Synthesis of Novel Protected Nα(ω-Drug) Amino Acid Building Units for Facile Preparation of Anticancer Drug-Conjugates

Int J Pept Res Ther

DOI 10.1007/s10989-015-9509-1

Synthesis of Novel Protected N_a(x-Drug) Amino Acid Building

Units for Facile Preparation of Anticancer Drug-Conjugates

Y. Gilad^1,2S. Waintraub²A. Albeck²G. Gellerman¹

•

Accepted: 29 December 2015

Ó Springer Science+Business Media New York 2016

Abstract We report about the preparation of novel pro-

tected N_a(x-drug) amino acid building units and their

straightforward incorporation in solid phase synthesis for

the preparation of peptide-drug conjugates. These building

units were synthesized applying various coupling methods

between anticancer drugs and the side chains of different

N_aprotected amino acids. Subsequent incorporation of

these amino acid-drug motifs into linear and cyclic integrin

RGD and NGR containing ligands enabled a non-lin-

ear/divergent synthetic pathway of medicinally potential

peptide-drug conjugates. The synthetic routes reported in

this work are both general and applicable, and signiﬁcantly

expand the scope of the conjugation capabilities for peptide

drug conjugates. For the preliminary in vitro evaluation of

the novel peptide-drug conjugates reported herein, selec-

tive cytotoxicity of two representatives—one linear and

one cyclic RGD—camptothecin conjugates were evaluated

on a_vb₃integrin overexpressed cancer cell lines.

Graphical Abstract

O

NH-C

Linkage

Site

N

H-C

Drug

O

SPPS

O

peptide

Cyclic

Drug

ligand

HO₂C

NHFmoc

-

NH

C

O

Keywords Amino acid building units Á Anticancer

drugs Á Solid phase synthesis Á Peptide drug conjugates

Electronic supplementary material The online version of this

article (doi:10.1007/s10989-015-9509-1) contains supplementary

material, which is available to authorized users.

Abbreviations

AABU Amino acid building units

& G. Gellerman

garyg@ariel.ac.il

BU

Building unit

N,N⁰-dicyclohexylcarbodiimide

1

Department of Biological Chemistry, Ariel University,

40700 Ariel, Israel

DCC

DCM

Dichloromethane

2

DIPEA N,N⁰-diisopropylethylamine

The Julius Spokojny Bioorganic Chemistry Laboratory,

Department of Chemistry, Bar Ilan University,

52900 Ramat Gan, Israel

DMF Dimethylformamide

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Int J Pept Res Ther

EDC

1-Ethyl-3-(3-Dimethylaminopropyl)

Carbodiimide Hydrochloride

N-(9-Fluorenylmethoxycarbonyl)

Isobutyl Chloroformate

Not evaluated

amino acids responsible for maintaining of high binding

afﬁnity often allows a higher degree of ﬂexibility in the

design of linker length, its composition and conjugation

chemistry to the drug. Peptide conjugation can then include

a wide range of functional groups encompassing amides,

carboxylic acid esters, hydrazones, thioethers and carba-

mates (Majumdar and Siahaan 2012).

Fmoc

IBCF

NE

PNFC

SPPS

TFA

THF

TMS

para-Nitro phenyl chloroformate

Solid phase peptide synthesis

Triﬂuoroacetic acid

Based on the above, we decided to develop an efﬁcient

synthesis of premade ready-to-use Na-Fmoc protected

amino acid building units that bear anticancer drugs through

various biodegradable bonds, for incorporation in parallel

solid phase synthesis of peptide-drug conjugates.

Tetrahydrofuran

Trimethylsilyl

The rationale behind the concept presented herein is based

on the following: (1) premade Na-Fmoc protected AA-drug

building units can be incorporated into peptide sequence

using the convenient standard Fmoc chemistry protocol,

enabling efﬁcient high throughput parallel divergent syn-

thesis (Friligou et al. 2013) of linear and cyclic peptide-drug

conjugates; (2) Fmoc–AA(drug)–OH building units con-

taining biodegradable moieties: ester, carbamate and amide,

which potentiate enzymatic cleavage from the conjugate

in vivo (Vig et al. 2013); (3) The drug release proﬁle will

depend on the nature of the linker (AA side chain), positionin

the peptide sequence, linking moiety and the drug itself, as

we have already show in our previous study (Gilad et al.

2014). Consequently, drugs can be linked to the peptide

carrier through the appropriate building unit at various

positions in the peptide sequence for fast evaluation of

positioning and drug release parameters.

Introduction

Signiﬁcant research efforts have been invested in recent

years in the development of peptide therapeutics. Continuing

progress in synthetic peptide chemistry over the ensuing

years has largely solved many of the problems to the extent

that the ﬁeld is ﬂourishing today (Bellmann-Sickert and

Beck-Sickinger 2010). Recent surveys show that over 50

peptide drugs have been approved for clinical use. Another

several hundred peptides are in various stages of clinical

assessment (Firer and Gellerman 2012). This data gives a

strong impetus to using peptides as drug carriers for a variety

of medicinal applications and especially for cancer therapy.

Angiogenesis and metastasis are cell adhesion processes

mediated by heterodimeric transmembrane glycoprotein

receptors of the integrin family (Haubner et al. 1997;

Ruoslahti 1996). Tumor-induced angiogenesis is a conse-

quence of ligation by extracellular matrix proteins to the

a_vb₃integrin (Plow et al. 2000), which is highly expressed

on many tumor cells (Lafrenie et al. 1994). A common

binding motif in these matrix proteins is the amino acid

sequence arginine-glycine-aspartic acid (RGD). Another

binding motif with afﬁnity to tumor vasculature is CD13

receptor ligand, asparagine-glycine-arginine (NGR) (Arap

et al. 1998; Colombo et al. 2002; Pasqualini et al. 2000;

Soudy et al. 2012; van Hensbergen et al. 2002). Blocking

tumor-induced angiogenesis by inhibition of the a_vb₃

integrin is now a major target for cancer chemotherapy

(Cheng et al. 2014; Goodman and Picard 2012), and many

RGD-containing peptides have been evaluated as antago-

nists of integrins (Danhier et al. 2012).

By optimizing these parameters in the design of peptide

carrier-drug conjugates we hope to release the payload

speciﬁcally in the target cancer cells and thereby avoid

exposure of benign tissues to the cytotoxic treatment (Cohen

et al. 2007). We anticipate that the utilization of the Fmoc–

AA(drug) building units can be integrated into the rational

design and application of targeted drug delivery strategies

and ultimately expand the scope of the conjugation capa-

bilities for PDCs in general. Preparation of extended reper-

toire of Fmoc–AA(drug)–OH, their incorporation into other

peptide carriers and investigation of targeted therapeutic

properties is an ongoing process in our lab.

Results and Discussion

We are also investigating a_vb₃integrin peptidic antag-

onists as delivery vehicles for highly cytotoxic warheads,

while focusing on the important question of appropriate

carrier-linker-drug design and synthesis of peptide drug

conjugates (PDCs). The site of conjugation between the

peptide and the drug molecule can have a profound effect

on maintaining peptide binding afﬁnity, drug release,

activity and conjugate stability (Gilad et al. 2014). How-

ever, exact knowledge of the peptide sequence and the

Synthesis of N_a(x-Drug) Amino Acid (AA) Building

Units (BUs)

In several recent reports we discussed the synthesis of

functional amino acid building blocks for peptidomimetic

assembly and as scaffolds bearing various bioactive

modalities. (Gellerman et al. 2001). Kriek et al. reported on

uridylylated tyrosine and serine building blocks for stepwise

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Int J Pept Res Ther

NH₂

O

N

O

Cl

NH₂

N

O

OH

O

N

O

HO

N

HO

O

Cl

O

N

O

N

HO

O

N

O

OH

Deacetyl-colchicine

Amonafide

Cytarabine

Chlorambucil

Camptothecin

Fig. 1 Drugs that used for the preparation of the AABUs

solid phase synthesis of viral genome-linked peptides (Kriek

et al. 2003) using tritylation or allylation of metal-salt Fmoc–

AA–OH for regioselective esteriﬁcation of free carboxylic

acids. The following functionalization of the side chain and

consequent deprotection yielded the desired building blocks.

We decided to implement the same strategy, but with several

modiﬁcations, for preparing an extended repertoire of

Fmoc–AA(drug)–OH building units. The drugs that were

selected to assess the feasibility of this mission were ﬁve—

mechanistically distinguished and with different chemical

functions: (1) old DNA mustard alkylating drug—chloram-

bucil (CLB) with free carboxylic group; (2) topoisomerase II

intercalating agent—amonaﬁde (AM) with free aromatic

NH₂; (3) topoisomerase I inhibitor camptothecin (CPT), with

tertiary OH; (4) Microtubulin poison—colchicine, which

after deacetylation (Bagnato et al. 2004) revealed a primary

amine; (5) cytarabine—antimetabolite which is mainly used

(alone or together with other drugs) in treatment of cancers of

white blood cells—with the cytosine aromatic amine.

Structures of the mentioned drugs are shown in Fig. 1.

First, Fmoc amino acids that possess hydroxyl func-

tionality at their side chain (Ser, Thr and Tyr) were sub-

jected to the tritylation with 2-chlorotrityl chloride using

optimized conditions (as described in the experimental

section) leading to the intermediate esters 2a–

c (Scheme 1). Then, 2a–c were directly reacted with active

Cl

N

O

Cl

O

HO

1a (82% after 3 steps)

NHFmoc

b, c

Cl

O

N

R

b, c

a

O

Cl

R

O

ClTrt O

HO

NHFmoc

O

NHFmoc

HO

1b (73% after 3 steps)

2a-c

NHFmoc

b, c

Cl

O

N

O

Cl

1c (75% after 3 steps)

HO

FmocHN

Scheme 1 Synthesis of ester BUs 1a–c: CLB-Ser, -Thr and -Tyr AA BUs. a 2-ClTrt-Cl, DIPA, 2 h, rt; b Chlorambucil, HOBt, DCC, DMAP,

overnight, rt; c 3 % TFA/DCM

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Int J Pept Res Ther

ester of CLB, followed by acidic hydrolysis to recover—

after puriﬁcation—the corresponding Fmoc–Ser(CLB)–OH

1a, Fmoc–Thr(CLB)–OH 1b and Fmoc–Tyr(CLB)–OH 1c

building units in good yields. Notably, the DNA alkylating

drug CLB is linked to the side chains of these BUs through

different biodegradable esters: namely, in Ser BU 1a

through a primary alkyl ester, in Thr BU 1b through a

secondary alkyl ester and in Tyr BU 1c through a phenolic

ester. Such structurally controllable linkage of CLB will

deﬁne a more adequate and adjustable release proﬁle of the

drug from the peptide conjugate (Goldshaid et al. 2010).

