Polypeptide-based molecular platform and its docetaxel/sulfo-cy5-containing conjugate for targeted delivery to prostate specific membrane antigen

molecules

Article

Polypeptide-Based Molecular Platform and Its

Docetaxel/Sulfo-Cy5-Containing Conjugate for

Targeted Delivery to Prostate Speciﬁc

Membrane Antigen

Stanislav A. Petrov ¹ , Aleksei E. Machulkin

1,2

, Anastasia A. Uspenskaya , Nikolay Y. Zyk ,

1

Ekaterina A. Nimenko , Anastasia S. Garanina

, Rostislav A. Petrov

,

Vladimir I. Polshakov ³ , Yuri K. Grishin , Vitaly A. Roznyatovsky

1

, Nikolay V. Zyk ,

1

Alexander G. Majouga ¹

,2,4

and Elena K. Beloglazkina *

1,

1

Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-3,

19991 Moscow, Russia; stanislavpetrovsh1994@gmail.com (S.A.P.); alekseymachulkin@rambler.ru (A.E.M.);

1

uspenskaya.n@gmail.com (A.A.U.); zyknikola@gmail.com (N.Y.Z.); nimenkoea@mail.ru (E.A.N.);

anastasiacit@gmail.com (A.S.G.); petrovrostaleks@gmail.com (R.A.P.); grishin@nmr.chem.msu.ru (Y.K.G.);

Vit.Rozn@nmr.chem.msu.su (V.A.R.); zyk@org.chem.msu.ru (N.V.Z.);

alexander.majouga@gmail.com (A.G.M.)

Laboratory of Biomedical Nanomaterials, National University of Science and Technology MISiS,

Leninskiy pr., 4, 119049 Moscow, Russia

2

3

Faculty of Fundamental Medicine, Lomonosov Moscow State University, Lomonosovsky Ave., 27-1,

1

19991 Moscow, Russia; vpolsha@mail.ru

4

Mendeleev University of Chemical Technology of Russia, Miusskaya sq. 9, 125947 Moscow, Russia

Correspondence: bel@org.chem.msu.ru

*

ꢀ

ꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editors: Andrea Trabocchi and Elena Lenci

Received: 14 November 2020; Accepted: 6 December 2020; Published: 8 December 2020

ꢀ

ꢁꢂꢃꢄꢅꢆ

Abstract: A strategy for stereoselective synthesis of molecular platform for targeted delivery of

bimodal therapeutic or theranostic agents to the prostate-speciﬁc membrane antigen (PSMA) receptor

was developed. The proposed platform contains a urea-based, PSMA-targeting Glu-Urea-Lys (EuK)

fragment as a vector moiety and tripeptide linker with terminal amide and azide groups for subsequent

addition of two diﬀerent therapeutic and diagnostic agents. The optimal method for this molecular

platform synthesis includes (a) solid-phase assembly of the polypeptide linker, (b) coupling of this

linker with the vector fragment, (c) attachment of 3-aminopropylazide, and (d) amide and carboxylic

groups deprotection. A bimodal theranostic conjugate of the proposed platform with a cytostatic

drug (docetaxel) and a ﬂuorescent label (Sulfo-Cy5) was synthesized to demonstrate its possible

sequential conjugation with diﬀerent functional molecules.

Keywords: theranostic agent; peptide synthesis; prostate cancer; anticancer drugs; PSMA conjugate;

docetaxel; Sulfo-Cy5

1

. Introduction

Prostate cancer (PCa) is one of the most commonly diagnosed men’s cancers and remains one of

the leading causes of cancer death. In 2018, approximately 1,276,106 new cases and 358,989 suspected

deaths were diagnosed worldwide [1,2].

Depending on the stage of the cancer and its severity, various imaging techniques, such as

computed tomography (CT), transrectal ultrasound, and relatively recent methods such as magnetic

resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron

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emission tomography (PET) are used to assess prostate cancer [

3]. However, the speciﬁcity of existing

imaging methods in evaluating metastases is limited [4,5]. The selected method for the treatment

of PC usually depends on the stage of the disease. For example, for localized PC, the options range

from radical prostatectomy to radiation therapy. Metastatic PC is preferably treated with androgen

deprivation therapy (ADT). When tumors develop resistance to androgens, the options are reduced

to alternative hormone therapy or chemotherapy. Preferred therapeutic agents are taxanes, such as

docetaxel [6,7]. However, so far, no active treatment for PC showed superiority in survival rates.

Treatment options diﬀer only concerning their side eﬀects.

One of the promising methods in PCa therapy and diagnostics is targeted delivery of medicinal

and diagnostic drugs to cancer cells, as well as delivery of theranostics. Theranostic conjugates

are drugs for the simultaneous solving of therapeutic and diagnostic problems. These compounds

are composed of several functional units, one of which is an early diagnostic tool and the other

is a therapeutic agent. This allows diagnosis and treatment of the disease simultaneously [8–10].

The advantage of this approach is the decrease in side eﬀects and injection dose in order to improve

diagnosis and treatment of the disease [11]. In addition, theranostic agents can minimize the inevitable

diﬀerences in biodistribution and selectivity that exist between diagnostic and therapeutic materials

for a particular disease [12]. This is particularly important in the case of cancer pathologies that are

highly heterogeneous [13].

Due to the high expression of prostate-speciﬁc membrane antigen on the cell membrane of prostate

cancer cells, this protein is an attractive molecular target for PCa theranostics [14]. Urea-based EuK

inhibitor is currently the reference in the development of targeted delivery systems to a prostatic

speciﬁc membrane antigen due to its stability, high aﬃnity, and good bioavailability [15].

The study of the prostate-speciﬁc membrane antigen (PSMA) crystal structure showed that access

to the binding site of this enzyme is provided by a ligand through a 20-Å-long, narrow tunnel with two

hydrophobic pockets and an arginine cluster [16,17]. Therefore, to construct a sterically unhindered

conjugate with optimal complementarity to the contours and chemical composition of the tunnel,

the ligand must be associated with diagnostic and therapeutic fragments via a linker of a certain

length and chemical composition. According to previous studies, relatively short lipophilic linkers are

preferable, as well as the presence of aryl moieties in the linker, which signiﬁcantly increase aﬃnity.

However, too much lipophilicity may adversely aﬀect the selectivity of accumulation. In general,

careful selection of the linker fragment allows optimization of the eﬃciency of PSMA inhibition, cellular

internalization, accumulation in nontarget tissues (kidneys, liver, spleen, etc.), as well as the quality

of visualization in vivo [18]. Thus, the chemical structure of the linker fragment has a signiﬁcant

inﬂuence on the aﬃnity to PSMA, as well as on the pharmacokinetic properties of PSMA-oriented

theranostic conjugates.

In this work, the development of synthetic approaches to create a molecular platform based

on the EuK ligand for targeted delivery of bimodal therapeutic or theranostic agents speciﬁc to the

PSMA receptor was carried out. The structures of synthesized compounds are shown in Figure 1.

The proposed molecular platform for PSMA delivery consists of two parts: (1) a vector fragment

providing conjugate-directed delivery to the prostate cancer cells and (2) a polypeptide linker providing

a possibility of subsequent conjugation with therapeutic and diagnostic (or two therapeutic) agents and

increasing aﬃnity to the PSMA receptor. As part of the work, a comparison of liquid- and solid-phase

techniques for synthesis of the delivery molecule was made.

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Figure 1. EuK-based ligand 12 with terminal amino and azido groups, synthesized in this work,

and bimodal conjugate 19 on its base.

To demonstrate the possibility of the synthesized bifunctional probe application for the stepwise

attachment of diagnostic and therapeutic agents, a double conjugate with docetaxel (DTX) and a

ﬂuorescent dye Sulfo-Cyanine5 (Sulfo-Cy5) (Figure 1) was synthesized. This obtained compound was

tested for cytotoxic activity and cell staining.

2

. Results and Discussion

Previously, we described therapeutic conjugates of doxorubicin [19] and paclitaxel [20] with PSMA

ligands of structurally related types, and it was shown that, for maximum aﬃnity to the receptor,

conjugate polypeptide fragments should contain aromatic substituents of diﬀerent nature in the -NH₂

position of Lys-amino acid of the PSMA ligand and the dipeptide fragment Phe(L)-Phe(L) in the linker

structure [18 19]. In this article, two functional groups of diﬀerent nature were introduced into the

linker fragment for further stage-by-stage conjugation, with diagnostic and therapeutic moieties at

orthogonal conditions to obtain the bimodal theranostic agents. These groups were NH , which allows

ζ

,

2

attachment of the additional structural fragments using peptide synthesis reactions, and N , which can

3

be entered into azide–alkyne cycloaddition (Figure 1).

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To obtain the target PSMA ligand with peptide fragments, we developed the synthetic scheme,

including the following stages: (1) synthesis of EuK vector with modiﬁed urea fragment (Scheme 1),

2) synthesis of the tripeptide linker using liquid-phase techniques (Scheme 2), (3) alternative synthesis

6

(

of the tripeptide linker using solid-phase peptide synthesis (SPPS) techniques (Scheme 3), (4) coupling

of the vector fragment with the linker with the formation of compound 12 (Schemes 2 and 3),

(5) modiﬁcation of docetaxel with hex-5-ynoic acid giving intermediate 17 (Scheme 4), and (6) click

reaction between the compounds 17 and 12 and the subsequent conjugation of the resulting compound

with a ﬂuorescent label (Scheme 4).

Scheme 1. Synthesis of prostate-specific membrane antigen (PSMA)-vector fragment. Reagents and

◦

conditions: (

a

) (1) Thriphosgene, DCM (dichloromethane),

−

78 C; (2) H-Lys(Cbz)-O-tBu·HCl, Et N,

3

◦

2

0 C; (

b

) H , Pd/C (10%), MeOH; (

c

) (1) 3-Cl–C H -CHO, DCM (2) NaBH ; (

d

) PyBOP (benzotriazol-

2

6

4

1

-yl-oxytripyrrolidinophosphonium hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF,

◦

N (CH ) COOH; (e) THF/H O, Ph P, 50 C; (f) (1) succinic anhydride, DCM, DIPEA; (2) MeOH; (3) HCl

2 3

3

2 5

(0.1M). All amino acids have L-configuration.

Scheme 2. Synthesis of the vector fragment with the peptide linker by the liquid-phase technique.

Reagents and conditions: ( ) (1) HBTU (hexaﬂuorophosphate benzotriazole tetramethyl uranium),

a

HOBt (1-hydroxybenzotriazole), DIPEA, DMF; (2) N (CH )3NH ; (b) Et NH, DMF; (c) HBTU, HOBt,

3

2

DIPEA, FmocPhePhe-OH, DMF; (

d

) Et NH, DMF; (

e) (1) 6, HBTU, HOBt, DIPEA, DMF; (2) 10;

2

(

f) DCM/TFA (triﬂuoroacetic acid). All amino acids have L-conﬁguration.

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Scheme 3. Synthesis of the vector fragment with the peptide linker by SPPS. Reagents and conditions:

) (1)FmocLys(L)(NHBoc), DIPEA, DMF; (2) 4-methylpiperidine/DMF; ( ) (1) FmocPhe-OH(L),

HBTU, HOBt, DIPEA; (2) 4-methylpiperidine/DMF; ( ) (1) FmocPhe-OH(L), HBTU, HOBt, DIPEA;

) (1) 6, HBTU, HOBt, DIPEA, DMF; (2) DCM/TFA (99.25%/0.75%);

) (1) HBTU, HOBt, DIPEA, DMF; N (CH ) NH ; ( ) DCM/TFA/TIPS/H O. All amino acids

(

a

b

c

(

2) 4-methylpiperidine/DMF; (d

e

f

3

2 3

2

have L-conﬁguration.

