5′-trityl-substituted thymidine derivatives as a novel class of antileishmanial agents: Leishmania infantum EndoG as a potential target

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DOI: 10.1002/cmdc.201300129

5’-Trityl-Substituted Thymidine Derivatives as a Novel

Class of Antileishmanial Agents: Leishmania infantum

EndoG as a Potential Target

Elena Casanova,^[a]David Moreno,^[b]Alba Gigante,^[a]Eva Rico,^[b]Carlos Mario Genes,^[b]

Cristina Oliva,^[b]Marꢀa-Josꢁ Camarasa,^[a]Federico Gago,^[c]Antonio Jimꢁnez-Ruiz,^[b]and Marꢀa-

Jesﬄs Pꢁrez-Pꢁrez*^[a]

Two series of 5’-triphenylmethyl (trityl)-substituted thymidine

derivatives were synthesized and tested against Leishmania in-

fantum axenic promastigotes and amastigotes. Several of these

compounds show significant antileishmanial activity, with IC₅₀

values in the low micromolar range. Among these, 3’-O-(isoleu-

cylisoleucyl)-5’-O-(3,3,3-triphenylpropanoyl)thymidine displays

particularly good activity against intracellular amastigotes.

Assays performed to characterize the nature of parasite cell

death in the presence of the tritylthymidines indicated signifi-

cant alterations in mitochondrial transmembrane potential, an

increase in superoxide concentrations, and also significant de-

creases in DNA degradation during the cell death process. Re-

sults point to the mitochondrial nuclease LiEndoG as a target

for the action of this family of compounds.

Introduction

Leishmaniasis, one of the most prevalent neglected diseases, is

a major health problem in tropical and subtropical countries,

with more than 12 million people affected and about 300 mil-

lion persons at risk of infection. The manifestation of this dis-

ease can be cutaneous, diffuse cutaneous, mucocutaneous, or

visceral.^[1]It is transmitted to humans by the bite of an infected

insect vector, the female Phlebotominae sand fly. The Leishma-

nia parasite is present in two different forms: the promastigote

form in the vector, and the amastigote form in the mammalian

host.

ment of resistance, which is a serious problem common to

many of these drugs.^[4]Therefore, there is an urgent need for

the development of new antileishmanial agents based on mo-

lecular scaffolds distinct from those explored thus far. It can be

speculated that by exploring different molecular frameworks,

we may obtain bioactive compounds with a mechanism of

action different from those of drugs in current use.

Focusing on nucleoside metabolism, Leishmania species lack

the metabolic pathway for de novo synthesis of purine nucleo-

sides. Therefore, salvage of purines is an obligatory pathway

and has been the focus of considerable interest.^[5]In contrast,

the situation is quite different for pyrimidines, the metabolism

of which has generally been considered less susceptible to

therapeutics than the pathway of purine salvage.^[6]Indeed, to

the best of our knowledge, there are very few examples of pyr-

imidine nucleosides such as 5-substituted pyrimidine nucleo-

sides^[7–10]with reported antileishmanial activity in vitro. None-

theless, it is reasonable to expect that improved knowledge

about the enzymes involved in pyrimidine biosynthesis may

afford new targets. In this respect, a thymidine kinase in

L. major was recently identified and shown to be involved in

flagellum formation, promastigote shape and growth, and viru-

lence.^[11]Another interesting example comes from the search

for inhibitors of the parasitic dUTPase, in which 5’-O-trityl-3’-O-

TBDMSi-2’-deoxyuridine was identified as an inhibitor of L. do-

novani replication, with an IC₅₀value of 17 mm. However, fur-

ther careful research revealed that this activity could not be as-

cribed to the inhibition of the related L. major dUTPase, as the

compound was virtually inactive against the isolated enzyme,

indirectly suggesting the involvement of a yet unidentified

molecular target.^[12]

Unfortunately, the arsenal of available drugs to combat the

various forms of leishmaniasis is very limited. Pentavalent anti-

monials, pentamidine, amphotericin B, and miltefosine are cur-

rently the most effective compounds against this family of dis-

eases.^[2,3]However, treatment with most of these is usually ac-

companied by significant side effects. These include severe

acute pancreatitis and cardiac arrhythmia for the antimonials;

nephrotoxicity or even anaphylaxis for amphotericin B; renal,

pancreatic, and hepatic toxicities for pentamidine; and an ex-

tremely long half-life for miltefosine that favors the develop-

[a] Dr. E. Casanova, A. Gigante, Prof. M.-J. Camarasa, Prof. M.-J. Pꢀrez-Pꢀrez

Instituto de Quꢁmica Mꢀdica (IQM-CSIC)

Juan de la Cierva 3, 28006, Madrid (Spain)

E-mail: mjperez@iqm.csic.es

[b] Dr. D. Moreno, Dr. E. Rico, C. M. Genes, C. Oliva, Prof. A. Jimꢀnez-Ruiz

Departamento de Bioquꢁmica y Biologꢁa Molecular

Unidad Asociada de I+D+i al CSIC

Campus Universitario, Universidad de Alcalꢂ

Alcalꢂ de Henares, 28871 Madrid (Spain)

[c] Prof. F. Gago

Departamento de Farmacologꢁa

Unidad Asociada de I+D+i al CSIC, Campus Universitario

Universidad de Alcalꢂ, Alcalꢂ de Henares (Spain)

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Based on these findings, we decided to evaluate our own

collection of 5’-trimethylphenyl (trityl)-substituted pyrimidine

nucleosides and analogues against L. infantum promastigotes.

We found that 5’-tritylthymidine derivatives with a valine ester

at the 3’-position (compounds 1 and 2, Figure 1) were able to

efficiently inhibit promastigote replication at a concentration

of 12.5 mm. Interestingly, the corresponding 3’-hydroxy ana-

logues 3 and 4 (Figure 1) were virtually inactive.

Scheme 1. Reagents and conditions: a) triphenylpropionic acid (2.0 equiv),

PPh₃(2.0 equiv), DBAD (2.0 equiv), DMF, 08C, 1.5 h, 88%; b) 1. Boc-Xaa-OH

(2.0 equiv), PyBOP (1.2 equiv), Et₃N (2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA

(23 equiv), CH₂Cl₂, RT, 3–5 h (1, 63%; 6, 63%; 7, 52%; 8, 62%; 9, 52%).

Me₂tBu)-OH. After coupling, treatment with trifluoroacetic acid

(TFA) removed the Boc protecting group together with the Si-

Me₂tBu group in the side chain of the Ser derivative to afford

the NH₂-free compounds 1 and 6–9. Based on the results ob-

tained and synthetic accessibility, the amino acids of choice for

the next series of compounds were l-Val and l-Ile.

Our next aim was to modify the linker at the 5’-position con-

necting the nucleoside and the trityl substituent by replace-

ment of the ester group at the 5’-position by the correspond-

ing amide. This required the synthesis of 5’-amino-5’-deoxythy-

midine as the key intermediate that had been described by Tel-

zlaff et al.^[13]Thus, treatment of thymidine with tetrachloroph-

thalimide under Mitsunobu conditions, as described, afforded

tetrachlorophthalimido compound 10 (Scheme 2). However,

treatment of 10 with hydrazine hydrate to obtain 5’-amino-5’-

deoxythymidine gave, in our hands, a compound difficult to

isolate and handle. We therefore decided to introduce a pro-

tecting group at the 3’-position of 10 prior to its transforma-

tion into the amino derivative.

Figure 1. 5’-Trityl nucleosides initially tested against L. infantum promasti-

gotes.

The work described herein includes the synthesis and bio-

logical evaluation of various analogues of our prototype com-

pounds 1 and 2 by varying the nature of the amino acid at the

3’-position, by modifying the nexus at the 5’-position connect-

ing the nucleoside and the trityl substituent, by introducing

subtle changes at the base moiety, and by elongating the sub-

stituent at the 3’-position either by preparing dipeptidyl deriv-

atives or by including a spacer between the 3’-hydroxy group

and the amino acid residue. The observed changes in mito-

chondrial activity, together with the surprisingly low percent-

age of hypoploid cells in the population of treated parasites,

led us to the identification of L. infantum endonuclease G

(LiEndoG), a mitochondrial nuclease, as a target for these mole-

cules.

Thus, compound 10 was allowed to react with tBuMe₂SiCl in

the presence of imidazole to provide the 3’-protected nucleo-

side 11 (Scheme 2). Reaction of 11 with hydrazine hydrate led

to the 5’-amino derivative 12 in 96% yield. This amino nucleo-

side was subjected to a BOP-mediated coupling with 3,3,3-tri-

phenylpropionic acid to afford amide 13 in excellent yield. Re-

moval of the 3’-O-silyl protecting group (compound 14) fol-

lowed by coupling of the amino acids at the 3’-position and

Boc deprotection afforded the desired nucleosides 15 and 16,

with Val and Ile residues at the 3’-position, respectively

(Scheme 2).

Results and Discussion

Chemistry

The synthesis of prototype compound 1 and 3’-O-aminoacyl

analogues 6–9 is illustrated in Scheme 1. Reaction of thymidine

5 with 3,3,3-triphenylpropionic acid under Mitsunobu condi-

tions (Ph₃P and DBAB) effectively afforded the 5’-substituted

compound 3 in 88% yield. The coupling of 3 with various

amino acids protected as their corresponding Boc derivatives

was performed in the presence of PyBOP and triethylamine.

The amino acid derivatives incorporated were Boc-Val-OH, Boc-

Ile-OH, Boc-cHexGly-OH, Boc-d-Val-OH, and Boc-Ser(OSi-

Our next goal was to evaluate how subtle modifications at

the pyrimidine base of the nucleoside affects their activity

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Scheme 4. Reagents and conditions: a) diphenylpropionic acid or 3,3,3-tris(4-

chlorophenyl)propionic acid (2.0 equiv), PPh₃(2.0 equiv), DBAD (2.0 equiv),

DMF, 08C, 1.5 h (22, 51%; 23, 61%); b) 1. Boc-Ile-OH or Boc-Val-OH

(2.0 equiv), PyBOP (1.2 equiv), Et₃N (2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA

(23 equiv), CH₂Cl₂, RT, 3–5 h (24, 58%; 25, 55%; 26, 66%).

Scheme 2. Reagents and conditions: a) TBDMSCl (1.5 equiv), imidazole

(3.0 equiv), DMF, RT, overnight, 77%; b) H₂NNH₂H₂O (3.0 equiv), EtOH, reflux,

1 h, 96%; c) triphenylpropionic acid (1.5 equiv), BOP (1.5 equiv), Et₃N

(1.5 equiv), CH₂Cl₂, RT, overnight 95%; d) 1n HCl, THF, RT, overnight, 93%;

e) 1. Boc-Val-OH or Boc-Ile-OH (2.0 equiv), PyBOP (1.2 equiv), Et₃N

(2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA (23 equiv), CH₂Cl₂, RT, 3–5 h (15,

60%; 16, 70%).

tion at 1008C for 1 h led to the 3-N-methyl analogue 19 in

85% yield. Both nucleosides 18 and 19 were subjected to

PyBOP-mediated coupling with Boc-Ile-OH followed by treat-

ment with TFA to yield the respective Ile derivatives 20 and

21.

We also modified the ester group at the 5’-position of the

nucleoside by replacing the triphenylpropanoyl group with

structurally related analogues. For this purpose, we selected

two commercial acids: 3,3-diphenylpropionic acid and 3,3,3-

tris(4-chlorophenyl)propionic acid. Thus, reaction of thymidine

5 with the above-mentioned acids under Mitsunobu conditions

afforded the 5’-substituted thymidine derivatives 22 and 23 in

51 and 61% yields, respectively (Scheme 4). Reaction

of 22 with Boc-Ile-OH followed by treatment with

TFA yielded 24 (58% yield). Similarly, reaction of 23

with Boc-Val-OH and Boc-Ile-OH, followed by Boc re-

moval, led to the target compounds 25 and 26 in 55

and 66% respective yields.

against in vitro parasite growth. Reaction of 2’-deoxyuridine

(17, Scheme 3) with 3,3,3-triphenylpropionic acid under Mitsu-

nobu conditions afforded the 5’-substituted nucleoside 18 in

70% yield. Alternatively, reaction of the 5’-substituted thymi-

dine derivative 3 with methyl iodide in acetone in the pres-

ence of potassium carbonate under microwave (MW) irradia-

We next focused our interest on the 3’-position of

the nucleoside. Starting from the 3’-O-aminoacyl de-

rivatives 1 and 6, we decided to incorporate an addi-

tional amino acid to prepare dipeptidyl derivatives.

Thus, reaction of 1 with Boc-Ile-OH and treatment

with TFA afforded the dipeptidyl nucleoside 27

(Scheme 5). Similarly, reaction of the isoleucyl deriva-

tive 6 with Boc-Ile-OH or Boc-Val-OH, followed by

Boc removal, yielded the Ile-Ile compound 28 and

the Val-Ile analogue 29.

