Synthesis and structure-activity relationships of phenylenebis(methylene)-linked bis-azamacrocycles that inhibit HIV-1 and HIV- 2 replication by antagonism of the chemokine receptor CXCR4

Article abstract of DOI:10.1021/jm990211i

Bis-tetraazamacrocycles such as the bicyclam AMD3100 are a class of potent and selective anti-HIV-1 and HIV-2 agents that inhibit virus replication by binding to the chemokine receptor CXCR4, the co-receptor for entry of X4 viruses. With the aim of optimi

Full text of DOI:10.1021/jm990211i

J . Med. Chem. 1999, 42, 3971-3981
3971
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of P h en ylen ebis(m eth ylen e)-
Lin k ed Bis-a za m a cr ocycles Th a t In h ibit HIV-1 a n d HIV-2 Rep lica tion by
An ta gon ism of th e Ch em ok in e Recep tor CXCR4
Gary J . Bridger,*,† Renato T. Skerlj,† Sreenivasan Padmanabhan,‡ Stephen A. Martellucci,‡
Geoffrey W. Henson,† Sofie Struyf,§ Myriam Witvrouw,§ Dominique Schols,§ and Erik De Clercq§
AnorMED Inc., 200-20353 64th Avenue, Langley, British Columbia V2Y 1N5, Canada, J ohnson Matthey Pharmaceutical
Research, 1401 King Road, West Chester, Pennsylvania 19380, and Rega Institute for Medical Research, Katholieke
Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received April 27, 1999
Bis-tetraazamacrocycles such as the bicyclam AMD3100 are a class of potent and selective
anti-HIV-1 and HIV-2 agents that inhibit virus replication by binding to the chemokine receptor
CXCR4, the co-receptor for entry of X4 viruses. With the aim of optimizing the anti-HIV-1 and
HIV-2 activity of bis-azamacrocycles, a series of analogues were synthesized which contain
neutral heteroatom (oxygen, sulfur) or heteroaromatic (of lower pKa than a secondary amine)
replacements for the amino groups of AMD3100. The introduction of one or more heteroatoms
such as oxygen or sulfur into the macrocyclic ring of p-phenylenebis(methylene)-linked dimers
(to give N3X or N2X2 bis-macrocycles) gave analogues with substantially reduced anti-HIV-1
(IIIB) and anti-HIV-2 (ROD) potency. In addition, the bis-sulfur analogue was also markedly
more cytotoxic to MT-4 cells. However, bis-tetraazamacrocycles featuring a single pyridine group
incorporated within the macrocyclic framework exhibited anti-HIV-1 and HIV-2 potency
comparable to that of their saturated, aliphatic counterparts. The p-phenylenebis(methylene)-
linked dimer of the py[14]aneN4 macrocycle inhibited HIV-1 replication at a 50% effective
concentration (EC50) of 0.5 µM while remaining nontoxic to MT-4 cells at concentrations
approaching 200 µM. A series of analogues containing macrocyclic heteroaromatic groups of
varying pKa were also synthesized, and their ability to inhibit HIV replication was evaluated.
Replacing the pyridine moiety of the py[14]aneN4 macrocyclic ring with pyrazine or pyridine
groups substituted in the 4-position (with electron-withdrawing or -donating groups) either
reduced antiviral potency or increased cytotoxicity to MT-4 cells. Finally, we synthesized a
series of analogues in which the ring size of the bis-pyridyl macrocycles was varied between
12 and 16 members per ring including the py[iso-14]aneN4 ring system, an isomer of the py-
[14]aneN4 macrocycle. The p-phenylenebis(methylene)-linked dimer of the py[iso-14]aneN4
(AMD3329) displayed the highest antiviral activity of the bis-azamacrocyclic analogues reported
to date, exhibiting EC50’s against the cytopathic effects of HIV-1 and HIV-2 replication of 0.8
and 1.6 nM, respectively, that is, about 3-5-fold lower than the EC50 of AMD3100. AMD3329
also inhibited the binding of a specific CXCR4 mAb and the Ca2+ flux induced by SDF-1R, the
natural ligand for CXCR4, more potently than AMD3100. Furthermore, AMD3329 also
interfered with virus-induced syncytium formation at an EC50 of 12 nM.
In tr od u ction
We have previously reported that bis-azamacrocycles
such as the bicyclam AMD3100 exhibit potent and
selective inhibition of HIV-1 and HIV-2 replication in
vitro2-7 by binding to the chemokine receptor CXCR4,8-13
the co-receptor utilized by T-tropic (X4) HIV viruses for
membrane fusion and subsequent entry of the virus into
the cell. Recent reports indicate that antiviral agents
targeted at CXCR4 may have several significant thera-
peutic advantages. For example, CCR5 and CXCR4
were found to be the only physiologically relevant
chemokine co-receptors used for viral entry of both
laboratory and primary strains of HIV-1 isolated from
blood, and progression to X4 using (syncytium-inducing)
HIV-1 strains was associated with a more rapid disease
course and a faster CD4+ T-cell decline.14 Furthermore,
Verdin et al.15 and Hesselgesser et al.16 have observed
apoptosis of CD8+ T-cells and neuronal cells, respec-
tively, induced by direct interaction of the HIV-1
The formally approved chemotherapeutic agents for
the treatment of HIV infections such as the reverse
transcriptase inhibitors, HIV protease inhibitors, and,
more recently, nonnucleoside reverse transcriptase in-
hibitors interfere with critical steps in the HIV-repli-
cative cycle following release of the viral RNA into the
cell.1 Nevertheless, the identification of potent anti-HIV
agents that disrupt early steps in the HIV-replicative
cycle preceding the reverse transcription (RT) step
remains an important goal to both ameliorate existing
therapies and possibly impede the development of drug-
resistant strains.
* To whom correspondence should be addressed. Tel: (604)-532-
4652. Fax: (604)-530-0976. E-mail: gbridger@anormed.com.
† AnorMED Inc.
‡ J ohnson Matthey Pharmaceutical Research.
§ Rega Institute for Medical Research.
10.1021/jm990211i CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/26/1999

3972 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Bridger et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (a) (EtO)2POCl, Et3N, CH2Cl2; (b) H2, Ra Ni, NH3,
MeOH; (c) Ts-Cl, Et3N, CH2Cl2; (d) Cs2CO3, DMF, 65-70 °C; (e)
HBr, HOAc, rt; (f) 0.5 equiv p-dibromoxylene, K2CO3, CH3CN,
reflux; (g) 48% aq HBr, HOAc, reflux.
a
Ch em istr y
Reagents: (a) Et3N, CH2Cl2, rt; (b) BH3‚THF; (c) Ts-Cl, Et3N,
CH2Cl2; (d) ethylene glycol ditosylate, Cs2CO3, DMF, 60-70 °C;
(e) Na/Hg amalgam; (f) 1.0 equiv Ts-Cl, Et3N, CH2Cl2; (g) 0.5 equiv
p-dibromoxylene, K2CO3, CH3CN reflux.
The synthetic route used to prepare phenylenebis-
(methylene)-linked [14]aneN2X2 dimers is shown in
Scheme 1. The left-hand portion of the dithia ring
system was prepared by reaction of ethanedithiol and
bromopropionitrile in CH2Cl2 containing Et3N to give
the dithia-dinitrile 1 in 88% yield. A straightforward
reduction of the dinitrile with BH3‚THF and subsequent
derivatization with p-toluenesulfonyl chloride under
standard conditions gave the ditosylate 3a . Macrocy-
clization of 3a was accomplished using our previously
established conditions.4,18 Dropwise addition of a DMF
solution of ethyleneglycol ditosylate into a solution of
3a in DMF containing Cs2CO3 gave the fully protected
[14]aneN2S2 macrocycle 4a in 42% yield following
purification by column chromatography on silica gel. To
complete the synthesis of the symmetrical dimer 7a , the
ditosylated macrocycle 4a was converted to the mono-
tosylated intermediate 5a by a two-step procedure
involving deprotection of the tosyl groups with 3%
sodium amalgam followed by monoamine protection.
Dimerization of the available secondary amine in 5a
with R,R′-dibromo-p-xylene in refluxing CH3CN in the
presence of K2CO3, as previously described,4,5 gave the
ditosyl-protected dimer 6a which was deprotected with
1% sodium amalgam to give the free base of 7a .
Inexplicably, deprotection of 6a with 3% sodium amal-
gam led to decomposition. The free base was finally
converted to the tetrahydrochloride salt giving 7a . The
corresponding [14]aneN2O2 bis-azamacrocycle 7b was
prepared in a manner similar to that for 7a starting
from the commercially available dioxo-diamine 2b.
Bis-azamacrocycles featuring a single heteroatom
substitution per macrocyclic ring (N3X macrocycles)
were synthesized as illustrated in Scheme 2. Due to the
envelope glycoprotein gp120 with CXCR4. Thus, CXCR4
antagonists may exhibit potent antiviral activity and
concomitant protection of the immune system in vivo.
To demonstrate that inhibition of viral entry into cells
is a legitimate target for antiviral chemotherapy of HIV
infection, the inhibitory effect of AMD3100 was evalu-
ated in intrathymically HIV-1-infected SCID-hu (Thy/
Liv) mice.17 Once-daily, subcutaneous injections of
AMD3100 at nontoxic doses caused a significant reduc-
tion in p24 antigen expression, a dose-dependent de-
crease in viremia, and a protection of the decrease in
CD4/CD8 T-cell ratio. Antiviral efficacy was also poten-
tiated by the combined administration of AMD3100 and
AZT or ddI. Therefore, AMD3100 exhibits potent anti-
viral efficacy in vitro and in vivo, clearly establishing
inhibition of CXCR4 and virus entry into cells as a
target for chemotherapeutic intervention with antiviral
agents.
In the present study, we report the results of our
continuing efforts to optimize the anti-HIV activity of
bis-azamacrocycles. To this end, a series of compounds
in which the secondary amine groups of AMD3100 were
replaced by neutral heteroatoms or heteroaromatic
groups were synthesized and evaluated for their inhibi-
tory effects on HIV-1 and HIV-2 replication in vitro. This
approach led to the identification of AMD3329, a bis-
tetraazamacrocycle featuring a pyridine group incorpo-
rated within the macrocyclic ring, as the most potent
inhibitor of HIV-1 and HIV-2 replication among the bis-
azamacrocycles reported to date.