Scheme 4. Synthesis of the docetaxel/Cy5-containing conjugate. Reagents and conditions: (

5

a

) Hex-

-ynoic acid, DMAP (4-Dimethylaminopyridine), DCM; (2) DIC (N,N -Diisopropylcarbodiimide);

) (1)12 17, CuSO *5H O, sodium ascorbate, DMF, H O; (2) EDTA (Ethylenediaminetetraacetic acid);

c) (1) DIPEA, DMF; (2) Sulfo-Cy5-NHS ester.

0

(

b

,

4

2

(

2

.1. The Assembly of the Peptide Sequence

The initial stages of the synthesis of the vector fragment

described methods [21]. Compound 6 was prepared by coupling of succinic anhydrides with compound

(Scheme 1); the resulting products contained a free carboxylic group suitable for further addition of

the peptide fragment.

Tripeptide (Phe(L)-Phe(L)-Lys(L)-(CH ) -N ) was synthesized from L-phenylalanine (F) and

6

(Scheme 1) were realized by previously

5

2

3

L-lysine (K) to obtain highly speciﬁc PSMA vectors. Phe(L)–Phe(L) dipeptide fragments in the

linker improve the binding to the receptor [18 19]; the dipeptide nature of linkers further improves

biodegradability and reduces the unsystematic toxicity of PSMA vectors [22 23]. The coupling of

,

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additional lysine amino acid with an azide-containing fragment to the Phe(L)–Phe(L) linker provides

the possibility of further modiﬁcation with therapeutic and diagnostic drugs in orthogonal conditions.

2

.1.1. Synthesis of Tripeptide Sequence by Liquid-Phase Technique

The assembly of the peptide sequence was performed in the following manner (Scheme 2):

N

α

-Fmoc-N

compound , from which Fmoc was subsequently removed; as a result, the product containing a free amino

group was isolated. At the next step, a peptide synthesis between compounds and Fmoc-PhePhe-OH

was performed to synthesize the compound . The removal of Fmoc protection allowed the desired

compound 10 to be obtained as an individual stereoisomer (see Supplementary Information, Figure S4).

ε-Boc-l-lysine was introduced into the reaction with 3-aminopropylazide to obtain

7

8

9

2

.1.2. Synthesis of Tripeptide Sequence by SPPS Technique

The assembly of the peptide sequence was also realized using solid phase peptide synthesis (SPPS)

on 2-chlorotrityl chloride resin (2-CTC). This reaction sequence is presented as a classical peptide

synthesis scheme: (1) immobilization of N-substituted amino acid on a solid-phase polymer substrate,

(

2) removal of the protective group, (3) modiﬁcation of the NH -group of the amino acid (stages 2 and

2

3

were repeated to get the desired peptide sequence), and (4) removal of obtained peptide from the

polymer substrate [24].

Subsequently, the operations were performed with the necessary amino acids to obtain compound

1

5 (Scheme 3).

The 2-CTC resin allows application of the Fmoc SPPS concept and minimizes the adverse reactions.

Furthermore, it keeps labile acid functional groups intact, since the amino acid sequence is removed

from the polymer substrate under mild conditions (in our case DCM/TFA—99.25%/0.75%, V/V;

this system does not aﬀect labile acid actions of the NHBoc and COOtBu groups) [25].

2

.2. Synthesis of DCL-Modiﬁed Tripeptide 12

For the coupling of the vector fragments with peptide sequences by liquid-phase technique vector

compound

6 was dissolved in DMF and preactivated using the HOBt/HBTU/DIPEA system for 2 h

(Scheme2). Then, compound10wasaddedandthemixturewasstirredfor24h. Thereactionproduct11was

isolated by column chromatography and further converted to compound 12 (see Section 2.4). All substances

were obtained as individual stereoisomers (see Supplementary Information, Figures S7 and S8).

During the SPPS sequence (Scheme 3), vector fragment

6 was attached to tripeptide 15, mounted

on 2-CTC. After that, the modiﬁed tripeptide was removed from the polymer carrier by treatment

with DCM/TFA. As a result, compounds 16 were isolated as individual stereoisomers according to the

1

13

H NMR, C NMR LCMS, HRMS data (see Supplementary Information, Figures S5 and S6).

Further, 3-aminopropylazide was attached to the free carboxyl group of compounds 16. Based on

published data, these reactions may be carried out by one of three possible procedures [26]:

1

.

Addition of a coupling reagent (carbodiimide, EEDQ (N-Ethoxycarbonyl-2-ethoxy-1,2-

dihydroquinoline), phosphonium and carbenium salts, trisubstituted phosphates, etc.) and

a tertiary amine, if necessary, to a mixture of the acid and the amine nucleophile to be combined;

Addition of the amine nucleophile to a solution of the coupling reagent and the acid only after

they were reacted and an activated compound was generated;

2

3

.

Addition of the amine nucleophile to one of the activated forms of the acid (activated ester,

acyl azide, anhydrides, etc.) to which it was to be combined.

Considering method 1, it is necessary to note that the activated agent (HBTU) is capable of reacting

with N-terminal amino component, leading to a guanidine derivative; this side process may compete

for peptide chain elongation. To avoid this side reaction, the preliminary activation of the carboxylic

acid component is recommended [27]. We performed method 1 (addition of a coupling reagent

and tertiary amine to a mixture of the acid and the amine). Applying this technique to the reaction

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of compound 16 with 3-aminopropylazide, we obtained the individual stereoisomer of desirable

substance 11, as conﬁrmed by NMR spectroscopy (see Supplementary Information, Figure S8).

Taking into account the racemization taking place during the discussed reactions, we concluded

that method 2 (adding amine to the activating agent solution, tertiary amine, and acid) was not optimal

for stereoselective syntheses of individual stereoisomer of target peptide due to possible intermediate

formation of achiral oxazolone intermediate [26]. However, method 2 could be successfully used

to obtain compound 11 by the liquid-phase technique (Scheme 2). This is explained by the fact

that in the case of the liquid-phase technique, the carboxylic group involved in the formation of a

peptide bond is in a vector fragment and does not have a stereocenter in the α-position. Therefore,

the possible formation of oxazolone during the reaction does not lead to racemization. NMR spectra of

compound 11 obtained by the liquid-phase technique are given in the Supplementary Information

(Figure S7).

When using method 3, it should be noted that there is no general method for activated amino

acid creation. Also, it is necessary to activate the acid with this method, and then isolate the activated

form, which adds an extra stage of synthesis and may lead to undesirable reactions with inappropriate

functional groups [26]. For this reason, we did not test method 3 to obtain compound 12.

The next stage of the synthesis was the removal of the protective tert-butyl groups from carboxyl

fragments and the Boc group from ε-NH of terminal lysine moiety (Schemes 2 and 3). The deprotection

2

was performed by two methods, i.e., by treating of compound 11 with TFA/DCM or DCM/TFA/TIPS/H O

2

mixture. As a result, target compound 12 was obtained, and its structure was conﬁrmed by HRMSm as

1

13

well as H/ C NMR (see Supplementary Information, Figures S9–S12).

The data obtained for diﬀerent methods of vector peptide 12 synthesis are summarized in Table 1.

The total yields of the target compounds based on the starting amino acid for linker formation and the

starting Boc-Fmoc-protected lysine (Scheme 1) were evaluated. The laboriousness of the syntheses was

compared, taking into account the total number of synthetic stages and the number of stages with

chromatographic isolation of the target product.

Table 1. Comparison of synthetical approaches to obtain target compound 12.

Liquid-Phase Technique (Scheme 2)

5%

SPPS Technique (Scheme 3)

Yield based on starting

Boc-Fmoc-Lysine

2

45%

37%

Yield based on compound 6

55%

The total number of

synthesis steps (stages with

chromatographic separation)

13 (10)

16 (7)

In summary, the liquid-phase technique (Scheme 2) using method 2 (addition of the amine

nucleophile to a solution of the coupling reagent and the acid only after they were reacted and

generated an activated compound) to create a peptide bond between compound 10 and a vector

fragment of ligand 6 was characterized by a maximum yield based on compound 11, but the minimum

yield counting of the initial amino acid. The total number of stages using laborious chromatographic

isolation was also large.

SPPS technique (Scheme 3) using method 1 (addition of a coupling reagent and tertiary amine to

a mixture of the acid and the amine) to create a peptide bond between 16 and 3-aminopropylazide,

showed the best yield on the initial amino acid and good yield on compound 6, which seemed to be

optimal. Also, this technique showed further advantages over the liquid-phase technique, namely,

a less time-consuming process of target platforms obtaining, product isolation simplicity, and the

absence of additional puriﬁcation stages, both for intermediate compounds and the target substance.

However, it should be noted that the liquid-phase technique allows the obtainment of large

amounts of target compounds, although it is more laborious and time-consuming than the SPPS

Molecules 2020, 25, 5784

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approaches. At the same time, obtainment by the SPPS technique may be convenient for the rapid

preparation of the libraries of similar compounds, although the reactions proceed with lower yields

and require a large excess of amino acids.

2

.3. Synthesis of the Bimodal Conjugate 19

At the next stage of the work, to demonstrate the possibility of compound 12 use as a molecular

platform for bimodal agent preparation, we synthesized its double conjugate with the anticancer drug

docetaxel and a ﬂuorescent dye Sulfo-Cy5. Docetaxel is a taxane-derivative diterpenoid and is one

of the most widely used anticancer agents in clinical practice today [28]. Analysis of literature data

on the eﬀect of modiﬁcations of various structural fragments of docetaxel on its activity suggested

that the most appropriate strategy for introducing a linker is to form an ester bond with one of the

secondary hydroxyl groups [29]. In cells, the ester bond is known to hydrolyze with the extrication of

free drug. We carried out the reaction of docetaxel with hex-5-ynoic acid, and the obtained adduct 17

was also introduced into the azide–alkyne cycloaddition with peptide 12. The standard procedure

for ester formation in the presence of diisopropylcarbodiimide (DIC) and a catalytic amount of

4

-(dimethylamino)pyridine (DMAP) gave compound 17 with reasonable yield (Scheme 4). 2D NMR

1

13

1

13

spectroscopy (HSQC H- C, HMBC H- C) made it possible to make complete signal correlation in

the spectra of compound 17 (Supplementary Information, Figures S13–S16, Tables S1 and S2).

To obtain conjugate 18 from azide 12 and alkyne 17, we chose the click-reaction of 1,3-dipolar

cycloaddition catalyzed by copper(I). This reaction is widely used in synthesizing biologically

active organic compounds, in particular, agents against tuberculosis and peptide–carbohydrate

conjugates [30]. The complete correlation of signals in NMR spectra of compound 18 was made using

1

13

1

13

2

D NMR spectroscopy (HSQC H- C, HMBC H- C; see Supplementary Information, Figures S17–S20,

Tables S3 and S4).

At the next step, the NHS-activated ester of the ﬂuorescent label Sulfo-Cy5 was attached to the

free NH group of compound 18. Near-infrared ﬂuorescence (NIRF) imaging agents, like Sulfo-Cy5,

2

have high extinction coeﬃcients, large Stokes’ shifts, and are able to generate strong ﬂuorescence

emission oﬀering the possibility of in vivo cancer diagnosis. Their considerable advantages for in vivo

imaging include stronger ligand labeling, signal strength, and tissue absorbance, a wider range of

imaging materials for coupling, and less background ﬂuorescence. The far-red cyanine dye Sulfo-Cy5

(λex 640 nm, λem 656 nm), with high detection sensitivity (0.05 vs. 3.15 mM for Indocyanine green

(ICG)), tissue penetration (9 vs. 6 mm for ICG), and brightness (quantum yield, 28% vs. 0.3% for

ICG) [31], was chosen as the ﬂuorescent label for conjugation with 18.

As a result, the target bimodal conjugate 19 was obtained, and its structure was conﬁrmed

1

by HRMS, LCMS, and H-NMR data. Moreover, initial biological experiments on the synthesized

conjugate interaction with human cells diﬀering in the level of PSMA expression were carried out in

order to preliminary estimate its possibility and potential for biomedical application.

