Strained cyclophane macrocycles: Impact of progressive ring size reduction on synthesis and structure

Article abstract of DOI:10.1021/ja208503y

The synthesis, X-ray crystal structures, and calculated strain energies are reported for a homologous series of 11- to 14-membered drug-like cyclophane macrocycles, representing an unusual region of chemical space that can be difficult to access synthetic

Full text of DOI:10.1021/ja208503y

Article
pubs.acs.org/JACS
Strained Cyclophane Macrocycles: Impact of Progressive Ring Size
Reduction on Synthesis and Structure
Andrew R. Bogdan,† Steven V. Jerome,‡ K. N. Houk,*,‡ and Keith James*,†
†The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569, United States
S
* Supporting Information
ABSTRACT: The synthesis, X-ray crystal structures, and
calculated strain energies are reported for a homologous
series of 11- to 14-membered drug-like cyclophane macrocycles,
representing an unusual region of chemical space that can be
difficult to access synthetically. The ratio of macrocycle to dimer,
generated via a copper catalyzed azide−alkyne cycloaddition
macrocyclization in flow at elevated temperature, could be
rationalized in terms of the strain energy in the macrocyclic
product. The progressive increase in strain resulting from reduc-
tion in macrocycle ring size, or the introduction of additional conformational constraints, results in marked deviations from
typical geometries. These strained cyclophane macrocyclic systems provide access to spatial orientations of functionality that
would not be readily available in unstrained or acyclic analogs. The most strained system prepared represents the first report of
an 11-membered cyclophane containing a 1,4-disubstituted 1,2,3-triazole ring and establishes a limit to the ring strain that can be
generated using this macrocycle synthesis methodology.
is also the chemistry of oligomerization. Consequently,
macrocyclizations are typically run at high (ca. 1 mM) or
very high (<ca. 1 mM) dilution in order to slow the rate of
intermolecular reactions.7 However, this is inefficient in terms
of solvent consumption and reaction time, particularly on a
large scale. The problem is amplified in the case of
macrocycles at the lower end of the defined ring size, where
the strain in the macrocyclic product results in a slower
macrocyclization rate and therefore a low yield of macrocycle
compared to the product of the undesired, but less strained,
intermolecular reaction. In some cases, this can render the
macrocyclization unobservable.9 The strain energy of these
‘small ring’ macrocycles results from distortion of standard
bond angles and lengths associated with enclosure in a ring.
In addition, rings of this size can experience unfavorable
transannular interactions between substituents on opposite
sides of the ring.
Consequently, small strained macrocycles represent an
intriguing region of chemical space that can be difficult to
access chemically. They have the potential to yield unusual
molecular shapes, unconventional bond geometries, and spatial
orientations of functional groups that are quite different from
unstrained or acyclic systems. When such macrocycles also
possess the functionality and physicochemical properties typical
of drug molecules, they offer access to drug-like molecular
diversity that is quite distinct from that usually found in
corporate or public domain screening files. These strained
INTRODUCTION
■
There is growing interest in the design of macrocyclic drugs as
a strategy to address challenging biological targets, such as
protein−protein interfaces.1 Although often biologically com-
pelling, these targets are rarely amenable to small-molecule
modulation.2 Macrocycles offer both pharmacological and
physicochemical advantages over acyclic molecules with regard
to modulation of these problematic molecular targets. The
pharmacological advantage stems from the preorganization of
bioactive conformations, thereby avoiding entropic losses upon
binding that would decrease affinity. Numerous examples of the
successful application of this concept have been reported.3 The
physicochemical advantages of macrocycles result from their
shape,1 lower rotatable bond count,4 and, in some cases, their
ability to conceal polar functionality by forming stable,
intramolecular hydrogen bonds in low dielectric environments.5
However, despite these potential advantages, the design of
macrocycle-based drugs represents an emerging field, since the
synthetic challenges of constructing macrocycles have impeded
progress in the area.6 All current macrocyclic drugs are either
natural product-based or cyclic peptides.7
An impressive recent chemo-informatic analysis of bio-
logically active macrocycles defines them as organic molecules
containing a nonbridged cycle of 12 or more covalently
connected atoms.8 This analysis also revealed that macrocycles
represented 2.8% of a database of 130 000 natural products and
that, of these, rings containing 14 atoms were most common
and fewer than 1% had ring sizes larger than 40 atoms. The
construction of macrocyclic rings via cyclization of a linear
precursor is problematic in that the chemistry of the cyclization
Received: September 8, 2011
Published: December 1, 2011
© 2011 American Chemical Society
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Figure 1. Macrocycles prepared by CuAAC macrocyclization in flow. Ring size and yield indicated in parentheses. No specific amide bond geometry
is intended.
macrocycles therefore have potential value in tackling novel
types of molecular targets.
norephedrine respectively, with which we could examine the
effect of changes in stereochemistry as well as transannular
interactions. The synthesis of each macrocycle precursor
followed an analogous route (Scheme 1a). The only departures
involved the 10-membered system, where the azido-group was
We now report a systematic study of a series of strained,
drug-like cyclophane macrocycles. We have explored the consequences
of progressively increasing macrocyclic ring strain for their
synthesis and structure and have established a basis for pre-
dicting which members of a larger virtual library of macrocycles
would be synthetically accessible via this route.
Scheme 1. Synthetic Routes to Azido-alkyne Precursors for
a
CuAAC Macrocyclizations
RESULTS AND DISCUSSION
■
We recently reported a method for generating what we defined
as drug-like macrocycles via an intramolecular copper catalyzed
azide−alkyne cycloaddition (CuAAC) reaction conducted in
flow, using a heated copper tube.7 This protocol allows the
macrocycles to be prepared in useful quantities under modest
dilution and has been used successfully to generate macrocycles
as large as 29-membered rings in good yield. The reaction
generates a 1,4-disubstituted 1,2,3-triazole, a stable, drug-like
functional group possessing geometric and H-bond acceptor
properties which render it isosteric with a trans-amide bond.10
We have now applied this methodology to the synthesis of a
homologous series of macrocycles containing 10- to 14-
membered rings, in order to determine the structural properties
of these systems and the lower limit of macrocycle ring size
accessible via this approach (Figure 1). We presumed that this
lower limit would be dependent upon the substitution pattern
of the ring and would be governed by the point at which the
incipient ring strain in the macrocycle would significantly
reduce the rate of the macrocyclization reaction in comparison
to the dimerization. The flow synthetic approach used was ideal
for taking precise measurements of the macrocycle-to-dimer
ratio, via UV spectroscopy, of reaction segments emerging from
the flow reactor.
a
(a) N3-(CH2)n-CO2H, PyBOP, DIPEA, DMF. (b) NaH, propargyl
bromide, THF. (c) Chloroacetic anhydride, TEA, CH2Cl2. (d) NaN3,
MeCN (aq). (e) N-Boc-β-alanine, PyBOP, DIPEA, DMF. (f) HCl,
Dioxane. (g) Imidazole 1-sulphonyl azide hydrochloride, Cu-
SO4·5H2O, K2CO3, MeOH.