Phenylenebis(methylene)-Linked Bis-azamacrocycles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3973
Sch em e 3a
Sch em e 4a
a
Reagents: (a) MeOH, cat. H2SO4; (b) when R ) H: PCl5,
CHCl3, reflux then MeOH; or R ) Me: Tf2O, pyridine; (c)
PhB(OH)2, K2PO3, KBr, Pd(PPh3)4, dioxane, reflux; (d) NaOMe,
MeOH, reflux; (e) NaBH4, EtOH, reflux; (f) Ms-Cl, Et3N, CH2Cl2.
difficulties associated with the synthesis of “unsym-
metrical” N3X macrocycles (for example, construction of
the corresponding cyclam ring in which a single aza
group has been replaced with a heteroatom), a short
series of compounds based on the [iso-14]aneN4 ring
system were prepared using a combination of diethoxy-
phosphoryl (Dep) and p-toluenesulfonyl groups.4 For the
synthesis of the bis-[iso-14]aneN3O macrocycle 16a ,
iminodipropionitrile (8) was chosen as a convenient
starting material. Protection of the amino group of 8
with diethyl chlorophosphate in CH2Cl2 in the presence
of Et3N gave the Dep-protected dinitrile 9 in 64% yield.
Reduction of the nitrile groups by hydrogenation over
Raney Ni in a saturated solution of NH3 in MeOH gave
the diamine 10 followed by derivatization of the corre-
sponding amines with p-toluenesulfonyl chloride under
standard conditions provided the required precursor 11.
Using our standard macrocyclization conditions,4,18 cy-
clization of the ditosylate 11 with 2-bromoethyl ether
(12a ) was highly efficient, giving the protected N3O
macrocycle 13a in a 79% isolated yield following silica
gel column purification. To liberate a single secondary
amine of known regiochemistry for the impending
dimerization reaction, the Dep group in 13a was sub-
sequently deprotected under conditions in which the
tosyl group is stable. Thus, reaction of 13a with HBr/
acetic acid at room temperature for 2-3 h gave the bis-
tosyl macrocycle 14a . The synthesis of the ditosyl
[14]aneN3 macrocycle 14b was accomplished in a man-
ner similar to that described for 14a by replacing
2-bromoethyl ether with 1,5-dibromopentane (12b) in
the macrocyclization reaction with 11. The dimerization
and final deprotection reactions were performed as
illustrated in Scheme 2.
a
Reagents: (a) Cs2CO3, DMF, 65 °C; (b) HBr, HOAc, rt; (c) 0.5
equiv p-dibromoxylene, K2CO3, CH3CN, reflux; (d) 48% aq HBr,
HOAc, reflux or concd H2SO4, 110 °C.
quenched with methanol to give the corresponding
4-chloropyridine-2,6-dicarboxylate dimethyl ester (21a ).
Reduction of the diester with NaBH4 in refluxing
ethanol gave the diol 22a in 65% yield which was
subsequently reacted with methanesulfonyl chloride in
CH2Cl2 in the presence of Et3N. Contrary to our
expectations, we obtained the bis-chloromethyl inter-
mediate 23a rather than the desired bis-mesylate under
these conditions (by in situ nucleophilic substitution of
the initially formed mesylate by chloride), even though
the reaction time was relatively short (30 min at 0 °C).
Since a bis(chloromethyl)pyridine intermediate is an
equally suitable precursor for the macrocyclization
reaction, subsequent conversions of diols to bis-electro-
philes using this procedure were allowed a minimum
reaction time of 30 min to complete the conversion to
the respective dichlorides (23b,c). To prepare the 4-meth-
oxypyridine precursor, the 4-chloropyridine intermedi-
ate 21a was reacted with freshly prepared sodium
methoxide in refluxing MeOH to give 21d . In a similar
two-step reduction and chlorination procedure utilized
for the preparation of 23a , the methoxy compound 21d
gave 23c. Finally, the 4-phenylpyridine intermediate
23b was also prepared from chelidamic acid (20a ).
Esterification of 20a with MeOH and catalytic concen-
trated H2SO4 gave the dimethyl ester 20b. The 4-hy-
droxy substituent on the pyridine ring was then con-
verted to the corresponding triflate by treatment with
Tf2O in pyridine to give 21b. Palladium-catalyzed cross-
coupling of the triflate 21b with phenylboronic acid in
the presence of K3PO4 and KBr introduced the 4-phenyl
substituent to give 21c, which was subsequently con-
verted to the desired bis(chloromethyl) precursor 23b
as previously described.
For construction of azamacrocyclic rings containing
an aromatic or heteroaromatic group (pyridine or pyra-
zine) within the macrocyclic framework, the Dep-
protected ditosylate 11 was macrocyclized with a series
of bis-electrophiles 17-19 and 23a -c as shown in
Schemes 3 and 4. Compounds 18 and 19 were prepared
as previously described,4,5 and compounds 23a -c, which
feature a 4-pyridine substituent, were synthesized from
a common starting material, chelidamic acid (20a )
(Scheme 3). To introduce the 4-chloro substituent, 20a
was reacted with PCl5 in chloroform at reflux and

3974 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Bridger et al.
Sch em e 5a
Ta ble 1. Anti-HIV Activity Data for Bis-azamacrocycles
EC50 (µM)b
c
HIV-1
(IIIB)
HIV-2
(ROD)
CC50
(µM)
compd
formulaa
7a
7b
7c
C
C
C
28H50N4S4‚4HCl
>6.8357 >6.8357 6.8
28H50N4O4‚4HCl‚2‚5H2O >363
>363
>363
>421
28H54N8‚8HCl‚2H2O
(AMD3100)
28H52N6O2‚6HBr‚H2O‚
HOAc
0.0042d
0.0059
16a
C
4.4919
NTe
>244
16b
16c
27a
27b
27c
27d
27e
27f
27g
C
C
C
C
C
C
C
30H56N6‚6HBr‚5H2O
28H54N8‚8HBr‚2H2O
36H52N6‚6HBr‚2H2O
34H50N8‚6HBr‚5H2O
32H48N10‚7HBr‚HOAc
9.6078
0.0337d
7.9605
0.5342
30.942
NT
0.0422
NT
133
>421
133
199
209
17.8
19.3
216
200
NT
20.741
0.7593
NT
8.4605
199.97
34H48N8Cl2‚6HBr‚HOAc 0.7509
46H58N8‚8HBr‚HOAc
1.7547
3.0979
174.37
C36H54N8O2‚6HBr‚2H2O
C
34H50N8O2‚8HBr
a
b
Microanalyses were within (0.4 of theoretical values. 50%
antiviral effective concentration. c 50% cytotoxic concentration. The
greater than symbol (>) is used to indicate the highest concentra-
d
tions at which the compounds were tested. Anti-HIV-1 (IIIB) and
anti-HIV-2 (ROD) data from ref 4. e NT, not tested.
bis-electrophiles 29 and 30,4 respectively, which contain
a Dep group targeted for the selective deprotection
reaction. The key intermediate 28 was prepared in three
steps from 2,6-bis(bromomethyl)pyridine (18). Alterna-
tively, the dimesylate 30 can be converted to the
corresponding bis-tosylamide 31 and macrocyclized with
18 to give the corresponding py[12]aneN4 macrocyclic
ring, which provided 32 on selective removal of the Dep
group. The aforementioned macrocyclic intermediates
32-34 were used to prepare analogues 35a ,b, 36, and
37 (Table 2, isolated as their octahydrobromide salts)
using the appropriately substituted R,R′-dibromo-p-
xylene intermediate and the procedures shown in
Schemes 1, 2, and 4.
a
Reagents: (a) NaCN, cetyltrimethylammonium bromide, ben-
zene, water, reflux; (b) H2/Ra Ni, NH3, MeOH; (c) Ts-Cl, Et3N,
CH2Cl2; (d) NaN3, DMF, 80 °C, 18 h; (e) H2/Pd/C, EtOAc; (f)
Cs2CO3, 18 or 28, DMF, 65 °C; (g) HBr, HOAc, rt.
Macrocycles were constructed by cyclization of 11 with
the appropriate bis-electrophile under established con-
ditions affording 24a -f (Scheme 4). Following removal
of the Dep group post-macrocyclization (to give 25a -f)
and the penultimate step of dimerization with R,R′-
dibromo-p-xylene, the tosyl-protecting groups of 26a -f
were subjected to hydrolytic deprotection to give the
desired bis-azamacrocycles 27a-g. However, the 4-meth-
oxypyridine precursor 26f provided two final products,
27f,g, depending on the choice of conditions used for the
final deprotection step. Treatment of 26f with 48%
aqueous HBr and acetic acid at reflux simultaneously
cleaved the tosyl and methyl ether groups giving the
4-hydroxypyridine analogue 27g, whereas reaction of
26f with concentrated H2SO4 at 100-110 °C for 2 h
selectively deprotected the tosyl groups in the presence
of the methyl ether giving the 4-methoxypyridine ana-
logue 27f.
Finally, a series of analogues were prepared in which
the ring size of the pyridine-containing tetraazamacro-
cycle was varied between 12 and 16 members per ring,
including the py[iso-14]aneN4 ring system, an isomer
of the py[14]aneN4 macrocycle featured in compound
27b [compare structure 33 (Scheme 5) to 27b (Scheme
4)]. This synthetic goal was accomplished by straight-
forward manipulation of our collection of macrocyclic
precursors as illustrated in Scheme 5. For assembly of
the protected py[iso-14]aneN4 (33) and py[16]aneN4 (34)
rings, the N,N′-bis-tosyl derivative of 2,6-(2-aminoethyl)-
pyridine (28) was macrocyclized with the appropriate
Biologica l Resu lts a n d Discu ssion
The bis-azamacrocycles reported in Tables 1 and 2
were tested for their inhibitory effects on HIV-1 (IIIB)
and HIV-2 (ROD) replication in MT-4 cells according
to published procedures. These assays are considered
representative of inhibition via the co-receptor CXCR4
due to the following observations. First, whereas HIV-1
(IIIB) predominantly uses CXCR4 for entry, HIV-2
(ROD) is multitropic and can use CCR3 and CCR5 in
addition to CXCR4 to enter cells.13 However, MT-4 cells
are CD4+ and CXCR4+ but do not express detectable
levels of CCR3 or CCR5 (D. Schols, unpublished data),
and both viral strains are inhibited by SDF-1R, the
natural ligand for CXCR4, in MT-4 cells at comparable
EC50’s (20-100 ng/mL). Second, while AMD3100 dose-
dependently inhibited HIV-1 (IIIB) and HIV-2 (ROD)
infection of U373MG-CD4 cells stably transfected with
the chemokine receptor CXCR4, AMD3100 failed to
inhibit infection of cells stably transfected with the
chemokine receptors CCR5, CCR3, BOB, and Bonzo and
cells transiently expressing US28.13 These receptors are
members of a growing family of chemokine receptors
reported to mediate infection by diverse HIV and SIV
strains. Finally, we recently reported a mechanism-of-
action study on a diverse series of bicyclam analogues,11
which indicated a close correlation between antiviral
potency against X4 strains of HIV-1, inhibition of

Phenylenebis(methylene)-Linked Bis-azamacrocycles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3975
Ta ble 2. Anti-HIV Activity Data for Pyridine-Containing Bis-azamacrocycles
EC50 (µM)b
HIV-2 (ROD)
compda
structure
HIV-1 (IIIB)
0.5238
CC50c (µM)
35a , para
0.1273
8.5
35b, meta
36, n ) 1
0.0971
0.0008
0.0025
0.0016
30
194
37, n ) 2
0.4213
1.2068
142
a
All compounds were tested as their octahydrobromide salts (see Experimental Section). Microanalyses were within (0.4 of theoretical
values. 50% antiviral effective concentration. c 50% cytotoxic concentration.
b
Ta ble 3. Data for Inhibition of CXCR4-Specific mAb (12G5)
Binding to CXCR4 by Selected Compounds from Tables 1 and 2
binding of the CXCR4-specific monoclonal antibody
12G5 to CXCR4, and inhibition of the intracellular Ca2+
signal induced by the natural ligand SDF-1R (stromal
cell-derived factor 1R). Thus, it can be concluded that
MT-4 cells do not express functional co-receptors that
are resistant to inhibition by AMD3100 and that assays
which measure viral replication of HIV-1 (IIIB) and
HIV-2 (ROD) strains in MT-4 cells are representative
of inhibition via antagonism of CXCR4.