At this stage we moved to the two-step synthesis of

Fmoc–Asp/Glu(drug)–OH amide-BUs 3a–f, starting from

commercially available Fmoc–AA mono esters

(Scheme 2). At the beginning, N_a-Fmoc-protected Asp or

Glu bearing orthogonal protecting group on their alpha

carboxylic group, were subjected to coupling reaction with

AM, cytarabine (Cyt) and deacetylated colchicine using

either standard peptide coupling conditions, utilizing

PyBop coupling reagent, or via asymmetric anhydride

(Manfredini et al. 2000) resulting intermediates 4a–l (for

yields see Table 1 in Supplementary Information). The

purpose of this study was to compare coupling conditions

for affording optimized AA drug coupling protocol. After

their isolation, the C-terminal protected intermediates were

subjected to an appropriate deprotection conditions, lead-

ing to the desired BUs 3a–f (for yields see Table 2 in

Supplementary Information). Overall, asymmetric anhy-

dride i-butyl chloroformate (IBCF) coupling method using

Fmoc–AA–OtBu starting materials was found as more

effective.

Next, commercially available Fmoc–Lys(Boc)–OH was

employed in the two-step synthesis of Fmoc–Lys(CPT)–

OH building block 5 (Scheme 3), in which CPT is linked to

the side chain amine through a carbamate bond.

Encouraged by the facile and successful synthesis of the

above building blocks, we decided to use them for a

preparation of peptide-drug conjugates. Therefore, standard

Fmoc SPPS, including a step of BU incorporation was

utilized. This resulted in a serial of four cyclic and one

linear RGD-containing peptide-drug conjugates, as

described below.

Synthesis of a Linear RGD-NGR Peptide-Drug

Conjugate

First we demonstrated the ease and effectiveness of

incorporation of our building unit into a linear peptide. For

this purpose we chose as a model two different tumor

vessels targeting motifs—RGD, (integrin a_vb₃targeting

moiety) (Arap et al. 1998; Chen and Chen 2011), and NGR

(CD 13 targeting moiety) (Pasqualini et al. 2000)—which

are both routinely employed for developing tumor vascu-

lature targeted delivery systems and imaging agents in

various peptide conjugates (Corti et al. 2008; Danhier et al.

2012; Ruoslahti 2002). Such an approach of covalent

fusion of two distinct targeting motifs—addressing differ-

ent receptors of the tumor vasculature—may increase the

targeting effectivity of this kind of delivery systems.

Moreover, the NGR motif resembles the a_vb₃ligand—

RGD, and it is well documented that under physiological

conditions a spontaneous rearrangement turns NGR into

O

b

O

R = tBu.

(88-94%)

NHFmoc

RO

HO

a

n

H

₂C

OH

n

H

₂C

NH

n

H

₂C

Drug

NH

Drug

(63-84%)

c

O

3a-f

R = Allyl

(37-82%)

4a-l

3a, n = 1 Drug = Deacetyl colchicine

3b, n = 2 Drug = Deacetyl colchicine

3c, n = 1 Drug = Amonafide

4a,

R =

n = 1 Drug = Deacetyl colchicine

n = 2 Drug = Deacetyl colchicine

n = 1 Drug = Amonafide

4b,

4c,

4d,

4e,

4f,

3d, n = 2 Drug = Amonafide

n = 2 Drug = Amonafide

3e,

3f,

n = 1 Drug = Cytarabine

n = 2 Drug = Cytarabine

n = 1 Drug = Cytarabine

n = 2 Drug = Cytarabine

4g,

4h,

4i,

n = 1 Drug = Deacetyl colchicine

n = 2 Drug = Deacetyl colchicine

n = 1 Drug = Amonafide

n = 2 Drug = Amonafide

n = 1 Drug = Cytarabine

4j,

4k,

4l,

n = 2 Drug = Cytarabine

Scheme 2 Synthesis of amide BUs 3a–f: amonaﬁde/cytarabine/deacetyl colchicine, -Asp, -Glu AA BU. a Drug, PyBop, HOBt, DIPA/DMF or

Drug, IBCF, TEA; b TFA/DCM; c Pd(PPh3)4, DMBA/DCM, 4 h, rt

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O

a

b

NHFmoc

NHBoc

NHFmoc

HN

HO

NH₂

O

6

N

O

N

O

5 (83% after 2 steps)

Scheme 3 Synthesis of the carbamate AABU 5. a 90 % TFA/DCM, 90 min, rt; b i. CPT, p-nitrophenyl chloroformate, DMF. ii. DIPA/DMF

Synthesis of Cyclic RGD Peptide-Drug Conjugates

isoDGR (Corti and Curnis 2011; Reissner and Aswad

2003; Robinson et al. 2004), which also exhibits relatively

high binding afﬁnity to the a_vb₃integrin (Curnis et al.

2006).

The cyclic pentapeptide c(RGDfK), ﬁrst designed and

synthesized by Kessler and co-workers (Haubner et al.

1997), was selected as a prototypical delivery vehicle

because the lysine side-chain provides a convenient handle

for attachment and since diverse relatives are amenable to

parallel synthesis (Dai et al. 2000). Apparently, the 5th

amino acid side chain residue of c(RGDfK) is not part of

the pharmacophore in this peptide (Arap et al. 1998;

Goldshaid et al. 2010; Haubner et al. 1997), and therefore it

can be substituted with other amino acids like serine,

threonine, and aspartic acid, having functional side chains

Therefore, for demonstrating the applicability of our

BUs as connecting elements between two distinct peptidic

sequences, we decided to construct the linear peptide 7,

where RGD and NGR sequences are connected through

Fmoc–Lys(CPT)–OH building unit 5 (Scheme 4). A linear

hepta-peptide-drug conjugate was resulted, containing two

different targeting moieties with Topoisomerase I interca-

lating agent CPT linked through the biodegradable carba-

mate ester (Vig et al. 2013).

Scheme 4 SPPS of the liner peptide-drug conjugate 7. a 4, PyBop, DIPA/DMF, 3 h, rt; b TFA/H₂O/Phenol/TIPS (90:5:3:2), 3 h, rt

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Scheme 5 SPPS of cyclic conjugates 8a–d. a–d HATU, DIPA/DMF,

3 h, rt, 1a for a, 5 for b, 3b for c, 3d for d; e 20 % piperidine/DMF;

f Fmoc-^D-Phe, HATU, DIPA/DMF; g Pd(PPh₃)₄(0.3 eq.), DMBA

(6 eq.);

overnight, rt; i TFA/H₂O/Phenol/TIPS (90 %/5 %/3 %/2 %), 3 h, rt

h

PyBop (5 eq.), HOBt (5 eq.), DIPA (10 eq.)/NMP,

Cell Cytotoxicity

suitable for drug linkage. We now describe a practical and

efﬁcient solid-phase synthesis of ‘Kessler type’ c(RGDfK),

c(RGDfS) and c(RGDfE) peptide-drug conjugates, based

on standard Fmoc chemistry protocols, using our BUs.

Noteworthy, our proposed BU approach can be utilized for

facile incorporation of drugs into peptide conjugates by

throughput parallel synthesis.

In order to compare the speciﬁc cytotoxic effects of the

linear conjugate 7 with its cyclic counterpart 8b, cytotox-

icity assays were performed using three different cell lines:

a human non-small cell lung carcinoma cell line H-1299

and a human prostate cancer cell line PC-3, both of which

overexpress a_vb₃integrins (68 and 69 % respectively,

Fig. 2), and a HEK-293 (Human Embryonic Kidney 293)

cell line, which served as a negative control with 2 % of

a_vb₃expression (Fig. 2). The level of a_vb₃integrin

expression was determined by direct immunoﬂuorescence

assay (Fig. 2) as described in materials and methods.

Cells were seeded in micro wells and allowed to adhere

during a 24 h incubation period, after which the cultures

were washed, fresh medium containing different concen-

trations of the tested substances was added and subse-

quently the cultures were incubated for additional 6 h.

After removal of the substances containing medium (to

avoid non-receptor dependent activity), fresh medium was

added and the cells were reincubated for additional 24 h.

At the end of the second 24 h incubation period, the

mitochondrial activity of the cells was measured by XTT

assay. The effect of drug treatment on the mitochondrial

activity was expressed as % of growth inhibition (GI)

The synthesis of cyclic conjugates 8a–d, which covers

all three drug-BU linkage functionalities—namely ester,

carbamate and amide—were carried out by using four

representative AABUs: BU 1 for the ester conjugate, BU 5

for the carbamate conjugate and BUs 3b and 3d for the

amide conjugates (Scheme 5). Notably, analogs of our

cyclic RGD–camptothecin conjugate 8b were previously

described (Dal Pozzo et al. 2010). In that work Dal Pozzo

et al. reported on a conjugation of Namitecan and its

aldehyde derivatives through an amide and hydrazine

linkage moiety to the cyclic RGDfK peptide carrier in

solution. We report, on the other hand, on complete SPPS

of the peptide-drug conjugate in which camptothecins’

linkage to the peptide carrier is through carbamate func-

tionality, thus yielding structurally different conjugate.

The extension of our BUs repertoire, evaluation of

peptide/drug/sequence/release proﬁling and bioactivity of

peptide-drug conjugates is currently under investigation.

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Int J Pept Res Ther

Fig. 2 Flow cytometry analysis of a_vb₃integrin expression in a H1299, b PC-3 and c HEK cell lines. In each graph the x axis represents the

intensity of ﬂuorescence and the y axis represents the number of counted cells

compared to cells which were exposed only to DMSO

(0.05 %) containing medium.

(GI) of 8b in PC-3 cells was even higher than the per-

centage of growth inhibition of a free drug. This tendency

was observed as well in H1299 cells, but only at concen-

trations higher than 50 lM.

In the in vitro cytotoxicity assay, the free CPT exerted

non-speciﬁc dose dependent manner of action towards all

the tested cell lines, namely H1299, PC-3 and HEK

(Fig. 3). Conjugation of this Topo I inhibitor (CPT) to the

linear RGD-NGR peptide 7 signiﬁcantly reduced its toxi-

city in tested cancer cell lines, but still preserving moderate

potency probably due to low afﬁnity of the linear RGD and

NGR sequences on a_vb₃integrin receptor (Gurrath et al.

1992; Shabbir et al. 2010). On the other hand, the cyclic

RGD-CPT conjugate 8b presented signiﬁcant toxicity

reduction towards a_vb₃negative HEK cells, but not

towards a_vb₃integrin overexpressing H-1299 and PC-3

tumor cells. Moreover, the percentage of growth inhibition

Afﬁnity Assay of Conjugate 8b to a_vb₃Integrin

In order to assess the correlation between the selective

cytotoxicity of conjugate 8b towards cells with overex-

pression of the a_vb₃integrin and with the afﬁnity of this

conjugate (8b) to the relevant receptor (a_vb₃integrin), an

immunoﬂuorescence assay was performed: Cells with

overexpressed level of a_vb₃were ﬁrst incubated with

solutions containing different concentrations of conjugate

Fig. 3 XTT growth inhibition

assay—the effect on cell growth

of peptide-CPT-conjugates vs a

free CPT was studied in three

cell lines: a H1299 and b PC-3

cell lines, which over-express

integrin a_vb₃and c HEK cell

line, which is a_vb₃-negative.

