Synthesis and structure-activity relationships of phenylenebis(methylene)-linked bis-tetraazamacrocycles that inhibit human immunodeficiency virus replication. 2. Effect of heteroaromatic linkers on the activity of bicyclams

Article abstract of DOI:10.1021/jm950584t

A series of bicyclam analogs connected through a heteroaromatic linker have been synthesized and evaluated for their inhibitory effects on HIV-1 (III_B) and HIV-2 (ROD) replication in MT-4 cells. The activity of pyridine- and pyrazine-linked bicyclams was found to be highly dependent upon the substitution of the heteroaromatic linker connecting the cyclam rings. For example, 2,6- and 3,5-pyridine-linked bicyclams were potent inhibitors of HIV-1 and HIV-2 replication, whereas the 2,5- and 2,4-substituted pyridine-linked compounds exhibited substantially reduced activity and, in addition, were found to be highly toxic to MT-4 cells. We have subsequently discovered that these effects are not unique; amino-substituted linkers also have the potential to deactivate phenylenebis(methylene)-linked bicyclams. A model is proposed to explain the deactivating effects of the pyridine group in certain substitution patterns based on the ability of the pyridine nitrogen to participate in pendant conformations (complexation) with the adjacent azamacrocyclic ring, which may involve hydrogen bonding or coordination to a transition metal. The introduction of a sterically hindering group such as phenyl at the 6-position of the 2,4-substituted pyridine-linked bicyclam appears to prevent pendant conformations, providing an analog with comparable anti-HIV-1 and anti-HIV-2 activities to the parent m-phenylenebis(methylene)-linked bicyclam. The results of this study have been used to develop a quantitative structure-activity relationship model with improved predictive capability in order to aid the design of antiviral bis-azamacrocyclic analogs.

Full text of DOI:10.1021/jm950584t

J . Med. Chem. 1996, 39, 109-119
109
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-tetr a a za m a cr ocycles Th a t In h ibit Hu m a n Im m u n od eficien cy Vir u s
Rep lica tion . 2. Effect of Heter oa r om a tic Lin k er s on th e Activity of Bicycla m s
Gary J . Bridger,*,† Renato T. Skerlj,† Sreenivasan Padmanabhan,† Stephen A. Martellucci,†
Geoffrey W. Henson,† Michael J . Abrams,† Heidi C. J oao,‡ Myriam Witvrouw,§ Karen De Vreese,§
Rudi Pauwels,§ and Erik De Clercq§
J ohnson Matthey Pharmaceutical Research, 1401 King Road, West Chester, Pennsylvania 19380, Sandoz Research Institute,
Brunner Strasse 59, A-1235 Vienna, Austria, and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received August 7, 1995X
A series of bicyclam analogs connected through a heteroaromatic linker have been synthesized
and evaluated for their inhibitory effects on HIV-1 (IIIB) and HIV-2 (ROD) replication in MT-4
cells. The activity of pyridine- and pyrazine-linked bicyclams was found to be highly dependent
upon the substitution of the heteroaromatic linker connecting the cyclam rings. For example,
2,6- and 3,5-pyridine-linked bicyclams were potent inhibitors of HIV-1 and HIV-2 replication,
whereas the 2,5- and 2,4-substituted pyridine-linked compounds exhibited substantially reduced
activity and, in addition, were found to be highly toxic to MT-4 cells. We have subsequently
discovered that these effects are not unique; amino-substituted linkers also have the potential
to deactivate phenylenebis(methylene)-linked bicyclams. A model is proposed to explain the
deactivating effects of the pyridine group in certain substitution patterns based on the ability
of the pyridine nitrogen to participate in pendant conformations (complexation) with the
adjacent azamacrocyclic ring, which may involve hydrogen bonding or coordination to a
transition metal. The introduction of a sterically hindering group such as phenyl at the
6-position of the 2,4-substituted pyridine-linked bicyclam appears to prevent pendant conforma-
tions, providing an analog with comparable anti-HIV-1 and anti-HIV-2 activities to the parent
m-phenylenebis(methylene)-linked bicyclam. The results of this study have been used to develop
a quantitative structure-activity relationship model with improved predictive capability in
order to aid the design of antiviral bis-azamacrocyclic analogs.
In tr od u ction
Bicyclams are a novel class of antiviral agents that
exhibit potent inhibitory effects on HIV-1 and HIV-2
replication with high selectivity.1,2 From previous
structure-activity relationship studies of aliphatic linked
bicyclams such as J M2763 (1)1 (Figure 1) and aromatic
linked bis-tetraazamacrocycles that vary with ring size,3
it was discovered that dimers of the 1,4,8,11-tetraaza-
cyclotetradecane (cyclam) ring connected via a 1,4-
phenylenebis(methylene) linker provided optimum anti-
HIV-1 and anti-HIV-2 activity in vitro. Thus, J M3100
(10d , isolated as the octahydrochloride salt, Figure 1)
was identified as a lead candidate for further structure-
activity elaboration.2,3
F igu r e 1. Structures of the bicyclam analogs J M2763 and
The anti-HIV activity exhibited by bicyclams is par-
J M3100.
ticularly intriguing due to the possibility that binding
bicyclams exhibit antiviral activity.3,5 In this manner,
to the molecular target at the HIV-inhibitory step is
10d could be viewed as a prodrug for a transition metal
complex in a similar manner to the antitumor agent
bleomycin.6 However, definitively proving the involve-
ment (or lack of involvement) of transition metal ions
in the anti-HIV activity of bicyclams is complicated by
the fact that upon protonation (which occurs at physi-
ological pH) azamacrocycles can also complex anions.7,8
During the course of our ongoing investigations to
determine the structural features required for potent
anti-HIV activity in this class of compounds, we pre-
pared a series of compounds in which the cyclam rings
are connected by a heteroaromatic linker. Depending
upon the substitution pattern of pyridine and pyrazine
(and aniline) linkers, these analogs exhibited a striking
transition metal-mediated. For example, in coincidental
but unrelated recent publications, Arnold and co-work-
ers4 have proposed that bis-transition metal complexes
of 10d can provide complementary coordinating sites
for the chelating amino acid residues on a protein
surface. This type of mechanism is not implausable in
our system since 10d will almost assuredly complex free
transition metals in vitro, and a variety of preformed
transition metal complexes of 10d and several other
* Author to whom correspondence should be addressed.
† J ohnson Matthey Pharmaceutical Research.
‡ Sandoz Research Institute.
§ Rega Institute for Medical Research.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0109$12.00/0 © 1996 American Chemical Society

110 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bridger et al.
Sch em e 1a
(chloromethyl)pyrazine and 2,5-bis(bromomethyl)pyra-
zine were prepared by halogenation of dimethylpyrazine
isomers with NCS or NBS, respectively. The amino
analogs 10a and 11a were obtained from the nitro
derivatives 6a and 7a by reduction with Fe/concentrated
aqueous HCl11 followed by deprotection.
Resu lts a n d Discu ssion
Bicyclam analogs containing a pyridine linker (8a -
d ; Table 1) were tested for their inhibitory effects upon
HIV-1 (IIIB) and HIV-2 (ROD) replication in MT-4 cells
according to known procedures.3 Their anti-HIV activ-
ity was found to be highly dependent upon the substitu-
tion pattern of the pyridyl moiety connecting the cyclam
rings. For example, both the 2,6- (8a ) and 3,5- (8b)
substituted pyridine analogs exhibited comparable anti-
HIV-1 and anti-HIV-2 activity to the simple m-phenyl-
enebis(methylene)-linked analog 11c [50% effective
concentrations (EC50s) against HIV-1 for 8a ,b and 11c
were found to be 0.0245, 0.0316, and 0.0337 µM,
respectively], whereas the 2,4- (8c) and 2,5- (8d ) sub-
stituted pyridyl-linked analogs exhibited substantially
reduced activity against HIV-1 and HIV-2. Both the
2,4-substituted (8c) and 2,5-substituted (8d ) pyridine
analogs were found to be 2 orders of magnitude less
active against HIV-1 and HIV-2 replication than the
parent phenylenebis(methylene) analogs 11c and 10d ,
respectively. Furthermore, 8c,d also displayed a sig-
nificantly increased cytotoxicity to MT-4 cells. We
subsequently discovered that these effects were not
unique to pyridine-linked bicyclams. Replacing the
pyridine linker with the weaker base pyrazine (9a ,b)
(pKa of pyridine is 5.25 and pKa of pyrazine is 0.6512)
1
or a nonheterocyclic base of comparable pKa to pyridine
such as aniline (pKa of 4.65) (to give compounds 10a
and 11a ) gave similar results: The 2,6-pyrazine analog
9a exhibited comparable anti-HIV-1 and anti-HIV-2
activity to 11c, whereas the 2,5-analog 9b was 3 orders
of magnitude less active against HIV-1 and HIV-2
replication than 11c. Although the effect of the aniline
linkers was less pronounced, 10a proved to be 44- and
15-fold less effective in inhibiting HIV-1 and HIV-2
replication than 11a ,c.
