a single agent for comparison. The results are summarized in Table 1. As Table 1 shows, the cytotoxicity
of taxoid 1 was 148-846 times stronger than camptothecin against four cancer cell lines examined (entries
1 and 2). The most significant and interesting observation was the fact that the order of exposure to these
two drugs, i.e., (a) simultaneous exposure (entry 3), (b) taxoid first for 24 h, followed by camptothecin
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(entry 4) and (c) camptothecin first for 24 h, followed by taxoid (entry 5), exhibited a marked difference
in the potency. When cancer cells were exposed to camptothecin first for 24 h, followed by equimolar
concentrations of taxoid 1 (entry 5), drastic increases in the IC50 values as compared to those values for
simultaneous exposure (entry 3) were observed. These IC50 values (entry 5) were even higher than those
for camptothecin alone except for that against the ID8 cell line (entry 2). The results clearly indicate that
these two drugs act antagonistically in this case and camptothecin appears to block the action of taxoid 1.
This is probably ascribed to the cell cycle control, e.g., synchronization, by camptothecin, which causes
detrimental effects on the action of taxoid 1.42 In sharp contrast, when cancer cells were exposed to
equimolar mixture of the two drugs (entry 3) or to taxoid 1 first for 24 h, followed by equimolar
concentrations of camptothecin (entry 4), IC50 values were essentially the same as those for taxoid 1 alone
within errors. (entry 1). The results indicate that in these two cases, these two drugs act at least
cooperatively, and taxoid 1 might be potentiating camptothecin.21
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Table 1. Cytotoxicities (IC50, nM) of Taxoid 1, Camptothecin (CPT), and Their Equimolar Combinations
against Cancer Cell Lines Overexpressing Biotin Receptor (BR+)
Entry
1e
Drug
1
Time
72 h
72 h
72 h
MX-1a
MCF-7b
ID8c
L1210FRd
2.14 ± 1.30
510 ± 139
4.14 ± 0.46
3.51 ± 0.27
949 ± 116
2.01 ± 0.52
1,700 ± 200
1.88 ± 0.14
1.98 ± 0.29
>5,000
0.44 ± 0.18
65.1 ± 12.3
0.40 ± 0.09
0.33 ± 0.10
79.2 ± 12.4
1.11 ± 0.26
474 ± 101
1.02 ± 0.17
0.96 ± 0.14
110 ± 12
2e
CPT
3f
1:CPT (1:1)
1
24 h
48 h
24 h
48 h
4g
then CPT
CPT
then 1
5h
b
c
aHuman breast carcinoma cell line (BR+); Human breast carcinoma cell line (BR+); Murine ovarian carcinoma cell line
d
e
o
f
(BR+); Murine lymphocytic leukemia cell line (BR+); Cells were incubated with a drug for 72 h at 37 C; Cells were
incubated with an equimolar mixture of two drugs for 72 h at 37 oC; gCells were incubated with 1 at a given concentration for
24 h at 37 oC. Then, an equimolar amount of CPT was added and the cells were incubated for additional 48 h at 37 oC. hCells
were incubated with CPT at a given concentration for 24 h at 37 oC. Then, an equimolar amount of 1 was added and the cells
were incubated for additional 48 h at 37 oC.
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Biological Evaluation of Dual-Warhead Drug Conjugates.
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Internalization of dual-warhead conjugate 2 by CFM. We have shown the efficient
internalization of fluorescence-labeled biotin probe 7 into several cancer cell lines via RME (see Figures
4 and 5). We have also previously reported the proofs for highly efficient RME of a biotin-linker-(taxoid-
fluorescein) conjugate11 and a (biotin)n-SWNT-(taxoid-fluorescein)m conjugate.32 In this study, we
examined the receptor-mediated internalization of dual-warhead conjugate 2 by taking advantage of the
intrinsic fluorescence of camptothecin, using CFM. ID8 and MCF-7 cancers were incubated with
conjugate 2 (10 M) at 37 oC for 10 h. After ample washing of the drug media, the cells were analyzed
by CFM. As Figure 6 shows, the intense signature blue fluorescence of camptothecin was observed in
both cancer cells, confirming the anticipated efficient internalization of conjugate 2.
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Figure 6. Confocal fluorescence microscopy images showing
internalization of conjugate 2 in ID8 (left) and MCF-7 (right)
after incubation at 37 oC for 10 h.
Biological evaluations of dual-warhead conjugates. Cytotoxicities of novel dual-warhead
conjugates, 2 and 3, as well as their single-warhead surrogates, 4 and 5, were evaluated in two ways against
four BR(+) cancer cell lines, MX-1, MCF-7, ID8 and L1210FR, as well as normal human lung fibroblast
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cell line, WI38, using the standard MTT assay. Results are summarized in Tables 2 and 3. Taxoid 1 and
camptothecin were also examined for comparison (Table 2, entries 1 and 2, respectively).
7
8
9
In the first experiment, MX-1 (BR++), MCF7 (BR+++), ID8 (BR+++) and L1210FR (BR+++) cancer
cells were incubated with conjugate 2-5 for 72 h, and the corresponding IC50 values were determined. As
Table 2 shows, the cytotoxicities of conjugates 2 and 3 based on their IC50 values were found to be in a
range of 19-65 nM (entries 3 and 4), which makes a sharp contrast with that against normal cell line WI38
(IC50 742-868 nM) (entries 3 and 4). The results indicate that these conjugates were selectively
internalized into BR(+) cancer cells via RME and the drug was partially released inside cancer cells.
