Synthesis and anti-HIV activity of [d4U]-[trovirdine analogue] and [d4T]-[trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse transcriptase.

Article abstract of DOI:10.1081/NCN-120015066

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a-e) and (N-3)d4T-Trovirdine conjugates (10a-f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.

Full text of DOI:10.1081/NCN-120015066

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SYNTHESIS AND ANTI-HIV ACTIVITY OF [D4U]-
[TROVIRDINE ANALOGUE] AND [D4T]-[TROVIRDINE
ANALOGUE] HETERODIMERS AS INHIBITORS OF HIV-1
REVERSE TRANSCRIPTASE
D. Gavriliu a , C. Fossey a , A. Ciurea a , Z. Delbederi a , E. Sugeac a , D. Ladurée a , S.
Schmidt b , G. Laumond b & A. M. Aubertin b
a Centre d'Etudes et de Recherche sur le Médicament de Normandie , U.F.R. des Sciences
Pharmaceutiques , 5 Rue Vaubénard, Caen, 14032 Caen Cedex, France
b I.N.S.E.R.M. , 3 Rue Koeberlé, Strasbourg, 67000, France
Published online: 07 Feb 2007.
To cite this article: D. Gavriliu , C. Fossey , A. Ciurea , Z. Delbederi , E. Sugeac , D. Ladurée , S. Schmidt , G. Laumond
& A. M. Aubertin (2002) SYNTHESIS AND ANTI-HIV ACTIVITY OF [D4U]-[TROVIRDINE ANALOGUE] AND [D4T]-[TROVIRDINE
ANALOGUE] HETERODIMERS AS INHIBITORS OF HIV-1 REVERSE TRANSCRIPTASE, Nucleosides, Nucleotides and Nucleic Acids,
21:8-9, 505-533, DOI: 10.1081/NCN-120015066
To link to this article: http://dx.doi.org/10.1081/NCN-120015066
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©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 8 & 9, pp. 505–533, 2002
SYNTHESIS AND ANTI-HIV ACTIVITY OF
[D4U]-[TROVIRDINE ANALOGUE] AND
[D4T]-[TROVIRDINE ANALOGUE]
HETERODIMERS AS INHIBITORS OF
HIV-1 REVERSE TRANSCRIPTASE
D. Gavriliu,1 C. Fossey,1 A. Ciurea,1 Z. Delbederi,1 E. Sugeac,1
1,
D. Laduree, * S. Schmidt, G. Laumond, and A. M. Aubertin
2
2
2
´
1Centre d’Etudes et de Recherche sur le Me
´
dicament de Normandie
nard,
U.F.R. des Sciences Pharmaceutiques, 5 Rue Vaube
14032 Caen Cedex, France
2I.N.S.E.R.M., 3 Rue Koeberle
, 67000 Strasbourg, France
´
´
ABSTRACT
A series of eleven heterodimers containing both a nucleoside analogue
(d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue)
were synthesized and evaluated for their ability to inhibit HIV replica-
tion. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a^e) and
(N-3)d4T-Trovirdine conjugates (10a^f) were found to be inactive sug-
gesting that the two individual inhibitor compounds do not bind simul-
taneously in their respective sites.
*Corresponding author. E-mail: laduree@pharmacie.unicaen.fr
505
DOI: 10.1081=NCN-120015066
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

506
GAVRILIU ET AL.
INTRODUCTION
In addition to the Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
(AZT, d4T, ddC, ddI and 3TC) that are used clinically for the treatment of
AIDS,[1–3] the structural diverse non-nucleoside RT inhibitors (NNRTIs)
(HEPT, TIBO, nevirapine, pyridinone, BHAP, TSAO, a-APA, PETT,
quinoxaline) have been identified as highly specific to HIV-1 RT.[4–8] In
contrast to the NRTIs that interact to the substrate binding site after con-
version to the triphosphate form, the NNRTIs do not need any metabolic
conversion and are targeted at an allosteric non-substrate binding site of the
HIV-1 RT which is functionally and spatially associated with the substrate
binding site.
Combination chemotherapy of different anti-HIV agents has been
widely used to treat AIDS patients to circumvent or delay the emergence of
drug-resistant mutants.[9–12] Certain combinations of nucleoside and non-
nucleoside RT inhibitors lead to a synergistic inhibition of HIV replication
in vitro and in clinical trials [nevirapine=AZT=ddI,[13] Merck’s pyridinone
L-697,661=AZT,[14,15] delavirdine=AZT=ddI,[16,17] and MKC-442=AZT=
ddI[18,19]]. The rationale for the NRTI-NNRTI combination therapy includes
that a synergistic drug activity will lead to greater efficacy and that the
emergence of resistance to individual agents will be forestalled.[12]
Furthermore, the cooperative interaction between the NNRTI-binding
[20]
pocket and the substrate-binding site led Spence et al.
and Rittinger
et al. [21]to postulate that an inhibitor combining the functionalities of a non-
nucleoside inhibitor and a nucleotide analogue could bind very tightly and
specifically to the HIV-RT and could be effective in the treatment of AIDS.
One alternative approach to combination therapy has been earlier
suggested by Arnold and co-workers with the use of dimers resulting from
the linking of a NRTI and a 20,30-dideoxynucleoside (ddN) through an
appropriate spacer in an attempt to combine the inhibitory capacity of these
two different classes of molecules.[22] The NNRTIs interact noncompetitively
with the enzyme at an allosteric and highly hydrophobic nonsubstrate
binding site that is distinct from, but functionally and also spatially asso-
ciated with, the substrate binding site.[23] In 1995, Vela
´
zquez and co-workers
reported for the first time novel [AZT]-[TSAO-T] and [AZT]-[HEPT] het-
erodimers linked through flexible polymethylene spacers between the N-3
positions of thymidine bases of both compounds.[24] The most active com-
pound of this series was the [TSAO-T]-(CH2)3-[AZT] heterodimer (EC50
0.10 mM against HIV-1 in CEM=0 cells) but nevertheless less potent than the
parent compound from which it was derived.
With this aim, we also reported the synthesis and anti-HIV activity of a
series of heterodimers of the general formula [d4T]-NH-(CH2)n-NH-
[imidazo[1,5-b]pyridazine] (n ¼ 6 to 12).[25] The in vitro inhibitory activities
of these heterodimers were found to be comparable to that of d4T in

ANTI-HIV ACTIVITY OF HETERODIMERS
507
HIV-infected cells. The antiviral activities of d4T (IC50 0.059 mM against
HIV-1LAI in CEM-SS cells; IC50 0.28 mM against HIV-1IIIB in MT-4 cells)
and the heterodimers (IC50 0.025 to 0.06 mM against HIV-1LAI in CEM-SS
cells; IC50 0.09 to 0.36 mM against HIV-1IIIB in MT-4 cells) were of the same
magnitude. Moreover the [d4T]-NH-(CH2)n-NH-[imidazo[1,5-b]pyridazine]
heterodimers showed pronounced activity against HIV-2 (IC50 0.1 to 0.31 mM
against HIV-2D194 in PBMC cells) by comparison to d4T (IC50 0.18 mM
against HIV-2D194 in PBMC cells). None of the heterodimers proved
markedly cytotoxic to CEM-SS or MT-4 cells. In fact, cytotoxicity was not
observed in either cell line at concentrations equal or below 10 mM.
Recently, Velazquez et al. reported novel [AZT]-[TSAO-T] and [d4T]-
´
[TSAO-T] heterodimers modified in the linker and in the dideoxynucleoside
moiety.[26] The [d4T]-(CH2)3-[TSAO-T] heterodimer was ꢀ5- to 10-fold more
inhibitory to HIV-1 in MT-4 and CEM cells cultures than the corresponding
AZT heterodimer prototype and even 2-fold more potent than the unsub-
stituted TSAO-T parent compound. Furthermore, Pontikis et al. reported the
[AZT]-[HEPT] and [ddC]-[HEPT] conjugates as inhibitors of HIV reverse
transcriptase.[27] The [AZT]-[HEPT] conjugates displayed 2–5 mM anti-HIV
activity, but they had no effect on the replication of HIV-2. The [ddC]-
[HEPT] molecule displayed the same potency (EC50 0.45 mM) against HIV-1
(wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell
culture. No synergistic effect was observed for these bi-substrate inhibitors.
All these findings prompted us to report our results concerning the
synthesis and the anti-HIV evaluation of a series of mixed inhibitors resulting
from the linking of a known nucleoside analogue inhibitor (d4T) and a non-
nucleoside RT inhibitor through a functionalized tethering arm. The
tethering arm was varied in length and composition containing between 3 to
6 atoms in order to obtain a quite flexible dimer possessing an optimum
distance between both active principles (NRTI and NNRTI). In fact, in the
light of further structural studies it appears that, although the distance
between the catalytic site and the hydrophobic binding pocket is relatively
small (10–15 A), the two sites are distinct.[28] Such a flexible molecule
would be potentially have the right conformational freedom to interact with
the RT.
For the NRTI, we have chosen a d4T analogue since d4T is used in the
clinical treatment of AIDS. The choice of the attachment site of the tether on
the N-3 position in the pyrimidine ring was based upon the observation that
derivatives of AZT bearing a variety of N-3 side chains display potent anti-
HIV-1 activity.[29]
For the NNRTI, we have chosen a phenethyl thiazolyl thiourea (PETT)
derivative (Sch. 1).[30–33] The representative compound of this series is Tro-
virdine. Indeed the N-[2-(1-piperazinyl)ethyl]-N0-2-(5-bromopyridyl)]thiourea
(4) was found more active than AZT [IC50 ¼ 6 nM for inhibition of HIV
replication (measured by p24 production in HIV-infected human peripheral

508
GAVRILIU ET AL.
Scheme 1. Phenethylthiazolylthiourea (PETT) derivatives.
blood mononuclear cells) and SI ¼ 8000] or Trovirdine (IC50[p24] ¼ 7 nM in
PBMC cells, SI > 104) (Sch. 1).[34] Furthermore, the Trovirdine derivative (4)
inhibit HIV replication at nanomolar concentrations (IC50[p24] ¼ 2 nM)
without evidence of cytotoxicity (no inhibition of cellular proliferation at
concentrations as high as 100 mM).[34] In addition, the NH of the piperazine
ring allowed further reactions with halogeno derivatives.
Since we have an ongoing program on the synthesis of many modified
d4T analogues involving the formation of the key intermediate: the 50-O-
acetyl-20,30-didehydro-20,30-dideoxyuridine (50-O-acetyl-d4U), the first objec-
tive of this project was to design and synthesize a series of covalently linked
[d4U]-spacer-[Trovirdine derivative] heterodimers (9a^f) with an expanded
range of spacers of different nature and conformational freedom (Sch. 2).[35]
Then, we focused on the extension of the heterodimer approach with repla-
cing the d4U by d4T leading to a series of [d4T]-spacer-[Trovirdine deriva-
tive] heterodimers (10a^ e) in an attempt to combine the inhibitory capacity
of these two different classes of molecules.

ANTI-HIV ACTIVITY OF HETERODIMERS
509
Scheme 2. Modifications carried out on the [d4U]-(CH2)3-[Trovirdine derivative]
heterodimer.
RESULTS AND DISCUSSION
Chemical
Our general strategy for the synthesis of the [d4U or d4T]N3-spacer-
N
piperazinyl[Trovirdine analogue] target heterodimers (9a^f and 10a^ e) was
based on method previously described.[25]
Firstly, we have developed a two-step sequence to the versatile
unsaturated nucleoside precursor 2 using a strategy similar to that pre-
viously used in our laboratory.[35] This intermediate 2 was also synthesized
by Shiragami et al. according to a modified Eastwoodþdeoxygenation
procedure [mp 128–128.5ꢁC (from ethanol); m=z 253 (MH )].[36] In order
to develop a convenient synthetic route to the 20,30-didehydro-20,30-
dideoxy-uridine (2), we have used the method described in the literature
by Marumoto et al. for the conversion of uridine to acetoxybromo-
nucleoside (1) (Sch. 3).[37] Treatment of the resulting bromoacetate 1 with
zinc in anhydrous ethanol under irradiation of 35 KHz ultrasound (150 W)
for 30 min provided the corresponding 50-O-acetyl d4U (2) which was
isolated as crystalline solid in 44% yield after silica gel chromato-
graphy.[35] This method was attractive since the olefinic nucleoside was
obtained in neutral medium. In fact, the presence of acid promotes the
decomposition of the unsaturated nucleosides.
Initial experiments were aimed at introducing the spacer moiety into the
unsaturated precursor. Thus, reaction of 2 with commercially available
dibromo- or dichloro-alkyl reagents [(CH2)nBr2 (n ¼ 3, 4 and 6), 2-bro-
moethyl ether, 1,4-dichlorobutyne and 1,4-dibromobutene] in anhydrous
acetone, DMF or acetone:DMF (1:1) in the presence of potassium carbonate
afforded respectively the d4U intermediates 5a^f. After chromatography,
these N-3-substituted derivatives 5a^e were isolated with acceptable yields

510
GAVRILIU ET AL.
Scheme 3. Synthesis of [d4U]-spacer-[Trovirdine analogue] (9a–f) and [d4T]-spacer-[Tro-
virdine analogue] (10a–e).