Compared to the corresponding bis-tetraazamacro-
cycle 7c (AMD3100), replacing two aza groups of the
macrocyclic ring with heteroatoms such as sulfur or
oxygen gave analogues of substantially reduced antivi-
ral potency. For example, both the [14]aneN2S2 (7a ) and
[14]aneN2O2 (7b) bis-macrocycles exhibited 50% effec-
tive concentrations (EC50’s) against HIV-1 that were
similar to their respective 50% cytotoxic concentrations
(CC50’s) in MT-4 cells, thereby negating selectivity for
inhibition of virus replication. Furthermore, the intro-
duction of sulfur rather than oxygen heteroatoms sig-
nificantly affected cytotoxicity: 7a displayed a greater
than 50-fold lower CC50 than 7b (or 7c).
A second short series of analogues were prepared in
which a single amine group of the [iso-14]aneN4 ring
system (featured in 16c, Scheme 2) was replaced by
oxygen or a methylene group (to give the [14]aneN3 bis-
macrocycle 16b). The results are also shown in Table
1. Although the [iso-14]aneN3O bis-macrocycle 16a
exhibited a modest selectivity for inhibition of HIV-1
replication in MT-4 cells [EC50 against HIV-1 of 4.4919
µM, CC50 of >244 µM, selectivity index (SI) of >54]
compared to the [14]aneN2X2 bis-macrocycles 7a ,b, its
EC50 value was only 2-fold lower than that of the
methylene analogue 16b. Moreover, both 16a ,b were
still significantly less potent than the parent tet-
raazamacrocyclic dimer 16c, exhibiting EC50’s against
HIV-1 replication that were 2 orders of magnitude
higher than the concentration of 16c required to inhibit
HIV-1 replication by 50%. These data clearly indicate
that all four amine groups of the tetraazamacrocyclic
rings of 7c or 16c are required to achieve high antiviral
potency.
12G5 mAb
inhibition IC50
(µM)a
12G5 mAb
inhibition IC50
(µM)a
compd
compd
7a
>35
27c
27d
27g
35a
36c
>35
7b
>35
5.737
>35
7cb
16b
16c
0.007
>35
0.067
4.363
0.001
a
50% inhibitory concentration. The greater than symbol (>) is
used to indicate the highest concentrations at which the com-
pounds were tested. b AMD3100. c Tested as the octahydrochloride
salt.
(AMD3100)] but contain a more powerful hydrogen-bond
acceptor than the ether oxygen of 16a . To this end, we
prepared a phenylenebis(methylene)-linked dimer of a
tetraazamacrocyclic ring containing a pyridine group
incorporated within the macrocyclic framework (bis-py-
[14]aneN4, 27b). Compound 27b was found to inhibit
HIV-1 replication at submicromolar concentrations
(EC50: 0.5342 µM) while remaining nontoxic to MT-4
cells at a CC50 approaching 200 µM. The corresponding
phenyl analogue 27a was ca. 15-fold less potent against
HIV-1 replication than 27b, thereby confirming the
beneficial effect of the pyridine-N in the macrocyclic
rings of 27b on antiviral activity. We further prepared
a series of compounds in which the basicity of the
pyridine-N was systematically varied by incorporation
of substituents in the 4-position of the pyridine ring.
Analogue 27d , containing an electron-withdrawing chlo-
ro substituent (that lowers the pKa and therefore
basicity of the pyridine-N), displayed comparable anti-
viral potency to the unsubstituted pyridine analogue
27b (exhibiting an EC50 against HIV-1 replication of
0.7509 µM) but was ca. 11-fold more cytotoxic to MT-4
cells. However, electron-donating substituents (which
increase the basicity of the pyridine-N) had a detrimen-
tal effect on antiviral activity. The 4-phenyl and 4-meth-
oxy analogues 27e,f exhibited EC50’s against HIV-1
replication that were approximately 3- and 6-fold higher,
respectively, than those of compounds 27b,g. The latter
was essentially inactive, exhibiting a EC50 against
HIV-1 replication that was comparable to its CC50 in
MT-4 cells. Although we are unable to fully explain
these results at the present time, the most likely
explanation for the variation in antiviral activity with
pyridine basicity is that the pyridine pKa controls the
In view of these results, we reasoned that bis-
tetraazamacrocycles featuring a heteroaromatic replace-
ment for a secondary amine of 16c may also provide
analogues that are doubly protonated at physiological
pH [in a manner similar to the cyclam rings5 of 7c

3976 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Bridger et al.
F igu r e 1. Inhibition of the binding of an anti-CXCR4 mAb (12G5) to SUP-T1 cells in the presence of compound 36 or AMD3100
at different concentrations (200, 40, 8, and 1.6 ng/mL). NC is the isotype control mAb, and PC is the specific staining obtained
with 12G5 in the absence of test compound. The percentage of positive cells and the MFI values are indicated in each histogram.
position and degree of protonation on the azamacrocyclic
ring.19 However, the substantial reduction in antiviral
activity observed with the hydroxyl analogue 27g is
probably due to dominance of the pyridone (rather than
pyridine) tautomeric form under the pH conditions of
the assay. Since pyridine analogues of lower basicity
(such as the chloro analogue 27d ) exhibited comparable
activity to the unsubstituted parent compound 27b, we
decided to incorporate an aromatic heterocycle that is
substantially less basic than pyridine into the macro-
cyclic framework. For this purpose we chose to prepare
the pyrazine analogue 27c (pKa of pyridine is 5.25 and
pKa1 of pyrazine is 0.655). Although we expected that
27c would exhibit (at the very least) an HIV-1 activity
profile that was similar to that of analogues 27a ,b, we
were disappointed to find that the incorporation of a
pyrazine group reduced antiviral potency: the EC50 of
27c against HIV-1 was 30.942 µM, a 3- and 58-fold
increase in the concentrations of 27a ,b , respectively,
required to inhibit HIV-1 replication by 50%.
Having established that a pyridine group incorporated
into the azamacrocyclic ring has a favorable effect on
antiviral activity, we prepared a series of compounds
in which the macrocyclic ring contained 12 and 16 ring
members (py[12]aneN4 and py[16]aneN4). Unlike the
structure-activity relationship observed in the series
of aliphatic tetraazamacrocyclic compounds,4 the p-
phenylenebis(methylene)-linked dimers of the py[12]-
aneN4 (35a ), py[14]aneN4 (27b), and py[16]aneN4 (37)
ring systems exhibited comparable EC50’s for inhibition
of HIV-1 (and HIV-2) replication: EC50’s for 35a , 27b,
and 37 were 0.5238, 0.5342, and 0.4213 µM, respec-
tively. However, compounds 27b and 37 were signifi-
cantly less cytotoxic to MT-4 cells than 35a : the CC50
of 35a (8.5 µM) was approximately 23- and 17-fold lower
than that of analogues 27b and 37, to give a selectivity
index for 35a against HIV-1 replication of only >16. In
contrast, the corresponding m-phenylenebis(methylene)-
linked dimer of the py[12]aneN4 macrocycle (35b)
displayed higher antiviral potency for inhibition of
HIV-1 and HIV-2 replication than the corresponding
para-analogue 35a (and the activity against HIV-2 was
greater than against HIV-1), consistent with the struc-
ture-activity relationship of the aliphatic tetraazamac-
rocycles previously reported.4 Compound 35b exhibited
EC50’s against HIV-1 (0.0971 µM) and HIV-2 (0.0025
µM) replication that were ca. 5- and 50-fold lower than
the concentration of 35a required to inhibit HIV-1 and
HIV-2 replication by 50%. Finally, we synthesized the
p-phenylenebis(methylene)-linked dimer of the py[iso-
14]aneN4 macrocycle (36), an isomer of the macrocyclic
ring in analogue 27b. To our surprise, compound 36
exhibited an EC50 against HIV-1 replication that was 3
orders of magnitude lower than the EC50 of 27b: EC50’s
of 36 against HIV-1 and HIV-2 replication were 0.8 and
1.6 nM; the CC50 in MT-4 cells was 199 µM, which gives
a selectivity index for 36 against HIV-1 of greater than
2.4 × 105. In addition, 36 inhibited HIV-1 and HIV-2
replication at a 3-5-fold lower concentration than the
concentration of AMD3100 (7c) required to inhibit viral

Phenylenebis(methylene)-Linked Bis-azamacrocycles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3977
(AMD3329) with CXCR4, consistent with its higher
antiviral activity.
In summary, we have prepared a series of bis-
azamacrocycles in which selected secondary amines of
the bis-tetraazamacrocycle AMD3100 (7c) have been
replaced by neutral heteroatoms or heteroaromatic
groups. This approach to optimizing the anti-HIV activ-
ity has led to the identification of AMD3329 (36), a
p-phenylenebis(methylene)-linked dimer of the py[iso-
14]aneN4 macrocycle, as the most potent inhibitor of
HIV-1 and HIV-2 replication among the bis-azamacro-
cycles reported to date.
Exp er im en ta l Section
AMD3100 is 1,1′-[1,4-phenylenebis(methylene]bis(1,4,8,11-
tetraazacyclo tetradecane)octahydrochloride, dihydrate3,4 (for-
mula weight ) 830.51).
F igu r e 2. Inhibition of SDF-1R-induced Ca2+ flux in SUP-T1
cells by compound 36 (8 and 1.6 ng/mL) and AMD3100 (200,
40, 8, and 1.6 ng/mL). Test compounds or buffer was added at
50 s (4), and SDF-1R was given as a second stimulus at a
concentration of 30 ng/mL (2), 100 s after addition of the
compound.
1
General experimental procedures are provided in ref 4. H
and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, on a Bruker AC300 spectrometer. Fast atom
bombardment mass spectral analysis was carried out by
M-Scan (West Chester, PA). Microanalyses for C, H, N, and
halogen were performed by Atlantic Microlabs (Norcross, GA)
and were within (0.4% of theoretical values.
replication by 50%. We also compared 36 and 7c for
their ability to interfere with virus-induced syncytium
formation between persistently HIV-1 (IIIB)-infected
HUT-78 cells and uninfected MOLT-4 cells. In these
assays, the concentration of 36 required to inhibit
syncytium formation by 50% was 90-fold lower than that
of 7c (AMD3100) (EC50’s for 36 and 7c were 0.012 and
1.130 µM,3 respectively).