Single-warhead surrogate 4 showed a similar potency as that of conjugate 3 (entry 5), while the other
surrogate 5 did not exhibit appreciable cytotoxicity (MX-1 and ID8) or very weak potency (MCF-7,
WI38), with L1210FR as an exception (entry 6). For conjugates 2-4 bearing taxoid 1 as a warhead, the
cytotoxicity level appeared to be markedly reduced as compared to that for free taxoid 1. Since we have
confirmed that these four cancer cell lines overexpress BR (see Figures 3 and 4), and also observed marked
selectivity for BR+ cancer cell lines as compared to normal WI38 cell line (BR-), the internalization of
conjugate 2-5 via RME should not be the major issue for these cancer cell lines. Thus, it is very likely that
the level of GSH (and/or other endogenous thiols) is not sufficient to release the warhead(s) via disulfide
linker cleavage under in vitro conditions. This situation makes a sharp contrast to that in vivo where there
are sufficient endogenous thiols (e.g., GSH) in tumors, which are under hypoxic conditions.
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In order to clarify these points, glutathione ethyl ester (GSH-OEt) (6 mole equiv. to a conjugate)
was added to the re-suspended cancer cells after the cells had been incubated with a conjugate for 24 h,
followed by thorough washing with phosphate-buffered saline (PBS), and further incubated for 48 h in
the second experiment (Table 3). It should be noted that the re-suspended cancer cells only included a
conjugate internalized in the first 24 h period. As Table 3 shows, this protocol, including washing of cells
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and addition of GSH-OEt, did not make any appreciable difference in the cytotoxicity of these conjugates
against WI38 normal fibroblast cells, based on the comparison of the results in Table 2 with those in Table
3. Conjugate 2 showed IC50 values of 3.2-9.8 nM against four BR+ cancer cell lines, but that against WI38
was 742 nM (entry 1). Thus, there is two-order of magnitude difference in cytotoxicity between cancer
cells and normal cells, which is unambiguously attributed to the highly efficient receptor-targeting by
conjugate 2. There was also considerable difference in potency between conjugate 2 and conjugate 3
(entries 1 and 2). This difference is attributable to full release of both taxoid 1 and camptothecin in
conjugate 2 via cleavage of a disulfide linkage versus insufficient release of camptothecin in conjugate 3,
due to a more stable ester linkage between the linker and the drug, under the conditions examined. The
results clearly indicate that there were synergistic or cooperative effects between taxoid 1 and
camptothecin when these two drugs were delivered to cancer cells simultaneously and released inside
cancer cells. Since surrogate 4, bearing taxoid 1 and phenol, exhibited a similar but systematically slightly
weaker potency that that of conjugate 3 (entry 3), it is reasonable to assume that the release of campothecin
occurred to some extent under the experimental conditions examined.
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It appears that all four BR+ cancer cell lines are highly drug resistant to camptothecin and the
potency was further decreased substantially when camptothecin and phenol were delivered to MX-1,
MCF-7 and ID8 cells via RME and released inside cancer cells (Table 3, entry 4) as compared to that
observed in the standard MTT assay of camptothecin (Table 2, entry 2). The results may indicate that
phenol interferes with the action of camptothecin in those cancer cells. Interestingly, surrogate 5 exhibited
higher potency than camptothecin against L1210FR leukemia cell line (IC50 319 nM in Table 2 and 177
nM in Table 3 for 5 vs. 510 nM for CPT in Table 2). The results may suggest that phenol has cytotoxic
effect on this leukemia cell line.
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Table 2. Cytotoxicities (IC50, nM) of Conjugates 2-5 against BR+ and BR- Cell Lines
5
6
7
8
9
Entry
1f
Conjugate/Drug
MX-1a
2.01 ± 0.52
1700 ± 200
51.7 ± 5.1
65.1 ± 9.7
66.9 ± 4.4
>5,000
MCF-7b
0.44 ± 0.18
65.1 ± 12.3
19.0 ± 3.4
21.1 ± 2.9
23.1 ± 1.8
1,260 ± 172
ID8c
L1210FRd
2.14 ± 1.30
510 ± 139
39.1 ± 9.1
42.5 ± 8.9
50.9 ± 15.9
319 ± 139
WI38e
1
CPT
2
1.11 ± 0.26
474 ± 101
23.4 ± 4.7
25.6 ± 3.9
26.7 ± 4.2
>5,000
5.89 ± 2.38
786 ± 309
742 ± 166
868 ± 264
941 ± 298
1,440 ± 364
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2 f
3 f
4 f
3
5 f
4
6 f
5
a-dSee captions for cell lines in Table 1; eHuman lung fibroblast cell line (BR-); fCells were incubated with a drug or a
conjugate at 37 oC for 72 h.
Table 3. Cytotoxicities (IC50, nM) of Conjugates 2-5 in the Presence of GSH-OEt after Internalization
Entry
Conjugate
MX-1a
9.8 ± 1.4
20.8 ± 2.2
23.6 ± 4.6
>5000
MCF-7b
3.2 ± 0.8
9.4 ± 1.3
9.9 ± 2.0
538 ± 105
ID8c
L1210FRd
7.40 ± 3.48
13.7 ± 1.5
14.4 ± 4.2
177 ± 33
WI38e
1
2
3
4
2
3
4
5
5.7 ± 1.0
705 ± 114
782 ± 132
850 ± 100
10.3 ± 1.7
15.6 ± 3.0
2,390 ± 405
1230 ± 219
*Cells were initially incubated with conjugates 2-5 for 24 h, followed by washing of the drug media with PBS, then addition
of GSH-OEt (6 equiv. to a conjugate) for drug release and additional incubation at 37 oC for 48 h.
a-eSee captions for cell lines in Table 1.
