Synthesis and structure - Activity relationships of chiral allosteric modifiers of hemoglobin

Article abstract of DOI:10.1021/jm000199q

A series of allosteric effectors of hemoglobin, 2-(aryloxy)-2-alkanoic acids, was prepared to investigate the effect of the stereocenter on allosteric activity. The chiral analogues were based on the lead compound, RSR13 (3b), with different alkyl/alkanoic and cycloalkyl/cycloalkanoic groups positioned at the acidic chiral center. Of the 23 racemic molecules synthesized, 5 were selected for resolution based on structure - activity relationships. One chiral analogue, (-)-(1R,2R)-1-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylcycl opentanecarboxylic acid (11), exhibited greater in vitro activity in hemoglobin solutions than its antipode, racemate, and RSR13. Compound (-)-(1R,2R)-11 was equipotent with RSR13 in whole blood, is a candidate for in vivo animal studies, and if efficacious and safe has a potential for use in humans. In general, it was found that chirality affects allosteric effector activity with measurable differences observed between enantiomers and the racemates.

Full text of DOI:10.1021/jm000199q

4726
J . Med. Chem. 2000, 43, 4726-4737
Articles
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Ch ir a l Alloster ic Mod ifier s
of Hem oglobin #
Melissa Phelps Grella,† Richmond Danso-Danquah,† Martin K. Safo,† Gajanan S. J oshi,§ J ean Kister,‡
Michael Marden,‡ Stephen J . Hoffman,§ and Donald J . Abraham*,†
Department of Medicinal Chemistry, School of Pharmacy, Institute for Structural Biology and Drug Discovery,
Virginia Commonwealth University, Richmond, Virginia 23298-0133, Allos Therapeutics, Inc., 7000 North Broadway,
Suite 400, Denver, Colorado 80221, and Physiologie et Physiopathologie Moleculaires, INSERM U 299, Du Globule Rouge,
78 Rue du General Leclerc, 94275 Le Kremlin Bicetre, France
Received May 8, 2000
A series of allosteric effectors of hemoglobin, 2-(aryloxy)-2-alkanoic acids, was prepared to
investigate the effect of the stereocenter on allosteric activity. The chiral analogues were based
on the lead compound, RSR13 (3b), with different alkyl/alkanoic and cycloalkyl/cycloalkanoic
groups positioned at the acidic chiral center. Of the 23 racemic molecules synthesized, 5 were
selected for resolution based on structure-activity relationships. One chiral analogue, (-)-
(1R,2R)-1-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylcyclopentanecarboxyl-
ic acid (11), exhibited greater in vitro activity in hemoglobin solutions than its antipode,
racemate, and RSR13. Compound (-)-(1R,2R)-11 was equipotent with RSR13 in whole blood,
is a candidate for in vivo animal studies, and if efficacious and safe has a potential for use in
humans. In general, it was found that chirality affects allosteric effector activity with
measurable differences observed between enantiomers and the racemates.
In tr od u ction
Human hemoglobin (Hb) is a tetrameric allosteric
protein composed of two R-chains and two â-chains. The
tetramer equilibrates between the deoxy tense (T) state
and oxy relaxed (R) state. The subunits are bisected by
a molecular 2-fold axis of symmetry creating a central
water cavity, which narrows in the R state. The allo-
steric equilibrium is influenced by allosteric modifiers.
The major regulatory allosteric effector in vivo in
humans and most mammals is 2,3-diphosphoglycerate
(2,3-DPG). Other naturally occurring Hb effectors in-
clude chloride ions, protons, and temperature. Modifiers
that decrease the oxygen affinity such as 2,3-DPG act
by adding constraints (intersubunit salt bridge interac-
tions) to the T state.1 Oxygen affinity decreasing agents
have several potential clinical applications in the treat-
ment of diseases or illnesses associated with hypoxia,
i.e., stroke, angina, trauma, etc., as well as for the
radiosensitization of hypoxic tumors and to increase the
shelf life of stored blood.2,3
Several synthetic agents have been reported to lower
the oxygen affinity of Hb (Figure 1). In the search for
an antisickling agent, Abraham and co-workers discov-
ered that the antilipidemic drug clofibric acid (1)
* To whom correspondence should be addressed. Phone: 804-828-
8183. Fax: 804-827-3664. E-mail: dabraham@hsc.vcu.edu.
† Virginia Commonwealth University.
§ Allos Therapeutics, Inc.
F igu r e 1. Structures of allosteric modifiers that decrease
oxygen affinity. Phenyl rings have been denoted A and B.
lowered the oxygen affinity of Hb.4,5 Perutz and Poyart
followed with a report that bezafibrate (2), another
‡ INSERM U 299.
# Abbreviations: Hb, hemoglobin; DMSO, dimethyl sulfoxide; HPLC,
high-performance liquid chromatography; NMR, nuclear magnetic
resonance; TFA, trifluoroacetic acid; SAR, structure-activity relation-
ships.
10.1021/jm000199q CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/18/2000

Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4727
Sch em e 1a
a
Reagents and conditions: (i) xylene, reflux, 3 days; (ii) CHCl3, NaOH.
Sch em e 2a
a
Reagents and conditions: (i) xylene, reflux, 3 days; (ii) CHCl3, NaOH.
antilipidemic agent, produced a greater decrease in
oxygen affinity than clofibric acid.6 X-ray crystallogra-
phy studies of bezafibrate complexed with Hb by Perutz,
Abraham, and colleagues revealed that two symmetri-
cally related molecules bind to the protein near the top
of the R-subunits and extend to the R,â-interface.7,8
Lalezari and co-workers showed that shortening the
four-atom bridge to a three-atom urea bridge produced
even more potent allosteric modifiers, but their potential
as clinical agents was limited due to loss of activity in
the presence of serum albumin and plasma proteins.9-11
Abraham et al. designed and synthesized a series of
fibrate analogues that replaced the urea linkage with
an amide bridge and a variety of substitutions on ring
A (Figure 1, structure 3).12 Compounds RSR4 (3a ) and
RSR13 (3b) retained strong allosteric effector activity
in the presence of serum albumin and plasma proteins
making them candidates for clinical trials. RSR4 was a
stronger effector than RSR13 in Hb solution studies and
about equipotent in whole blood studies.13-17 However,
RSR13 was chosen as the candidate for phase I clinical
trials with the rationale that the phenyl methyl sub-
stitution might be better tolerated in vivo in animal
studies. This proved to be correct. Currently, RSR13 is
in phase III clinical trials for the treatment of brain
metastasis in conjunction with radiation therapy.
We have continued to search for a more potent
molecule than RSR13 by modifying (1) the phenyl ring
A, (2) the three-atom bridge between aromatic rings,
and (3) the gem-dimethyl group. Prior to this study, the
most potent analogues of RSR13 included a 3-methyl-
5-chloro derivative (KDD86, 3c), an indanyl derivative
(RSR46, 4), and a cyclopentyl derivative (J P7, 5).18,19
The effect of an asymmetric center on the allosteric
activity of Hb has not been investigated.
To determine the effect of a stereocenter on allosteric
activity, several chiral analogues of RSR13, RSR46, J P7,
and KDD86 were designed by replacing the gem-
dimethyl group with alkyl groups, substituted cycloalkyl
groups, and cycloalkyl groups with a heteroatom in the
ring. This study describes the synthesis of the chiral
allosteric modifiers, their resolution into enantiomers,
and measurement of their ability to shift the oxygen-
binding curve of Hb toward the low-affinity state.
Separation into enantiomers was based on degree of
activity and chemical diversity among the racemates.

4728 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Phelps Grella et al.
Sch em e 3a
a
Reagents and conditions: (i) EDAC, HOBt, 12 h, rt; (ii) CHCl3, NaOH.
Sch em e 4a
a
Reagents and conditions: (i) K2CO3, DMF; (ii) 10% NaOH, EtOH; (iii) cinchonidine, EtOH.
Racemates and enantiomers were subjected to in vitro
testing with Hb solutions and in whole blood for
structure-activity relationships (SARs).
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC)
were used to form the amide bond between 25 and the
4-hydroxyphenyl acetic acid (Scheme 3).