2,2′-(Eth ylen ed ith io)bis[2-p r op ion itr ile] (1). To a solu-
tion of 1,2-ethanedithiol (3.8 g, 40.3 mmol) and bromopropi-
onitrile (10.8 g, 80.7 mmol) in 100 mL of CH2Cl2 was added
triethylamine (12.23 g, 121 mmol) and the resulting white
slurry was stirred at room temperature for 4 h. The solution
was diluted with CH2Cl2 (100 mL), washed with H2O (2 × 50
mL) and brine (50 mL), dried (MgSO4), and concentrated to
give 1 (7.1 g, 88%) as a white solid: 1H NMR (CDCl3) δ 2.62-
2.70 (m, 4H), 2.79-2.90 (m, 8H).
1,12-Bis(p-tolu en esu lfon yl)-5,8-d ith ia -1,12-d ia za d od e-
ca n e (3a ). To a solution of 1 (7.1 g, 35.5 mmol) in anhydrous
THF (100 mL) was added BH3‚THF (1.0 M solution in THF,
180 mL) and the mixture was heated to reflux for 24 h. After
cooling, the excess borane was destroyed by addition of MeOH
(50 mL) and evaporation (repeated three times) affording a
viscous oil (2a ) which was used without purification in the
subsequent step: 1H NMR (CDCl3) δ 1.27 (br s, 4H), 1.72
(quintet, 4H, J ) 7.1 Hz), 2.61 (m, 4H), 2.70-2.85 (m, 8H).
To a vigorously stirred solution of the crude diamine 2a and
NaOH (3.55 g, 88.25 mmol) in H2O (150 mL) was added
dropwise a solution of p-toluenesulfonyl chloride (13.5 g, 71.0
mmol) in Et2O (100 mL) and the mixture was stirred for 20 h
at room temperature. The solution was diluted with CH2Cl2
(300 mL), washed twice with brine (100 mL), dried (MgSO4),
and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (EtOAc/
hexane, 1:1) to give 3a (7.44 g, 36%) as a white solid: 1H NMR
(CDCl3) δ 1.79 (quintet, 4H, J ) 7.1 Hz), 2.42 (s, 6H), 2.61 (t,
4H, J ) 7.1 Hz), 2.70 (s, 4H), 3.00-3.09 (m, 4H), 5.19 (t, 2H,
J ) 6.1 Hz), 7.30 (d, 4H, J ) 8.3 Hz), 7.74 (d, 4H, J ) 8.3 Hz).
Gen er a l P r oced u r e A: Ma cr ocycliza tion . 8,11-Bis(p-
t olu en esu lfon yl)-1,4-d it h ia -8,11-d ia za cyclot et r a d eca n e
(4a ). To a stirred solution of 3a (7.44 g, 14.42 mmol) in
anhydrous DMF (400 mL) containing cesium carbonate (14.1
g, 43.25 mmol) maintained at 65 °C was added a solution of
ethylene glycol bis-p-toluenesulfonate (5.33 g, 14.42 mmol) in
DMF (100 mL) dropwise over a period of 14-16 h. The reaction
mixture was stirred at 65 °C for a total of 24 h and then
allowed to cool to room temperature and evaporated in vacuo.
The residue was partitioned between CH2Cl2 (500 mL) and
brine (300 mL) and the aqueous layer was separated and
extracted with CH2Cl2 (2 × 75 mL). The combined organic
phases were dried (MgSO4) and evaporated in vacuo to give
the crude product as an orange oil. Purification by column
chromatography on silica gel (EtOAc/hexane, 3:7) gave 4a (3.3
g, 42%) as a white solid: 1H NMR (CDCl3) δ 1.89 (quintet,
The effects of the compounds described herein were
studied in more detail for their interactions with
CXCR4, the main co-receptor for entry of T-tropic HIV
strains. In this regard, a group of compounds that
exhibited varying EC50’s for HIV inhibition were se-
lected and tested for their ability to inhibit binding of a
CXCR4-specific mAb (12G5) to CXCR4 on SUP-T1 cells
(Table 3). A strong correlation between inhibition of
HIV-1 replication and inhibition of 12G5 binding to
CXCR4 was observed. For example, compounds 7c, 16c
(Table 1), and 36 (Table 2) exhibited EC50’s for inhibition
of HIV-1 replication that were comparable to their IC50’s
for inhibition of 12G5 binding, whereas compounds that
were ineffective antiviral agents did not inhibit 12G5
binding at concentrations exceeding 35 µM. As shown
in Figure 1, both 7c (AMD3100) and 36 (for these
experiments, isolated as the octahydrochloride salt)
dose-dependently inhibited binding of 12G5 to CXCR4.
Compound 36 at 200, 40, 8, and 1.6 ng/mL inhibited
12G5 binding by 97%, 93%, 83%, and 63%, respectively,
as calculated from the MFI values, whereas 7c at 200,
40, 8, and 1.6 ng/mL inhibited 12G5 binding by 94%,
83%, 63%, and 40%, respectively. From these data, the
calculated IC50 value for 36 was found to be 7-fold lower
than the concentration of 7c required to inhibit 12G5
binding by 50% (IC50’s for 36 and 7c were 0.001 and
0.007 µM, respectively).
Their effects on SDF-1R-induced Ca2+ flux ([Ca2+]i)
in SUP-T1 cells were also investigated (Figure 2).
Compound 36 completely blocked [Ca2+
] increases
i
induced by SDF-1R at 8 ng/mL, whereas at this con-
centration, 7c partially inhibited (42%) the [Ca2+
]
i
increase. AMD3100 (7c) completely blocked the re-
sponse at 100 ng/mL9 (Figure 2). These combined assays
clearly demonstrate the more potent interaction of 36

3978 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Bridger et al.
4H, J ) 7.1 Hz), 2.45 (s, 6H), 2.58 (t, 4H, J ) 7.1 Hz), 2.72 (s,
4H), 3.22 (t, 4H, J ) 7.1 Hz), 3.27 (s, 4H), 7.33 (d, 4H, J ) 7.9
Hz), 7.71 (d, 4H, J ) 7.9 Hz).
of the hydrochloride salt (as described for 7a ), 6b gave 7b (96%
overall yield) as a white solid: 1H NMR (D2O) δ 1.88 (m, 4H),
1.95 (m, 4H), 3.20 (t, 4H, J ) 5.3 Hz), 3.26 (m, 4H), 3.47-3.66
(m, 24H), 4.40 (s, 4H), 7.57 (s, 4H); 13C NMR (D2O) δ 23.71,
24.72, 42.55, 49.58, 49.95, 52.76, 58.39, 69.08, 69.45, 70.11,
70.83, 131.15, 132.65; FAB MS m/z 543 (MH + H35Cl, 66), 507
(M + H, 90), 307 (85), 203 (100). Anal. (C28H50N4O4‚4HCl‚
2.5H2O) C, H, N, Cl.
Gen er a l P r oced u r e B: Am a lga m Dep r otection . 8-(p-
T olu e n e su lfon y l)-1,4-d it h ia -8,11-d ia za c y c lo t e t r a d e -
ca n e (5a ). To a stirred solution of 4a (3.3 g, 6.09 mmol) in a
mixture of anhydrous THF (50 mL) and MeOH (50 mL) were
added dibasic sodium phosphate (6.2 g, 43.67 mmol) and
freshly prepared 3% sodium amalgam (50 g). The reaction
mixture was heated to reflux for 44 h then allowed to cool to
room temperature and the supernatant solution was decanted
from the amalgam and evaporated to dryness. The residue was
partitioned between CHCl3 (100 mL) and brine (25 mL), the
aqueous layer was separated and extracted with CHCl3 (2 ×
75 mL), and the combined organic extracts were dried (MgSO4)
and concentrated to give 1,4-dithia-8,11-diazacyclotetradecane
(1.4 g, 98%) as a pale yellow oil: 1H NMR (CDCl3) δ 1.78 (m,
4H), 2.68-2.81 (m, 16H); FAB MS m/z 235 (M + H, 100). This
was used without further purification.
N-Diet h oxyp h osp h or yl-3,3′-im in od ip r op ion it r ile (9).
To a solution of 3,3′-iminodipropionitrile (8) (2.0 g, 16 mmol)
and triethylamine (2.7 mL) in dichloromethane (50 mL) was
added dropwise with stirring, under argon, a solution of diethyl
chlorophosphate (2.8 g, 16 mmol) in dichloromethane (20 mL)
over approximately 30 min and the mixture was then allowed
to stir overnight at room temperature. The mixture was
washed with brine (50 mL), then dried (Na2SO4), and evapo-
rated in vacuo giving 9 (2.7 g, 64%) as a colorless oil: 1H NMR
(CDCl3) δ 1.36 (t, 6H, J ) 7.2 Hz), 2.66 (t, 4H, J ) 6.5 Hz),
3.45 (m, 4H), 4.15 (m, 4H).
The macrocycle from above (1.4 g, 5.98 mmol) was dissolved
in anhydrous CH2Cl2 (50 mL) containing Et3N (1.81 g, 17.95
mmol). To this solution was added a solution of p-toluene-
sulfonyl chloride (1.02 g, 5.58 mmol) in CH2Cl2 (50 mL)
dropwise with stirring over 2 h at 0 °C. The reaction mixture
was then allowed to warm to room temperature and stirred a
further 2 h. Brine (20 mL) was added to the reaction mixture
and the organic layer was separated, dried (MgSO4), and
evaporated to dryness to give the crude product as a white
solid. Purification by column chromatography on silica gel
(CH2Cl2/MeOH, 95:5) gave 5a (1.5 g, 65%) as a white solid:
1H NMR (CDCl3) δ 1.75-1.84 (m, 2H), 1.91-2.04 (m, 2H), 2.43
(s, 3H), 2.62-2.73 (m, 4H), 2.75 (s, 4H), 2.78 (t, 2H, J ) 5.5
Hz), 2.85 (t, 2H, J ) 5.5 Hz), 3.12 (m, 4H), 7.31 (d, 2H, J )
8.3 Hz), 7.67 (d, 2H, J ) 8.3 Hz); FAB MS m/z 389 (M + H,
100), 233 (16).