CONCLUSION
We have designed and successfully synthesized novel tumor-targeting dual-warhead conjugates, 2 and 3,
which consist of taxoid 1 and camptothecin as two warheads, self-immolative disulfide linkers for drug
release, biotin as the tumor-targeting moiety, and 1,3,5-triazine as the tripod splitter module. Conjugates,
2 and 3 incorporated tetraethylene glycol diamine moieties to increase the water-solubility of the
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conjugates. The functionalized 1,3,5-triazine-based TTDDS platform is versatile and applicable to a
variety of two-drug combinations as well as the use of various tumor-targeting modules. For the
introduction of the second warhead-linker module to the 1,3,5-triazine splitter module, a propargylamine
spacer was introduced first to the splitter module. Then, its acetylene moiety was used for click reaction
with the second drug-linker construct bearing an -azido group to install the second warhead module as
the third arm.
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Since biotin was used as the tumor-targeting moiety, we screened the expression levels of the
biotin receptor (BR) in various human breast cancer cell lines, MX-1, MCF-7, LCC6-WT, LCC6-MDR,
MDA-MB 231, SkBr3, which had not been reported previously in addition to the known BR-
overexpressing cancer cell lines, ID8 (human ovary) and L1210FR (murine leukemia), by means of flow
cytometry and confocal fluorescence microscopy (CFM). Then, we confirmed that these human breast
cancer cell lines overexpress BR at variable levels. Also, we confirmed that there was no appreciable
expression of BR in L1210 (murine leukemia) and WI38 (normal, human lung fibroblast) cell lines.
The potencies of conjugates, 2 and 3, were evaluated against MX-1, MCF-7, ID8, L1210FR and
WI38. It was found that the amount of endogenous thiols such as glutathione (GSH) was insufficient to
trigger the drug release via cleavage of the self-immolative disulfide linkers under in vitro assay conditions
(this is not an issue in vivo). Accordingly, glutathione ethyl ester (GSH-OEt) (6 equiv. to a cojugate) was
added after the cells were incubated with 2 or 3 for 24 h, thoroughly washed with saline (PBS), and re-
suspended. Then, the cells were further incubated for 48 h. Thus, only the conjugate molecules internalized
into the cancer cells in the first 24 h were treated with GSH-OEt in this protocol. The addition of GSH-
OEt showed remarkable effect, as anticipated, and conjugate 2 exhibited IC50 values of 3.22-9.80 nM
against all BR+ cancer cell lines, while that against normal cell line, WI38, was 705 nM. Thus, there were
two-orders of magnitude difference in selectivity to cancer cells, which was impressive. Also, the potency
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of 1 was 2-3 times as high as those of 3 and surrogate conjugate 4, which clearly indicates the synergistic
or cooperative effect of the taxoid–camptothecin combination. It is noteworthy that this substantial
enhancement in potency in this combination was observed only when these two drugs were internalized
via RME.
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The IC50 values observed for conjugate 2 were 3.5-7.3 times larger than that of taxoid 1. The
observed reduced potencies can be ascribed to insufficient internalization of 1 to those cells in the first 24
h, which can be attributed to (a) a saturation phenomenon for BR-binding and internalization, depending
on the expression level of BR on the cell surface and (b) slow recycling of the receptors in the first 24 h.
This is, however, not an issue in vivo.
Further studies along this line, as well as the evaluation of the efficacy of 2 and 3 in vivo are
actively underway in our laboratory.
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EXPERIMENTAL SECTION
4
5
6
7
8
Caution. Taxoids and camptothecins have been identified as potent cytotoxic agents. Thus, these
drugs and all structurally related compounds and derivatives must be considered as mutagens and potential
reproductive hazards for both males and females. Appropriate precautions, such as the use of gloves,
goggles, labware, and fume hood, must be taken while handling the compounds at all times.
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1
13
General Methods. H and C NMR spectra were measured on a Varian 300, 400, or 500 MHz
spectrometer or a Bruker 500 MHz NMR spectrometer. Melting points were measured on a Thomas-
Hoover capillary melting point apparatus and are uncorrected. TLC was performed on Sorbent
Technologies aluminum-backed Silica G TLC plates (Sorbent Technologies, 200 μm, 20 x 20 cm), and
column chromatography was carried out on silica gel 60 (Merck, 230-400 mesh ASTM). Purity was
determined with a Shimadzu L-2010A HPLC HT series HPLC assembly, using a Kinetex PFP column
(4.6 mm x 100 mm, 2.6 μm) with acetonitrile-water gradient solvent system. Two analytical conditions
were used and noted as a part of the characterization data and purity for literature unknown compounds,
i.e., HPLC (1): flow rate 0.4 mL/min, a gradient of 15→95% acetonitrile for the 0-12 min period; HPLC
(2): flow rate 0.5 mL/min, a gradient of 5-95% acetonitrile for the 0-12 min period and 95% acetonitrile
for the 11-15 min period. All new compounds had >95% purity. High resolution mass spectrometry
analysis was carried out on an Agilent LC-UV-TOF mass spectrometer at the Institute of Chemical
Biology and Drug Discovery, Stony Brook, NY or at the Mass Spectrometry Laboratory, University of
Illinois at Urbana-Champaign, Urbana, IL.