ANTI-HIV ACTIVITY OF HETERODIMERS
511
(39 to 80%) except for 5f (spacer: CH2CH ¼ CHCH2) which was isolated in a
rather low yield (18%).
Subsequent reaction of these intermediates with the Trovirdine analo-
gue 4 under basic conditions (K2CO3) gave the heterodimers 7a^f [in 39 to
71% yield except for compound 17 [spacer: (CH2)4) which was isolated in
24% yield]. The Trovirdine analogue 4 was prepared as described previously
by Vig et al.[33] Deacetylation of 7a^f was performed using NaCN at room
temperature and the [d4U]N3-spacer-Npiperazinyl[Trovirdine analogue] final
heterodimer targets 9a^f were isolated in 74 to 98% yield as white crystalline
solids after purification by silica gel column chromatography (dichloro-
methane=methanol 85:15).[38]
Next, we focused on modification on the nucleoside moiety in the model
heterodimer. Thus, for comparative studies, the synthesis of a series of
[d4T]N3-spacer-Npiperazinyl[Trovirdine analogue] heterodimers (10a^ e) was
carried out in a similar way starting from the corresponding d4T which was
provided by Bristol Myers Squibb (Sch. 3). In a first step, d4T was converted
to its 50-O-acetyl counterpart (3) by a standard procedure. This intermediate
3 was also synthesized by Shiragami et al. according to a reductive b-elimi-
nation method of the corresponding acetoxy-bromo nucleoside precursor.[39]
Reaction of 50-O-acetyl d4T (3) with the appropriate dibromo or dichloro
reagent [(CH2)nBr2 (n ¼ 3, 4 and 6), 2-bromoethyl ether and 1,4-dichloro-
butyne] in the presence of K2CO3 afforded the N-3 alkyl intermediates (6a^e)
[73 to 97% yield except for 6a (spacer: (CH2)3) isolated in 34% yield].
Treatment of these intermediates with the Trovirdine analogue (4) gave the
corresponding heterodimers 8a^e isolated in 23 to 48% yield. Finally,
compounds 8a^e were subjected to an ester hydrolysis to provide free het-
erodimers 10a^ e [89 to 98% yield except for 10d (spacer: (CH2)2-O-(CH2)2)
isolated in 40% yield].
Structure of the novel heterodimers 9a^f and 10a^ e were assigned on
the basis of their analytical and spectroscopic data. The full assignment of 1H
and 13C NMR spectra for compounds 5a, 5e, 7a, 7c, 7d, 7f, 8e, 9a and 9b
were achieved by one and two-dimensional (1D and 2D) NMR techniques.
Structures of the target heterodimers [d4T]-spacer-[Trovirdine analogue]
1
(10a^ e) were determined through comparison of their H and 13C NMR
spectra to those of the model compound 9a.
Biological
The novel [d4U]-[Trovirdine analogue] (9a^f) and [d4T]-[Trovirdine
analogue] (10a^ e) were evaluated by comparison to AZT for inhibition of
HIV-1 multiplication in lymphocytic cell lines (CEM-SS and MT-4). As
shown in Table 1, the heterodimers 9a^f and 10a^ e were unfortunately
devoid of antiviral activity at non toxic concentration. Overall, the activities

512
GAVRILIU ET AL.
Table 1.
HIV-1LAI in CEM-SS Cells HIV-1IIIB in MT-4 Cells HIV-1III B in PBMC Cells
CC50
Compd IC50 (mM)a (mM)b
CC50
SIc IC50 (mM) (mM)
CC50
SI IC50 (mM) (mM)
SI
d4T
4
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
0.059
13
> 100
> 100
15
> 100
> 73
> 100
> 100
19
> 100 > 1695
0.28
> 100 > 357
0.05
8.6
42 840
> 100 > 11.62
> 100 > 7.69
> 100 > 1
> 100 > 1
> 100 > 6.67
> 100 > 1
> 100 > 1.37
> 100 > 1
> 100 > 1
> 100 > 5.26
> 100 > 10
> 100 > 1.49
> 100 > 1
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
52
> 100 > 1
> 100 > 1
> 100 > 1
> 100 > 1
> 100 > 1
> 100 > 1
NDd
ND
88
ND
ND
98
ND
ND
ND
ND
ND
ND
ND
1.11
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
70
> 100
1
> 100 > 1
> 100 > 1
> 100 > 1
> 100 > 1.92
> 100 > 1
10
67
> 100
> 100 > 1.43
ND ND
> 100 > 2.22
ND
45
> 100
a
IC50 is the concentration required to inhibit HIV-1 multiplication by 50%.
CC50 is the concentration of drug which causes 50% cytotoxicity to uninfected cells.
SI corresponds to the ratio CC50=IC50.
b
c
d
ND : not determined.
All data represent the mean values of three separate experiments ꢂ SD.
observed for the conjugates 9a^f and 10a^ e were inferior to the values for
the parent nucleoside analogue and the NNRTI component. Indeed, the
differences in activities may result in differences in cell membrane perme-
ability and=or different levels of phosphorylation of the nucleoside subunit.
In fact, the lack of activity of these N-3-substituted d4T derivatives is most
likely due to the fact that the compounds are poorly recognized by cellular
kinases, and therefore they will become much less phosphorylated than the
parent d4T. Moreover, it is conceivable that the lack of activity for the
heterodimers 9a^f and 10a^ e is a consequence of a wrong positioning of
either the NRTI or the NNRTI component with their respective sites.
EXPERIMENTAL SECTION
Chemical Procedures
Reagent grade acetonitrile was refluxed and distilled from phosphorus
pentoxide. Anhydrous ethanol was prepared by using magnesium turnings.
Anhydrous methanol p.a., acetone, N,N-dimethylformamide and pyridine
were respectively purchased from E. Merck, Carlo Erba and Aldrich. Unless

ANTI-HIV ACTIVITY OF HETERODIMERS
513
otherwise stated, reactions were run under an atmosphere of argon and
monitored by thin-layer chromatography (TLC) using precoated silica gel 60
F254 sheets (0.2 mm layer) purchased from Macherey-Nagel, and compounds
were detected by UV absorption at 254 nm. Column chromatography were
effected by using Merck silica gel 60 (0.063–0.200 mm) and silica gel Si 60 for
flash chromatography (40–63 mm) was supplied by Merck. All samples were
kept in a drying oven at 30ꢁC over P2O5 for at least 24 h prior to analysis.
Melting points were determined on a Kofler apparatus and are uncor-
rected. IR spectra were recorded on a Fourier transform Mattson spectro-
meter Genesis DTGS using WinFIRSTTM Macros and ApProTM and only
1
noteworthy absorptions are listed. H and 13C NMR spectra were obtained
on a JEOL Lambda 400 using TMS as an internal standard. NH and OH
signals appeared as broad singlets exchangeable with D2O (s ¼ singlet,
b ¼ broad, d ¼ doublet, t ¼ triplet, q ¼ quadruplet, m ¼ multiplet). Mass
spectra were determined with a JEOL GC mate apparatus. Only the peak due
to the molecular ion Hþ is listed.
50-O-Acetyl-20,30-didehydro-20,30-dideoxythymidine (3). Acetic anhy-
dride (4 mL) was added dropwise to a stirred solution of d4T (500 mg,
2.23 mmol) in dry pyridine (50 mL) at 0ꢁC (ice=water bath). The reaction
mixture was stirred overnight and evaporated to dryness under reduced
pressure. The residue was coevaporated with EtOH (3 ꢃ 20 mL) to afford
590 mg (99%) of crude product as a white foam which was used immediately
1
for the next preparation without purification: Rf 0.52 [EtOAc (100%)]; H
NMR (DMSO-d6): d 1.76 (s, 3H, CH3-5), 2.01 (s, 3H, CH3COO), 4.16 (dd,
1H, H-50, J ¼ 12.2, J ¼ 2.7 Hz), 4.21 (dd, 1H, H-500, J ¼ 12.2, J ¼ 4.1 Hz), 4.96
(bs, 1H, H-40), 5.99 (d, 1H, H-20, J ¼ 5.8 Hz), 6.40 (d, 1H, H-30, J ¼ 5.8 Hz),
6.80 (bs, 1H, H-10), 7.25 (s, 1H, H-6), 11.38 (bs, 1H, NH); 13C NMR
(DMSO-d6): d 12.1 (CH3-5), 20.6 (CH3COO), 64.5 (C-50), 83.6 (C-40), 89.3
(C-10), 109.5 (C-5), 126.6 (C-20), 133.6 (C-30), 135.8 (C-6), 150.7 (C-2), 163.8
(C-4), 170.0 (CH3COO).
N-[2-(1-Piperazinylethyl)]-N0-[2-(5-bromopyridyl)]thiourea (4). The title
compound was prepared by the method reported by Vig et al.[33] The
remaining solid was purified by flash chromatography on silica gel with
methanol in dichloromethane (from 0% to 10%) to give compound 4 as
a white foam (overall yield:37%): Rf 0.20 [CH2Cl2-CH3OH (7:3)]; mp
1
178–180ꢁC, H NMR (DMSO-d6): d 2.35 (m, 4H, 2 ꢃ NCH2 of piperazinyl),
2.49 (m, 2H, NHCH2CH2N), 2.71 (m, 4H, 2 ꢃ CH2NH of piperazinyl), 3.65
(m, 2H, CSNHCH2), 7.12 (d, 1H, H-III, J ¼ 8.9 Hz), 7.98 (dd, 1H, H-IV,
J ¼ 8.9 Hz, J ¼ 2.4 Hz), 8.26 (d, 1H, H-VI, J ¼ 2.4 Hz), 10.69 (bs, 1H, NH),
11.36 (bs, 1H, NH); 13C NMR (DMSO-d6): d 41.9 (CSNHCH2), 45.8
(CH2NH of piperazinyl), 53.5 (2 ꢃ NCH2 of piperazinyl), 55,8 (NHCH2CH2),
111.8 (C-V), 114.5 (C-III), 141.3 (C-IV), 145.8 (C-VI), 152.4 (C-II), 178.7

514
GAVRILIU ET AL.
(CS); Anal. Calcd for C12 H18 Br N5 S: C, 41.86; H, 5.23; N, 20.35. Found: C,
41.92; H, 5.35; N, 20.42; MS m=z 345 (MHþ).
General Procedure for the Synthesis of 50 -O-Acetyl-3-N-(n-bromo or
n-chloro-spacer) Nucleosides Intermediates (5a–f) and (6a–e). To a solution
of the nucleoside [50-O-acetyl-d4U (2) or 50-O-acetyl-d4T (3)] (1 equiv.) in
acetone or DMF or acetone=DMF were added K2CO3 (1.1 equiv.) and 6
equiv. of the correspondant dibromoalkyl, alkenyl, aryl, or dichloroalkynyl
reagent. The reaction mixture in acetone was refluxed for 19 h, in DMF
heated at 80ꢁC for 1–5 h and in the mixture acetone=DMF heated at 70ꢁC for
12–24 h. After evaporation of the solvent, the residue was dissolved in ethyl
acetate (50 mL), washed with water (2 ꢃ 50 mL), dried (MgSO4), filtered, and
evaporated to dryness. The residue was purified by chromatography on a
silica gel column (ethyl acetate). The fractions containing the desired product
were pooled together, and the solvent was evaporated in vacuo. The reagent,
solvent of the reaction mixture, reaction time, chromatography eluent, yield
1
of the isolated products, and H NMR and 13C NMR data are indicated
below each compound.
50 -O-Acetyl-3-N-(3-bromopropyl)-20, 30-didehydro-20, 30-dideoxyuridine
(5a). reagent: 1,3-dibromopropane, solvent of the reaction mixture: ace-
tone; reaction time 19 h; eluent of the chromatography: ethyl acetate; yield of
5a (59%) as a syrup; Rf 0.67 [EtOAc (100%)]; 1H NMR (DMSO-d6): d 2.00
(s, 3H, CH3COO), 2.07 (s, 2H, CH2CH2N-3), 3.52 (t, 2H, CH2Br,
J ¼ 6.7 Hz), 3.91 (t, 2H, CH2N-3, J ¼ 7.1 Hz), 4.18 (d, 2H, H-50 and H-50,
J ¼ 3.4 Hz), 5.00 (bs, 1H, H-40), 5.80 (d, 1H, H-5, J ¼ 8.0 Hz), 6.01 (d, 1H, H-
20, J ¼ 6.1 Hz), 6.44 (d, 1H, H-30, J ¼ 6.1 Hz), 6.84 (bs, 1H, H-10), 7.49 (d, 1H,
H-6, J ¼ 8.0 Hz); 13C NMR (DMSO-d6): d 20.6 (CH3), 30.5 (CH2CH2N-3),
32.0 (CH2Br), 39.4 (CH2N-3), 64.5 (C-50), 83.9 (C-40), 90.4 (C-10), 101.1 (C-
5), 126.3 (C-20), 134.0 (C-30), 139.2 (C-6), 150.9 (C-2), 162.0 (C-4), 170.1
(CH3COO).
50 -O-Acetyl-3-N-(4-bromobutyl)-20, 30-didehydro-20, 30-dideoxyuridine
(5b). reagent: 1,4-dibromobutane, solvent of the reaction mixture: ace-
tone=DMF (1:1); reaction time 14 h; eluent of the chromatography: ethyl
acetate in dichloromethane (from 0% to 10 %); yield of 5b (39%) as a syrup;
Rf 0.68 [EtOAc (100%)]; 1H NMR (CDCl3): d 1.71 (m, 2H, CH2CH2Br), 1.80
(m, 2H, CH2CH2N-3), 1.96 (s, 3H, CH3COO), 3.34 (t, 2H, CH2Br,
J ¼ 6.5 Hz), 3.85 (t, 2H, CH2N-3, J ¼ 6.9 Hz), 4.15 (dd, 1H, H-50, J ¼ 12.3,
J ¼ 3.2 Hz), 4.22 (dd, 1H, H-500, J ¼ 12.3, J ¼ 3.8 Hz), 4.97 (bs, 1H, H-40), 5.64
(d, 1H, H-5, J ¼ 8.1 Hz), 5.83 (d, 1H, H-20, J ¼ 5.3 Hz), 6.22 (d, 1H, H-30,
J ¼ 5.3 Hz), 6.92 (bs, 1H, H-10), 7.33 (d, 1H, H-6, J ¼ 8.1 Hz); 13C NMR
(CDCl3): d 20.7 (CH3), 26.2 (CH2CH2N-3), 29.9 (CH2CH2Br), 33.2 (CH2Br),