Gen er a l P r oced u r e C: Dim er iza tion . 11,11′-[1,4-P h e-
n ylen ebis(m eth ylen e)]bis[8-(p-tolu en esu lfon yl)-1,4-dith ia-
8,11-d ia za cyclotetr a d eca n e] (6a ). To a stirred solution of
5a (1.5 g, 3.86 mmol) in dry CH3CN (75 mL) were added R,R′-
dibromo-p-xylene (510 mg, 1.93 mmol) and potassium carbon-
ate (1.61 g, 11.59 mmol) and the mixture was heated to reflux
for 22 h. The reaction mixture was allowed to cool to room
temperature then concentrated and the residue was parti-
tioned between CH2Cl2 (100 mL) and H2O (30 mL). The
aqueous phase was separated and extracted with two further
portions of CH2Cl2 (50 mL). The combined organic phases were
dried (MgSO4) and evaporated, and the residue was purified
by column chromatography on silica gel (EtOAc/hexane, 1:1)
to give 6a (1.25 g, 74%) as a white solid: 1H NMR (CDCl3) δ
1.73 (m, 4H), 1.93 (m, 4H), 2.42 (s, 6H), 2.44-2.53 (m, 8H),
2.57 (t, 4H, J ) 7.3 Hz), 2.69-2.76 (m, 12H), 3.13-3.27 (m,
8H), 3.56 (s, 4H), 7.23 (s, 4H), 7.27 (d, 4H, J ) 8.3 Hz), 7.61
(d, 4H, J ) 8.3 Hz); FAB MS m/z 879 (M + H, 100), 723 (70),
490 (25).
11,11′-[1,4-P h en ylen ebis(m eth ylen e)]bis[1,4-d ith ia -8,-
11-d ia za cyclo tetr a d eca n e] Tetr a h yd r och lor id e (7a ). Us-
ing general procedure B, deprotection of 6a (450 mg, 0.51
mmol) with 1% sodium amalgam gave 7a (free base) (262 mg,
90%) as a white solid: 1H NMR (CDCl3) δ 1.77 (m, 4H), 1.85
(m, 4H), 2.44 (t, 4H, J ) 5.7 Hz), 2.51-2.61 (m, 12H), 2.66-
2.81 (m, 16H), 3.52 (s, 4H), 7.22 (s, 4H).
To a solution of the free base (112 mg, 0.196 mmol) in EtOH
(8 mL) was passed HCl(g) resulting in the immediate forma-
tion of a white precipitate. The solution was concentrated to
dryness and dried in vacuo to give 7a hydrochloride (127 mg,
98%) as a white solid: 1H NMR (D2O) δ 1.94 (quintet, 8H, J )
6.5 Hz), 2.54 (t, 4H, J ) 6.5 Hz), 2.61 (t, 4H, J ) 6.5 Hz), 2.80
(s, 8H), 3.09-3.23 (m, 8H), 3.48 (s, 8H), 4.31 (s, 4H), 7.52 (s,
4H); 13C NMR (D2O) δ 23.00, 24.23, 27.79, 27.96, 31.11, 31.28,
39.58, 45.96, 46.56, 50.24, 58.78, 131.53, 132.29; FAB MS m/z
607 (MH + H35Cl, 58), 571 (M + H, 72), 339 (75), 235 (86),
185 (100). Anal. (C28H50N4S4‚4HCl) C, H, N, Cl.
N-Dieth oxyph osph or yl-3,3′-im in obis[pr opylam in e] (10).
To a solution of 9 (1.0 g, 4 mmol) in methanol (50 mL,
saturated with ammonia) was added Raney nickel (5.0 g,
excess) and the mixture was hydrogenated at 45 Psi and room
temperature for 48 h. The catalyst was filtered off and the
solvent evaporated in vacuo to give 10 (0.95 g, 92%) as a
colorless viscous oil: 1H NMR (CDCl3) δ 1.31 (t, 6H, J ) 7.2
Hz), 1.65 (m, 4H), 2.75 (m, 4H), 3.05 (m, 4H), 4.15 (m, 4H).
N-Diet h oxyp h osp h or yl-N′,N′′-b is(p -t olu en esu lfon yl)-
3,3′-im in obis[p r op yla m in e] (11). To a solution of 10 (1.0 g,
4 mmol) and triethylamine (1.2 mL) in dichloromethane (50
mL) was added dropwise with stirring a solution of p-
toluenesulfonyl chloride (1.6 g, 7 mmol, 2.2 equiv) in dichlo-
romethane (25 mL) over approximately 15 min and the
mixture was then allowed to stir at room temperature over-
night. The mixture was washed with dilute hydrochloric acid
(50 mL), saturated aqueous sodium bicarbonate (50 mL), and
brine (50 mL), then dried (Na2SO4), and evaporated in vacuo
to give a brown oil. The crude product was purified by column
chromatography on silica gel (CH2Cl2:MeOH, 97:3) to give 11
(0.9 g, 43%) as a white solid: 1H NMR (CDCl3) δ 1.21 (t, 6H,
J ) 7.2 Hz), 1.65 (m, 4H), 2.42 (s, 6H), 2.91 (m, 8H), 3.85 (m,
4H), 7.27 (d, 4H, J ) 8.2 Hz), 7.74 (d, 4H, J ) 8.2 Hz).
8-Diet h oxyp h osp h or yl-4,12-b is(p -t olu en esu lfon yl)-1-
oxa -4,8,12-tr ia za cyclotetr a d eca n e (13a ). Using general
procedure A, 11 (2.9 g, 5 mmol) and 2-bromoethyl ether (12a )
(Aldrich; 1.16 g, 5 mmol) gave 13a (1.1 g, 79%) as a colorless
oil: 1H NMR (CDCl3) δ 1.28 (t, 6H, J ) 7.0 Hz), 1.95 (m, 4H),
2.42 (s, 6H), 3.15-3.25 (m, 12H), 3.65 (m, 4H), 3.95 (m, 4H),
7.35 (d, 4H, J ) 8.2 Hz), 7.65 (d, 4H, J ) 8.2 Hz).
Gen er a l P r oced u r e D: Dieth oxyp h osp h or yl Dep r o-
tection . 4,12-Bis(p-tolu en esu lfon yl)-1-oxa -4,8,12-tr ia za -
cyclotetr a d eca n e (14a ). To a solution of 13a (750 mg) in
glacial acetic acid (4.0 mL) was added 30% HBr/acetic acid
(Aldrich; 2.0 mL) and the reaction mixture stirred at room
temperature for 2 h. Ether (100 mL) was added to precipitate
a white solid which was allowed to settle to the bottom of the
flask and the supernatant solution was decanted off. The solid
was then washed by decantation with ether three times and
the remaining traces of ether removed by evaporation under
reduced pressure. The solid was partitioned between sodium
hydroxide solution (10 mL, 10 N) and CH2Cl2 (150 mL) and
the organic layer was separated, dried (Na2SO4), and evapo-
rated in vacuo to give 14a (410 mg, 69%) as a white solid: 1H
NMR (CDCl3) δ 1.95 (m, 4H), 2.43 (s, 6H), 2.85 (t, 4H, J ) 6.5
Hz), 3.28 (m, 8H), 3.65 (t, 4H, J ) 6.5 Hz), 7.32 (d, 4H, J )
8.2 Hz), 7.67 (d, 4H, J ) 8.2 Hz); FAB MS m/z 510 (M + H,
100), 329 (38), 307 (36).
8,8′-[1,4-P h en ylen ebis(m eth ylen e)]bis[4,12-bis(p-tolu -
en esu lfon yl)-1-oxa -4,8,12-tr ia za cyclotetr a d eca n e] (15a ).
Using general procedure C, 14a (403 mg, 0.79 mmol) and R,R′-
dibromo-p-xylene (105 mg, 0.40 mmol) gave 15a (393 mg, 89%)
as a white solid: 1H NMR (CDCl3) δ 1.75 (m, 8H), 2.42 (s, 12H),
2.55 (m, 8H), 3.15-3.75 (m, 28H), 7.24 (s, 4H), 7.27 (d, 8H, J
11,11′-[1,4-P h en ylen eb is(m et h ylen e)]b is[1,4-d ioxa -8,-
11-d ia za cyclo tetr a d eca n e] Tetr a h yd r och lor id e Dih y-
d r a te (7b). Using general procedure B followed by formation

Phenylenebis(methylene)-Linked Bis-azamacrocycles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3979
) 8.2 Hz), 7.65 (d, 8H, J ) 8.2 Hz); FAB MS m/z 1121 (M +
H, 100), 965 (34), 611 (55), 354 (40).
1H NMR (CDCl3) δ 1.33-1.40 (m, 8H), 2.13 (br t, 8H, J ) 6.8
Hz), 2.43 (s, 12H), 3.11 (br t, 8H, J ) 7.5 Hz), 4.30 (s, 8H, 6.99
(s, 4H), 7.31 (d, 8H, J ) 8.2 Hz), 7.49 (s, 4H), 7.69 (d, 8H, J )
8.2 Hz); FAB MS m/z 1255 (M + H, 100), 1099 (34), 678 (36),
575 (64).
Gen er a l P r oced u r e E: HBr /Acetic Acid Dep r otection .
8,8′-[1,4-P h en ylen ebis(m eth ylen e)]bis[1-oxa-4,8,12-tr iaza-
cyclot et r a d eca n e] H exa h yd r ob r om id e Tet r a h yd r a t e
(16a ). To a solution of 15a (250 mg) in acetic acid (2.5 mL)
was added hydrobromic acid (Aldrich; 48% aqueous, 1.5 mL)
and the mixture was heated to reflux with stirring for 18 h.
The mixture was allowed to cool and ether (50 mL) was added
giving a white precipitate. The solid was allowed to settle to
the bottom of the flask and the supernatant solution was
decanted off. The solid was then washed by decantation with
ether three times and the remaining traces of ether removed
by evaporation under reduced pressure followed by drying in
vacuo overnight gave 16a as a white solid (221 mg, 62%): 1H
NMR (D2O) δ 2.05 (m, 8H), 3.15-3.35 (m, 24H), 3.75 (m, 8H),
4.25 (s, 4H), 7.55 (s, 4H); FAB MS m/z 587 (M + H81Br, 49),
585 (M + H79Br, 49), 506 (M + H, 100), 307 (41). Anal.
(C28H52N6O2‚6HBr‚H2O‚HOAc) C, H, N.
5,5′-[1,4-P h en ylen eb is(m et h ylen e)]b is[1,5,7-t r ia za cy-
clotetr a d eca n e] Hexa h yd r obr om id e P en ta h yd r a te (16b).
1H NMR (D2O) δ 1.60 (m, 8H), 2.81-3.22 (m, 16H), 4.19 (s,
4H), 4.30 (m, 8H), 7.31-7.65 (m, 12H); FAB MS m/z 583 (MH
+ H81Br, 20), 581 (MH + H79Br, 20), 501 (M + H, 36), 384
(20), 304 (58), 200 (66), 185 (10). Anal. (C30H56N6‚6HBr‚5H2O)
C, H, N, Br.
4-Ch lor o-2,6-bis(h yd r oxym et h yl)p yr id in e (22a ). To a
stirred solution of dimethyl 4-chloropyridine-2,6-dicarboxylate
(21a ) (5 g, 21.83 mmol) in 200 mL of anhydrous EtOH was
added sodium borohydride (3.31 g, 87.33 mmol) and the
mixture gently refluxed under an argon atmosphere for 16 h.