Materials. The chemicals were purchased from Sigma-Aldrich, Fisher Scientific, and VWR
International, and used as received or purified before use by standard methods. Tetrahydrofuran was
freshly distilled from sodium and benzophenone. Dichloromethane was also distilled immediately prior
to use under nitrogen from calcium hydride. 10-Deacetylbaccatin III was obtained from Indena, SpA,
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Italy. 2,2’-Dipyridyldisulfide,11 (2-sulfanylphenyl)acetic acid,11 taxoid 1,25 1-amino-11-azido-3,6,9-
trioxaundecane,35 N-Boc-ethylenediamine,43 N-biotinyl-PEG3-diamine36 and 4-sulfanylpentanoic acid
(8)37, 41 were prepared by literature methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was obtained from Sigma Chemical Co. Biological materials including RPMI-1640 and
DMEM cell culture media, fetal bovine serum, NuSerum, PenStrep, and TrypLE were obtained from
Gibco and VWR International, and used as received for cell-based assays.
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Biotin-NH-PEG3-(CH2)2-NH-FITC (7). To a solution of fluorescein isothiocyanate (FITC) (20
mg, 0.0502 mmol) and N-biotinyl‐3,6,9-trioxaundecamethylene-1,11-diamine (30 mg, 0.0717 mmol) in
DMF (0.6 mL) was added diisopropylethylamine (DIPEA) (14 μL). The mixture was allowed to react
for 48 h at room temperature in the dark with stirring, and the reaction mixture was directly loaded onto
a silica gel column. Purification of the reaction mixture by column chromatography on silica gel using
15% CH3OH in CH2Cl2 as eluent gave probe 7 (27 mg, 68% yield) as an orange solid: mp 191-192 oC;
1H NMR (500 MHz, CD3OD) δ 1.40 (m, 2H), 1.62 (m, 2H), 1.69 (m, 2H), 2.19 (t, J = 7.4 Hz, 2H), 2.67
(d, J = 12.7 Hz, 1H), 2.88 (dd, J = 5.0, 12.7 Hz, 1H), 3.15 (m, 1H), 3.32 (m, 2H), 3.51 (t, J = 5.2 Hz,
2H), 3.59 (m, 2H), 3.63 (m, 2H), 3.68 (s, 4H), 3.71 (t, J = 5.2 Hz, 2H), 3.82 (bs, 2H), 4.26 (dd, J = 4.5,
7.9 Hz, 1H), 4.45 (dd, J = 4.5, 7.9 Hz, 1H), 6.55 (dd, J = 2.4, 8.8 Hz, 2H), 6.67 (d, J = 2.4 Hz, 2H), 6.69
(d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.95 (bs, 1H), 8.15 (d, 1.7 Hz,
1H); 13C NMR (125 MHz, CD3OD) 7.21, 12.43, 15.43, 21.71, 24.82, 27.47, 27.73, 30.77, 34.74, 38.32,
39.05, 43.49, 45.89, 54.97, 59.61, 61.35, 68.58, 69.24, 69.32, 69.56, 69.61, 101.54, 109.74, 112.02,
+
123.99, 128.46, 140.43, 152.40, 164.11, 169.25, 174.15, 180.88; HRMS (TOF) for C39H46N5O16S2
calcd: 808.2681. Found: 808.2691 (Δ = 1.2 ppm). HPLC (1): t = 9.1 min, purity >97%.
Triisopropylsilyl 4-(pyridin-2-yldisulfanyl)pentanoate (10).41 To a solution of 9 (709 mg, 5.29
mmol) in ethanol (20 mL) was added 2,2’-dipyridinedisulfide (7.0 g, 31.7 mmol) in ethanol (100 mL),
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and the mixture was refluxed at 80 oC for 2 h. The reaction mixture was cooled to 25 oC and concentrated
in vacuo to afford a yellow precipitate. The crude product was purified by column chromatography on
silica gel to afford a mixture of 4-(pyridin-2-yldisulfanyl)pentanoic acid and pyridine-2-thiol. To a cooled
solution of this mixture in dichloromethane (40 mL) at 0 oC was added triethylamine (2.1 mL, 15.1 mmol)
and chlorotriisopropylsilane (1.93 mL, 9.04 mmol). The mixture was allowed to react for 24 h with
stirring and concentrated in vacuo to afford a crude product as an oil. Purification of the crude product by
column chromatography on silica gel with hexanes/ethyl acetate (2:1) as eluent gave 10 (2.71 g, 92% yield
for 2 steps), as a colorless oil: 1H NMR (600 MHz, CDCl3) δ 1.06 (m, 21 H), 1.34 (d, J = 6.8 Hz, 3H),
1.89 (m, 1H), 2.00 (m, 1H), 2.51 (m, 2H), 3.03 (m, 1H), 7.06 (ddd, J = 1.0, 4.8, 7.4 Hz, 1H), 7.62 (dt, J =
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1.5, 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 4.8 Hz, 1H). H NMR spectrum was consistent
with the reported data.41
2-(5-Oxo-triisopropylsiloxypentan-2-yl)disulfanylphenylacetic acid (11).41 To a cooled
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solution of 10 (300 mg, 0.752 mmol) in tetrahydrofuran (1.5 mL) at -10 C was added (2-
sulfanylphenyl)acetic acid (130 mg, 0.752 mmol) in tetrahydrofuran (1.5 mL), and the mixture was
allowed to react for 2 h at room temperature with stirring. The reaction mixture was concentrated in vacuo
to afford a yellow oil. Purification the crude produce by column chromatography on silica gel with
hexanes/ethyl acetate (3:1) as eluent gave 11 (166 mg, 68% yield) as an orange oil: 1H NMR (300 MHz,
CDCl3) δ 1.06 (m, 21H), 1.25 (d, J = 6.8, 3H), 1.81 (m, 1H), 1.92 (m, 1H), 2.44 (m, 2H), 2.90 (m, 1H),
3.90 (s, 2H), 7.24 (m, 2H), 7.32 (m, 1H), 7.82 (d, J = 7.7 Hz, 1H); HRMS (TOF) for C22H37O4S2Si+ calcd:
457.1897, found: 457.1892 (Δ = -1.1 ppm).