A model to explain the deactivating effects of het-
eroaromatic linkers in certain substitution patterns was
obtained from reviewing the coordination chemistry
literature. Azamacrocycles such as cyclam display a
rich coordination chemistry, forming complexes with a
variety of transition metals with high thermodynamic
stability.13 However, from a chelation standpoint, cy-
clam suffers from the disadvantage that it is unable to
fill all of the vacant coordination sites on the majority
of transition metals. One solution to this problem
developed by several groups is the attachment of a
single pendant-coordinating substituent such as an
amine,14,15 phenol,16,17 pyridine,18-20 imidazole,21 or car-
boxylate22 group to the periphery of the cyclam ring,
which increases the ligating ability of the macrocycle
by filling a remaining coordination site, thereby “wrap-
ping up” the transition metal.9 Furthermore, previous
studies also indicate that the pendant-coordinating arm
can assist in the kinetics of azamacrocyclic metal
complex formation by rapid capture of the metal ion by
the pendant arm preceeding the relatively slow step of
transfer to the azamacrocyclic cavity, which is hindered,
in part, by conformational changes in the ligand neces-
a
Reagents: (a) K2CO3, CH3CN, reflux; (b) when X ) NO2, Fe,
concentrated aqueous HCl, EtOH; (c) when R ) Ts, 48% aqueous
HBr, HOAc, reflux, when R ) Dep, HBr/HOAc, room temperature.
anti-HIV structure-activity relationship, consistent
with a model in which the heteroatom of the linker can
assume a “pendant-coordinating”9 conformation with the
adjacent cyclam ring that is unfavorable with respect
to anti-HIV activity. This intramolecular interaction
may involve hydrogen bonding of the linker heteroatom
with the azamacrocyclic ring, competition for the azamac-
rocyclic binding site, or, indeed, coordination to a
transition metal complexed within the azamacrocyclic
cavity.
In the present work, we propose a conformational
model to explain the deleterious effects of certain
heteroaromatic linkers on the anti-HIV activity of
bicyclam analogs that is clearly a necessary consider-
ation for further structural design.
Ch em istr y
Bicyclam analogs (8-11; Table 1) were prepared by
the reaction of the tris-N-protected cyclam derivatives
with the appropriate aromatic or heteroaromatic bis-
electrophiles as previously described (Scheme 1).3 The
bis(bromomethyl)pyridine electrophiles were obtained
from commercially available pyridine diacids by BH3‚
THF reduction to the corresponding diols followed by
treatment with 48% aqueous hydrobromic acid/acetic
anhydride according to literature procedures.10 2,6-Bis-

Phenylenebis(methylene)-Linked Bis-tetraazamacrocycles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 111
Ta ble 1. Anti-HIV Activity of Heteroaromatic Linked Bis-Cyclams
EC50 (µM)b
compd
structure
subst
2,6-
3,5-
2,4-
2,5-
2,6-
2,5-
X ) NH2
X ) CO2H
X ) CO2Me
X ) H
X ) NH2
X ) CO2H
X ) H
formulaa
HIV-1 (IIIB)
HIV-2 (ROD)
CC50c (µM)
8a
8b
8c
8d
9a
I
I
I
I
II
II
III
III
III
III
IV
IV
IV
C27H53N9‚8HBr‚3.5H2O
0.0245d
0.0316d
16.367
0.9081
0.0261
138.53
0.1869
200.30
0.6825
0.0042
0.0468
203.00
0.0337
0.0654
0.0710
16.367
2.1947
0.0174
105.88
0.0934
200.30
0.5904
0.0059
0.0390
203.00
0.0422
>409
>395
17
C
C
C
C
C
27H53N9‚9HBr‚2.5H2O
27H53N9‚8HBr‚4H2O
27H53N9‚9HBr‚5H2O
18
26H52N10‚8HBr‚H2O‚0.5HOAc
26H52N10‚8HCl‚3.4H2O‚HOAc
>203
>203
>179
>200
>198
>421e
>194
>200
>421e
9b
10a
10b
10c
10d
11a
11b
11c
C28H55N9‚9HBr‚H2O‚1.5HOAc
C29H54N8O2‚8HBr‚HOAc
C30H56N8O2‚8HBr‚3H2O
C28H55N9‚9HBr‚2.2H2O
C29H54N8O2‚8HBr‚2H2O
a
Microanalyses are within (0.4 of theoretical values. All compounds tested as their hydrobromide salts unless otherwise indicated.
50% antiviral effective concentration. c 50% cytotoxic concentration. The > symbol is used to indicate the highest concentrations at
b
which the compounds were tested and still found to be noncytotoxic. Anti-HIV-1 (IIIB) data from ref 2. e Data from ref 3.
d
which allow the pyridine group to participate in unfa-
vorable (with respect to anti-HIV activity) pendant
conformations without steric interference from the
second ring (or the methylene group; see Figure 2).
Conversely, the pyridyl-linked analog 8b has a 3,5-
substitution pattern which itself prevents pendant
conformations. To complete the model, the 2,6-substitu-
tion pattern of 8a must also prevent the pyridine
nitrogen from approaching the macrocyclic ring as if it
were the axial ligand of an octahedral metal complex.
This is not unreasonable since, in fact, 2,6-disubstituted
pyridines are poor ligands24 and 2,6-lutidine is used as
a hindered base for synthetic organic transformations.
Similar arguments can be applied to the pyrazyl (9a ,b)
and aniline (10a and 11a ) analogs: Both the 2,6-pyrazyl
(9a ) and 3,5-disubstituted aniline (11a ) analogs possess
substitution patterns which would prevent pendant
conformations (and therefore exhibit comparable activ-
ity to 11c), whereas the corresponding 2,5-pyrazyl (9b)
and 2,5-aniline (10a ) analogs in which pendant confor-
mations are allowed exhibit reduced anti-HIV activity.
Moreover, the reduction in anti-HIV-1 and anti-HIV-2
potency of the 2,5-pyrazyl-linked analog 9b compared
to the 2,5-pyridine-linked analog 8d and 1,4-phenylene-
linked analog 10d can be explained by assuming that
both nitrogens of the pyrazine group of 9b can partici-
pate in intramolecular pendant complexation with their
respective, adjacent macrocyclic rings25 (EC50s against
HIV-1 for 9b, 8d , and 10d were 138.53, 0.9081, and
0.0042 µM, respectively). But what is the nature of the
pendant interaction?
F igu r e 2. Proposed model of pyridine-linked bicyclams in
which the 2,4- and 2,5-isomers can adopt pendant conforma-
tions.
sary for complexation.20,23 Though the role of transition
complexation at the molecular target for bicyclam
activity at the HIV-inhibitory step has not been estab-
lished, we found that these observations explained the
reduced activity of particular bicyclam analogs by
assuming that the heteroatom of the linker can partici-
pate in pseudopendant conformations (complexation)
with the azamacrocycle ring dictated by the substitution
pattern. Presumably, in cases where pendant confor-
mations can occur, this results in the “wrong” molecular
shape for binding with the target for 11c and 10d .
Application of this hypothesis to the pyridyl-linked
bicyclams is depicted in Figure 2.
If the binding of bicyclams to the molecular target is
not transition metal-mediated, then the pendant het-
eroatom of the linker may hydrogen bond to the protons
shared between the nitrogen groups of the macrocyclic
ring thereby inducing an unfavorable conformation with
respect to anti-HIV activity and possibly competing for
the azamacrocyclic binding site. Cyclam has four pKas
measured at 11.5, 10.3, 1.6, and 0.9,13,26 which provide
an overall charge on the macrocyclic ring under physi-
The pyridyl-linked bicyclam analogs 8c,d , exhibiting
reduced anti-HIV activity, possess 2,4- and 2,5-substitu-
tion patterns connecting the tetraazamacrocyclic rings,

112 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bridger et al.