The majority of the macrocycles are based upon the
homochiral (1R,2S)-ephedrine fragment. This common core
provided a set of reference atoms through which we could
compare the structures of the homologous macrocycles. It also
constituted a useful bifunctional amino alcohol building block,
which allowed incorporation of the requisite alkynyl and azido-
functional groups via a short synthetic sequence. Additionally,
we could readily access the diastereoisomeric and homologous
homochiral fragments, (1R,2R)-pseudoephedrine and (1R,2S)-
introduced in the final step (Scheme 1b), and the 11-
membered system, where β-elimination issues necessitated
use of a diazo-transfer reagent on a β-amino acyl system
(Scheme 1c) (full experimental details are provided in the
Supporting Information).
The structures of all macrocycles successfully isolated (all but 6)
were determined by X-ray crystallography, in order to allow
accurate measurement of bond angles and lengths. Geometry
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optimizations were conducted for all the macrocycles at the
B3LYP/6-31+G(d) level of theory. It is known that B3LYP and
related density functionals do not include dispersion energy,
and this may introduce significant errors in comparing different
structures that have varying nonbonded interactions. Dispersion
corrections were therefore added through Grimme’s D3
procedure, since these provide considerably better energetics
for such systems. Gas-phase enthalpies for each compound were
computed from calculated vibrational frequencies at 298 K.
Using standard statistical formulas, gas-phase entropies were
also computed. Entropic effects have a minimal impact on
calculation of strain and are left for discussion in the Supporting
Information
Side-by-side comparison of computed (left) and experimen-
tal structures (right) for all compounds are displayed in
Figure 2. Several specific geometrical features from experiment
and theory are compared in Table 1. Where we could make a
comparison, the global minima were mostly in excellent
agreement with the experimental X-ray data. The exceptions
were as follows. In the 13-membered macrocycle 3, the
puckering of the ring and the distortion of the amide differed
significantly between X-ray and computed structures. Thus, we
find a 16.0° deviation from planarity of the amide in the X-ray
structure compared with only a 2.7° distortion in the computed
structure. In all other ephedrine derivatives possessing a
trans-amide bond (1−5 and 7−8), the deviation of the amide
was found not to exceed 5° between computed and X-ray
structures. This larger deviation in 3 cannot be readily
accounted for. Macrocycle 9 was the only case in which the
lowest energy computed structure contained a cis-amide bond,
whereas the X-ray structure exhibited a trans-amide bond.
Finally, macrocycles 2 and 7 exhibited a difference in the rotation
of the triazole between computed and X-ray structures. These cis-
versus trans-amide and triazole-rotamer issues are discussed more
fully below.
We also computed the strain energies of each of the
macrocyclic compounds in two ways. First, we compared the
energies of hydrogenation of each of the optimized cyclic
structures and compared those heats of hydrogenation with
those of an unstrained compound. The strain energies were also
calculated by computing the energies of reaction of the azide−
alkyne cycloadditions leading to macrocycles and by comparing
those to the energies of reaction for an acyclic system. Strain
Figure 2. Computed (left) and X-ray (right) global minimum structures. The perspective for each pair is aligned by superimposition of the amide
carbonyl, carbon, and nitrogen atoms. Only the calculated structure 6 is displayed.
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Table 1. Summary of Macrocycle Yields, Strain Energies, and Selected X-ray Crystallographic Measurements (Computed Values
in Parentheses)
Product
to
Strain via
Enthalpiesof
Hydrogenation
(kcal/mol)
Strain via
Enthalpies of
Reaction
Triazole
Distortion
(180-β)
(deg)
Amide
Distortion
(180-γ)
(deg)
c
d
Average
Strain
(kcal/mol)
Triazole
b
Ring
Compd Size
Dimer
Isolated
Yield (%)
Distortion
a
Ratio
(kcal/mol)
(180-α) (deg)
1
2
3
4
5
6
7
8
9
14
14
13
12
11
10
12
12
13
13.7:1
6.7:1
2.4:1
4.6:1
0.49:1
0.02:1
1.6:1
1.9:1
1.2:1
90
80
66
73
20
<1
51
54
35
−0.8
−2.9
0.0
−2.6
−1.9
0.7
−1.7
−2.4
0.4
2.0 (7.5)
9.2 (5.9)
4.0 (3.1)
7.0 (9.6)
25.5 (26.1)
(43.0)
0.4 (5.1)
6.6 (3.8)
1.6 (5.9)
5.9 (1.7)
2.6 (3.5)
16.0 (2.7)
4.4 (3.6)
5.9 (4.3)
(28.6)
1.0
2.2
1.6
12.0 (11.9)
18.1 (19.5)
(30.2)
4.1
6.0
5.0
21.9
−3.4
−2.9
4.4
24.3
−2.3
5.2
23.1
−2.8
1.2
10.6 (12.5)
13.4 (13.0)
21.5 (19.3)
9.1 (12.0)
14.3 (14.9)
8.8 (14.8)
2.2 (1.0)
10.5 (10.7)
30.6 (3.2)
11.1
7.7
a
b
Ratio of macrocycle to dimer determined by UV spectroscopy. Dihedral NNNC angle α (defined in Figure 4) measures out-of-plane bend of N1-
c
substituent on triazole. Numbers in parentheses are from calculations. Dihedral NNCC angle β (defined in Figure 4) measures out-of-plane bend of
C4-substituent on triazole. Numbers in parentheses are from calculations. Dihedral CCNC angle γ measures torsion about N−C(O) bond of amide.