ANTI-HIV ACTIVITY OF HETERODIMERS
515
40.1 (CH2N-3), 64.5 (C-50), 84.2 (C-40), 90.6 (C-10), 101.9 (C-5), 126.9 (C-20),
133.5 (C-30), 137.8 (C-6), 151.1 (C-2), 162.4 (C-4), 170.2 (CH3COO).
50 -O-Acetyl-3-N-(6-bromohexyl)-20, 30-didehydro-20, 30-dideoxyuridine
(5c). reagent: 1,6-dibromohexane, solvent of the reaction mixture: DMF;
reaction time 4 h; eluent of the chromatography: ethyl acetate in dichlor-
omethane (from 0 to 10%); yield of 5c (41%) as a syrup; Rf 0.73 [EtOAc
1
(100%)]; H NMR (CDCl3): d 1.32 (m, 2H, CH2(CH2)2Br), 1.41 (m, 2H,
CH2(CH2)2N-3), 1.58 (m, 2H, CH2CH2Br), 1.80 (m, 2H, CH2CH2N-3), 2.00
(s, 3H, CH3COO), 3.33 (t, 2H, CH2Br, J ¼ 6.8 Hz), 3.85 (t, 2H, CH2N-3,
J ¼ 6.6 Hz), 4.18 (dd, 1H, H-50,J ¼ 12.3, J ¼ 2.6 Hz), 4.27 (dd, 1H, H-500,
J ¼ 12.3, J ¼ 3.5 Hz), 5.00 (bs, 1H, H-40), 5.67 (d, 1H, H-5, J ¼ 8.0 Hz), 5.86
(d, 1H, H-20, J ¼ 5.6 Hz), 6.24 (d, 1H, H-30, J ¼ 5.7 Hz), 6.96 (bs, 1H, H-10),
7.35 (d, 1H, H-6, J ¼ 8.0 Hz); 13C NMR (CDCl3): d 20.6 (CH3), 25.9
(CH2(CH2)2N-3), 27.1 (CH2(CH2)2Br), 27.6 (CH2CH2N-3), 32.4
(CH2CH2Br), 33.7 (CH2Br), 40.9 (CH2N-3), 64.5 (C-50), 84.1 (C-40), 90.5
(C-10), 101.9 (C-5), 126.9 (C-20), 133.5 (C-30), 137.8 (C-6), 151.1 (C-2), 162.4
(C-4), 170.2 (CH3COO).
50 -O-Acetyl-3-N-[2-(2-bromoethoxy)ethyl]-20, 30-didehydro-20, 30-dideox-
yuridine (5d). reagent: 2-bromoethyl ether, solvent of the reaction mixture:
acetone=DMF (7:2); reaction time 24 h; eluent of the flash chromatography:
methanol in dichloromethane (from 0 to 3%); yield of 5d (67%) as a syrup;
1
Rf 0.57 [EtOAc (100%)]; H NMR (CDCl3): d 2.07 (s, 3H, CH3COO), 3.45
(t, 2H, CH2Br, J ¼ 6.0 Hz), 3.77 (t, 2H, BrCH2CH2O, J ¼ 6.0 Hz), 3.82 (t, 2H,
OCH2CH2N-3, J ¼ 6.0 Hz), 4.18 (m, 2H, CH2N-3), 4.25 (dd, 1H, H-50,
J ¼ 12.4, J ¼ 2.7 Hz), 4.34 (dd, 1H, H-500, J ¼ 12.4, J ¼ 3.6 Hz), 5.07 (bs, 1H,
H-40), 5.75 (d, 1H, H-50, J ¼ 8.0 Hz), 5.94 (d, 1H, H-20, J ¼ 5.7 Hz), 6.32
(d, 1H, H-30, J ¼ 5.7 Hz), 7.02 (bs, 1H, H-10), 7.44 (d, 1H, H-6, J ¼ 8.0 Hz).
50 -O-Acetyl-3-N-(4-chloro-2-butynyl)-20, 30-didehydro-20, 30-dideoxyur-
idine (5e). reagent: 1,4-dichloro-2-butyne, solvent of the reaction mixture:
acetone=DMF (1:1); reaction time 12 h; eluent of the chromatography:
methanol in dichloromethane (from 0 to 2%); yield of 5e (80%) as a syrup;
1
Rf 0.68 [EtOAc (100%)]; H NMR (CDCl3): d 1.96 (s, 3H, CH3COO), 4.02
(bs, 2H, CH2Cl), 4.15 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.6 Hz), 4.23 (dd, 1H, H-50,
J ¼ 12.4, J ¼ 3.6 Hz), 4.63 (bs, 2H, CH2N-3), 4.98 (bs, 1H, H-40), 5.68 (d, 1H,
H-5, J ¼ 8.1 Hz), 5.85 (d, 1H, H-20, J ¼ 5.6 Hz), 6.24 (d, 1H, H-30, J ¼ 5.6 Hz),
6.93 (bs, 1H, H-10), 7.39 (d, 1H, H-6, J ¼ 8.1 Hz); 13C NMR (CDCl3): d 20.7
(CH3COO), 30.4 (CH2Cl and CH2N-3), 64.5 (C-50), 77.0 (ClCH2CꢄC), 80.7
(CꢄCCH2N-3), 84.3 (C-40), 90.7 (C-10), 101.8 (C-5), 126.7 (C-20), 133.7
(C-30), 138.3 (C-6), 150.5 (C-2), 161.4 (C-4), 170.2 (CH3COO).

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GAVRILIU ET AL.
50 -O-Acetyl-3-N-(4-bromo-2-(E)butenyl)-20, 30-didehydro-20,30-dideoxyur-
idine (5f). reagent: 1,4-dibromo-2-butene, solvent of the reaction mixture:
DMF; reaction time 1 h 30 min; eluent of the chromatography: methanol in
dichloromethane (from 0 to 1%); yield of 5f (18%) as a syrup; Rf 0.77
1
[EtOAc (100%)]; H NMR (CDCl3): d 2.07 (s, 3H, CH3COO), 3.92 (d, 2H,
CH2Br, J ¼ 7.3 Hz), 4.34 (dd, 1H, H-50, J ¼ 12.4, J ¼ 3.0 Hz), 4.34 (dd, 1H, H-
500, J ¼ 12.4, J ¼ 3.7 Hz), 4.56 (d, 2H, CH2N-3, J ¼ 6.0 Hz), 5.07 (bs, 1H, H-
40), 5.76 (d, 1H, H-5, J ¼ 8.0 Hz), 5.93 (d, 1H, H-20, J ¼ 6.0 Hz), 5.95–6.00 (m,
2H, CH ¼ CH), 6.31 (d, 1H, H-30, J ¼ 6.0 Hz), 7.03 (m, 1H, H-10), 7.44 (d,
1H, H-6, J ¼ 8.0 Hz); 13C NMR (CDCl3): d 20.7 (CH3), 31.6 (CH2Br), 41.7
(CH2N-3), 64.6 (C-50), 84.3 (C-40), 90.7 (C-10), 102.0 (C-5), 127.0 (C-20), 128.3
(CHCH2Br), 130.4 (CHCH2N-3), 133.5 (C-30), 137.9 (C-6), 151.0 (C-2), 162.1
(C-4), 170.2 (CH3COO).
50 -O-Acetyl-3-N-(3-bromopropyl)-20, 30-didehydro-20, 30-dideoxythymi-
dine (6a). solvent of the reaction mixture: DMF; reaction time 5 h; eluent of
the chromatography: ethyl acetate in dichloromethane (from 0 to 10%); yield
of 6a (34%) as a syrup; Rf 0.65 [EtOAc (100%)];1H NMR (CDCl3): d 1.93 (s,
3H, CH3-5), 2.10 (s, 3H, CH3COO), 2.23 (t, 2H, CH2CH2N-3, J ¼ 6.9 Hz), 3.44
(t, 2H, CH2Br, J ¼ 6.9 Hz), 4.09(t, 2H, CH2N-3, J ¼ 6.9 Hz), 4.25(dd, 1H, H-50,
J ¼ 12.4, J ¼ 2.9 Hz), 4.39 (dd, 1H, H-500, J ¼ 12.4, J ¼ 4.0 Hz), 5.06 (bs, 1H,
H-40), 5.93 (d, 1H, H-20, J ¼ 5.8 Hz), 6.31 (d, 1H, H-30, J ¼ 5.8 Hz), 7.04 (bs, 1H,
H-10), 7.23 (s, 1H, H-6); 13C NMR (CDCl3): d 13.1 (CH3-5), 20.7 (CH3COO),
30.3 (CH2CH2N-3), 30.8 (CH2Br), 40.2 (CH2N-3), 64.5 (C-50), 84.0 (C-40), 90.3
(C-10), 109.9 (C-5), 127.1 (C-20), 133.1 (C-30), 133.5 (C-6), 151.1 (C-2), 163.0
(C-4), 170.1 (CH3COO).
50 -O-Acetyl-3-N-(4-bromobutyl)-20, 30-didehydro-20, 30-dideoxythymidine
(6b). solvent of the reaction mixture: DMF; reaction time 5 h; eluent of the
chromatography: ethyl acetate in dichloromethane (from 0 to 100%); yield of
6b (73%) as a syrup; Rf 0.77 [EtOAc (100%)];1H NMR (CDCl3): d 1.78–1.84
(m, 2H, CH2CH2Br), 1.88–1.96 (m, 2H, CH2CH2N-3), 1.93 (s, 3H, CH3-5),
2.10 (s, 3H, CH3COO), 3.45 (t, 2H, CH2Br, J ¼ 6.6 Hz), 3.99 (d, 2H, CH2N-3,
J ¼ 7.1 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.3, J ¼ 2.9 Hz), 4.39 (dd, 1H, H-500,
J ¼ 12.3, J ¼ 4.0 Hz), 5.06 (bs, 1H, H-40), 5.93 (d, 1H, H-20, J ¼ 6.0 Hz), 6.31
(d, 1H, H-30, J ¼ 6.0 Hz), 7.05 (bs, 1H, H-10), 7.22 (s, 1H, H-6); 13C NMR
(CDCl3): d 13.1 (CH3-5), 20.6 (CH3COO), 26.1 (CH2CH2N-3), 29.9
(CH2CH2Br), 33.0 (CH2Br), 40.2 (CH2N-3), 64.5 (C-50), 83.9 (C-40), 90.3
(C-10), 109.9 (C-5), 127.1 (C-20), 133.0 (C-30), 133.4 (C-6), 151.1 (C-2), 163.1
(C-4), 170.1 (CH3COO).
50 -O-Acetyl-3-N-(6-bromohexyl)-20, 30-didehydro-20, 30-dideoxythymidine
(6c). solvent of the reaction mixture: DMF; reaction time 1 h 45 min; eluent
of the chromatography: ethyl acetate in dichloromethane (from 0 to 10%);