Schemes 1, 2, and 3-5 were utilized to produce the
racemates. The previously reported R-aryloxyisobutyric
acid analogues were obtained via reaction of amidophe-
nols with acetone-chloroform in the presence of sodium
hydroxide.12,20,21 In this process, the appropriate ketone
was substituted for acetone in tetrahydrofuran to obtain
the proposed compounds 9-16, 19-21, and 27-29
(Schemes 1-3). The alternate method to prepare com-
pounds 30-38 followed Schemes 4 and 5. The latter
method employed condensation of the corresponding
amidophenol with an R-bromo ester in the presence of
base followed by base hydrolysis of the ester to give the
desired R-aryloxy acid product.22
Resu lts a n d Discu ssion
Syn th esis. The synthesis of the proposed compounds
involved central intermediate amidophenols 8, 18, and
26. Schemes 1 and 2 were followed to produce ami-
dophenols 8 and 18 where 3,5-dimethylaniline or 5-ami-
noindan was condensed with 4-hydroxyphenylacetic acid
in refluxing xylene over a 3-day period. While 3,5-
dimethylaniline and 5-aminoindan were both readily
available, 3,5-chloromethylaniline (25) was prepared by
following a literature procedure. Thus, a chlorination
of 4-methyl-2-nitroaniline with N-chlorosuccinimide,
followed by diazotization to remove the amino group,
gave 3-chloro-5-nitrotoluene, 24. Reduction of 24 using
tin(II) chloride dihydrate yielded the desired 3-chloro-
5-methylaniline, 25. 1Hydroxybenzotriazole (HOBt) and
Resolu tion a n d Ster eoch em istr y. Crystallization
from ethanol of (()-30 cinchonidine salt, the monometh-
yl analogue of RSR13, gave (-)-30. The optically pure

Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4729
Sch em e 5a
a
Reagents and conditions: (i) K2CO3, DMF; (ii) 10% NaOH,
EtOH.
antipode (+)-30 was recovered from the mother liquor
by crystallization. The same method was used to obtain
the monoethyl RSR13 analogue (-)-32. Enriched (+)-
32 was obtained from the mother liquor by crystalliza-
tion. Enantiomers of J P7 analogues 2-methylcyclopentyl
(11) and 2-methyltetrahydrofuran (13) and the meth-
ylethyl RSR13 analogue 10 were separated and isolated
using a Chiracel OD semipreparative HPLC column.23
The column was also used in the purification of enriched
(+)-32.
Analysis of the alkyl-substituted analogues 10, 30,
and 32 on the Chiracel OD column revealed that the
(-)-isomer eluted first and the (+)-isomer second (Fig-
ure 2). Cycloalkyl racemates 11 and 13 showed the
opposite pattern with the (+)-isomer eluting first and
the (-)-isomer second. Furthermore, HPLC chromato-
grams showed that racemates 11 and 13 were composed
of only two of the four possible stereoisomers. Sharp
melting points, optical rotation measurements, and
proton NMR of the purified enantiomers confirmed the
presence of only one set of diastereomers.
F igu r e 2. HPLC enantioseparation of (a) racemic mixture
(()-10, (b) (-)-10, and (c) (+)-10. Conditions: Chiracel OD
(0.46 × 25 cm); mobile phase 90:10 heptane/ethanol with 1%
TFA; flow rate 1.0 mL/min; detection wavelength 254 nm.
The absolute configurations of the enantiomers were
not established. Configurational studies on 2-phenoxy-
propionic acids have shown that isomers with (-)-
rotation have the (S)-configuration and the (+)-isomers
have the (R)-configuration.24,25 However, it is not pos-
sible by direct comparison of the optical rotation of the
structurally similar phenoxy acids to unequivocally
establish the stereochemical assignments of the alkyl-
substituted RSR13 analogues as (+)-(R) and (-)-(S).
The appearance of only one set of diastereomers for
the methylcyclopentyl or methylcyclotetrahydrofuran
derivatives 11 and 13 was rationalized in the following
way. Attack by the phenoxide ion (Figure 3) on the least
sterically hindered dichloroepoxide intermediates of 11
and 13 with the configuration (1R,2S) and (1S,2R)
(Figure 4) suggests that one set of diastereomers would
be favored (Figure 5). Crystallographic studies to be
published elsewhere confirm the stereochemistry of the
(()-11 diasteromeric pair suggested by the mechanism.
Biologica l Eva lu a tion . Allosteric SARs were deter-
mined for the synthetic chiral effectors comparing the
shift in the oxygen-binding curve of Hb solutions using
a Hemox analyzer. In vitro biological activity testing in
the presence of plasma proteins was also performed on
selected enantiomers and racemates (multipoint tonom-
etry and blood gas analysis) to screen for candidates
with potential clinical use. SAR studies compared the
degree in shift in P50 values, i.e., the partial pressure
of molecular oxygen necessary to half-saturate Hb. An
F igu r e 3. Mechanism for the ketone-chloroform reaction in
the synthesis of compound 11.
effector that decreases Hb oxygen affinity increases the
P50 value relative to the control. Thus, the activity or
potency of each analogue could be expressed by the ratio
P
50(effector)/P50(control). Tables 1 and 2 summarize the
P50 and the Hill coefficient values (n50) at saturation.
The slope of the logarithmic Hill plot is known as the
Hill coefficient. The Hill coefficient measures the degree

4730 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Phelps Grella et al.
F igu r e 4. Structure of proposed configurations for the enantiomers of compounds 11 and 13.
F igu r e 5. Close-up stereoview of the superimposed binding sites for (+)-(1S,2S)-11 and (-)-(1R,2R)-11 Hb complexes showing
the stereochemistry at the chiral centers of the effectors. The effectors (+)-(1S,2S)-11 and (-)-(1R,2R)-11 and their binding site
residues are shown in light blue and yellow, respectively. Oxygen and nitrogen atoms are dark blue and red, respectively.
of cooperativity of ligand binding to an allosteric protein;
the normal range for human blood is 2.7-3.2. Tables
1-3 present the results from Hb solution and whole
appears to be important, 2-methyl being > 3-methyl.
The 2-methylcyclopentyl compound (11) was nearly
twice as active as the 3-methyl derivative (9). A slight
loss in activity was observed when the size of the
cycloalkyl ring was increased to a six-membered ring.
Oxygen substitutions to alkyl chains and cycloalkyl
groups reduced activity. This observation was most
apparent with compound 13. Compounds 11 and 13 are
structurally similar except for an ether oxygen substitu-
tion in the cyclopentyl ring, yet 11 was nearly 3 times
more potent than 13. Compound 11 was the most potent
compound from the study exhibiting activity comparable
to that of RSR13 and J P7.
In general, the results of the enantiomers showed that
the stereocenter does have an effect on allosteric activity
(Table 2). A small difference in the P50 values was
observed between the enantiomers of 30: (+)-30 was
slightly more active than (-)-30. While the specific
activity for compound 30 is not high and the differences
in shifting the Hb solution oxygen-binding curve be-
tween enantiomers and the racemate are small, the
whole blood studies show the same trend: i.e., the (+)-
enantiomer exhibited stronger activity than the (-)-
enantiomer. The same trend was observed for the
isomers of 32. The (+)-isomer was nearly twice as active
as its mirror image, (-)-isomer. (+)-32 was also more
potent than (+)-30, which further indicated that the
longer alkyl group enhances activity. A similar differ-
ence in activity between the enantiomers of 10 was also
observed: (+)-10 > (-)-10. The difference in activity
blood studies given as P50, ∆P50, and Hill coefficient (n50
)
values.
SARs. The new analogues differ in their substitution
at the gem-dimethyl position R to the carboxylate group.
All of the synthesized derivatives from this study
increased P50 exhibiting a wide range of allosteric
effector activity (Table 1). In general, similarly substi-
tuted compounds from the 3,5-dimethylphenyl and 3,5-
chloromethylphenyl series appeared to be equal in
potency. Corresponding compounds with the indanyl
group substitution were less active. The majority of the
compounds showed good cooperativity in Hb solutions,
n50 ) 2.3-2.7 (Table 1).
Removal of one of the methyl groups from the gem-
dimethyl position, 30, significantly reduced activity.
Substitution of fluoro group for methyl resulted in
further decreased activity. The length of the monoalkyl
group enhances activity, as exhibited in compounds 30
and 32-34. The activity of compounds with a monoalkyl
substitution tended to increase with the size of the
group (butyl g propyl > ethyl > methyl). The effect
seemed to plateau going from the propyl to the butyl
group with only a slight increase in the P50. Replace-
ment of one of the methyl groups with an ethyl group
decreased activity by one-half, and increasing the length
of the chain further reduced P50 (Table 1). The position
of the methyl substitution on the cyclopentyl ring

Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4731
Ta ble 1. Results of Hb Solution Studies for Synthesized
Ta ble 2. Results of Hb Solution Studies for Resolved
Analoguesa
Enantiomersa
P50e/
d
no.