2

.4. Biological Evaluation

First, we investigated the selectivity of synthesized ﬂuorescent conjugate 19 modiﬁed with

PSMA vector toward three human prostate cancer cell lines, which diﬀer in the level of PSMA

expression—LNCaP (PSMA++), 22Rv1 (PSMA+), and PC-3 (PSMA-) [32]—using ﬂuorescent

microscopy. The results are presented in Figure 2.

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Figure 2. Interaction potency of the ﬂuorescent conjugate 19 by LNCaP, 22Rv1, and PC-3 cells after 2 h

0

of co-incubation. Cell nuclei are stained with blue 4 ,6-diamidino-2-phenylindole (DAPI), a ﬂuorescent

stain that binds strongly to DNA. Fluorescent microscopy.

We observed a homogeneous diﬀuse staining of all cells in the LNCaP line and a part of the

cell population in 22Rv1 culture after 2 h incubation with PSMA-Cy5. It must be noted that the

most intensive ﬂuorescence signal from PSMA-Cy5 conjugate was observed in the perinuclear area.

This fact indicated the intracellular localization of the PSMA-Cy5 conjugate. No ﬂuorescence signal

from PSMA-Cy5 was detected in cells of PSMA-negative PC-3 line. For compound 19, all LNCaP

cells were also positively stained. However, the nature of the staining was diﬀerent—we revealed the

ﬂuorescent signal of conjugate 19 to be mainly point-concentrated, presumably, in cell vesicles. At the

same time, less pronounced diﬀuse staining of the entire cells cytoplasm was found. A similar result

was obtained for the 22Rv1 cell line. However, ﬂuorescent signal from conjugate 19 was detected not

in all cells of population. Moreover, the presence of point-concentrated localization of compound 19 in

these cells was signiﬁcantly less than in the LNCaP line. Single accumulations of conjugate 19 were

identiﬁed predominantly in the lamellae of PC-3 cells. Thus, the obtained data demonstrated that the

eﬀectiveness of the conjugate 19 selective interaction with PSMA++ LNCaP cells was higher than with

PSMA+ 22Rv1 cells. Interaction of compound 19 with PSMA- PC-3 cells was signiﬁcantly lower than

with both investigated PSMA-positive cell lines. Some accumulations of conjugate 19 revealed in PC-3

cells could be due to its nonspeciﬁc interaction with the cells and penetration by diﬀusion presumed

for Docetaxel. The mechanism of this taxane penetration inside the cells was well studied only for

hepatocytes, while further investigations are required for other cell types [33].

Further, bimodal conjugate 19, as well as its synthetical precursors (conjugate 18, containing

docetaxel, but not containing a ﬂuorescent label, peptide vector 12 without imaging or therapeutic

agents, and free docetaxel as a comparison substance) were evaluated for in vitro cytotoxicity against

two PSMA-positive cell lines—LNCaP and 22Rv1 (Figure 3) [32]. The cargo Docetaxel and conjugate

1

8 were used as a positive control, whereas compound 12 was used as a negative control. As a result,

conjugates 18 and 19 showed good activity against both cell lines with a slightly more pronounced

eﬀect on LNCaP cells, where LNCaP IC₅₀= 100 nM and 200 nM, respectively, as well as 22Rv1

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IC textsubscript50 = 130 nM and >200 nM. Docetaxel by itself caused signiﬁcant cell death in both

cultures, where LNCaP IC₅₀= 1 nM and 22Rv1 IC₅₀= 2.1 nM. These data were consistent with the

selectivity of the resulting conjugates in relation to cell lines expressing PSMA. The lower toxicity of

conjugates 18 and 19 in comparison with free Docetaxel, could apparently be explained by the slow

release of the active drug from the conjugate, consistent with previously obtained results [19]. Vector

peptide 12, as expected, was not toxic for either of the cell lines.

(

a)

Figure 3. Cytotoxicity of compounds 12

) tumor cells. Results are shown as means

performed in triplicate.

(b)

,

18,

19, and docetaxel (DTX) against LNCaP (a) and 22Rv1

SD (t-test, * p < 0.05). MTS-assay. Experiments were

(

b

±

Based on these data, we can conclude that the designed vector is a perspective conjugate,

which demonstrated selectivity and toxicity against PSMA-positive cells and should be further

investigated in more detail for targeted drug delivery, at least in PSMA-overexpressed LNCaP cells.

3

. Materials and Methods

All used solvents were puriﬁed according to procedures described in [34]. All starting compounds

were commercially available reagents. The initial stages of the synthesis of the vector fragment 1–5

(

Scheme 1) were made by methods previously developed by our scientiﬁc group [21]. Spectral data of

1

the compounds

7

and

8

(Scheme 2) were described in [35]. H NMR was measured using a Bruker

1

Avance spectrometer operating at 400 MHz for H using CDCl and DMSO-d as solvents. Chemical

3

6

shifts were reported in

precision using residual solvent as an internal reference. C NMR was measured using a Bruker Avance

spectrometer operating at 100 MHz using DMSO-d as solvents. Chemical shifts were reported in

δ units to 0.01 ppm precision with coupling constants reported to 0.1 Hz

13

δ

6

units to 0.1 ppm precision using residual solvent as an internal reference. 2D NMR was measured using

1

13

an Agilent 600 spectrometer operating at 600 MHz for H and 100 MHz for ( C) using DMSO-d as the

6

solvent. As 2D NMR methods were used, such as heteronuclear single quantum coherence spectroscopy

1

13

1

13

H- C (gHSQC) and heteronuclear multiple bond correlation H- C (gHMBC). NMR spectra were

processed and analyzed using Mnova software (Mestrelab Research, Spain). High-resolution mass

spectra were recorded on the Orbitrap Elite high-resolution mass spectrometer. Solutions of samples in

acetonitrile with 1% formic acid were introduced into the ionization source by electrospray. For the

HPLC analysis system with Shimadzu Prominence, an LC-20 column and a convection fraction

collector connected with a single quadrupole mass spectrometer Shimadzu LCMS-2020 with dual

ionization source DUIS-ESI-APCI were used. The analytical and preparative column was Phenomenex

Luna 3u C18 100A. Preparative chromatographic separation of substances was carried out using the

INTERCHIM puriFlash 430 chromatograph.

Molecules 2020, 25, 5784

11 of 21

For better interpretation of the NMR spectra of target compound 19, the notation of structural

fragments is shown in Figure 4.

Figure 4. The notation of structural fragments of synthesized compounds (for conjugate 19).

E1 = glutamic amino acid residue, K2 = lysine amino acid residue etc.; a, b: diastereotopic protons;

m, n: notation for two forms of rotational isomers, m/n = 3/2; Hα = Ha, Hβ = Hb etc.

Cell Lines: LNCaP, 22Rv1, and PC-3 human prostate cancer cells were purchased from the

American Type Culture Collection (ATCC, Manassas, VA, USA).

Cell Cultivation: Cells were maintained in RPMI-1640 medium (gibco), supplemented with 10%

Fetal Bovine Serum (Sigma), 2 mM L-glutamine, and RPMI vitamin solution (Sigma). Cells were

◦

cultured at 37 C in a humidiﬁed incubator (Sanyo) supplied with 5% CO . Cells were seeded on

2

glass coverslips or in 96-well plates (Corning) at concentrations of 120,000 cells per mL for LNCaP,

2

00,000 cells per mL for 22Rv1, and 90,000 cells per mL for PC-3 in experiments. The counting of cells

was carried out using the automatic cell counter EVE.

Cell Incubation with Conjugates: A day after seeding the cells on glass coverslips, PSMA-Cy5

or ﬂuorescently labeled compound 19 were added in culture medium at a concentration of 30 nM

for 2 h. Later, cells were washed with PBS (pH 7.2–7.4) and ﬁxed with 4% formaldehyde (Sigma)

(

on PBS) for 15 min. Cell nuclei were stained with DAPI (Sigma) for 10 min. Obtained preparations

were imaged using an inverted ﬂuorescence microscope EVOS (life technologies, objective PlanFluor

2

0×/0.45). Further processing of the photos was carried out by ImageJ software.

Cytotoxicity Assay: A day after cell seeding in 96-well plates, serial dilutions of conjugates and

Docetaxel in culture medium were added to cells. Cells incubated in culture medium were used as

control. DMSO diluted in the cell medium (20%) was used as a positive control. Cells were incubated

◦

for 72 h at 37 C and 5% CO . Later, the culture medium from each well was removed and 20

µL of MTS

2

reagent (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega) was added to each

◦

well with 100

µ

L of new culture medium. After 4 h of incubation at 37 C in darkness, the absorbance

of the obtained solution was measured at 490 nm wavelength using the Thermo Scientiﬁc Multiskan

GO spectrometer. Cell viability was calculated as percent compared to cells incubated in culture

medium. MTS assay revealed 100% cell death after incubation with 20% DMSO (data not shown).

The absorbance of MTS reagent in culture medium without cells was taken as zero. Experiments were

performed in triplicate.

Molecules 2020, 25, 5784

12 of 21

Compound 6. To a solution of compound

1.4 eq; 244

was stirred for 12 h. After that, MeOH (2 eq.) was added and the resulting mixture was stirred for

h. Then, the solvent was removed under reduced pressure, and residue was dissolved in DCM and

extracted with (1) 0.1 M HCl (2 30 mL) and (2) brine (2 30 mL). Then, the organic fraction was dried

over Na SO , and concentrated under reduced pressure to obtain the ﬁnal compound as a yellow oil

5

(1 eq; 725 mg; 1.0 mmol) in 20 mL of DCM, DIPEA

(

µL; 1.4 mmol) and succinic anhydride (1.02 eq; 102 mg; 1.02 mmol) were added. The mixture

1

×

6

2

4

(

801 mg, yield 97%).

¹H-NMR (400 MHz, DMSO-d6,

δ): 12.06 (br.s., 1H, X4C(O)OH), 7.81 (t, J = 5.2 Hz, m) & 7.77

(t, J = 5.2 Hz, n) (1H, X3NHk, m + n, m/n = 3/2), 7.40 (t, J = 7.7 Hz, X8He, n), 7.37–7.27 (m, X8Hd +

X8He(m)), 7.26–7.21 (m, 1H, X8Ht, m + n), 7.19–7.10 (m, 1H, X8Hg, m + n), 6.34–6.20 (m, 2H, K2NH +

E1NH, m + n), 4.56 (s, n) & 4.48 (s, m) (2H, X8Ha, m + n, m/n = 3/2), 4.07–4.00 (m, 1H, E1Ha, m + n),

4

3

.00–3.90 (m, 1H, K2Ha, m + n), 3.22 (t, J = 7.3 Hz, n) & 3.19 (t, J = 7.3 Hz, m) (2H, K2He, m + n, m/n = 3/2),

.01 (q, J = 6.4, 12.7 Hz, m) & 2.96 (q, J = 6.4, 12.7 Hz, n) (2H, X3He, m + n, m/n = 3/2), 2.44–2.38 (m, 2H,

X4Hb, m + n), 2.36 (t, J = 7.4 Hz, X3Ha, m), 2.31–2.25 (m, 2H, X4Ha, m + n), 2.25–2.15 (m, E1Hg +

X3Ha(n)), 1.91–1.80 (m, 1H, E1Hb(a)), 1.72–1.63 (m, 1H, E1Hb(b)), 1.63–1.56 (m, 1H, K2Hb(a)), 1.40–1.35

(

m, 27H, tBu), 1.56–1.15 (m, 11H, K2Hb(b) + X3Hb + X3Hd+ K2Hd + K2Hg + X3Hg, m + n).