ANTI-HIV ACTIVITY OF HETERODIMERS
517
1
yield of 6c (87%) as a syrup; Rf 0.73 [EtOAc (100%)]; H NMR (CDCl3): d
1.40 (m, 2H, CH2(CH2)2Br), 1.49 (m, 2H, CH2(CH2)2N-3), 1.65 (m, 2H,
CH2CH2Br), 1.87 (m, 2H, CH2CH2N-3), 1.93 (s, 3H, CH3-5), 2.10 (s, 3H,
CH3COO), 3.40 (t, 2H, CH2Br, J ¼ 6.8 Hz), 3.94 (t, 2H, CH2N-3,
J ¼ 7.5 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.9 Hz), 4.38 (dd, 1H, H-500,
J ¼ 12.4, J ¼ 4.0 Hz), 5.06 (bs, 1H, H-40), 5.92 (d, 1H, H-20, J ¼ 6.0 Hz), 6.30
(d, 1H, H-30, J ¼ 6.0 Hz), 7.05 (bs, 1H, H-10), 7.21 (s, 1H, H-6); 13C NMR
(CDCl3): d 13.1 (CH3-5), 20.7 (CH3COO), 25.9 (CH2(CH2)2N-3), 27.1
(CH2(CH2)2Br), 27.6 (CH2CH2N-3), 32.4 (CH2CH2Br), 33.7 (CH2Br), 41.1
(CH2N-3), 64.5 (C-50), 83.9 (C-40), 90.3 (C-10), 109.9 (C-5), 127.2 (C-20), 133.0
(C-30), 133.3 (C-6), 151.1 (C-2), 163.1 (C-4), 170.1 (CH3COO).
50 -O-Acetyl-3-N-[2-(2-bromoethoxy)ethyl]-20, 30-didehydro-20, 30-dideox-
ythymidine (6d). Solvent of the reaction mixture: DMF; reaction time 1 h 30
min; eluent of the chromatography: methanol in dichloromethane (from 0 to
15%); yield of 6d (97%) as a syrup; Rf 0.63 [EtOAc (100%)];1H NMR
(CDCl3): d 1.78 (s, 3H, CH3-5), 1.95 (s, 3H, CH3COO), 3.30 (t, 2H, CH2Br,
J ¼ 5.6 Hz), 3.62 (t, 2H, BrCH2CH2O, J ¼ 5.6 Hz), 3.67 (m, 2H, OCH2CH2N-
3), 4.06 (m, 2H, CH2N-3), 4.09 (dd, 1H, H-50, J ¼ 12.1, J ¼ 2.2 Hz), 4.23 (dd,
1H, H-500, J ¼ 12.1, J ¼ 3.5 Hz), 4.91 (bs, 1H, H-40), 5.78 (d, 1H, H-20,
J ¼ 5.2 Hz), 6.16 (d, 1H, H-30, J ¼ 5.2 Hz), 6.89 (bs, 1H, H-10), 7.08 (s, 1H, H-
6); 13C NMR (CDCl3): d 13.2 (CH3-5), 20.7 (CH3COO), 30.6 (CH2Br), 39.7
(CH2N-3), 64.5 (C-50), 67.1 (OCH2CH2N-3), 70.1 (BrCH2CH2O), 83.9 (C-40),
90.3 (C-10), 109.8 (C-5), 127.1 (C-20), 133.1 (C-30), 133.6 (C-6), 151.2 (C-2),
163.1 (C-4), 170.1 (CH3COO).
50 -O-Acetyl-3-N-(4-chloro-2-butynyl)-20,
ythymidine (6e). solvent of the reaction mixture: DMF; reaction time 1 h;
30-didehydro-20,
30-dideox-
eluent of the chromatography: ethyl acetate in dichloromethane (from 0 to
1
10%); yield of 6e (76%) as a syrup; Rf 0.83 [EtOAc (100%)]; H NMR
(CDCl3): d 1.95 (s, 3H, CH3-5), 2.10 (s, 3H, CH3COO), 4.13 (t, 2H, CH2Cl,
J ¼ 1.9 Hz), 4.26 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.9 Hz), 4.40 (dd, 1H, H-500,
J ¼ 12.4, J ¼ 4.0 Hz), 4.77 (t, 2H, CH2N-3, J ¼ 1.9 Hz), 5.08 (bs, 1H, H-40),
5.95 (d, 1H, H-20, J ¼ 6.0 Hz), 6.34 (d, 1H, H-30, J ¼ 6.0 Hz), 7.05 (m, 1H, H-
10), 7.27 (s, 1H, H-6); 13C NMR (CDCl3): d 12.9 (CH3-5), 20.5 (CH3COO),
30.2 (CH2N-3), 30.4 (CH2Cl), 64.5 (C-50), 76.6 (ClCH2CꢄC), 80.7 (Cꢄ
CCH2N-3), 83.9 (C-40), 90.3 (C-10), 109.8 (C-5), 126.9 (C-20), 133.1 (C-30),
133.8 (C-6), 150.3 (C-2), 162.0 (C-4), 170.0 (CH3COO).
General Procedure for the Synthesis of the Heterodimers [50 -O-acetyl-
d4U or 50-O-acetyl-d4T]N3-spacer-Npiperazinyl[Trovirdine analogue] (7a–f) and
(8a–e). To a solution of the 50-O-acetyl-N-(n-bromo or n-chloro-spacer)-20,
30-didehydro-20, 30-dideoxy-nucleoside (5a^f and 6a^e) (1 equiv.) in dry
DMF were added K2CO3 (1.1 equiv.) and the Trovirdine analogue (4)

518
GAVRILIU ET AL.
(1.1 equiv.). The reaction mixture was heated at 80ꢁC for 8–16 h. After
evaporation of the solvent, the residue was dissolved in ethyl acetate (20 mL),
washed with water (2 ꢃ 20 mL), dried (MgSO4), filtered, and evaporated to
dryness. A flash chromatography of the residue was required to give the pure
heterodimers.
The reaction time, chromatography eluent, yield of the isolated pro-
1
ducts, H NMR and 13C NMR data, and elemental analysis are indicated
below each compound.
Heterodimer [50 -O-acetyl-d4U]N3-(CH2)3-Npiperazinyl[Trovirdine analo-
gue] (7a). reaction time 8 h; chromatography eluent: methanol in dichlor-
omethane (from 0% to 3%); yield of 7a (51%) as white crystals; Rf 0.50
1
[CH2Cl2-CH3OH (90:10)]; mp 139ꢁC; H NMR (DMSO-d6): d 1.68 (quint.,
2H, CH2CH2N-3, J ¼ 7.2 Hz), 2.00 (s, 3H, CH3COO), 2.32 (t, 2H,
CH2CH2CH2N-3, J ¼ 7.2 Hz), 2.34–2.48 (m, 8H, CH2 of piperazinyl), 2.49 (m,
2H, NHCH2CH2), 3.64 (q, 2H, NHCH2, J ¼ 5.2 Hz), 3.83 (t, 2H, CH2N-3,
J ¼ 7.2 Hz), 4.17 (d, 2H, H-50 and H-500, J ¼ 3.4 Hz), 5.00 (m, 1H, H-40), 5.79
(d, 1H, H-5, J ¼ 8.0 Hz), 6.00 (d, 1H, H-20, J ¼ 6.1 Hz), 6.44 (d, 1H, H-30,
J ¼ 6.1 Hz), 6.86 (m, 1H, H-10), 7.12 (d, 1H, H-III of pyridyl, J ¼ 9.0 Hz), 7.48
(d, 1H, H-6, J ¼ 8.0 Hz), 7.97 (dd, 1H, H-IV of pyridyl, J ¼ 9.0, J ¼ 2.4 Hz),
8.25 (d, 1H, H-VI of pyridyl, J ¼ 2.4 Hz), 10.69 (bs, 1H, ArNHCS), 11.34 (bs,
1H, CSNHCH2); 13C NMR (DMSO-d6): d 20.6 (CH3), 23.9 (CH2CH2N-3),
40.1 (CH2N-3), 42.0 (NHCH2), 52.4 (CH2 of piperazinyl), 52.8 (CH2 of
piperazinyl), 55.2 (NHCH2CH2), 55.5 (CH2CH2CH2N-3), 64.5 (C-50), 83.9
(C-40), 90.3 (C-10), 101.2 (C-5), 111.8 (C-V of pyridyl), 114.5 (C-III of pyridyl),
126.4 (C-20), 134.0 (C-30), 139.0 (C-6), 141.6 (C-IV of pyridyl), 145.9 (C-VI of
pyridyl), 150.4 (C-2), 152.4 (C-II of pyridyl), 162.0 (C-4), 170.1 (CH3COO),
178.7 (CS); Anal. Calcd for C26 H34 Br N7 O5 S: C, 49.06; H, 5.38; N, 15.40.
Found: C, 48.88; H, 5.32; N, 15.37.
Heterodimer [50 -O-acetyl-d4U]N3-(CH2)4-Npiperazinyl[Trovirdine analo-
gue] (7b). reaction time 6 h; chromatography eluent: methanol in dichlor-
omethane (from 0% to 4%); yield of 7b (24%) as white crystals; Rf 0.37
1
[CH2Cl2-CH3OH (90:10)]; mp 193ꢁC; H NMR (CDCl3): d 1.58 (m, 2H,
CH2(CH2)2N-3), 1.68 (m, 2H, CH2CH2N-3), 2.08 (s, 3H, CH3COO), 2.40
(t, 2H, CH2(CH2)3N-3, J ¼ 7.3 Hz), 2.55 (m, 8H, CH2 of piperazinyl),
2.65 (t, 2H, NHCH2CH2, J ¼ 5.7 Hz), 3.81 (m, 2H, NHCH2), 3.97 (t, 2H,
CH2N-3, J ¼ 6.6 Hz), 4.26 (dd, 1H, H-50, J ¼ 12.3, J ¼ 3.0 Hz), 4.35 (dd, 1H,
H-500, J ¼ 12.3, J ¼ 3.4 Hz), 5.07 (bs, 1H, H-40), 5.76 (d, 1H, H-5, J ¼ 8.0 Hz),
5.93 (d, 1H, H-20, J ¼ 5.7 Hz), 6.30 (d, 1H, H-30, J ¼ 5.7 Hz), 6.88 (d, 1H, H-
III of pyridyl, J ¼ 8.8 Hz), 7.04 (bs, 1H, H-10), 7.42 (d, 1H, H-6, J ¼ 8.0 Hz),
7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 1.8 Hz), 8.24 (d, 1H, H-VI of
pyridyl, J ¼ 1.8 Hz), 9.38 (bs, 1H, ArNHCS), 11.50 (bs, 1H, CSNHCH2);
13C NMR (CDCl3): d 20.7 (CH3), 24.2 (CH2CH2N-3), 25.5 (CH2(CH2)2N-3),

ANTI-HIV ACTIVITY OF HETERODIMERS
519
41.0 (CH2N-3), 42.8 (NHCH2), 52.6 (CH2 of piperazinyl), 53.4 (CH2 of
piperazinyl), 55.4 (NHCH2CH2), 58.2 (CH2(CH2)3N-3), 64.5 (C-50), 84.2
(C-40), 90.6 (C-10), 102.0 (C-5), 112.6 (C-V of pyridyl), 113.5 (C-III of pyridyl),
127.0 (C-20), 133.3 (C-30), 137.5 (C-6), 141.0 (C-IV of pyridyl), 146.5 (C-VI of
pyridyl), 151.2 (C-2), 151.8 (C-II of pyridyl), 162.5 (C-4), 170.2 (CH3COO),
178.5 (CS); Anal. Calcd for C27 H36 Br N7 O5 S: C, 49.85; H, 5.58; N, 15.07.
Found: C, 48.89; H, 5.49; N, 15.05.
Heterodimer [50 -O-acetyl-d4U]N3-(CH2)6-Npiperazinyl[Trovirdine analo-
gue] (7c). reaction time 14 h; chromatography eluent: methanol in dichloro-
methane (from 0% to 3%); yield of 7c (43%) as white crystals; Rf
0.42 [CH2Cl2-CH3OH (90:10)]; 1H NMR (CDCl3): d 1.41 (m, 4H,
(CH2)2(CH2)2N-3), 1.55 (m, 2H,CH2(CH2)5N-3), 1.68 (m, 2H, CH2CH2N-3),
2.10 (s, 3H, CH3COO), 2.38 (t, 2H, CH2(CH2)5N-3, J ¼ 8.0 Hz), 2.59 (m, 8H,
CH2 of piperazinyl), 2.68 (t, 2H, NHCH2CH2, J ¼ 6.3 Hz), 3.84 (m, 2H,
NHCH2), 3.95 (m, 2H, CH2N-3), 4.29 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.9 Hz),
4.37 (dd, 1H, H-500, J ¼ 12.4, J ¼ 3.6 Hz), 5.09 (m, 1H, H-40), 5.78 (d, 1H,
H-5, J ¼ 8.0 Hz), 5.95 (d, 1H, H-20, J ¼ 6.1 Hz), 6.32 (d, 1H, H-30, J ¼ 6.1 Hz),
6.84 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.07 (bs, 1H, H-10), 7.44 (d, 1H,
H-6, J ¼ 8.0 Hz), 7.75 (dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 2.2 Hz), 8.28 (d,
1H, H-VI of pyridyl, J ¼ 2.2 Hz), 9.13 (bs, 1H, ArNHCS), 11.50 (bs, 1H,
CSNHCH2); 13C NMR (CDCl3): d 21.0 (CH3), 27.0 (CH2(CH2)2N of spacer),
27.1 (CH2(CH2)2N of spacer), 27.5 (CH2CH2N of spacer), 27.7 (CH2CH2N of
spacer), 41.5 (CH2N-3), 43.2 (NHCH2), 53.0 (CH2 of piperazinyl), 53.7
(CH2 of piperazinyl), 55.8 (NHCH2CH2), 59.0 (CH2(CH2)5N-3), 64.9 (C-50),
84.5 (C-40), 91.0 (C-10), 102.4 (C-5), 113.0 (C-V of pyridyl), 113.7 (C-III
of pyridyl), 127.4 (C-20), 133.6 (C-30), 137.8 (C-6), 141.4 (C-IV of pyridyl),
147.0 (C-VI of pyridyl), 151.5 (C-2), 152.0 (C-II of pyridyl), 162.8 (C-4), 170.6
(CH3COO), 178.9 (CS); Anal. Calcd for C29 H40 Br N7 O5 S: C, 51.33;
H, 5.94; N, 14.45. Found: C, 51.19; H, 5.81; N, 14.37.
Heterodimer
[50 -O-acetyl-d4U]N3-CH2-CH2-O-CH2-CH2-Npiperazinyl
[Trovirdine analogue] (7d). reaction time 6 h; chromatography eluent:
methanol in dichloromethane (from 0% to 4%); yield of 7d (39%) as white
1
crystals; Rf 0.27 [CH2Cl2-CH3OH (90:10)]; mp 125ꢁC; H NMR (CDCl3): d
2.07 (s, 3H, CH3COO), 2.56 (m, 8H, CH2 of piperazinyl), 2.60 (t, 2H,
CH2CH2O(CH2)2N-3, J ¼ 5.7 Hz), 2.64 (t, 2H, NHCH2CH2, J ¼ 6.1 Hz), 3.66
(t, 2H, CH2O(CH2)2N-3, J ¼ 5.7 Hz), 3.71 (m, 2H, OCH2CH2N-3), 3.80 (m,
2H, NHCH2), 4.19 (t, 2H, CH2N-3, J ¼ 6.0 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.3,
J ¼ 3.0 Hz), 4.34 (dd, 1H, H-500, J ¼ 12.3, J ¼ 3.8 Hz), 5.06 (m, 1H, H-40), 5.74
(d, 1H, H-5, J ¼ 8.0 Hz), 5.91 (d, 1H, H-20, J ¼ 5.8 Hz), 6.29 (d, 1H, H-30,
J ¼ 5.8 Hz), 6.65 (d, 1H, H-III of pyridyl, J ¼ 9.0 Hz), 7.01 (m, 1H, H-10), 7.41
(d, 1H, H-6, J ¼ 8.0 Hz), 7.73 (dd, 1H, H-IV of pyridyl, J ¼ 9.0, J ¼ 2.4 Hz),
8.26 (d, 1H, H-VI of pyridyl, J ¼ 2.4 Hz), 8.41 (bs, 1H, ArNHCS), 11.41