The result shown for each

concentration point represents

the mean SD calculated from

a quadruplicate

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Int J Pept Res Ther

8b. Following this incubation, all the cell samples were

centrifuged, washed and then incubated again with uniform

amount of R-phycoerythrin-conjugated anti-human CD51/

CD61 antibody. At the end of the second incubation period

all the cell samples were centrifuged and washed again and

then tested for the bound level of CD51/CD61 antibody by

FACS cell analyzer (Fig. 2). As seen from Fig. 2, increase

in the amount of the conjugate 8b (ﬁrst step incubation)

results in decrease levels of the bound CD51/CD61 anti-

body (second step incubation). Being c(RGDfK) an high

afﬁnity ligand to the integrin a_vb₃receptor was supported

by this experiment, which shows that cells with overex-

pression of a_vb₃bound less a_vb₃speciﬁc antibody if pre-

viously incubated with the c(RGDfK)-CPT conjugate (8b).

Moreover, this observation conﬁrms as well, that also after

the conjugation of the drug (CPT) to the peptide core, the

afﬁnity of the resulted conjugate (8b) to the targeted

receptor is preserved. These results support our perception

that the selective cytotoxicity of 8b is mediated by a_vb₃

integrin receptor (Fig. 4).

Conclusions

In this paper we describe a facile preparation of Fmoc

protected amino acid—drug building units for using as

structural elements in the divergent synthesis of PDCs.

Their simple, practical and efﬁcient implementation in the

solid-phase synthesis of novel linear RGD-NGR as well as

a ‘Kessler like’ a_vb₃integrin antagonistic conjugates using

straight forward Fmoc chemistry was also exempliﬁed.

Moreover, our method utilizes the diversity of drug link-

ages by a facile incorporation into peptide conjugates,

applying convergent SPPS. The synthetic routes reported in

this work should be amenable to fast parallel synthesis of a

wide range of analogous peptide-drug conjugates for fast

assessment of drug release proﬁle that depends on the

nature of the linker (AA side chain), position in the peptide

sequence, linking moiety and the drug itself.

In the in vitro cytotoxicity study, the free CPT was non-

speciﬁcally cytotoxic to all cell lines, including negative

control HEK cells that lack a_vb₃integrins. Cyclic RGD-CPT

conjugate, on the other hand, exerted selective potency on

a_vb₃overexpressed cancer cell lines as compared to the

negative control HEK cells. In particular, cyclic RGD-CPT

was more toxic than free CPT on prostate cancer cells PC-3

in all concentrations. The well know phenomena of being

c(RGDfK) a high afﬁnity ligand to the integrin a_vb₃was

supported in this work by the FACS experiment. Further-

more, these results conﬁrm that after conjugating a drug

(CPT) to the peptidic core, the afﬁnity of the resulted con-

jugate to the targeted receptor is preserved.

A future perspective of incorporation of the discussed

AABUs in the backbone of targeted peptide-drug biocon-

jugates could be an attachment of several types of anti-

cancer drugs - through different linkages—within a single

conjugate. This may result in a synergistic action of the

drugs in the target and thereby could further enhance the

drug delivery for preclinical targeted cancer therapy

assessment.

Materials and Methods

General Methods and Cell Lines

Fig. 4 Flow cytometry analysis of four samples of PC-3 cells: gray—

unstained cells (control sample); solid red line—cells which were

incubated with 10 nM of conjugate 8b then washed and incubated

again with R-phycoerythrin-conjugated anti-human CD51/CD61

antibody; dashed red line—cells which were incubated with 1 nM

of conjugate 8b then washed and incubated again with R-phycoery-

thrin-conjugated anti-human CD51/CD61 antibody; black solid line—

cells which were incubated with R-phycoerythrin-conjugated anti-

human CD51/CD61 antibody without be previously incubated with

8b

The NMR spectral data was recorded in deuterated solvents

at 300 MHz—Bruker NMR spectrometer or 400 MHz—

Bruker Avance-III 400 MHz NMR spectrometer equipped

with a BBFO probe, and reported in d ppm relative to TMS

as an internal standard. Mass spectra was measured using

Autoﬂex III smartbeam (MALDI, Bruker) in linear or

reﬂectron mode, positive or negative or Q-TOF micro

(Waters) ESI (? or -). HPLC/LC–MS analyses were

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Int J Pept Res Ther

[Fmoc–Ser(CLB)–OH] (1a) Compound 1a was obtained

as brownish oil with 82 % yield Rf: 0.72 (25 %MeOH/

collected using C18, 2.1 9 50 mm, 1.8 lm column kept at

50 °C, with detection at 254 nm. The eluent solvents were

A (0.1 % TFA in H₂O) and B (0.1 % TFA in ACN). The

ﬂow rate was of 0.4 mL/min. The MS fragmentor was

tuned on 30 or 70 V on positive or negative mode. For the

elution gradient proﬁle see supporting information. All

HPLC puriﬁcations were done via reverse phase on semi-

preparative system with dual UV detection at 254 nm and

1

EtOAc); LC–MS m/z: 615 (M?H^?), RT = 10.80 min; H

NMR (300 MHz, DMSO-d₆): d 1.88 (t, J = 8.7 Hz, 2H),

2.32 (t, J = 8.7 Hz, 2H), 2.52 (t, J = 8.7 Hz, 2H), 3.58 (t,

J = 6.8 Hz, 4H), 3.68 (t, J = 6.8 Hz, 4H), 4.25 (t,

J = 8.1 Hz, 1H), 4.35 (d, J = 8.1 Hz, 2H), 4.45 (m, 1H),

4.65 (m, 1H), 5.65 (d, J = 8.0 Hz, 1H), 6.8 (d, J = 8.0 Hz,

2H), 7.2 (d, J = 8.0 Hz, 2H), 7.3 (t, J = 8.0 Hz, 2H), 7.39

(t, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.75 (d,

J = 8.0 Hz, 2H); HRMS: ESI–MS m/z calcd: 613.1832,

found: 613.1834 (MH^?).

230 nm. Phenomenex Gemini^Ò10 lm C18 110 A, LC

˚

250 9 21.2 mm prep column was utilized. The eluent

solvents were A (0.1 % TFA in H₂O) and B (0.1 % TFA in

ACN). The ﬂow rate was set up on 25 mL/min. For the

elution gradient proﬁle see supporting information. Thin

layer chromatography (TLC) was performed using

20 9 20 cm silica-gel plates (Merck silica gel, 60 F₂₅₄),

and visualized under UV ﬂuorescence (k_max= 254 nm

and/or 366 nm). CAMP and CLB were purchased from

Tzamal D-Chem Laboratories Ltd. Petah-Tikva, Israel. All

the cell lines were cultured in RPMI medium supplemented

with ^L-glutamine, 10 % fetal bovine serum and with

penicillin streptomycin (100 IU/mL of each) (cell culture

growth medium and all of its additives were purchased

from Biological Industries, Bet-Ha’emek, Israel). All cell

cultures were grown at a 37 °C incubator in an environ-

ment containing 6 % CO₂.

[Fmoc–Thr(CLB)–OH] (1b) Compound 1b was obtained

as brownish oil with 73 % yield Rf: 0.79 (25 %MeOH/

1

EtOAc); LC–MS m/z: 627 (M?H^?), RT = 11.05 min; H

NMR (400 MHz, DMSO-d₆): d 1.21 (br. s, 3H); 1.75 (br.

quint, 2H), 2.24 (br. q, 2H), 2.43 (br. q, 2H), 3.68 (s, 8H), 4.24

(m, 4H), 5.25 (m, 1H), 6.64 (d, J = 8.6 Hz, 2H), 7.00 (d,

J = 8.6 Hz, 2H), 7.32 (t, J = 6.9 Hz, 2H), 7.41 (t, J =

6.9 Hz, 2H), 7.77 (d, J = 6.9 Hz, 2H), 7.90 (d, J = 6.9 Hz,

2H); HRMS: ESI–MS m/z calcd: 649.189, found: 649.185

(M?Na^?), calcd: 665.159, 665.166 (M?K^?).

[Fmoc–Tyr(CLB)–OH] (1c) Compound 1c was obtained

as brownish oil with 75 % yield Rf: 0.80 (25 % MeOH/

1

EtOAc); LC–MS m/z: 689 (M?H^?), RT = 11.10 min; H

Chemistry

NMR (300 MHz, DMSO-d₆): d 1.88 (quint, J = 7.6 Hz

2H), 2.55 (m, 4H), 2.87 (dd, J = 14.0, 7.6 Hz 1H), 3.08

(dd, J = 14.0, 7.6 Hz 1H), 3.7 (s, 8H), 4.2 (m, 4H), 6.65 (d,

J = 8 Hz, 2H), 7.0 (d, J = 8 Hz, 2H), 7.06 (d, J = 8 Hz,

2H), 7.28 (m, 2H), 7.31 (m, 2H), 7.4 (m, 2H), 7.65 (t,

J = 8 Hz, 2H), 7.74 (d, J = 8 Hz, 1H), 7.87 (d, J = 8 Hz,

2H); HRMS: ESI–MS m/z calcd: 711.205, found: 711.193

(M?Na^?).

General Procedure for the Synthesis of 1a–c

To a suspension of Fmoc–AA–OH (0.5 mmol) in DCM

(25 mL) was added DIPA (1.1 eq.) (making the solution

clear), followed by addition of 2-Cl-Trt chloride

(1.05 mmol). After stirring at rt. for 2 h, the solvent was

evaporated, providing a cotton-like product. It was dissolved

in DCM and then CLB (1 eq.), HOBt (1.5 eq.), DMAP

(1 eq.) and DCC (1.5 eq.) were added and white precipitate

was observed after about 20 min. The mixture was allowed

to stir overnight. The white precipitate was removed by ﬁl-

tration resulting in a clear pale orange solution. The solvent

was removed, and the crude product was dissolved in DCM

(30 mL) with a small amount of MeOH. The organic solution

was washed several times with H₂O, brine and NaHCO₃

(5 %). The collected organic phase was dried over Na₂SO₄,

and evaporated, yielding beige oil. The crude was suspended

in DCM, TFA was added (to 3 % ﬁnal concentration) and the

beige suspension immediately turns into an orange solution.

The mixture was allowed to stir for 1 h. The solvent was

removed and the crude was puriﬁed by semi-preparative

HPLC. Clean fractions were lyophilized resulting oily

products in 82, 75, and 73 % yields for Ser, Tyr, and Thr

building units respectively.

General Procedure for the Synthesis of 4a–l: PyBop

Method

Fmoc–AA–OtBu/OAllyl (0.5 mol) was stirred in 10 mL of

DMF and then DIPA (3 eq.) was added. Into the resulted

clear solution of the AA carboxylate the coupling reagent

PyBop (1 eq.) was added. Into this stirred active ester

solution the drug with a free amine group was added

immediately and the mixture allowed to stir at rt. Aliquots

of the reaction solution were monitored by LC–MS and

after a completion of the reaction—typically after 3 h—the

reaction solution was directly subjected to puriﬁcation by

semi prep. HPLC. Clean fractions were lyophilized

resulting powdery product.

123

Int J Pept Res Ther

General Procedure for the Synthesis of 4a–l: IBCF

(Asymmetric Anhydride) Method

(CH₂), 107.62 (CH), 112.01 (CH), 120.03 (2 CH), 125.17

(2 CH), 125.33 (C), 126.98 (2 CH), 127.56 (2 CH), 130.26

(CH), 134.13 (C), 134.31 (CH), 135.07 (C), 140.63 (2 C),

143.70 (2 C), 143.71 (C), 150.34 (C), 150.66 (C), 152.84

(C), 156.00 (CO–NH-OR), 163.43 (C), 170.50 (CO-OR),

171.24 (CO-NHR), 177.89 (CO); LC–MS m/z: 765

(M?H^?), RT = 10.33 min.