A second series of modifications at the 3’-position

of the prototype compound 1 consisted of introduc-

ing a spacer between the 3’-hydroxy group of the

nucleoside and the amino acid derivative. The pro-

posed spacer incorporated a succinate and an ethyl-

ene glycol, as recently employed in the coupling of

amino acids to the antiviral cidofovir.^[14]Thus, reac-

tion of 3 with succinic anhydride in pyridine in the

presence of DMAP afforded the hemisuccinate 30 in

Scheme 3. Reagents and conditions: a) triphenylpropionic acid (2.0 equiv), PPh₃

(2.0 equiv), DBAD (2.0 equiv), DMF, 08C, 1.5 h, 70%; b) MeI (4.0 equiv), K₂CO₃(0.5 equiv),

acetone, 1008C, 1 h, MW, 85%; c) 1. Boc-Ile-OH (2.0 equiv), PyBOP (1.2 equiv), Et₃N

(2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA (23 equiv), CH₂Cl₂, RT, 3–5 h (20, 63%; 21,

80%).

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Scheme 5. Reagents and conditions: a) 1. Boc-Ile-OH or Boc-Val-OH

(2.0 equiv), PyBOP (1.2 equiv), Et₃N (2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA

(23 equiv), CH₂Cl₂, RT, 3–5 h (27, 54%; 28, 50%; 29, 82%).

82% yield (Scheme 6). Further reaction of 30 with ethylene

glycol using PyBOP as the condensation agent afforded alcohol

31 (53% yield). This alcohol was treated with Boc-Val-OH or

Boc-Ile-OH in the presence of PyBOP, followed by removal of

the Boc protecting group. In this way, conjugates 32 and 33

were obtained (36 and 35% respective yields for the two

steps).

The trityl ether 2 had also been identified as an interesting

hit in our initial screen of L. infantum promastigote replication

inhibitors. Based on the significant structural similarity be-

tween ester 1 and ether 2, in the trityl ether series we per-

formed only those modifications that led to the most success-

ful compounds in the ester series. Condensation of 5’-O-tri-

tylthymidine^[15](4, Scheme 7) with Fmoc-Val-OH or Fmoc-Ile-

OH was performed in the presence of PyBOP, followed by re-

moval of the Fmoc protecting group with piperidine. In this

way the 3’-O-Val 2 and 3’-O-Ile derivatives 34 were obtained

(74 and 65% yield, respectively). On the other hand, reaction

of 5’-O-tritylthymidine (4) with succinic anhydride in dichloro-

methane in the presence of triethylamine and DMAP afforded

Scheme 7. Reagents and conditions: a) 1. Fmoc-Val-OH or Fmoc-Ile-OH

(2.0 equiv), PyBOP (1.2 equiv), Et₃N (2.5 equiv), CH₂Cl₂, 08C, overnight; 2. pi-

peridine, CH₂Cl₂, RT, 1 h (2, 74%; 34, 65%); b) succinic anhydride (2.0 equiv),

DMAP (1.0 equiv), pyridine, RT, 7 h (37, 96%; 38, 71%).

the hemisuccinate 35 in 82% yield. BOP-mediated condensa-

tion with ethylene glycol gave alcohol 36 (92% yield), which

was coupled to Fmoc-Val-OH or Fmoc-Ile-OH, followed by pi-

peridine treatment to afford conjugates 37 and 38 (96 and

71% respective yields).

Biological evaluation

The synthesized compounds were initially screened

against the promastigote form of L. infantum. Those

compounds showing significant activity in this assay

(IC₅₀<25 mm) were also tested against amastigotes

and, to measure their cytotoxicity against human cell

lines, proliferating Jurkat cells. The IC₅₀values ob-

tained are listed in Table 1. Miltefosine was included

as a reference compound.

In the trityl ester series and concerning the evalua-

tion of the 3’-O-aminoacyl derivatives 1 and 6–9

against promastigotes, it is clear that the presence of

an amino acid is required for effective inhibition.

Moreover, the nature of the amino acid side chain

has a clear impact on the antileishmanial effect.

Whereas the l-Ser derivative 9 was virtually inactive

(IC₅₀>25 mm), moderate activity (5 mm<IC₅₀<10 mm)

was observed for l- and d-Val derivatives 1 and 8,

and good activity (IC₅₀<5 mm) was shown when l-Ile

and l-cHexGly were included at the 3’ position (com-

Scheme 6. Reagents and conditions: a) succinic anhydride (2.0 equiv), DMAP (1.0 equiv),

pyridine, RT, overnight (82%); b) ethylene glycol (10.0 equiv), PyBOP (1.3 equiv), Et₃N

(1.3 equiv), CH₂Cl₂, RT, overnight (53%); c) 1. Boc-Val-OH or Boc-Ile-OH (2.0 equiv), PyBOP

(1.2 equiv), Et₃N (2.5 equiv), CH₂Cl₂, 08C, overnight; 2. TFA (23 equiv), CH₂Cl₂, RT, 3–5 h

(32, 36%; 33, 35%).

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cant number of them show IC₅₀values in the low micromolar

range (<10 mm) against the amastigote form of the parasite.

Among the most active compounds are Ile derivative 6,

cHexGly derivative 7, 5’-tris(4-chlorophenyl)propanoyl com-

pounds with Val or Ile at the 3’-position (25 and 26, respective-

ly), those with dipeptides coupled at the 3’-position (com-

pound 28), and the succinate-glycol-Ile derivative 33.

Concerning the trityl ether derivatives 2, 4, 34, 37, and 38,

all of the 3’-substituted compounds were active against L. in-

fantum promastigotes, with IC₅₀values <5 mm. Except for com-

pound 2 (IC₅₀ꢀ10 mm), all of them also showed significant in-

hibitory activity against amastigotes, with IC₅₀values in the

5 mm range. Also in this case, IC₅₀values against Jurkat cells

were three- to fourfold higher than against the parasites.

Leishmanicidal activity was also assayed in amastigote-in-

fected phorbol myristate acetate (PMA)-differentiated THP-

1 cells by flow cytometry.^[16]Seven compounds were selected

on the basis of their high activity against axenic parasites and

moderate toxicity in Jurkat cells. Accordingly, compounds 2, 6,

28, 32, 33, 37, and 38 were assayed over the course of 48 h in

differentiated THP-1 cells infected with L. infantum amastigotes

expressing green-fluorescent protein (eGFP); infected cells

were identified by the green fluorescence emitted by their in-

tracellular amastigotes (Figure 2).

Table 1. In vitro activity of the synthesized trityl esters, amides, and

ethers against the promastigote form of L. infantum, the amastigote form

of L. infantum, and Jurkat cells.^[a]

IC₅₀[mm]

Compd

Promastigote

Amastigote

Jurkat

1

2

3

7.67ꢁ0.21

2.88ꢁ0.13

>25

10.63ꢁ0.44

ND

16.9ꢁ0.5

17.1ꢁ2.2

ND

4

>25

ND

6

7

8

9

3.4ꢁ0.6

7.33ꢁ0.04

6.33ꢁ0.01

22.60ꢁ1.52

ND

9.87ꢁ1.8

13.9ꢁ0.7

15.3ꢁ0.4

ND

3.5ꢁ0.6

6.51ꢁ0.03

>25

15

>25

ND

16

>25

ND

20

21

24

25

26

27

28

29

32

33

34

37

7.31ꢁ3.22

5.87ꢁ0.75

17.29ꢁ1.64

2.59ꢁ0.24

6.56ꢁ0.05

6.67ꢁ0.23

1.31ꢁ0.09

5.99ꢁ0.33

6.38ꢁ1.17

3.78ꢁ0.14

2.22ꢁ0.09

4.33ꢁ0.05

4.51ꢁ0.65

46.45ꢁ3.03

12.76ꢁ0.25

9.68ꢁ0.36

>25

9.3ꢁ0.3

10.6ꢁ0.9

22.4ꢁ1

6.83ꢁ0.1

9.9ꢁ0.3

16.2ꢁ1.1

15.9ꢁ0.6

8.28ꢁ1.0

24.9ꢁ2.3

20.9ꢁ1.2

7.0ꢁ0.9

17.2ꢁ0.9

15.6ꢁ1.5

48

4.49ꢁ0.52

7.07ꢁ0.02

22.33ꢁ0.31

11.75ꢁ0.74

13.42ꢁ0.14

14.52ꢁ1.40

8.65ꢁ0.51

6.11ꢁ0.01

6.44ꢁ0.01

7.37ꢁ0.02

3.96ꢁ0.53

38

miltefosine

[a] Values are expressed as the mean ꢁSEM of three independent experi-

ments; ND: not determined.

pounds 6 and 7, respectively). Therefore, it can be concluded

that a hydrophobic side chain of the amino acid is the pre-

ferred substituent for this position. Compounds 6 and 7 main-

tained good activity when assayed against amastigotes.

Interestingly, the antiparasitic effect was significantly de-

creased by changing the linker between the trityl substituent

and the 5’-position of the nucleoside from an ester to an

amide (compare esters 1 and 6 with the corresponding amides

15 and 16). On the other hand, subtle changes in the pyrimi-

dine base (while maintaining l-Ile at the 3’-position) decreased

activity against promastigotes, as evidenced by the fact that

both the uracil (20) and 3-N-methylthymine (21) derivatives

showed IC₅₀values >5 mm. The IC₅₀value increased significant-

ly if the triphenylmethyl ester was replaced by the correspond-

ing diphenylmethyl derivative (compound 24). On the other

hand, the 5’-tris(4-chlorophenyl)propanoyl compounds with

either Val or Ile at the 3’-position (25 and 26, respectively)

maintained IC₅₀values close to 5 mm in promastigotes, show-

ing a strikingly similar activity in amastigotes. IC₅₀values

against Jurkat cells are twofold higher than against the para-

sites. Moving back to the triphenylpropanoyl compounds, cou-

pling of dipeptides at the 3’-position (27–29), or incorporation

of a succinate-glycol spacer between the nucleoside and the

amino acid (32 and 33) caused minimal variations in IC₅₀

values relative to the single amino acid derivatives 1 and 6,

except that toxicity toward Jurkat cells was notably decreased

for most of them. In general, the compounds are less active

against amastigotes than against promastigotes, yet a signifi-

Figure 2. Inhibition of intracellular infection rates: PMA-differentiated THP-

1 cells were infected with amastigotes expressing green-fluorescent protein

(eGFP). Percentages of infection were evaluated by counting the number of

green-fluorescent PMA-differentiated THP-1 cells by flow cytometry. All com-

pounds were assayed at 15 mm. Edelfosine (Edel.) was assayed at 3 mm. Error

bars represent standard deviation; results are representative of three inde-

pendent experiments.

Most of the compounds assayed were able to decrease the

number of infected cells by approximately 20–40%, but only

compound 28 showed a leishmanicidal activity against intra-

cellular parasites similar to that observed for the control drug

edelfosine (87% decrease in the number of infected macro-

phages), with an estimated IC₅₀value of 8.0ꢁ0.15 mm. Accord-

ing to these results, compound 28 was selected for the study

of death-related processes in the parasites. Changes in the mi-

tochondrial transmembrane potential (DY_m) of logarithmically

growing L. infantum promastigotes were measured with the

potentiometric probe TMRM.^[17]The results presented in Fig-

ure 3a indicate a rapid but transient increase in the mean

DY_mwhen the parasites were incubated with 28 at 10 mm, as

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shown by the significant increase in the TMRM-derived fluores-

cence observed during the first 20 min of treatment. The mean

values of fluorescence are restored to that of the control para-

sites after 80 min exposure to the compound. The mitochon-

drial proton gradient uncoupler carbonyl cyanide m-chloro-

phenyl hydrazone (CCCP, 100 mm) was added as a positive con-

trol of mitochondrial depolarization.

mycin-treated cells, is indicative of an accumulation of O2^·ꢂ

inside the mitochondrion (Figure 3b). Accordingly, compound

28 induces changes in the activity of the single mitochondrion

of the parasites, causing its sudden hyperpolarization and

a concomitant increase in superoxide production that eventu-

ally leads to cell death.

Figure 4a shows bi-parametric plots of forward scatter (Fs)

and propidium iodide (PI) staining of control, staurosporine-,

and 28-treated parasites. The lower right quadrant (C4) repre-

sents healthy parasites displaying no alterations in shape or

plasma membrane integrity, which constitute 96% of the pop-

ulation of the untreated control parasites. This number is de-

creased to 54 and 56% as a consequence of 24 h treatment

with staurosporine (17 mm ) or 28 (10 mm), respectively. Events

in any of the other three quadrants are representative of para-

sites that are either dead or in the process of dying. DNA con-

tent in the treated parasites was analyzed by flow cytometry.