520
GAVRILIU ET AL.
(bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 20.8 (CH3), 39.8 (CH2N-3), 43.1
(NHCH2), 52.6 (CH2 of piperazinyl), 53.9 (CH2 of piperazinyl), 55.5
(NHCH2CH2), 58.0 (CH2CH2O(CH2)2N-3), 64.6 (C-50), 67.5 (OCH2CH2N-3),
68.1 (CH2O(CH2)2N-3), 84.3 (C-40), 90.7 (C-10), 102.0 (C-5), 112.8 (C-V of
pyridyl), 113.1 (C-III of pyridyl), 127.1 (C-20), 133.4 (C-30), 137.7 (C-6), 141.2
(C-IV of pyridyl), 146.9 (C-VI of pyridyl), 151.4 (C-2), 151.5 (C-II of pyridyl),
162.6 (C-4), 170.5 (CH3COO), 178.8 (CS); Anal. Calcd for C27 H36 Br N7 O6
S: C, 48.65; H, 5.44; N, 14.71. Found: C, 48.59; H, 5.49; N, 14.60.
Heterodimer [50 -O-acetyl-d4U]N3-CH2-CꢄC-CH2-Npiperazinyl[Trovirdine
analogue] (7e). reaction time 2 h; chromatography eluent: methanol in
dichloromethane (from 0% to 3%); yield of 7e (42%) as white crystals; Rf
1
0.46 [CH2Cl2-CH3OH (90:10)]; mp 124ꢁC; H NMR (CDCl3): d 2.09 (s, 3H,
CH3COO), 2.59 (m, 8H, CH2 of piperazinyl), 2.66 (t, 2H, NHCH2CH2,
J ¼ 6.0 Hz), 3.28 (bs, 2H, CH2CꢄCCH2N-3), 3.83 (q, 2H, NHCH2,
J ¼ 5.6 Hz), 4.27 (dd, 1H, H-50, J ¼ 12.4, J ¼ 3.0 Hz), 4.36 (dd, 1H, H-500,
J ¼ 12.4, J ¼ 3.8 Hz), 4.77 (bs, 2H, CH2N-3), 5.09 (bs, 1H, H-40), 5.82 (d, 1H,
H-5, J ¼ 8.0 Hz), 5.95 (d, 1H, H-20, J ¼ 6.0 Hz), 6.33 (d, 1H, H-30, J ¼ 6.0 Hz),
6.97 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.05 (m, 1H, H-10), 7.47 (d, 1H, H-
6, J ¼ 8.0 Hz), 7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 2.4 Hz), 8.27 (d, 1H,
H-VI of pyridyl, J ¼ 2.4 Hz), 9.67 (bs, 1H, ArNHCS), 11.51 (bs, 1H,
CSNHCH2); 13C NMR (CDCl3): d 20.6 (CH3COO), 30.4 (CH2N-3), 42.6
(NHCH2), 47.0 (CH2CꢄCCH2N-3), 52.2 (CH2 of piperazinyl), 52.3 (CH2 of
piperazinyl), 55.3 (NHCH2CH2), 64.4 (C-50), 78.9 (CꢄC), 84.1 (C-40), 90.5 (C-
10), 101.7 (C-5), 112.4 (C-V of pyridyl), 113.5 (C-III of pyridyl), 126.7 (C-20),
133.3 (C-30), 137.9 (C-6), 140.8 (C-IV of pyridyl), 146.3 (C-VI of pyridyl),
150.4 (C-2), 151.7 (C-II of pyridyl), 161.4 (C-4), 170.1 (CH3COO), 178.3 (CS);
Anal. Calcd for C27 H32 Br N7 O5 S: C, 50.16; H, 4.99; N, 15.16. Found:
C, 50.22; H, 5.02; N, 14.98.
Heterodimer [50 -O-acetyl-d4U]N3-CH2-CH ¼ CH-CH2-Npiperazinyl[Trovir-
dine analogue] (7f). reaction time 5 h; chromatography eluent: methanol in
dichloromethane (from 0% to 3%); yield of 7f (71%) as white crystals; Rf 0.36
1
[CH2Cl2-CH3OH (90:10)]; mp 128ꢁC; H NMR (CDCl3): d 2.08 (s, 3H,
CH3COO), 2.55 (m, 8H, CH2 of piperazinyl), 2.65 (t, 2H, NHCH2CH2,
J¼ 6.1 Hz), 3.00 (d, 2H, CH2CH¼CHCH2N-3, J ¼ 4.6 Hz), 3.81 (q, 2H,
NHCH2, J ¼ 6.0 Hz), 4.26 (dd, 1H, H-50, J¼ 12.4, J ¼ 3.0 Hz), 4.35 (dd, 1H, H-
500, J ¼ 12.4, J ¼ 3.6 Hz), 4.56 (d, 2H, CH2N-3, J ¼ 3.9 Hz), 5.07 (m, 1H, H-40),
5.74 (m, 2H, CH¼CH), 5.78 (d, 1H, H-5, J ¼ 8.0 Hz), 5.93 (d, 1H, H-20,
J¼ 6.1Hz), 6.31 (d, 1H, H-30, J ¼ 6.1 Hz), 6.88 (d, 1H, H-III of pyridyl,
J¼ 8.8 Hz), 7.04 (m, 1H, H-10), 7.44 (d, 1H, H-6, J ¼ 8.0 Hz), 7.72 (dd, 1H, H-IV
of pyridyl, J¼ 8.8, J ¼ 2.4 Hz), 8.24 (d, 1H, H-VI of pyridyl, J ¼ 2.4 Hz), 9.44
(bs, 1H, ArNHCS), 11.48 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 20.7
(CH3), 42.1 (CH2N-3), 42.8 (NHCH2), 52.5 (CH2 of piperazinyl), 53.2 (CH2 of

ANTI-HIV ACTIVITY OF HETERODIMERS
521
piperazinyl), 55.4 (NHCH2CH2), 60.2 (CH2CH¼CHCH2N-3), 64.5 (C-50), 84.2
(C-40), 90.6 (C-10), 102.0 (C-5), 112.6 (C-V of pyridyl), 113.5 (C-III of pyridyl),
126.8 (C-20), 126.9 (CH¼CH), 129.9 (CH ¼ CH), 133.3 (C-30), 137.7 (C-6), 141.0
(C-IV of pyridyl), 146.5 (C-VI of pyridyl), 151.0 (C-2), 151.8 (C-II of pyridyl),
162.1 (C-4), 170.2 (CH3COO), 178.5 (CS); Anal. Calcd for C27 H34 Br N7 O5 S:
C, 50.00; H, 5.28; N, 15.12. Found: C, 49.87; H, 5.12; N, 15.17.
Heterodimer [50 -O-acetyl-d4T]N3-(CH2)3-Npiperazinyl[Trovirdine analogue]
(8a). reaction time 20 h; chromatography eluent: methanol in dichlor-
omethane (from 0% to 3%); yield of 8a (38%) as white crystals; Rf 0.33
[CH2Cl2-CH3OH (90:10)]; 1H NMR (CDCl3): d 1.86 (quint., 2H, CH2CH2N-
3, J ¼ 7.0 Hz), 1.93 (s, 3H, CH3-5), 2.10 (s, 3H, CH3COO), 2.48 (t, 2H,
CH2CH2CH2N-3, J ¼ 7.0 Hz), 2.53 (m, 8H, CH2 of piperazinyl), 2.64 (t, 2H,
NHCH2CH2, J ¼ 5.9 Hz), 3.81 (m, 2H, NHCH2), 4.03 (t, 2H, CH2N-3,
J ¼ 7.0 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.7 Hz), 4.39 (dd, 1H, H-500,
J ¼ 12.4, J ¼ 3.8 Hz), 5.05 (bs, 1H, H-40), 5.92 (d, 1H, H-20, J ¼ 5.7 Hz), 6.29
(d, 1H, H-30, J ¼ 5.7 Hz), 6.85 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.06 (bs,
1H, H-10), 7.20 (s, 1H, H-6), 7.72 (dd, 1H, H-IV of pyridyl, J ¼ 2.1,
J ¼ 8.8 Hz), 8.24 (d, 1H, H-VI of pyridyl, J ¼ 2.1 Hz), 9.29 (bs, 1H,
ArNHCS), 11.47 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.3 (CH3-5),
20.8 (CH3COO), 24.5 (CH2CH2N-3), 39.9 (CH2N-3), 42.8 (NHCH2), 52.6
(CH2 of piperazinyl), 53.2 (CH2 of piperazinyl), 55.5 (NHCH2CH2), 55.8
(CH2CH2CH2N-3), 64.7 (C-50), 84.0 (C-40), 90.4 (C-10), 110.5 (C-5), 112.6
(C-V of pyridyl), 113.5 (C-III of pyridyl), 127.3 (C-20), 133.0 (C-30), 133.4
(C-6), 141.0 (C-IV of pyridyl), 146.6 (C-VI of pyridyl), 151.3 (C-2), 151.8
(C-II of pyridyl), 163.4 (C-4), 170.3 (CH3COO), 178.6 (CS); Anal. Calcd for
C27 H36 Br N7 O5 S: C, 49.85; H, 5.58; N, 15.07. Found: C, 49.74; H, 5.42;
N, 14.99.
Heterodimer [50-O-acetyl-d4T]N3-(CH2)4-Npiperazinyl[Trovirdine analogue]
(8b). reaction time 20 h; chromatography eluent: methanol in dichlor-
omethane (from 0% to 3%); yield of 8b (39%) as white crystals; Rf 0.25
1
[CH2Cl2-CH3OH (90:10)]; H NMR (CDCl3): d 1.56 (m, 2H, CH2(CH2)2N-
3), 1.68 (m, 2H, CH2CH2N-3), 1.93 (s, 3H, CH3-5), 2.10 (s, 3H, CH3COO),
2.40 (t, 2H, CH2(CH2)3N-3, J ¼ 7.2 Hz), 2.55 (m, 8H, CH2 of piperazinyl),
2.65 (t, 2H, NHCH2CH2, J ¼ 5.7 Hz), 3.82 (m, 2H, NHCH2), 3.99 (t, 2H,
CH2N-3, J ¼ 6.5 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.3, J ¼ 2.0 Hz), 4.39 (dd, 1H,
H-500, J ¼ 12.3, J ¼ 3.5 Hz), 5.05 (bs, 1H, H-40), 5.92 (d, 1H, H-20, J ¼ 5.7 Hz),
6.29 (d, 1H, H-30, J ¼ 5.7 Hz), 6.91 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.07
(bs, 1H, H-10), 7.20 (s, 1H, H-6), 7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8,
J ¼ 1.8 Hz), 8.24 (d, 1H, H-VI of pyridyl, J ¼ 1.8 Hz), 9.49 (bs, 1H,
ArNHCS), 11.50 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.2 (CH3-5),
20.7 (CH3COO), 24.2 (CH2CH2N-3), 25.5 (CH2(CH2)2N-3), 41.2 (CH2N-3),
42.8 (NHCH2), 52.6 (CH2 of piperazinyl), 53.3 (CH2 of piperazinyl), 55.4