Sch em e 2
ological conditions of +2/ring. The third and fourth
protonations of cyclam become increasingly difficult (as
indicated by their extremely low pKa values) due to the
mutually repulsive effects of the two positive charges
already confined in a relatively constrained macrocyclic
framework.13 Kimura and co-workers have demon-
strated that when a pyridine group is attached to the
periphery of the cyclam ring in a pendant fashion, the
pKa of the pyridine nitrogen is dramatically reduced
when appended in proximity to the macrocyclic ring. For
example, the measured pKa of the pyridine nitrogen in
3
the 6-ethylpyridine cyclam derivative 1218 (Scheme 2)
is 5.32 (which compares with pyridine, pKa ) 5.25),
whereas the 5-pyridylcyclam derivatives 1319 and 1420
exhibit pKa s for the pyridine nitrogen of <3 and 3.6,
3
respectively. This observation (for 14) has been inter-
preted as evidence for an intramolecular hydrogen bond
between the pyridine N and a shared proton complexed
within the azamacrocyclic cavity in aqueous solution.20
On the basis of the assumption that the reduced pKa
value of the pyridine groups in 13 and 14 reflects the
presence of an intramolecular hydrogen bond, we at-
tempted to measure the pKa of the pyridine moiety in
15 (Scheme 2), which represents the pendant-coordinat-
ing portion of the pyridyl analogs 8c,d . Titration of an
aqueous solution of 15 (0.1 mM, 50 mL) and 4 equiv of
HClO4 at 25 °C and I ) 0.1 M (NaClO4) with 0.1 M
NaOH gave the titration curve shown in Figure 3A.
From this curve, the pKa of the pyridine group could
only be estimated to be <3, a value indistinguishable
F igu r e 3. (A) pH titration curve of 15 (0.1 mM, 50 mL) and
4.0 equiv of HClO4 at 25 °C and I ) 0.1 M (NaClO4) vs equiv
of NaOH. (B) UV absorption spectrum of 15 (0.17 mM) at 25
°C and I ) 0.1 M (NaClO4) at varying pHs.
anion at physiological pH. Under basic conditions (pH
10-11), this derivative formed pendant-coordinated
complexes with Cu(II) and a weak interaction of the
carboxylate anion with the Ni(II) complex. The corre-
sponding p-toluic acid derivative was, as expected,
unable to form a complex in which the carboxylate is
coordinated to Cu(II) or Ni(II). Furthermore, an X-ray
crystal structure of a complex between four molecules
of 4-tert-butylbenzoic acid and the cyclam ring has
recently been published.8 The detailed structure re-
vealed that each of only two molecules of 4-tert-butyl-
benzoic acid interacts directly with the cyclam ring on
opposing faces of the macrocycle through a network of
hydrogen bonds (following a two-proton transfer to the
macrocyclic ring) spanning diagonal amine groups.
These combined results suggest that the carboxylate
group has the propensity to pendant coordinate to a
transition metal complex of the tetraazamacrocycle or
simply form a direct complex with the protonated ring.
By analogy, therefore, one would expect that bicyclam
from the pKa of the cyclam ring. That protonation of
3
the pyridine group only occurs at a pH of <3 was more
accurately confirmed by monitoring the UV absorption
spectrum of the pyridine moiety in 15 [0.17 mM at I )
0.1 M (NaClO4)] at varying pHs (Figure 3B). The
characteristic hyperchromic shift of the pyridine chro-
mophore which occurs upon protonation begins to ap-
pear at pH 3 and increases with decreasing pH to a
maximum at pH 1. Thus, the protonation constant of
the pyridine moiety in 15 is entirely consistent with the
hydrogen-bonding proposal of Kimura.20
Also of interest were the studies performed by Kaden
et al.22 on the chelating properties of the o-toluic acid
cyclam derivative 16 (Scheme 2). In this case, the pKa
of the pendant carboxylic acid was found to be 3.85,
which would provide a negatively charged carboxylate

Phenylenebis(methylene)-Linked Bis-tetraazamacrocycles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 113
Ta ble 2. Anti-HIV Activity of Substituted Pyridine-Linked Bis-Cyclamsa
EC50 (µM)
compd
subst
structure
formula
HIV-1 (IIIB)
HIV-2 (ROD)
CC50 (µM)
17a
17b
X ) N
X ) CH
C31H55N9‚9HBr‚5.5H2O
3.7716
0.1572
4.2744
0.0786
>419
207b
25a
25b
X ) N
X ) CH
C33H57N9‚9HBr
0.0398
0.2060
0.0200
0.0246
>203
>198b
a
b
See footnotes to Table 1. Data from ref 3.
analogs containing a strategically placed carboxyl group
on the aromatic linker would provide an identical anti-
HIV structure-activity relationship to the pyridine-
linked systems described above. Analogs 10b and 11b
were prepared and tested for their inhibitory effects on
HIV-1 and HIV-2 replication as shown in Table 1. To
our surprise, neither 10b (pendant conformation al-
lowed) nor 11b (pendant conformation disallowed)
inhibited HIV replication in MT-4 cells at concentrations
exceeding 200 µM. One possible explanation that could
account for these results is that the negative charge of
the carboxylate on the linker of 10b or 11b has a net
repulsive interaction with the target for bicyclam activ-
ity. This was confirmed by testing the methyl ester
derivative 10c (Table 1) which inhibited HIV-1 and
HIV-2 replication at 50% effective concentrations of
0.6825 and 0.5904 µM, respectively.
Sch em e 3a
In order to test our hypothesis that the proximal
pyridine nitrogen was responsible for the antiviral
inactivation of the pyridine-linked bicyclams 8c,d , we
reasoned that incorporation of a sterically demanding
group at the 6-position of the 2,4-pyridine-linked bicy-
clam 8c should prevent this analog from adopting
pendant conformations (giving a 2,6-disubstituted py-
ridine linker such as the 2,6-pyridyl analog 8a ; see
Figure 2) thereby restoring anti-HIV activity compa-
rable to that of the m-phenylenebis(methylene) analog
11c. To this end, two analogs were synthesized, the 2,4-
quinoline-linked analog in which the peri-hydrogen to
the quinoline nitrogen has moderate blocking ability
(17a ; Table 2) and the phenyl analog (25a ; Table 2)
which, by examination of molecular models, should
provide a total restriction on pendant conformations.
The synthesis of 25a was accomplished in seven steps
from the commercially available 2-amino-4,6-dimeth-
ylpyridine precursor 18 as illustrated in Scheme 3.
Diazotization27 of 18 in the presence of bromine gave
the bromide 19 which was subjected to palladium-
catalyzed cross-coupling28 with phenylboronic acid in
order to introduce the phenyl substituent giving 20.
Conversion of 20 to the corresponding bis-electrophile
23 proved straightforward, and this reagent was used
to prepare the bicyclam 25a using previously estab-
lished methodology (see also Scheme 1).
a
Reagents: (a) NaNO2, Br2, 48% aqueous HBr; (b) 1.1 equiv of
PhB(OH)2, 2.0 equiv of Na2CO3, toluene, EtOH, H2O, 5 mol %
Pd(PPh3)4, reflux; (c) 10.0 equiv of KMnO4, t-BuOH, H2O, reflux;
(d) BH3‚THF; (e) 48% aqueous HBr, Ac2O, reflux; (f) 2.0 equiv of
2a , K2CO3, CH3CN, reflux; (g) 48% aqueous HBr, HOAc, reflux.
were 3.7716 and 0.1572 µM, respectively). However, the
6-phenylpyridine analog 25a proved equally potent at
inhibition of HIV replication as 11c, exhibiting EC50s
against HIV-1 and HIV-2 of 0.0398 and 0.0200 µM,
respectively, which are around 400-fold lower than the
concentration of 8c required to inhibit viral replication
by 50%. In addition, 25a was also 11-fold less cytotoxic
to the host cells than 8c. The inactivity of the pyridine-
linked bicyclams can therefore be directly assigned to
the proximal pendant pyridine moiety.
In support of an alternate, transition metal-mediated
anti-HIV inactivation mechanism for the proposed
model, it was also necessary to demonstrate that forma-
tion of a pendant-coordinated transition metal complex
is achievable for the pyridine analogs 8c,d . Interest-
ingly, of the pyridylcyclam literature examples shown
in Scheme 2, only 13 and 14 formed pendant-coordi-
nated complexes with Ni(II), consistent with several
other examples of 14-29 and 16-membered30 tetraazamac-
rocyclic ligands featuring pendant amino or pyridine
groups. The existence of a 5-coordinate, low-spin, d7 Ni-
(III) complex of 12 in which the pyridine group was
axially coordinated was only observed in the ESR
spectrum when the 12:Ni(II) complex was chemically
oxidized.18 Clearly, the conformational requirements for
formation of a pendant transition metal complex are
Upon anti-HIV-1 and anti-HIV-2 evaluation in MT-4
cells (Table 2), we found that activity modestly improved
by incorporation of the 2,4-quinoline linker (17a ) com-
pared to the 2,4-pyridine-linked bicyclam analog 8c.