Surprisingly, although staurosporine and compound 28 at the

indicated concentrations cause a similar deleterious effect in

the population (Figure 4a), the process of DNA degradation as-

sociated with cell death is much lower in 28-treated parasites

(19% of hypoploid cells) than in staurosporine-treated para-

sites (39% of hypoploid cells) (Figure 4b). The decreased per-

centage of hypoploid parasites after treatment with 28, to-

Changes in mitochondrial transmembrane potential are fre-

C^ꢂ

quently associated with variations in superoxide (O2 ) produc-

tion that can be detected by flow cytometry with specific

probes such as MitoSOX^TM. The increase in MitoSOX-derived

fluorescence over the first 20 min of treatment with compound

28, similar to that observed during the same period in the anti-

C^ꢂ

gether with its effect on DY_mand O2 production, led us to

postulate that the target for this compound could be a mito-

chondrial protein that mediates nuclear DNA degradation

during the cell-death process. The recently characterized LiEn-

doG protein fulfills both requisites.^[18]

Accordingly, the nuclease activity of this enzyme in the pres-

ence of increasing concentrations of 28 was evaluated in a fluo-

rimetric assay. Purified recombinant LiEndoG was incubated

with a double-stranded DNA probe (16 nucleotides long) that

contains 6-carboxyfluorescein (FAM) attached to both 3’ ends

and tetramethylrhodamine (TAMRA) bound to both 5’ ends. In

the undigested probe, FAM-derived fluorescence is quenched

by the proximity of TAMRA. Digestion of any of the two oligo-

nucleotides causes separation of the fluorophore from the

quencher, so that a fluorescent signal can then be emitted.

Fluorescence generated as a consequence of the nuclease ac-

tivity of LiEndoG clearly diminishes in the presence of increas-

ing concentrations of compound 28 (Figure 5a). This inhibitory

effect on LiEndoG was also assayed for compounds 3, 4, 6, and

25. Reactions in the presence of compounds 3 and 4 at 25 mm

were very slightly inhibited relative to the control reaction,

which indicates that the substituent at the 3’ position is impor-

tant for LiEndoG inhibitory activity. Compound 6 showed mod-

erate inhibitory activity, whereas compounds 25 and 28 dis-

played high capacity for LiEndoG inhibition. Thus, the ability of

the compounds to inhibit LiEndoG activity strongly correlates

with their leishmanicidal capacity, and the presence of sub-

stituents at the 3’-position is essential for both activities.

Figure 3. a) Analysis of mitochondrial transmembrane potential (DY_m):

TMRM-derived fluorescence in L. infantum promastigotes treated with com-

pound 28 (10 mm) or with the uncoupler CCCP (100 mm). Y-axis values repre-

sent the mean fluorescence (F) of the entire population of living cells. Para-

sites treated with an equivalent concentration of DMSO used as solvent are

included as a control (DMSO). Fluorescence was analyzed by flow cytometry

(n=3). Samples were collected and analyzed at 20, 50, 80, and 110 min of

treatment. Error bars represent standard deviation; results are representative

of three independent experiments. b) Analysis of intra-mitochondrial super-

oxide concentration: MitoSOX-derived fluorescence in L. infantum promasti-

gotes treated with compound 28 (10 mm) or with antimycin A (20 mm). Y-axis

values represent the mean fluorescence (F) of the entire population of living

cells. Parasites treated with an equivalent concentration of DMSO used as

solvent are included as a control (DMSO). Fluorescence was analyzed by

flow cytometry (n=3). Samples were collected and analyzed at 20, 50, and

90 min of treatment. Error bars represent standard deviation; results are rep-

resentative of three independent experiments. The mean value of the treat-

ments differ from that of the DMSO control with p values <0.05 (**) or

0.005 (***).

Based on the ability of compound 28 to inhibit LiEndoG ac-

tivity and also to decrease intracellular infection rates without

causing severe cell damage, we decided to gauge the evolu-

tion of this compound in PMA-differentiated THP-1 cells. Thus

a sample of 28 was incubated with in cell-free extract of these

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Conclusions

Two series of 5’-trityl-substitut-

ed thymidine derivatives were

synthesized and assayed against

L. infantum promastigotes and

amastigotes.

A

significant

number of these compounds

showed pronounced inhibitory

activity against L. infantum pro-

mastigotes, and had slightly

higher IC₅₀values against amas-

tigotes. Because of its good ac-

tivity against intracellular amas-

tigotes, compound 28 was se-

lected for analysis of its cellular

effects. Profound changes in mi-

Figure 4. DNA degradation in treated parasites: a) Bi-parametric plots of forward scatter (Fs) and propidium

iodide (PI) staining of DMSO (Control), 17 mm staurosporine- and 10 mm compound-28-treated parasites. Healthy

parasites without alterations in shape or plasma membrane integrity are located in the lower right quadrant (C4).

b) DNA content in the parasite populations shown in panel a). Cell-cycle distributions of the parasites along the

G₁, S, or G₂/M phases are indicated. Percentages of parasites with decreased DNA content (hypoploid parasites)

are indicated (H); n=number of events; PI=propidium iodide-derived fluorescence.

C^ꢂ

tochondrial DY_mand O₂pro-

duction were observed as early

as 20 min after compound addi-

tion. Because of the low per-

centage of hypoploid parasites

cells at 378C, and the stability of the compound was moni-

tored by HPLC at various time points. Compound 28 evolved

toward the Ile derivative 6 and further to the free 3’-hydroxy

compound 3. In contrast, compound 28 is perfectly stable in

PBS after 24 h incubation at 378C, and no hydrolysis products

were detected.

observed after 28-induced cell death, the nuclease LiEndoG

was assayed as a putative target for this family of compounds.

The activity of this enzyme in the presence of selected com-

pounds was significantly inhibited. The varying activity of the

derivatives against intracellular amastigotes strongly correlates

with their ability to inhibit this enzyme. Taken together, LiEn-

doG can be considered a macromolecular target responsible

for the toxic activity of these compounds on L. infantum.

Therefore, it can be hypothesized that the presence of the

dipeptide at the 3’-position in compound 28 favors entrance

into the differentiated THP-1 cells, where it exerts its leishmani-

cidal effect until it is enzymatically cleaved toward the Ile de-

rivative 6 and eventually to 3. It must be pointed out that

compound 6 also has significant leishmanicidal activity and is

able to cause moderate inhibition of LiEndoG, which may

extend the initial activity of compound 28 once it has been

modified by endogenous esterases.

Experimental Section

Chemistry

General procedures: Melting points were obtained on a Reichert-

Jung Kofler apparatus and are uncorrected. Microanalysis was ob-

tained with a Heraeus CHN-O-RAPID instrument. The elemental

compositions of the compounds agreed to within ꢁ0.4% of the

calculated values. Electrospray mass spectra were measured on

a quadrupole mass spectrometer equipped with an electrospray

The results reported above suggest that LiEndoG is a likely

target for the compounds described herein. EndoGs are mem-

bers of a family of DNA/RNA nonspecific bba metal nucleases

that have been demonstrated to take part in the apoptotic

process, whereby they translocate to the nucleus and contrib-

ute to the degradation of genomic DNA into oligonucleosomal

fragments.^[18]Apart from their involvement in the death pro-

cess, these nucleases have been shown to be necessary for the

proper growth of several unicellular organisms, indicating

a dual (pro-life and pro-death) function, most likely due to

their postulated role in mitochondrial DNA recombination.^[19–22]

A recent study reveals that EndoG^ꢂ/ꢂmice have impaired mito-

chondrial respiration and increased production of reactive

oxygen species, also associated with a decrease in the ratios of

mitochondrial to genomic DNA.^[23]Accordingly, blockage of

these pro-life activities of LiEndoG may be responsible for the

antileishmanial activity of this family of compounds.

1

source (Hewlett–Packard, LC–MS HP 1100). H and ¹³C NMR spectra

were recorded on a Varian INNOVA 300 operating at 299 (¹H) and

75 MHz (¹³C), and a Varian INNOVA-400 operating at 399 (¹H) and

99 MHz (¹³C).

Analytical TLC was performed on silica gel 60 F₂₅₄(Merck) pre-

coated plates (0.2 mm). Spots were detected under UV light

(254 nm) and/or by charring with phosphomolybdic acid. Separa-

tions on silica gel were performed by preparative centrifugal circu-

lar thin-layer chromatography (CCTLC) on a Chromatotron (Kiesel-

gel 60 PF₂₅₄gipshaltig, Merck), with a layer thickness of 1 and

2 mm and flow rate of 4 or 8 mLmin^ꢂ1, respectively. Flash chroma-

tography was performed using a force flow Horizon HPFG system

(Biotage) with Flash 25 or 40 silica gel cartridges.

All experiments involving water-sensitive compounds were con-

ducted under scrupulously dry conditions. Et₃N and CH₃CN were

dried by holding at reflux over CaH. THF was dried by reflux over

sodium/benzophenone. Anhydrous N,N’-dimethylformamide (DMF)

was purchased from Aldrich. Microwave reactions were performed

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MgSO₄, filtered, and evaporated. The residue was purified by flash

chromatography (CH₂Cl₂/MeOH, 100:0!96:4) to yield 463 mg

(88%) of 3 as a white solid; mp: 94–968C (CH₂Cl₂/MeOH); MS

(ES, +) m/z 549 [M+Na]⁺; ¹H NMR (CDCl₃): d=1.85 (d, J=1.0 Hz,

3H, 5-CH₃), 1.95 (m, 1H, H-2’), 2.27 (m, 1H, H-2’’), 3.67 (d, J=

15.6 Hz, 1H, CH₂CO), 3.74 (m, 1H, H-4’), 3.82 (m, 1H, H-3’), 3.91 (d,

J=15.6 Hz, 1H, CH₂CO), 3.94 (dd, J=12.1, 4.1 Hz, 1H, H-5’), 4.17

(dd, J=12.1, 4.3 Hz, 1H, H-5’’), 6.15 (pseudo-t, J=6.4 Hz, 1H, H-1’),

7.15 (d, J=1.1 Hz, 1H, H-6), 7.20–7.29 (m, 15H, CPh₃), 8.31 ppm

(brs, 1H, 3-NH); Anal. calcd for C₃₁H₃₀N₂O₆: C 70.71, H 5.74, N 5.32,

found: C 70.54, H 5.66, N 5.19.

General procedure for the synthesis of 3’-amino acid ester deriv-

atives: To a solution of the nucleoside derivative (1.0 mmol) in

CH₂Cl₂(5 mL) at 08C, the corresponding Boc-protected amino acid

(2.0 mmol), PyBOP (624 mg, 1.2 mmol), and Et₃N (345 mL, 2.5 mmol)

were added, and the solution was adjusted to pH 11 by the addi-

tion of DMAP. The reaction mixture was stirred overnight, diluted

with CH₂Cl₂(50 mL), and washed with a 10% aqueous solution of

citric acid (320 mL), H₂O (320 mL), a 5% aqueous solution of

NaHCO₃(320 mL), and brine (320 mL). The organic phase was

dried on anhydrous MgSO₄, filtered, and evaporated. The residue

was purified as specified. The fractions containing the desired com-

pound were evaporated. A solution of the Boc-protected com-

pound (1.0 mmol) in CH₂Cl₂(10 mL) was treated with trifluoroacetic

acid (TFA; 1.7 mL, 23 mmol). The reaction mixture was stirred at

room temperature for 3–5 h, and volatiles were removed. The resi-

due was purified by CCTLC in the Chromatotron, as specified in

each case.

5’-O-(3,3,3-Triphenylpropanoyl)-3’-O-valylthymidine (1): Reaction

of 3 (100 mg, 0.19 mmol) with Boc-Val-OH (82 mg, 0.38 mmol),

PyBOP (118 mg, 0.23 mmol), and Et₃N (66 mL, 0.47 mmol) afforded

a residue that was purified in the Chromatotron (hexane/EtOAc,

1:1) to yield 103 mg of the Boc-protected ester [MS (ES, +) m/z 726

[M+1]⁺]. Treatment of this compound with TFA, as described in

the general procedure, afforded a residue that was purified in the

Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield 75 mg (63%, two

steps) of 1 as a white solid; mp: 85–868C (CH₂Cl₂/MeOH); MS

(ES, +) m/z 626 [M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.81 (d, J=

6.8 Hz, 3H, CH₃-g), 0.86 (d, J=6.8 Hz, 3H, CH₃-g), 1.72 (s, 3H, 5-

CH₃), 1.85 (m, 1H, CH-b), 2.17 (m, 1H, H-2’), 2.34 (m, 1H, H-2’’), 3.14

(d, J=5.2 Hz, 1H, CH-a), 3.84 (s, 2H, CH₂CO), 3.87–4.00 (m, 3H, H-

4’, H-5’), 4.97 (m, 1H, H-3’), 6.12 (m, 1H, H-1’), 7.15–7.27 (m, 15H,

CPh₃), 7.44 (s, 1H, H-6), 11.37 ppm (brs, 1H, NH); Anal. calcd for

C₃₆H₃₉N₃O₇: C 69.10, H 6.28, N 6.72, found: C 68.97, H 6.31, N 6.80.

Figure 5. Inhibition of LiEndoG activity was assayed by the cleavage of

a dual-labeled double-stranded probe. a) LiEndoG-mediated DNA degrada-

tion is followed by an increase in fluorescence (F) observed over 40 min of

probe digestion in the presence of increasing amounts of compound 28.

b) LiEndoG-mediated DNA degradation in the presence of compounds 3, 4,

6, 25, and 28 at 25 mm. The slopes of the linear section of the curves differ

from that of the DMSO control with p values <10^ꢂ6(**) or 10^ꢂ3(*).