(+)-10
(-)-10
R1
R2
P50eb 50cc n50
P
P50e/
P
P50eb 50cc n50
d
no.
R1
R2
CH3
CH3
CH2CH3 17.4 3.5 2.4
CH2CH3 10.5 2.1 2.3
19.9 4.0 2.6
3b (RSR13) CH3
3c (KDD86) Cl
4 (RSR46) CH3
CH3
CH3
CH3
23.4 4.7 2.3
21.0 4.4 2.4
15.3 3.0 2.6
25.7 5.1 2.4
12.9 2.6 2.5
13.3 2.7 2.4
21.6 4.3 2.3
7.6 1.5 2.4
7.6 1.5 2.4
(+)-(1S,2S)-11 2-methylcyclopentyl
(-)-(1R,2R)-11 2-methylcyclopentyl
30.7 6.1 2.4
7.7 1.5 2.8
(+)-13
2-methyltetrahydro-
5 (J P7)
cyclopentyl
3-methylcyclopentyl
CH3
2-methylcyclopentyl
4-tetrahydropyran
2-methyltetrahydro-
furan
furan
2-methyltetrahydro-
furan
9
(-)-13
9.3 1.9 2.6
10
11
12
13
CH2CH3
(+)-30
(-)-30
(+)-32
(-)-32
CH3
CH3
CH2CH3
CH2CH3
H
H
H
H
9.8 2.0 2.6
6.6 1.3 2.5
18.0 3.6 2.7
9.8 2.0 2.8
14
15
16
19
20
21
27
28
29
30
31
32
33
34
35
36
37
38
CH3
CH3
CH2OCH3
8.4 1.7 2.4
CH2CH2CH3 11.7 2.3 2.7
10.3 2.1 2.5
a
All studies were carried out at 50-60 mM heme concentration
in the presence of 0.5 mM effector concentration. All solutions were
prepared in 100 mM bis-Tris buffer, pH 7.2. See Experimental
3-methylcyclohexyl
3-methylcyclopentyl
CH3
2-methylcyclopentyl
3-methylcyclopentyl
CH3
12.7 2.5 2.6
b
CH2CH3
CH2CH3
10.6 2.1 2.3
13.8 2.8 2.6
14.0 2.8 2.4
16.6 3.3 2.5
21.9 4.4 2.4
9.3 1.9 2.6
5.9 1.2 2.5
13.0 2.6 2.7
16.1 3.2 2.5
17.0 3.4 2.5
9.4 1.9 2.6
13.1 2.6 2.6
6.3 1.3 2.7
9.1 1.8 2.6
Section for more details. P50e is the oxygen pressure in mmHg
at which Hb is 50% saturated with oxygen in the presence of the
effector. c Ratio of P50e to P50c (P50 control value with no effector
d
present, 5.0 mmHg). The Hill coefficient at 50% saturation (n50
)
2-methylcyclopentyl
CH3
is calculated from the Hill equation by linear regression analysis
of data points between 40% and 60% oxygen saturation (n50 control
value with no effector present, 2.7).
H
H
H
H
H
H
H
H
H
F
CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
CH3
more active isomer from both 11 and 30 was only
slightly more active than the racemic mixture. This
suggests that there are pharmacokinetic and/or bio-
availability factors involved, possibly enantioselective
plasma protein binding. Enantioselective plasma protein
binding has been observed for several drugs including
ibuprofen, warfarin, and propranolol where the enan-
tiomers differ in their affinity for plasma proteins
resulting in differing free fractions of the isomers.27-30
Further studies need to be initiated to investigate this
possibility for these chiral allosteric effectors.
CH2CH3
CH3
CH2CH3
a
All studies were carried out at 50-60 mM heme concentration
in the presence of 0.5 mM effector concentration. All solutions were
prepared in 100 mM bis-Tris buffer, pH 7.2. See Experimental
b
Section for more details. P50e is the oxygen pressure in mmHg
at which Hb is 50% saturated with oxygen in the presence of the
effector. c Ratio of P50e to P50c (P50 control value with no effector
d
present, 5.0 mmHg). The Hill coefficient at 50% saturation (n50
)
is calculated from the Hill equation by linear regression analysis
of data points between 40% and 60% oxygen saturation (n50 control
value with no effector present, 2.7).
Su m m a r y a n d Con clu sion s
The chiral allosteric effectors synthesized in this study
decreased the oxygen affinity of Hb. SARs showed that
modifying the gem-dimethyl group of RSR13 with vari-
ous alkyl groups reduced activity. The 2-methylcyclo-
pentyl derivative of RSR13 was the most potent allo-
steric effector from this study demonstrating that
methyl substitution on the cyclopentyl ring was better
tolerated in the 2-position than the 3-position. Further-
more, ether oxygen substitutions for methylene groups
significantly decreased activity.
Most importantly, this investigation revealed that
enantiomeric selectivity produces differences in allo-
steric activity. Enantiomers were observed to possess
different degrees of potency. The replacement of the
gem-dimethyl group on RSR13 (3b) with alkyl substit-
uents showed that the (+)-isomer was more potent than
the (-)-isomer. In whole blood, (-)-(1S,2S)-11 exhibited
strong allosteric activity, making it one of the most
potent chiral allosteric effector reported to date. Since
the Hb solution studies do not include DPG, a natural
allosteric effector, they show a large change in P50
compared to the whole blood. Therefore, the amplitude
of the P50 effects cannot be compared directly with whole
between the enantiomers of compound 13 did not appear
to be as significant. The most interesting observation
among the enantiomers involved compound 11. The
results showed that (-)-(1R,2R)-11 was more potent
than the (+)-(1S,2S)-11 isomer, and furthermore, (-)-
11 was more potent in Hb solutions than both RSR13
and J P7 with a P50 of 30.7 mmHg (Table 2). The activity
observed for some of the racemates was not an average
of the P50 value for the enantiomeric pair. The difference
in activity may be related to an intrinsic activity factor
discussed in one of our earlier papers.26
The racemates and the enantiomers of compounds 11
and 30 were also analyzed in vitro using human whole
blood. Results from the whole blood study revealed the
same general trend in activity as observed in Hb
solutions. The compounds lowered the n50 which means
to a small extent all of the compounds reduce the
cooperativity of Hb. Generally, compounds with a high
P50 value cause the n50 to decrease. (+)-30 was more
potent than (-)-30. In addition, (-)-11 was significantly
more active than (+)-11 and equipotent to RSR13 and
J P7. However, the whole blood results revealed that the

4732 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Ta ble 3. Results of in Vitro Whole Blood Studiesa
Phelps Grella et al.
no.
(()-11
R1
R2
P50cb
P50ec
∆P50 ( SDd
40.1 ( 0.9
n50 ( SDe
1.6 ( 0.09
2-methylcyclopentyl
26.5
66.0
67.2
49.7
46.8
42.4
78.5
72.5
47.2
43.5
47.4
48.7
39.7
40.5
76.5
73.4
74.7
67.7
(+)-(1S,2S)-11
2-methylcyclopentyl
25.7
20.6 ( 3.6
47.6 ( 4.2
18.2 ( 2.6
20.7 ( 0.7
12.3 ( 0.6
45.2 ( 2.2
42.3 ( 4.9
1.8 ( 0.12
1.6 ( 0.04
2.0 ( 0.03
1.9 ( 0.04
2.2 ( 0.05
1.47 ( 0.05
1.66 ( 0.3
(-)-(1R,2R)-11
(()-30
2-methylcyclopentyl
27.9
27.2
27.3
27.8
29.8
28.9
CH3
H
(+)-30
CH3
H
(-)-30
CH3
H
3b (RSR13)
5 (J P7)
CH3
CH3
cyclopentyl
a
All studies were carried out at 2.5 mM Hb concentration in the presence of 5.0 mM effector concentration. All solutions were prepared
b
c
in DMSO. See Experimental Section for more details. P50c is the control value in mmHg (average P50c value ) 27.6 ( 1.3, n ) 8). P50
e
is the value in the presence of the effector in mmHg. ∆P50 ) (P50e - P50c) in mmHg. e The Hill coefficient at 50% saturation (n50) in the
d
presence of effector (average n50 control value ) 2.7 ( 0.1, n ) 8).
anhydrous tetrahydrofuran (30 mL). After 15 min, 3-methyl-
cyclopentanone (4.9 g, 50 mmol) was added, the mixture was
cooled to 0 °C and anhydrous chloroform (2.4 g, 20 mmol) was
added dropwise. The reaction mixture was maintained at 0
°C for 2 h and then allowed to come to room temperature while
stirring overnight. Tetrahydrofuran was removed under re-
duced pressure. The residue was dissolved in water (150 mL)
and washed with ethyl acetate (2 × 30 mL). The aqueous layer
was acidified (pH 2) with concentrated HCl and extracted with
ethyl acetate (3 × 40 mL). The combined organic fractions were
washed with brine, dried over anhydrous MgSO4, and the
solvent was removed under reduced pressure. The brown oil
was purified by flash chromatography (eluent: hexane/ethyl
acetate, 2:1) to afford a pale yellow powdery solid, 0.89 g, 47%.