Compound 17a was ca. 4-fold more effective at inhibit-
ing HIV-1 and HIV-2 replication than 8c but was still
significantly less active than the nonheteroaromatic
naphthyl analog 17b (EC50s for 17a ,b against HIV-1

114 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bridger et al.
rigid. Confirmation of the pendant-coordinating capa-
bilities of the pyridyl analogs 8c,d was accomplished
by preparation of the Ni(II) complex of 15. Addition of
1 equiv of Ni(II) perchlorate in n-butanol, which had
been previously distilled to one-half volume to remove
water, to an ethanol solution of 15 gave a bright purple
precipitate which analyzed to the formula 15:Ni(ClO4)2
(26). In full agreement with the literature,14,29,30 the
UV absorption spectrum of 26 (9.0 mM) in aqueous 1
M NaClO4 (pH 6.9) gave three bands at 327 (sh), 531 (ꢀ
) 10) and 934 (ꢀ ) 9.1) nm, consistent with a 5-coordi-
nate, high-spin Ni(II) complex in which the pyridine
moiety is pendant-coordinated. Dissolving the solid 26
(9.0 mM) in 5 M aqueous perchloric acid gave an
immediate color change from purple (26) to orange (27)
(this concentration of HClO4 was required for complete
conversion of 26 to 27), which was found to be reversible
upon alternate additions of sodium hydroxide and
perchloric acid. The UV absorption spectrum of the
orange solution of 27 gave a new band at 462 (ꢀ ) 40)
nm which is typical of low-spin Ni(II) tetraamine
complexes,14 while the high-spin chromophore was
absent. This equilibrium represents a competition
between the nickel cation and the proton for the
pyridine nitrogen as illustrated in Figure 4 and estab-
lishes that formation of a pendant-coordinated transi-
tion metal complex can occur.
Finally, the anti-HIV virucidal properties of the
pyridine (8a -d ), pyrazine (9a ,b), aniline (10a and 11a ),
and phenyl (10d ) analogs from Table 1 were tested. Rice
and co-workers31 have recently demonstrated that treat-
ing HIV-1 virions with a series of 3-nitrosobenzamide
derivatives causes a loss of zinc ions and viral inactiva-
tion. Although the mechanism of this inactivation is
not through metal chelation but oxidative damage of the
cysteine thiolates of the NCp7 nucleocapsid protein, the
ultimate result of treating free virus with chelating
agents would be identical, that is, loss of metal ions and
viral inactivation. Since, from a metal chelation stand-
point, the pendant group can assist in the kinetics of
metal complex formation and subsequently form com-
plexes of higher kinetic stability then the parent un-
substituted cyclam (as described above), then it would
be feasible that the observed anti-HIV EC50s of the
heteroaromatic linked bicyclam analogs of reduced
activity reflected a virucidal effect caused by the extru-
sion of metal ions from free virus rather than a
structure-activity relationship related to the molecular
target for 10d which has been previously shown not to
exhibit a virucidal effect.2 However, in all cases tested,
preincubation of the bicyclam analogs with HIV-1 (IIIB)
at concentrations up to 50 µM failed to reduce the
infectivity of the virus in MT-4 cells.32 We conclude
therefore that the observed structure-activity relation-
ship of heteroaromatic linked bicyclams appears to be
directly related to the target for the anti-HIV activity
of 10d (or J M3100; Figure 1) at the HIV-inhibitory step.
F igu r e 4. Reversible protonation equilibrium of 26: (- - -) 26
(9.0 mM) in 1 M NaClO4 (pH 6.9) and (s) 26 (9.0 mM)
dissolved in 5 M HClO4 (giving 27).
with a high predictive capacity (as evident from the
predictive r2 value of 0.79). Partial least-squares (PLS)
analysis of the descriptors used in this model led to the
identification of a number of necessary features required
for this class of compounds to display potent antiviral
activity. For each analog included in the model, all
sterically allowed conformations were included in the
analysis, thus taking the flexibility of the bicyclams into
account. The main descriptors used in the model are
as follows. For each analog, a dummy atom was defined
at the center of each macrocyclic ring to represent the
position of a metal ion (or proton) complexed within the
azamacrocyclic cavity. Planes were defined to represent
the face of each macrocyclic ring (such that the four
nitrogen atoms in each ring lay approximately in the
plane), with the angles and torsions between these two
planes subsequently being measured for every confor-
mation generated in the conformational search (see
Figure 5). These two descriptors are referred to as the
“plane angle” and “plane torsion”, respectively. In
addition, the distance between the two dummy atoms
(located at the metal coordination center of each mac-
rocyclic ring) was measured for all conformations gener-
P r ed ictive Mod el Ba sed on a QSAR Stu d y of Bis-
tetr a a za m a cr ocycles. In an earlier study, a quantita-
tive structure-activity relationship (QSAR-1) of bis-
tetraazamacrocyclic compounds, including the potent
anti-HIV-1 and anti-HIV-2 inhibitor 10d , was de-
scribed.33 In this QSAR study, a correlation between
several structural features of these compounds with
anti-HIV activity was observed, resulting in a model

Phenylenebis(methylene)-Linked Bis-tetraazamacrocycles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 115
Ta ble 3. Experimental vs Predicted EC50s against HIV-1
Using the Refined QSAR-2 Model for Anti-HIV
Bis-azamacrocycles
EC50 (µM)
compd
predicted
experimental
8a
8b
8c
8d
9a
0.06
0.10
1.00
2.50
0.04
88.0
0.10
0.02
0.03
16.4
0.91
0.03
139
9b
11c
0.03
with a maximum in the distribution (ca. 40% of all
conformations) being in the range 80-110°, and plane
tortion angles are in the range -180-180°. However,
since the N atom in the aromatic linker region of 8c is
capable of pendant complexation with the adjacent
cyclam ring, the conformational search for sterically
allowed conformations was repeated using the refined
QSAR-2 model. A starting pendant conformation was
chosen in which the distance between the dummy atom
and the N atom in the linker was fixed at 2.11 Å (an
average value for the N-Ni distance of the pendant
group in several published crystal structures of pendant-
coordinated complexes15,19,20,29c but which also approxi-
mates the length of a hydrogen bond), and only the two
rotatable bonds of the linker to the cyclam ring not
involved in pendant complexation were allowed to rotate
at 10° increments (see compound 8c in Figure 2). In
striking contrast to the search results for 8c using
QSAR-1, the search results using QSAR-2 show that 8c
prefers conformations in which the metal-metal dis-
tance is 9.1-9.3 Å, the plane angles are in the range
70-120°, and the plane torsions are in the range 30-
84°. The pendant conformation with the lowest energy
is observed to have a metal-metal distance of 9.3 Å, a
plane angle of 70°, and a plane torsion of 84°. Compar-
ing these structural features with those of 11c and the
general descriptor requirements found to be necessary
for potent antiviral activity in our earlier study with
QSAR-1,33 namely, (a) distance between metal-binding
centers must be 9.5-11.5 Å, (b) plane torsions of -60-
30° and 120-140° are allowed, and (c) plane angles of
40-70° and 110-140° are allowed, shows that none of
these three structural features can be satisfied in the
case of a pendant-complexed conformer of 8c, and it
would therefore be expected to have poor antiviral
activity. Using the QSAR-2 model in which the pendant
coordination indicator variable is given a value of 1, the
EC50 for inhibition of HIV-1 by bicyclam 8c was pre-
dicted to be 1 µM, while the experimentally observed
activity is 16.4 µM (Table 3). A series of compounds
from Table 1 were selected, and their predicted anti-
HIV-1 EC50s were calculated using the refined QSAR-2
model. The results show a strong correlation of the
predicted versus the experimentally observed values
(Table 3).