3’-O-Isoleucyl-5’-O-(3,3,3-triphenylpropanoyl)thymidine (6): Fol-

lowing the general procedure, reaction of 3 (100 mg, 0.19 mmol)

with Boc-Ile-OH (87 mg, 0.38 mmol), PyBOP (118 mg, 0.23 mmol),

and Et₃N (66 mL, 0.47 mmol) afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield the Boc-protected

ester [MS (ES, +) m/z: 762 [M+Na]⁺]. Treatment of this compound

with TFA afforded a residue that was purified in the Chromatotron

(CH₂Cl₂/MeOH, 10:1) to yield 76 mg (63%, two steps) of 6 as

a white solid; mp: 78–798C (CH₂Cl₂/MeOH); MS (ES, +) m/z 640

with a Biotage Initiator 2.0 single-mode cavity instrument from Bi-

otage (Uppsala, Sweden). Experiments were carried out in sealed

microwave process vials using the standard absorbance level

(400 W maximum power). The temperature was measured with an

IR sensor on the outside of the reaction vessel.

5’-O-(3,3,3-Triphenylpropanoyl)thymidine (3): A solution of dry

di-tert-butylazodicarboxylate (DBAD; 460 mg, 2.0 mmol) in DMF

(2.5 mL) was added dropwise to a solution containing thymidine

(5; 242 mg, 1.0 mmol), triphenylpropionic acid (604 mg, 2.0 mmol),

and Ph₃P (525 mg, 2.0 mmol) in dry DMF (2.5 mL) at 08C. The mix-

ture was stirred at 08C under argon for 1.5 h. MeOH (2.5 mL) was

added, and volatiles were removed. The residue was treated with

4n HCl in dioxane (5 mL), and stirred for an additional hour. After

removal of the solvent in vacuo, the residue was dissolved in

CH₂Cl₂(50 mL) and washed with 4n HCl (30 mL) and a solution of

NaHCO₃(30 mL). The organic phase was dried on anhydrous

1

[M+1]⁺; H NMR ([D₆]DMSO): d=0.79–0.84 (m, 6H, CH₃-g, CH₃-d),

1.12 (m, 1H, CH’-g), 1.40 (m, 1H, CH’’-g), 1.58 (m, 1H, CH-b), 1.72 (s,

3H, 5-CH₃), 2.18 (m, 1H, H-2’), 2.34 (m, 1H, H-2’’), 3.18 (d, J=

5.6 Hz, 1H, CH-a), 3.83 (s, 2H, CH₂CO), 3.88–4.00 (m, 3H, H-4’, H-5’),

4.96 (m, 1H, H-3’), 6.12 (dd, J=6.6, 6.3 Hz, 1H, H-1’), 7.14–7.30 (m,

15H, CPh₃), 7.44 (s, 1H, H-6), 11.36 ppm (brs, 1H, NH); Anal. calcd

for C₃₇H₄₁N₃O₇: C 69.47, H 6.46, N 6.57, found: C 69.19, H 6.44, N

6.51.

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3’-O-Cyclohexylglycyl-5’-O-(3,3,3-triphenylpropanoyl)thymidine

3’, H-4’), 6.19 (dd, J=8.0, 5.5 Hz, 1H, H-1’), 7.39 (d, J=1.0 Hz, 1H,

(7): Following the general procedure, reaction of

3

(90 mg,

H-6), 8.14 ppm (s, 1H, 3-NH).

0.17 mmol) with Boc-cHexGly-OH (88 mg, 0.34 mmol), PyBOP

(107 mg, 0.20 mmol) and Et₃N (59 mL, 0.43 mmol) afforded a residue

that was purified in the Chromatotron (CH₂Cl₂/MeOH, 20:1) to yield

the corresponding ester [MS (ES, +) m/z 788 [M+Na]⁺]. Treatment

of this compound with TFA and purification in the Chromatotron

(CH₂Cl₂/MeOH, 10:1) afforded 59 mg (52%, two steps) of 7 as

a white solid; mp: 84–868C (CH₂Cl₂/MeOH); MS (ES, +) m/z 666

[M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.97–1.16 (m, 5H, cHex), 1.48–

1.70 (m, 6H, cHex), 1.72 (d, J=1.0 Hz, 3H, 5-CH₃), 2.18 (m, 1H, H-

2’), 2.32 (m, 1H, H-2’’), 3.19 (d, J=5.6 Hz, 1H, CH-a), 3.83 (s, 2H,

CH₂CO), 3.88 (m, 1H, H-4’), 3.95 (m, 2H, H-5’), 4.96 (m, 1H, H-3’),

6.12 (dd, J=7.9, 6.4 Hz, 1H, H-1’), 7.14–7.27 (m, 15H, CPh₃), 7.44 (d,

J=1.0 Hz, 1H, H-6), 11.33 ppm (s, 1H, 3-NH); Anal. calcd for

C₃₉H₄₃N₃O₇: C 70.36, H 6.76, N 4.59, found: C 70.12, H 6.91, N 4.62.

5’-Amino-3’-O-(tert-butyldimethylsilyl)-5’-deoxythymidine (12):

To a suspension of 11 (195 mg, 0.31 mmol) in EtOH (5 mL),

H₂NNH₂H₂O (49 mL, 1.00 mmol) was added. The reaction mixture

was stirred at reflux for 1 h. The white solid precipitated was fil-

tered off. The filtrate was evaporated, and the residue was purified

by CCTLC in the Chromatotron (CH₂Cl₂/MeOH, 5:1) to afford

107 mg (96%) of 12 as a colorless oil. The analytical and spectro-

scopic data are identical to those previously described.^[25]

3’-O-(tert-Butyldimethylsilyl)-5’-(3,3,3-triphenylpropanamide)-5’-

deoxythymidine (13): To a solution of 12 (109 mg, 0.31 mmol) in

CH₂Cl₂(1.5 mL), 3,3,3-triphenylpropionic acid (139 mg, 0.46 mmol),

BOP (204 mg, 0.46 mmol), and Et₃N (64 mL, 0.46 mmol) were added.

The reaction mixture was stirred at room temperature overnight,

and volatiles were removed. The residue was purified by CCTLC in

the Chromatotron (EtOAc/hexane, 1:2!1:1) to yield 186 mg (95%)

of 13 as a white solid; mp: 94–968C (CH₂Cl₂/MeOH); MS (ES, +) m/

5’-O-(3,3,3-Triphenylpropanoyl)-3’-O-d-valylthymidine (8): Follow-

ing the general procedure, reaction of 3 (122 mg, 0.23 mmol) with

Boc-d-Val-OH (100 mg, 0.46 mmol), PyBOP (140 mg, 0.27 mmol),

and Et₃N (80 mL, 0.56 mmol) afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 30:1) to yield the desired ester

[MS (ES,+) m/z 748 [M+Na]⁺]. Treatment of the Boc-protected

ester with TFA afforded a residue that was purified in the Chroma-

totron (CH₂Cl₂/MeOH, 10:1) to yield 89 mg (62%, two steps) of 8 as

a white solid; mp: 66–688C (CH₂Cl₂/MeOH); MS (ES, +) m/z 626

[M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.84 (d, J=6.8 Hz, 3H, CH₃-g),

0.88 (d, J=6.8 Hz, 3H, CH₃-g), 1.72 (d, J=1.2 Hz, 3H, 5-CH₃), 1.85

(m, 1H, CH-b), 2.14 (m, 1H, H-2’), 2.32 (m, 1H, H-2’’), 3.16 (d, J=

5.4 Hz, 1H, CH-a), 3.81 (d, J=15.9 Hz, 1H, CH₂CO’), 3.88 (d, J=

15.9 Hz, 1H, CH₂CO’’), 3.93–4.00 (m, 3H, H-4’, H-5’), 4.93 (m, 1H, H-

3’), 6.12 (dd, J=8.5, 6.0 Hz, 1H, H-1’), 7.13–7.27 (m, 15H, CPh₃),

7.43 (d, J=1.2 Hz, 1H, H-6), 11.37 ppm (s, 1H, 3-NH); Anal. calcd

for C₃₆H₃₉N₃O₇: C 68.88, H 6.38, N 6.69, found: C 69.05, H 6.66, N

6.71.

1

z 641 [M+1]⁺; H NMR ([D₆]DMSO): d=0.84 (s, 9H, (CH₃)₃), 1.72 (s,

3H, 5-CH₃), 1.91 (m, 1H, H-2’), 2.02 (m, 1H, H-2’’), 3.03 (m, 2H, H-

5’), 3.49 (m, 1H, H-4’), 3.60 (s, 2H, CH₂), 4.14 (m, 1H, H-3’), 6.19

(pseudo-t, J=7.1 Hz, 1H, H-1’), 7.11–7.23 (m, 15H, CPh₃), 7.41 (s,

1H, H-6), 7.33 (t, J=5.6 Hz, 1H, NH), 11.30 ppm (s, 1H, 3-NH).

5’-(3,3,3-Triphenylpropanamide)-5’-deoxythymidine (14): A sus-

pension of 13 (140 mg, 0.22 mmol) in THF (2 mL) was treated with

1n HCl (0.8 mL). The reaction mixture was stirred at room temper-

ature overnight. The reaction was diluted with EtOAc (25 mL) and

washed with a solution of NaHCO₃(210 mL) and H₂O (210 mL).

The organic phase was dried on anhydrous MgSO₄, filtered, and

evaporated. The residue was purified by CCTLC in the Chromato-

tron (CH₂Cl₂/MeOH, 10:1) to afford 106 mg (93%) of 14 as a white

solid; mp: 116–1188C (CH₂Cl₂/MeOH); MS (ES, +) m/z 526 [M+1]⁺;

¹H NMR ([D₆]DMSO): d=1.72 (s, 3H, 5-CH₃), 1.97 (m, 2H, H-2’), 3.04

(m, 2H, H-5’), 3.49 (m, 1H, H-4’), 3.60 (s, 2H, CH₂), 3.93 (m, 1H, H-

3’), 5.16 (d, J=4.0 Hz, 1H, OH), 6.19 (pseudo-t, J=6.7 Hz, 1H, H-1’),

7.11–7.24 (m, 15H, CPh₃), 7.39 (s, 1H, H-6), 7.78 (t, J=5.7 Hz, 1H,

NH), 11.28 ppm (s, 1H, 3-NH); Anal. calcd for C₃₁H₃₁N₃O₅·2H₂O: C

66.30, H 6.28, N 7.48, found: C 65.94, H 6.54, N 7.58.

3’-O-Seryl-5’-O-(3,3,3-triphenylpropanoyl)thymidine (9): Follow-

ing the general procedure, reaction of 3 (82 mg, 0.16 mmol) with

Boc-Ser(TBDMSi)-OH^[24](100 mg, 0.31 mmol), PyBOP (97 mg,

0.19 mmol), and Et₃N (55 mL, 0.40 mmol) afforded a residue that

was purified in the Chromatotron (CH₂Cl₂/MeOH, 30:1) to yield the

protected ester [MS (ES, +) m/z 850 [M+Na]⁺]. Treatment of this

compound with TFA afforded a residue that was purified in the

Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield 51 mg (52%, two

steps) of 9 as a white solid; mp: 85–878C (CH₂Cl₂/MeOH); MS

(ES, +) m/z 614 [M+1]⁺; ¹H NMR ([D₆]DMSO): d=1.72 (s, 3H, 5-

CH₃), 2.18 (m, 1H, H-2’), 2.32 (m, 1H, H-2’’), 3.39 (t, J=4.6 Hz, 1H,

CH-a), 3.56 (m, 2H, CH₂-b), 3.85 (s, 2H, CH₂CO), 3.94–4.00 (m, 3H,

H-4’, H-5’), 4.87 (brs, 1H, OH), 4.95 (m, 1H, H-3’), 6.13 (dd, J=8.2,

6.2 Hz, 1H, H-1’), 7.15–7.27 (m, 15H, CPh₃), 7.43 ppm (s, 1H, H-6);

Anal. calcd for C₃₄H₃₅N₃O₈·0.5H₂O: C 65.58, H 5.83, N 6.75, found: C

65.36, H 6.16, N 6.78.