The solution was cooled to room temperature and concentrated
to dryness. The residue was partitioned between ethyl acetate
(50 mL) and H2O (50 mL) and the aqueous layer was separated
and extracted with ethyl acetate (×3). The combined organic
extracts were dried (MgSO4) and concentrated in vacuo to give
22a (2.41 g, 64%) as a white solid: 1H NMR (DMSO-d6) δ 4.51
(d, 4H, J ) 6.7 Hz), 5.53 (t, 2H, J ) 6.7 Hz), 7.35 (s, 2H).
4-Ch lor o-2,6-b is(ch lor om et h yl)p yr id in e (23a ). To a
stirred solution of 22a (2.41 g, 13.93 mmol) and Et3N (7.8 mL,
55.72 mmol) in CH2Cl2 (100 mL) and CHCl3 (50 mL) under an
argon atmosphere, cooled to 0 °C, was added methanesulfonyl
chloride (3.2 mL, 41.79 mmol). The solution was stirred at 0
°C for 30 min, then allowed to warm to room temperature,
and stirred for a further 36 h. The reaction mixture was
quenched with water (50 mL) and the aqueous phase was
separated and extracted with CH2Cl2. The combined organic
extracts were dried (MgSO4) and concentrated in vacuo to
afford a red-orange oil. The product was purified by column
chromatography on a short plug of silica gel (CH2Cl2) to give
23a (1.9 g, 65%) as a pale yellow solid: 1H NMR (DMSO-d6) δ
4.77 (s, 4H), 7.69 (s, 2H).
15-Ch lor o-7-d iet h oxyp h osp h or yl-3,11-b is(p -t olu en e-
su lfon yl)-3,7,11,17-tetr aazabicyclo[13.3.1]h eptadeca-1(17),-
13,15-tr ien e (24d ). Using general procedure A, 11 (1.8 g, 3.13
mmol) and 23a (660 mg, 3.13 mmol) gave 24d (640 mg, 29%)
as a fluffy white solid: 1H NMR (CDCl3) δ 1.22 (t, 3H, J ) 7.8
Hz), 1.25 (t, 3H, J ) 7.8 Hz), 1.51 (quintet, 4H, J ) 7.6 Hz),
2.43 (s, 6H), 2.70-2.83 (m, 4H), 3.11 (t, 4H, J ) 7.6 Hz), 3.80-
4.01 (m, 4H), 4.25 (4H), 7.33 (d, 4H, J ) 9 Hz), 7.55 (s, 2H),
7.69 (d, 4H, J ) 9 Hz); FAB MS m/z 713 (M + H, 100), 557
(55).
15-Ch lor o-3,11-b is(p -t olu e n e su lfon yl)-3,7,11,17-t e t -
r a a za bicyclo [13.3.1]h ep ta d eca -1(17),13,15-tr ien e (25d ).
Using general procedure D, 24d (640 mg, 0.899 mmol) gave
25d (440 mg, 85%) as a white solid: 1H NMR (CDCl3) δ 1.52
(quintet, 4H, J ) 7.6 Hz), 2.31-2.37 (m, 4H), 2.44 (s, 6H), 3.17
(t, 4H, J ) 7.6 Hz), 4.29 (s, 4H), 7.33 (d, 4H, J ) 8.7 Hz), 7.43
(s, 2H), 7.72 (d, 4H); FAB MS m/z 577 (M + H, 100), 421 (16).
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[15-ch lor o-3,11-
bis(p-tolu en esu lfon yl)-3,7,11,17-tetr a a za bicyclo[13.3.1]-
h ep ta d eca -1(17),13,15-tr ien e] (26d ). Using general proce-
dure C, 25d (430 mg, 0.746 mmol) and R,R′-dibromo-p-xylene
(99 mg, 0.373 mmol) gave 26d (280 mg, 60%) as a white solid:
Gen er a l P r oced u r e F : Su lfu r ic Acid Dep r otection .
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[15-ch lor o-3,7,11,-
17-tetr a a za bicyclo [13.3.1]h ep ta d eca -1(17),13,15-tr ien e]
Hexa h yd r obr om id e (27d ). A solution of 26d (270 mg, 0.215
mmol) in concentrated H2SO4 (3 mL) was stirred at 110 °C for
2 h. The dark brown solution was allowed to cool to room
temperature and the pH adjusted to pH 14 with 10 N NaOH.
The aqueous solution was extracted with CHCl3 (3 × 20 mL)
and the combined organic extracts were dried (MgSO4) and
concentrated in vacuo to give a pale yellow oil. This compound
was converted to the hydrobromide salt using the following
procedure:
To a stirred solution of the oil in anhydrous EtOH (5 mL)
was passed HBr(g). The resulting tan solid was collected by
filtration under argon and washed with acetic acid and then
Et2O. The solid was dissolved in H2O (5 mL) and treated with
charcoal (120 mg) and the mixture was heated to 80 °C for 30
min. The hot solution was filtered through Celite and the
filtrate was concentrated to approximately 2 mL, after which
glacial acetic acid was added resulting in the immediate
formation of a white precipitate. The precipitate was collected
by filtration, washed with Et2O, and dried in vacuo giving 27d
(90 mg, 35%) as a white solid: 1H NMR (D2O) δ 2.10-2.24
(m, 8H), 3.00-3.12 (m, 8H), 3.12-3.24 (m, 8H), 4.21 (s, 4H),
4.40 (s, 8H), 7.39 (s, 4H), 7.53 (s, 4H); 13C NMR (D2O) δ 19.46,
43.22, 48.33, 48.75, 58.38, 125.09, 130.6, 132.1, 147.1, 151.6;
FAB MS m/z 721 (MH + H81Br, 51), 719 (MH + H79Br, 38)
639 (M + H, 100), 372 (18). Anal. (C34H48N8Cl2‚6HBr‚HOAc)
C, H, N, Hal.
7,7′-[1,4-P h en ylen eb is(m et h ylen e)]b is[3,7,11-t r ia za -
bicyclo[13.3.1]h ep ta d eca -1(17),13,15-tr ien e] Hexa h yd r o-
br om id e Dih yd r a te (27a ). Using general procedure E, 26a
(70 mg, 0.06 mmol) gave 27a (63 mg, 90%) as a white solid:
1H NMR (D2O) δ 1.65-1.75 (m, 8H), 2.95-3.15 (m, 16H), 4.23
(s, 4H), 4.34 (s, 8H), 7.43 (s, 4H), 7.54-7.74 (m, 8H); FAB MS
m/z 651 (MH + H81Br, 18), 649 (MH + H79Br, 18), 569 (M +
H, 45), 535 (15), 370 (75), 338 (100). Anal. (C36H52N6.6HBr‚
2H2O) C, H, N, Br.
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[3,7,11,17-tetr a -
a za bicyclo[13.3.1]h ep ta d eca -1(17),13,15-tr ien e] Hexa h y-
d r obr om id e Hexa h yd r a te (27b). Using general procedure
E, 26b (150 mg, 0.13 mmol) gave 27b (60 mg, 39%) as a white
solid: 1H NMR (D2O) δ 2.22 (m, 8H), 3.03 (m, 8H), 3.27 (m,
8H), 4.35 (s, 4H), 4.43 (s, 8H), 7.44 (d, 4H, J ) 7.5 Hz), 7.49
(s, 4H), 7.85 (t, 2H, J ) 7.5 Hz); FAB MS m/z 653 (MH + H81
-
Br, 13), 651 (MH + H79Br, 13), 571 (M + H, 48), 339 (58), 235
(100). Anal. (C34H50N8‚6HBr‚6H2O‚0.5HOAc) C, H, N, Br.
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[3,7,11,15,17-pen ta-
a za bicyclo[13.3.1]h ep ta d eca -1(17),13,15-tr ien e] Hep ta h y-
d r obr om id e (27c). Using general procedure F, 26c (350 mg,
0.295 mmol) gave 27c (220 mg, 62%) as a pale yellow solid:
1H NMR (D2O) δ 2.27 (m, 8H), 3.14 (m, 8H), 3.30 (m, 8H), 4.38
(s, 4H), 4.57 (s, 8H), 7.51 (s, 4H), 8.67 (s, 4H); 13C NMR (D2O)
δ 19.45, 43.46, 46.92, 48.39, 58.78, 130.64, 132.52, 145.44,
146.54; FAB MS m/z 655 (MH + H81Br, 12), 653 (MH + H79
Br, 10), 573 (M + H, 20), 340 (13), 236 (56). Anal. (C32H48N10
7HBr‚HOAc) C, H, N, Br.
-
‚
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[15-p h en yl-3,7,-
11,17-t e t r a a za b ic y c lo [13.3.1]h e p t a d e c a -1(17),13,15-
tr ien e] Octa h yd r obr om id e (27e). Using general procedure
E, 26e (420 mg, 0.314 mmol) gave 27e (330 mg, 74%) as a
white solid: 1H NMR (D2O) δ 2.24 (m, 8H), 3.10 (m, 8H), 3.28
(m, 8H), 4.33 (s, 4H), 4.48 (s, 8H), 7.36-7.45 (m, 6H), 7.48 (s,
4H), 7.60-7.66 (m, 4H), 7.71 (s, 4H); 13C NMR (D2O) δ 19.68,
43.40, 48.67, 49.55, 58.73, 122.76, 127.48, 129.69, 130.50,
130.56, 132.59, 136.32, 150.88, 151.83; FAB MS m/z 805 (MH
+ H81Br, 5), 803 (MH + H79Br, 4), 723 (M + H, 60), 415 (34),
311 (100). Anal. (C46H58N8‚8HBr‚HOAc) C, H, N, Br.

3980 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Bridger et al.
7,7′-[1,4-P h e n yle n e b is(m e t h yle n e )]b is[15-m e t h oxy-
3,7,11,17-tetr aazabicyclo[13.3.1]h eptadeca-1(17),13,15-tr i-
en e] Hexa h yd r obr om id e Dih yd r a te (27f). Using general
procedure F, 26f (160 mg, 0.128 mmol) gave 27f (65 mg, 44%)
as a white solid: IR (KBr) ν 1607 cm-1 (CdC-OMe); 1H NMR
(D2O) δ 2.14-2.26 (m, 8H), 3.00-3.10 (m, 8H), 3.12-3.23 (m,
8H), 3.80 (s, 6H), 4.25 (s, 4H), 4.35 (s, 8H), 7.01 (s, 4H), 7.43
(s, 4H); 13C NMR (D2O) δ 19.25, 42.98, 48.21, 49.12, 55.99,
58.26, 110.80, 130.46, 132.16, 151.63, 167.99: FAB MS m/z
713 (MH + H81Br, 41), 711 (MH + H79Br, 40), 631 (M + H,
100), 617 (14), 416 (12), 368 (13). Anal. (C36H54N8‚6HBr‚
2.5H2O) C, H, N, Br.