Fmoc–AA–OtBu/OAllyl (0.5 mol) was dissolved in 5 mL

of THF containing 1.1 eq. of TEA. The carboxylate solu-

tion was cooled to (-15) °C to (-20) °C following IBCF

addition. After 15 min to the cool white suspended solution

the drug with a free amine group—dissolved in a minimal

amount of DMSO—was added and the mixture stirred for

30 min in the cool bath following 3–7 h of stirring at rt. If

needed, portions of DMSO were added in order to result

clear solution. After the LC–MS monitoring indicated that

the reaction was completed, the mixture was subjected to

puriﬁcation by semi prep. HPLC. Clean fractions were

lyophilized resulting crystalline powders.

[Fmoc–Asp(Amonaﬁde)–OtBu] (4c) Compound 4c was

obtained as orange solid with 70 % yield by PyBop cou-

pling method or 75 % yield by IBCF asymmetric anhy-

1

dride method. H NMR: (400 MHz, DMSO-d₆): d 1.36 (s,

9H), 2.77 (dd, J = 16.0, 6.0 Hz, 1H), 2.91 (s, 3H), 2.92 (s,

3H), 2.96 (dd, J = 16.0, 6.0 Hz, 1H), 3.44–3.49 (br. q, 2H),

4.23 (t, 7.0 Hz, 1H), 4.30–4.36 (m, 2H), 4.39 (t,

J = 5.0 Hz, 2H), 4.67 (q, J = 7.0 Hz, 1H), 7.31 (t,

J = 7.0 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.71 (dd,

J = 8.0, 1.0 Hz, 2H), 7.82–7.86 (m, 2H), 7.89 (d,

J = 7.0 Hz, 2H), 8.70 (d, J = 2.0 Hz, 1H), 8.77 (d,

J = 2.0 Hz, 1H), 9.26 (s, 1H), 10.71 (s, 1H); LC–MS m/z:

677 (M?H^?), RT = 9.49 min.

[Fmoc–Asp(Deacetyl Colchicine)–OtBu] (4a) Compound

4a was obtained as yellow solid with 63 % yield by PyBop

coupling method or 78 % yield by IBCF asymmetric

anhydride method. ¹H NMR: (400 MHz, DMSO-d₆): d

1.32 (s, 9H), 1.79–1.87 (m, 1H), 1.96–2.02 (m, 1H),

2.18–2.27 (m, 1H), 2.54–2.61 (m, 3H), 3.53 (s, 3H), 3.78

(s, 3H), 3.82 (s, 3H), 3.86 (s, 3H), 4.15–4.29 (m, 4H),

4.30–4.40 (m, 1H), 6.76 (s, 1H), 7.01–7.13 (m, 2H), 7.16

(s, 1H), 7.40 (t, J = 8.0 Hz, 2H) 7.59 (d, J = 7.0 Hz, 1H),

7.66 (dd, 7.5, 3 Hz, 2H) 7.88 (d, J = 7.0 Hz, 2H), 8.58 (d,

J = 7.0 Hz, 1H); ¹³C and DEPTQ (¹³C) NMR (101 MHz,

DMSO-d₆): d 27.39 (3 CH₃), 29.10 (CH₂), 30.31 (C), 35.79

(CH₂), 36.83 (CH₂), 46.46 (CH), 51.12 (CH), 51.26 (CH),

55.72 (CH₃), 55.93 (CH₃), 60.58 (CH₃), 60.72 (CH₃), 65.53

(CH₂), 107.72 (CH), 111.96 (CH), 120.00 (2 CH),

125.08(CH), 125.10 (CH), 125.30 (C), 126.92 (2 CH),

127.50 (CH), 127.51 (CH), 130.39 (CH), 134.06 (C),

134.27 (CH), 134.98 (C), 140.59 (C), 140.65 (C), 143.64 (2

C), 143.68 (C), 150.32 (C), 150.35 (C), 152.86 (C), 156.60

(CO–NH-OR), 163.42 (C), 168.07 (CO-OR), 170.55 (CO-

NHR), 177.89 (CO); LC–MS m/z: 751 (M?H^?),

RT = 10.70 min.

[Fmoc–Glu(Amonaﬁde)–OtBu] (4d) Compound 4d was

obtained as orange solid with 78 % yield by PyBop cou-

pling method or 81 % yield by IBCF asymmetric anhy-

1

dride method. H NMR: (400 MHz, DMSO-d₆): d 1.42 (s,

9H), 1.89–1.95 (m, 1H), 2.11–2.16 (m, 1H), 2.56 (t,

J = 5.5 Hz, 2H), 2.91 (s, 3H), 2.92 (s, 3H), 3.43–3.48 (m,

2H), 4.01–4.06 (m, 1H), 4.19–4.36 (m, 3H), 4.38 (t,

J = 5.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H) 7.42 (t,

J = 7.5 Hz, 2H), 7.73 (d, J = 7.5 Hz, 2H), 7.77–7.85 (m,

2H), 7.89 (d, J = 7.5 Hz, 2H), 8.37–8.41 (m, 2H), 8.68 (d,

J = 2 Hz, 1H), 8.77 (d, J = 2 Hz, 1H), 9.30 (s, 1H), 10.61

(s, 1H); ¹³C and DEPTQ (¹³C) NMR (101 MHz, DMSO-

d₆): d 26.04 (CH₂), 27.56 (3 CH₃), 32.50 (CH₂), 35.07

(CH₂), 42.67 (2 CH₃), 46.55 (CH), 53.85 (CH), 54.84

(CH₂), 64.53 (CH₂), 80.59 (C), 120.03 (2 CH), 120.75

(CH), 121.85 (C), 122.64 (C), 123.80 (CH), 124.00 (C),

125.13(2 CH), 126.96 (2 CH), 127.51 (CH), 127.55 (2 CH),

128.94 (CH), 132.01 (C), 133.81 (CH), 137.90 (C), 140.62

(C), 140.63 (C), 143.67 (C), 143.71 (C), 156.07 (CO–NH–

OR), 163.70 (CO–NR–CO), 163.95 (CO–NR–CO), 171.04

(CO–OH), 171.29 (CO–NHR); LC–MS m/z: 691 (M?H^?),

RT = 9.30 min.

[Fmoc–Glu(Deacetyl colchicine)–OtBu] (4b) Compound

4b was obtained as yellow solid with 64 % yield by PyBop

coupling method or 80 % yield by IBCF asymmetric

anhydride method. ¹H NMR: (400 MHz, DMSO-d₆): d

1.36 (s, 9H), 1.65–1.75 (m, 1H), 1.78–1.92 (m, 2H),

1.96–2.06 (m, 1H), 2.17–2.30 (m, 3H), 2.59 (dd, J = 13.0,

7.0 Hz, 2H), 3.53 (s, 3H), 3.79 (s, 3H), 3.83 (s, 3H), 3.87

(s, 3H), 4.20–4.37 (m, 4H), 6.67 (s, 1H) 7.01–7.14 (m, 3H),

7.33 (t, J = 7.5 Hz, 2H) 7.43 (t, J = 7.5 Hz, 2H), 7.69 (s,

1H) 7.72 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H),

8.58 (d, J = 5.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-

d₆): d 26.30 (CH₂), 27.51 (3 CH₃), 29.11 (CH₂), 31.31 (C),

35.65 (CH₂), 46.55 (CH), 51.13 (CH), 51.93 (CH), 55.74

(CH₃), 55.93 (CH₃), 60.59 (CH₃), 60.75 (CH₃), 65.53

[Fmoc–Asp(Cytarabine)–OtBu] (4e) Compound 4e was

obtained as white solid with 72 % yield by IBCF asym-

1

metric anhydride method. H NMR: (400 MHz, DMSO-

d₆): d 1.39 (s, 9H), 2.74 (dd, J = 15, 6 Hz), 2.91 (dd,

J = 15, 6 Hz), 3.62 (d, J = 5.4 Hz, 2H), 3.81–3.86 (m,

1H), 3.93 (t, J = 3.1 Hz, 1H), 4.07 (dd, J = 4, 2.5 Hz, 1H),

4.23 (d, J = 7.0 Hz, 1H), 4.29 (s, 1H), 4.31 (br quart, 1H),

4.39 (dd, J = 15.0, 7.0 Hz, 1H), 5.00–5.90 (br s, 3 [OH]),

123

Int J Pept Res Ther

6.05 (d, J = 4.0 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.32

(dt, J = 7.5, 1.2 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.71 (d,

J = 7.5 Hz, 2H), 7.80 (d, J = 7.5 Hz, 1H), 7.89 (d,

J = 7.5 Hz, 2H), 8.08 (d, J = 7.5 Hz, 1H), 10.95 (s, 1H);

¹³C and DEPTQ (¹³C) NMR (400 MHz, DMSO-d₆): d

27.43 (3 CH₃), 35.75 (CH₂), 38.37 (CH₂), 46.48 (CH),

50.67 (CH), 60.91 (CH₂), 65.59 (CH₂), 74.47 (CH), 76.02

(CH), 80.81 (C), 85.69 (CH), 86.93 (CH), 94.22 (CH),

120.02 (2 CH), 125.12 (2 CH), 126.96 (2 CH), 127.54 (2

CH), 140.61 (2 C), 143.66 (2 C), 146.76 (CH), 154.31 (CO-

(NR₂)₂), 155.72 (CO-NHR-OR), 161.94 (C), 170.15 (CO-

OR), 170.49 (CO-NHR); LC–MS m/z: 637 (M?H^?),

RT = 9.58 min.

(CH₂), 126.94 (2 CH), 127.52 (2 CH), 130.36 (CH), 132.16

(CH), 134.07 (C), 134.33 (CH), 134.99 (C), 140.60 (2 C),

140.65 (C), 143.62 (C), 143.66 (C), 150.32 (C), 150.35 (C),

152.86 (C), 155.65 (CO–NH-OR), 163.43 (C), 167.94 (CO-

OR), 171.05 (CO-NHR), 177.92 (CO); LC–MS m/z: 735

(M?H^?), RT = 10.26 min.