522
GAVRILIU ET AL.
(NHCH2CH2), 58.2 (CH2(CH2)3N-3), 64.6 (C-50), 83.9 (C-40), 90.3 (C-10),
110.0 (C-5), 112.6 (C-V of pyridyl), 113.5 (C-III of pyridyl), 127.2 (C-20), 133.0
(C-30), 133.6 (C-6), 140.9 (C-IV of pyridyl), 146.5 (C-VI of pyridyl), 151.2
(C-2), 151.8 (C-II of pyridyl), 163.2 (C-4), 170.2 (CH3COO), 178.4 (CS); Anal.
Calcd for C28 H38 Br N7 O5 S: C, 50.60; H, 5.76; N, 14.75. Found: C, 50.49;
H, 5.74; N, 14.69.
Heterodimer [50-O-acetyl-d4T]N3-(CH2)6-Npiperazinyl[Trovirdine analogue]
(8c). reaction time 24 h; chromatography eluent: methanol in dichlor-
omethane (from 0% to 3%); yield of 8c (23%) as white crystals; Rf 0.30
[CH2Cl2-CH3OH (90:10)]; 1H NMR (CDCl3): d 1.38 (m, 4H, (CH2)2
(CH2)2N-3), 1.52 (m, 2H, CH2(CH2)4N-3), 1.64 (m, 2H, CH2CH2N-3), 1.93
(s, 3H, CH3-5), 2.10 (s, 3H, CH3COO), 2.36 (t, 2H, CH2(CH2)5N-3,
J ¼ 7.6 Hz), 2.57 (m, 8H, CH2 of piperazinyl), 2.65 (t, 2H, NHCH2CH2,
J ¼ 5.9 Hz), 3.82 (m, 2H, NHCH2), 3.95 (m, 2H, CH2N-3), 4.25 (dd, 1H,
H-50, J ¼ 12.3, J ¼ 2.7 Hz), 4.39 (dd, 1H, H-500, J ¼ 12.3, J ¼ 3.9 Hz), 5.05 (bs,
1H, H-40), 5.92 (d, 1H, H-20, J ¼ 5.9 Hz), 6.28 (d, 1H, H-30, J ¼ 5.9 Hz), 6.87
(d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.07 (bs, 1H, H-10), 7.20 (s, 1H, H-6),
7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 2.2 Hz), 8.25 (d, 1H, H-VI of
pyridyl, J ¼ 2.2 Hz), 9.35 (bs, 1H, ArNHCS), 11.49 (bs, 1H, CSNHCH2); 13
C
NMR (CDCl3): d 13.3 (CH3-5), 20.8 (CH3COO), 26.6 (CH2(CH2)2N of
spacer), 26.8 (CH2(CH2)2N of spacer), 27.1 (CH2CH2N of spacer), 27.4
(CH2CH2N of spacer), 41.4 (CH2N-3), 42.8 (NHCH2), 52.6 (CH2 of piper-
azinyl), 53.4 (CH2 of piperazinyl), 55.4 (NHCH2CH2), 58.6 (CH2(CH2)5N-3),
64.7 (C-50), 84.0 (C-40), 90.3 (C-10), 110.1 (C-5), 112.6 (C-V of pyridyl), 113.5
(C-III of pyridyl), 127.4 (C-20), 133.0 (C-30), 133.3 (C-6), 141.0 (C-IV of
pyridyl), 146.6 (C-VI of pyridyl), 151.2 (C-2), 151.8 (C-II of pyridyl), 163.2
(C-4), 170.3 (CH3COO), 178.5 (CS); Anal. Calcd for C30 H42 Br N7 O5 S: C,
52.02; H, 6.11; N, 14.15. Found: C, 51.89; H, 6.14; N, 13.99.
Heterodimer
[50-O-acetyl-d4T]N3-CH2-CH2-O-CH2-CH2-Npiperazinyl
[Trovirdine analogue] (8d). reaction time 30 h; chromatography eluent:
methanol in dichloromethane (from 0% to 3%); yield of 8d (42%) as white
1
crystals; Rf 0.37 [CH2Cl2-CH3OH (90:10)]; mp 112ꢁC; H NMR (CDCl3): d
1.92 (s, 3H, CH3-5), 2.10 (s, 3H, CH3COO), 2.55 (m, 8H, CH2 of piperazinyl),
2.59 (t, 2H, CH2CH2O(CH2)2N-3, J ¼ 5.7 Hz), 2.64 (t, 2H, NHCH2CH2,
J ¼ 6.0 Hz), 3.66 (t, 2H, CH2O(CH2)2N-3, J ¼ 5.7 Hz), 3.72 (m, 2H,
OCH2CH2N-3), 3.81 (q, 2H, NHCH2, J ¼ 6.0 Hz), 4.20 (t, 2H, CH2N-3,
J ¼ 5.8 Hz), 4.24 (dd, 1H, H-50, J ¼ 12.3, J ¼ 2.6 Hz), 4.38 (dd, 1H, H-50,
J ¼ 12.3, J ¼ 3.9 Hz), 5.04 (bs, 1H, H-40), 5.90 (d, 1H, H-20, J ¼ 5.7 Hz), 6.28
(d, 1H, H-30, J ¼ 5.7 Hz), 6.80 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.03 (bs,
1H, H-10), 7.20 (s, 1H, H-6), 7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8,
J ¼ 2.2 Hz), 8.25 (d, 1H, H-VI of pyridyl, J ¼ 2.2 Hz), 9.04 (bs, 1H,
ArNHCS), 11.45 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.3 (CH3-5),

ANTI-HIV ACTIVITY OF HETERODIMERS
523
20.8 (CH3COO), 40.0 (CH2N-3), 42.9 (NHCH2), 52.5 (CH2 of piperazinyl),
53.8 (CH2 of piperazinyl), 55.5 (NHCH2CH2), 57.9 (CH2CH2O(CH2)2N-3),
64.7 (C-50), 67.4 (OCH2CH2N-3), 68.0 (CH2O(CH2)2N-3), 84.0 (C-40), 90.4
(C-10), 110.1 (C-5), 112.7 (C-V of pyridyl), 113.4 (C-III of pyridyl), 127.3
(C-20), 133.1(C-30), 133.5 (C-6), 141.1 (C-IV of pyridyl), 146.7 (C-VI of pyr-
idyl), 151.3 (C-2), 151.7 (C-II of pyridyl), 163.3 (C-4), 170.2 (CH3COO), 178.6
(CS); Anal. Calcd for C28 H38 Br N7 O6 S: C, 49.41; H, 5.63; N, 14.41. Found:
C, 49.32; H, 5.67; N, 14.29.
Heterodimer [50-O-acetyl-d4T]N3-CH2-CꢄC-CH2-Npiperazinyl[Trovirdine
analogue] (8e). reaction time 16 h; chromatography eluent: methanol in
dichloromethane (from 0% to 3%); yield of 8e (48%) as white crystals; Rf
1
0.46 [CH2Cl2-CH3OH (90:10)]; H NMR (CDCl3): d 1.94 (s, 3H, CH3-5),
2.10 (s, 3H, CH3COO), 2.59 (m, 8H, CH2 of piperazinyl), 2.66 (t, 2H,
NHCH2CH2, J ¼ 6.0 Hz), 3.26 (bs, 2H, CH2CꢄCCH2N-3), 3.82 (q, 2H,
NHCH2, J ¼ 5.6 Hz), 4.25 (dd, 1H, H-50, J ¼ 12.4, J ¼ 2.9 Hz), 4.39 (dd, 1H,
H-500, J ¼ 12.4, J ¼ 4.1 Hz), 4.78 (bs, 2H, CH2N-3), 5.05 (m, 1H, H-40), 5.92
(d, 1H, H-20, J ¼ 6.0 Hz), 6.29 (d, 1H, H-30, J ¼ 6.0 Hz), 6.85 (d, 1H, H-III of
pyridyl, J ¼ 8.8 Hz), 7.06 (m, 1H, H-10), 7.22 (s, 1H, H-6), 7.72 (dd, 1H, H-IV
of pyridyl, J ¼ 8.8, J ¼ 2.4 Hz), 8.27 (d, 1H, H-VI of pyridyl, J ¼ 2.4 Hz), 9.23
(bs, 1H, ArNHCS), 11.46 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.3
(CH3-5), 20.9 (CH3COO), 30.9 (CH2N-3), 43.0 (NHCH2), 47.3
(CH2CꢄCCH2N-3), 52.5 (CH2 of piperazinyl), 52.6 (CH2 of piperazinyl), 55.6
(NHCH2CH2), 64.7 (C-50), 77.5 (CꢄC), 79.4 (CꢄC), 84.2 (C-40), 90.6 (C-10),
110.3 (C-5), 112.8 (C-V of pyridyl), 113.6 (C-III of pyridyl), 127.4 (C-20), 133.2
(C-30), 133.8 (C-6), 141.1 (C-IV of pyridyl), 146.7 (C-VI of pyridyl), 150.7
(C-2), 151.9 (C-II of pyridyl), 162.4 (C-4), 170.4 (CH3COO), 178.8 (CS); Anal.
Calcd for C28 H34 Br N7 O5 S: C, 50.91; H, 5.19; N, 14.84. Found: C, 50.84;
H, 5.22; N, 14.87.
General Procedure for the Synthesis of the Heterodimers [d4U or d4T]
N3-spacer-Npiperazinyl[Trovirdine analogue] (9a–f) and (10a–e). To a solution
of the protected heterodimers (7a^f) and (8a^e) in methanol was added a
few crystals of NaCN. The reaction mixture was stirred at room temperature
overnight. The solvent was evaporated under reduced pressure and the oily
residue was purified by column chromatography (dichloromethane=methanol
85:15) to give the free heterodimers (9a^f) and (10a^ e) as white crystals.
1
The yield of the isolated products, H NMR and 13C NMR data, and
elemental analysis are indicated below each compound.
Heterodimer [d4U]N3-(CH2)3-Npiperazinyl[Trovirdine analogue] (9a). Yield
1
of 9a (91%); Rf 0.18 [CH2Cl2-CH3OH (90:10)]; H NMR (CDCl3): d 1.78
(quint., 2H, CH2CH2N-3, J ¼ 7.0 Hz), 2.40 (t, 2H, CH2CH2CH2N-3,
J ¼ 7.0 Hz), 2.47 (m, 8H, CH2 of piperazinyl), 2.58 (t, 2H, NHCH2CH2,