Mp: 148-153 °C. 1H NMR (CDCl3): δ 1.03 (d, 3H, J ) 6.6
Hz), 1.27-2.31 (m, 6H), 2.24 (s, 6H), 2.40-2.66 (m, 1H), 3.60
(s, 2H), 6.73 (s, 1H), 6.78 (d, 2H, J ) 8.5 Hz), 7.06 (s, 2H),
7.18 (d, 2H, J ) 8.4 Hz). Anal. (C23H27NO4‚0.25H2O) C, H, N.
blood studies. Plasma protein binding could be a sig-
nificant factor in whole blood studies since that affects
the red cell membrane permeability of racemates and
enantiomers and hence the Hb allosteric activity. SARs
and differences in binding interactions based on high-
resolution X-ray crystallography studies with enanti-
omers bound to Hb have been investigated and will be
discussed in detail in a future manuscript.
Exp er im en ta l Section
Ch em istr y. All reagents and starting materials used in the
syntheses were purchased from Aldrich, Fluka, or Sigma and
used without purification. All solvents were purchased from
Aldrich or Fisher. Silica gel-coated plates (0.25-mm thickness)
from Analtech, Inc. were used for thin-layer chromatography
(TLC). Separations were visualized by ultraviolet (UV) lamp
or iodine exposure. Column chromatography was performed
on silica gel (Merck, grade 9385, 230-400 mesh). Melting
points (mp) were determined on a Thomas-Hoover melting
point apparatus and are uncorrected. Proton nuclear magnetic
resonance (1H NMR) spectra were obtained on a Varian
Gemini 300-MHz spectrophotometer and are reported in parts
per million (δ, ppm) with tetramethylsilane as the internal
standard. Elemental analyses were performed by Atlantic
Microlab, Inc. (Norcross, GA), and results are within (0.4%
of the theoretical value. All intermediate compounds were
analyzed but are not reported. Their purity was determined
by TLC and 1H NMR.
4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en ol (8).
A mixture of 4-hydroxyphenylacetic acid (20.0 g, 131 mmol)
and 3,5-dimethylaniline (15.9 g, 131 mmol) in xylene (100 mL)
was stirred for 3 days at 160 °C with a Dean-Stark trap. The
mixture was cooled to room temperature and filtered. The solid
product obtained was washed with hexane (200 mL), 10%
sodium bicarbonate solution (250 mL), water (200 mL), 10%
hydrochloric acid (200 mL), and then water (200 mL). The
beige solid was air-dried to yield 27.7 g, 82.7%. Mp: 183-185
°C. 1H NMR (CDCl3): δ 2.25 (s, 6H), 3.60 (s, 2H), 6.71 (s, 1H),
6.82 (d, 2H, J ) 8.5 Hz), 7.05 (s, 2H), 7.13 (d, 2H, J ) 8.4 Hz).
1-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
3-m eth ylcyclop en ta n eca r boxylic Acid (9). Sodium hydrox-
ide (1.8 g, 45 mmol) was added to a stirred solution of 4-[[(3,5-
dimethylanilino)carbonyl]methyl]phenol (1.27 g, 5 mmol) in
Compounds 10-16 were prepared using the same procedure
as described above for 9.
2-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
2-m eth ylbu ta n oic Acid (10). Compound 10 was prepared
by reacting 8 (1.50 g, 5.88 mmol) and 2-butanone (4.23 g, 58.8
mmol) to yield a brown oil. The crude product was purified by
flash chromatography (eluent: hexane/ethyl acetate 2:1) to
obtain a yellow oil. Recrystallization from methylene chloride
and hexane gave yellow crystals, 0.59 g, 28%. Mp: 131-133
°C. 1H NMR (CD3OD): δ 1.02 (t, 3H, J ) 7.3 Hz), 1.51 (s, 3H),
1.99 (m, 2H), 2.27 (s, 6H), 3.63 (s, 2H), 6.75 (s, 1H), 6.92 (d,
2H, J ) 7.0 Hz), 7.09 (s, 2H), 7.22 (d, 2H, J ) 7.1 Hz). Anal.
(C21H25NO4) C, H, N.
1-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
2-m eth ylcyclop en ta n eca r boxylic Acid (11). 2-Methylcy-
clopentanone (3.46 g, 35.3 mmol) and 8 (1.0 g, 3.9 mmol) were
reacted to yield a brown oil. The oil was purified by flash
chromatography (eluent: hexane/ethyl acetate 3:1) to give a
yellow solid. Recrystallization from methylene chloride and
1
hexane gave a white solid, 0.30 g, 20%. Mp: 184-186 °C. H
NMR (CD3OD): δ 1.02 (d, 3H, J ) 7.2 Hz), 1.43-2.46 (m, 6H),
2.25 (s, 6H), 2.48-2.54 (m, 1H), 3.56 (s, 2H), 6.53 (s, 1H), 6.55
(d, 2H, J ) 8.3 Hz), 6.94 (s, 2H), 7.00 (d, 2H, J ) 8.4 Hz).
Anal. (C23H27NO4‚0.25H2O) C, H, N.
4-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en oxy]-
tetr a h yd r o-2H-4-p yr a n ca r boxylic Acid (12). Tetrahydro-

Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4733
4H-pyran-4-one (4.5 g, 45 mmol) and 8 (1.3 g, 5 mmol) were
reacted to yield a yellow-brown oil. The product was purified
by flash chromatography (eluent: hexane/ethyl acetate, 2:1)
to afford a pale yellow solid. Recrystallization with methylene
chloride and hexane gave a white solid, 0.85 g, 45%. Mp: 186-
(2.0 g, 7.5 mmol) were reacted to yield a brown oil. The product
was purified by flash chromatography (eluent: hexane/ethyl
acetate, 2:1) to give a yellow solid. Recrystallization from
methylene chloride and hexane afforded a pale yellow solid,
0.85 g, 31%. Mp: 160-161 °C. 1H NMR (CDCl3): δ 1.01 (t,
3H, J ) 7.5 Hz), 1.49 (s, 3H), 1.95 (m, 2H), 2.04 (m, 2H), 2.85
(m, 4H), 3.60 (s, 2H), 6.91 (d, 2H, J ) 8.5 Hz), 7.12 (s, 2H),
7.21 (d, 2H, J ) 8.5 Hz), 7.37 (s, 1H). Anal. (C22H25NO4) C, H,
N.
1
188 °C. H NMR (CD3OD): δ 2.05-2.23 (m, 4H), 2.29 (s, 6H),
3.63 (s, 2H), 3.79 (m, 4H), 6.79 (s, 1H), 6.92 (d, 2H, J ) 8.6
Hz), 7.19 (s, 2H), 7.30 (d, 2H, J ) 8.6 Hz). Anal. (C22H25NO5‚
0.25H2O) C, H, N.
3-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
2-m eth yltetr a h yd r o-3-fu r a n ca r boxylic Acid (13). 2-Meth-
yltetrahydrofuran-3-one (3.5 g, 35 mmol) and 8 (1.0 g, 3.9
mmol) were reacted to give an orange oil. Purification by
column chromatography (eluent: hexane/ethyl acetate 3:1f1:
2) afforded a yellow oil which upon recrystallization from
methylene chloride and hexane gave a white solid, 0.47 g, 32%.
Mp: 187-190 °C. 1H NMR (DMSO-d6): δ 1.16 (d, 3H, J ) 6.5
Hz), 2.15 (m, 1H), 2.21 (s, 6H), 2.75 (m, 1H), 3.51 (s, 2H), 3.73
(q, 1H, J ) 7.5 Hz), 3.97-4.14 (m, 2H), 6.67 (s, 1H), 6.71 (d,
2H, J ) 8.6 Hz), 7.20 (s, 2H), 7.22 (d, 2H, J ) 8.3 Hz). Anal.
(C22H25NO5) C, H, N.