13

C-NMR (100 MHz, DMSO-d₆, δ): 173.93 (X4Cg), 172.26 (K2C(n)), 172.23 (K2C(m)), 172.22 (X3C(n)),

172.19 (X3C(m)), 171.95 (E1C), 171.47 (E1Cg), 170.76 (X4C(m)), 170.73 (X4C(n)), 157.18 (U(m)),

157.16 (U(n)), 141.20 (X9Cb(m)), 140.80 (X9Cb(n)), 133.45 (X9Ce(n)), 133.10 (X9Ce(m)), 130.63 (X9Cd(n)),

130.26 (X9Cd(m)), 127.24 (X9Ct(m)), 127.17 (X9Ck(n)), 126.88 (X9Ck(m)), 126.34 (X9Ct(n)),

126.08 (X9Cg(m)), 124.99 (X9Cg(n)), 80.59 (E1tBu), 80.42 (K2tBu(m)), 80.33 (K2tBu(n)), 79.77 (E1dtBu),

5

4

3

2

3.01 (K2Ca(n)), 52.88 (K2Ca(m)), 52.20 (E1Ca(m)), 52.18 (E1Ca(n)) 49.63 (X9Ca(n)), 47.11 (X9Ca(m)),

6.83 (K2Ce(m)), 45.20 (K2Ce(n)),38.49 (X3Ce(m)), 38.43 (X3Ce(n)), 32.34 (X3Ca(n)), 31.95 (X3Ca(m)),

1.83 (K2Cb), 30.93 (E1Cg), 30.06 (X4Ca), 29.25 (X4Cb), 29.13 (X3Cd(m)), 29.04 (X3Cd(n)), 27.75 (tBuE1),

7.69 (K2Cd(m)), 27.66 (tBuK2), 27.64 (tBuE1g + E1Cb), 26.72 (K2Cd(n)), 26.23 (X3Cg(m)), 26.15 (X3Cg(n)),

4.76 (X3Cb(m)), 24.63 (X3Cb(n)), 22.45 (K2Cg(n)), 22.27 (K2Cg(m)).

+

ESI-MS C H ClN O : m/z calcd. for [M + H ] : 825.44, found: 825.45.

41

65

4

11

Compound 7. To a solution of FmocLys(L)(NHBoc)-OH (1 eq.; 1000 mg; 2.134 mmol) in DMF

20 mL), DIPEA (1.5 eq.; 556 L; 3.2 mmol), HOBt (1.2 eq.; 344 mg; 2.56 mmol), and HBTU (1.2 eq.;

71 mg; 2.56 mmol), were added, then the resulting mixture was purged with Ar and stirred for 60 min.

(

9

µ

Then, NH -(CH ) -N (2 eq.; 38 mg; 0.38 mmol) was added and the mixture was stirred for 24 h under

2

2 3

3

Ar atmosphere. At the next step, the solvent was removed under reduced pressure and re-evaporated

with DCM twice. The residue was dissolved in DCM (50 mL) and extracted with 1) H O (2 50 mL)

50 mL). Then, the organic fraction was dried over Na SO . After the solvent was

×

2

and 2) brine (2

removed under reduced pressure, the residue was puriﬁed by column chromatography (Puriﬂash 15

0g, eluent: Hex(100%)/EtOAc(0%) => Hex(0%)/EtOAc(100%) for 30 min. The eluent for TLC was

×

2

4

µ

4

EtOAc/Hex = 1:1. Compound 7 was obtained as a yellow oil (950 mg, 81% yield).

1

H-NMR (400 MHz, CDCl₃, δ): 7.76 (d, J = 7.5 Hz, 2H, Fmoc), 7.58 (d, J = 7.5 Hz, 2H, Fmoc), 7.40

(

4

t, J = 7.4 Hz, 2H, Fmoc), 7.31 (t, J = 7.4 Hz, 2H, Fmoc), 6.43 (br.s., 1H, X8NH), 5.54 (br.d., 1H, K7NH),

.63 (br.s., 1H, K7NHk), 4.40 (d, J = 6.8 Hz, 2H, Fmoc), 4.20 (t, J = 6.8 Hz, 1H, Fmoc), 4.15–4.01 (m, 1H,

K7Ha), 3.40–3.25 (m, 4H, X8Hg + X8Ha), 3.18–3.01 (m, 2H, K7He), 1.92–1.81 (m, 1H, K7Hb(a)), 1.80–1.71

(

m, 2H, X8Hb), 1.70–1.57 (m,1H, K7Hb(b)), 1.56–1.46 (m, 2H, K7Hd), 1.43 (s, 9H, tBu), 1.39–1.29

m, 2H, K7Hg).

Compound 8. To a solution of

7 (1 eq.; 792 mg; 1.44 mmol) in DMF (10 mL), Et NH (10 eq.; 1053 µL;

2

14.4 mmol) was added, then the resulting mixture was purged with Ar and stirred for 1 h. The control

of the reaction was performed with TLC. The eluent for TLC was EtOAc/Hex = 1:1. At the next step,

the solvent was removed under reduced pressure and re-evaporated with DCM twice. The residue

was puriﬁed by column chromatography (Puriﬂash 15µ 25g, eluent: DCM(100%)/MeOH(0%) =>

DCM(85%)/MeOH(15%) for 30 min, after MeOH (100%) for 5 min. Compound

8 was obtained as a

yellow oil (469 mg, 99% yield).

Molecules 2020, 25, 5784

13 of 21

¹H-NMR (400 MHz, CDCl₃,

δ): 4.59 (br.s., 1H, K7NHk), 3.40–3.25 (m, 5H, X8Hg + K7Ha + X8Ha),

3

.19–3.02 (m, 2H, K7He), 1.91–1. 1.70 (m, 4H, K7Hb(a) + X8Hb + K7Hb(b)), 1.56–1.46 (m, 2H, K7Hd),

1

.44 (s, 9H, tBu), 1.39–1.32 (m, 2H, K7Hg).

Compound 9. To a solution of FmocFF (1 eq.; 770 mg; 1.44 mmol) in DMF (20 mL), DIPEA (1.2 eq.;

3

(

01

1 eq.; 469 mg; 1.43 mmol) were added, then the resulting mixture was purged with Ar and stirred

for 16 h. The control of the reaction was performed with TLC. The eluent for TLC was DCM/MeOH

19:1. At the next step, the solvent was removed under reduced pressure and re-evaporated with

DCM twice. The residue was dissolved in DCM (50 mL), and extracted with 1) H O (2 50 mL), 2)

50 mL). Then, the organic fraction was dried over Na SO . After the solvent was removed

µL; 1.73 mmol), HOBt (1.2 eq.; 233 mg; 1.73 mmol), HBTU (1.2 eq.; 655 mg; 1.73 mmol), and 8

=

×

2

brine (2

×

2

4

under reduced pressure, the residue was puriﬁed by column chromatography (Puriﬂash 15

µ

40g,

eluent: DCM(100%)/MeOH(0%) => DCM(90%)/MeOH(10%) for 30 min, after MeOH (100%) for 5 min.

Compound 9 was obtained as a yellow oil (853 mg, 70% yield).

Compound 10. To a solution of

9 (1 eq.; 840 mg; 0.995 mmol) in DMF (7 mL), Et NH (10 eq.; 1029 µL;

2

9.95 mmol) was added, then the resulting mixture was purged with Ar and stirred for 1 h. The control

of the reaction was performed with TLC. The eluent for TLC was DCM/MeOH = 19:1. At the next step,

the solvent was removed under reduced pressure and re-evaporated with DCM twice. The residue

was puriﬁed by column chromatography (Puriﬂash 15µ 25g, eluent: DCM(100%)/MeOH(0%) =>

DCM(90%)/MeOH(10%) for 30 min, after MeOH (100%) for 5 min. Compound 10 was obtained as a

yellow oil (490 mg, 79% yield).

1

H-NMR (400 MHz, DMSO-d₆, δ): 8.14 (d, J = 7.9 Hz, 1H, F6NH), 8.06 (d, J = 7.7 Hz, 1H, K7NH),

7

(

6

(

.90 (t, J = 5.7 Hz, 1H, X8NH), 7.29–7.09 (m, 10H, Ph + Ph), 6.77 (t, J = 5.5 Hz, 1H, K7NHk), 4.64–4.54

m, 1H, F6Ha), 4.21–4.09 (m, 1H, K7Ha), 3.40–3.29 (m, 4H, F5Ha + F6Hb(a) + X8Hg), 3.11 (q, J = 6.0,

.5 Hz, 2H, X8Ha), 2.97 (dd, J = 13.8, 4.9 Hz, 1H, F6Hb(b)), 2.91–2.78 (m, 4H, F5Hb(ab) + K7He), 1.73

br.s., 2H, F5NH ), 1.69–1.56 (m, 3H, X8Hb + K7Hb(a)), 1.56–1.44 (m, 1H, K7Hb(b)), 1.36 (s, 9H, tBu),

2

1

.35–1.29 (m, 2H, K7Hd), 1.28–1.11 (m, 2H, K7Hg).

Compound 15. Activation of 2-CTC. The mixture of 2-CTC (1 eq.; 1 g; 1.2–1.4 mmol/g; 100–200 mesh)

in DCM (10 mL) was stirred for 10 min, then the mixture was purged with Ar, then SOCl (3 eq.; 305

µ

◦

L;

2

4.2 mmol) was added dropwise, and then DMF (16

µ

L; 5% V/V to SOCl ) was added and stirred at 40 C

2

for 4 h. After that, the resin was filtered and transferred to a polypropylene reactor and washed with

DMF (3 × 10 mL, 1 min) and DCM (3 × 10 mL, 1 min).

The addition of FmocLys(L)(NHBoc)-OH. To the mixture of CTC-2 (1 equiv; 1 g; 1.2–1.4 mmol/g;

1

2

00–200 mesh) in DMF (10 mL), FmocLys(NHBoc)-OH (2 eq.; 1.312 g; 2.8 mmol) and DIPEA (10 eq.;

.44 mL; 14 mmol) were added, and the mixture was stirred for 2 h. Then, the resin was ﬁltered oﬀ and

washed with MeOH (3 10 mL, 5 min), DCM (3 0 mL, 1 min), DMF (3 10 mL, 1 min), and DCM

×

(

3 × 10 mL, 1 min).

Deprotection of Fmoc. FmocK(NHBoc) on a 2-CTC resin (1 eq.) was washed with DMF (2

min), then 4-methylpiperidine in DMF (20%/80% V/V, 15 mL) was added and stirred for 15 min,

15 mL, 1 min), then 4-methylpiperidine

×

15 mL,

1

then the resin was ﬁltered oﬀ and washed with DMF (3

×

in DMF (20%/80% V/V,15 mL) was added and stirred for 15 min. After the resin was ﬁltered oﬀ,

the resulting solution was washed with DMF (3 × 15 mL, 1 min) and DCM (3 × 15 mL, 1 min).

The addition of FmocPhe(L)-OH. To the mixture of NH -K(NHBoc) on a CTC-2 resin (1 eq.) in

2

DMF (15 mL), FmocPhe(L)-OH (2 eq.; 1.085 g; 2.8 mmol), HOBt (0.5 eq.; 95 mg; 0.7 mmol), HBTU (2 eq.;

1

.062 g; 2.8 mmol), and DIPEA (3 eq.; 0.73 mL; 4.2 mmol) were added and stirred for 2 h. Then the

resin was ﬁltered oﬀ and washed with DMF (3 × 15 mL, 1 min) and DCM (3 × 15 mL, 1 min).

Deprotection of Fmoc. FmocFK(NHBoc) on a CTC-2 resin (1 eq.) was washed with DMF (2

min), then 4-methylpiperidineine in DMF (20%/80% V/V, 15 mL) was added and stirred for 15 min,

then the resin was ﬁltered oﬀ and washed with DMF (3 15 mL, 1 min), then 4-methylpiperidine in

DMF (20%/80% V/V, 15 mL) was added and stirred for 15 min. After the resin was ﬁltered oﬀ, DMF

3 × 15 mL, 1 min) and DCM (3 × 15 mL, 1 min) wash was carried out.