524
GAVRILIU ET AL.
J¼ 5.8 Hz), 3.73–3.76 (m, 3H, NHCH2 and H-50), 3.83 (dd, 1H, H-500, J¼ 11.9,
J¼ 2.2 Hz), 3.91 (t, 2H, CH2N-3, J ¼ 7.0 Hz), 4.88 (bs, 1H, H-40), 5.65 (d, 1H,
H-5, J ¼ 8.0 Hz), 5.81 (d, 1H, H-20, J ¼ 5.7 Hz), 6.29 (d, 1H, H-30, J ¼ 5.7 Hz),
6.84 (d, 1H, H-III of pyridyl, J ¼ 8.9Hz), 6.97 (bs, 1H, H-10), 7.64–7.68 (m, 2H,
H-6 and H-IV of pyridyl), 8.16 (d, 1H, H-VI of pyridyl, J ¼ 2.0 Hz), 9.47 (bs, 1H,
ArNHCS), 11.43 (bs, 1H, CSNHCH2); 13C NMR (CDCl3) d 24.3 (CH2CH2N-
3), 39.5 (CH2N-3), 42.6 (NHCH2), 52.4 (CH2 of piperazinyl), 53.0 (CH2 of
piperazinyl), 55.4 (NHCH2CH2), 55.7 (CH2CH2CH2N-3), 62.9 (C-50), 87.2
(C-40), 90.6 (C-10), 101.6 (C-5), 112.6 (C-V of pyridyl), 113.5 (C-III of pyridyl),
126.1 (C-20), 134.4 (C-30), 138.8 (C-6), 140.9 (C-IV of pyridyl), 146.4 (C-VI of
pyridyl), 151.3 (C-2), 151.7 (C-II of pyridyl), 162.8 (C-4), 178.4 (CS); Anal. Calcd
for C24 H32 Br N7 O4 S: C, 48.49; H, 5.42; N, 16.49. Found: C, 48.47; H, 5.40; N,
16.41.
Heterodimer [d4U]N3-(CH2)4-Npiperazinyl[Trovirdine analogue] (9b). Yield
of 9b (98%); Rf 0.20 [CH2Cl2-CH3OH (85:15)]; mp 181ꢁC; 1H NMR (CDCl3):
d 1.54–1.60 (m, 2H, CH2(CH2)2N-3), 1.63–1.69 (m, 2H, CH2CH2N-3), 2.40 (t,
2H, CH2(CH2)3N-3, J ¼ 7.4 Hz), 2.55 (m, 8H, CH2 of piperazinyl), 2.65 (t, 2H,
NHCH2CH2, J ¼ 6.0 Hz), 3.78–3.82 (m, 3H, H-50 and NHCH2), 3.90–3.97 (m,
3H, H-500 and CH2N-3), 4.94 (m, 1H, H-40), 5.72 (d, 1H, H-5, J¼ 8.0 Hz), 5.87
(d, 1H, H-20, J ¼ 5.8 Hz), 6.34 (d, 1H, H-30, J ¼ 5.8 Hz), 6.79 (d, 1H, H-III of
pyridyl, J ¼ 8.8 Hz), 7.03 (m, 1H, H-10), 7.63 (d, 1H, H-6, J¼ 8.0 Hz), 7.72 (dd,
1H, H-IV of pyridyl, J ¼ 8.8, J¼ 2.4 Hz), 8.25 (d, 1H, H-VI of pyridyl,
J¼ 2.4 Hz), 9.02 (bs, 1H, ArNHCS), 11.46 (bs, 1H, CSNHCH2); 13C NMR
(CDCl3): d 24.1 (CH2CH2N-3), 25.5 (CH2(CH2)2N-3), 41.0 (CH2N-3), 42.9
(NHCH2), 52.6 (CH2 of piperazinyl), 53.4 (CH2 of piperazinyl), 55.5
(NHCH2CH2), 58.2 (CH2(CH2)3N-3), 63.5 (C-50), 87.1 (C-40), 90.8 (C-10), 102.0
(C-5), 112.8 (C-V of pyridyl), 113.4 (C-III of pyridyl), 126.4 (C-20), 134.4 (C-30),
138.5 (C-6), 141.1 (C-IV of pyridyl), 146.7 (C-VI of pyridyl), 151.4 (C-2), 151.7
(C-II of pyridyl), 162.8 (C-4), 178.6 (CS); Anal. Calcd for C25 H34 Br N7 O4 S: C,
49.34; H, 5.63; N, 16.11. Found: C, 49.30; H, 5.60; N, 16.08.
Heterodimer [d4U]N3-(CH2)6-Npiperazinyl[Trovirdine analogue] (9c). Yield
1
of 9c (90%); Rf 0.20 [CH2Cl2-CH3OH (90:10)]; H NMR (CDCl3): d 1.36
(m, 4H, (CH2)2(CH2)2N-3), 1.52 (m, 2H, CH2(CH2)4N-3), 1.63 (m, 2H,
CH2CH2N-3), 2.37 (t, 2H, CH2(CH2)5N-3, J ¼ 7.7 Hz), 2.58 (m, 8H, CH2 of
piperazinyl), 2.66 (t, 2H, NHCH2CH2, J ¼ 6.1 Hz), 3.77–3.82 (m, 3H, H-50 and
NHCH2), 3.89–3.93 (m, 3H, H-500 and CH2N-3), 4.94 (bs, 1H, H-40), 5.72 (d, 1H,
H-5, J ¼ 8.0 Hz), 5.87 (d, 1H, H-20, J ¼ 5.8 Hz), 6.34 (d, 1H, H-30, J ¼ 5.8 Hz),
6.83 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.04 (bs, 1H, H-10), 7.67 (d, 1H, H-6,
J¼ 8.0 Hz), 7.72 (dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 2.2 Hz), 8.25 (d, 1H, H-VI
of pyridyl, J ¼ 2.2 Hz), 9.21 (bs, 1H, ArNHCS), 11.47 (bs, 1H, CSNHCH2); 13
C
NMR (CDCl3): d 26.3 (CH2(CH2)2N of spacer), 26.7 (CH2(CH2)2N of spacer),
27.1 (CH2CH2N of spacer), 27.3 (CH2CH2N of spacer), 41.1 (CH2N-3), 42.8

ANTI-HIV ACTIVITY OF HETERODIMERS
525
(NHCH2), 52.4 (CH2 of piperazinyl), 53.3 (CH2 of piperazinyl), 55.4
(NHCH2CH2), 58.5 (CH2(CH2)5N-3), 63.2 (C-50), 87.2 (C-40), 90.8 (C-10), 101.9
(C-5), 112.7 (C-V of pyridyl), 113.5 (C-III of pyridyl), 126.3 (C-20), 134.4 (C-30),
138.6 (C-6), 141.1 (C-IV of pyridyl), 146.6 (C-VI of pyridyl), 151.4 (C-2), 151.7
(C-II of pyridyl), 162.8 (C-4), 178.6 (CS); Anal. Calcd for C27 H38 Br N7 O4 S: C,
50.94; H, 6.02; N, 15.40. Found: C, 50.90; H, 5.97; N, 15.37.
Heterodimer [d4U]N3-CH2-CH2-O-CH2-CH2-Npiperazinyl[Trovirdine ana-
logue] (9d). Yield of 9d (91%); Rf 0.21 [CH2Cl2-CH3OH (90:10)]; mp
1
120ꢁC; H NMR (CDCl3): d 2.48–2.53 (m, 10H, CH2 of piperazinyl and
CH2CH2O(CH2)2N-3), 2.58 (t, 2H, NHCH2CH2, J ¼ 5.5 Hz), 3.58 (t,
2H, CH2O(CH2)2N-3, J ¼ 4.6 Hz), 3.64 (t, 2H, OCH2CH2N-3, J ¼ 5.2 Hz),
3.69–3.75 (m, 3H, H-50 and NHCH2), 3.82 (dd, 1H, H-50, J ¼ 12.0,
J ¼ 2.9 Hz), 4.12 (m, 2H, CH2N-3), 4.86 (bs, 1H, H-40), 5.64 (d, 1H, H-5,
J ¼ 8.0 Hz), 5.79 (d, 1H, H-20, J ¼ 5.8 Hz), 6.29 (d, 1H, H-30, J ¼ 5.8 Hz), and
H-IV of pyridyl), 8.17 (bs, 1H, H-VI of pyridyl), 9.46 (bs, 1H, ArNHCS),
11.42 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 39.7 (CH2N-3), 42.6
(NHCH2), 52.3 (CH2 of piperazinyl), 53.4 (CH2 of piperazinyl), 55.4
(NHCH2CH2), 57.6 (CH2CH2O(CH2)2N-3), 62.8 (C-50), 67.1 (OCH2CH2N-
3), 68.1 (CH2O(CH2)2N-3), 87.2 (C-40), 90.6 (C-10), 101.4 (C-5), 112.6 (C-V of
pyridyl), 113.6 (C-III of pyridyl), 125.9 (C-20), 134.6 (C-30), 138.9 (C-6), 140.9
(C-IV of pyridyl), 146.4 (C-VI of pyridyl), 151.4 (C-2), 151.5 (C-II of pyridyl),
162.8 (C-4), 178.5 (CS); Anal. Calcd for C25 H34 Br N7 O5 S: C, 48.08; H,
5.49; N, 15.70. Found: C, 48.00; H, 5.47; N, 15.67.
Heterodimer [d4U]N3-CH2-CꢄC-CH2-Npiperazinyl[Trovirdine analogue]
1
(9e). Yield of 9e (82%); Rf 0.36 [CH2Cl2-CH3OH (90:10)]; mp 150ꢁC; H
NMR (CDCl3): d 2.59 (m, 8H, CH2 of piperazinyl), 2.65 (t, 2H, NHCH2CH2,
J ¼ 6.1 Hz), 3.26 (bs, 2H, CH2CꢄCCH2N-3), 3.76–3.84 (m, 3H, H-50 and
NHCH2), 3.91 (dd, 1H, H-500, J ¼ 12.2, J ¼ 2.5 Hz), 4.74 (bs, 2H, CH2N-3),
4.94 (bs, 1H, H-40), 5.74 (d, 1H, H-5, J ¼ 8.1 Hz), 5.87 (d, 1H, H-20,
J ¼ 5.9 Hz), 6.35 (d, 1H, H-30, J ¼ 5.9 Hz), 6.82 (d, 1H, H-III of pyridyl,
J ¼ 8.8 Hz), 7.03 (bs, 1H, H-10), 7.72 (m, 2H, H-6 and H-IV of pyridyl), 8.27
(d, 1H, H-VI of pyridyl, J ¼ 2.1 Hz), 9.19 (bs, 1H, ArNHCS), 11.44 (bs, 1H,
CSNHCH2); 13C NMR (CDCl3): d 30.6 (CH2N-3), 42.9 (NHCH2), 47.1
(CH2CꢄCCH2N-3), 52.3 (CH2 of piperazinyl), 52.5 (CH2 of piperazinyl), 55.5
(NHCH2CH2), 63.1 (C-50), 79.3 (CꢄC), 87.2 (C-40), 90.8 (C-10), 101.7 (C-5),
112.7 (C-V of pyridyl), 113.5 (C-III of pyridyl), 126.3 (C-20), 134.5 (C-30),
139.1 (C-6), 141.1 (C-IV of pyridyl), 146.6 (C-VI of pyridyl), 150.8 (C-2),
151.7 (C-II of pyridyl), 161.8 (C-4), 178.4 (CS); Anal. Calcd for C25 H30 Br N7
O4 S: C, 49.67; H, 5.00; N, 16.22. Found: C, 49.60; H, 4.94; N, 16.19.
Heterodimer [d4U]N3-CH2-CH¼CH-CH2-Npiperazinyl[Trovirdine analo-
gue] (9f). Yield of 9f (74%); Rf 069 [CH2Cl2-CH3OH (80:20)]; mp 126ꢁC;

526
GAVRILIU ET AL.
1H NMR (CDCl3): d 2.56 (m, 8H, CH2 of piperazinyl), 2.64 (m, 2H,
NHCH2CH2), 3.01 (m, 2H, CH2CH¼CHCH2N-3), 3.80–3.95 (m, 4H, H-
50,H-500 and NHCH2), 4.55 (m, 2H, CH2N-3), 4.96 (m, 1H, H-40), 5.77 (m,
3H, CH¼CH and H-5), 5.89 (d, 1H, H-20, J ¼ 6.8 Hz), 6.37 (d, 1H, H-30,
J ¼ 6.8 Hz), 6.83 (d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.04 (m, 1H, H-10), 7.73
(m, 2H, H-6 and H-IV of pyridyl), 8.26 (d, 1H, H-VI of pyridyl, J ¼ 2.2 Hz),
9.23 (bs, 1H, ArNHCS), 11.49 (bs, 1H, CSNHCH2); 13C NMR (CDCl3):
d 42.1 (CH2N-3), 42.8 (NHCH2), 52.5 (CH2 of piperazinyl), 53.2 (CH2 of
piperazinyl), 55.5 (NHCH2CH2), 60.2 (CH2CH¼CHCH2N-3), 63.1 (C-50),
87.2 (C-40), 90.8 (C-10), 101.8 (C-5), 112.7 (C-V of pyridyl), 113.5 (C-III of
pyridyl), 126.2 (C-20), 127.1 (CH¼CH), 129.7 (CH¼CH), 134.5 (C-30), 138.8
(C-IV of pyridyl), 141.1 (C-6), 146.6 (C-VI of pyridyl), 151.2 (C-2), 151.7 (C-II
of pyridyl), 162.5 (C-4), 178.7 (CS); Anal. Calcd for C25 H32 Br N7 O4 S: C,
49.51; H, 5.32; N, 16.17. Found: C, 49.45; H, 5.27; N, 15.98.
Heterodimer [d4T]N3-(CH2)3-Npiperazinyl[Trovirdine analogue] (10a). Yield
of 10a (89%); Rf 0.21 [CH2Cl2-CH3OH (90:10)]; 1H NMR (CDCl3): d 1.85 (m,
2H, CH2CH2N-3), 1.88 (s, 3H, CH3-5), 2.47 (t, 2H, CH2CH2CH2N-3,
J¼ 7.1 Hz), 2.54 (m, 8H, CH2 of piperazinyl), 2.64 (t, 2H, NHCH2CH2,
J¼ 5.9 Hz), 3.78–3.82 (m, 3H, NHCH2 and H-50), 3.91 (dd, 1H, H-500, J¼ 12.1,
J¼ 2.5 Hz), 4.00 (t, 2H, CH2N-3, J ¼ 7.2 Hz), 4.92 (bs, 1H, H-40), 5.87 (d, 1H,
H-20, J ¼ 5.7 Hz), 6.34 (d, 1H, H-30, J¼ 5.7 Hz), 6.81 (d, 1H, H-III of pyridyl,
J¼ 8.8 Hz), 7.05 (bs, 1H, H-10), 7.48 (s, 1H, H-6), 7.72 (dd, 1H, H-IV of pyridyl,
J¼ 8.8, J ¼ 2.1 Hz), 8.23 (d, 1H, H-VI of pyridyl, J ¼ 2.1Hz), 9.20 (bs, 1H,
ArNHCS), 11.46 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.1 (CH3-5), 24.5
(CH2CH2N-3), 39.8 (CH2N-3), 42.8 (NHCH2), 52.6 (CH2 of piperazinyl), 53.1
(CH2 of piperazinyl), 55.5 (NHCH2CH2), 55.8 (CH2CH2CH2N-3), 63.5 (C-50),
87.0 (C-40), 90.6 (C-10), 110.0 (C-5), 112.7 (C-V of pyridyl), 113.5 (C-III of pyri-
dyl), 126.5 (C-20), 134.2 (C-30), 134.6 (C-6), 141.0 (C-IV of pyridyl), 146.6 (C-VI
of pyridyl), 151.5 (C-2), 151.7 (C-II of pyridyl), 163.5 (C-4), 178.6 (CS); Anal.
Calcd for C25 H34 Br N7 O4 S: C, 49.34; H, 5.63; N, 16.11. Found: C, 49.27; H,
5.58; N, 15.96.
Heterodimer [d4T]N3-(CH2)4-Npiperazinyl[Trovirdine analogue] (10b). Yield
of 10b (98%); Rf 0.20 [CH2Cl2-CH3OH (90:10)]; mp 186ꢁC; 1H NMR (CDCl3):
d 1.52–1.57 (m, 2H, CH2(CH2)2N-3), 1.60–1.66 (m, 2H, CH2CH2N-3), 1.88 (s,
3H, CH3-5), 2.38 (t, 2H, CH2(CH2)3N-3, J¼ 7.2 Hz), 2.55 (m, 8H, CH2 of
piperazinyl ), 2.64 (t, 2H, NHCH2CH2, J¼ 5.9 Hz), 3.77–3.83 (m, 3H, H-50 and
NHCH2), 3.89–3.98 (m, 3H, H-500 and CH2N-3), 4.93 (bs, 1H, H-40), 5.86 (d, 1H,
H-20, J ¼ 5.7 Hz), 6.34 (d, 1H, H-30, J¼ 5.7 Hz), 6.86 (d, 1H, H-III of pyridyl,
J¼ 8.8 Hz), 7.06 (bs, 1H, H-10), 7.45 (s, 1H, H-6), 7.72 (dd, 1H, H-IV of pyridyl,
J¼ 8.8, J ¼ 2.1 Hz), 8.24 (d, 1H, H-VI of pyridyl, J ¼ 2.1Hz), 9.29 (bs, 1H,
ArNHCS), 11.47 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.1 (CH3-5), 24.1
(CH2CH2N-3), 25.5 (CH2(CH2)2N-3), 41.2 (CH2N-3), 42.8 (NHCH2), 52.6