2-[4-[[(5-In d a n yl)ca r bon yl]m eth yl]p h en oxy]-2-m eth yl-
cyclop en ta n eca r boxylic Acid (21). 2-Methylcyclopentanone
(3.30 g, 33.7 mmol) and 18 (1.0 g, 3.7 mmol) were reacted
together as described for 9. The product was recrystallized
from acetone and ether to yield a tan amorphous solid, 0.40 g,
27%. Mp: 191-193 °C. 1H NMR (CD3OD, free acid): δ 1.02
(d, 3H, J ) 7.1 Hz), 1.43-2.46 (m, 6H,), 2.05 (m, 2H), 2.48-
2.55 (m, 1H), 2.84 (m, 4H), 3.57 (s, 2H), 6.75 (d, 2H, J ) 8.7
Hz), 7.13 (s, 2H), 7.22 (d, 2H, J ) 8.7 Hz), 7.41 (s, 1H). Anal.
(C24H27NO4‚0.25H2O) C, H, N.
2-Ch lor o-4-m et h yl-6-n it r oa n ilin e (23). A mixture of
4-methyl-2-nitroaniline (50.0 g, 329 mmol) and N-chlorosuc-
cinimide (70.3 g, 526 mmol) was refluxed in acetonitrile (200
mL) overnight. Removal of acetonitrile under reduced pres-
sure, afforded a dark red crude oil mixture. The mixture was
purified by flash chromatography (eluent: hexane/ethyl ac-
etate 7:1f6:1) to obtain a dark orange solid, 17.6 g, 29%. Mp:
2-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
3-m eth oxy-2-m eth ylp r op a n oic Acid (14). Methoxyacetone
(10.0 g, 113 mmol) and 8 (3.22 g, 12.6 mmol) were reacted
together as described for compound 9 to yield an orange-brown
semisolid. The product was recrystallized from methylene
chloride and hexane to give a pale yellow solid, 2.27 g, 48%.
1
55-58 °C. H NMR (CDCl3): δ 2.28 (s, 3H), 6.41 (s, 2H), 7.38
1
Mp: 170-172 °C. H NMR (CD3OD): δ 1.46 (s, 3H), 2.25 (s,
(s, 1H), 7.90 (s, 1H).
6H), 3.37 (s, 3H), 3.59 (s, 2H), 3.66 (s, 2H), 6.74 (s, 1H), 6.95
(d, 2H, J ) 8.5 Hz), 7.15 (s, 2H), 7.24 (d, 2H, J ) 8.5 Hz).
Anal. (C21H25NO5) C, H, N.
3-Ch lor o-5-n itr otolu en e (24). A solution of 23 (17.5 g, 94.4
mmol) in ethanol (100 mL) was allowed to cool to 0 °C and
concentrated sulfuric acid was added (20 mL). Sodium nitrite
(40 wt % solution in water, 13.0 g, 189 mmol) was added
dropwise to the solution, and the reaction mixture was allowed
to warm to room temperature and stirred for 30 min. Then
the mixture was refluxed until the evolution of nitrogen gas
ceased. The reaction mixture was concentrated under reduced
pressure and then diluted with water (100 mL) and extracted
with ethyl acetate (4 × 75 mL). The combined organic fractions
were washed with brine, dried over anhydrous MgSO4, and
the solvent was removed under reduced pressure to afford a
dark red oil. The product was purified by flash chromatography
(eluent: hexane/ethyl acetate 7:1) to give an orange solid, 11.2
2-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
2-m eth ylp en ta n oic Acid (15). Using 8 (1.5 g, 5.9 mmol) and
2-pentanone (4.55 g, 52.9 mmol), compound 15 was prepared
as described for compound 9. The brown semisolid obtained
was purified by flash chromatography (eluent: hexane/ethyl
acetate 3:1f1:1). Recrystallization from methylene chloride
and hexane gave a white amorphous solid, 0.70 g, 32%. Mp:
145-147 °C. 1H NMR (CD3OD): δ 0.94 (t, 3H, J ) 7.3 Hz),
1.46 (s, 3H), 1.47 (m, 2H), 1.88 (m, 2H), 2.25 (s, 6H), 3.58 (s,
2H), 6.74 (s, 1H), 6.87 (d, 2H, J ) 8.6 Hz), 7.15 (s, 2H), 7.23
(d, 2H, J ) 8.6 Hz). Anal. (C22H27NO4) C, H, N.
1
1-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
3-m eth ylcycloh exa n eca r boxylic Acid (16). 3-Methylcyclo-
hexanone (4.39 g, 39.2 mmol) and 8 (1.0 g, 3.9 mmol) were
reacted together to give a light brown oil. The product was
purified by flash chromatography (eluent: hexane/ethyl ac-
etate 2:1), followed by recrystallization from methylene chlo-
ride and hexane to obtain white fluffy crystals, 0.08 g, 5%.
g, 69%. Mp: 55-57 °C. H NMR (CDCl3): δ 2.46 (s, 3H), 7.50
(s, 1H), 7.94 (s, 1H), 8.03 (s, 1H).
3-Ch lor o-5-m eth yla n ilin e (25). To a solution of 24 (7.0 g,
41 mmol) in ethanol (100 mL) was added tin(II) chloride
dihydrate (45.8 g, 204 mmol) and the mixture was refluxed
for 3 h. Upon cooling, ice was added to the reaction mixture
and basified with 2 N NaOH. The mixture was filtered and
the filtrate was concentrated under reduced pressure. The
aqueous solution was extracted with ethyl acetate (4 × 50 mL).
The organic layers were combined, washed with brine, dried
over anhydrous MgSO4 and the solvent was removed under
reduced pressure to obtain a yellow-orange liquid, 5.4 g, 93%.
1H NMR (CDCl3): δ 2.22 (s, 3H), 3.66 (s, 2H), 6.36 (s, 1H),
6.48 (s, 1H), 6.56 (s, 1H).
1
Mp: 175-177 °C. H NMR (CD3OD): δ 0.93 (d, 3H), J ) 6.3
Hz), 1.20 (m, 1H), 1.45-1.80 and 2.37 (m, 8H), 2.26 (s, 6H),
3.58 (s, 2H), 6.75 (s, 1H), 6.90 (d, 2H, J ) 8.6 Hz), 7.15 (s,
2H), 7.22 (d, 2H, J ) 8.6 Hz). Anal. (C24H29NO4) C, H, N.
4-[[(5-In dan yl)car bon yl]m eth yl]ph en ol (18). Using 5-ami-
noindan (10.0 g, 75.2 mmol) and 4-hydroxyphenylacetic acid,
the amide was synthesized as described for 8 to give a brown
solid, 18.1 g, 90%. Mp: 148-151 °C. 1H NMR (CDCl3): δ 2.04
(m, 2H), 2.84 (m, 4H), 3.60 (s, 2H), 6.83 (d, 2H, J ) 8.5 Hz),
7.13 (s, 2H), 7.15 (d, 2H, J ) 8.5 Hz), 7.35 (s, 1H).
4-[[(3-Ch lor o-5-m et h yla n ilin o)ca r b on yl]m et h yl]p h e-
n ol (26). To a solution of 4-hyroxyphenylacetic acid (5.9 g, 39
mmol) and 1-hydroxybenzotriazole hydrate (5.8 g, 43 mmol)
in dimethylformamide (40 mL) were added 3-chloro-5-meth-
ylaniline (5.5 g, 39 mmol) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (9.0 g, 47 mmol). The reaction
mixture was stirred overnight at room temperature, then
diluted with ethyl acetate (100 mL). The mixture was washed
with water (3 × 50 mL) and 10% potassium hydrogen sulfate
(3 × 50 mL). The organic layers were combined and washed
with brine, then dried over anhydrous MgSO4. The solvent was
removed under reduced pressure to give a brown oil. Recrys-
tallization from methylene chloride and hexane afforded a
Compounds 19-21 were prepared using the same procedure
as described above for 9.
1-[4-[[(5-In d a n yl)ca r bon yl]m eth yl]p h en oxy]-3-m eth yl-
cyclop en ta n eca r boxylic Acid (19). 3-Methylcyclopentanone
(5.9 g. 60 mmol) and 18 (1.6 g, 6.0 mmol) were reacted to yield
a brown oil. Purification by flash chromatography (eluent:
hexane/ethyl acetate 2:1) afforded a yellow oil. Recrystalliza-
tion with ether and hexane gave yellow crystals, 0.87 g, 37%.
1
Mp: 148-151 °C. H NMR (CD3OD): δ 1.06 (d, 3H, J ) 6.6
Hz), 1.32-2.32 (m, 6H), 2.05 (m, 2H), 2.42-2.67 (m, 1H), 2.84
(m, 4H), 3.56 (s, 2H), 6.78 (d, 2H, J ) 8.5 Hz), 7.12 (s, 2H),
7.20 (d, 2H, J ) 8.5 Hz), 7.37 (s, 1H). Anal. (C24H27NO4) C, H,
N.