[Fmoc–Glu(Deacetyl Colchicine)–OAllyl] (4h) Com-

pound 4h was obtained as yellow solid with 70 % yield by

PyBop coupling method or 74 % yield by IBCF asym-

1

metric anhydride method. H NMR: (400 MHz, DMSO-

d₆): d 1.71–1.85 (m, 2H), 1.95–2.04 (m, 2H), 2.17–2.24 (m,

1H), 2.28 (t, 8 Hz 2H) 2.54–2.61 (m, 1H), 3.53 (s, 3H),

3.79 (s, 3H), 3.83 (s, 3H), 3.87 (s, 3H), 4.00–4.06 (m, 1H),

4.20–4.37 (m, 4H), 4.55 (td, 5, 1.5 Hz, 2H), 5.10 (dq,

J = 11.0, 2.0 Hz, 1H), 5.23 (dq, J = 17.0, 2.0 Hz, 1H),

5.82–5.91 (m, 1H), 6.77 (s, 1H), 7.02 (d, 7.0 Hz, 1H), 7.11

(d, 7.0 Hz, 1H), 7.13 (s, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.42

(t, J = 7.5 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.84 (d,

J = 7.0 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.59 (d,

J = 7.0 Hz, 1H); DEPTQ (¹³C) NMR (400 MHz, DMSO-

d₆): d 26.08 (CH₂), 28.92 (CH₂), 31.06 (CH₂), 35.60 (CH₂),

46.50 (CH), 51.10 (CH), 53.30 (CH), 55.70 (CH₃), 55.92

(CH₃), 60.59 (CH₃), 60.76 (CH₃), 64.72 (CH₂), 65.60

(CH₂), 107.62 (CH), 112.01 (CH), 117.60 (CH), 120.02(2

CH), 125.15 (2 CH), 125.30 (CH₂), 126.99 (2 CH), 127.56

(2 CH), 130.25 (CH), 132.24 (CH), 134.12 (C), 134.33

(CH), 135.06 (C), 140.61 (2 C), 143.67 (2 C), 150.32 (C),

150.65 (2 C), 152.83 (C), 156.03 (CO–NH-OR), 163.43

(C), 170.39 (CO-OR), 171.71 (CO–NHR), 177.88 (CO);

LC–MS m/z: 749 (M?H^?), RT = 10.43 min.

[Fmoc–Glu(Cytarabine)–OtBu] (4f) Compound 4f was

obtained as white solid with 76 % yield by IBCF asym-

1

metric anhydride method. H NMR: (400 MHz, DMSO-

d₆): d 1.40 (s, 9H), 1.73–1.90 (m, 1H), 1.93–2.06 (m, 1H),

2.51–2.55 (m, 2H), 3.62 (d, J = 5.4 Hz, 2H), 3.70–4.10 (br

s, 3 [OH]), 3.81–3.86 (m, 1H), 3.93 (t, J = 3.1 Hz, 1H),

4.07 (dd, J = 4.0, 2.5 Hz, 1H), 4.20–4.36 (m, 3H), 6.06 (d,

4.0 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.34 (dt, J = 7.5,

1.2 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.70 (d, J = 7.5 Hz,

1H), 7.73 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H),

8.07 (d, J = 7.5 Hz, 1H), 10.98 (s, 1H); ¹³C and DEPTQ

(¹³C) NMR (400 MHz, DMSO-d₆): d 25.60 (CH₂), 27.52

(3 CH₃), 32.67 (CH₂), 46.53 (CH), 53.68 (CH), 60.91

(CH₂), 65.52 (CH₂), 74.48 (CH), 76.01 (CH), 80.59 (C),

85.66 (CH), 86.88 (CH), 94.19 (CH), 120.03(2 CH), 125.14

(2 CH), 126.98 (2 CH), 127.55 (2 CH), 140.63 (2 C),

143.68 (C), 143.69 (C), 146.66 (CH), 154.34 (CO–(NR₂)₂),

155.98 (CO–NHR–OR), 161.97 (C), 171.18 (CO–OR),

172.78 (CO–NHR); LC–MS m/z: 651 (M?H^?),

RT = 9.58 min.

[Fmoc–Asp(Amonaﬁde)–OAllyl] (4i) Compound 4i was

obtained as brownish solid with 82 % yield by PyBop

coupling method or 84 % yield by IBCF asymmetric

anhydride method. ¹HNMR: (400 MHz, DMSO-d₆): d 2.88

(dd, J = 16.0, 6.0 Hz, 1H), 2.91 (s, 3H), 2.92 (s, 3H), 3.04

(dd, J = 16.0, 6.0 Hz, 1H), 3.44–3.49 (br. q, 2H), 4.22 (t,

7.0 Hz, 1H), 4.31–4.36 (m, 2H), 4.39 (t, J = 5.0 Hz, 2H),

4.61 (td, J = 5.0, 1.5 Hz, 2H), 4.67 (q, J = 7.0 Hz, 1H),

5.17 (dq, J = 11.0, 2.0 Hz, 1H), 5.31 (dq, J = 11.0,

2.0 Hz, 1H), 5.83–5.93 (m, 1H), 7.30 (t, J = 7.5, 2H) 7.39

(t, J = 7.5 Hz, 2H), 7.70 (dd, J = 7.0, 3.0 Hz, 2H), 7.84 (t,

J = 7.0 Hz, 1H), 7.88 (d, J = 7.0 Hz, 2H), 7.99 (d,

J = 7.0 Hz, 2H), 8.40 (dt, J = 8.0, 1.0 Hz, 2H), 8.68 (d,

J = 2.0 Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H), 9.31 (s, 1H),

10.74 (s, 1H); ¹³C and DEPTQ (¹³C) NMR (101 MHz,

DMSO-d₆): d 35.08 (CH₂), 38.03 (CH₂), 42.67 (2 CH₃),

46.67 (CH), 50.39 (CH), 54.84 (CH₂), 64.98 (CH₂), 65.66

(CH₂), 117.42 (CH₂), 120.02 (2 CH), 120.93 (CH), 121.87

(C), 122.66 (C), 123.80 (CH), 124.10 (C), 125.06(2 CH),

126.96 (2 CH), 127.53 (2 CH), 127.56 (CH), 129.04 (CH),

131.98 (C), 132.16 (CH), 133.86 (CH). 137.70 (C), 140.61

[Fmoc–Asp(Deacetyl Colchicine)–OAllyl] (4g) Com-

pound 4g was obtained as yellow solid with 72 % yield by

PyBop coupling method or 70 % yield by IBCF asym-

metric anhydride method.¹H NMR: (400 MHz, DMSO-d₆):

d 1.78–1.87 (m, 1H), 1.95–2.05 (m, 1H), 2.17–2.26 (m,

1H), 2.54–2.72 (m, 3H), 3.53 (s, 3H), 3.79 (s, 3H), 3.83 (s,

3H), 3.87 (s, 3H), 4.18–4.29 (m, 3H), 4.34–4.44 (m, 2H),

4.50–4.60 (m, 2H), 5.10 (dq, J = 11.0, 2.0 Hz, 1H), 5.23

(dq, J = 17.0, 2.0 Hz, 1H), 5.75–5.85 (m, 1H), 6.76 (s,

1H), 7.02 (d, 7.0 Hz, 1H), 7.12 (d, 7.0 Hz, 1H), 7.16 (s,

1H), 7.25–7.30 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H) 7.66 (dd,

J = 7.5, 2.5 Hz, 2H), 7.76 (d, 7.0 Hz, 1H), 7.88 (d,

J = 7.0 Hz, 2H), 8.58 (d, J = 7.0 Hz, 1H); ¹³C and

DEPTQ (¹³C) NMR (400 MHz, DMSO-d₆): d 29.10 (CH₂),

35.75 (CH₂), 36.72 (CH₂), 46.44 (CH), 50.40 (CH), 51.25

(CH), 55.73 (CH₃), 55.94 (CH₃), 60.59 (CH₃), 60.76

(CH₃), 64.85 (CH₂), 65.61 (CH₂), 107.62 (CH), 111.98

(CH), 117.38 (CH), 120.02(2 CH), 125.08 (2 CH), 125.30

123

Int J Pept Res Ther

[Fmoc–Glu(Cytarabine)–OAllyl] (4l) Compound 4l was

obtained as white solid with 84 % yield by IBCF asym-

1

metric anhydride method. H NMR: (400 MHz, DMSO-

(2 C), 143.60 (C), 143.64 (C), 155.81 (CO–NH–OR),

163.68 (CO–NR–CO), 163.94 (CO-NR-CO), 167.57 (CO-

OR), 170.95 (CO–NHR); LC–MS m/z: 661 (M?H^?),

RT = 9.26 min.

d₆): d 1.81–1.91 (m, 1H), 2.03–2.12 (m, 1H), 2.52–2.56 (m,

2H), 3.62 (d, J = 5.5 Hz, 2H), 3.82–3.85 (m, 1H), 3.93 (t,

J = 2.5 Hz, 1H), 4.06–4.12 (m, 2H), 4.24 (d, J = 7 Hz,

1H), 4.29–4.35 (m, 2H), 4.50 (br s, [3 OH]), 4.58 (td,

J = 5.0, 2.0 Hz, 2H), 5.20 (dq, J = 10.5, 1.5 Hz, 1H), 5.31

(dq, J = 17.5, 1.5 Hz, 1H), 5.85–5.95 (m, 1H), 6.06 (d,

5.0 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.34 (tt, J = 7.5,

1.2 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.72 (d, J = 7.5 Hz,

2H), 7.85 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H),

8.08 (d, J = 7.5 Hz, 1H), 10.88 (s, 1H); ¹³C and DEPTQ

(¹³C) NMR (101 MHz, DMSO-d₆): d 37.98 (CH₂), 46.46

(CH), 50.00 (CH), 60.89 (CH₂), 64.97 (CH₂), 65.71 (CH₂),

74.49 (CH), 76.02 (CH), 85.70 (CH), 86.95 (CH), 94.28

(CH), 117.41 (CH₂), 120.03 (2 CH), 125.10 (2 CH), 126.98

(2 CH), 127.55 (2 CH), 132.11 (CH) 140.62 (2 C), 143.72

(2 C), 146.77 (CH), 154.22 (CO–(NR₂)₂), 155.68 (CO–

NHR–OR), 161.96 (C), 170.26 (CO–OR), 170.71 (CO–

NHR); LC–MS m/z: 635 (M?H^?), RT = 9.30 min.

[Fmoc–Glu(Amonaﬁde)–OAllyl] (4j) Compound 4j was

obtained as brownish solid with 79 % yield by PyBop

coupling method or 83 % yield by IBCF asymmetric

anhydride method. ¹H NMR: (400 MHz, DMSO-d₆): d

1.93–2.01 (m, 1H), 2.16–2.24 (m, 1H), 2.59 (t, J = 5.5 Hz,

2H), 2.91 (s, 3H), 2.92 (s, 3H), 3.38–3.44 (br. q, 2H),

4.19–4.36 (m, 4H), 4.39 (t, J = 5.5 Hz, 2H), 4.62 (td,

J = 5, 2 Hz, 3H), 5.21 (dq, J = 11.0, 2.0 Hz, 1H), 5.33

(dq, J = 11.0, 2.0 Hz, 1H), 5.87–5.97 (m, 1H), 7.33 (t,

J = 7.5 Hz, 2H) 7.42 (t, J = 7.5 Hz, 2H), 7.72 (d,

J = 7.5 Hz, 2H), 7.83 (t, J = 7.5 Hz, 1H), 7.89 (d,

J = 7.5 Hz, 2H), 7.93 (d, J = 7.5 Hz, 1H), 8.37–8.41 (m,

2H), 8.68 (d, J = 2 Hz, 1H), 8.76 (d, J = 2 Hz, 1H), 9.34

(s, 1H), 10.63 (s, 1H); ¹³C and DEPTQ (¹³C) NMR

(101 MHz, DMSO-d₆): d 25.97 (CH₂), 32.44 (CH₂), 35.07

(CH₂), 42.63 (2 CH₃), 46.51 (CH), 53.30 (CH), 54.82

(CH₂), 64.81 (CH₂), 65.62 (CH₂), 117.63 (CH₂), 120.03 (2

CH), 120.75 (CH), 121.84 (C), 122.63 (C), 123.79 (CH),

124.00 (C), 125.11(2 CH), 126.97 (2 CH), 127.50 (CH),

127.55 (2 CH), 128.93 (CH), 132.00 (C), 132.28 (CH),

133.82 (CH). 137.88 (C), 140.62 (C), 140.63 (C), 143.64

(C), 143.67 (C), 156.12 (CO–NH-OR), 163.69 (CO-NR-

CO), 163.94 (CO-NR-CO), 170.98 (CO-OR), 171.75 (CO-

NHR); LC–MS m/z: 676 (M?H^?), RT = 9.29 min.