Numbers in parentheses are from calculations.
d
was then calculated using the equation ΔΔH(Strain) = ΔHrxn-
(Unstrained Model) − ΔHrxn(Macrocycle) (Figure 3).
Figure 4. Dihedral angles used to calculate distortion of triazole and
amide.
(compared with 1 in Figure 5) is the N-methylated analog of 1.
It was obtained in slightly lower, but still high, yield (80%).
Figure 5. Superposition of macrocycles 1 (green) and 2 at the amide
oxygen, carbon, and nitrogen atoms (X-ray).
The strain energy of this system is comparable. Interestingly,
Figure 3. (a) Reference hydrogenation reactions used to calculate
there is now a 15.8° distortion from planarity evident across the
triazole system (sum of columns 8 and 9 in Table 1), and the
macrocycle has adopted a slightly different pucker in order to
macrocycle strain energies. (b) Reference cycloaddition reactions used
to calculate strain energies.
avoid a steric clash between the triazole C-5 proton and the
amide N-methyl group now present in this system, as illustrated
in Figure 5. The distance between the amide nitrogen atom and
the triazole C-5 carbon has increased from 3.6 Å in the
norephedrine-derived macrocycle to 4.3 Å in the more
congested ephedrine-derived macrocycle.
The next lower homologue 3 in the ephedrine series,
containing a 13-membered ring, shows a modest increment in
strain energy (average 2.8 kcal/mol) compared to the 14-
membered homologue. As illustrated in Figure 6, the relative
orientation of the triazole ring and amide bond have now
reversed compared to the higher homologue, presumably, in
order to avoid an even more pronounced steric clash between
the triazole C-5 proton and the amide N-methyl group noted
above. Despite the slight increase in strain energy compared to
its higher homologue, the distortion of the triazole ring is less
Inspection of the X-ray crystal structures revealed distortions
in the macrocyclic ring, which became more pronounced as the
strain energy increased. In particular, the bonds to the triazole
ring are bent out of plane and the amide is distorted from
planarity. The distortion observed for these functional groups,
the strain energies computed via both methods, an average
strain energy (from the two different calculation methods) for
each molecule, macrocycle-to-dimer ratios, and isolated yields
are summarized in Table 1. The angular distortions in the last
three columns are discussed below.
Impact of Ring Size. The norephedrine-derived 14-
membered macrocycle 1 represents a low strain system in
which the triazole ring and amide bond are both almost planar.
The dihedral angles used to determine distortion are defined in
Figure 4. It is notable that it was generated in 90% isolated
yield. The ephedrine-derived 14-membered macrocycle 2,
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Figure 6. Overlap of 13-membered macrocycle 3 and 14-membered
macrocycle 2 (green). Molecules are superimposed at the amide
oxygen, carbon, and nitrogen atoms (X-ray).
Figure 8. Edge-on view of 11-membered macrocycle 5, illustrating the
significant distortion across the triazole ring. Dihedral angles α = 155°
and β =162° (X-ray).
pronounced in this 13-membered ring, perhaps because of the
flip in orientation. Instead, there is much greater deformation of
the amide bond, which is 16° out of planarity.
an 11-membered ring containing a 1,5-disubstituted 1,2,3-
triazole, which was the surprising product of a CuAAC reaction
expected to produce a 12-membered 1,4-triazole system.11 In
that case, it was concluded that the nonbridged 11-membered
ring was less strained than the intended bridged 12-membered
ring, although the generation of a 1,5-disubstituted system
under copper catalyzed conditions is clearly highly unusual.
Our copper catalyzed reaction yielded the expected 1,4-triazole
product, which we would expect to be more strained than the
11-membered ring generated in this literature report, by virtue
of the additional challenge of accommodating the 1,4-
disubstitution pattern across the triazole ring.
Since we had now observed two different conformations of
the triazole ring in relation to the amide bond, we have adopted
a convention of describing the triazole conformer which places
the triazole N-2 and N-3 atoms on the same face of the
macrocycle as the amide carbonyl oxygen as syn (as in the
norephedrine 14-mer and ephedrine 14-mer X-ray structures)
and the confomer which places these groups on opposite faces
of the macrocycle as anti (as in the ephedrine 13-mer X-ray
structure).
Our attempt to generate 6, possessing a 10-membered ring,
was unsuccessful. Although a small peak was visible in the LC/
MS trace of the reaction segment, it represented a less than 1%
yield, with the starting material having been converted to a
dimer and other higher-order oligomers. The average calculated
strain energy for this system is now >25 kcal/mol higher than
the 14-membered homologue, and it appears that the azido-
alkyne substrate proceeds almost exclusively along alternative
intermolecular reaction pathways to yield less strained
products.
The 12-membered system 4 was also generated in good
yield and exhibits a broadly similar conformation to the
13-membered macrocycle 3 (Figure 7). Strain has once again
Impact of Stereochemistry. Since changes in substrate
stereochemistry are known to affect the outcome of macro-
cyclizations,12 we also explored the impact of changing the
configuration at the stereogenic center bearing a methyl group
in the 12-membered ephedrine-derived macrocycle 4. Thus, the
corresponding pseudoephedrine-derived macrocycle 7 was
generated, albeit in slightly lower yield than its diastereoisomer 4.
As illustrated in Figure 9, its X-ray structure did indeed reveal a
Figure 7. Overlap of 12-membered macrocycle 4, 11-membered
macrocycle 5 (green), and 13-membered macrocycle 3 (blue),
illustrating progressive distortion across the triazole ring (viewed
edge on). Molecules are superimposed at the amide oxygen, carbon,
and nitrogen atoms (X-ray).
increased slightly, although it still modest. Upon examination of
the crystal structures, it is surprising, however, that in this case
the triazole ring exhibits most of the distortion resulting from
the increase in strain (19° deviation from planarity), with the
amide bond appearing much closer to planarity (4° deviation)
than in the 13-membered ring.