5’-(3,3,3-Triphenylpropanamide)-3’-O-valyl-5’-deoxythymidine

(15): Following the general procedure for the synthesis of 3’-amino

acid ester derivatives, reaction of 14 (57 mg, 0.11 mmol) with Boc-

Val-OH (47 mg, 0.22 mmol), PyBOP (67 mg, 0.13 mmol), and Et₃N

(37 mL, 0.27 mmol) afforded a residue that was purified in the Chro-

matotron (CH₂Cl₂/MeOH, 10:1) to yield the coupling derivative [MS

(ES,+) m/z 725 [M+1]⁺], that was treated with TFA for Boc depro-

tection. The final residue was purified in the Chromatotron (CH₂Cl₂/

MeOH, 10:1) to yield 42 mg (60%, two steps) of 15 as a white

solid; mp: 124–1258C (CH₂Cl₂/MeOH); MS (ES, +) m/z 625 [M+1]⁺;

¹H NMR ([D₆]DMSO): d=0.93 (d, J=6.8 Hz, 3H, CH₃-g), 0.94 (d, J=

6.8 Hz, 3H, CH₃-g), 1.73 (s, 3H, 5-CH₃), 2.05–2.25 (m, 3H, H-2’, CH-

b), 3.09–3.17 (m, 3H, H-5’, CH-a), 3.61 (s, 2H, CH₂CO), 3.77 (m, 1H,

H-4’), 5.08 (m, 1H, H-3’), 6.10 (dd, J=8.2, 6.1 Hz, 1H, H-1’), 7.14–

7.24 (m, 15H, CPh₃), 7.46 (s, 1H, H-6), 7.87 (t, J=4.8 Hz, 1H, NH),

11.35 ppm (s, 1H, 3-NH); Anal. calcd for C₃₆H₄₀N₄O₆: C 69.21, H

6.45, N 8.97, found: C 69.44, H 6.69, N 9.01.

5’-(1,3-Dihydro-1,3-dioxo-4,5,6,7-tetrachloroisoindol-2-yl)-3’-O-

(tert-butyldimethylsilyl)-5’-deoxythymidine (11): To

a solution

containing tert-butyldimethylsilyl chloride (107 mg, 0.71 mmol) and

imidazole (96 mg, 1.41 mmol) in dry DMF (2 mL), compound 10^[14]

(240 mg, 0.47 mmol) was added. The reaction mixture was stirred

at room temperature overnight, and volatiles were removed. The

residue was purified by CCTLC in the Chromatotron (EtOAc/

hexane, 1:2!1:1) to afford 225 mg (77%) of 11 as a white solid;

mp: 110–1128C (CH₂Cl₂/MeOH); MS (ES, +) m/z 622 [M+1]⁺with

4Cl isotopic pattern; ¹H NMR ([D₆]DMSO): d=0.90 (s, 9H, (CH₃)₃),

2.03 (s, 3H, 5-CH₃), 2.09 (m, 1H, H-2’), 2.31 (ddd, J=13.5, 5.5,

2.4 Hz, 1H, H-2’’), 3.86 (d, J=7.0 Hz, 2H, H-5’), 4.24–4.30 (m, 2H, H-

3’-O-Isoleucyl-5’-(3,3,3-triphenylpropanamide)-5’-deoxythymi-

dine (16): Following the general procedure for the synthesis of 3’-

amino acid ester derivatives, reaction of 14 (44 mg, 0.08 mmol)

with Boc-Ile-OH (39 mg, 0.17 mmol), PyBOP (52 mg, 0.10 mmol),

and Et₃N (29 mL, 0.21 mmol) afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 10:1) to provide the coupling

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product [MS (ES,+) m/z 739 [M+1]⁺]. This was treated with TFA

for Boc deprotection. The final residue was purified in the Chroma-

totron (CH₂Cl₂/MeOH, 10:1) to yield 36 mg (70%, two steps) of 16

as a white solid; mp: 83–858C (CH₂Cl₂/MeOH); MS (ES, +) m/z 639

with Boc-Ile-OH (127 mg, 0.55 mmol), PyBOP (171 mg, 0.33 mmol),

and Et₃N (95 mL, 0.68 mmol) afforded a residue that was purified

by flash chromatography (CH₂Cl₂/MeOH, 100: 96:4) to yield 174 mg

of the coupling product [MS (ES, +) m/z 776 [M+Na]⁺]. This was

deprotected by treatment with TFA. After removal of volatiles, the

residue was purified by flash chromatography (CH₂Cl₂/MeOH,

100.0!92:8) to yield 141 mg (80%, two steps) of 21 as a white

solid; mp: 67–698C (CH₂Cl₂/MeOH); MS (ES, +) m/z 654 [M+1]⁺;

¹H NMR ([D₆]DMSO): d=0.86–0.91 (m, 6H, CH₃-g, CH₃-d), 1.27 (m,

1H, CH’-g), 1.43 (m, 1H, CH’’-g), 1.79 (s, 3H, 5-CH₃), 1.87 (m, 1H,

CH-b), 2.27 (m, 1H, H-2’), 2.39 (m, 1H, H-2’’), 3.16 (m, 4H, N-CH₃,

CH-a), 3.81 (d, J=16.3 Hz, 1H, CH₂CO’), 3.87 (d, J=16.3 Hz, 1H,

CH₂CO’’), 3.99–4.03 (m, 3H, H-4’, H-5’), 5.07 (m, 1H, H-3’), 6.21

(pseudo-t, J=7.1 Hz, 1H, H-1’), 7.14–7.27 (m, 15H, CPh₃), 7.50 (s,

1H, H-6), 8.34 ppm (brs, 2H, NH₂); Anal. calcd for C₃₈H₄₃N₃O₇: C

69.81, H 6.63, N 6.43, found: C 69.72, H 6.78, N 6.52.

1

[M+1]⁺; H NMR ([D₆]DMSO): d=0.80–0.84 (m, 6H, CH₃-g, CH₃-d),

1.15 (m, 1H, CH’-g), 1.40 (m, 1H, CH’’-g), 1.58 (m, 1H, CH-b), 1.72 (s,

3H, 5-CH₃), 2.06–2.26 (m, 2H, H-2’), 3.11 (m, 2H, H-5’), 3.17 (d, J=

5.5 Hz, 1H, CH-a), 3.60 (s, 2H, CH₂CO), 3.69 (m, 1H, H-4’), 4.98 (m,

1H, H-3’), 6.06 (dd, J=8.3, 6.1 Hz, 1H, H-1’), 7.11–7.23 (m, 15H,

CPh₃), 7.47 (s, 1H, H-6), 7.82 (t, J=5.4 Hz, 1H, NH), 11.34 ppm (s,

1H, 3-NH); Anal. calcd for C₃₇H₄₂N₄O₆: C 69.57, H 6.63, N 8.77,

found: C 69.66, H 6.54, N 8.57.

2’-Deoxy-5’-O-(3,3,3-triphenylpropanoyl)uridine (18): To a solution

containing 2’-deoxyuridine (17; 100 mg, 0.44 mmol), 3,3,3-triphe-

nylpropionic acid (302 mg, 0.87 mmol) and Ph₃P (262 mg,

0,87 mmol) in dry DMF (1.1 mL), a solution of DBAD (230 mg,

0.87 mmol) in DMF (1.1 mL) was added dropwise. The mixture was

stirred at 08C under argon for 1.5 h. MeOH (2.5 mL) was added,

and volatiles were removed. The work-up was performed as de-

scribed for 3. The residue that was purified by HPFC (CH₂Cl₂/

MeOH, 100:0!92:8) to yield 158 mg (70%) of 18 as an amorphous

5’-O-(3,3-Diphenylpropanoyl)thymidine (22): As described for the

synthesis of 3, thymidine (5; 100 mg, 0.41 mmol) reacted with 3,3-

diphenylpropionic acid (186 mg, 0.82 mmol) in the presence of

Ph₃P (215 mg, 0.82 mmol) and DBAD (189 mg, 0.82 mmol). The res-

idue was purified by flash chromatography (CH₂Cl₂/MeOH, 100:0!

96:4) to yield 93 mg (51%) of 22 as an amorphous white solid. MS

(ES, +) m/z 451 [M+1]⁺; ¹H NMR ([D₆]DMSO): d=1.74 (s, 3H, 5-

CH₃), 1.99–2.18 (m, 2H, H-2’), 3.17 (m, 2H, CH₂CO), 3.80 (m, 1H, H-

4’), 4.08–4.15 (m, 3H, H-3’, H-5’, H-5’’), 4.43 (t, J=8.0 Hz, 1H,

CHPh₂), 5.37 (d, J=4.1 Hz, 1H, OH), 6.15 (pseudo-t, J=6.8 Hz, 1H,

H-1’), 7.14–7.32 (m, 10H, CPh₂), 7.40 (s, 1H, H-6), 11.33 ppm (brs,

1H, 3-NH).

1

white solid. MS (ES, +) m/z 513 [M+1]⁺; H NMR ([D₆]DMSO): d=

2.04 (m, 2H, H-2’), 3.69 (m, 1H, H-4’), 3.80 (brs, 2H, CH₂CO), 3.87

(m, 1H, H-3’), 3.94 (m, 2H, H-5’), 5.30 (d, J=4.2 Hz, 1H, OH), 5.57

(d, J=8.0 Hz, 1H, H-5), 6.07 (pseudo-t, J=6.7 Hz, 1H, H-1’), 7.15–

7.28 (m, 15H, CPh₃), 7.51 (d, J=8.1 Hz, 1H, H-6), 11.31 ppm (brs,

1H, 3-NH).

N³-Methyl-5’-O-(3,3,3-triphenylpropanoyl)thymidine (19): To a so-

lution of 3 (125 mg, 0.24 mmol) in acetone (2 mL) in 5 mL Pyrex

microwave process vial, K₂CO₃(16 mg, 0.12 mmol) and MeI (60 mL,

0.95 mmol) were added. The reaction vessel was sealed, stirred,

and subsequently irradiated for 60 min at 1008C in a single-mode

microwave reactor. The mixture was purified by flash chromatogra-

phy (CH₂Cl₂/MeOH, 100:0!96:4) to afford 152 mg (85%) of 19 as

a white solid; mp: 56–588C (CH₂Cl₂/MeOH); MS (ES, +) m/z 541

[M+1]⁺; ¹H NMR ([D₆]DMSO): d=1.75 (d, J=1.0 Hz, 3H, 5-CH₃),

2.05 (m, 1H, H-2’) 3.15 (s, 3H, NCH₃), 3.41 (m, 1H, H-2’’), 3.72 (m,

1H, H-4’), 3.82 (s, 2H, CH₂CO), 3.90–4.01 (m, 3H, H-5’, H-3’), 5.31 (d,

J=4.2 Hz, 1H, OH), 6.16 (pseudo-t, J=6.8 Hz, 1H, H-1’), 7.14–7.27

(m, 15H, CPh₃), 7.43 ppm (d, J=1.0 Hz 1H, H-6).

5’-O-[3,3,3-Tris(4-chlorophenyl)propanoyl]thymidine (23): As de-

scribed for the synthesis of 3, thymidine (5; 100 mg, 0.41 mmol) re-

acted with 3,3,3-tris(4-chlorophenyl)propionic acid (332 mg,

0.82 mmol) in the presence of Ph₃P (215 mg, 0.82 mmol) and

DBAD (189 mg, 0.82 mmol). The final residue was purified by flash

chromatography (CH₂Cl₂/MeOH, 100:0!96:4) to yield 156 mg

(61%) of 23 as an amorphous white solid. MS (ES, +) m/z 652 [M+

Na]⁺with 3Cl isotopic pattern; ¹H NMR ([D₆]DMSO): d=1.70 (s,

3H, 5-CH₃), 2.01–2.15 (m, 2H, H-2’), 3.69 (m, 1H, H-4’), 3.80 (d, J=

17.0 Hz, 1H, CH₂CO’), 3.87 (d, J=17.0 Hz, 1H, CH₂CO’’), 3.92–4.04

(m, 3H, H-3’, H-5’), 5.33 (d, J=4.2 Hz, 1H, OH), 6.12 (pseudo-t, J=

6.4 Hz, 1H, H-1’), 7.16 (d, J=8.5 Hz, 6H, CPh₃), 7.32 (d, J=8.5 Hz,

6H, CPh₃), 7.37 (s, 1H, H-6), 11.32 ppm (s, 1H, 3-NH).

2’-Deoxy-3’-O-isoleucyl-5’-O-(3,3,3-triphenylpropanoyl)uridine

(20): Following the general procedure for the synthesis of 3’-amino

acid ester derivatives, reaction of 18 (67 mg, 0.13 mmol) with Boc-

Ile-OH (60 mg, 0.26 mmol), PyBOP (81 mg, 0.16 mmol), and Et₃N

(46 mL, 0.33 mmol) afforded a residue that was purified in the Chro-

matotron (CH₂Cl₂/MeOH, 30:1) to yield 72 mg of the coupling

product [MS (ES, +) m/z 748 [M+Na]⁺]. This compound was treat-

ed with TFA for removal of the Boc protecting group. After remov-

al of volatiles, the residue was purified in the Chromatotron

(CH₂Cl₂/MeOH, 10:1) to yield 51 mg (63%, two steps) of 20 as

a white solid; mp: 81–838C (CH₂Cl₂/MeOH); MS (ES, +) m/z 626

3’-O-Isoleucyl-5’-O-(3,3-diphenylpropanoyl)thymidine (24): Fol-

lowing the general procedure for the synthesis of 3’-amino acid

ester derivatives, reaction of 22 (80 mg, 0.18 mmol) with Boc-Ile-

OH (82 mg, 0.36 mmol), PyBOP (111 mg, 0.21 mmol), and Et₃N

(62 mL, 0.44 mmol) afforded a residue that was purified in the Chro-

matotron (CH₂Cl₂/MeOH, 10:1) to yield the coupling product [MS

(ES, +) m/z 687 [M+Na]⁺]. Treatment with TFA followed by re-

moval of volatiles afforded a residue that was purified in the Chro-

matotron (CH₂Cl₂/MeOH, 10:1) to yield 59 mg (58%, two steps) of

24 as a white solid; mp: 58–608C (CH₂Cl₂/MeOH); MS (ES, +) m/z

564 [M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.80–0.84 (m, 6H, CH₃-g,

CH₃-d), 1.12 (m, 1H, CH’-g), 1.39 (s, m, 1H, CH’’-g), 1.58 (m, 1H, CH-

b), 1.75 (d, J=1.1 Hz, 3H, 5-CH₃), 2.22 (m, 1H, H-2’), 2.40 (m, 1H, H-

2’’), 3.18 (m, 3H, CH₂CO, CH-a), 4.01 (m, 1H, H-4’), 4.15 (m, 2H, H-

5’), 4.43 (t, J=8.0 Hz, 1H, CHPh₂), 5.09 (m, 1H, H-3’), 6.15 (dd, J=

8.2, 6.2 Hz, 1H, H-1’), 7.10–7.17 (m, 2H, CHPh₂), 7.21–7.32 (m, 8H,

CHPh₂), 7.47 (d, J=1.1 Hz, 1H, H-6), 11.38 ppm (s, 1H, 3-NH); Anal.

calcd for C₃₁H₃₇N₃O₇·2H₂O: C 62.21, H 7.21, N 7.30, found: C 62.09,

H 6.89, N 7.01.