General Procedures of the Synthesis of 3a–f

For compounds 4a–4f, the puriﬁed Fmoc–AA(drug)–OtBu

was dissolved in 4–7 mL of TFA/DCM 9/1. The concen-

trated TFA solution was stirred for 2 h, after which the

solvent was removed under a gentle N₂ﬂow resulting in an

oily crude. Fmoc–AA(drug)–OH was obtained in a form of

crystal powder after precipitation from diethyl ether.

For compounds 4g–4l, the puriﬁed Fmoc–AA(drug)–

OAllyl was dissolved in 10 mL of DCM. Then 0.3 eq. of

Pd(PPh₃)₄and 3 eq. of DMBA were added, and the mix-

ture was allowed to stir for 4 h at rt after what the solvent

was removed under reduced pressure and the resulted oily

crude was redissolved in ACN or DMF and then subjected

to puriﬁcation by semi prep. HPLC. Clean fractions where

lyophilized to resulting Fmoc–AA(drug)–OH BUs as

crystalline powder.

[Fmoc–Asp(Cytarabine)–OAllyl] (4k) Compound 4k was

obtained as white solid with 81 % yield by IBCF asym-

1

metric anhydride method. H NMR: (400 MHz, DMSO-

d₆): d 2.84 (dd, J = 14, 8 Hz, 1H), 3.00 (dd, J = 16, 6 Hz,

1H), 3.63 (d, J = 5.4 Hz, 2H), 3.83–3.87 (m, 1H), 3.94 (t,

J = 3.1 Hz, 1H), 4.08 (dd, J = 4, 2.5 Hz, 1H), 4.24 (d,

J = 7 Hz, 1H), 4.31 (br q, 1H), 4.33 (br q, 1H), 4.58 (td,

J = 5.0, 2.0 Hz, 2H), 4.76 (br s [3 OH]), 5.18 (dq,

J = 10.5, 1.5 Hz, 1H), 5.30 (dq, J = 17.5, 1.7 Hz, 1H),

5.82–5.91 (m, 1H), 6.07 (d, 4.0 Hz, 1H), 7.18 (d,

J = 7.5 Hz, 1H), 7.32 (dt, J = 7.5, 1.2 Hz, 2H), 7.42 (t,

J = 7.5 Hz, 2H), 7.7 (d, J = 7.5 Hz, 2H), 7.89 (d,

J = 7.5 Hz, 2H), 7.94 (d, J = 8.1 Hz, 1H), 8.09 (d,

J = 7.5 Hz, 1H), 10.98 (s, 1H); DEPTQ (¹³C) NMR

(101 MHz, DMSO-d₆): d 25.52 (CH₂), 32.61 (CH₂), 46.51

(CH), 53.12 (CH), 60.90 (CH₂), 64.81 (CH₂), 65.61 (CH₂),

74.48 (CH), 76.00 (CH), 85.67 (CH), 86.90 (CH), 94.20

(CH), 117.66 (CH₂), 120.04(2 CH), 125.13 (2 CH), 127.00

(2 CH), 127.56 (2 CH), 132.25 (CH) 140.63 (2 C), 143.66

(2 C), 146.70 (CH), 154.31 (CO-(NR₂)₂), 156.06 (CO-

NHR-OR), 161.93 (C), 171.67 (CO–OR), 172.76 (CO–

NHR); LC–MS m/z: 621 (M?H^?), RT = 9.23 min.

[Fmoc–Asp(Deacetyl Colchicine)–OH] (3a) Compound

3a was obtained as pale yellow solid with 90 % yield from

4a or with 58 % yield from 4 g. ¹H NMR: (400 MHz,

DMSO-d₆): d 1.76–1.90 (m, 1H), 1.91–2.04 (m, 1H),

2.14–2.29 (m, 1H), 2.53–2.64 (m, 3H), 3.53 (s, 3H), 3.78

(s, 3H), 3.82 (s, 3H), 3.86 (s, 3H), 4.16–4.21 (m, 4H),

4.31–4.39 (m, 1H), 6.75 (s, 1H), 7.01 (d, J = 11.5 Hz, 1H),

7.10 (d, J = 11.5 Hz, 1H), 7.16 (s, 1H), 7.26 (m, 2H), 7.39

(t, J = 8.0 Hz, 2H) 7.55 (d, J = 7.0 Hz, 1H), 7.66 (dd, 7.5,

3.0 Hz, 2H) 7.88 (d, J = 7.0 Hz, 2H), 8.58 (d, J = 7.0 Hz,

1H); DEPTQ (¹³C) NMR (101 MHz, DMSO-d₆): d 29.10

(CH₂), 35.75 (CH₂), 36.66 (CH₂), 46.44 (CH), 50.26 (CH),

55.70 (CH₃), 55.92 (CH₃), 60.58 (CH₃), 60.77 (CH₃), 65.54

123

Int J Pept Res Ther

[Fmoc–Glu(Amonaﬁde)–OH] (3d) Compound 3d was

obtained as brownish solid with 88 % yield from 4d or

with 74 % yield from 4j. ¹H NMR: (400 MHz, DMSO-d₆):

d 1.89–2.00 (m, 1H), 2.14–2.23 (m, 1H), 2.58 (t, 7.5 Hz,

2H), 2.91 (s, 3H), 2.92 (s, 3H), 3.42–3.50 (m, 2H),

4.06–4.12 (m, 1H), 4.21 (d, J = 7.0 Hz, 1H), 4.26–4.32 (m,

2H), 4.38 (t, 5.0 Hz, 2H), 7.33 (t, J = 7.5, 2H) 7.41 (t,

J = 7.5 Hz, 2H), 7.73 (d, J = 7.5 Hz, 2H), 7.77 (d,

J = 7.5 Hz, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.89 (d,

J = 7.5 Hz, 2H), 8.36–8.40 (m, 2H), 8.67 (d, 2 Hz, 1H),

8.76 (d, 2 Hz, 1H), 9.36 (s, 1H), 10.62 (s, 1H); ¹³C and

DEPTQ (¹³C) NMR (101 MHz, DMSO-d₆): d 26.19 (CH₂),

32.74 (CH₂), 35.06 (CH₂), 42.66 (2 CH₃), 46.53 (CH),

53.22 (CH), 54.82 (CH₂), 64.83 (CH₂), 65.59 (CH₂),

120.01 (CH), 120.03 (CH), 120.74 (CH), 121.93 (C),

122.61 (C), 123.83 (CH), 123.99 (C), 125.17(CH), 125.20

(CH), 126.98 (2 CH), 127.49 (CH), 127.55 (2 CH), 128.92

(CH), 130.00 (C), 133.81 (CH), 137.92 (C), 140.60 (C),

140.62 (C), 143.68 (C), 143.73 (C), 156.13 (CO–NH–OR),

163.69 (CO–NR–CO), 163.94 (CO–NR–CO), 171.14 (CO–

OH), 173.59 (CO–NHR); LC–MS m/z: 635 (M?H^?),

RT = 8.73 min.

(CH₂), 107.60 (CH), 111.94 (CH), 119.99 (2 CH),

125.11(CH), 125.14 (CH), 125.27 (C), 126.93 (2 CH),

127.49 (2 CH), 130.35 (CH), 134.06 (C), 134.27 (CH),

134.97 (C), 140.56 (2 C), 140.63 (C), 143.64 (C), 143.71

(C) 150.29 (C), 150.34 (C), 152.85 (C), 155.61 (CO–NH–

OR), 163.40 (C), 168.30 (CO–OH), 172.93 (CO–NHR),

177.91 (CO); LC–MS m/z: 695 (M?H^?), RT = 9.19 min.

[Fmoc–Glu(Deacetyl Colchicine)–OH] (3b) Compound

3b was obtained as pale yellow solid with 91 % yield from

4b or with 51 % yield from 4 h. ¹H NMR: (400 MHz,

DMSO-d₆): d 1.66–1.88 (m, 2H), 1.88–2.09 (m, 2H),

2.16–2.24 (m, 1H), 2.27 (t, J = 8.0 Hz, 2H), 2.59 (dd,

J = 12.0, 6.0 Hz, 1H), 3.53 (s, 3H), 3.79 (s, 3H), 3.83 (s,

3H), 3.87 (s, 3H), 4.20–4.28 (m, 4H), 4.32–4.38 (m, 1H),

6.77 (s, 1H) 7.03 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 7.5 Hz,

1H), 7.14 (s, 1H), 7.33 (t, J = 7.5 Hz, 2H) 7.43 (t,

J = 7.5 Hz, 2H), 7.69 (d, J = 7.5 Hz, 1H), 7.73 (d,

J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.58 (d,

J = 5.5 Hz, 1H); ¹³C and DEPTQ (¹³C) NMR (101 MHz,

DMSO-d₆): d 26.35 (CH₂), 29.11 (CH₂), 31.44 (CH₂),

35.63 (CH₂), 46.53 (CH), 51.11 (CH), 53.24 (CH), 55.73

(CH₃), 55.94 (CH₃), 60.59 (CH₃), 60.76 (CH₃), 65.57

(CH₂), 107.63 (CH), 112.04 (CH), 120.02 (2 CH),

125.21(CH), 125.23 (C), 125.33 (CH), 127.01 (2 CH),

127.56 (2 CH), 130.27 (CH), 134.13 (C), 134.33 (CH),

135.10 (C), 140.60 (C), 140.62 (2 C), 143.72 (C), 143.73

(C) 150.35 (C), 150.71 (C), 152.84 (C), 156.05 (CO–NH–

OR), 163.43 (C), 170.57 (CO–OH), 173.53 (CO–NHR),

177.90 (CO); LC–MS m/z: 709 (M?H^?), RT = 9.59 min.

[Fmoc–Asp(Cytarabine)–OH] (3e) Compound 3e was

obtained as white solid with 94 % yield from 4e or with

1

45 % yield from 4 k. H NMR: (400 MHz, DMSO-d₆): d

2.78 (dd, 15, 6 Hz), 2.95 (dd, 15, 6 Hz), 3.62 (d,

J = 5.4 Hz, 2H), 3.82–3.85 (m, 1H), 3.93 (t, J = 3.5 Hz,

1H), 4.06–4.08 (m, 1H), 4.20–4.30 (m, 3H), 4.40–4.47 (m,

1H), 4.73–5.85 (br s, 3 [OH]), 6.06 (d, 4.0 Hz, 1H), 7.16 (d,

J = 7.5 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.41 (t,

J = 7.5 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.76 (d,

J = 7.5 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 8.08 (d,

J = 7.5 Hz, 1H), 10.93 (s, 1H); DEPTQ (¹³C) NMR

(400 MHz, DMSO-d₆): d 38.09 (CH₂), 46.46 (CH), 49.86

(CH), 60.97 (CH₂), 65.60 (CH₂), 74.47 (CH), 76.01 (CH),

85.69 (CH), 86.92 (CH), 94.23 (CH), 120.01 (2 CH),

125.14 (CH), 125.16 (CH), 126.97 (2 CH), 127.53 (2 CH),

140.59 (2 C), 143.67 (2 C), 146.72 (CH), 154.29 (CO–

(NR₂)₂), 155.72 (CO–NHR–OR), 161.92 (C), 170.53 (CO–

OR), 172.69 (CO–NHR); LC–MS m/z: 581 (M?H^?),

RT = 8.79 min.