The 11-membered macrocycle 5, which by the earlier definition
is strictly a medium-ring system, proved to be significantly more
strained and much more challenging to synthesize. The
macrocyclization reaction was further diluted in this case in
order to improve the isolated yield, but even so, the macrocycle
was now generated in only 20% yield. The average strain energy
is >7 kcal/mol higher than the 14-membered homologue, and
the X-ray structure of this system now shows significant
deviation from planarity across the triazole system (43°) in
order to accommodate this strain (Figure 8). The relative
orientation of triazole and amide bonds matches that of the 12-
and 13-membered rings (Figure 7). This molecule is the first
11-membered ring cyclophane reported containing a 1,4-
disubstituted 1,2,3-triazole. There has been one earlier report of
Figure 9. Overlap of 12-membered macrocycle 7 derived from
pseudoephedrine (green) and 12-membered macrocycle 4 derived
from ephedrine. Molecules are superimposed at the amide oxygen,
carbon, and nitrogen atoms (X-ray).
significant conformational change in the 12-membered macro-
cycle. Relative to the amide bond, the triazole ring has flipped
from the anticonformer into the syn-arrangement that had
hitherto been observed in the larger, 14-membered rings. In
this case it is difficult to rationalize why this change in
stereochemistry at one ring position should lead to the
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Table 2. Relative Enthalpies of Cis versus Trans Amide and Syn versus Anti Triazole Conformers (kcal/mol), Computed with
a
B3LYP/6-31+G(d)
Cis-Syn
Relative
Enthalpy
(kcal/mol)
Cis-Anti
Relative
Enthalpy
(kcal/mol)
Trans-Syn
Relative
Enthalpy
(kcal/mol)
Trans-Anti
Relative
Enthalpy
(kcal/mol)
ΔH(Cis)-
ΔH(Trans)
(kcal/mol)
ΔH(Syn)-
ΔH(Anti)
(kcal/mol)
Ring
Size
Compd
1
2
3
4
5
6
7
8
9
14
14
13
12
11
10
12
12
13
13.7
12.2
9.6
12.2
11.0
11.0
11.2
13.7
9.9
0.0*
1.5*
6.0
0.2
12.2
11.0
9.6
−0.2
1.5
0.0
0.0*
0.0*
0.0*
0.0
6.0
12.6
15.1
11.7
9.0
2.5
11.2
13.7
9.9
2.5
6.2
6.2
4.5
4.5
5.4
1.1*
4.1
0.0
5.4
1.1
15.1
0.0
14.0
11.4
0.0*
0.5
14.0
−0.5
4.1
0.7*
−0.5
a
Lowest energy conformer for each compound defined as 0 kcal/mol. X-ray conformation indicated by asterisk.
adoption of such a different conformation for the 12-membered
ring. The strain energy of this system is low, and therefore we
attribute the lower yield observed relative to its diastereoisomer
as a kinetic effect, whereby the arrangement of methyl, phenyl,
azido, and alkynyl substituents in the linear precursor is less
favorable for achievement of the transition state for cyclization
in the case of macrocycle 7 compared with macrocycle 4.
In order to explore this change in triazole conformation in
more detail, we calculated the relative energies of both triazole
conformers for all nine macrocycles (Table 2). Additionally, in
order to ensure a comprehensive picture of conformational
preferences in these systems, we also determined the relative
energies of the cis-amide rotamer for each triazole conformer. It
is noteworthy that all the X-ray structures exhibited a trans-
geometry for the amide bond, even though the amide is tertiary
in all but 1, and so the energy cost of adopting a cis-
conformation would be minimized. As Table 2 illustrates, apart
from molecules 1 and 9, the majority of the macrocycles would
be expected to exhibit a preference for the trans-anti conformer.
However, in the case of macrocycles 2 and 7, where the trans-
syn conformer is observed in the X-ray structure, the energy
difference is small and could readily be compensated for by
crystal packing forces. Thus, the apparent difference in triazole
conformation observed upon changing the stereochemistry of
macrocycle 4 (derived from ephedrine) to yield macrocycle 7
(derived from pseudoephedrine) is probably an artifact of
crystal packing.
Figure 10. Overlap of 12-membered macrocycle 4 and 12-membered
benzo-fused macrocycle 8 (green). Molecules are superimposed at the
amide oxygen, carbon, and nitrogen atoms (X-ray).
behavior. Thus, the benzo-bridged system 9 was generated via
incorporation of a m-(azidomethyl)benzoyl substituent in the
macrocyclization precursor. This yielded 13-membered macro-
cycle 9 in low yield, although still slightly higher than that of the
11-membered system. Its average strain energy is >7 kcal/mol
higher than the nonbridged 13-membered macrocycle 3. As
illustrated in Figure 11, like the 11-membered macrocycle 5,
Impact of Conformational Constraint. Since it was not
possible to increase ring strain further by ring contraction or a
change in stereochemistry, we examined an alternative
approach, involving conformational constraint of the ring
atoms. The benzo-fused system 8 was prepared by incorpo-
ration of an o-(azidomethyl)benzoyl substituent into the
macrocyclization precursor in place of a simple azido-alkanoyl
moiety. This yielded a 12-membered macrocycle in which two
carbon atoms were also part of a benzene ring. Surprisingly,
although generated in a slightly lower yield than the
corresponding nonbenzo-fused system 4, macrocycle 8 did
not appear to be more strained (Table 1, entry 8 versus 4).
Comparison of the X-ray structures, as illustrated in Figure 10,
showed a very close correspondence, indicating that the
transformation of the two adjacent sp3 centers into sp2 centers
by incorporation of the ortho-fused benzene ring does not
represent a significant perturbation for the macrocyclic ring.
In contrast, incorporation of a m-bridged benzene ring
resulted in a profound change in macrocycle conformation and
Figure 11. Overlap of 13-membered m-benzo-bridged macrocycle 9
and 13-membered macrocycle 3 (green). Molecules are superimposed
at the amide oxygen, carbon, and nitrogen atoms (X-ray).
compound 9 shows significant distortion of both the triazole ring
and amide bond (>30° deviation from planarity in both cases). In
addition, the amide carbonyl is not conjugated with the aromatic
ring, due to rotation around the (aryl)−C−(CO)C bond.
Relationship of Structure to Conformational Flexi-
bility. Although access to X-ray crystallographic data has enabled
us to understand the impact of ring size on strain energy and
structural deformation within this series of macrocycles, NMR
data, particularly for macrocycle 9, suggested that the static X-ray
structures provided only a partial description of these systems,
which appeared to exhibit dynamic exchange behavior in solution.
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Figure 12. Continuum of conformational exchange rates indicating qualitative differences in behavior between macrocycles with differing degrees of
conformational constraint.