1

[M+1]⁺; H NMR ([D₆]DMSO): d=0.79–0.84 (m, 6H, CH₃-g, CH₃-d),

1.12 (m, 1H, CH’-g), 1.38 (m, 1H, CH’’-g), 1.58 (m, 1H, CH-b), 2.17–

2.36 (m, 2H, H-2’), 3.18 (d, J=5.6 Hz, CH-a), 3.82 (s, 2H, CH₂CO),

3.88–4.98 (m, 3H, H-4, H-5’), 4.94 (m, 1H, H-3’), 5.62 (d, J=8.0 Hz,

1H, H-5), 6.07 (m, 1H, H-1’), 7.14–7.27 (m, 15H, CPh₃), 7.56 (d, J=

8.1 Hz, 1H, H-6), 11.35 ppm (brs, 1H, 3-NH); Anal. calcd for

C₃₆H₃₉N₃O₇: C 69.10, H 6.28, N 6.72, found: C 68.94, H 6.06, N 6.68.

3’-O-Isoleucyl-N³-methyl-5’-O-(3,3,3-triphenylpropanoyl)thymi-

dine (21): Following the general procedure for the synthesis of 3’-

amino acid ester derivatives, reaction of 19 (148 mg, 0.27 mmol)

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3’-O-Valyl-5’-O-[3,3,3-tris(4-chlorophenyl)propanoyl]thymidine

(25): Following the general procedure the synthesis of 3’-amino

acid ester derivatives, reaction of 23 (93 mg, 0.15 mmol) with Boc-

Val-OH (64 mg, 0.30 mmol), PyBOP (92 mg, 0.18 mmol), and Et₃N

(51 mL, 0.37 mmol) afforded a residue that was purified in the Chro-

matotron (CH₂Cl₂/MeOH, 10:1) to yield the corresponding ester

[MS (ES, +) m/z 852 [M+Na]⁺with a 3Cl isotopic pattern]. Treat-

ment with TFA and removal of volatiles afforded a residue that was

purified in the Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield 60 mg

(55%, two steps) of 25 as a white solid; mp: 105–1078C (CH₂Cl₂/

MeOH); MS (ES, +) m/z 728 [M+1]⁺with a 3Cl isotopic pattern;

¹H NMR ([D₆]DMSO): d=0.81 (d, J=6.8 Hz, 3H, CH₃-g), 0.86 (d, J=

6.8 Hz, 3H, CH₃-g), 1.72 (d, J=0.9 Hz, 3H, 5-CH₃), 1.83 (m, 1H, CH-

b), 2.21 (m, 1H, H-2’), 2.40 (m, 1H, H-2’’), 3.13 (d, J=5.4 Hz, 1H,

CH-a), 3.79–4.09 (m, 5H, H-4’, H-5’, CH₂CO), 5.02 (m, 1H, H-3’), 6.13

(dd, J=7.6, 6.2 Hz, 1H, H-1’), 7.17 (d, J=8.7 Hz, 6H, CPh₃), 7.32 (d,

J=8.7 Hz, 6H, CPh₃), 7.45 (d, J=0.9 Hz, 1H, H-6), 11.36 ppm (brs,

1H, NH); Anal. calcd for C₃₆H₃₆Cl₃N₃O₇: C 59.31, H 4.98 N, 5.76, Cl

14.59, found: C 59.18, H 4.91, N 5.99, Cl 14.26.

and Et₃N (49 mL, 0.35 mmol) afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 30:1) to yield the coupling prod-

uct [MS (ES, +) m/z 876 [M+Na]⁺]. Treatment with TFA and purifi-

cation of the residue in the Chromatotron (CH₂Cl₂/MeOH, 10:1) af-

forded 58 mg (55%, two steps) of 28 as a white solid; mp: 111–

113 8C (CH₂Cl₂/MeOH); MS (ES, +) m/z 754 [M+1]⁺; ¹H NMR

([D₆]DMSO): d=0.80–0.91 (m, 12H, CH₃-g-Ile, CH₃-d-Ile), 1.11 (m,

2H, CH’-g-Ile), 1.23 (m, 2H, CH’-g-Ile), 1.72 (s, 3H, 5-CH₃,), 1.69–1.88

(m, 2H,CH-b-Ile), 2.14 (m, 1H, H-2’), 2.23 (m, 1H, H-2’’), 3.56 (d, J=

5.8 Hz, 1H, CH-a-Ile), 3.78–4.02 (m, 5H, CH₂CO, H-4’, H-5’), 4.25

(pseudo-t, J=6.5 Hz, 1H, CH-a-Ile), 4.96 (m, 1H, H-3’), 6.13 (dd, J=

8.1, 6.2 Hz, 1H, H-1’), 7.13–7.27 (m, 15H, CPh₃), 7.43 (s,1H, H-6),

8.15 (d, J=6.3 Hz, 1H, NH-Ile), 11.37 ppm (s, 1H, 3-NH); Anal. calcd

for C₄₃H₅₂N₄O₈: C 68.60, H 6.90, N 7.44, found: C 68.81, H 7.05, N

7.31.

3’-O-(Valyisoleucyl)-5’-O-(3,3,3-triphenylpropanoyl)thymidine

(29): As described for 27, reaction of 6 (155 mg, 0.25 mmol) with

Boc-Val-OH (108 mg, 0.50 mmol), PyBOP (155 mg, 0.30 mmol), and

Et₃N (86 mL, 0.62 mmol) afforded a residue that was purified by

HPFC (CH₂Cl₂/MeOH, 100:0!96:4) to yield the coupling product

[MS (ES, +) m/z 836 [M+1]⁺]. Treatment with TFA and removal of

volatiles afforded a residue that was purified by HPFC (CH₂Cl₂/

MeOH, 100:0!90:10) to yield 151 mg (82%, two steps) of 29 as

a white solid: mp: 132–1338C (CH₂Cl₂/MeOH); MS (ES, +) m/z 739

3’-O-Isoleucyl-5’-O-[3,3,3-tris(4-chlorophenyl)propanoyl]thymi-

dine (26): Following the general procedure the synthesis of 3’-

amino acid ester derivatives, reaction of 23 (115 mg, 0.18 mmol)

with Boc-Ile-OH (84 mg, 0.36 mmol), PyBOP (112 mg, 0.22 mmol),

and Et₃N (62 mL, 0.45 mmol) afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield 106 mg of the cou-

pling product [MS (ES, +) m/z 866 [M+Na]⁺with a 3Cl isotopic

pattern]. After treatment with TFA and removal of volatiles, the

residue was purified in the Chromatotron (CH₂Cl₂/MeOH, 10:1) to

yield 88 mg (66%, two steps) of 26 as a white solid; mp: 122–

1258C (CH₂Cl₂/MeOH); MS (ES, +) m/z 744 [M+1]⁺with a 3Cl iso-

topic pattern; ¹H NMR ([D₆]DMSO): d=0.84–0.89 (m, 6H, CH₃-g,

CH₃-d), 1.20 (m, 1H, CH’-g), 1.43 (m, 1H, CH’’-g), 1.73 (s, 3H, 5-CH₃),

1.80 (m, 1H, CH-b), 2.24 (m, 1H, H-2’), 2.41 (m, 1H, H-2’’), 3.80–3.85

(m, 2H, CH₂CO’, CH-a), 3.90 (d, J=16.4 Hz, 1H, CH₂CO’’), 3.95–4.10

(m, 3H, H-4, H-5’), 5.10 (m, 1H, H-3’), 6.18 (dd, J=7.8, 6.6 Hz, 1H,

H-1’), 7.17 (d, J=8.7 Hz, 6H, CPh₃), 7.32 (d, J=8.7 Hz, 6H, CPh₃),

7.45 (s, 1H, H-6), 11.39 ppm (s, 1H, 3-NH); Anal. calcd for

C₃₇H₃₈Cl₃N₄O₆: C 59.81, H 5.15, Cl 14.31, N 5.65, found: C 59.72, H

5.09, Cl 14.23, N 5.82.

1

[M+1]⁺; H NMR ([D₆]DMSO): d=0.82–0.96 (m, 12H, CH₃-g-Ile, CH₃-

g-Val, CH₃-d-Ile), 1.15 (m, 1H, CH’-g-Ile), 1.45 (m, 1H, CH’’-g-Ile), 1.72

(s, 3H, 5-CH₃), 1.83 (m, 1H, CH-b-Ile), 2.03 (m, 1H, CH-b-Val), 2.17

(m, 1H, H-2’), 2.34 (m, 1H, H-2’’), 3.65 (d, J=5.6 Hz, 1H, CH-a-Val),

3.78–3.90 (m, 3H, CH₂CO, H-4’), 3.94–4.00 (m, 2H, H-5’), 4.25

(pseudo-t, J=6.4 Hz, 1H, CH-a-Ile), 4.96 (m, 1H, H-3’), 6.13 (dd, J=

8.4, 6.1 Hz, 1H, H-1’), 7.13–7.27 (m, 15H, CPh₃)_,7.43 (d, J=1.1 Hz,

1H, H-6), 7.95 (brs, 2H, NH₂), 8.65 (d, J=6.8 Hz, 1H, NH-Ile),

11.38 ppm (s, 1H, 3-NH); Anal. calcd for C₄₂H₅₀N₄O₈·2H₂O: C 68.27,

H 6.82, N 7.58, found: C 68.10, H 6.98, N 7.39.

4-[(2R,3S,5R)-5-(Thymin-1-yl)-2-(3,3,3-triphenylpropanoyloxyme-

thyl)tetrahydrofuran-3-yloxy]-4-oxobutanoic acid (30): To a solu-

tion of 3 (132 mg, 0.25 mmol) in anhydrous pyridine (4 mL), DMAP

(31 mg, 0.25 mmol), and succinic anhydride (50 mg, 0.50 mmol)

were added. The reaction mixture was stirred at room temperature

overnight, diluted with CH₂Cl₂(50 mL), and washed with a 10%

aqueous solution of KH₂PO₄(230 mL), and H₂O (30 mL). The or-

ganic phase was dried on anhydrous MgSO₄, filtered, and evaporat-

ed. The residue was purified by flash chromatography (CH₂Cl₂/

MeOH, 100:0!90:10) to afford 129 mg (82%) of 30 as an amor-

3’-O-(Isoleucylvalyl)-5’-O-(3,3,3-triphenylpropanoyl)thymidine

(27): Following a similar procedure to that described for the syn-

thesis of 3’-amino acid ester derivatives, reaction of 1 (48 mg,

0.08 mmol) with Boc-Ile-OH (35 mg, 0.15 mmol), PyBOP (47 mg,

0.09 mmol), and Et₃N (27 mL, 0.19 mmol) afforded a residue that

was purified in the Chromatotron (CH₂Cl₂/MeOH, 30:1) to yield the

coupling product [MS (ES,+) m/z 839 [M+1]⁺]. Treatment with

TFA and removal of volatiles afforded a residue that was purified in

the Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield 32 mg (54%, two

steps) of 27 as a white solid; mp: 93–958C (CH₂Cl₂/MeOH); MS

(ES, +) m/z 740 [M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.78–0.95 (m,

12H, CH₃-g-Ile, CH₃-g-Val, CH₃-d-Ile), 1.08 (m, 1H, CH’-g-Ile), 1.43 (m,

1H, CH’’-g-Ile), 1.57 (m, 1H, CH-b-Ile), 1.72 (s, 3H, 5-CH₃), 2.03 (m,

1H, CH-b-Val), 2.17 (m, 1H, H-2’), 2.34 (m, 1H, H-2’’), 3.06 (d, J=

5.7 Hz, 1H, CH-a-Ile), 3.78–4.00 (m, 5H, CH₂CO, H-4’, H-5’), 4.16

(pseudo-t, J=6.6 Hz, 1H, CH-a-Val), 4.97 (m, 1H, H-3’), 6.12 (dd, J=

8.0, 6.4 Hz, 1H, H-1’), 7.14–7.27 (m, 15H, CPh₃)_,7.43 (d, J=0.9 Hz,

1H, H-6), 7.95 (brs, 2H, NH₂), 8.12 (d, J=7.4 Hz, 1H, NH-Val),

11.25 ppm (s, 1H, 3-NH); Anal. calcd for C₄₂H₅₀N₄O₈·1.5H₂O: C

65.86, H 6.97, N 7.32, found: C 65.93, H 6.86, N 7.39.