Taxoid-(SS-Linker)-OTIPS (12T).41 To a solution of taxoid 1 (600 mg, 0.703 mmol), 11 (350
mg, 0.768 mmol), and 4-N,N-dimethylaminopyridine (DMAP) (38 mg, 0.308 mmol) in CH2Cl2 (13 mL)
was added N,N’-diisopropylcarbodiimide (DIC) (135 μL, 0.877 mmol), and the mixture was allowed to
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react for 24 h at room temperature with stirring. The reaction was quenched with saturated NH4Cl (10
mL), and the reaction mixture was diluted with water (20 mL x 3), washed with brine (20 mL x 3), dried
over MgSO4, and concentrated in vacuo to afford a yellow oil. The crude product was dissolved in ether
(20 mL) and the insoluble urea byproduct was removed by gravity filtration on a filter paper. Purification
of the crude product by column chromatography on silica gel with hexanes/ethyl acetate (3:1) as eluent
gave 12T (697 mg, 77% yield), as a white solid: 1H NMR (400 MHz, CDCl3) δ 0.97 (m, 2H), 1.06 (d, J =
7.2 Hz, 18H), 1.14 (m, 5H), 1.25 (s, 6H), 1.30 (d, J = 6.8 Hz, 3H), 1.33 (s, 9H), 1.66 (s, 3H), 1.69 (s, 3H),
1.72 (s, 3H), 1.76 (m, 2H), 1.85 (m, 1H), 1.90 (s, 3H), 1.93 (m, 2H), 2.30 (m, 2H), 2.35 (s, 3H), 2.42 (m,
2H), 2.53 (m, 2H), 2.59 (m, 1H), 2.94 (m, 1H), 3.81 (d, J = 7.2 Hz, 1H), 3.94 (dd, J = 1.7, 16.8 Hz, 1H),
4.10 (dd, J = 1.7, 16.8 Hz, 1H), 4.18 (d, J = 8.4 Hz, 1H), 4.30 (d, J = 8.4 Hz, 1H), 4.42 (m, 1H), 4.79 (d, J
= 8.4 Hz, 1H), 4.92 (s, 1H), 4.96 (d, J = 7.2 Hz, 1H), 5.07 (d, J = 8.4 Hz, 1H), 5.67 (d, J = 7.2 Hz, 1H),
6.19 (t, J = 8.7 Hz, 1H), 6.28 (s, 1H), 7.26 (m, 3H), 7.47 (t, J = 7.9 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.80
(d, J = 7.4 Hz, 1H), 8.11 (d, J = 7.1 Hz, 2H); HRMS (TOF) for C67H94NO18S2Si+ calcd: 1292.5676. Found:
1292.5676 (Δ = 0 ppm).
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Camptothecin-(SS-Linker)-OTIPS (12C). To a solution of camptothecin (158 mg, 0.431 mmol),
10 (489 mg, 1.072 mmol), and DMAP (53 mg, 0.431 mmol) in CH2Cl2 (20 mL) was added DIC (73 μL,
0.474 mmol), and the mixture was allowed to react for 16 h at room temperature with stirring. The reaction
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mixture was cooled to 0 C and the urea by-product was removed via filtration. The filtrate was
concentrated in vacuo to give a green solid. Purification of the crude product by column chromatography
on silica gel with 1.5% CH3OH in CH2Cl2 as eluent gave 12C (192 mg, 57% yield) as an off-white solid;
1H NMR (500 MHz, CDCl3) δ 0.98 (t, J = 7.5 Hz, 3H), 1.05 (m, 21H), 1.45 (d, J = 6.8 Hz, 2H), 1.85 (m,
1H), 2.18 (m, 1H), 2.33 (m, 2H), 2.87 (m, 1H), 4.00 (dd, J = 4.5, 16.5 Hz, 1H), 4.02 (d, J = 22.0 Hz, 1H),
4.17 (dd, J = 9.2, 16.5 Hz, 1H), 5.21 (m, 2H), 5.38 (d, J = 17.5 Hz, 1H), 5.64 (d, J = 17.2 Hz, 1H), 7.21
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(d, J = 2.5 Hz, 1H), 7.26 (m, 4H), 7.66 (t, J = 7.5 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz,
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1H), 8.27 (d, J = 8.5 Hz, 1H), 8.35 (s, 1H); C NMR (125 MHz, CDCl3) δ 7.60, 11.85, 17.78, 20.39,
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20.98, 22.55, 23.53, 30.94, 31.78, 32.96, 38.59, 45.82, 49.92, 53.51, 67.13, 96.26, 120.18, 127.62, 127.67,
127.77, 127.92, 128.05, 128.13, 128.23, 128.27, 128.42, 129.69, 129.72, 129.94, 130.04, 130.40, 130.54,
130.59, 130.86, 130.89, 131.06, 132.56, 132.63, 134.65, 137.09, 137.44, 145.60, 146.20, 148.83, 152.30,
153.82, 157.27, 167.33, 169.91, 172.91; HRMS (TOF) for C42H51N2O7S2Si+ calcd: 787.2901; found:
787.2897 (Δ = -0.5 ppm).