Comparison of 1H and 13C NMR spectra for members of the series
revealed differences in line shape, the presence of multiple species,
and response to elevated temperature, which were indicative of
differing degrees of conformational mobility (see Supporting
Information for spectra and qualitative analysis). In particular,
although only a single conformer of the triazole ring was observed
in the unit cell for each of the X-ray structures (although the
particular conformer was variable), it appeared that under some
circumstances both possible conformers could be present in
solution. Based upon these observations, we constructed a
qualitative representation of where members of this macrocyclic
series sit on a hypothetical continuum of conformational exchange
rates, as illustrated in Figure 12.
at 30 °C with a mixing time of 300 ms (together with a
reference ‘zero’ mixing time of 3 ms), as illustrated in Figure 13c.
This shows the clear presence of cross-peaks, indicating
exchange between the three species, designated A, B, and C,
which are slowly interconverting with the rate constants shown
Figure 13d.
Based upon the sum of structural and energetic data gathered
on this entire series of macrocycles, we envisaged the
equilibrium illustrated in Figure 14 to account for these
observations. Since, as discussed above, the triazole ring can
exist in either syn or anti orientation with respect to the
macrocyclic ring, and the tertiary amide bond can exist in either
cis or trans conformations, there are four plausible con-
formations available to this molecule, A, B, C (observed by
NMR) and D (not observed by NMR). These can interchange
via a series of bond rotations, which result in flipping of the
triazole ring and/or cis−trans isomerism of the amide bond.
Since only three of the four possible conformers are observed in
solution, we presume that the fourth, conformer D, is too high
in energy to be populated to any extent. In order to
unambiguously assign the identities of these four conformers,
we have used a combination of computation and spectroscopic
analysis.
As illustrated in Table 2 above, the calculated energies for
these four possible conformations of macrocycle 9 reveal that
while three of them are relatively close in energy, the cis-anti
conformer is significantly higher in energy. This result is
therefore consistent with the experimental observation of only
three species in solution and identifies the nonobserved species
D as the cis-anti conformer. Since the calculated energy
differences between the other three conformers were small, we
did not feel confident assigning their identities based only upon
these data. We therefore looked for unique NMR signatures,
which would be indicative of these structures. We hypothesized
that the most abundant species was likely to be the trans-syn
conformer observed in the X-ray structure. This assignment is
supported by a trans-annular NOE between the proton
attached to the triazole C-5 position and the protons of the
amide N-methyl group. Based upon modeled conformations,
this is the only species in which these protons are close enough
to enable this interaction. We therefore assigned species A as
the trans-syn conformer.
Macrocycle 9 occupies approximately the center of this
continuum based upon the following analysis. Although the
X-ray crystal structure of macrocycle 9 contains a single molecular
1
species in the unit cell, it was evident from the H NMR
spectrum of this molecule that there were three distinct species
present in solution in an approximate ratio of 7:1:1. Figure 13a
illustrates the upfield region of the 400 MHz 1H NMR
spectrum in d6-DMSO at 30 °C, in which three signals arising
from the methyl group are visible. A similar pattern of three
related signals was also evident in other regions of the spectrum
(see Supporting Information for full spectroscopic details).
This suggested the presence of three conformers of similar but
unequal energy, which undergo slow conformational exchange
on the NMR time scale. In order to characterize this unusual
system, we therefore conducted a series of additional NMR
experiments.
First, we performed a variable temperature (VT) NMR
experiment in which the solution was heated and spectra were
measured at temperature increments from 30 to 80 °C, as
illustrated in Figure 13b. The series of spectra illustrate that as
the temperature is raised, the signals coalesce and that by 80 °C
there is a single signal, corresponding to an averaging of the
original signals, indicative of rapid conformational exchange
now between the three species present. The coalescence point
for the exchange appears to be between 70 and 80 °C.
Although it is possible to derive kinetic information about such
an exchange process from VT NMR studies on simple, binary
systems,13 the complexity of the current system (three
interconverting species in unequal proportions) meant that
this was not possible. We therefore ran an EXSY experiment14
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Figure 13. Dynamic exchange behavior of macrocycle 9. (a) 400 MHz 1H NMR spectrum at 30 °C in d6-DMSO for macrocycle 9. Expanded upfield
region illustrating methyl group doublets from three interconverting species shown as insert. (b) Variable temperature 400 MHz 1H NMR spectrum
for macrocycle 9 in d6-DMSO from 30 to 80 °C in 10 °C increments. (c) Expanded region of 400 MHz 1H−1H EXSY NMR spectrum at 30 °C in
d6-DMSO for macrocycle 9 (Tm 300 ms) illustrating cross-peaks and associated integrals arising from conformational exchange between methyl
signals arising from three interconverting conformers. (d) Conformational equilibrium and rate constants for exchange between three conformers of
macrocycle 9, denoted A, B, and C.
We based the assignment of species B and C on the shifts of the
signal for the methyl group doublet (Figure 13a). In the cis-syn
conformer, this methyl group is close to the m-bridged aromatic
ring. This results in shielding, which moves the signal corresponding
to this methyl group upfield to −0.05 ppm. The corresponding
methyl group in the other, trans-conformers is too far away from
aromatic rings to experience such a shielding effect. We have
therefore assigned species C as cis-syn and species B as trans-anti.
Table 3 shows the computed rate constants for the
conformational exchange of macrocycle 9. The data were
Table 3. Rate Constants for Conformational Exchange in
Macrocycle 9 at 30 °C
rate
Conformational
Exchange
constant
(s−1
)
k1
A → B
B → A
B → C
C → B
C → A
A → C
0.95
6.88
3.43
3.39
19.80
2.56
k−1
k2
Figure 14. Perspective representations of the four possible conformers
of macrocycle 9 (triazole nitrogens omitted for clarity), illustrating that
whereas conformers A, B, and C are observed and in equilibrium with
each other, conformer D is not observed.