ANTI-HIV ACTIVITY OF HETERODIMERS
527
(CH2 of piperazinyl), 53.4 (CH2 of piperazinyl), 55.5 (NHCH2CH2), 58.2
(CH2(CH2)3N-3), 63.5 (C-50), 87.0 (C-40), 90.6 (C-10), 110.0 (C-5), 112.7 (C-V of
pyridyl), 113.5 (C-III of pyridyl), 126.5 (C-20), 134.2 (C-30), 134.4 (C-6), 141.0
(C-IV of pyridyl), 146.5 (C-VI of pyridyl), 151.4 (C-2), 151.8 (C-II of pyridyl),
163.4 (C-4), 178.5 (CS); Anal. Calcd for C26 H36 Br N7 O4 S: C, 50.16; H, 5.83; N,
15.75. Found: C, 50.10; H, 5.74; N, 15.67.
Heterodimer [d4T]N3-(CH2)6-Npiperazinyl[Trovirdine analogue] (10c). Yield
1
of 10c (98%); Rf 0.15 [CH2Cl2-CH3OH (90:10)]; H NMR (CDCl3): d 1.36
(m, 4H, (CH2)2(CH2)2N-3), 1.52 (m, 2H, CH2(CH2)4N-3), 1.62 (m, 2H,
CH2CH2N-3), 1.88 (s, 3H, CH3-5), 2.37 (t, 2H, CH2(CH2)5N-3, J ¼ 7.6 Hz), 2.58
(m, 8H, CH2 of piperazinyl), 2.65 (t, 2H, NHCH2CH2, J ¼ 5.9 Hz), 3.76–3.82 (m,
3H, H-50 and NHCH2), 3.87–3.93 (m, 3H, H-500 and CH2N-3), 4.92 (bs, 1H, H-
40), 5.85 (d, 1H, H-20, J ¼ 5.8 Hz), 6.33 (d, 1H, H-30, J ¼ 5.8 Hz), 6.79 (d, 1H, H-
III of pyridyl, J ¼ 8.8 Hz), 7.05 (bs, 1H, H-10), 7.46 (s, 1H, H-6), 7.72 (dd, 1H, H-
IV of pyridyl, J¼ 8.8, J ¼ 2.0 Hz), 8.24 (bs, 1H, H-VI of pyridyl), 9.15 (bs, 1H,
ArNHCS), 11.45 (bs, 1H, CSNHCH2); 13C NMR (CDCl3): d 13.1 (CH3-5), 26.3
(CH2(CH2)2N of spacer), 26.7 (CH2(CH2)2N of spacer), 27.1 (CH2CH2N of
spacer), 27.3 (CH2CH2N of spacer), 41.3 (CH2N-3), 42.8 (NHCH2), 52.3 (CH2 of
piperazinyl), 53.2 (CH2 of piperazinyl), 55.4 (NHCH2CH2), 58.5 (CH2(CH2)5N-
3), 63.3 (C-50), 87.0 (C-40), 90.6 (C-10), 110.0 (C-5), 112.7 (C-V of pyridyl), 113.5
(C-III of pyridyl), 126.5 (C-20), 134.2 (C-30), 134.5 (C-6), 141.1 (C-IV of pyridyl),
146.6 (C-VI of pyridyl), 151.5 (C-2), 151.7 (C-II of pyridyl), 163.5 (C-4), 178.7
(CS); Anal. Calcd for C28 H40 Br N7 O4 S: C, 51.69; H, 6.20; N, 15.07. Found:
C, 51.60; H, 6.14; N, 14.92.
Heterodimer [d4T]N3-CH2-CH2-O-CH2-CH2-Npiperazinyl[Trovirdine ana-
logue] (10d). Yield of 10d (40%); Rf 0.14 [CH2Cl2-CH3OH (90:10)]; mp
1
130ꢁC; H NMR (CDCl3): d 1.87 (s, 3H, CH3-5), 2.53 (m, 8H, CH2 of
piperazinyl), 2.57 (t, 2H, CH2CH2O(CH2)2N-3, J ¼ 5.3 Hz), 2.63 (t, 2H,
NHCH2CH2, J ¼ 6.1 Hz), 3.64 (t, 2H, CH2O(CH2)2N-3, J ¼ 5.2 Hz), 3.71
(t, 2H, OCH2CH2N-3, J ¼ 5.7 Hz), 3.76–3.82 (m, 3H, H-50 and NHCH2),
3.90 (dd, 1H, H-500, J ¼ 12.2, J ¼ 2.1 Hz), 4.20 (m, 2H, CH2N-3), 4.92 (bs, 1H,
H-40), 5.85 (d, 1H, H-20, J ¼ 5.8 Hz), 6.33 (d, 1H, H-30, J ¼ 5.8 Hz), 6.79 (d,
1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.03 (bs, 1H, H-10), 7.44 (s, 1H, H-6), 7.72
(dd, 1H, H-IV of pyridyl, J ¼ 8.8, J ¼ 2.2 Hz), 8.25 (d, 1H, H-VI of pyridyl,
J ¼ 1.9 Hz), 8.99 (bs, 1H, ArNHCS), 11.43 (bs, 1H, CSNHCH2); 13C NMR
(CDCl3): d 13.1 (CH3-5), 40.0 (CH2N-3), 42.9 (NHCH2), 52.6 (CH2 of piper-
azinyl), 53.6 (CH2 of piperazinyl), 55.6 (NHCH2CH2), 57.8 (CH2CH2O-
(CH2)2N-3), 63.3 (C-50), 67.3 (OCH2CH2N-3), 68.4 (CH2O(CH2)2N-3), 87.0
(C-40), 90.7 (C-10), 109.9 (C-5), 112.8 (C-V of pyridyl), 113.4 (C-III of pyridyl),
126.5 (C-20), 134.3 (C-30), 134.6 (C-6), 141.1 (C-IV of pyridyl), 146.7 (C-VI of
pyridyl), 151.4 (C-2), 151.7 (C-II of pyridyl), 163.3 (C-4), 178.6 (CS); Anal.

528
GAVRILIU ET AL.
Calcd for C26 H36 Br N7 O5 S: C, 48.90; H, 5.68; N, 15.35. Found: C, 48.81;
H, 5.74; N, 15.48.
Heterodimer [d4T]N3-CH2-CꢄC-CH2-Npiperazinyl[Trovirdine analogue]
(10e). Yield of 10e (95%); Rf 0.36 [CH2Cl2-CH3OH (90:10)]; mp 119ꢁC;
1H NMR (CDCl3): d 1.89 (s, 3H, CH3-5), 2.60 (m, 8H, CH2 of piperazinyl),
2.66 (t, 2H, NHCH2CH2, J ¼ 6.0 Hz), 3.26 (bs, 2H, CH2CꢄCCH2N-3),
3.78–3.83 (m, 4H, H-50 and NHCH2), 4.76 (bs, 2H, CH2N-3), 4.92 (m, 1H,
H-40), 5.86 (d, 1H, H-20, J ¼ 5.7 Hz), 6.36 (d, 1H, H-30, J ¼ 5.7 Hz), 6.87
(d, 1H, H-III of pyridyl, J ¼ 8.8 Hz), 7.05 (bs, 1H, H-10), 7.59 (s, 1H, H-6),
7.73 (dd, 1H, H-IV of pyridyl, J ¼ 8.7, J ¼ 2.0 Hz), 8.26 (bs, 1H, H-VI of
pyridyl), 9.44 (bs, 1H, ArNHCS), 11.49 (bs, 1H, CSNHCH2); 13C NMR
(CDCl3): d 13.2 (CH3-5), 31.0 (CH2N-3), 42.9 (NHCH2), 47.3 (CH2Cꢄ
CCH2N-3), 52.4 (CH2 of piperazinyl), 52.6 (CH2 of piperazinyl), 55.7
(NHCH2CH2), 63.3 (C-50), 77.3 (CꢄC), 79.8 (CꢄC), 87.4 (C-40), 90.8 (C-10),
110.1 (C-5), 112.9 (C-V of pyridyl), 113.9 (C-III of pyridyl), 126.5 (C-20), 134.7
(C-30), 135.3 (C-6), 141.3 (C-IV of pyridyl), 146.7 (C-VI of pyridyl), 151.0
(C-2), 152.1 (C-II of pyridyl), 163.0 (C-4), 179.0 (CS); Anal. Calcd for C26 H32
Br N7 O4 S: C, 50.49; H, 5.21; N, 15.85. Found: C, 50.29; H, 4.98; N, 15.73.
Antiviral Test Procedures
The cultures of CEM-SS and MT4 cells were maintained at 37ꢁC in
a 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10%
complement-depleted foetal bovine serum (FBS). The antiviral HIV-1 activity
of a given compound in CEM-SS cells was measured by quantification of the
reverse transcriptase activity (RT) associated with particles released from
HIV-1LAI-infected cells in the culture medium. CEM-SS cells were infected
with 100 TCDI50 (the virus stock was titrated under the same experimental
conditions); after 30 nm adsorption, free virus particles were washed out and
cells were re-suspended in RPMI-1640 plus 10% foetal calf serum at a final
concentration of 105 cells mLꢅ1 in the presence of different concentrations of
test compounds. After 5 days, virus production was measured by RT assay as
already described.[40] The 50% inhibitory concentration (IC50) was derived
from the computer generated median effect plot of the dose-effect data.[41]
The cytotoxicity of the drugs was evaluated in parallel by incubating unin-
fected cells in the presence of different concentrations of antiviral products.
The cell viability was determined by a measure of mitochondrial dehy-
drogenase activity, enzymes reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) into formazan (whose quantity was measured
by the absorbance at 540 nm).[42] The 50% cytotoxic concentration (CC50) is
the concentration of drug which reduces cell viability by 50% and was cal-
culated with the program used in the determination of the IC50. The assays
using different cells and virus isolates were done according to previously

ANTI-HIV ACTIVITY OF HETERODIMERS
529
published protocols;[40,43] virus production was quantified by the RT activity
associated to virus particles released from the cells in the culture medium.
Conditions in which the inhibitory properties were measured on HIV-1
reverse transcriptase in vitro has also been described.[40] In vitro RT inhibi-
tion was also described.[40] The CEM-SS cells were obtained from P. Nara
through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH (Bethesda, Md., USA).
ACKNOWLEDGMENTS
We thank S. Schmidt and G. Albrecht for excellent technical assistance
in the antiviral and cytotoxic assays. This work was supported by Ensemble
Contre le SIDA grants.
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Received September 5, 2001
Accepted June 14, 2002