4H), 3.65 (m, 4H), 7.05 (d, 2H, J ) 7.5 Hz), 7.31 (d, 4H, J )
8.2 Hz), 7.55 (t, 1H, J ) 7.5 Hz), 7.67 (d, 4H, J ) 8.2 Hz).
4,12-Bis(p-tolu en esu lfon yl)-4,8,12,19-tetr a a za bicyclo-
[15.3.1]n on a d eca -1(19),15,17-tr ien e (34). Macrocyclization
of 28 and 29 using general procedure A (48% yield) followed
by deprotection of the diethoxyphosphoryl group using general
procedure D (97% yield) gave 34 as a white solid: 1H NMR
(CDCl3) δ 1.55 (m, 4H), 2.42 (s, 6H), 2.35 (t, 4H, J ) 6.2 Hz),
3.05 (t, 4H, J ) 6.2 Hz), 3.15 (t, 4H, J ) 6.2 Hz), 3.60 (t, 4H,
J ) 6.2 Hz), 7.05 (d, 2H, J ) 7.5 Hz), 7.35 (d, 4H, J ) 8.2 Hz),
7.55 (t, 1H, J ) 7.5 Hz), 7.75 (d, 4H, J ) 8.2 Hz); FAB MS m/z
571 (M + H, 100), 415 (20).
6,6′-[1,4-P h e n y le n e b i s (m e t h y le n e )]b i s [3,6,9,15-
tetraazabicyclo[11.3.1]pentadeca-1(15),11,13-triene]Hexahy-
d r obr om id e Tr ih yd r a te (35a ). General procedure F gave
35a (33% yield) as a white solid: 1H NMR (D2O) δ 2.65 (m,
8H), 3.05 (m, 8H), 3.75 (s, 4H), 4.45 (s, 8H), 7.20 (d, 4H, J )
7.5 Hz), 7.26 (s, 4H), 7.85 (t, 2H, J ) 7.5 Hz); FAB MS m/z
597 (MH + H81Br, 9), 595 (MH + H79Br, 9), 515 (M + H, 57),
440 (48), 223 (100). Anal. (C30H42N8‚6HBr‚3H2O‚0.75HOAc) C,
H, N.
6,6′-[1,3-P h e n y le n e b i s (m e t h y le n e )]b i s [3,6,9,15-
tetraazabicyclo[11.3.1]pentadeca-1(15),11,13-triene]Hexahy-
d r obr om id e Tr ih yd r a te (35b). General procedure F gave
35b (45% yield) as a white solid: 1H NMR (D2O) δ 2.66 (m,
8H), 3.11 (m, 8H), 3.76 (s, 4H), 4.45 (s, 8H), 7.26-7.29 (m, 8H),
7.78 (t, 2H, J ) 7.5 Hz); FAB MS m/z 597 (MH + H81Br, 9),
595 (MH + H79Br, 9), 515 (M + H, 100). Anal. (C30H42N8‚6HBr‚
3H2O‚HOAc) C, H, N, Br.
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[4,7,10,17-tetr a -
a za bicyclo[13.3.1]h ep ta d eca -1(17),13,15-tr ien e] Octa h y-
d r obr om id e Tetr a h yd r a te (36). General procedure F gave
36 (72% yield) as a white solid: 1H NMR (D2O) δ 2.75 (m, 8H),
3.05-3.65 (m, 28H), 6.88 (s, 4H), 7.15 (d, 4H, J ) 7.5 Hz),
7.65 (t, 2H, J ) 7.5 Hz); FAB MS m/z 653 (MH + H81Br, 22),
651 (MH + H79Br, 22), 571 (M+H, 31), 339 (21), 235 (100).
Anal. (C34H50N8‚8HBr‚4H2O‚0.5HOAc) C, H, N, Br.
8,8′-[1,4-P h en ylen ebis(m eth ylen e)]bis[4,8,12,19-tetr a -
a za bicyclo[15.3.1]n on a d eca -1(19),15,17-tr ien e] Non a h y-
d r obr om id e Tr ih yd r a te (37). General procedure E gave 37
(65% yield) as a white solid: 1H NMR (D2O) δ 2.13 (m, 8H),
3.06-3.39 (m, 32H), 4.41 (s, 4H), 7.13 (d, 4H, J ) 7.5 Hz),
7.51 (s, 4H), 7.65 (t, 2H, J ) 7.5 Hz); FAB MS m/z 709 (MH +
H81Br, 33), 707 (MH + H79Br, 33), 627 (M + H, 83), 367 (100).
Anal. (C38H58N8‚8HBr‚3.75H2O) C, H, N.
7,7′-[1,4-P h en ylen ebis(m eth ylen e)]bis[3,7,11,17-tetr a -
a za bicyclo[13.3.1]h ep ta d eca -13,16-tr ien -15-on e] Octa h y-
d r obr om id e (27g). Using general procedure E, 26f (150 mg,
0.12 mmol) gave 27g (95 mg, 64%) as a white solid: IR (KBr)
ν 1629 cm-1 (CdO); 1H NMR (D2O) δ 2.16-2.29 (m, 8H), 3.06
(t, 8H, J ) 7.6 Hz), 3.27 (t, 8H, J ) 7.6 Hz), 4.31 (s, 8H), 4.34
(s, 4H), 6.85 (s, 4H), 7.48 (s, 4H); 13C NMR (D2O) δ 19.1, 42.94,
48.20, 49.03, 58.30, 112.19, 130.29, 132.24, 151.63, 165.71; FAB
MS m/z 685 (MH + H81Br, 5), 683 (MH + H79Br, 5), 603 (M +
H, 100), 460 (12), 329 (16). Anal. (C34H50N8O2‚8HBr) C, H, N,
Br.
2,6-Bis(N ,N ′-p -t olu e n e su lfon yl-2-a m in oe t h yl)p yr i-
d in e (28). A stirred solution of 2,6-bis(bromomethyl)pyridine
hydrobromide (18) (6.0 g, 17 mmol), NaCN (5.1 g, 104 mmol),
and cetyltrimethylammonium bromide (633 mg, 1.7 mmol) in
benzene (50 mL) and H2O (25 mL) was heated to reflux for 4
h. Upon cooling, the aqueous layer was separated and ex-
tracted with benzene (50 mL) and dichloromethane (75 mL)
and the combined organic phases were dried (Na2SO4) and
evaporated in vacuo to give a brown solid. Filtration of a CH2-
Cl2 solution of the brown residue through a short bed of basic
alumina gave the crude dinitrile (2.4 g, 86%) as a white solid:
1H NMR (CDCl3) δ 3.94 (s, 4H), 7.45 (d, 2H, J ) 7.5 Hz), 7.85
(t, 1H, J ) 7.5 Hz).
To a solution of the dinitrile from above (4.3 g, 27 mmol) in
MeOH (75 mL, saturated with ammonia) was added Raney
nickel (10.0 g, excess) and the mixture was hydrogenated at
45 psi and room temperature for 48 h. The catalyst was
removed by filtration through Celite and the filtrate was
evaporated in vacuo to give the pyridyldiamine as a brown
viscous oil (3.7 g, 83%): 1H NMR (CDCl3) δ 2.91 (m, 4H), 3.01
(m, 4H), 7.01 (d, 2H, J ) 7.5 Hz), 7.55 (t, 1H, J ) 7.5 Hz).
This was used without further purification.
An ti-HIV Activity Assa ys. Inhibition of HIV-1 (IIIB) and
HIV-2 (ROD) replication assays was performed as previously
described.2-5 Anti-HIV activity and cytotoxicity measurements
were carried out in parallel. They were based on the viability
of MT-4 cells that had been infected with HIV in the presence
of various concentrations of the test compounds. After the
MT-4 cells were allowed to proliferate for 5 days, the number
of viable cells was quantified by a tetrazolium-based colori-
metric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) procedure in 96-well microtrays. In all of these
assays, viral input (viral multiplicity of infection, MOI) was
0.01, or 100 times the 50% cell culture infective dose (CCID50).
The EC50 was defined as the concentration required to protect
50% of the virus-infected cells against viral cytopathicity. The
50% cytotoxic concentration (CC50) was defined as the com-
pound concentration required to reduce the viability of mock-
infected cells by 50%. The greater than symbol (>) is used to
indicate the highest concentrations at which the compounds
were tested and still found to be noncytotoxic. Average EC50
and CC50 values for several separate experiments are pre-
sented as defined above. As a rule, the individual values did
not deviate by more than 2-fold up or down from the EC50 and
CC50 values indicated in Tables 1 and 2.
To a stirred solution of the brown oil and Et3N (7.0 mL) in
CH2Cl2 (75 mL) was added dropwise a solution of p-toluene-
sulfonyl chloride (8.7 g, 49 mmol) in CH2Cl2 (25 mL) over
approximately 15 min and the reaction mixture was then
allowed to stir at room temperature overnight. The solution
was washed with saturated aqueous sodium bicarbonate (50
mL) and brine (50 mL), then dried (Na2SO4), and evaporated
in vacuo to give a pale yellow viscous oil. The product was
purified by column chromatography on silica gel (CH2Cl2/
MeOH, 98:2) to give 28 (8.0 g, 75%) as a white solid: 1H NMR
(CDCl3) δ 2.41 (s, 6H), 2.89 (t, 4H, J ) 6.5 Hz), 3.25 (m, 4H),
5.65 (t, 2H), 6.95 (d, 2H, J ) 7.5 Hz), 7.25 (d, 4H, J ) 8.2 Hz),
7.45 (t, 1H, J ) 7.5 Hz), 7.71 (d, 4H, J ) 8.2 Hz).
3,9-Bis(p -t olu e n e su lfon yl)-3,6,9,15-t e t r a a za b icyclo-
[11.3.1]p en ta d eca -1(15),11,13-tr ien e (32). Macrocyclization
of 18 and 31 using general procedure A (68% yield) followed
by deprotection of the diethoxyphosphoryl group using general
procedure D (80% yield) gave 32 as a white solid: 1H NMR
(CDCl3) δ 2.44 (s, 6H), 2.65 (m, 4H), 3.35 (m, 4H), 4.31 (s, 4H),
7.15 (d, 2H, J ) 7.5 Hz), 7.35 (d, 4H, J ) 8.2 Hz), 7.65 (d, 4H,
J ) 8.2 Hz), 7.85 (t, 1H, J ) 8.2 Hz); FAB MS m/z 537 (M +
Na, 26), 515 (M + H, 100), 359 (9).