k−2
k3
k−3
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obtained using an 1H−1H EXSY pulse sequence on a 400 MHz
spectrometer at 30 °C in d6-DMSO (Figure 13c) and analyzed
using the EXSYCalc program.15 The exchange rates for
transformation of conformations B and C to conformation A
are more rapid than the conversion of conformation A to B and
C, consistent with the higher abundance of A compared to B
and C at 30 °C.16 When the Onsager equation is applied to the
rate data in Table 3 as a self-consistency check, the product is
1.08 (theoretical product = 1.0).17
continuum, since the ring size and flexibility in these cases are
sufficient to allow more rapid interconversion of triazole
conformers at room temperature, which is manifest as a single
set of signals in the NMR spectrum. Heating macrocycle 1 has
no impact on NMR line shape up to 100 °C. The 11-membered
macrocycle 5 occupies the left-hand end of the continuum. Even
though it exhibits sharp signals in the 1H NMR spectrum, it is much
more rigid than 9 and cannot undergo conformational exchange. In
fact, since there is only one species present in solution (i.e., the X-
ray structure), it has been formed as a single atropisomer. The 12-
membered macrocycle 4 sits on the continuum between the more
flexible 14-membered macrocycle 1, which is in fast exchange, and
the less flexible, constrained 13-membered macrocycle 9, which is in
slow exchange. Thus, it shows broadening of lines in the NMR
spectrum, but at room temperature it lies above its coalescence
point, since heating results in line sharpening. We therefore placed
the conformationally constrained 12-membered macrocycle 8
toward the left-hand end of the continuum, since it is more rigid
than macrocycle 4 and yet shows a single sharp set of signals in the
1H NMR spectrum. Once again, this macrocycle appears to have
been formed as a single atropisomer.
In order determine how these rate constants changed with
temperature, it was necessary to examine a lower temperature
range,16 which necessitated a switch of solvent to CD3OD.
1
Consequently, H−1H EXSY experiments were conducted at
10° increments from 273 to 303 K, generating a series of rate
constants shown in Table 4. These were then used to calculate
Table 4. Rate Constants for Conformational Exchange in
Macrocycle 9 between 273 and 303 K Determined by
Variable Temperature EXSY Experiments
T
(K)
k1
k2
k3
k4
k5
k6
(s−1
)
(s−1
)
(s−1
)
(s−1
)
(s−1
)
(s−1
)
The analysis suggests that, in this series, the rate of
conformational exchange is governed by both macrocycle size
and rigidity, with smaller, more rigid macrocycles trending
toward slower exchange rates. It appears not to be a function of
macrocycle strain energy per se, since macrocycle 8 appears to
exhibit low conformational flexibility and very little ring strain.
In those cases where a single conformer has been formed in the
reaction, it appears that there is a significant energy difference
between the transition states for the two possible atropisomeric
macrocyclic products.
Impact of Strain on Macrocyclization Efficiency. The
efficiency of the macrocyclization reactions at a given reaction
concentration is reflected in both the product-to-dimer ratios
(observed by UV spectroscopy) and the isolated yields of the
macrocycle products after chromatography. A plot of product-
to-dimer ratios and the isolated yields is illustrated in Figure 15,
implying a logarithmic relationship. Since the UV method was
based on an assumption that the extinction coefficients for
macrocycle and dimer are equivalent, which cannot be
guaranteed, we have used isolated yield as the preferred
measure of macrocyclization efficiency. Isolated yields for
cyclophane macrocycles were therefore plotted against the
average strain energy (in kcal/mol) or the structural distortion
(in degrees), as determined from the X-ray structure.
The variation in yield resulting from changes in ring strain
across the homologous series is illustrated in Figure 16. There
is a correlation with both average strain energy (Figure 16a)
and structural distortion (Figure 16b). This correlation suggests
that product structure/energy is a significant determinant of
reaction outcome. Thus, as the strain energy of the product,
and the transition-state, increase, the reaction is more likely to
proceed along an alternative, intermolecular pathway, yielding
dimer and higher-order oligomers. The equation describing the
linear relationship between average strain energy and yield for
this homologous series in Figure 16a is
303
293
283
273
1.24
0.39
0.12
0.03
3.18
1.07
0.32
0.07
0.61
0.53
0.22
0.07
2.4
11.92
7.93
2.75
0.74
1.53
1.10
0.43
0.13
1.51
0.48
0.12
activation parameters for the conformational exchanges in
macrocycle 9 via the Eyring equation.18 These are summarized
in Table 5. The results reveal a significant negative ΔS‡ value
Table 5. Activation Parameters for Conformational
Exchange in Macrocycle 9, Calculated by Application of the
Eyring Equation to Data in Table 4
Eact
(kcal
mol−1
ΔH‡
(kcal
ΔS‡
ΔG‡
303
(kcal
Conformational
Exchange
(cal mol−1
)
mol−1
)
K−1
)
mol−1
)
A → B
B → A
B → C
C → B
C → A
A → C
20.5
21.0
12.4
16.8
15.5
13.9
19.9
20.4
11.8
16.2
14.0
13.2
7.6
11.3
17.6
17.0
17.9
17.1
16.1
17.4
−20.0
−2.8
−3.8
−13.5
for the two transformations, B → C and A → C, both involving
a trans to cis amide bond isomerization within the macrocycle
ring. This suggests a requirement for a greater degree of
organization in the transition states for these transformations,
potentially involving a reduction in rotational degrees of
freedom within the macrocyclic ring. The B → C exchange also
involves a simultaneous flip of the triazole ring, perhaps in a
concerted manner with isomerization of the amide. Despite
these negative entropic changes, the overall free energy of
activation for these two conformational changes is comparable
with those associated with A → B and B → A exchanges, as a
result of lower enthalpies of activation. This appears therefore
to be an example of enthalpic/entropic compensation.19
In light of these spectroscopic findings, we have therefore
placed macrocycle 9 just left of center upon the continuum
depicted in Figure 12, since at room temperature it lies below
its coalescence point in the 400 Mz NMR spectrum. In
contrast, the 14-membered macrocycles 1, 2 and the 13-
membered macrocycle 3 occupy the right-hand end of the
%Yield = − 3.0(Average Strain Energy) + 62.9
2
(R = 0.69)
This suggests that the yield of a macrocyclization would
reach 10% at a strain energy of 18 kcal/mol and 1% at a strain
energy of 21 kcal/mol. Although we were not able to isolate the
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designed to enable systematic exploration of incremental
structural variations, which would not be straightforward in a
natural product-based system, using a combination of X-ray
crystallographic, spectroscopic, and computational approaches.
Decreasing the size of the ring, or introducing certain
conformational constraints, resulted in diminished experimental
yields and increased computed strain energies. The correlation
between strain and experimental yield, shown in Figure 16a,
while only qualitative, illustrates the eventual failure to generate
the macrocyclic product when strain energy becomes too large.