F igu r e 5. Schematic representation of compound 8c showing
the structural descriptors used for the QSAR analysis. (a)
Plane anglessPlanes were defined in which the four nitrogen
atoms of an azamacrocyclic ring lie in the plane. The plane
angle is the smallest angle formed upon intersection of the
two planes. (b) Plane torsionsNormals to each plane were
also defined, passing through a dummy atom placed at the
center of each ring, having a length of 2 Å above and below
the plane. This allowed measurement of the plane torsion (or
twist) between the azamacrocyclic rings, illustrated by the
connectivity 1-2-3-4 (using the shortest distance between the
end points of the two normals (or points 2 and 3) to describe
the torsion). (c) Metal-metal distancesDefined by the dis-
tance between the dummy atoms, points 1 and 4. Additional
descriptors are defined in ref 33. The bonds in the linker
region which were allowed to rotate during the conformational
search are indicated by the curved arrows.
ated (referred to as the “metal-metal distance”). The
binding affinity of each ring in the bis-azamacrocycles
was included as further important descriptors (where
zinc was chosen as the representative metal).
In the QSAR-1 model for antiviral activity (against
HIV-1 and HIV-2), no provisions were made for analogs
which might be able to adopt “pendant” conformations.
It is clear, however, that if analogs capable of forming
pendant complexes are included, a correction of the
QSAR-1 model is required in order to retain its predic-
tive capacity, taking into account both the metal affinity
parameter and the reduction in conformational freedom.
This was achieved by extention of the QSAR-1 model
to include an additional indicator variable, indicating
whether or not the analog in question can take up a
“pendant” conformation. “Pendant” was defined as a
conformation in which an additional complex between
a donor atom (typically nitrogen) in the linker and the
dummy atom in the center of the adjacent macrocyclic
ring is possible. A value equal to the number of
additional donor atoms was assigned to the pendant
indicator variable for that analog giving a refined model,
QSAR-2.
Analysis of the descriptors after a conformational
search of compound 8c using QSAR-1 reveals that this
analog adopts conformations in which the metal-metal
distance range is 9-10.4 Å, the plane angles are in the
range 40-160° (with ca. 70% of all conformations having
a plane angle in the range 80-110°), and the plane
torsions are in the range -150-150°. For comparison,
we repeated the conformational search of the m-phen-
ylenebis(methylene)-linked analog 11c using QSAR-1.
Not surprisingly, 11c exhibits similar conformational
descriptors to 8c: Metal-metal distances are in the
range 8.8-10.4 Å, with the distribution maximum
(including ∼85% of all conformations) lying between 9.5
and 10.2 Å. Plane angles are in the range 15-175°,
The results for all bicyclam analogs using the refined
QSAR-2 model which includes compounds able to take
up pendant conformations resulted in an overall im-
proved predictive model as reflected in an improvement
in the cross-validated (predictive) r2 value from 0.79
(QSAR-1) to 0.84 (QSAR-2). It is also evident that for
the bicyclam analogs a quantitative relationship exists
between the pendant indicator variable parameter and

116 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bridger et al.
50.43, 50.89, 56.15, 57.41, 127.14, 128.29, 142.20, 152.66,
158.89; FAB MS m/z (rel intensity) 586 (MH + H81Br, 39), 584
(MH + H79Br, 40), 504 (M + H, 76), 305 (40), 201 (100). Anal.
(C27H53N9‚8HBr‚4H2O) C, H, N, Br.
antiviral activity and that all pendant conformations
should be avoided if high antiviral activity is to be
achieved.
1,1′-[2,5-P yr id ylbis(m eth ylen e)]bis[1,4,8,11-tetr a a za cy-
clotetr a d eca n e] Non a h yd r obr om id e P en ta h yd r a te (8d ).
Using general procedure A, 1,1′-[2,5-pyridylbis(methylene)]bis-
[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraazacyclotetrade-
cane] (4d ) (350 mg, 0.245 mmol) gave 8d (180 mg, 56%) as a
Su m m a r y
The anti-HIV activity of a series of heteroaromatic
linked bicyclam analogs has been evaluated. Depending
upon the substitution pattern of the linker connecting
the tetraazamacrocyclic rings, we observed a striking
anti-HIV structure-activity relationship, which is con-
sistent with a model in which the heteroatom of the
linker can participate in pendant conformations (com-
plexation) with the adjacent azamacrocyclic ring. We
suggest that this interaction provides the wrong mo-
lecular shape for binding to the target for bicyclam
activity at the HIV-inhibitory step. We have provided
preliminary evidence that the intramolecular interac-
tion responsible for the inactivation may be due to the
involvement of transition metal chelation or hydrogen
bonding of the linker heteroatom with the shared
protons of the azamacrocyclic ring at physiological pH.
In either event, this is clearly an important molecular
design consideration. Further work is in progress to
resolve the apparent mechanistic paradox.
1
white solid: mp 241-243 °C dec; H NMR (D2O) δ 1.89 (m,
4H), 2.10 (m, 4H), 2.61-2.72 (m, 4H), 2.82-2.97 (m, 4H), 3.12-
3.58 (m, 24H), 3.96 (s, 2H), 4.02 (s, 2H), 7.87 (d, 1H, J ) 9
Hz), 8.39 (d, 1H, J ) 9 Hz), 8.67 (s, 1H); 13C NMR (D2O) δ
19.09, 19.89, 20.91, 20.97, 38.29, 38.71 (3C), 39.02, 40.67,
41.81, 42.07, 42.26, 42.42, 42.46, 43.13, 46.59, 47.81, 49.24,
50.81, 54.72, 56.28, 128.03, 132.87, 144.10, 148.17, 153.14; FAB
MS m/z (rel intensity) 586 (MH + H81Br, 25), 584 (MH + H79
-
Br, 27), 504 (M + H, 68), 306 (37), 201 (100). Anal. (C27H53N9‚
9HBr‚5H2O) C, H, N, Br.
1,1′-[2,6-P yr a zylb is(m et h ylen e)]b is[1,4,8,11-t et r a a za -
cyclotetr adecan e] Octah ydr obr om ide Mon oh ydr ate (9a).
Using general procedure A, 1,1′-[2,6-pyrazylbis(methylene)]-
bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraazacyclotetrade-
cane] (5a ) (500 mg, 0.35 mmol) gave 9a (95 mg, 23%) as a white
solid: mp 214-217 °C dec; 1H NMR (D2O) δ 1.83 (m, 4H), 1.91
(m, 4H), 2.63 (m, 4H), 2.71 (m, 4H), 2.82-3.32 (m, 24H), 3.82
(s, 4H), 8.42 (s, 2H); 13C NMR (D2O) δ 20.25, 20.92, 38.71,
39.65, 41.13, 42.56, 42.78 (2C), 47.27, 49.86, 55.84, 144.27,
150.35; FAB MS m/z (rel intensity) 587 (MH + H81Br, 60), 585
(MH + H79Br, 66), 505 (M + H, 100). Anal. (C26H52N10
8HBr‚H2O‚0.5HOAc) C, H, N, Br.
‚
Exp er im en ta l Section
General experimental procedures for the synthesis of bicy-
clams have been previously published.3
1,1′-[2,5-P yr a zylb is(m et h ylen e)]b is[1,4,8,11-t et r a a za -
cyclotetr a d eca n e] Octa h yd r och lor id e Tr ih yd r a te (9b).
To a stirred solution of 1,1′-[2,5-pyrazylbis(methylene)]bis-
[4,8,11-tris(diethoxyphosphoryl)-1,4,8,11-tetraazacyclotetrade-
cane] (5b) (240 mg, 0.181 mmol) in THF (10 mL) was bubbled
HClg, and the pale yellow solution was stirred at room
temperature for 20 h, during which time a solid precipitated.
The reaction mixture was concentrated to dryness, and the
residue was partitioned between H2O (10 mL) and CH2Cl2 (10
mL). The aqueous phase was then separated and treated with
charcoal (100 mg) with heating to 80 °C for 30 min. The hot
solution was filtered through Celite, and the filtrate was
concentrated to ca. 1 mL, after which acetic acid was added
resulting in the immediate formation of a white precipitate.
The solid was collected by filtration to give 9b (80 mg, 50%):
1H NMR (D2O) δ 1.78-2.10 (m, 8H), 2.71 (m, 8H), 2.89-3.37
(m, 22H), 3.42 (s, 2H), 3.81 (s, 4H), 8.58 (s, 2H); 13C NMR (D2O)
δ 19.13, 21.71, 38.30, 40.37, 41.97, 42.43, 43.82, 44.55, 49.41,
51.89, 55.38, 145.04, 151.18; FAB MS m/z (rel intensity) 542
(MH + H37Cl, 58), 506 (M + H, 44), 306 (32), 201 (100), 93
(100). Anal. (C26H52N10‚8HCl‚3.4H2O‚HOAc) C, H, N, Cl.