1
beige solid, 6.26 g, 58%. Mp: 176-178 °C. H NMR (CDCl3):
δ 2.26 (s, 3H), 3.54 (s, 2H), 6.74 (d, 2H, J ) 8.6 Hz), 6.91 (s,
1H), 7.14 (d, 2H, J ) 8.6 Hz), 7.23 (s, 1H), 7.52 (s, 1H).
2-[4-[[(5-In d a n yl)ca r b on yl]m et h yl]p h en oxy]-2-m et h -
ylbu ta n oic Acid (20). 2-Butanone (5.4 g, 75 mmol) and 18
Compounds 27-29 were prepared using the same procedure
as described above for 9.

4734 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Phelps Grella et al.
1-[4-[[(3-Ch lor o-5-m eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]-3-m et h ylcyclop en t a n eca r b oxylic Acid (27). 3-
Methylcyclopentanone (3.57 g, 36.4 mmol) and 25 (1.0 g, 3.6
mmol) were reacted together to give a brown oil. The impure
product was purified by flash chromatography (eluent: hexane/
ethyl acetate 3:1f2:1) to give a yellow semisolid. Recrystal-
lization from methylene chloride and hexane yielded a white
solid, 0.38 g, 26%. Mp: 164-166 °C. 1H NMR (CD3OD): δ 1.04
(d, 3H, J ) 6.6 Hz), 1.36-2.32 (m, 6H), 2.29 (s, 3H), 2.43-
2.67 (m, 1H), 3.58 (s, 2H), 6.73 (d, 2H, J ) 8.6 Hz), 6.91 (s,
1H), 7.20 (d, 2H, J ) 8.7 Hz), 7.22 (s, 1H), 7.51 (s, 1H). Anal.
(C22H24ClNO4) C, H, Cl, N.
methanol was removed by rotavap. A white solid precipitated
and was collected by filtration to obtain 1.3 g. Mp: 169-171
°C. Optical rotation measured at 21 °C: [R]D -25.6 (c ) 1,
methanol). Anal. (C19H21NO4‚0.5H2O) C, H, N.
(+)-2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]p r op ion ic Acid (30). The enriched mother liquor
obtained after the first crystallization of (-)-30 was concen-
trated under reduced pressure. The residue was neutralized
as described for (-)-30 to give a white solid, 2.3 g of optically
pure (+)-30. Mp: 169-171 °C. Optical rotation measured at
21 °C: [R]D +25.1 (c ) 1, methanol). Anal. (C19H21NO4) C, H,
N.
2-[4-[[(3-Ch lor o-5-m eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]-2-m eth ylbu ta n oic Acid (28). 2-Butanone (4.64 g, 64.4
mmol) and 25 (1.77 g, 6.44 mmol) were reacted together to
give a brown oil. Purification by flash chromatography (elu-
ent: hexane/ethyl acetate 3:1f2:1) followed by recrystalliza-
tion from methylene chloride and hexane afforded a beige solid,
0.44 g, 18%. A small portion of the product was purified for
analytical purposes via esterification which was purified by
flash chromatography (eluent: hexane/ethyl acetate 4:1) then
hydrolyzed to the acid to give white fluffy crystals. Mp: 101-
Compounds 31-38 were prepared using the same procedure
as described above for 30.
2-[4-[[(3,5-Dim eth ylan ilin o)car bon yl]m eth yl]ph en oxy]-
2-flu or oa cetic Acid (31). Using ethyl bromofluoroacetate (2.9
g, 16 mmol), 8 (2.0 g, 7.8 mmol), and potassium carbonate (2.16
g, 15.6 mmol), compound 31 was prepared as described for 30,
except the reaction went for 2 days. The brown oil obtained
was purified by flash chromatography (eluent: hexane/ethyl
acetate, 2:1) to afford a yellow oil. Ester hydrolysis afforded a
yellow oil. The product was recrystallized from methylene
chloride and hexane to yield a pale yellow powder, 0.47 g, 18%.
1
103 °C. H NMR (CD3OD): δ 0.99 (t, 3H, J ) 7.4 Hz), 1.45 (s,
1
3H), 1.96 (m, 2H), 2.29 (s, 3H), 3.59 (s, 2H), 6.87 (d, 2H, J )
8.6 Hz), 6.92 (s, 1H), 7.22 (d, 2H, J ) 8.5 Hz), 7.24 (s, 1H),
7.51 (s, 1H). Anal. (C20H22ClNO4) C, H, Cl, N.
Mp: 123-127 °C. H NMR (CD3OD): δ 2.26 (s, 6H), 3.64 (s,
2H), 6.10 (d, 1H, J ) 59.7 Hz), 6.75 (s, 1H), 7.10 (d, 2H, J )
8.6 Hz), 7.16 (s, 2H), 7.34 (d, 2H, J ) 8.6 Hz). Anal. (C18H18
FNO4) C, H, N.
-
1-[4-[[(3-Ch lor o-5-m eth yla n ilin o)ca r bon yl]m eth yl]ph e-
n oxy]-2-m et h ylcyclop en t a n eca r b oxylic Acid (29). 2-
Methylcyclopentanone (3.57 g, 36.4 mmol) and 25 (1.0 g, 3.6
mmol) were reacted together to give a brown oil. Purification
by flash chromatography (eluent: hexane/ethyl acetate 3:1f2:
1) gave a yellow solid. Recrystallization from methylene
chloride and hexane yielded a white solid, 0.15 g, 10%. Mp:
180-182 °C. 1H NMR (CD3OD): δ 1.02 (d, 3H, J ) 7.1 Hz),
1.43-2.48 (m, 6H), 2.52-2.57 (m, 1H), 3.58 (s, 2H), 6.76 (d,
2H, J ) 8.6 Hz), 6.92 (s, 1H), 7.21 (d, 2H, J ) 8.7 Hz), 7.24 (s,
1H), 7.52 (s, 1H). Anal. (C22H24ClNO4‚1.0H2O) C, H, Cl, N.
2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en oxy]-
bu ta n oic Acid (32). Ethyl 2-bromobutyrate (3.9 g, 20 mmol)
and 8 (2.5 g, 10 mmol) were reacted to yield the ethyl ester of
32, a yellow oil. Ester hydrolysis afforded a yellow solid, which
upon recrystallization from methylene chloride and hexane
gave a white solid, 2.86 g, 84%. Mp: 173-175 °C. 1H NMR
(CDCl3): δ 1.08 (t, 3H, J ) 7.5 Hz), 1.95 (m, 2H), 2.25 (s, 6H),
3.56 (s, 2H), 4.61 (t, 1H, J ) 7.0 Hz), 6.74 (s, 1H), 6.86 (d, 2H,
J ) 8.6 Hz), 7.15 (s, 2H), 7.25 (d, 2H, J ) 8.6 Hz). Anal. (C20H23
NO4) C, H, N.
-
2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en oxy]-
p r op ion ic Acid (30). Ethyl 2-bromopropionate (1.8 g, 10
mmol) was added to a stirred mixture of 8 (1.27 g, 5.00 mmol)
and potassium carbonate (1.4 g, 10 mmol) in dry dimethyl-
formamide (30 mL). The mixture was heated overnight at 80
°C, cooled to room temperature, and diluted with ethyl acetate
(100 mL). The mixture was washed with water (3 × 40 mL)
followed by brine. The organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure to afford a
yellow oil. Without further purification the ester was hydro-
lyzed using 10% sodium hydroxide (10 mL) in ethanol (25 mL)
and allowing the reaction to stir overnight at room tempera-
ture. Ethanol was removed under reduced pressure at room
temperature. The residual product was dissolved in water (100
mL) and washed with ethyl acetate (3 × 40 mL). The aqueous
layer was acidified (pH 2) with concentrated hydrochloric acid
and extracted with ethyl acetate (3 × 40 mL), dried over
anhydrous MgSO4, and evaporated to dryness to obtain a
yellow solid. The product was recrystallized from a mixture
of methylene chloride and hexane to yield a white powder, 1.07
g, 66%. Mp: 183-187 °C. 1H NMR (CDCl3): δ 1.55 (d, 3H,
J ) 6.9 Hz), 2.17 (s, 6H), 3.52 (s, 2H), 4.65 (q, 1H, J ) 6.7 Hz),
6.65 (s, 1H), 6.81 (d, 2H, J ) 8.6 Hz), 6.98 (s, 2H), 7.15 (d, 2H,
J ) 8.6 Hz). Anal. (C19H21NO4) C, H, N.