We observed this directly, since, for each of the macrocycles
successfully synthesized, we predict strain energies less than
10 kcal/mol. In the case of the 10-membered ring macrocycle 6,
for which we calculate an average strain energy of >23 kcal/mol,
macrocyclization is not observed. The smallest ring size successfully
prepared, macrocycle 5, is the first reported example of an 11-
membered cyclophane containing a 1,4-triazole bridge and was
generated as a single atropisomer with regard to the conformation
of the triazole ring. Although we observed a qualitative correlation
between macrocycle strain energy and isolated yield, there are
clearly additional factors, such as kinetics, that impact the cyclization,
since a stereochemical change did not increase macrocycle strain
but nevertheless decreased yield. Our results suggest that, for
CuAAC-macrocyclizations under these conditions, as strain energy
approaches ∼21 kcal/mol, the reaction is likely to yield only
oligomeric products.
The 13-membered macrocycle 9 beautifully illustrates the
unusual properties of a strained macrocycle of small ring size.
Our calculations show that it is the most strained macrocycle
we were able to generate. Consistent with the correlation
between calculated strain energy and X-ray derived structural
distortion data, illustrated in Figure 16c, this molecule also
shows the most significant perturbations in triazole and amide
geometries. A consequence of this strain/distortion has been a
closing of the energy gap between cis/trans and syn/anti
conformers. As a result, it exists as a mixture of slowly
interconverting conformers in solution at room temperature,
which we have been able to characterize spectroscopically. This
molecule thus approximately occupies the midpoint on a
continuum of conformational exchange rates, as illustrated in
Figure 12. Relaxing the degree of conformational constraint, or
increasing macrocycle ring size, results in a rightward shift along
the continuum to rapid conformational exchange rates where
only single species are observed by NMR as a result of signal
averaging. Tightening the degree of conformational constraint
further, or decreasing macrocycle ring size, results in a leftward
shift along the continuum to slow conformational exchange
rates. Again, only single species are observed by NMR, but in
this case because only a single atropisomer has been generated
in the cycloaddition reaction.
Figure 15. (a) Plot of isolated yield versus product-to-dimer ratio for
compounds 1−5 and 7−9 (%Yield = 14.5*log(Ratio) + 46.3). (b) Plot
of isolated yield versus logarithm of product-to-dimer ratio for
compounds 1−5 and 7−9 (%Yield = 50.3*log(Ratio) + 38.0, R2 =
0.95).
product of the macrocyclization to yield a 10-membered ring,
we estimated its average strain energy by calculation to be >23
kcal/mol, which would be consistent with its production in
only trace amounts. We therefore conclude that the generation
of cyclophane macrocycles via a CuAAC macrocyclization is
unlikely to be practical as the strain energy of the intended
product approaches ∼21 kcal/mol.
Since both methods of assessing strain provide comparable
correlations with yield, we conclude that the strain energy of
these macrocyclic systems can be largely accounted for by
perturbation of the triazole and amide geometries. This is
demonstrated in Figure 16c, which shows a correlation (R2 =
0.93) between the total distortion of the amide and triazole
groups and the average computed strain energies. Finally, a plot
of isolated yield versus enthalpy of reaction (Figure 16d)
provides a similar picture to that obtained by plotting isolated
yield versus strain energy. This is related to a Bronsted or
Marcus relationship, where the rate of a reaction increases as
the reaction becomes more exothermic.
CONCLUSION
■
The unusual architecture offered by the strained macrocycles
generated via this procedure opens up new opportunities for
the construction of geometrically novel molecules of potential
pharmaceutical interest. Libraries based upon such systems
would enhance molecular diversity by introducing distinct
geometries, and therefore disposition of macrocycle substitu-
ents, rather than by the conventional means of connecting
together atoms in a novel but strain-free configuration. Future
studies will explore the utility of libraries of strained
macrocycles in the discovery of leads against therapeutically
important protein−protein interaction targets.
Small macrocyclic rings represent an intriguing region of
chemical space in which the intrinsic conformational constraint
possessed by macrocycles is superimposed upon the geometric
distortion exhibited by strained molecules. Although this
domain has been explored to some extent in the context of
natural product synthesis,20 our results constitute an effort to
systematically characterize how progressive changes in ring size
and shape within a related series of macrocycles affect the
likelihood of closing the macrocyclic ring and the structure of
the resultant products. Our studies are based upon a
homologous series of drug-like cyclophane macrocycles,
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Figure 16. (a) Plot of isolated yield versus computed average strain energy for examples 1−9 (%Yield = −3.0*Strain Energy + 62.9, R2 = 0.69).
(b) Plot of isolated yield versus total distortion in X-ray structures for examples 1−5, 7−9 (%Yield = −1.2*Distortion + 93.9, R2 = 0.77). (c) Plot of
computed average strain energies versus sum of amide and triazole distortion. Distortion calculated from experimental structures 1−5, 7−9
represented by black circles and distortion from calculated structures 1−9 by triangles. (Strain Energy = 0.3*Experimental Distortion −6.8, R2 =
0.93). (d) Plot of isolated yield for examples 1−9 versus enthalpy of reaction in kcal/mol (%Yield = −2.9*ΔHrxn − 134.24, R2 = 0.72).
this manner were collected in a round-bottom flask. Upon completion,
the reaction mixture was concentrated and dried in vacuo. The crude
reaction mixture was purified using a Biotage Horizon automated flash
EXPERIMENTAL SECTION
■
General Considerations. All reagents and solvents were used as
received. THF was distilled from sodium. NMR spectra were recorded
column chromatography system (silica gel, EtOAc, Rf = 0.23) to yield
on a Bruker DRX-600, Bruker DRX-500, or Bruker AMX-400
instrument using a residual solvent peak as a reference. Data are
reported as s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad. LC/MS analyses were carried out using an
Agilent Technologies HPLC (Agilent Technologies 1100 Series diode
array detector, Agilent Technologies 1100 Series column heater,
Agilent 1100 Series pump, and Agilent 1100 Series degasser)
interfaced with an Agilent Technologies 6110 Quadrupole LC/MS.
Column chromatography was performed using a Biotage Horizon
automated flash chromatography system equipped with a Biotage
Horizon detector, fraction collector, and pump where noteed.