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Phenol-(SS-Linker)-OTIPS (12P). To a solution of phenol (135 mg, 1.434 mmol), 11 (545 mg,
1.195 mmol) and DMAP (14 mg, 0.119 mmol) in CH2Cl2 (15 mL) was added DIC (210 μL, 1.314 mmol),
and the mixture was allowed to react for 10 h at room temperature with stirring. The reaction was
quenched with saturated NH4Cl (10 mL), and the reaction mixture was diluted with water (5 mL),
extracted with CH2Cl2 (20 mL x 3). The combined organic layers were dried over MgSO4 and concentrated
in vacuo to afford a yellow oil. Purification of the crude product by column chromatography on silica gel
with 3% ethyl acetate in hexanes as eluent gave 12P (505 mg, 79% yield) as a colorless oil: 1H NMR (400
MHz, CDCl3) δ 1.06 (m, 21H, TIPS), 1.30 (d, J= 6.8 Hz, 3H), 1.85 (m, 1H), 1.93 (m, 1H), 2.42 (m, 2H),
2.96 (m, 1H), 4.11 (s, 2H), 7.10 (d, J=7.4 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H), 7.26 (t, J = 7.3 Hz, 1H), 7.31
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(t, J = 7.3 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.82 (d, J = 7.9, 1H); C NMR (100 MHz, CDCl3) 11.89,
17.79, 20.57, 30.99, 33.03, 39.41, 46.11, 121.48, 125.84, 127.65, 128.27, 129.36, 130.23, 130.83, 133.38,
137.67, 150.79, 169.40, 173.01; HRMS (TOF) for C28H41O4S2Si+ calcd: 533.2210. Found: 533.2222 (Δ
= 2.3 ppm).
Taxoid-(SS-Linker)-COOH (13T).41 To a solution of 12T (649 mg, 0.502 mmol) in a 1:1 mixture
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of CH3CN-pyridine (1:1) (50 mL) and cooled to 0 C under nitrogen was added HF/pyridine (6.5 mL),
slowly, and the mixture was stirred for 24 h at room temperature. The reaction was quenched with 10%
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citric acid (10 mL), and the reaction mixture was diluted with ethyl acetate (20 mL). The reaction mixture
was extracted with ethyl acetate (20 mL x 2), washed with saturated CuSO4 (3 x 20 mL) and brine (3 x 20
mL). The organic layer was dried over MgSO4 and concentrated in vacuo to afford a clear oil. Purification
of the crude produce by column chromatography on silica gel with hexanes/ethyl acetate (1:4) as eluent
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gave 13T (516 mg, 91% yield) as a white solid: H NMR (500 MHz, CDCl3) δ 1.02 (m, 2H), 1.16 (m,
2H), 1.18 (m, 3H), 1.29 (s, 6H), 1.39 (s, 9H), 1.70 (s, 3H), 1.75 (s, 3H), 1.76 (s, 3H), 1.80 (m, 2H), 1.89
(bt, 1H), 1.93 (s, 3H) 1.94 (m, 2H), 2.39 (m, 2H), 2.42 (s, 3H), 2.56 (m, 1H), 3.03 (m, 1H), 3.83 (m, 1H),
3.96 (dd, J = 1.7, 16.8 Hz, 1H), 4.15 (dd, J = 1.7, 16.8 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H), 4.34 (d, J = 8.5
Hz, 1H), 4.45 (m, 1H), 5.00 (m, 3H), 5.18 (d, J = 7.1 Hz, 1H), 5.70 (d, J = 7.2 Hz, 1H), 6.25 (m, 1H),
6.32 (m, 1H), 7.33 (m, 3H), 7.51 (t, J = 7.8 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 8.14
+
(d, J = 7.1 Hz, 2H), 8.66 (bs, 1H); HRMS for (TOF) C58H74NO18S2 calcd: 1136.4342. Found: 1136.4365
(Δ = 2.0 ppm).
Camptothecin-Me-SS-Linker-COOH (13C). To a cooled solution of 12C (192 mg, 0.244 mmol)
in CH3CN-pyridine (1:1) (30 mL) at 0 oC was added 70% HF/pyridine (3 mL) dropwise, and the mixture
was allowed to gradually warm to room temperature over 5 h with stirring. The reaction was quenched
with 10% citric acid (10 mL), the reaction mixture was diluted with water (10 mL). The reaction mixture
was extracted with CH2Cl2 (3 x 20 mL), and the combined organic layers were washed with saturated
CuSO4 (60 mL), water (60 mL), and brine (60 mL). The organic layers were dried over MgSO4 and
concentrated in vacuo to give a green solid. Purification of the crude product by column chromatography
on silica gel with 1% CH3OH in CH2Cl2 as eluent gave 13C (131 mg, 85% yield) as a pale-green solid:
1H NMR (500 MHz, DMSO-d6) δ 0.91 (t, J = 7.5 Hz, 3H), 1.05 (dd, J = 6.8, 8.4 Hz, 3 H), 1.53 (m, 1H),
1.70 (m, 1H), 2.18 (t, J = 7.5 Hz, 2H), 2.20 (m, 2H), 2.88 (m, 1H), 4.05 (d, J = 16.5 Hz, 1H), 4.20 (dd, J
= 4.0, 16.5 Hz, 1H), 5.30 (m, 2H), 5.46 (m, 2H), 7.10 (s, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.35 (m, 2H), 7.74
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(m, 2H), 7.91 (t, J = 7.3 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.70 (s, 1H), 12.10 (s,
1H); 13C NMR (125 MHz, DMSO-d6) δ 8.00, 20.13, 20.24, 30.69, 30.79, 30.86, 31.29, 31.35, 38.57, 45.75,
45.83, 50.70, 55.40, 66.78, 76.61, 95.61, 119.37, 127.95, 127.97, 128.17, 128.46, 128.89, 129.00, 129.57,
129.78, 129.86, 130.16, 130.84, 131.70, 131.96, 133.11, 133.16, 137.59, 137.63, 145.48, 146.41, 148.43,
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152.71, 156.98, 167.51, 169.84, 174.22; HRMS (TOF) for C33H31N2O7S2 calcd: 631.1567; found:
631.1561 (Δ = -0.9 ppm).