×

15 mL,

1

×

(

Molecules 2020, 25, 5784

14 of 21

Addition of FmocPhe(L)-OH. To the mixture of NH -FK(NHBoc) on a CTC-2 resin (1 eq.) in

2

DMF (15 mL), FmocPhe(L)-OH (2 eq.; 1.085 g; 2.8 mmol), HOBt (0.5 eq.; 95 mg; 0.7 mmol), HBTU

(2 eq.; 1.062 g; 2.8 mmol), and DIPEA (3 eq.; 0.73 mL; 4.2 mmol) were added and stirred for 2 h. Then,

the resin was ﬁltered oﬀ and washed with DMF (3 × 15 mL, 1 min) and DCM (3 × 15 mL, 1 min).

Deprotection of Fmoc. FmocFFK(NHBoc) on a CTC-2 resin (1 eq.) was washed with DMF

(

2

×

15 mL, 1 min), then 4-methylpiperidineine in DMF (20%/80% V/V, 15 mL) was added and stirred

for 15 min. Then, the resin was ﬁltered oﬀ, washed with DMF (3 15 mL, 1 min), then 4-methylpiperidine

in DMF (20%/80% V/V, 15 mL) was added and stirred for 15 min. After the resin was ﬁltered oﬀ, DMF

15 mL, 1 min) and DCM (3 15 mL, 1 min) wash was carried out. Thus, the NH -FFK(NHBoc)

tripeptide was obtained on 2-CTC resin (1.95 g, ~1.4 mmol).

Compound 16. To the mixture of tripeptide 15 NH -F5F6K7(NHBoc) on 2-CTC resin (1 eq.;

×

(

3

×

2

4

63 mg; 0,33 mmol) in DMF (5 mL) in a polypropylene reactor, compound

mmol), HOBt (0.5 eq.; 22 mg; 0.165 mmol), HBTU (2 eq.; 250 mg; 0.66 mmol), and DIPEA (3 eq.; 172

.99 mmol) were added. The mixture was stirred for 2 h. Then, the solvent was removed by ﬁltration

6

(1.2 eq.; 327 mg; 0,396

µL;

0

on a porous reactor ﬁlter and the resin was washed with DMF (3

dried from residue of solvents.

×

5 mL), DCM (3

×

5 mL), and then

After that, a mixture of DCM/TFA (99.25%/0.75%, 6.5 mL) was added to the resin and stirred for

5 min, then the solution was ﬁltered oﬀ from the resin. The solvent was removed under reduced

1

pressure and the residue was re-evaporated three times with DCM. The product was puriﬁed by column

chromatography (Puriﬂash, column of PF-15C18AQ-F0025 (15 40g), eluent: H O(80%)/MeCN(20%)

> H O(0%)/MeCN (100%) for 15 min after MeCN (100%) for 5 min. Compound 16 was obtained as a

µ

2

=

2

colorless oil (338 mg, 76% yield).

1

H-NMR (400 MHz, DMSO-d₆, δ): 12.53 (br.s., 1H, K7COOH), 8.18 (d, J = 7.5 Hz, 2H, F5NH +

F6NH), 7.99–7.90 (m, 1H, K7NH), 7.89 (t, J = 5.2 Hz, m) & 7.86 (t, J = 5.2 Hz, n) (1H, X3NHk, m + n, m/n

3/2), 7.42–7.08 (m, 14H, Ph + Ph + X9H), 6.79 (t, J = 5.1 Hz, 1H,K7NHk), 6.35–6.22 (m, 2H, K2NH +

=

E1NH, m + n), 4.60–4.48 (m, F6Ha + X9Ha(n)), 4.48 (s, X9Ha(m), m + n, m/n = 3/2), 4.40–4.30 (m, 1H,

F5Ha), 4.20–4.09 (m, 1H, K7Ha), 4.08–4.00 (m, 1H, E1Ha), 4.00–3.90 (m, 1H, K2Ha), 3.22 (t, J = 7.3 Hz, n)

&

3.17 (t, J = 7.3 Hz, m) (2H, K2He, m + n, m/n = 3/2), 3.14–3.06 (m, 1H, F6Hb(a)), 3.05–2.82 (m, 6H,

F6Hb(b) + X3He + K7He + F5Hb(a)), 2.70–2.57 (m, 1H, F5Hb(b)), 2.37–2.11 (m, 8H, X4Hb + E1Hg

X4Ha + X3Ha), 1.91–1.80 (m, 1H, E1Hb(a)), 1.77–1.12 (m, 19H, E1Hb(b) + K7Hb(a) + K2Hb(a) +

K7Hb(b) + K2Hb(b) + X3Hb + X3Hd + K2Hd + K2Hg + X3Hg, m + n), 1.41–1.32 (m, 36H, tBu).

+

13

C-NMR (100 MHz, DMSO-d₆,

δ): 173.35 (K7C), 172.24 (K2C(n)), 172.20 (K2C(m)), 172.14

(X3C(n)+ F6C(m)), 172.12 (X3C(m)), 172.08 (F6C(n)), 171.92 (E1C), 171.45 (E1Cd), 171.39 (X4Cg(mn)),

171.07 (F5C(mn)), 171.00 (X4C(mn)), 157.14 (U(m)), 157.12 (U(n)), 155.60 (K7Boc), 141.17 (X9Cb(m)),

140.77 (X9Cb(n)),138.13 (F6Cg), 137.99 (F5Cg), 133.43 (X9Ce(n)), 133.07 (X9Ce(m)), 130.60 (X9Cd(n)),

130.27 (X9Cd(m)), 129.18 (F6Cd), 129.07 (F5Cd), 128.09 (F6Ce), 128.03 (F5Ce), 127.21 (X9Ct(m)), 127.15

(X9Ck(n)), 126.86 (X9Ck(m)), 126.31 (F6Ck), 126.25 (F5Ck), 126.19 (X9Ct(n)),126.06 (X9Cg(m)), 124.95

(X9Cg(n)), 80.58 (E1tBu), 80.41 (K2tBu(m)), 80.32 (K2tBu(n)), 79.77 (E1dtBu), 77.37 (K7BoctBu), 54.43

(F5Ca), 53.79 (F6Ca(m)), 52.99 (K2Ca(n)), 52.86 (K2Ca(m)), 52.18 (E1Ca), 52.00 (K7Ca), 49.60 (X9Ca(n)),

4

3

+

2

7.09 (X9Ca(m)), 46.79 (K2Ce(m)), 45.20 (K2Ce(n)), 39.10 (K7Ce(mn), 38.61 (X3Ce(m)), 38.55 (X3Ce(n)),

7.07 (F5Cb), 36.95 (F6Cb), 32.32 (X3Ca(n)), 31.95 (X3Ca(m)), 31.82 (K2Cb), 30.91 (E1Cg), 30.77 (X4Ca

K7Cb), 30.63 (X4Cb), 29.19 (K7Cd), 29.09 (X3Cd(m)), 28.99 (X3Cd(n)), 28.29 (tBuK7), 27.75 (tBuE1),

7.66 (tBuK2+ K2Cd(m)), 27.63 (tBuE1d+ E1Cb), 26.71 (K2Cd(n)), 26.31 (X3Cg(m)), 26.22 (X3Cg(n)),

4.75 (X3Cb(m)), 24.60 (X3Cb(n)), 22.73 (K7Cg), 22.44 (K2Cg(n)) 22.26 (K2Cg(m)).

+

ESI-MS C H ClN O : m/z calcd. for [M + H ] :1347.72, found:1347.55.

7

0

103

8

16

+

HRMS (m/z, ESI): calcd for C H ClN O -[M + H] 1347.7253, found: 1347.7236, 1369.7073

70

103

8

16

+

[

M + Na] , 1385.6801 [M + K] .

Compound 11. Scheme 2. Method 2. To a solution of compound

DMF (15 mL), DIPEA (1.5 eq.; 66 L; 0.385 mmol), HOBt(Cl) (1.2 eq.; 44 mg; 0.308 mmol), and HBTU

1.2 eq.; 97 mg; 0.308 mmol) were added, then the resulting mixture was purged with Ar and stirred

6

(1 eq.; 245 mg; 0.257 mmol) in

µ

(

Molecules 2020, 25, 5784

15 of 21

for 120 min, then compound 10 (1 equiv; 160 mg; 0.257 mmol) was added and the mixture was

stirred for 24 h under Ar atmosphere. Then, the solvent was removed under reduced pressure and

the residue was dissolved in DCM (25 mL), then extraction was carried out: (1) H O (2

×

30 mL),

2

(

2) brine (2

×

30 mL). Then, the organic fraction was dried over Na SO . After the solvent was removed

2

4

under reduced pressure, the residue was puriﬁed by column chromatography (Puriﬂash 15

µ

25g,

eluent: DCM(98%)/MeOH(2%) => DCM(92%)/MeOH(8%) for 40 min, where the eluent for TLC was

DCM/MeOH = 19:1. As a result, several fractions were obtained with the content of the claimed

substance from 21% to 57%. Re-puriﬁcation was performed using column chromatography (Puriﬂash on

the column PF-15C18HP-F0035 (15

µ 35g); eluent: H O(70%)/MeCN(30%) => H O(0%)/MeCN(100%) for

2 2

1

5 min, after MeCN (100%) for 5 min. Compound 12 was obtained as a colorless oil (244 mg, 66% yield).

1

H-NMR (400 MHz, CDCl₃,

m, 15H, X9H(mn) + F6NH + Ph + Ph), 6.98–6.88 (m, 1H, X8NH(mn), 6.30–6.14 (m, 1H, K7NH(m + n),

.52–5.27 (m, 2H, K2NH(m + n)+ E1NH(m + n), 5.05–4.90 (m, 1H, K7NHk(m + n)), 4.60–4.18 (m, 7H,

δ): 8.02–7.93 (m, 1H, F5NH), 7.87–7.72 (m, 1H, X3NH(mn)), 7.43–6.98

(

5

X9Ha(n) + F6Ha + X9Ha(m) + F5Ha + K7Ha + E1Ha + K2Ha), 3.41–3.00 (m, 10H, X8Hg + K2He +

X8Ha + X3He + K7He), 2.97–2.85 (m, 1H, F6Hb(a)), 2.84–2.72 (m, 1H, F6Hb(b)), 2.71–2.59 (m, 1H,

F5Hb(a)), 2.47–2.38 (m, 1H, F5Hb(b)), 2.36–2.11 (m, 8H, X4Hb(mn) + X4Ha + X3Ha(mn) + E1Hg),

2

1

.12–1.97 (m, 1H, E1Hb(a)), 1.91–1.77 (m, 3H, X8Hb + E1Hb(b)), 1.77–1.68 (m, 2H, K7Hb(a) + K2Hb(a)),

.68–1.10 (m, 16H, K7Hb(b) + K2Hb(b) + X3Hb + X3Hd + K7Hd + K2Hd + K7Hg + K2Hg + X3Hg,

m + n), 1.46–1.38 (m, 36H, tBu).

ESI-MS C H ClN O : m/z calcd. for [M + H ] : 1429.79, found: 1430.60.

+

73

109

12 15

Scheme 3. Method 1. To a solution of compound 16 (1 eq.; 30 mg; 0.022 mmol) in DMF (3 mL)

NH -(CH ) -N (2 eq.; 4 mg; 0.044 mmol), HOBt (1.2 eq.; 4 mg; 0.0264 mmol), HBTU (1.2 eq.; 10 mg;

2

2 3

3

0.0264 mmol), and DIPEA (1.5 eq.; 6 µL; 0.033 mmol) were added. The mixture was stirred for 24 h

in an inert atmosphere. Then, the solvent was removed under reduced pressure and was twice

reevaporated with DCM. The residue was puriﬁed by column chromatography (Puriﬂash on column

PF-15C18AQ-F0004 (15µ 4g)); eluent: system H O(80%)/MeCN(20%) => H O(0%)/MeCN (100%) for

2 2

1

0 min, after MeCN (100%) for 5 min. Compound 11 was obtained as a colorless oil (21 mg, yield 67%).