1,1′-[2-Am in o-1,4-p h en ylen eb is(m et h ylen e)]b is[1,4,8,
11-tetr a a za cyclotetr a d eca n e] Non a h yd r obr om id e Mon o-
h yd r a te (10a ). To a solution of 1,1′-[2-nitro-1,4-phenylenebis-
(methylene)]bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraaza-
cyclotetradecane] (6a ) (900 mg, 0.61 mmol) in ethanol (75 mL)
were added iron powder (900 mg) and concentrated HCl (1
mL), and the mixture was heated to reflux for 2.5 h with rapid
stirring. The hot solution was made basic by the dropwise
addition of aqueous NaOH solution (10 M). The solution was
filtered hot and the solvent evaporated under reduced pres-
sure. The residue was dissolved in CH2Cl2 (75 mL), boiled with
charcoal (50 mg), and filtered and the solvent removed under
reduced pressure to give 1,1′-[2-amino-1,4-phenylenebis-
(methylene)]bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraazacy-
clotetradecane] (680 mg, 77%) as a pale yellow solid: 1H NMR
(CDCl3) δ 1.55-1.75 (m, 4H), 1.85-2.05 (m, 4H), 2.25-2.45
(m, 22H), 2.55-2.75 (m, 4H), 2.85-3.35 (m, 24H), 3.47 (s, 2H),
3.57 (s, 2H), 6.55 (d, 1H, J ) 7.5 Hz), 6.59 (s, 1H), 6.89 (d, 1H,
J ) 7.5 Hz), 7.25-7.45 (m, 12H), 7.50-7.80 (m, 12H); FAB
MS m/z (rel intensity) 1443 (M + H, 27), 1287 (18), 708 (100),
663 (41), 624 (25). This was used without further purification.
The amino compound from above (500 mg, 0.35 mmol) was
deprotected using general procedure A to give 10a (250 mg,
Gen er a l P r oced u r e A. 1,1′-[2,6-P yr id ylbis(m eth ylen e)]-
b is[1,4,8,11-t et r a a za cyclot et r a d eca n e] Oct a h yd r ob r o-
m id e Tr ih yd r a te (8a ). To a solution of 1,1′-[2,6-pyridylbis-
(methylene)]bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraaza-
cyclotetradecane] (4a ) (500 mg, 0.35 mmol) in acetic acid (16
mL) was added 48% aqueous HBr (Aldrich; 12 mL), and the
mixture was heated to 110 °C with stirring for 48 h during
which time a white crystalline solid precipitated from a dark
brown solution. Upon cooling, the solid was collected by
filtration, washed with acetic acid and then ether, and dried
in vacuo to give 8a (280 mg, 65%) as a white solid: mp 229-
1
231 °C dec; H NMR (D2O) δ 1.89 (m, 4H), 2.01 (m, 4H), 2.69
(m, 4H), 2.90 (m, 4H), 3.04-3.45 (m, 24H), 3.98 (s, 4H), 7.68
(d, 2H, J ) 8 Hz), 8.19 (t, 1H, J ) 8 Hz); 13C NMR (D2O) δ
19.35, 19.78, 37.77, 37.95, 39.07, 41.47, 41.70, 41.89, 46.17,
49.41, 58.23, 126.35, 143.22, 151.02; FAB MS m/z (rel inten-
sity) 586 (MH + H81Br, 47), 584 (MH + H79Br, 50), 504 (M +
H, 100), 306 (19), 201 (60). Anal. (C27H53N9‚8HBr‚3.5H2O)
C, H, N, Br.
1,1′-[3,5-P yr id ylbis(m eth ylen e)]bis[1,4,8,11-tetr a a za cy-
clotetr a d eca n e] Non a h yd r obr om id e Dih yd r a te (8b). Us-
ing general procedure A, 1,1′-[3,5-pyridylbis(methylene)]bis-
[4,8,11-t r is(p-t olylsu lfon yl)-1,4,8,11-t et r a a za cyclot et r a -
decane] (4b) (350 mg, 0.245 mmol) gave 8b (150 mg, 53%) as
a white solid: mp 252-254 °C dec; 1H NMR (D2O) δ 1.92 (m,
4H), 2.04 (m, 4H), 2.57 (m, 4H), 2.74 (m, 4H), 3.07-3.46 (m,
24H), 3.92 (s, 4H), 8.58 (s, 1H), 8.69 (s, 2H); 13C NMR (D2O) δ
20.11, 22.09, 38.89, 39.45, 42.26, 42.56, 42.73, 43.36, 47.42,
49.75, 54.38, 136.80, 141.92, 149.55; FAB MS m/z (rel inten-
sity) 586 (MH + H81Br, 40), 584 (MH + H79Br, 41), 504 (M +
H, 60), 305 (20), 201 (100). Anal. (C27H53N9‚9HBr‚2.5H2O)
C, H, N, Br.
1,1′-[2,4-P yr id ylbis(m eth ylen e)]bis[1,4,8,11-tetr a a za cy-
clotetr a d eca n e] Octa h yd r obr om id e Tetr a h yd r a te (8c).
Using general procedure A, 1,1′-[2,4-pyridylbis(methylene)]bis-
[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraazacyclotetrade-
cane] (4c) (270 mg, 0.189 mmol) gave 8c (120 mg, 52%) as a
white solid: mp 220-222 °C dec; 1H NMR (D2O) δ 1.79-1.96
(m, 4H), 2.03-2.21 (m, 4H), 2.39 (m, 4H), 2.78-2.95 (m, 4H),
3.04-3.56 (m, 24H), 3.93 (s, 2H), 4.02 (s, 2H), 7.86 (d, 1H, J )
8 Hz), 7.92 (s, 1H), 8.59 (d, 1H, J ) 8 Hz); 13C NMR (D2O) δ
19.88, 20.01, 22.18, 22.38, 38.74 (2C), 38.79, 38.99, 39.19,
42.06, 42.13, 42.42, 42.55, 42.72, 42.95, 43.28, 47.73, 47.95,
1
58%) as a white solid: mp 255-260 °C dec; H NMR (D2O) δ

Phenylenebis(methylene)-Linked Bis-tetraazamacrocycles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 117
1.75-2.15 (m, 8H), 2.75-3.45 (m, 32H), 3.89 (s, 2H), 4.06 (s,
2H), 7.11-7.25 (m, 2H), 7.34 (d, 1H, J ) 8.2 Hz); 13C NMR
(D2O) δ 23.57, 23.67, 24.15, 25.35, 41.89, 42.08, 42.35 (3C),
43.67, 45.64, 45.76, 46.04 (2C), 46.22, 46.50, 49.04, 50.05,
52.38, 53.16, 60.47, 63.12, 128.62, 130.07, 132.15, 136.14,
138.06, 143.07; FAB MS m/z (rel intensity) 601 (MH + H81Br,
91), 599 (MH + H79Br, 91), 519 (M + H, 77), 473 (55). Anal.
(C28H55N9‚9HBr‚H2O‚1.5HOAc) C, H, N, Br.
132.53, 133.78, 138.30, 168.84; FAB MS m/z (rel intensity) 629
(M + H81Br, 14), 627 (M + H79Br, 15), 548 (M + H, 58), 185
(45), 93 (100). Anal. (C29H54N8O2‚8HBr‚2H2O) C, H, N, Br.
1,1′-[2,4-Qu in olin ylbis(m eth ylen e)]bis[1,4,8,11-tetr aaza-
cyclot et r a d eca n e] Non a h yd r ob r om id e P en t a h yd r a t e
(17a ). Using general procedure B, 1,1′-[2,4-quinolinylbis-
(methylene)]bis[4,8,11-tris(diethoxyphosphoryl)-1,4,8,11-tet-
raazacyclotetradecane] (170 mg, 0.13 mmol) gave 17a (0.15 g,
1
1,1′-[2-C a r b o x y -1,4-p h e n y le n e b is (m e t h y le n e )]b is -
[1,4,8,11-tetr a a za cyclotetr a d eca n e] Octa h yd r obr om id e
(10b). Using general procedure A, 1,1′-[2-carbomethoxy-1,4-
ph en ylen ebis(m et h ylen e)]bis[4,8,11-t r is(p-t olylsu lfon yl)-
1,4,8,11-tetraazacyclotetradecane] (6b) (280 mg, 0.189 mmol)
gave 10b (160 mg, 68%) as a white solid: mp 247-251 °C dec;
94%) as a white solid: mp 222-224 °C dec; H NMR (D2O) δ
1.75-2.15 (m, 8H), 2.55-3.45 (m, 32H), 4.24 (s, 2H), 4.41 (s,
2H), 7.80 (t, 1H, J ) 7.5 Hz), 7.96 (t, 1H, J ) 7.8 Hz), 8.13 (s,
1H), 8.22 (d, 1H, J ) 8.4 Hz), 8.31 (d, 1H, J ) 8.4 Hz); FAB
MS m/z (rel intensity) 636 (MH + H81Br, 11), 634 (MH +
H79Br, 11), 554 (M + H, 13), 356 (14), 229 (37), 201 (100). Anal.