Phenol-(SS-Linker)-COOH (13P). To a cooled solution of 12P (505 mg, 0.947 mmol) in
CH3CN-pyridine (1:1) (80 mL) at 0 oC, was added 70% HF/pyridine (5 mL), and the mixture was stirred
for 6 h at room temperature. The reaction was quenched with 10% citric acid and extracted with ethyl
acetate (50 mL x 3). The combined organic layers were washed with saturated CuSO4 (50 mL x 3) and
brine (50 mL x 3), dried over MgSO4, and concentrated in vacuo to afford a pale-yellow oil. Purification
of the crude product by column chromatography on silica gel with 50% ethyl acetate in hexanes as eluent
gave 14P (314 mg, 88% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) δ 1.29 (d, J = 6.8 Hz, 3H,
CH3), 1.86 (m, 1H), 1.95 (m, 1H), 2.42 (m, 2H), 2.94 (m, 2H), 4.12 (s, 2H), 7.11 (d, J = 7.5 Hz, 2H), 7.21
(t, J = 7.5 Hz, 1H), 7.26 (m, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.81 (d, J = 7.5 Hz,
1H); 13C NMR (400 MHz, CDCl3) δ 20.48, 30.35, 31.03, 39.42, 45.90, 121.47, 125.88, 127.80, 128.31,
+
129.39, 130.45, 130.94, 133.53, 137.51, 150.77, 169.50, 178.29; HRMS (TOF) for C19H21O4S2 calcd:
377.0876. Found: 377.0888 (Δ = 3.2 ppm).
Taxoid-(SS-Linker)-OSu (14T). To a solution of 13T (500 mg, 0.441 mmol) and N-
hydroxysuccinimide (HOSu) (0.160 g, 1.32 mmol) in THF-pyridine (1:1) (9 mL) was added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) (0.100 g, 0.485 mmol), and the mixture
was allowed to react for 36 h at room temperature with stirring. The reaction was quenched with saturated
NH4Cl (10 mL), washed with brine (3 x 20 mL), dried over MgSO4, and concentrated in vacuo to afford
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a yellow oil. Purification of the crude product by column chromatography on silica gel with hexanes/ethyl
acetate (1:1) as eluent gave 14T (376 mg, 70% yield) as a sticky white solid: 1H NMR (500 MHz, CDCl3)
δ 0.98 (m, 2H), 1.12 (m, 2H), 1.14 (s, 3H), 1.25 (m, 3H), 1.31 (dd, J = 4.8, 6.8 Hz, 3H), 1.33 (s, 2H), 1.34
(s, 9H), 1.66 (s, 3H), 1.71 (s, 3H), 1.73 (s, 3H), 1.86 (t, J = 13.8 Hz, 1H), 1.90 (s, 3H), 1.97 (m, 2H), 2.32
(m, 2H), 2.35 (s, 3H), 2.53 (m, 1H), 2.64 (t, J = 7.0 Hz, 2H), 2.83 (s, 4H), 2.99 (m, 1H), 3.80 (d, J = 7.2
Hz, 1H), 3.97 (dd, J = 1.7, 16.8 Hz, 1H), 4.08 (dd, J = 1.7, 16.8 Hz, 1H), 4.13 (m, 1H), 4.18 (d, J = 8.4 Hz,
1H), 4.30 (d, J = 8.4 Hz, 1H), 4.42 (m, 1H), 4.81 (m, 1H), 4.92 (s, 1H), 4.96 (d, J = 8.5 Hz, 1H), 5.09 (d,
J = 8.5 Hz, 1H), 5.67 (d, J = 7.2 Hz, 1H), 6.18 (t, J = 8.5 Hz, 1H), 6.28 (s, 1H), 7.30 (m, 3H), 7.47 (t, J =
7.8 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 8.11 (d, J = 7.5 Hz, 2H). 13C NMR (500
MHz, CDCl3) δ 9.19, 9.39, 9.58, 13.04, 13.75, 14.23, 14.87, 18.54, 19.15, 20.40, 22.25, 22.45, 23.51,
25.61, 25.75, 26.70, 28.24, 30.26, 30.66, 35.45, 35.51, 38.80, 42.29, 43.20, 45.60, 58.48, 64.40, 71.83,
72.16, 75.01, 75.22, 75.49, 76.42, 79.31, 80.97, 84.52, 119.98, 128.03, 128.48, 128.66, 129.29, 130.21,
130.64, 130.69, 131.00, 132.40, 133.45, 133.63, 154.94, 156.89, 167.01, 168.16, 169.10, 169.64, 170.19,
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170.21, 175.12, 204.18; HRMS (TOF) for C62H77N2O20S2 calcd: 1233.4506. Found: 1233.4496 (Δ = -
0.8 ppm). HPLC (2): t = 14.1 min, purity >98%.