1

H-NMR (400 MHz, DMSO-d₆, δ): 8.34 (d, J = 7.2 Hz, 1H, F5NH), 8.19 (d, J = 7.5 Hz, 1H, F6NH),

8

7

.01–7.89 (m, 1H, X3NHk(mn), m/n = 3/2), 7.77–7.67 (m, 1H, K7NH(mn)), 7.65–7.56 (m, 1H, X8NH(mn)),

.42–7.09 (m, 14H, Ph + Ph + X9H(mn)), 6.79 (t, J = 5.1 Hz, 1H,K7NHk), 6.34–6.21 (m, K2NH(mn)

E1NH(mn)), 4.59–4.39 (m, 3H, X9Ha(n) + X9Ha(m) + F6Ha), 4.37–4.25 (m, 1H, F5Ha), 4.17–4.07

+

(

m, 1H, K7Ha), 4.05–4.00 (m, 1H, E1Ha), 4.00–3.91 (m, 1H, K2Ha), 3.33 (t, J = 6.9 Hz, 2H, X8Hg), 3.21

t, J = 7.3 Hz, n) & 3.16 (t, J = 7.3 Hz, m) (2H, K2He, m + n, m/n = 3/2), 3.14–2.81 (m, 9H, X8Ha + F6Hb(a)

+

F6Hb(b) + X3He(mn) + K7He + F5Hb(a)), 2.71–2.60 (m, 1H, F5Hb(b)), 2.38–2.12 (m, 8H, X4Hb +

E1Hg + X4Ha + X3Ha), 1.93–1.80 (m, 1H, E1Hb(a)), 1.72–1.60 (m, 4H, E1Hb(b) + X8Hb + K7Hb(a)),

1.60–1.10 (m, 17H, K2Hb(a) + K7Hb(b) + K2Hb(b) + X3Hb + X3Hd + K2Hd + K2Hg + X3Hg, m + n),

1

.41–1.32 (m, 36H, tBu).

+

−

ESI-MS C H ClN O : m/z calcd. for[M − H ] : 1429.79, found: 1430.70.

73

109

12 15

+

HRMS (m/z, ESI): calcd. for C H ClN O -[M + Na] : 1451.7748, found: 1451.7716.

73

109

12 15

Compound 12. Scheme 2. Compound 11 (1 eq.; 243 mg; 0.17 mmol) was dissolved in mixture

of DCM/TFA (9 mL of DCM, 1 mL of TFA). The mixture was stirred for 12 h, then the solvent

was removed under reduced pressure and re-evaporated with DCM three times. The product was

precipitated with Et O and washed twice with Et O (10 mL). After, the residue was puriﬁed by column

2

chromatography (Puriﬂash on a column of PF-15C18AQ-F0025 (15µ 25g), eluent: H O(80%)/MeCN(20%)

2

=

> H O(0%)/MeCN(100%) for 15 min after MeCN (100%) for 5 min. Compound 12 was obtained as a

2

colorless oil (166 mg, yield 84%).

1

H-NMR (400 MHz, DMSO-d₆, δ): 8.76–8.68 (m, F5NH(m)), 8.60–8.53 (m, F5NH(n)), 8.54–8.43

(

6

m, F6NH(m) + X3NHk(m)), 8.42–8.37 (m, F6NH(n)), 8.36–8.27 (m, X3NHk(n), m/n = 3/2), 7.76–7.62

m, 1H, K7NH(mn)), 7.59–7.46 (m, 1H, X8NH(n) + X8NH(m)), 7.43–7.07 (m, 14H, Ph + Ph + X9H(mn))

.43–6.23 (m, K2NH(mn) + E1NH(mn)), 4.59–4.44 (m, 2H, X9Ha(n) + X9Ha(m)), 4.43–4.33 (m, 1H, F6Ha),

Molecules 2020, 25, 5784

16 of 21

4

2

.26–4.16 (m, 1H, F5Ha), 4.16–4.05 (m, 1H, K7Ha), 4.04–3.91 (m, 2H, E1Ha + K2Ha), 3.33 (t, J = 6,9 Hz,

H, X8Hg), 3.25–2.95 (m, 8H, K2He(mn) + X8Ha + F6Hb(a) + F6Hb(b) + X3He(mn), 2.94–2.82 (m, 1H,

F5H(a)), 2.73 (t, J = 7.5 Hz, 2H, K7He), 2.70–2.61 (m, 1H, F5Hb(b)), 2.44–2.26 (m, X4Hb(mn) + X4Ha(a) +

X3Ha(m)), 2.25–2.11 (m, E1Hg + X3Ha(n) + X4Ha(b)), 1.84-1.69 (m, 3H, E1Hb(a) + E1Hb(b) + K7Hb(a)),

1

.69–1.60 (m, 3H, X8Hb + K2Hb(a)), 1.60–1.31 (m, 10H, K7Hb(b) + K2Hb(b) + K7Hd + X3Hb + X3Hd +

K2Hd), 1.30–1.12 (m, 6H, K2Hg + K7Hg + X3Hg).

13

C-NMR (100 MHz, DMSO-d₆,

δ): 175.56 (K2C(m)), 175.32 (K2C(n)), 175.06 (E1C(m)), 174.99

(

E1C(n)), 174.62 (E1Cd(mn)), 173.66 (X4Cg(m)), 173.40 (X4Cg(n)), 172.32 (F5C(mn)), 172.23 (X3C(m)),

1

72.13 (X3C(n)), 171.97 (X4C(m)), 171.84 (X4C(n)), 171.26 (K7C(mn)), 171.16 (F6C(m)), 171.08 (F6C(n)),

57.38 (U(mn)), 141.22 (X9Cb(m)), 140.79 (X9Cb(n)), 138.16 (F6Cg(m)), 138.08 (F6Cg(n)), 137.89

(F5Cg(mn)), 133.40 (X9Ce(n)), 133.04 (X9Ce(m)), 130.61 (X9Cd(n)), 130.24 (X9Cd(m)), 129.00 (F6Cd),

1

28.93 (F5Cd), 128.27 (F6Ce), 128.14 (F5Ce), 127.21 (X9Ct(m)), 127.13 (X9Ck(n)), 126.84 (X9Ck(m)),

26.38 (F6Ck), 126.34 (F5Ck), 126.28 (X9Ct(n)), 126.09 (X9Cg(m)), 125.00 (X9Cg(n)), 55.97 (F5Ca(m)),

55.65 (F5Ca(n)), 55.27 (F6Ca(m)), 55.07 (F6Ca(n)), 53.17 (K2Ca(m)), 52.88 (K2Ca(n) + E1Ca(mn) +

K7Ca(mn)), 49.54 (X9Ca(n)), 48.20 (X8Cg), 47.05 (X9Ca(m)), 46.94 (K2Ce(m)), 44.89 (K2Ce(n)), 38.76

(

X3Ce(m)), 38.68 (X3Ce(n)), 38.50 (K7Ce(mn), 36.62 (F5Cb(mn)), 36.25 (F6Cb(n)), 36.10 (F6Cb(m)), 35.87

X8Ca), 32.51 (K2Cb(m)), 32.28 (K2Cb(n) + X3Ca(n)), 31.76 (X3Ca(m)), 31.17 (K7Cb + E1Cg), 30.72

X4Ca), 30.46 (X4Cb), 28.93 (E1Cb + X3Cd(n)), 28.73 (X3Cd(m)), 28.20 (X8Cb), 27.93 (K2Cd(m)), 26.89

K2Cd(n) + K7Cd(mn)), 26.13 (X3Cg(mn)), 24.63 (X3Cb(mn)), 22.66 (K7Cg(m)), 22.58 (K7Cg(n)), 22.40

K2Cg(n)), 22.33 (K2Cg(m)).

+

ESI-MS C H ClN O : m/z calcd. for [M + H ] :1161.55, found: 1161.55.

56

77

12 13

Scheme 3. 11 (1 eq.; 39 mg; 0.027 mmol) was dissolved in the system of DCM/TFA/TIPS/H O

2

(46.25%/46.25%/2.5%/5%; V/V respectively, 2 mL). The mixture was stirred for 3 h, then the solvent

was removed under reduced pressure and re-evaporated with DCM three times. The product was

precipitated with Et O and washed twice with Et O (1 mL). After, the compound was puriﬁed by column

2

chromatography (Puriﬂash on the column PF-15C18AQ-F0004 (15µ 4g), eluent: H O(80%)/MeCN

2

(

20%) => H O(0%)/MeCN (100%) for 15 min, after MeCN (100%) for 5 min. Individual 12 was obtained

2

as a colorless oil (28 mg, yield 88%).

1

H-NMR (400 MHz, DMSO-d₆, δ): 8.76–8.67 (m, F5NH(m)), 8.60–8.53 (m, F5NH(n)), 8.52–8.42

(

m, F6NH(m) + X3NHk(m)), 8.42–8.35 (m, F6NH(n)), 8.35–8.26 (m, X3NHk(n), m/n = 3/2), 7.75–7.62

m, 1H, K7NH(mn)), 7.59–7.41 (m, 1H, X8NH(n) + X8NH(m)), 7.43–7.07 (m, 14H, Ph + Ph + X9H(mn))

6.43–6.23 (m, K2NH(mn) + E1NH(mn)), 4.54–4.44 (m, 2H, X9Ha(n) + X9Ha(m)), 4.43–4.33 (m, 1H, F6Ha),

4.27–4.17 (m, 1H, F5Ha), 4.17–4.07 (m, 1H, K7Ha), 4.04–3.91 (m, 2H, E1Ha + K2Ha), 3.33 (t, J = 6,9 Hz,

2H, X8Hg), 3.25–2.95 (m, 8H, K2He(mn) + X8Ha + F6Hb(a) + F6Hb(b) + X3He(mn), 2.94–2.85 (m, 1H,

F5H(a)), 2.73 (t, J = 7.5 Hz, 2H, K7He), 2.70–2.61 (m, 1H, F5Hb(b)), 2.44–2.26 (m, X4Hb(mn) + X4Ha(a) +

X3Ha(m)), 2.25–2.11 (m, E1Hg + X3Ha(n) + X4Ha(b)), 1.84–1.69 (m, 3H, E1Hb(a) + E1Hb(b) + K7Hb(a)),

1.69–1.60 (m, 3H, X8Hb + K2Hb(a)), 1.60–1.31 (m, 10H, K7Hd + K7Hb(b) + K2Hb(b) + X3Hb(m) +

X3Hb(n) + X3Hd + K2Hd), 1.30–1.12 (m, 6H, K2Hg + K7Hg + X3Hg).

13

C-NMR (100 MHz, DMSO-d₆,

E1C(n)), 174.75 (E1Cd(m)), 174.71 (E1Cd(n)), 173.54 (X4Cg(m)), 173.33 (X4Cg(n)), 172.30 (F5C(mn) +

X3C(m)), 172.17 (X3C(n)), 171.99 (X4C(m)), 171.89 (X4C(n)), 171.39 (K7C(mn)), 171.23 (F6C(m)), 171.18

δ): 175.62 (K2C(m)), 175.46 (K2C(n)), 175.22 (E1C(m)), 175.15

(

F6C(n)), 157.51 (U(mn)), 141.26 (X9Cb(m)), 140.84 (X9Cb(n)), 138.18 (F6Cg(m)), 138.12 (F6Cg(n)), 137.96

F5Cg(mn)), 133.50 (X9Ce(n)), 133.14 (X9Ce(m)), 130.65 (X9Cd(n)), 130.29 (X9Cd(m)), 129.11 (F6Cd),

1

29.03 (F5Cd), 128.31 (F6Ce), 128.20 (F5Ce), 127.26 (X9Ct(m)), 127.20 (X9Ck(n)), 126.90 (X9Ck(m)),

26.40 (F6Ck+ F5Ck), 126.32 (X9Ct(n)), 126.14 (X9Cg(m)), 125.03 (X9Cg(n)), 55.83 (F5Ca(m)), 55.60

(

4

F5Ca(n)), 55.24 (F6Ca(m)), 55.10 (F6Ca(n)), 53.20 (K2Ca(m)), 52.95 (K2Ca(n) + E1Ca(mn) + K7Ca(mn)),

9.72 (X9Ca(n)), 48.29 (X8Cg), 47.19 (X9Ca(m)), 47.07 (K2Ce(m)), 45.26 (K2Ce(n)), 38.79 (X3Ce(m)), 38.69

(X3Ce(n)), 38.62 (K7Ce(mn), 36.78 (F5Cb(mn)), 36.42 (F6Cb(n)), 36.31 (F6Cb(m)), 35.95 (X8Ca), 32.55

(K2Cb(m)), 32.38 (K2Cb(n) + X3Ca(n)), 31.90 (X3Ca(m)), 31.23 (K7Cb), 31.05 (E1Cg), 30.81 (X4Ca), 30.58

(X4Cb), 28.96 (E1Cb), 28.90 (X3Cd), 28.29 (X8Cb), 28.05 (K2Cd(m)), 26.91 (K2Cd(n) + K7Cd(mn)), 26.28

Molecules 2020, 25, 5784

17 of 21

(

X3Cg(mn)), 24.76 (X3Cb(m)), 24.68 (X3Cb(n)), 22.68 (K7Cg(m)), 22.58 (K7Cg(n)), 22.53 (K2Cg(n)) 22.45

K2Cg(m)).