(-)-2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]p r op ion ic Acid (30). A solution of cinchonidine (4.50
g, 15.3 mmol) in hot ethanol (70 mL) was added to a solution
of (()-30 (5.00 g, 15.3 mmol) in hot ethanol. The mixture was
cooled to room temperature and a portion of the solvent was
removed under reduced pressure. Crystals obtained were
collected by filtration, 4.6 g. Mp: 198-200 °C. Optical rotation
measured at 25 °C: [R]D -69.8° (c ) 0.2, methanol). The salt
was recrystallized from ethanol to give pale yellow crystals,
2.86 g. Mp: 200-202 °C. Optical rotation measured at 25 °C:
[R]D -71.0° (c ) 0.2, methanol). 2.7 g of the salt was dissolved
in warm methanol (65 mL) and acidified to pH 2 with 1 N
HCl. The solution stirred for 1 h and then the majority of the
(-)-2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]bu ta n oic Acid (32). Following the same procedure as
described for (-)-(30), cinchonidine (8.61 g, 29.3 mmol) in hot
ethanol (175 mL) was added to a solution of (()-32 (10.0 g,
29.3 mmol) in hot ethanol. The solution was allowed to cool to
room temperature and a portion of the solvent was removed
under reduced pressure. Crystals obtained were collected by
filtration, 6.6 g. Mp: 204-205 °C. Optical rotation measured
at 21 °C: [R]D -73.3° (c ) 0.5, methanol). The salt was
recrystallized from ethanol to give fluffy white crystals, 3.7 g.
Mp: 206-207 °C. Optical rotation measured at 21 °C: [R]D
-74.2° (c ) 0.5, methanol). The acid was recovered from salt,
as described previously for (-)-30, to obtain a white solid, 1.7
g. Mp: 150-151 °C. Optical rotation measured at 21 °C: [R]D
-28.4° (c ) 1.2, methanol). Anal. (C20H23NO4) C, H, N.
2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en oxy]-
p en ta n oic Acid (33). Ethyl 2-bromovalerate (4.2 g, 20 mmol)
and 8 (2.5 g, 10 mmol) were reacted in the presence of
potassium carbonate (2.76 g, 20.0 mmol) as described for
compound 30 except that the solvent used was ethanol (50
mL). The mixture was refluxed overnight. The potassium
carbonate was removed by filtration and washed with ethyl
acetate. Concentration under reduced pressure afforded a
light-brown oil. Hydrolysis afforded a yellow solid. Recrystal-
lization from methylene chloride and hexane gave a white
solid, 1.38 g, 39%. Mp: 164-166 °C. 1H NMR (CD3OD): δ 0.98
(t, 3H, J ) 7.3 Hz), 1.55 (m, 2H), 1.89 (m, 2H), 2.25 (s, 6H),
3.57 (s, 2H), 4.64 (t, 1H, J ) 7.4 Hz), 6.74 (s, 1H), 6.86 (d, 2H,
J ) 8.6 Hz), 7.15 (s, 2H), 7.24 (d, 2H, J ) 8.6 Hz). Anal. (C21H25
NO4‚0.25H2O) C, H, N.
-
2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h en oxy]-
h exa n oic Acid (34). Ethyl 2-bromohexanoate (1.95 g, 10.0
mmol) and 8 (1.3 g, 5.0 mmol) were reacted together to give a
yellow oil. A white solid was obtained by flash chromatography
(eluent: hexane/ethyl acetate 5:1). Ester hydrolysis yielded a
white amorphous solid, 1.2 g, 65%. Mp: 153-155 °C. 1H NMR
(CD3OD): δ 0.94 (t, 3H, J ) 7.3 Hz), 1.36-1.54 (m, 4H), 1.91

Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4735
(m, 2H), 2.25 (s, 6H), 3.57 (s, 2H), 4.64 (t, 1H, J ) 6.1 Hz),
6.74 (s, 1H), 6.85 (d, 2H, J ) 8.7 Hz), 7.15 (d, 2H), 7.24 (d, 2H,
J ) 8.7 Hz). Anal. (C22H27NO4) C, H, N.
Anal. (C21H24NO4‚0.75H2O‚0.17TFA) C, H, F, N. (+)-10 was
collected as a yellow solid, 0.10 g. Mp: 127-129 °C. Optical
rotation measured at 20 °C: [R]D +11.0 (c ) 0.3, methanol).
1H NMR (CD3OD): δ 0.99 (t, 3H, J ) 7.4 Hz), 1.45 (s, 3H),
1.96 (m, 2H), 2.25 (s, 6H), 3.58 (s, 2H), 6.74 (s, 1H), 6.87 (d,
2H, J ) 8.6 Hz), 7.15 (d, 2H), 7.24 (d, 2H, J ) 8.6 Hz). Anal.
(C21H24NO4‚0.5H2O‚0.25TFA) C, H, F, N.
2-[4-[[(3-Ch lor o-5-m eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]p r op ion ic Acid (35). Ethyl 2-bromopropionate (1.2 g,
6.5 mmol) and 26 (0.90 g, 3.27 mmol) were reacted together
to yield the ethyl ester, a yellow-orange oil. Hydrolysis of the
ester was carried out as described to obtain a yellow semisolid.
Recrystallization from methylene chloride and hexane yielded
(+)-2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]bu ta n oic Acid (32). The mother liquor from the re-
crystallization of (-)-32 cinchonidine salt was concentrated
under reduced pressure. The enriched (+)-32 isomer was
obtained from the mother liquor by neutralizing the salt to
obtain the free acid, as described previously for compound 30.
The optical rotation showed that the isomer was approximately
80% enantiomeric excess. Optical rotation measured at 21
°C: [R]D +18.2° (c ) 1.2, methanol). A stock solution (1 mg/
mL) of the racemic mixture (32) was prepared in ethanol/
mobile phase (1:1). The injected sample volume was 250 µL.
The compound was eluted with a mobile phase of heptane/
ethanol with 1% TFA (88:12) at a flow rate of 2.5 mL/min.
Under these conditions, the (-)-isomer eluted at 15.8 min and
the (+)-isomer eluted at 18.2 min retention times. The collected
fractions were concentrated under reduced pressure at room
temperature. (+)-32 was collected as a white solid, 0.14 g.
Mp: 146-148 °C. Optical rotation measured at 21 °C: [R]D
1
a yellow solid, 0.86 g, 76%. Mp: 163-165 °C. H NMR (CD3-
OD): δ 1.56 (d, 3H, J ) 6.9 Hz), 2.29 (s, 3H), 3.59 (s, 2H), 4.78
(q, 1H, J ) 6.7 Hz), 6.86 (d, 2H, J ) 8.7 Hz), 6.92 (s, 1H), 7.22
(s, 1H), 7.24 (d, 2H, J ) 8.7 Hz), 7.50 (s, 1H). Anal. (C18H18
ClNO4) C, H, Cl, N.
-
2-[4-[[(3-Ch lor o-5-m eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]bu ta n oic Acid (36). Following the procedure given for
30, ethyl 2-bromobutyrate (1.4 g, 7.3 mmol) and 26 (1.0 g, 3.6
mmol) were reacted together to give a yellow oil. The ester
was hydrolyzed as described for 13 to give a white powder,
1
0.90 g, 69%. Mp: 168-170 °C. H NMR (CD3OD): δ 1.07 (t,
3H, J ) 7.6 Hz), 1.95 (m, 2H), 2.29 (s, 3H), 3.59 (s, 2H), 4.60
(t, 1H, J ) 6.9 Hz), 6.86 (d, 2H, J ) 8.7 Hz), 6.92 (s, 1H), 7.23
(s, 1H), 7.24 (d, 2H, J ) 8.7 Hz), 7.51 (s, 1H). Anal. (C19H20
ClNO4) C, H, Cl, N.
-
2-[4-[[(5-In d a n yl)ca r b on yl]m et h yl]p h en oxy]p r op ion -
ic Acid (37). Ethyl 2-bromopropionate (1.8 g, 10 mmol) and
18 (1.3 g, 5.0 mmol) were reacted to yield a brown oil. A brown
oil was obtained from the hydrolysis, which upon recrystalli-
zation from methylene chloride and hexane yielded a white
solid, 0.77 g, 45.5%. Mp: 133-136 °C. 1H NMR (CDCl3): δ
1.64 (d, 3H, J ) 6.9 Hz), 2.04 (m, 2H), 2.85 (m, 4H), 3.63 (s,
2H), 4.74 (q, 1H, J ) 6.9 Hz), 6.89 (d, 2H, J ) 8.6 Hz), 7.12 (s,
2H), 7.24 (d, 2H, J ) 8.6 Hz), 7.37 (s, 1H). Anal. (C20H21NO4)
C, H, N.