[Fmoc–Asp(Amonaﬁde)–OH] (3c) Compound 3c was

obtained as brownish solid with 93 % yield from 4c or with

82 % yield from 4i. ¹H NMR: (400 MHz, DMSO-d₆): 2.84

(dd, J = 16.0, 6.0 Hz, 1H), 2.90 (s, 3H), 2.91 (s, 3H), 3.01

(dd, J = 16.0, 6.0 Hz, 1H), 3.44–3.48 (br. q, 2H), 4.22 (t,

7.0 Hz, 1H), 4.29–4.31 (m, 2H), 4.39 (t, J = 5.0 Hz, 2H),

4.56 (m, 1H), 7.29 (t, J = 7.5, 1.5 Hz, 2H), 7.39 (t,

J = 7.5 Hz, 2H), 7.71 (dd, J = 8.0, 1.0 Hz, 2H), 7.81–7.86

(m, 2H), 7.88 (d, J = 7.0 Hz, 2H), 8.38–8.41 (m, 2H), 8.69

(d, J = 2.0 Hz, 1H), 8.77 (d, J = 2.0 Hz, 1H), 9.36 (s, 1H),

10.71 (s, 1H); DEPTQ (¹³C) NMR (101 MHz, DMSO-d₆):

d 35.07 (CH₂), 38.22 (CH₂), 42.64 (2 CH₃), 46.48 (CH),

50.30 (CH), 54.73 (CH₂), 65.66 (CH₂), 120.01 (2 CH),

120.85 (CH), 121.87 (C), 122.65 (C), 123.79 (CH), 124.03

(C), 125.11(CH), 125.14 (CH), 126.96 (2 CH), 127.52 (3

CH), 128.99 (CH), 131.99 (C), 133.84 (CH), 137.77 (C),

140.59 (2 C), 143.64 (C), 143.68 (C), 155.81 (CO–NH–

OR), 163.68 (CO–NR–CO), 163.94 (CO-NR-CO), 168.91

(CO–OH), 172.84 (CO–NHR); HRMS: ESI–MS m/z calcd:

643.217, found: 643.210 (M?Na^?), calcd: 659.191

(M?K^?), found: 659.182 (M?K^?); LC–MS m/z: 621

(M?H^?), RT = 8.62 min.

[Fmoc–Glu(Cytarabine)–OH] (3f) Compound 3f was

obtained as white solid with 93 % yield from 4f or with

1

37 % yield from 4 l. H NMR: (400 MHz, DMSO-d₆): d

1.77–1.87 (m, 1H), 2.02–2.10 (m, 1H), 2.51–2.55 (m, 2H),

3.62 (d, J = 5.0 Hz, 2H), 3.81–3.85 (m, 1H), 3.93 (t,

J = 3.5 Hz, 1H), 4.06 (dd, J = 4.0, 2.5 Hz, 1H), 4.21–4.30

(m, 3H), 4.36–5.00 (br s, 3 [OH]), 6.06 (d, 4.0 Hz, 1H),

7.19 (d, J = 7.5 Hz, 1H), 7.34 (tt, J = 7.5, 1.5 Hz, 2H),

7.42 (t, J = 7.5 Hz, 2H), 7.68 (d, J = 7.5 Hz, 1H), 7.73 (d,

J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.07 (d,

123

Int J Pept Res Ther

J = 7.5 Hz, 1H), 10.98 (s, 1H); ¹³C and DEPTQ (¹³C)

NMR (400 MHz, DMSO-d₆): d 25.66 (CH₂), 32.81 (CH₂),

46.52 (CH), 52.98 (CH), 60.90 (CH₂), 65.61 (CH₂), 74.48

(CH), 76.00 (CH), 85.67 (CH), 86.88 (CH), 94.21 (CH),

120.02 (CH), 120.03 (CH), 125.16 (CH), 125.19 (CH),

126.99 (2 CH), 127.54 (2 CH), 140.60 (C), 140.62 (C),

143.69 (C), 143.71 (C), 146.65 (CH), 154.31 (CO-(NR₂)₂),

156.06 (CO-NHR-OR), 161.96 (C), 172.88 (CO-OR),

173.48 (CO-NHR); LC–MS m/z: 595 (M?H^?),

RT = 8.83 min.

Fmoc protecting group was removed from the N-terminal

and then the resin was thoroughly washed and dried, and

the crude product was cleaved from it by a TFA cocktail

[TFA/H₂O/phenol/TIPS (90:5:3:2)], 3 h, rt. The crude was

puriﬁed by semi-prep HPLC and clean fractions were

lyophilized resulting conjugate 7 as yellow powder. LC–

MS m/z: 1176 (M?H^?), RT = 7.79 min, purity [95 %;

HRMS: ESI–MS m/z calcd: 1176.519, found: 1176.526

(M?H^?).

General Procedure for the Synthesis of Cyclic c(RGDf-AA)

Synthetic Procedure of 5

Drug Conjugates 8a–d

[Fmoc–Lys(CPT)–OH] (5): Fmoc–Lys(Boc) (234 mg,

0.5 mmol) was treated with 90 % TFA in DCM (10 mL).

TLC analysis (5 % MeOH in EtOAc) indicated the com-

pletion of the reaction after 90 min. The solvent was

removed by a nitrogen ﬂow and the crude was precipitated

from ether to provide pale orange oil, which was dissolved

in DMF. Addition of DIPA (6 eq.) turned the clear solution

into a suspension, which was treated dropwise with a

dioxane solution of para-nitrophenyl chloroformate. The

yellow slurry solution was stirred at rt for 4 h. The solvents

were removed under reduced pressure. Precipitation from

ether resulted in a brown-yellow powder, which was

puriﬁed by semi-prep. HPLC, affording brown powder in

83 % yield; Rf: 0.70 (25 %MeOH/EtOAc); LC–MS:

2-Chlorotrityl chloride resin with loading capacity of

1.12 mmol/g was placed in a reactor and suspended in

DCM under nitrogen atmosphere. A mixture of Fmoc–

Asp–OAll (2 eq.) and DIPA (8 eq.) in DCM was added.

The resin loading reaction was allowed to proceed for 4 h

and subsequently the resin was capped by an addition of

0.5 mL of methanol. The Fmoc protecting group was

removed with 20 % piperidine/DMF (10 min 9 3) and

then a linear SPPS was applied using standard Fmoc pro-

cedures. All the couplings were performed in DMF mix-

tures of Fmoc AA (2 eq.), HATU (2 eq.), and DIPA

(6 eq.). Each coupling cycle lasted for 2–3 h. The com-

pletion of each coupling reaction or Fmoc removal was

monitored by the ninhydrin test. After the coupling of the

last AA, the C-terminal allyl ester was removed by

exposing the peptidyl resin to a mixture of Pd(PPh₃)₄

(0.3 eq.) and DMBA (6 eq.) in DCM for 4 h, after which

the resin was thoroughly washed with 0.5 M diethyl-

dithiocarbamic acid sodium salt—DMF solution. Finally,

the Fmoc protecting group was removed from the N-ter-

minal and the cyclization reaction was performed by add-

ing a mixture of PyBop (10 eq.), HOBt (10 eq.), DIPA

(20 eq.) in NMP and shaking gently for 5 h. After the resin

was thoroughly washed and dried, the crude product was

cleaved from the resin by a TFA cocktail [TFA/H₂O/Phe-

nol/TIPS (90:5:3:2)], 3 h, rt. The crude was puriﬁed by

semi-prep HPLC and clean fractions were lyophilized to

result the peptide-drug conjugates as crystalline powders.

1

RT = 9.88 min; H NMR (400 MHz, DMSO-d₆): d 0.92

(t, J = 10.4 Hz, 2H), 1.35 (m, 2H), 1.49 (m, 2H), 1.62 (m,

2H), 2.1 (m, 2H), 2.88 (m, 2H), 4.2 (m, 4H), 5.30 (s, 2H),

5.45 (s, 2H), 7.04 (s, 1H), 7.29 (t, J = 7.3 Hz, 2H), 7.38 (t,

J = 7.3 Hz, 2H), 7.52 (d, J = 8.3 Hz, 1H), 7.68 (m, 3H),

7.77 (t, J = 6.2 Hz, 1H), 7.85 (m, 3H), 8.11 (d,

J = 8.3 Hz, 1H), 8.18 (d, J = 8.3 Hz, 1H), 8.68 (s, 1H);

HRMS: ESI–MS m/z calcd:765.254, found: 765.258

(M?Na^?), calcd: 781.228, 781.238 (M?K^?).

Synthetic Procedure of 7

[RGD-Lys(CPT)-NGR] (7) 2-Chlorotrityl chloride resin

with loading capacity of 1.12 mmol/g was placed in a

reactor and suspended in DCM under nitrogen atmosphere.

A mixture of Fmoc–Asp(OtBu)–OH (2 eq.) and DIPA

(8 eq.) in DCM was added. The resin loading reaction was

allowed to proceed for 4 h and subsequently the resin was

capped by an addition of 0.5 mL of methanol. The Fmoc

protecting group was removed with 20 % piperidine/DMF

(3 9 7 min) and then a linear SPPS was applied using

standard Fmoc procedures. All the couplings were per-

formed in DMF mixtures of Fmoc AA (2 eq.), HATU

(2 eq.), and DIPA (6 eq.). Each coupling cycle lasted for

2–3 h. The completion of each coupling reaction or Fmoc

removal was monitored by the ninhydrin test. Finally, the

[c(RGDfS)-CLB] (8a) Compound 8a was obtained as

pale yellow solid, 62 % yield. LC–MS m/z: 848 (M?H^?),

RT = 9.11 min, purity[95 %; HRMS: ESI–MS m/z calcd:

848.327, found: 848.320 (M?H).

[c(RGDfK)-CPT] (8b) Compound 8b was obtained as

yellow solid, 53 % yield. LC–MS m/z: 978 (M?H^?),

RT = 8.71 min, purity[95 %; HRMS: ESI–MS m/z calcd:

HRMS: ESI–MS m/z calcd: 978.411 found: 978.411

(M?H), calcd: 1000.393, found: 1000.388 (M?Na).

123

Int J Pept Res Ther

[c(RGDfE)-Deacetyl Colchicine] (8c) Compound 8c was

obtained as pale yellow solid, 49 % yield. LC–MS m/z: 944

(M?H^?), RT = 8.41 min, purity[95 %; HRMS: ESI–MS

m/z calcd: 944.415, found: 944.415 (MH^?).

argon-ion laser (15 W) at 488 nm with a 530/30 DF ﬁlter.