1

phous solid. MS (ES, +) m/z 627 [M+1]⁺; H NMR ([D₆]DMSO): d=

1.71 (s, 3H, 5-CH₃), 2.15 (m, 1H, H-2’), 2.30 (m, 1H, H-2’’), 2.59–2.72

(m, 4H, CH₂CH₂), 3.78–4.05 (m, 5H, H-4’, H-5’, CH₂CO), 4.92 (m, 1H,

H-3’), 6.11 (dd, J=8.1, 6.2 Hz, 1H, H-1’), 7.17–7.27 (m, 15H, CPh₃),

7.43 (s, 1H, H-6), 11.38 ppm (s, 1H, 3-NH).

2-Hydroxyethyl-[(2R,3S,5R)-5-(thymin-1-yl)-2-(3,3,3-triphenylpro-

panoyloxymethyl)tetrahydrofuran-3-yl] succinate (31): To a solu-

tion of 30 (85 mg, 0.13 mmol) in CH₂Cl₂(1.0 mL), ethylene glycol

(76 mL, 1.35 mmol), PyBOP (88 mg, 0.17 mmol), and Et₃N (23 mL,

0.17 mmol) were added. The reaction mixture was stirred at room

temperature overnight, diluted with CH₂Cl₂(25 mL), and washed

with a 5% aqueous solution of citric acid (10 mL), NaHCO₃solution

(10 mL), and brine. The organic phase was dried on anhydrous

MgSO₄, filtered, and evaporated. The residue was purified by flash

chromatography (CH₂Cl₂/MeOH, 100:0!95:5) to afford 48 mg

(53%) of 31 [MS (ES, +) m/z 671 [M+Na]⁺]. This compound was

used as such for the subsequent steps.

3’-O-(Isoleucylisoleucyl)-5’-O-(3,3,3-triphenylpropanoyl)thymi-

dine (28): As described for 27, reaction of 6 (90 mg, 0.14 mmol)

with Boc-Ile-OH (65 mg, 0.28 mmol), PyBOP (87 mg, 0.17 mmol),

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2-[(S)-2-Amino-3-methylbutanoyloxy]ethyl-[(2R,3S,5R)-5-(thymin-

1-yl)-2-(3,3,3-triphenylpropanoyloxymethyl)tetrahydrofuran-3-yl]

succinate (32): Following a similar procedure to that described for

the synthesis of 3’-amino acid ester derivatives, reaction of 31

(80 mg, 0.12 mmol) with Boc-Val-OH (52 mg, 0.24 mmol), PyBOP

(74 mg, 0.14 mmol), and Et₃N (42 mL, 0.30 mmol) afforded a residue

that was purified in the Chromatotron (CH₂Cl₂/MeOH, 10:1) to yield

the coupling product [MS (ES, +) m/z 893 [M+Na]⁺]. Treatment

with TFA and purification of the residue in the Chromatotron

(CH₂Cl₂/MeOH, 10:1) yielded 33 mg (36%, two steps) of 32 as

a white solid; mp: 63–658C (CH₂Cl₂/MeOH); MS (ES, +) m/z 771

[M+1]⁺; ¹H NMR ([D₆]DMSO): d=0.86 (pseudo-t, J=6.5 Hz, 6H,

CH₃-g), 1.72 (s, 3H, 5-CH₃), 1.90 (m, 1H, CH-b), 2.15 (m, 1H, H-2’),

2.30 (m, 1H, H-2’’), 2.58 (m, 4H, OCCH₂CH₂CO), 3.20 (d, J=5.0 Hz,

1H, CH-a), 3.85 (m, 2H, CH₂CO), 3.90–3.99 (m, 3H, H-4’, H-5’), 4.20–

4.33 (m, 4H, OCH₂CH₂O), 4.93 (m, 1H, H-3’), 6.10 (dd, J=8.4, 6.1 Hz,

1H, H-1’), 7.14–7.27 (m, 15H, CPh₃), 7.43 (s, 1H, H-6), 11.36 ppm (s,

1H, 3-NH); Anal. calcd for C₄₂H₄₇N₃O₁₁: C 65.53, H 6.15, N 5.46,

found: C 65.80, H 6.34, N 5.22.

10:1) yielded 81 mg (65%, two steps) of 34 as a white solid; mp:

84–868C (CH₂Cl₂/MeOH); MS (ES, +) m/z 620 [M+Na]⁺; ¹H NMR

([D₆]DMSO): d=0.76–0.87 (m, 6H, CH₃-g, CH₃-d), 1.03 (m, 1H, CH’-

g), 1.23 (m, 1H, CH’’-g), 1.47 (s, 3H, 5-CH₃), 1.53 (m, 1H, CH-b),

2.30–2.50 (m, 2H, H-2’), 3.15 (d, J=5.5 Hz, 1H, CH-a), 3.26 (m, 2H,

H-5’), 4.03 (m, 1H, H-4’), 5.30 (m, 1H, H-3’), 6.21 (pseudo-t, J=

7.0 Hz, 1H, H-1’), 7.27–7.38 (m, 15H, CPh₃), 7.52 (s, 1H, H-6),

11.37 ppm (brs, 1H, 3-NH); Anal. calcd for C₃₅H₃₉N₃O₆: C 70.33, H

6.58, N 7.03, found: C 70.12, H 6.57, N 6.98.

4-[(2R,3S,5R)-5-(Thymin-1-yl)-2-(trityloxymethyl)tetrahydrofuran-

3-yloxy]-4-oxobutanoic acid (35): To a suspension of 5’-O-tritylthy-

midine^[16](4; 150 mg, 0.31 mmol) in CH₂Cl₂(7 mL), Et₃N (86 mL,

0.62 mmol), DMAP (37 mg, 0.31 mmol), and succinic anhydride

(62 mg, 0.62 mmol) were added. The reaction mixture was stirred

at room temperature under argon for 7 h, diluted with CH₂Cl₂

(30 mL), and washed with a 10% aqueous solution of KH₂PO₄(2

15 mL), and H₂O (215 mL). The organic phase was dried on anhy-

drous MgSO₄, filtered, and evaporated. The residue was purified in

a

SPE silica cartridge (CH₂Cl₂/MeOH, 300:1!200:1) to afford

2-[(S)-2-Amino-3-methylpentanoyloxy]ethyl-[(2R,3S,5R)-5-

149 mg (82%) of 35 as a white solid; mp: 97–998C (CH₂Cl₂/MeOH);

MS (ES, +) m/z 584 [M]⁺; ¹H NMR (CDCl₃): d=1.38 (s, 3H, 5-CH₃),

2.41 (m, 1H, H-2’), 2.51 (m, 1H, H-2’’), 2.59–2.72 (m, 4H,

OCH₂CH₂O), 3.42, 3.48 (dd, J=10.6, 2.5 Hz, 2H, H-5’), 4.17 (m, 1H,

H-4’), 5.46 (m, 1H, H-3’), 6.38 (dd, J=9.0, 5.4 Hz, 1H, H-1’), 7.24–

7.41 (m, 15H, CPh₃), 7.61 (s, 1H, H-6), 9.72 ppm (s, 1H, 3-NH).

(thymin-1-yl)-2-(3,3,3-triphenylpropanoyloxymethyl)tetrahydro-

furan-3-yl] succinate (33): As described for the synthesis of 32, re-

action of 31 (67 mg, 0.10 mmol) with Boc-Ile-OH (46 mg,

0.20 mmol), PyBOP (62 mg, 0.12 mmol) and Et₃N (34 mL, 0.25 mmol)

afforded a residue that was purified in the Chromatotron (CH₂Cl₂/

EtOAc, 6:4) to yield 57 mg of the coupling product [MS (ES, +) m/z

884 [M+1]⁺]. Treatment with TFA and purification of the residue

in the Chromatotron (CH₂Cl₂/MeOH, 10:1) yielded 28 mg (35%, two

steps) of 33 as a white solid; mp: 58–598C (CH₂Cl₂/MeOH); MS

2-Hydroxyethyl-[(2R,3S,5R)-5-(thymin-1-yl)-2-(trityoxymethyl)te-

trahydrofuran-3-yl] succinate (36): To a solution of 35 (130 mg,

0.22 mmol) in CH₂Cl₂(1 mL) was added ethylene glycol (50 mL,

0.89 mmol), BOP (119 mg, 0.27 mmol), and Et₃N (37 mL, 0.27 mmol).

The reaction mixture was stirred at room temperature for 3 h, and

diluted with EtOAc (25 mL), washed with a 5% aqueous solution of

citric acid (10 mL), a NaHCO₃solution (10 mL), and brine. The or-

ganic phase was dried on anhydrous MgSO₄, filtered, and evaporat-

ed. The residue was purified by CCTLC in the Chromatotron

(CH₂Cl₂/MeOH, 20:1) to afford 128 mg (92%) of 36 as a white solid;

mp: 73–748C (CH₂Cl₂/MeOH); MS (ES, +) m/z 651 [M+Na]⁺;

¹H NMR (CDCl₃): d=1.36 (d, J=0.9 Hz, 3H, 5-CH₃), 2.47 (m, 2H, H-

2’), 2.67 (m, 4H, COCH₂CH₂CO), 3.47 (d, J=2.4 Hz, 2H, H-5’), 3.82

(m, 2H, CH₂OH), 4.14 (m, 1H, H-4’), 4.24 (m, 2H, OCH₂), 5.49 (m,

1H, H-3’), 6.40 (dd, J=8.8, 5.7 Hz, 1H, H-1’), 7.24–7.40 (m, 15H,

CPh₃), 7.60 (d, J=1.1 Hz, 1H, H-6), 8.24 ppm (s, 1H, 3-NH).

1

(ES, +) m/z 784 [M+1]⁺; H NMR ([D₆]DMSO): d=0.79–0.83 (m, 6H,

CH₃-g, CH₃-d), 1.14 (m, 1H, CH’-g), 1.40 (m, 1H, CH’’-g), 1.58 (m, 1H,

CH-b), 1.71 (s, 3H, 5-CH₃), 2.15 (m, 1H, H-2’), 2.30 (m, 1H, H-2’’),

2.58 (m, 4H, OCCH₂CH₂CO), 3.24 (d, J=5.4 Hz, 1H, CH-a), 3.78–4.00

(m, 5H, H-4’, H-5’, CH₂CO), 4.20–4.32 (m, 4H, OCH₂CH₂O), 4.92 (m,

1H, H-3’), 6.10 (dd, J=8.2, 6.3 Hz, 1H, H-1’), 7.14–7.27 (m, 15H,

CPh₃), 7.43 (s, 1H, H-6), 11.36 ppm (s, 1H, 3-NH); Anal. calcd for

C₄₃H₄₉N₃O₁₁: C 65.89, H 6.30, N, 5.36, found: 65.88, H 6.45, N 5.29.

5’-O-Trityl-3’-O-valylthymidine (2): Following the general proce-

dure the synthesis of 3’-amino acid ester derivatives, reaction of 5-

O-tritythymidine^[16](4; 150 mg, 0.31 mmol), with Fmoc-Val-OH

(210 mg, 0.62 mmol), PyBOP (193 mg, 0.37 mmol), and Et₃N

(107 mL, 0.77 mmol) afforded a residue that was purified in the

Chromatotron (CH₂Cl₂) to yield the coupling product [MS (ES, +)

m/z 829 [M+Na]⁺]. Treatment with piperidine (85 mL) and removal

of volatiles afforded a residue that was purified in the Chromato-

tron (CH₂Cl₂/MeOH, 10:1) to yield 133 mg (74%, two steps) of 2 as

a white solid: mp: 88–908C (CH₂Cl₂/MeOH); MS (ES, +) m/z 607

[M+Na]⁺; ¹H NMR ([D₆]DMSO): d=0.74 (d, J=6.5 Hz, 3H, CH₃-g),

0.82 (d, J=6.5 Hz, 3H, CH₃-g), 1.46 (s, 3H, 5-CH₃), 1.78 (m, 1H, CH-

b), 2.30 (m, 2H, H-2’), 3.10 (d, J=5.1 Hz, CH-a), 3.25 (m, 2H, H-5’),

4.02 (m, 1H, H-4’), 5.32 (m, 1H, H-3’), 6.21 (pseudo-t, J=6.8 Hz, 1H,

H-1’), 7.27–7.39 (m, 15H, CPh₃), 7.52 (s, 1H, H-6), 11.38 ppm (brs,

1H, 3-NH); Anal. calcd for C₃₄H₃₇N₃O₆: C 69.96, H 6.39, N 7.20,

found: C 69.85, H 6.21, N 7.05.