LC/MS Analysis. HPLC analyses was performed using a water
(formic acid 0.1% w/v/ammonium formate 0.05% w/v) and MeCN
(water 5% v/v, formic acid 0.1% v/v, ammonium formate 0.05% w/v)
based gradient from 0 to 100% MeCN over 4 min. A Waters XBridge
C18 2.5 μm (3.0 mm × 30 mm) column was used at 80 °C with a flow
rate of 2.4 mL min−1. Injections were made from diluted reaction
mixtures, and ionization was monitored in positive or negative mode.
General Procedure for Preparative Scale Flow Macro-
cyclization. Azido-alkyne (0.10 M in EtOH, 100 μL, 0.010 mmol,
1.0 equiv), TTTA (0.01 M in EtOH, 100 μL, 0.001 mmol, 0.10 equiv),
DIPEA (0.1 M in EtOH, 200 μL, 0.020 mmol, 2.0 equiv), and EtOH
(200 μL) were aspirated from their respective source vials, mixed
through a PFA mixing tube (0.2 mm inner diameter) and loaded into
an injection loop. The reaction segment was injected into the flow
reactor set at 150 °C and passed through the reactor at 300 μL min−1
(5 min residence time). A total of 40 reaction segments prepared in
1
1 as an off-white solid (117.5 mg, 90% yield): H NMR (600 MHz,
CDCl3): δ 7.72 (s, 1 H), 7.34−7.43 (m, 4 H), 7.27−7.31 (m, 1 H),
5.71 (d, J = 8.3 Hz, 1 H), 4.87 (d, J = 13.6 Hz, 1 H), 4.45−4.52 (m,
1 H), 4.38−4.44 (m, 2 H), 4.35 (d, J = 13.6 Hz, 1 H), 2.15−2.23 (m,
1 H), 1.77−2.00 (m, 4 H), 1.39−1.51 (m, 1 H), 1.21−1.30 (m, 2 H),
0.89 (d, J = 7.0 Hz, 3 H), 0.73−0.83 (m, 1 H); 13C NMR (150 MHz,
CDCl3): δ 171.2, 145.5, 138.7, 128.4, 127.5, 126.6, 122.6, 80.1, 61.3,
50.1, 49.9, 34.5, 28.4, 23.6, 23.4, 12.3; HRMS (ESI-TOF):
C18H24N4O2: [M + H]+: calculated 329.1972, found 329.1975.
Computational Methods. Geometry optimizations were per-
formed with the Gaussian0921 electronic structure program at the
B3LYP/6-31+G(d) level of theory. All single-point energies are
corrected for dispersion interactions using the DFT-D3 method of
Grimme and co-workers.22 Frequency calculations verified that each
structure was a minimum and provided thermal corrections to the
electronic energies. All absolute energies are reported in hartrees, and
relative electronic energies aregiven in kcal/mol. Energies of reaction
are also reported in kcal/mol.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and spectra for all molecules used in this
study; X-ray crystallographic data for all macrocycles; 1H-EXSY
spectra and structural assignment for conformer of macrocycle 9;
qualitative NMR data analysis of macrocycles, used to derive
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(16) Because of the large difference in abundance of the trans-syn
conformer relative to the cis-syn conformer, and therefore the relatively
high rate constant for the conformational change from cis-syn to trans-
syn, our data are on the limit of what can be processed using the
EXSYCalc algorithm. Thus, conducting the EXSY experiment at 40 °C
generated data that could not be processed, presumably because the
rate of conformational change is now outside the limits of the
algorithm.
Figure 12; full list of authors for ref 21; spreadsheet of variable
temperature H-EXSY data used to generate Tables 4 and 5;
coordinates for all computed and X-ray crystallographically
determined macrocycle structures. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
AUTHOR INFORMATION
■
(17) (a) Blackmond, D. G. Angew. Chem., Int. Ed. 2009, 48, 2648−
2654. (b) Onsager, L. Phys. Rev. 1931, 37, 405.
Corresponding Author
kjames@scripps.edu; houk@chem.ucla.edu
(18) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic
Chemistry; University Science Books: Sausalito, CA, 2006.
(19) For discussion of this concept, see: (a) Exner, O. Chem.
Commun. 2000, 11665−1656. (b) Sharp, K. Protein Sci. 2001, 10,
661−667.
(20) For selected examples, see: (a) Braddock, D. C.; Millan, D. S.;
́
Yolanda Perez-Fuertes, Y.; Pouwer, R. H.; Sheppard, R. N.; Solanki, S.;
ACKNOWLEDGMENTS
■
We are grateful to the National Institutes of General Medical
Sciences, the National Institutes of Health and Pfizer
Worldwide R&D for their support and Academic Technology
Services (ATS) at UCLA for access to the Hoffman2 cluster.
We are also grateful to Jason Ewanicki (Pfizer) and Wei Wang
(Pfizer) for assistance with VT and EXSY NMR experiments
and Professor Tammy Dwyer (University of San Diego) and
White, A. J. P. J. Org. Chem. 2009, 74, 1835−1841. (b) Speicher, A.;
Backes, T.; Hesidens, K.; Kolz, J. Beilstein J. Org. Chem. 2009, 5, 71.
(c) Brown, M. K.; Hoveyda, A. H. J. Am. Chem. Soc. 2008, 130,
12904−12906. (d) Cluzeau, J.; Oishi, S.; Ohno, H.; Wang, Z.; Evans,
B.; Peiperb, S. C.; Fujii, N. Org. Biomol. Chem. 2007, 5, 1915−1923.
(e) Matsumura, T.; Akiba, M.; Arai, S.; Nakagawa, M.; Nishida, A.
Tetrahedron Lett. 2007, 48, 1265−1268. (f) Tanabe, K.; Fujie, A.;
Ohmori, N.; Hiraga, Y.; Kojima, S.; Ohkata, K. Bull. Chem. Soc. Jpn.
2007, 80, 1597−1604. (g) Collins, S. K. J. Organomet. Chem. 2006,
691, 5122−5128. (h) Wipf, P.; Furegati, M. Org. Lett. 2006, 8, 1901−
1904. (i) Venkatraman, S.; Njoroge, F. G.; Girijavallabhan, V.
Tetrahedron 2002, 58, 5433−5458.
́
Dr Jordi Bures (The Scripps Research Institute) for advice
on EXSY calculations. We thank Curtis Moore and Arnold
Rheingold (UCSD Small-Molecule Lab) for X-ray crystallo-
graphic data.
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