For each sample *10⁴cells were analyzed. FlowJo soft-

ware was used to analyze the data.

Acknowledgments The authors thank Mrs. Cherna Moskowitz for

generous stipend to Y.G. Financial support was provided by Ariel

University Internal Research and Development Program.

[c(RGDfE)-Amonaﬁde] (8d) Compound 8d was obtained

as orange solid, 65 % yield. LC–MS m/z: 870 (M?H^?),

RT = 7.77 min, purity 95 %; HRMS: ESI–MS m/z calcd:

870.3898, found: 870.3892 (M?H).

Compliance with Ethical Standards

Conﬂict of Interest The authors declare that they have no conﬂict

of interest.

Cytotoxicity Tests

Ethical approval This article does not contain any studies with

human participants or animals performed by any of the authors.

The cytotoxicity of the compounds was determined by

measuring the mitochondrial enzymatic activity, using a

commercial XTT assay kit. All samples contained DMSO

at ﬁnal concentration \0.05 %.

Informed consent Informed consent was obtained from all indi-

vidual participants included in the study.

Cells were cultured in micro wells in a concentration of

2–4 9 10⁴cells/well and allowed to adhere during ﬁrst

24 h incubation period. At the end of the ﬁrst 24 h incu-

bation period the cells were washed, given a fresh medium

containing different concentrations of the tested substances

and subsequently incubated for additional 6 h. At the end

of the incubation period with the substances, the medium

was removed, all the wells were washed with PBS, given

with fresh medium and ﬁnally incubated for a second 24 h

period. At the end of the second 24 h incubation period the

cells were washed again and given a fresh medium con-

taining the XTT reagent after which the cells were re-in-

cubated for 2–4 h. During that time the absorbencies in the

wells were measured with a TECAN Inﬁnite M200 ELISA

reader at both 480 and 680 nm—the last is the background

absorbance. The difference between these measurements

was used for calculating the % of Growth Inhibition (GI) in

test wells compared to the cells that were exposed only to

the medium with 0.05 % DMSO. All the tests were done in

tetra-plicate.

References

Arap W, Pasqualini R, Ruoslahti E (1998) Cancer treatment by

targeted drug delivery to tumor vasculature in a mouse model.

Science 279:377–380. doi:10.1126/science.279.5349.377

Bagnato JD, Eilers AL, Horton RA, Grissom CB (2004) Synthesis and

characterization of a cobalamin–colchicine conjugate as a novel

tumor-targeted cytotoxin. J Org Chem 69:8987–8996. doi:10.

1021/jo049953w

Bellmann-Sickert K, Beck-Sickinger AG (2010) Peptide drugs to

target G protein-coupled receptors. Trends Pharmacol Sci

31:434–441. doi:10.1016/j.tips.2010.06.003

Chen K, Chen X (2011) Integrin targeted delivery of chemothera-

peutics. Theranostics 1:189–200

Cheng NC, van Zandwijk N, Reid G (2014) Cilengitide inhibits

attachment and invasion of malignant pleural mesothelioma cells

through antagonism of integrins avb3 and avb5. PLoS One

9:e90374. doi:10.1371/journal.pone.0090374

Cohen S, Cahan R, Ben-Dov E, Nisnevitch M, Zaritsky A, Firer MA

(2007) Speciﬁc targeting to murine myeloma cells of Cyt1Aa

toxin from bacillus thuringiensis subspecies israelensis. J Biol

Chem 282:28301–28308. doi:10.1074/jbc.M703567200

Colombo G et al (2002) Structure-activity relationships of linear and

cyclic peptides containing the NGR tumor-homing motif. J Biol

Chem 277:47891–47897. doi:10.1074/jbc.M207500200

Corti A, Curnis F (2011) Isoaspartate-dependent molecular switches

for integrin–ligand recognition. J Cell Sci 124:515–522. doi:10.

1242/jcs.077172

Direct Immunoﬂuorescence Assay and Flow

Cytometry

Corti A, Curnis F, Arap W, Pasqualini R (2008) The neovasculature

homing motif NGR: more than meets the eye. Blood

112:2628–2635. doi:10.1182/blood-2008-04-150862

Curnis F, Longhi R, Crippa L, Cattaneo A, Dondossola E, Bachi A,

Corti A (2006) Spontaneous formation of ^L-isoaspartate and gain

of function in ﬁbronectin. J Biol Chem 281:36466–36476.

doi:10.1074/jbc.M604812200

Dai X, Su Z, Liu JO (2000) An improved synthesis of a selective

avb3-integrin antagonist cyclo(-RGDfK-). Tetrahedron Lett

41:6295–6298. doi:10.1016/S0040-4039(00)01060-1

Dal Pozzo A et al (2010) Novel tumor-targeted RGD peptide–

camptothecin conjugates: synthesis and biological evaluation.

Bioorg Med Chem 18:64–72. doi:10.1016/j.bmc.2009.11.019

For measuring the level of a_vb₃integrin expression the

cells were washed and then scraped from the culture ﬂacks.

For each cell line two separate samples of 10⁶cells were

suspended in 200 lL of PBS and incubated in 4 °C—one

sample was incubated together with 20 lL pre-diluted

mouse 23C6 monoclonal antibody (which is anti-human

CD51/CD61) conjugated with R-phycoerythrin (PE) (BD

Bioscience), the second sample was free of labeling agent

and served as control. During the incubation period the

cells were gently shacked every 15 min. After centrifuging

and then washing couple of times with PBS, the cells were

resuspended in 400 lL of PBS and analyzed using a cell

analyzer (Becton Dickson FACSCalibur) equipped with an

´

Danhier F, Breton AL, Preat V (2012) RGD-based strategies to

target alpha(v) beta(3) integrin in cancer therapy and diagnosis.

Mol Pharm 9:2961–2973. doi:10.1021/mp3002733

123

Int J Pept Res Ther

Firer M, Gellerman G (2012) Targeted drug delivery for cancer

therapy: the other side of antibodies. J Hematol Oncol 5:70

Friligou I et al (2013) Divergent and convergent synthesis of

polymannosylated dibranched antigenic peptide of the immun-

odominant epitope MBP(83–99). Bioorg Med Chem

21:6718–6725. doi:10.1016/j.bmc.2013.08.008

Gellerman G, Elgavi A, Salitra Y, Kramer M (2001) Facile synthesis

of orthogonally protected amino acid building blocks for

combinatorial N-backbone cyclic peptide chemistry. J Pept Res

57:277–291. doi:10.1046/j.1397-002x.2000.0780.x

Manfredini S et al (2000) Peptide T-araC conjugates: solid-phase

synthesis and biological activity of N4-(acylpeptidyl)-araC.

Bioorg Med

0896(99)00317-X

Chem

8:539–547.

doi:10.1016/S0968-

Pasqualini R et al (2000) Aminopeptidase N is a receptor for tumor-

homing peptides and a target for inhibiting angiogenesis. Cancer

Res 60:722–727

Plow EF, Haas TA, Zhang L, Loftus J, Smith JW (2000) Ligand

binding to integrins. J Biol Chem 275:21785–21788. doi:10.

1074/jbc.R000003200

Gilad Y, Firer MA, Rozovsky A, Ragozin E, Redko B, Albeck A,

Gellerman G (2014) ‘‘Switch off/switch on’’ regulation of drug

cytotoxicity by conjugation to a cell targeting peptide. Eur J Med

Chem 85:139–146. doi:10.1016/j.ejmech.2014.07.073

Reissner KJ, Aswad DW (2003) Deamidation and isoaspartate

formation in proteins: unwanted alterations or surreptitious

signals? Cell Mol Life Sci 60:1281–1295. doi:10.1007/s00018-

003-2287-5

Goldshaid L et al (2010) Novel design principles enable speciﬁc

targeting of imaging and therapeutic agents to necrotic domains

in breast tumors. Breast Cancer Res 12:R29

Goodman SL, Picard M (2012) Integrins as therapeutic targets.

Trends Pharmacol Sci 33:405–412. doi:10.1016/j.tips.2012.04.

002

Gurrath M, Muller G, Kessler H, Aumailley M, Timpl R (1992)

Conformation/activity studies of rationally designed potent anti-

adhesive RGD peptides. Eur J Biochem 210:911–921. doi:10.

1111/j.1432-1033.1992.tb17495.x

Robinson NE, Robinson ZW, Robinson BR, Robinson AL, Robinson

JA, Robinson ML, Robinson AB (2004) Structure-dependent

nonenzymatic deamidation of glutaminyl and asparaginyl pen-

tapeptides. J Pept Res 63:426–436. doi:10.1111/j.1399-3011.

2004.00151.x

Ruoslahti E (1996) RGD and other recognition sequences for

integrins. Annu Rev Cell Dev Biol 12:697–715. doi:10.1146/

annurev.cellbio.12.1.697

Ruoslahti E (2002) Specialization of tumour vasculature. Nat Rev

Cancer 2:83–90

Haubner R, Finsinger D, Kessler H (1997) Stereoisomeric peptide

libraries and peptidomimetics for designing selective inhibitors

of the avb3 integrin for a new cancer therapy. Angew Chem Int

Ed 36:1374–1389. doi:10.1002/anie.199713741

Kriek NMAJ, Filippov DV, van den Elst H, Meeuwenoord NJ, Tesser

GI, van Boom JH, van der Marel GA (2003) Stepwise solid

phase synthesis of uridylylated viral genome-linked peptides

using uridylylated amino acid building blocks. Tetrahedron

59:1589–1597. doi:10.1016/S0040-4020(03)00042-5

Lafrenie RM, Gallo S, Podor TJ, Buchanan MR, Orr FW (1994) The

relative roles of vitronectin receptor, E-selectin and a4b1 in

cancer cell adhesion to interleukin-1-treated endothelial cells.

Eur J Cancer 30:2151–2158. doi:10.1016/0959-8049(94)00354-8

Majumdar S, Siahaan TJ (2012) Peptide-mediated targeted drug

delivery. Med Res Rev 32:637–658. doi:10.1002/med.20225

Shabbir SH, Eisenberg JL, Mrksich M (2010) An inhibitor of a cell

adhesion receptor stimulates cell migration. Angew Chem Int Ed

49:7706–7709. doi:10.1002/anie.201002699

Soudy R, Ahmed S, Kaur K (2012) NGR Peptide ligands for targeting

CD13/APN identiﬁed through peptide array screening resemble

ﬁbronectin sequences. ACS Comb Sci 14:590–599. doi:10.1021/

co300055s

van Hensbergen Y et al (2002) A doxorubicin-CNGRC-peptide

conjugate with prodrug properties. Biochem Pharmacol

63:897–908. doi:10.1016/S0006-2952(01)00928-5

Vig BS, Huttunen KM, Laine K, Rautio J (2013) Amino acids as

promoieties in prodrug design and development. Adv Drug Deliv

Rev 65:1370–1385. doi:10.1016/j.addr.2012.10.001

123

Article Doi

Synthesis of Novel Protected Nα(ω-Drug) Amino Acid Building Units for Facile Preparation of Anticancer Drug-Conjugates

DOI: 10.1007/s10989-015-9509-1

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