(C31H55N9‚9HBr‚5.5H2O) C, H, N, Br.
1
IR (CsI) ν 1705, 1615, 1576, 1456, 1208, 1076 cm-1; H NMR
(D2O) δ 1.92-2.07 (m, 8H), 2.97 (m, 2H), 3.02-3.43 (m, 26H),
3.47 (s, 4H), 4.05 (s, 2H), 4.36 (s, 2H), 7.50 (m, 2H), 7.81 (s,
1H); 13C NMR (D2O) δ 19.01, 19.20, 19.50, 19.58, 37.82 (4C),
37.98, 38.28, 41.38, 41.70 (3C), 42.12, 42.27, 45.39, 45.61,
48.51, 48.65, 58.42, 59.01, 131.91, 133.17, 133.98, 134.73,
135.44, 135.97, 171.25; FAB MS m/z (rel intensity) 629 (MH
+ H81Br, 5), 627 (MH + H79Br, 5), 547 (M + H, 82), 349 (40),
201 (100). Anal. (C29H64N8O2‚8HBr‚HOAc) C, H, N, Br.
Gen er a l P r oced u r e B. 1,1′-[2-Ca r bom eth oxy-1,4-p h en -
ylen eb is(m et h ylen e)]b is[1,4,8,11-t et r a a za cyclot et r a d e-
ca n e] Octa h yd r obr om id e Tr ih yd r a te (10c). To a stirred
solution of 1,1′-[2-carbomethoxy-1,4-phenylenebis(methylene)]-
bis[4,8,11-tris(diethoxyphosphoryl)-1,4,8,11-tetraazacyclotet-
radecane] (6c) (250 mg, 0.182 mmol) in acetic acid (2 mL) was
added 30% HBr in acetic acid (Aldrich; 6 mL), and the solution
was stirred at room temperature for 3 h. The resulting
precipitate was collected by filtration, washed with acetic acid
and ether, and dried in vacuo giving 10c (130 mg, 57%) as a
white powder: mp 212-214 °C dec; IR (CsI) ν 1715, 1615,
2,4-Dim eth yl-6-p h en ylp yr id in e (20). To a solution of
2-bromo-4,6-dimethylpyridine (19) (3.0 g, 16.1 mmol), phenyl-
boric acid (2.16 g, 17.7 mmol, 1.1 equiv), and sodium carbonate
(3.59 g, 33.9 mmol, 2.1 equiv) in toluene (200 mL), ethanol
(50 mL), and water (50 mL) was added Pd(PPh3)4 (932 mg, 5
mol %), and the mixture was heated to reflux overnight with
rapid stirring. Upon cooling, the reaction mixture was diluted
with CH2Cl2, washed with a solution of saturated aqueous
sodium bicarbonate, dried (MgSO4), and evaporated to dryness.
The residue was dissolved in boiling hexane, and the solid
which formed was removed by filtration. The filtrate was
allowed to cool, during which time a white solid precipitated
(triphenylphosphine) which was also removed by filtration.
This procedure was repeated until all of the triphenylphos-
phine had been removed giving 20 as a light yellow liquid upon
evaporation (2.7 g, 90%): 1H NMR (CDCl3) δ 2.36 (s, 3H), 2.58
(s, 3H), 7.33 (s, 1H), 7.37-7.47 (m, 3H), 7.95 (dd, 2H, J ) 8.3,
1.6 Hz). This was used without further purification.
6-P h en yl-2,4-p yr id in ed ica r boxylic Acid (21). To a solu-
tion of 20 (2.0 g, 10.9 mmol) in a mixture of H2O (30 mL) and
t-BuOH (60 mL) maintained at a temperature of 100 °C with
stirring was added KMnO4 (10.34 g, 6.0 equiv) in one portion.
The reaction mixture was heated overnight, during which time
a brown solid precipitated which was removed by hot filtration
through Celite. The filtrate was evaporated to a small volume
and then acidified to pH 4 with concentrated aqueous HCl
which precipitated a white solid. The solid was collected by
filtration and dried in vacuo to give 21 (0.95 g, 3.91 mmol,
36%): 1H NMR (DMSO-d6) δ 7.42-7.60 (m, 3H), 8.20 (dd, 2H,
J ) 7.9, 1.4 Hz), 8.32 (d, 1H, J ) 1.2 Hz), 8.46 (d, 1H, J ) 1.2
Hz).
6-P h en yl-2,4-p yr id in ed im eth a n ol (22). To a solution of
21 (0.92 g, 3.79 mmol) in anhydrous THF (15 mL) with stirring
was added BH3‚THF (Aldrich; 1.0 M solution in THF, 37.8 mL,
10.0 equiv), and the mixture was heated to 60 °C with stirring
overnight. The mixture was evaporated to dryness and the
residue dissolved in anhydrous methanol and evaporated once
again (repeated three times). The residue was dissolved in 1
N HCl and then made basic with 10 N sodium hydroxide to
pH 14, during which time a white solid precipitated. The
aqueous solution was extracted with CH2Cl2 (3 × 50 mL) and
then dried (MgSO4) and evaporated to give 22 as a white solid
(0.75 g, 3.49 mmol, 93%): 1H NMR (DMSO-d6) δ 4.60-4.62
(m, 4H), 5.45 (m, 2H), 7.38-7.48 (m, 4H), 7.70 (s, 1H), 8.05
(d, 2H, J ) 7.8 Hz).
1
1575, 1476, 1215, 1078 cm-1; H NMR (D2O) δ 1.90-1.99 (m,
8H), 2.83 (m, 2H), 2.93-3.64 (m, 30H), 3.87 (s, 3H), 3.97 (s,
2H), 4.54 (s, 2H), 7.59 (m, 2H), 8.02 (s, 1H); 13C NMR (D2O) δ
18.65, 19.24, 19.81, 20.07, 37.38, 37.55, 37.65, 38.65, 39.17
(2C), 41.13, 41.43 (2C), 41.92, 42.39, 42.83, 45.31, 46.15, 48.48,
48.92, 54.10, 57.95, 58.99, 131.63, 134.37, 134.66, 135.55,
136.42 (2C), 168.75; FAB MS m/z (rel intensity) 643 (M +
H81Br, 25), 641 (M + H79Br, 24), 562 (M + H, 66), 363 (100).
Anal. (C30H53N8O2‚8HBr‚3H2O) C, H, N, Br.
1,1′-[5-Am i n o -1,3-p h e n y le n e b i s (m e t h y le n e )]b i s -
[1,4,8,11-tetr a a za cyclotetr a d eca n e] Non a h yd r obr om id e
Dih yd r a te (11a ). In a similar manner to the preparation of
10a described above, 1,1′-[5-nitro-1,3-phenylenebis(methyl-
ene)]bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-tetraazacyclotet-
radecane] (7a ) (300 mg, 0.2 mmol) gave 1,1′-[5-amino-1,3-
phenylenebis(methylene)]bis[4,8,11-tris(p-tolylsulfonyl)-1,4,8,11-
tetraazacyclotetradecane] (240 mg, 83%) as a pale yellow
solid: 1H NMR (CDCl3) δ 1.65-1.85 (m, 4H), 1.90-2.05 (m,
4H), 2.25-2.55 (m, 22H), 2.65-2.75 (m, 4H), 2.95-3.35 (m,
24H), 3.42 (s, 4H), 6.49 (s, 1H), 6.55 (s, 2H), 7.15-7.45 (m,
12H), 7.55-7.75 (m, 12H); FAB MS m/z (rel intensity) 1442
(M, 100), 1286 (64), 1130 (13), 663 (36). This was used without
further purification.
The 5-amino compound from above (120 mg, 0.08 mmol) was
deprotected using general procedure A to give 11a (80 mg,
1
77%) as a white solid: mp 235-237 °C dec; H NMR (D2O) δ
1.85-2.15 (m, 8H), 3.15-3.65 (m, 32H), 4.36 (s, 4H), 7.52 (s,
2H), 7.67 (s, 1H); 13C NMR (D2O) δ 18.99, 19.09, 37.44, 37.59,
37.71, 38.00, 41.28, 41.66, 44.89, 48.09, 57.78, 126.96, 132.91,
133.32, 133.69; FAB MS m/z (rel intensity) 600 (M + H81Br,
19), 598 (M + H79Br, 19), 519 (M + H, 24), 320 (18), 201 (100).