Assa y for In h ibition of Syn cytiu m F or m a tion .3,20 Per-
sistently HIV-1 (IIIB)-infected HUT-78 cells (washed twice to
remove free virus) were cocultured in 96-well microtrays in
the presence of various concentrations of the test compounds
with uninfected MOLT-4 cells, both cell types at a ratio of 5 ×
105 cells each in a total volume of 200 µL. After incubation for
4,10-Bis(p-tolu en esu lfon yl)-4,7,10,17-tetr a a za bicyclo-
[13.3.1]h ep ta d eca -1(17),13,15-tr ien e (33). Macrocyclization
of 28 and 30 using general procedure A (23% yield) followed
by deprotection of the diethoxyphosphoryl group using general
procedure D (92% yield) gave 33 as a white solid: 1H NMR
(CDCl3) δ 2.42 (s, 6H), 2.55 (m, 4H), 2.99 (m, 4H), 3.15 (m,

Phenylenebis(methylene)-Linked Bis-azamacrocycles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3981
activity relationships of phenylenebis(methylene) linked bis-
tetraazamacrocycles that inhibit HIV replication 2. Effect of
heteroaromatic linkers on the activity of bicyclams. J . Med.
Chem. 1996, 39, 109-119.
20-24 h at 37 °C in a CO2-controlled atmosphere, syncytia
were evaluated microscopically. The 50% inhibitory concentra-
tion was defined as the compound concentration required to
inhibit syncytium formation by 50%.
(6) J oao, H. C.; De Vreese, K.; Pauwels, R.; De Clercq, E.; Henson,
G. W.; Bridger, G. J . Quantitative structural activity relationship
study of bis-tetraazacyclic compounds - a novel series of HIV-1
and HIV-2 inhibitors. J . Med. Chem. 1995, 38, 3865-3873.
(7) De Vreese, K.; Reymen, D.; Griffin, P.; Steinkasserer, A.; Werner,
G.; Bridger, G. J .; Este, J .; J ames, W.; Henson, G. W.; Desmyter,
J .; Anne, J .; De Clercq, E. The bicyclams, a new class of potent
human immunodeficiency virus inhibitors, block viral entry after
binding. Antiviral Res. 1996, 29, 209-219.
(8) Donzella, G. A.; Schols, D.; Lin, S. W.; Este´, A. J .; Nagashima,
K. A.; Maddon, P. J .; Allaway, G. P.; Sakmar, P. T.; Henson, G.;
De Clercq, E.; Moore, J . P. AMD3100, a small molecule inhibitor
of HIV-1 entry via the CXCR4 co-receptor. Nature Medicine
1998, 4, 72-76.
(9) Schols, D.; Struyf, S.; Van Damme, J .; Este´, J . A.; Henson, G.;
De Clercq, E. Inhibition of T-tropic HIV strains by selective
antagonization of the chemokine receptor CXCR4. J . Exp. Med.
1997, 186, 1383-1388.
(10) Schols, D.; Este´, J . A.; Henson, G.; De Clercq, E. Bicyclams, a
class of potent anti-HIV agents, are targeted at the HIV
coreceptor Fusin/CXCR4. Antiviral Res. 1997, 35, 147-156.
(11) Este´, J . A.; Cabrera, C.; De Clercq, E.; Struyf, S.; Van Damme,
J .; Bridger, G.; Skerlj, R. T.; Abrams, M. J .; Henson, G.;
Gutierrez, A.; Clotet, B.; Schols, D. Activity of different bicyclam
derivatives against Human Immunodeficiency Virus depends on
their interaction with the CXCR4 chemokine receptor. Mol.
Pharmacol. 1999, 55, 67-73.
(12) Simmons, G.; Reeves, J . D.; McKnight, A.; Dejucq, N.; Hibbitts,
S.; Power, C. A.; Aarons, E.; Schols, D.; De Clercq, E.; Proudfoot,
A. E.; Clapham, P. R. CXCR4 as a functional coreceptor for
Human Immunodeficiency Virus Type 1 infection of primary
macrophages. J . Virol. 1998, 72, 8453-8457.
(13) Labrosse, B.; Brelot, A.; Heveker, N.; Sol, N.; Schols, D.; De
Clercq, E.; Alizon, M. Determinants for sensitivity of Human
Immunodeficiency Virus coreceptor CXCR4 to the bicyclam
AMD3100. J . Virol. 1998, 72, 6381-6388.
(14) Zhang, L.; He, T.; Huang, Y.; Chen, Z.; Guo, Y.; Wu, S.;
Kunstman, J .; Clark Brown, R.; Phair, J . P.; Neumann, A. U.;
Ho, D. D.; Wolinsky, S. M. Chemokine coreceptor usage by
diverse primary isolates of Human Immunodeficiency Virus Type
1. J . Virol. 1998, 72, 9307-9312.
(15) Herbein, G.; Malhiknecht, U.; Batliwalla, F.; Gregersen, P.;
Pappas, T.; Butler, J .; O′Brien, W. A.; Verdin, E. Apoptosis of
CD8 T cells is mediated by macrophages through interaction of
HIV gp 120 with chemokine receptor CXCR4. Nature 1998, 395,
189-194.
An a lysis of In h ibition of CXCR4-Sp ecific m Ab 12G5
Bin d in g to CXCR4 by Bis-a za m a cr ocycles. The specific
effects on CXCR4 were determined as described previously.9
Briefly, the lymphocytic SUP-T1 cells were incubated with
compounds or PBS for 15 min at room temperature. The cells
were washed with PBS and the 12G5 mAb (R&D Systems)
was added for 30 min at room temperature. The cells were
washed twice, then incubated with FITC-conjugated goat anti-
mouse Ab (Caltag Labs) for 30 min at room temperature, and
then washed twice with PBS. The percentage of positive cells
and mean fluorescence intensity (NFI) values are indicated
in each histogram and were analyzed by a FACScan flow
cytometer (Becton Dickinson).
Mea su r em en t of In tr a cellu la r Ca lciu m Con cen tr a -
tion s. The determinations of intracellular calcium concentra-
tions [Ca2+]i were carried out as previously described.9,21 SUP-
T1 cells were loaded on a Fura-2 (Molecular Probes). Fura-2
fluorescence was measured in a luminescence spectrophotom-
eter fitted with a water-thermostatable, stirred four-position
cuvette holder (Perkin-Elmer). Cells were first stimulated with
dilution buffer (control) or test compounds [AMD3100 or
AMD3329 (compound 36)] at different concentrations. As a
second stimulus, SDF-1R (R&D Systems) was used at an
optimal concentration to induce a maximal [Ca2+]i increase.
The second stimulus was added 100 s after the first stimulus.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the editorial assistance of Susan O’Hara (J ohnson
Matthey Pharmaceutical Research) and Genevieve Kr-
eye (AnorMED Inc.). We also thank Christophe Pan-
necouque (Rega Institute) for critical reading of the
manuscript. Investigations at the Rega Institute were
supported by the Biomedical Research Program of the
European Commision, the Belgian Nationaal Fonds voor
Wetenschappelijk Onderzoek, and the Geconcerteerde
Onderzoeksacties. We also thank Sandra Claes, Kristien
Erven, and Cindy Heens for excellent technical as-
sistance.
(16) Hesselgesser, J .; Taub, D.; Baskar, P.; Greenberg, M.; Hoxie,
J .; Kolson, D. L.; Horuk, R. Neuronal apoptosis induced by HIV-1
gp120 and the chemokine SDF-1R is mediated by the chemokine
receptor CXCR4. Curr. Biol. 1998, 8, 595-598.
Refer en ces
(1) De Clercq, E. Toward improved anti-HIV chemotherapy: thera-
peutic strategies for intervention with HIV infections. J . Med.
Chem. 1995, 38, 2491-2517.
(2) De Clercq, E.; Yamamoto, N.; Pauwels, R.; Baba, M.; Schols, D.;
Nakashima, H.; Balzarini, J .; Debyser, Z.; Murrer, B. A.;
Schwartz, D.; Thornton, D.; Bridger, G.; Fricker, S.; Henson, G.;
Abrams, M.; Picker, D. Potent and selective inhibition of human
immunodeficiency virus (HIV)-1 and HIV-2 replication by a class
of bicyclams interacting with a viral uncoating event. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 5286-5290.
(3) De Clercq, E.; Yamamoto, N.; Pauwels, R.; Balzarini, J.; Witvrouw,
M.; De Vreese, K.; Debyser, Z.; Rosenwirth, B.; Peichl, P.;
Datema, R.; Thornton, D.; Skerlj, R.; Gaul, F.; Padmanabhan,
S.; Bridger, G.; Henson, G.; Abrams, M. Highly potent and
selective inhibition of human immunodeficiency virus by the
bicyclam derivative J M3100. Antimicrob. Agents Chemother.
1994, 38, 668-674.
(4) Bridger, G. J .; Skerlj, R. T.; Thornton, D.; Padmanabhan, S.;
Martellucci, S. A.; Henson, G. W.; Abrams, M. J .; Yamamoto,
N.; De Vreese, K.; Pauwels, R.; De Clercq, E. Synthesis and
structure-activity relationships of phenylenebis(methylene)
linked bis-tetraazamacrocycles that inhibit HIV replication.
Effects of macrocyclic ring size and substituents on the aromatic
linker. J . Med. Chem. 1995, 38, 366-378.
(5) Bridger, G. J .; Skerlj, R. T.; Padmanabhan, S.; Martellucci, S.
A.; Henson, G. W.; Abrams, M. J .; J oao, H.; Witvrouw, M.; De
Vreese, K.; Pauwels, R.; De Clercq, E. Synthesis and structure-
(17) Datema, R.; Rabin, L.; Hincenbergs, M.; Moreno, M. B.; Warren,
S.; Linquist, V.; Rosenwirth, B.; Seifert, J .; McCune, J . M.
Antiviral efficacy in vivo of the anti-Human Immunodeficiency
Virus bicyclam SDZ SID 791 (J M 3100), an inhibitor of infectious
cell entry. Antimicrob. Agents Chemother. 1996, 40, 750-754.
(18) Bridger, G.; Skerlj, R. T.; Padmanabhan, S.; Thornton, D. A
versatile intermediate for the preparation of C-functionalized
azamacrocycles and application to the synthesis of the potent
anti-HIV agent (() J M2936. J . Org. Chem. 1996, 61, 1519-1522.
(19) Costa, J .; Delgado, R. Metal complexes of macrocyclic ligands
containing pyridine. Inorg. Chem. 1993, 32, 5257-5265.
(20) Schols, D.; Pauwels, R.; Baba, M.; Desmyter, J .; De Clercq, E.
Syncytium formation and destruction of bystander CD4+ cells
cocultured with T-cells persistently infected with Human Im-
munodeficiency Virus as demonstrated by flow cytometry. J .
Gen. Virol. 1989, 70, 2397-2408.
(21) Struyf, S.; Proost, P.; Schols, D.; De Clercq, E.; Opdenakker, G.;
Lenaerts, J .-P.; Detheux, M.; Parmentier, M.; De Meester, I.;
Scharpe, S.; Van Damme, J . CD26/Dipeptidyl-peptidase IV
down-regulates the eosinophil chemotactic potency, but not
the anti-HIV activity of human eotaxin by affecting its interac-
tion with CC chemokine receptor 3. J . Immunol. 1999, 162,
4903-4909.
J M990211I