ESI-MS C H ClN O : m/z calcd. for [M − H ] :1161.55, found:1161.55.

+

−

56

77

12 13

+

HRMS (m/z, ESI): calcd. for C H ClN O -[M + H] 1161.5494, found: 1161,5505.

56

77

12 13

Compound 17. A solution of docetaxel (1 eq.; 500 mg; 0.619 mmol), hex-5-ynoic acid (1.1 eq.;

◦

7

1

6 mg; 0.68 mmol), and DMAP (0.1 eq.; 7 mg; 0.062 mmol) in DCM was cooled to 0 C. DIC (1.5 eq.;

◦

17 mg; 0.928 mmol)) was then added dropwise. The reaction mixture was stirred for 4 h at 0 C and

then stirred at room temperature overnight. The solvent was evaporated under reduced pressure.

The crude product was puriﬁed by chromatography ((Puriﬂash on column PF-15C18HP-F0040 (15

0g), eluent: Hex(95%)/EtOAc(5%) => Hex(0%)/EtOAc(100%) for 25 min after EtOAc (100%) for 5 min.).

µ

4

Compound 17 was obtained as a white crystalline powder (335 mg, yield 60%).

1

H-NMR (400 MHz, DMSO-d₆, δ): 7.99 (d, J = 7.2 Hz, 2H, 25 + 29), 7.89 (d, J = 8.9 Hz, 1H, NHBoc),

7.73 (t, J = 7.3 Hz, 1H, 27), 7.65 (t, J = 7.5 Hz, 2H, 26 + 28), 7.42 (t, J = 7.5 Hz, 2H, 35 + 37), 7.39–7.32

(

m, 2H, 34 + 38), 7.17 (t, J = 7.2 Hz, 1H, 36), 5.83–5.70 (m, 1H, 13), 5.40 (d, J = 6.5 Hz, 1H, 2), 5.14–5.04

m, 3H, 31 + 10 + 32), 5.02 (d, J = 7.2 Hz, 1H, 7OH), 4.93 (d, J = 2.5 Hz, 1H, 10OH), 4.92–4.87 (m, 1H, 5),

4

.45(br.s, 1H, 1OH), 4.10–3.98 (m, 3H, 7 + 20a + 20b), 3.63 (d, J = 6.2 Hz, 1H, 3), 2.83 (t, J = 2.6 Hz, 1H,

X10Hk), 2.55–2.50 (m, 2H, X10Hb), 2.32–2.25 (m, 1H, 6Hb), 2.24 (s, 3H, 22), 2.23–2.16 (m, 2H, X10Hd),

.87–1.78 (m, 1H, 14Hb), 1.77–1.71 (m, 2H, X10Hg), 1.69 (s, 3H, 18), 1.67–1.59 (m, 1H, 6Ha), 1.55–1.46

m, 4H, 19 + 14Ha), 1.38 (s, 9H, tBu), 0.98 (s, 6H, 16 + 17).

1

(

13

C-NMR (100 MHz, DMSO-d₆,

δ): 209.35 (9), 171.81 (X10Ca), 169.61 (21), 169.11 (30), 165.32 (23),

155.21 (C(O)Boc), 137.49 (33), 136.93 (11), 135.93 (12), 133.46 (27), 130.06 (24), 129.60 (25/29), 128.71

(

26/28), 128.61 (35/37), 128.10 (36), 127.46 (34/38), 83.80 (5), 83.51 (X10Ce), 80.30 (4), 78.52 (CBoc), 76.81

(1), 75.43 (20), 75.11 (31), 74.81 (2), 73.76 (10), 71.91 (X10Ck), 71.21 (13), 70.77 (7), 57.00 (8), 55.14 (32),

4

2

5.98 (3), 42.91 (15), 36.50 (6), 34.71 (14), 32.10 (X10Cb), 28.16 (tBu), 26.46 (16), 23.47 (X10Cg), 22.53 (22),

0.79 (17), 17.03 (X10Cd), 13.68 (18), 9.83 (19).

+

HRMS (m/z, ESI): calcd. for C H N O -[M + H] 902.3957, found: 902.3981.

49

59 15 13

Compound 18. Compounds 12 (1 eq.; 162 mg; 0.127 mmol) and 17 (1 eq.; 115 mg; 0.127 mmol),

CuSO ·5H O (0.4 eq.; 13 mg; 0.05 mmol) were dissolved in DMF/H O (6 mL/1 mL). After, the system

4

2

was purged with argon. To the mixture, sodium ascorbate (1.2 eq.; 30 mg; 0.152 mmol) was added in

H O (1 mL) with a syringe. The resulting solution was stirred for 24 h in an inert atmosphere. After,

2

EDTA (0.8 eq.; 30 mg; 0.1 mmol) was added. The mixture was stirred for 3 h. After the reaction,

the mixture was ﬁltered from the precipitate and the solvent was removed under reduced pressure.

The product was precipitated with MeCN and washed twice with MeCN (2 mL). After, the residue

was puriﬁed by column chromatography (Puriﬂash on a column of PF-15C18AQ-F0025 (15

µ 25g),

eluent: H O(90%)/MeCN(10%) => H O(0%)/MeCN(100%) for 20 min after MeCN (100%) for 5 min.

2

Compound 18 was obtained as a pink powder (99 mg, yield 38%).

1

H-NMR (600 MHz, DMSO-d₆, δ): 8.70-8.62 (m, F5NH(m)), 8.55-8.48 (m, F5NH(n)), 8.48–8.43

(

m, F6NH(m)), 8.43–8.37 (X3NHk(m)), 8.37–8.31 (m, F6NH(n)), 8.28–8.20 (m, X3NHk(n)), 7.98

d, J = 7.2 Hz, 2H, 25 + 29), 7.87 (d, J = 8.9 Hz, 1H, NHBoc), 7.83 (s, 1H, X10Hk), 7.76–7.68

m, 2H, 27 + K7NH(mn)), 7.68–7.57 (m, 3H, 26 + 28 + X8NH(n) + X8NH(m)), 7.40 (t, J = 7.5 Hz, 2H,

3

5 + 37), 7.38–7.34 (m, 2H, 34 + 38), 7.35–7.06 (m, 15H, Ph + Ph + X9H(mn) + 36), 6.43–6.20 (m, 2H,

K2NH(mn) + E1NH(mn)), 5.82–5.72 (m, 1H, 13), 5.39 (d, J = 6.5 Hz, 1H, 2), 5.13–5.00 (m, 3H, 31 + 10 +

2), 4.94 (br.s., 1H, OH), 4.90 (d, J = 9.4 Hz, 1H, 5), 4.57–4.47 (m, 2H, X9Ha(n) + X9Ha(m)), 4.45-4.37

m, 2H, OH + F6Ha), 4.30 (t, J = 6.9 Hz,2H, X8Hg), 4.27-4.18 (m, 1H, F5Ha), 4.18–4.10 (m, 1H, K7Ha),

3

(

4.08–3.94 (m, 5H, 7 + 20a + E1Ha + 20b + K2Ha), 3.63 (d, J = 6.2 Hz, 1H, 3), 3.22–2.95 (m, 8H, K2He(mn)

+ F6Hb(a) + X8Ha +F6Hb(b) + X3He(mn)), 2.93–2.84 (m, 1H, F5Hb(a)), 2.74 (t, J = 7.5 Hz, 2H, K7He),

2.71–2.65 (m, 1H, F5Hb(b)), 2.62 (t, J = 7.0 Hz, 2H, X10Hd), 2.45 (t, J = 7.0 Hz, 2H, X10Hb), 2.42–2.11

(

1

m, 9H, X4Hb(mn) + X4Ha(a) + X3Ha(m) + 6Hb + E1Hg + X3Ha(n) + X4Ha(b)), 2.23 (s. 3H, 22),

.99–1.90 (m, 2H, X8Hb), 1.90–1.84 (m, 2H, X10Hg), 1.84–1.69 (m, 4H, 14Hb + E1Hb(a) + E1Hb(b)

K7Hb(a)), 1.70 (s. 3H, 18), 1.68–1.58 (m, 3H, 14Ha + 6Ha + K2Hb(a)), 1.58–1.31 (m, 10H, K7Hd +

+

Molecules 2020, 25, 5784

18 of 21

K7Hb(b) + K2Hb(b) + X3Hb(m) + X3Hb(n) +X3Hd(mn) + K2Hd(mn)), 1.50 (s. 3H, 19), 1.33 (s. 9H,

tBu), 1.30–1.16 (m, 6H, K2Hg + K7Hg + X3Hg), 0.97 (s. 6H, 16 + 17).

13

C-NMR (100 MHz, DMSO-d₆,

74.76 (E1C(n)), 174.34 (E1Cd(m)), 174.25 (E1Cd(n)), 173.59 (X4Cg(mn)), 172.30 (F5C(mn)), 172.21

X3C(mn)), 172.07 (X10Ca + X3C(n)), 171.92 (X4C(m)), 171.87 (X4C(n)), 171.33 (K7C(mn)), 171.15

δ): 209.31 (9), 175.25 (K2C(m)), 175.21 (K2C(n)), 174.81 (E1C(m)),

1

(

F6C(m)), 171.10 (F6C(n)), 169.57 (21), 169.11 (30), 165.29 (23), 157.30 (U(mn)), 155.19 (C(O)Boc), 145.87

X10Ce), 141.19 (X9Cb(m)), 140.76 (X9Cb(n)), 138.10 (F6Cg(m)), 138.02 (F6Cg(n)), 137.78 (F5Cg(mn)),

137.50 (33), 136.89 (11), 135.93 (12), 133.43 (27), 133.37 (X9Ce(n)), 133.01 (X9Ce(m)), 130.59 (X9Cd(n)),

130.22 (X9Cd(m)), 130.03 (24), 129.56 (25/29), 128.97 (F6Cd), 128.89 (F5Cd), 128.68 (26/28), 128.57 (35/37),

128.23 (F6Ce), 128.11 (F5Ce), 128.06 (36), 127.41 (34/38), 127.20 (X9Ct(m)), 127.15 (X9Ck(n)), 126.82

(

X9Ck(m)), 126.36 (F6Ck), 126.30 (X9Ct(n) + F5Ck), 126.08 (X9Cg(m)), 124.99 (X9Cg(n)), 122.03 (X10Ck),

83.76 (5), 80.27 (4), 78.46 (CBoc), 76.80 (1), 75.40 (20), 75.01 (31), 74.79 (2), 73.72 (10), 71.16 (13), 70.74 (7),

56.97 (8), 55.89 (F5Ca(mn)), 55.20 F6Ca(mn)), 55.11 (32), 52.85 (K2Ca(mn) + E1Ca(mn)), 52.65 (K7Ca(mn)),

49.60 (X9Ca(n)), 47.00 (X9Ca(m)), 46.85 (X8Cg+ K2Ce(m)), 45.96 (3), 45.31 (K2Ce(n)), 42.88 (15), 38.71

(X3Ce(m)), 38.61 (X3Ce(n)), 38.50 (K7Ce(mn), 36.66 (F5Cb(mn)), 36.47 (6 + F6Cb(mn)), 35.82 (X8Ca), 34.69

(14), 32.65 (X10Cb), 32.30 (K2Cb(m)), 32.25 (K2Cb(n) + X3Ca(n)), 31.74 (X3Ca(m)), 31.09 (K7Cb), 30.92

(E1Cg), 30.70 (X4Ca), 30.48 (X4Cb), 29.81 (X8Cb), 28.66(E1Cb + X3Cd), 28.08 (tBu), 27.83 (K2Cd(m)),

26.85 (K2Cd(n) + K7Cd(mn)), 26.44 (16), 26.08 (X3Cg(mn)), 24.59 (X3Cb(m)), 24.56 (X3Cb(n)), 24.29

(

X10Cg), 24.18 (X10Cd), 22.60 (K7Cg(mn)), 22.50 (22), 22.27 (K2Cg(mn)), 20.77 (17), 13.65 (18), 9.79 (19).