Anal. (C28H55N9‚9HBr‚2.2H2O) C, H, N, Br.
1,1′-[5-C a r b o x y -1,3-p h e n y le n e b is (m e t h y le n e )]b is -
[1,4,8,11-tetr a a za cyclotetr a d eca n e] Octa h yd r obr om id e
Dih yd r a te (11b). Using general procedure A, 1,1′-[5-car-
bomethoxy-1,3-phenylenebis(methylene)]bis[4,8,11-tris(p-tolyl-
sulfonyl)-1,4,8,11-tetraazacyclotetradecane] (7b) (810 mg, 0.543
mmol) gave 11b (350 mg, 52%) as a white solid: mp 234-237
°C dec; IR (CsI) ν 1715, 1609, 1576, 1454, 1202, 1065 cm-1; 1H
NMR (D2O) δ 2.03 (m, 8H), 3.04-3.66 (m, 32H), 4.35 (s, 4H),
7.81 (s, 1H), 8.07 (s, 2H); 13C NMR (D2O) δ 18.71, 19.06, 37.41,
37.61 (2C), 41.12 (2C), 41.65, 44.69, 48.00, 58.23, 131.43,
6-P h en yl-2,4-b is(b r om om et h yl)p yr id in e H yd r ob r o-
m id e (23). To a solution of the diol 22 (160 mg, 0.74 mmol)
in acetic anhydride (6 mL) cooled to 0 °C was added hydro-
bromic acid (Aldrich; 48% aqueous solution, 5 mL) dropwise
with stirring, and the mixture was then heated to reflux
overnight. Upon cooling the solution was triturated with ether
to give 23 as a white crystalline solid (150 mg, 0.36 mmol,
47%): 1H NMR (DMSO-d6) δ 4.74 (s, 2H), 4.77 (s, 2H), 7.40-
7.56 (m, 3H), 7.59 (s, 1H), 7.96 (s, 1H), 8.10 (m, 2H).
1,1′-[6-P h en yl-2,4-pyr idylbis(m eth ylen e)]bis[4,8,11-tr is-
(p-tolylsu lfon yl)-1,4,8,11-tetr a a za cyclotetr a d eca n e] (24).
Dimerization of 2a (444 mg, 0.65 mmol) with 23 (125 mg, 0.3
mmol) under standard conditions3 and purification of the crude
product by column chromatography on silica gel (CH2Cl2/
MeOH/Et3N, 98:2:1) gave 24 (420 mg, 0.28 mmol, 94%) as a
white foam: 1H NMR (CDCl3) δ 1.78 (m, 2H), 1.92 (m, 2H),

118 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bridger et al.
2.32-2.43 (multiple s, 18H), 2.50-2.80 (m, 8H), 3.11-3.23 (m,
24H), 3.66 (s, 2H), 3.81 (s, 2H), 7.16 (d, 2H, J ) 7.8 Hz), 7.22-
7.69 (m, 27H), 7.98 (m, 2H); FAB MS m/z (rel intensity) 1503
(M + H, 100), 1348 (18).
1,1′-[6-P h en yl-2,4-p yr id ylb is(m et h ylen e)]b is[1,4,8,11-
t et r a a za cyclot et r a d eca n e] Non a h yd r ob r om id e (25a ).
Deprotection of 24 (400 mg, 0.27 mmol) using general proce-
dure A gave 25a (55 mg, 16%) as a white solid: 1H NMR (D2O)
δ 1.65 (m, 2H), 1.90 (m, 6H), 2.60 (m, 2H), 2.72 (m, 2H), 2.80
(m, 6H), 2.98 (m, 8H), 3.11-3.21 (m, 12H), 3.29 (m, 2H), 3.89
(m, 4H), 7.49 (m, 3H), 7.55 (s, 1H), 7.63-7.65 (m, 3H); FAB
MS m/z (rel intensity) 662 (MH + H81Br, 17), 660 (MH +
H79Br, 16), 580 (M + H, 57), 382 (38), 201 (100). Anal.
(C33H57N9‚9HBr) C, H, N, Br.
tions at the Rega Institute were supported by the
Biomedical Research Programme of the European Com-
munity, the Belgian Fonds voor Geneeskundig Weten-
schappelijk Onderzoek, the Belgian Nationaal Fonds
voor Wetenschappelijk Onderzoek, the Belgian Gecon-
certeerde Onderzoeksacties, and the J anssen Research
Foundation. We thank Hilde Azijn, Barbara Van Re-
moortel, and Patrick Seldeslachts for excellent technical
assistance.
Refer en ces
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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.
1-(2-P yr id ylm eth ylen e)-1,4,8,11-tetr a a za cyclotetr a d e-
ca n e Nick el Dip er ch lor a te (26). To a stirred solution of
1-(2-pyridylmethylene)-1,4,8,11-tetraazacyclotetradecane (15)
(236 mg, 0.81 mmol) in dry ethanol (5 mL) was added a
solution of nickel(II) perchlorate (296 mg, 1.0 equiv) in
n-butanol (which had been partially distilled to remove most
of the hydrated water, leaving ca. 5 mL). During the addition
a purple precipitate formed from the virtually colorless solu-
tion. The mixture was allowed to stir for 30 min, and the solid
was then collected by filtration, washed with ethanol and then
ether, and dried in vacuo giving 26 (380 mg, 86%) as a purple
powder: IR (KBr) ν 3427, 3269, 3151, 2924, 2868, 1606, 1442,
(2) De Clercq, E.; Yamamoto, N.; Pauwels, R.; Balzarini, J.; Witvrouw,
M.; De Vreese, K.; Debyser, Z.; Rosenwirth, B.; Peichl, P.;
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selective inhibition of human immunodeficiency virus by the
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(12) For comparison, the pKa values quoted are for the unsubstituted
bases. These values were obtained from the CRC Handbook of
Chemistry and Physics, 71st ed.; Lide, E. R., Ed.; CRC Press
Inc.: Boca Raton, FL, 1990-1991; pp 8-33.
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1141, 1111, 1087, 763, 626, 426 cm-1
NiCl2O8) C, H, N, Cl.
. Anal. (C16H29N5-
P oten tiom etr ic Titr a tion s. An aqueous (50 mL) solution
of the compound (0.1 mM) and 4 equiv of HClO4 at 25 °C and
I ) 0.1 M (NaClO4) was titrated with 0.1 M NaOH according
to literature procedures.20 The pH values were read with a
Corning 345 pH meter. The electrode was calibrated with pH
4.00 and 7.00 buffer solutions and checked with a theoretical
titration curve of 0.4 mM HClO4 and I ) 0.1 M (NaClO4) with
0.1 M NaOH solution. The perchloric acid was calibrated by
titration against standard NaOH solution.
UV Absor p tion Sp ectr a a t Va r yin g p Hs. A stock solu-
tion of 15 (3.4 mM) in 0.1 M NaClO4 was prepared. Aliquots
of 100 µL of the stock solution were diluted with varying
amounts of 0.4 M HClO4 (5-500 µL), and the mixture was
brought to an overall volume of 2 mL with 0.1 M NaClO4. This
gave pH values in the range 1-7 and kept the concentration
of 15 at 0.17 mM. The UV absorption spectra of these
solutions were recorded on a Perkin-Elmer Lambda 2 UV/vis
double-beam spectrometer with a cell path length of 1 cm and
a scan range of 400-200 nm.
An ti-HIV Activity Assa ys. The human immunodeficiency
virus strains used were HIV-1 (IIIB) and HIV-2 (ROD) whose
origin has been described previously.1 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 and then exposed to various concentrations of the
test compounds. After the MT-4 cells were allowed to prolifer-
ate for 5 days, the number of viable cells was quantified by a
tetrazolium-based colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) procedure in 96-well
microtrays.34 In all of these assays, viral input (viral multi-
plicity of infection, MOI) was 0.01, or 100 times the 50% cell
culture infective dose (CCID50). The 50% antivirally effective
concentration (EC50) was defined as the compound concentra-
tion required to protect 50% of the virus-infected cells against
viral cytopathicity. The 50% cytotoxic concentration (CC50
)
was defined as the compound concentration required to reduce
the viability of mock-infected cells by 50%. The > symbol is
used to indicate the highest concentration at which the
compounds were tested and still found noncytotoxic. Average
EC50 and CC50 values for several separate experiments are
presented 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 the tables.
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