2-[4-[[(5-In d a n yl)ca r b on yl]m et h yl]p h en oxy]b u t a n o-
ic Acid (38). Ethyl 2-bromobutyrate (3.9 g, 20 mmol) and 18
(2.67 g, 10.0 mmol) were reacted to yield a dark brown oil.
Purification by column chromatography (eluent: hexane/ethyl
acetate, 4:1f2:1) gave a beige solid. Hydrolysis was carried
out to yield a tan oil. Recrystallization from methylene chloride
and hexane gave a white solid, 1.77 g, 50%. Mp: 160-162 °C.
1H NMR (CD3OD): δ 1.07 (t, 3H, J ) 7.3 Hz), 1.95 (m, 2H),
2.05 (m, 2H), 2.84 (m, 4H), 3.57 (s, 2H), 4.60 (t, 1H, J ) 7.4
Hz), 6.86 (d, 2H, J ) 8.6 Hz), 7.12 (s, 2H), 7.25 (d, 2H, J ) 8.6
Hz), 7.40 (s, 1H). Anal. (C21H23NO4‚0.25H2O) C, H, N.
1
+25.3° (c ) 0.5, methanol). H NMR (CD3OD): δ 1.07 (t, 3H,
J ) 7.4 Hz), 1.95 (m, 2H), 2.25 (s, 6H), 3.57 (s, 2H), 4.60 (t,
1H, J ) 6.7 Hz), 6.74 (s, 1H), 6.87 (d, 2H, J ) 8.6 Hz), 7.15 (d,
2H), 7.24 (d, 2H, J ) 8.5 Hz). Anal. (C20H23NO4‚0.75H2O) C,
H, N.
(()-3-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]-2-m eth yltetr a h yd r o-3-fu r a n ca r boxylic Acid (13).
A stock solution (1 mg/mL) of the compound 13 was prepared
in ethanol/mobile phase (1:1). The racemic compound after
loading on HPLC was eluted with a mobile phase of heptane/
ethanol with 1% TFA (85:15) at a flow rate of 2.75 mL/min.
The injection sample volume was 1000 µL. Under these
conditions, the (+)-isomer eluted at 11.4 min and the (-)-
isomer eluted at 17.5 min retention times. The collected
fractions were concentrated under reduced pressure at room
temperature. (+)-13, a pale yellow solid, was collected by
filtration and washed with ether, 0.11 g. Mp: 164-167 °C.
Optical rotation measured at 21 °C: [R]D +67.5° (c ) 0.5,
methanol). 1H NMR (CD3OD): δ 1.26 (d, 3H, J ) 6.5 Hz), 2.25
(s, 6H), 2.28 (m, 1H), 2.85 (m, 1H), 3.57 (s, 2H), 3.85 (q, 1H,
J ) 7.3 Hz), 4.07 and 4.19 (m, 1H), 6.74 (s, 1H), 6.78 (d, 2H,
En a n tiom er ic Resolu tion by HP LC. The resolution of
compounds 10, 11, and 13 and the purification of 32 were
performed using a chiral semipreparative HPLC column
(Chiracel OD, 1 cm × 25 cm) packed with cellulose tris(3,5-
J ) 8.6 Hz), 7.15 (s, 2H), 7.24 (d, 2H, J ) 8.6 Hz). Anal. (C22H25
-
NO5‚0.75H2O‚0.125TFA) C, H, F, N. (-)-13 was collected by
filtration and washed with ether to give a beige solid, 0.10 g.
Mp: 168-171 °C. Optical rotation measured at 21 °C: [R]D
dimethylphenylcarbamate) on
a silica gel substrate. The
1
-70.6° (c ) 0.3, methanol). H NMR (CD3OD): δ 1.26 (d, 3H,
samples were injected using a Waters 712 WISP automated
injector system and detected with a Waters Lambda Max
(model 481) variable wavelength detector. All of the compounds
were detected at 254 nm. The solvent delivery was controlled
with a Waters automated gradient controller (model 660). A
Hewlett-Packard integrator (HP 3393A) was used to integrate
the peaks and to plot the chromatograms. The peak fractions
were collected using a Spectrum CF-1 fraction collector. All
solvents used for HPLC separation were purchased from
Aldrich Chemical Co. as HPLC grade and filtered prior to use.
(()-2-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]-2-m eth ylbu ta n oic Acid (10). The compound was
eluted with a mobile phase of heptane/ethanol with 1% TFA
(89:11) at a flow rate of 2.0 mL/min. A stock solution (1 mg/
mL) of the compound was prepared in ethanol/mobile phase
(1:1). The injection sample volume was 250 µL. Under these
conditions, the (-)-isomer eluted at 20.7 min and the (+)-
isomer eluted at 22.9 min. The collected fractions were
concentrated under reduced pressure at room temperature.
(-)-10 was collected as a yellow solid, 0.11 g. Mp: 125-127
°C. Optical rotation measured at 20 °C: [R]D -11.6° (c ) 0.3,
methanol). 1H NMR (CD3OD): δ 0.99 (t, 3H, J ) 7.4 Hz), 1.45
(s, 3H), 1.96 (m, 2H), 2.25 (s, 6H), 3.58 (s, 2H), 6.74 (s, 1H),
6.87 (d, 2H, J ) 8.6 Hz), 7.15 (d, 2H), 7.24 (d, 2H, J ) 8.5 Hz).
J ) 6.5 Hz), 2.25 (s, 6H, ArCH3), 2.28 (m, 1H), 2.87 (m, 1H),
3.57 (s, 2H), 3.85 (q, 1H, J ) 8.1 Hz), 4.07 and 4.19 (m, 1H),
6.74 (s, 1H), 6.78 (d, 2H, J ) 8.6 Hz), 7.15 (s, 2H), 7.24 (d, 2H,
J ) 8.4 Hz). Anal. (C22H25NO5‚0.75H2O‚0.125TFA) C, H, F,
N.
(()-1-[4-[[(3,5-Dim eth yla n ilin o)ca r bon yl]m eth yl]p h e-
n oxy]-2-m eth ylcyclop en ta n eca r boxylic Acid (11). A stock
solution (1 mg/mL) of the compound was prepared in ethanol/
mobile phase (1:1). The injection sample volume was 500 µL.
The mixture was eluted with a mobile phase of heptane/
ethanol with 1% TFA (90:10) at a flow rate of 2.0 mL/min.
Under these conditions, the (+) isomer eluted at 18.9 min and
the (-)-isomer eluted at 26.2 min. retention times. The
collected fractions were concentrated under reduced pressure
at room temperature. (+)-11, a white solid, was collected by
filtration and washed with ether, 0.11 g. Mp: 186-187 °C.
Optical rotation measured at 21 °C: [R]D +64.8° (c ) 0.5,
methanol). 1H NMR (CD3OD): δ 1.02 (d, 3H, J ) 7.2 Hz),
1.43-2.48 (m, 6H), 2.25 (s, 6H), 2.51-2.57 (m, 1H), 3.57 (s,
2H), 6.74 (s, 1H), 6.77 (d, 2H, J ) 8.6 Hz), 7.15 (s, 2H), 7.22
(d, 2H, J ) 8.6 Hz). Anal. (C23H27NO4‚0.25H2O) C, H, N. The
fractions for (-)-11 were collected and concentrated under
reduced pressure. The white solid was collected by filtration

4736 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Phelps Grella et al.
and washed with ether, 0.11 g. Mp: 184-186 °C. Optical
rotation measured at 21 °C: [R]D -60.2° (c ) 0.5, methanol).
1H NMR (CD3OD): δ 1.02 (d, 3H, J ) 7.1 Hz), 1.42-2.47 (m,
6H), 2.25 (s, 6H), 2.49-2.57 (m, 1H), 3.56 (s, 2H), 6.74 (s, 1H),
6.76 (d, 2H, J ) 8.6 Hz), 7.15 (s, 2H), 7.22 (d, 2H, J ) 8.6 Hz).
Anal. (C23H27NO4‚0.25H2O) C, H, N.
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Oxygen Equ ilibr iu m Stu d ies. 1. Hb Solu tion Stu d ies.
The effectors were prepared as 10 mM stock solutions in 100
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Chiral Allosteric Modifiers of Hemoglobin
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