+

2+

ESI-MS C₁₀₅H₁₃₆ClN O : m/z calcd. for [M + 2H ] 1031.97, found:1032.60.

13

28

2+

HRMS (m/z, ESI): calcd. for C₁₀₅H₁₃₆ClN O - [M + 2H] 1031.9726, found: 1031,9761.

13

28

Compound 19. Compound 18 (1 eq.; 14 mg; 6.43

were dissolved in DMF (2 mL). After, the system was purged with argon. To the mixture, Sulfo-Cy5

NHS-ester (1 eq.; 5 mg; 6.43 mol) was added. The mixture was stirred for 6 h. After, the solvent was

µmol) and DIPEA (8 eq.; 6.7 mg; 51.4 µmol)

µ

evaporated under reduced pressure. The product was precipitated with MeCN and washed twice with

MeCN (2 mL). After, the residue was puriﬁed by column chromatography (Puriﬂash on a column of

PF-15C18AQ-F0025 (15µ 25g), eluent: H O(90%)/MeCN(10%) => H O(0%)/MeCN(100%) for 20 min

2 2

after MeCN (100%) for 5 min. Compound 19 was obtained as a blue powder (15.8 mg, yield 92%).

1

H-NMR (600 MHz, DMSO-d₆, δ): 8.41–8.29 (m, 2H, ArSulfoCy5), 8.24–8.17 (m, 1H, ArSulfoCy5),

7

(

.97 (d, J = 7.2 Hz, 2H, 25 + 29), 7.87 (d, J = 8.9 Hz, 1H, NHBoc), 7.84 (s, 1H, X10Hk), 7.82–7.79

m, 1H, ArSulfoCy5), 7.79–7.73 (m, 1H, 27), 7.73–7.68 (m, 1H, ArSulfoCy5), 7.68–7.61 (m, 3H, 26 + 28

ArSulfoCy5), 7.40 (t, J = 7.5 Hz, 2H, 35 + 37), 7.37–7.33 (m, 2H, 34 + 38), 7.34–7.06 (m, 16H, Ph +

+

Ph + X9H(mn) + 36 + SulfoCy5(C=C)), 6.55 (t, J = 12.6 Hz, 1H, SulfoCy5(C=C)), 6.42–6.20 (m, 3H,

SulfoCy5(C=C)), 5.82–5.74 (m, 1H, 13), 5.38 (d, J = 6.5 Hz, 1H, 2), 5.12–5.00 (m, 3H, 31 + 10 + 32), 4.95

(

br.s., 1H, OH), 4.89 (d, J = 9.4 Hz, 1H, 5), 4.57–4.44 (m, 2H, X9Ha(n) + X9Ha(m)), 4.45–4.37 (m, 2H, OH

+

F6Ha), 4.35–4.23 (m, 3H, X8Hg + F5Ha), 4.17–3.94 (m, 6H, K7Ha + 7+20a + E1Ha + 20b + K2Ha),

0

3.62 (d, J = 6.2 Hz, 1H, 3), 3.57 (s, 3H, 28 ), 3.36 (br.s, 1H, OH), 3.53–2.95 (m, 12H, 6 + K2He(mn) +

F6Hb(a) + X8Ha+ K7He + F6Hb(b) + X3He(mn)), 2.93–2.84 (m, 1H, F5H(a)), 2.69–2.63 (m, 1H, F5Hb(b)),

2

.61 (t, J = 7.0 Hz, 2H, X10Hd), 2.46–2.40 (m, 2H, X10Hb), 2.37–2.11 (m, 9H, X4Hb(mn) + X4Ha(a) +

0

X3Ha(m) + 6Hb + E1Hg + X3Ha(n) +X4Ha(b)), 2.22 (s. 3H, 22), 2.01 (t, J = 7.0 Hz, 2H, 2 ), 1.96–1.90

(

1

m, 2H, X8Hb), 1.90–1.84 (m, 2H, X10Hg), 1.84–1.70 (m, 4H, 14Hb + E1Hb(a) + E1Hb(b) + K7Hb(a)),

0 0 0 0

.70–1.62 (m. 15H, 18 + 29 + 30 + 31 + 32 ), 1.68–1.58 (m, 3H, 14Ha + 6Ha + K2Hb(a)), 1.58–1.31

(

1

m, 10H, K7Hd + K7Hb(b) + K2Hb(b) + X3Hb(m) + X3Hb(n) +X3Hd(mn) + K2Hd(mn)), 1.49 (s. 3H,

0 0 0

9), 1.32 (s. 9H, tBu), 1.30–1.16 (m, 12H, 5 + 3 + 4 + K2Hg + K7Hg +X3Hg), 0.97 (s. 6H, 16 + 17).

+

2+

ESI-MS C₁₃₇H₁₇₂ClN O S : m/z calcd. for [M + 2H ] : 1344.57, found: 1345.05.

15

35 2

+

2+

HRMS (m/z, ESI): calcd. for C₁₃₇H₁₇₂ClN O S -[M + 2H ] 1344.5725, found: 1344.5768.

15

35 2

4

. Conclusions

Herein, we designed and synthesized a new PSMA-targeting, DCL-based molecular platform 12

for bimodal or theranostic agent delivery to prostate cancer cells. Its conjugate 19 with docetaxel and

Molecules 2020, 25, 5784

19 of 21

ﬂuorescent label Sulfo-Cy5 was also synthesized, demonstrating the possibility to stepwise conjugate

the proposed vector molecule with two diﬀerent functional moieties in orthogonal chemical conditions.

Two alternative methods to obtain polypeptide-based compound 12 using liquid- and solid-phase

techniques, including 13 to 16 sequential stages, were compared. The optimal method for stereoselective

synthesis of molecular platform 12 consists in solid-phase synthesis of a peptide sequence of the linker,

coupling of a polypeptide to a DCL vector fragment, subsequent attachment of 3-aminopropylazide

under optimized conditions, and ﬁnal removal of the protective groups.

The obtained compounds were characterized by NMR spectroscopy and high-resolution mass

spectrometry; complete assignment of signals in the NMR spectra of the compounds 12 and 18 was

made using two-dimensional NMR sequences. The reasonable cytotoxicity of vector molecule 12

,

its conjugate with docetaxel 18, and docetaxel/Sulfo-Cy5 19 against PSMA-expressing cell lines were

found during initial in vitro study as well as their selective interaction with cells. However, further

in vitro as far as in vivo investigations of the conjugates are required for a more explicit demonstration

of their eﬃcacy and selectivity for PSMA-expressing cells and tumors. Anyhow, conjugate 19 can be

used as a convenient starting point appropriate for the follow-up structure optimization study.

1

Supplementary Materials: The following are available online. Figure S1: H-NMR spectrum of compound

1

3

1

6

in DMSO-d ; Figure S2: C-NMR spectrum of compound

6

in DMSO-d ; Figure S3: H-NMR spectrum

6

1

of compound 7 in CDCl ; Figure S4: H-NMR spectrum of compound 10 in DMSO-d ; Figure S5: H-NMR

spectrum of compound 16 in DMSO-d ; Figure S6: C NMR spectrum of compound 16 in DMSO-d ; Figure S7:

H-NMR spectrum of compound 11 in CDCl₃_.Liquid-phase technique. Method 2; Figure S8: H-NMR spectrum

of compound 11 in DMSO-d . SPPS technique. Method 1: Figure S9: H-NMR spectrum of compound 12 in

DMSO-d . Liquid-phase technique; Figure S10: C NMR spectrum of compound 12 in DMSO-d . Liquid-phase

technique: Figure S11: H-NMR spectrum of compound 12 in DMSO-d . SPPS technique: Figure S12: C-NMR

spectrum of compound 12 in DMSO-d . SPPS technique; Figure S13: H-NMR spectrum of compound 17 in

DMSO-d ; Figure S14: C-NMR spectrum of compound 17 in DMSO-d ; Figure S15: HSQC H- C spectrum of

3

6

1

3

6

1

6

1

3

6

1

13

6

1

6

13

1

13

6

1

13

compound 17 in DMSO-d , T = 296 K; Figure S16: HMBC H- C spectrum of compound 17 in DMSO-d , T = 296 K;

Figure S17: H-NMR spectrum of compound 18 in DMSO-d ; Figure S18: C-NMR spectrum of compound 18

in DMSO-d ; Figure S19: HSQC H- C spectrum of compound 18 in DMSO-d , T = 296 K; Figure S20: HMBC

6

1

13

6

1

13

6

1

13

1

H- C spectrum of compound 18 in DMSO-d , T = 296 K; Figure S21: H-NMR spectrum of compound 19 in

6

13

1

13

1

DMSO-d ; Table S1: HSQC (

(

C

∆δ/ H∆δ (ppm/ppm)) of 17 in DMSO-d , T = 296 K; Table S2: HMBC (

C

∆δ/ H∆δ

ppm/ppm)) of 17 in DMSO-d , T = 296 K; Table S3: HSQC ( C∆δ/ H∆δ (ppm/ppm)) of 18 in DMSO-d , T = 296 K;

6

1

3

1

6

13

1

Table S4: HMBC ( C∆δ/ H∆δ (ppm/ppm)) of 18 in DMSO-d , T = 296 K.

6

Author Contributions: Conceptualization, E.K.B., A.E.M., and A.G.M.; methodology, S.A.P. and A.E.M.; validation,

E.K.B., S.A.P., and A.E.M.; formal analysis, R.A.P., Y.K.G., V.A.R., V.I.P., and A.S.G.; investigation, S.A.P. and

N.Y.Z.; data curation, R.A.P., Y.K.G., V.A.R., V.I.P., and A.S.G.; writing—original draft preparation, E.A.N., S.A.P.,

and A.S.G.; writing—review and editing, E.K.B., A.E.M., S.A.P., and A.S.G.; visualization, A.A.U.; supervision,

N.V.Z., E.K.B., and A.G.M.; project administration, E.K.B.; funding acquisition, E.K.B., S.A.P., A.E.M., and V.I.P.

All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by RFBR, grant number 19-33-90145 (Full scope of liquid phase and solid phase

synthesis of tripeptide sequences, study of cytotoxicity). This research was also funded by RFBR, grant number

2

0-33-70089 (cell staining investigations). NMR studies were supported by the Russian Science Foundation, grant

number 19-14-00115.

Acknowledgments: Authors are grateful to the Moscow State University (Russia) for the opportunity to use the

NMR facilities and to Oleg Saveliev for the expert technical assistance in the NMR measurements.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Polypeptide-based molecular platform and its docetaxel/sulfo-cy5-containing conjugate for targeted delivery to prostate specific membrane antigen

DOI: 10.3390/molecules25245784

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