2-[(S)-2-Amino-3-methylbutanoyloxy]ethyl-[(2R,3S,5R)-5-(thymin-

1-yl)-2-(trityloximethyl)tetrahydrofuran-3-yl] succinate (37): Fol-

lowing a similar procedure to that described for the synthesis of

3’-amino acid ester derivatives, compound 36 (85 mg, 0.14 mmol)

reacted with Fmoc-Val-OH (130 mg, 0.38 mmol), PyBOP (187 mg,

0.30 mmol), and Et₃N (37 mL, 0.30 mmol). The crude was dissolved

in DMF (1 mL) and treated with piperidine (72 mL). Removal of vola-

tiles afforded a residue that was purified in the Chromatotron

(CH₂Cl₂/MeOH, 10:1) to yield 60 mg (96%) of 37 as a white solid;

1

mp: 62–648C (CH₂Cl₂/MeOH); MS (ES, +) m/z 728 [M+1]⁺; H NMR

(CDCl₃): d=0.90 (d, J=6.8 Hz, 3H, CH₃-g), 0.97 (d, J=6.8 Hz, 3H,

CH₃-g), 1.36 (s, 3H, 5-CH₃), 2.01 (m, 1H, CH-b), 2.45 (m, 2H, H-2’),

2.67 (m, 4H, COCH₂CH₂CO), 3.33 (d, J=4.9 Hz, 1H, CH-a), 3.47 (d,

J=2.2 Hz, 2H, H-5’), 4.13 (m, 1H, H-4’), 4.32 (m, 4H, CH₂O), 5.50 (m,

1H, H-3’), 6.40 (dd, J=8.2, 6.2 Hz, 1H, H-1’), 7.25–7.43 (m, 15H,

CPh₃), 7.58 ppm (s, 1H, H-6); Anal. calcd for C₄₀H₄₅N₃O₁₀· H₂O): C

64.42, H 6.35, N 5.63, found: C 64.18, H 6.03, N 5.70.

3’-O-Isoleucyl-5’-O-tritylthymidine (34): Following the general

procedure the synthesis of 3’-amino acid ester derivatives, reaction

of 5’-O-tritythymidine^[16](4; 100 mg, 0.21 mmol), with Fmoc-Ile-OH

(146 mg, 0.41 mmol), PyBOP (129 mg, 0.25 mmol), and Et₃N (72 mL,

0.52 mmol) afforded a residue that was purified in the Chromato-

tron (CH₂Cl₂/MeOH, 30:1) to yield the coupling product [MS (ES, +)

m/z 842 [M+Na]⁺]. Treatment with piperidine (72 mL) followed by

purification of the residue in the Chromatotron (CH₂Cl₂/MeOH,

2-[(S)-2-Amino-3-methylpentanoyloxy]ethyl-[(2R,3S,5R)-5-

(thymin-1-yl)-2-(trityloximethyl)tetrahydrofuran-3-yl] succinate

(38): Following a reaction sequence similar to that described for

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37, alcohol 36 (110 mg, 0.17 mmol) reacted with Fmoc-Ile-OH

(93 mg, 0.26 mmol), PyBOP (109 mg, 0.21 mmol), and Et₃N (30 mL,

0.21 mmol), and was deprotected with piperidine (85 mL). The resi-

due was purified by HPFC (CH₂Cl₂/MeOH, 100:0!96:4) to yield

91 mg (71%) of 38; mp: 57–598C; MS (ES, +) m/z 742 [M+1]⁺;

¹H NMR (CDCl₃): d=0.77–0.85 (m, 6H, CH₃-g, CH₃-d), 1.08 (m, 1H,

CH’-g), 1.36 (m, 1H, CH’’-g), 1.43 (s, 3H, 5-CH₃), 1.55 (m, 1H, CH-b),

2.31–2.50 (m, 2H, H-2’), 2.57 (m, 4H, COCH₂CH₂CO), 3.14–3.19 (m,

2H, H-5’, CH-a), 3.30 (m, 1H, H-5’’), 4.05 (m, 1H, H-4’), 4.16–4.29

(m, 4H, CH₂O), 5.30 (m, 1H, H-3’), 6.40 (dd, J=8.3, 6.2 Hz, 1H, H-1’),

7.27–7.39 (m, 15H, CPh₃), 7.50 (s, 1H, H-6), 11.36 ppm (brs, 1H,

NH); Anal. calcd for C₄₁H₄₇N₃O₁₀: C 66.38, H 6.39, N 5.66, found: C

66.09, H 6.60, N 5.91.

of living cells was evaluated by flow cytometry by the propidium

iodide (PI) exclusion method.

Hydrolysis studies in cell extracts: Cell extracts from PMA-differenti-

ated THP-1 were prepared according to the procedure described

by Saboulard et al.^[26]Hydrolysis studies of 28 were performed as

follows: Aliquots of 200 mL of compound 28 (100 mm) were mixed

with 10 mL cell extract, and the samples were incubated at 378C.

At various time points (0, 0.5, 1, 2, 4, and 24 h), MeOH (100 mL) was

added to inactivate the enzymes. These samples were centrifuged,

and the supernatants were injected on HPLC. Identification of the

hydrolysis products was based on the retention times of the refer-

ence compounds and co-elution experiments under identical ana-

lytical conditions.

HPLC analysis of hydrolysis products: Analysis of the hydrolysis

products after cell extract incubation was performed by HPLC in-

jecting 30 mL of the supernatant. HPLC spectra were recorded on

an Aligent 1120 Compact LC using a diode array detector (l 230–

400 nm), and an analytical ACE5 C₁₈-300 column (1504.6 mm,

300 ). Solvents used were CH₃CN (0.05% TFA) for A, and H₂O

(0.05% TFA) for B, and the flow rate was 1 mLmin^ꢂ1. The gradient

used was 30!60% A over 20 min. The retention times of the ana-

lytes were: 16.8 min for compound 28, 14.2 min for compound 6,

and 13.5 min for compound 1.

Biology

Cells and culture conditions: L. infantum promastigotes (MCAN/ES/

89/IPZ229/1/89) were grown in RPMI-1640 medium (Sigma–Aldrich,

St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal

calf serum (FCS), antibiotics, and 25 mm HEPES (pH 7.2) at 268C.

L. infantum axenic amastigotes were grown in M199 medium (Invi-

trogen, Leiden, The Netherlands) supplemented with 10% heat-in-

activated FCS, 1 gL^ꢂ1b-alanine, 100 mgL^ꢂ1l-asparagine,

200 mgL^ꢂ1sucrose, 50 mgL^ꢂ1sodium pyruvate, 320 mgL^ꢂ1malic

acid, 40 mgL^ꢂ1fumaric acid, 70 mgL^ꢂ1succinic acid, 200 mgL^ꢂ1a-

ketoglutaric acid, 300 mgL^ꢂ1citric acid, 1.1 gL^ꢂ1sodium bicarbon-

ate, 5 gL^ꢂ1MES, 0.4 mgL^ꢂ1hemin, and 10 mgL^ꢂ1gentamicine,

pH 5.4 at 378C. Jurkat cells were grown in RPMI-1640 medium

(Sigma–Aldrich) supplemented with 10% heat-inactivated FCS, an-

tibiotics, and 10 mm HEPES, pH 7.2 at 378C and 5% CO₂. THP-

1 cells were grown in RPMI-1640 medium (Gibco, Leiden, The

Netherlands) supplemented with 10% heat-inactivated FCS, antibi-

otics, 1 mm HEPES, 2 mm glutamine, and 1 mm sodium pyruvate,

pH 7.2 at 378C and 5% CO₂.

Mitochondrial transmembrane potential: Parasites were incubated

in RPMI medium with 10% FCS at 1.510⁶mL with 50 nm tetrame-

thylrhodamine methyl ester perchlorate (TMRM, Sigma Cat.

#T5428) probe for 15 min at 268C and then analyzed by flow cy-

tometry. A positive control of mitochondrial depolarization was ob-

tained in parallel by incubating logarithmic promastigotes at 1.5

10⁶mL with the protonophore (H⁺ionophore) and uncoupler of

oxidative phosphorylation in mitochondria, carbonyl cyanide 3-

chlorophenylhydrazone (CCCP) during the indicated times and

then stained with TMRM as described above.

Cell proliferation assays: Drug treatment of promastigotes and

amastigotes was performed during logarithmic growth phase at

a concentration of 210⁶parasitesmL^ꢂ1at 268C or 110⁶parasi-

tesmL^ꢂ1at 378C for 24 h, respectively. Drug treatment of Jurkat

and THP-1 cells was performed during the logarithmic growth

phase at a concentration of 410⁵cellsmL^ꢂ1at 378C and 5% CO₂

for 24 h. The percentage of living cells was evaluated by flow cy-

tometry by the propidium iodide (PI) exclusion method.^[16]Briefly,

treated parasites were stained for 10 min with 5 mgmL^ꢂ1PI. The

number of PI-positive parasites was determined in a Beckman–

Colulter FC500 flow cytometer.

Mitochondrial superoxide detection: Parasites were preloaded for

10 min with 2 mm MitoSOX, and then fluorescence emission was

measured at the indicated times in a Beckman Coulter FC500 flow

cytometer.

DNA content analysis by flow cytometry: Parasites (1.510⁶) were

centrifuged at 1000 g for 5 min, the pellet was resuspended in

100 mL PBS, cold (ꢂ208C) 70% EtOH (600 mL) was added, and then

parasites were incubated on ice for 30 min. After incubation, the

parasites were washed with 1 mL PBS/50 mm EDTA, pelleted at

1000 g, and then resuspended in 400 mL PBS/50 mm EDTA/

50 mgmL^ꢂ1RNase and incubated for 30 min at 378C. Propidium

Leishmania infection assays: THP-1 cells were seeded at

120000 cellsmL^ꢂ1in 24-multiwell plates (Nunc, Roskilde, Denmark)

and differentiated to macrophages for 24 h in 1 mL RPMI-1640

medium containing 10 ngmL^ꢂ1phorbol 12-myristate 13-acetate

(PMA, Sigma–Aldrich). The culture medium was removed, and L. in-

fantum amastigotes (1.210⁶in 1 mL THP-1 medium) were added

to each well; 3 h later, all medium with non-infecting amastigotes

was removed, washed three times with 1 phosphate-buffered

saline (PBS) and replaced with new THP-1 medium and the corre-

sponding treatment. After 48 h of treatment, medium was re-

moved; THP-1 cells were washed three times with 1 PBS and de-

tached with TrypLE Express (Invitrogen, Leiden, The Netherlands)

according to the manufacturer’s indications. Infection was mea-

sured by flow cytometry.

iodide (PI) was then added to a final concentration of 5 mgmL^ꢂ1

,

and the parasites were immediately analyzed for PI fluorescence in

a Beckman Coulter FC500 flow cytometer.

LiEndoG activity: The nuclease activity of LiEndoG in the presence

of selected compounds was monitored by measuring the increase

in fluorescence derived from the digestion of a double-stranded

probe. 6-Carboxyfluorescein (FAM)-derived fluorescence is

quenched by the proximity of tetramethylrhodamine (TAMRA) in

the undigested probe constructed by hybridization of the oligonu-

cleotides FAM-5’-CTG TCG CTA CCT GTG G-3’-TAMRA and FAM-5’-

CCA CAG GTA GCG ACA G-3’-TAMRA. Digestion of any of the two

oligonucleotides causes separation of the fluorophore and quench-

er, and then a fluorescent signal can be emitted; 30 pmol of the

Cytotoxicity assays in human cells: Drug treatment of Jurkat cells

was performed during logarithmic growth phase at a concentration

of 410⁵cellsmL^ꢂ1at 378C and 5% CO₂for 24 h. The percentage

double-stranded probe were digested with LiEndoG 2.5 ngmL^ꢂ1

.

After digestion, both fluorophores separate, and FAM-derived fluo-

rescence can be detected. Reactions were monitored in a Victor

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E.C. thanks the Comunidad de Madrid and the Fondo Social Eu-

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Received: March 21, 2013

Published online on && &&, 0000

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FULL PAPERS

LiEndoG as drug target? Thymidine de-

rivatives with a triphenylmethyl sub-

stituent at the 5’-position exhibit signifi-

cant activity against Leishmania infan-

tum parasites. Characterization of cell

death points toward the mitochondrial

endonuclease G (LiEndoG) as a target.

E. Casanova, D. Moreno, A. Gigante,

E. Rico, C. M. Genes, C. Oliva,

M.-J. Camarasa, F. Gago, A. Jimꢀnez-Ruiz,

M.-J. Pꢀrez-Pꢀrez*

&& – &&

5’-Trityl-Substituted Thymidine

Derivatives as a Novel Class of

Antileishmanial Agents: Leishmania

infantum EndoG as a Potential Target

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Article Doi

5′-trityl-substituted thymidine derivatives as a novel class of antileishmanial agents: Leishmania infantum EndoG as a potential target

DOI: 10.1002/cmdc.201300129

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