Phenol-(SS-Linker)-OSu (14P). To a solution of 13P (154 mg, 0.410 mmol) and HOSu (0.141 g,
1.23 mmol) in CH2Cl2 (1.6 mL) was added EDC•HCl (94 mg, 0.493 mmol) in CH2Cl2 (1 mL), and the
mixture was allowed to react for 16 h at room temperature with stirring. The reaction was quenched with
saturated NH4Cl (10 mL), and the reaction mixture was diluted with water (10 mL), extracted with CH2Cl2
(20 mL x 3) and washed with brine (20 mL x 3). The combined organic layers were dried over MgSO4
and concentrated in vacuo to afford a yellow oil. Purification the crude product by column
chromatography on silica gel with 50% ethyl acetate in hexanes as eluent gave 14P (162 mg, 84% yield)
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as a colorless oil: H NMR (500 MHz, CDCl3) δ 1.30 (d, J = 6.8 Hz, 3H), 1.98 (m, 1H), 2.06 (m, 1H),
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2.66 (t, J = 7.8 Hz, 2H), 2.82 (bs, 4H), 3.00 (m, 1H), 4.13 (s, 2H), 7.10 (d, J = 7.5 Hz, 2H), 7.21 (m, 1H),
7.26 (m, 1H), 7.34 (m, 4H), 7.82 (d, J = 7.5 Hz, 1H); 13C NMR (500 MHz, CDCl3) δ 20.41, 25.60, 28.31,
30.28, 39.44, 45.60, 121.50, 125.88, 127.92, 128.42, 129.41, 130.56, 130.94, 133.57, 137.43, 150.76,
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168.16, 169.01, 169.46; HRMS (TOF) for C23H24NO6S2 calcd: 474.1040. Found: 474.1044 (Δ = 0.8
ppm).
Camptothecin-(SS-Linker)-NH-PEG3-(CH2)2N3 (15C). To a solution of 13C (110 mg, 0.175 mmol) and
1-amino-11-azido-3,6,9-trioxaundecane (76 mg, 0.349 mmol) in CH2Cl2 (60 mL) was added DIC (35 μL, 0.229
mmol), and the mixture was allowed to react for 4 h at room temperature with stirring. The reaction mixture was
concentrated in vacuo and directly loaded on to a silica gel column. Purification of the reaction mixture by column
chromatography on silica gel with 2% CH3OH in CH2Cl2 as eluent gave 15C (103 mg, 71% yield) as a pale-white
solid: mp 104-105 oC; 1H NMR (500 MHz, CDCl3) δ 1.00 (m, 3H), 1.18 (d, J = 6.5 Hz, 3H), 1.74 (m, 4H), 2.04 (m,
1H), 2.21 (m, 1H), 2.33 (m, 1H), 2.82 (m, 1H), 3.34 (m, 1H), 3.40 (m, 2H), 3.49 (m, 2H), 3.61 (m, 2H), 3.69 (m,
10H), 4.04 (d, J = 16.5 Hz, 1H), 4.22 (dd, J = 4.0, 16.5 Hz, 1H), 5.31 (s, 2H), 5.42 (d, J = 17.3 Hz, 1H), 5.66 (dd, J
= 1.4, 17.3 Hz, 1H), 5.98 (m, 1H), 7.25 (m, 5H), 7.72 (m, 2H), 7.89 (t, J = 7.4 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H),
8.30 (m, 1H), 8.42 (s, 1H); 13C NMR (400 MHz, CDCl3) δ 7.57, 14.20, 20.60, 20.73, 21.05, 23.50, 31.21, 31.42,
31.82, 33.50, 38.56, 39.10, 42.15, 46.36, 49.95, 50.67, 53.44, 60.39, 67.15, 69.74, 70.03, 70.23, 70.56, 70.61, 70.69,
96.30, 120.20, 120.29, 127.89, 128.01, 128.17, 128.19, 128.24, 128.46, 129.71, 130.62, 130.69, 131.12, 131.15,
131.21, 133.07, 137.55, 145.59, 146.22, 148.86, 152.32, 157.29, 167.32, 170.20, 171.14, 172.05, 172.12; HRMS
+
(TOF) for C41H47N6O9S2 calcd: 831.2840; found: 831.2838 (Δ = -0.2 ppm). HPLC (2): t = 12.4 min, purity >95%.
Phenol-(SS-Linker)-PEG3-(CH2)2N3 (15P). To a solution of 13P (149 mg, 0.396 mmol) and 1-amino-11-
azido-3,6,9-trioxaundecane (259 mg, 1.19 mmol) in CH2Cl2 (1 mL) was added DIC (67 μL, 0.436 mmol), and the
mixture was stirred for 10 h at room temperature. The reaction mixture was concentrated in vacuo to give a yellow
oil. Purification of the crude product by column chromatography on silica gel with 1.5% CH3OH in CH2Cl2 as
eluent gave 15P (0.127 g, 56%), as a colorless oil: 1H NMR (500 MHz, CDCl3) δ 1.34 (d, J = 6.8 Hz, 3H), 1.95 (m,
2H), 2.17 (t, J = 7.6 Hz, 1H), 2.57 (m, 1H), 2.94 (m, 1H), 3.40 (m, 2H), 3.49 (t, J = 5.3 Hz, 2H), 3.75 (m, 12H),
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