Synthesis and X-ray studies of chiral allosteric modifiers of hemoglobin

Article abstract of DOI:10.1021/jm010358l

This study was designed to investigate the effect of chirality on the allosteric activity of a series of Hb allosteric modifiers. The chiral analogues were based on the lead compound (4), JP7, {1-[4-(((3,5-dimethylanilino)carbonyl)methyl)phenoxy]cyclopentanecarboxylic acid} with different D- and L-amino acids conjugated to the JP7 acid moiety. The D-isomers were the most potent in vitro effectors in Hb solutions as well as with whole blood. In general, this study demonstrated that the chirality of extended amino acid side chains in JP7 conjugates plays an important role in observed degree of allosteric activity. The binding site interactions for four analogues were determined by single crystallographic diffraction studies. Conclusions show that the chiral configuration of some of the D-isomers enable the effectors to bind with a greater number of interactions with the protein residues, D- and L-isomers with equivalent or near equivalent allosteric activity did not show any significant differences or interactions between their amino acid side chains and the protein. The most potent effectors, in vitro, were compounds 15 and 19, D-isomers of leucine and phenylalanine, respectively. Compounds 21, 22, 30, and 32 were more potent in vitro in Hb solutions than JP7.

Full text of DOI:10.1021/jm010358l

1184
J . Med. Chem. 2002, 45, 1184-1195
Articles
Syn th esis a n d X-r a y Stu d ies of Ch ir a l Alloster ic Mod ifier s of Hem oglobin
Amal Mamdouh Youssef,† Martin K. Safo,† Richmond Danso-Danquah,† Gajanan S. J oshi,§ J ean Kister,‡
Michael C. Marden,‡ 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., 11080 CirclePoint Road,
Suite 200, Westminster, Colorado 80020, and Physiologie et Physiopathologie Moleculaires, INSERM U 473,
84 Rue du General Leclerc, 94276 Le Kremlin Bicetre Cedex, France
Received J uly 30, 2001
This study was designed to investigate the effect of chirality on the allosteric activity of a
series of Hb allosteric modifiers. The chiral analogues were based on the lead compound (4),
J P7, {1-[4-(((3,5-dimethylanilino)carbonyl)methyl)phenoxy]cyclopentanecarboxylic acid} with
different D- and L-amino acids conjugated to the J P7 acid moiety. The D-isomers were the most
potent in vitro effectors in Hb solutions as well as with whole blood. In general, this study
demonstrated that the chirality of extended amino acid side chains in J P7 conjugates plays an
important role in observed degree of allosteric activity. The binding site interactions for four
analogues were determined by single crystallographic diffraction studies. Conclusions show
that the chiral configuration of some of the D-isomers enable the effectors to bind with a greater
number of interactions with the protein residues. D- and L-isomers with equivalent or near
equivalent allosteric activity did not show any significant differences or interactions between
their amino acid side chains and the protein. The most potent effectors, in vitro, were compounds
15 and 19, D-isomers of leucine and phenylalanine, respectively. Compounds 21, 22, 30, and
32 were more potent in vitro in Hb solutions than J P7.
In tr od u ction
Hemoglobin (Hb) is a tetrameric allosteric protein
that consists of two R chains and two â chains arranged
around a central water cavity that is bisected by a
molecular 2-fold axis. It exists in two states: a relaxed
(R) conformation when oxygenated and a tense (T)
conformation in the deoxygenated state. The allosteric
equilibrium can be shifted in either direction by allos-
teric effectors. When the allosteric equilibrium is shifted
toward the R-state in the oxygen binding curve (OBC),
a high-affinity Hb is obtained that more readily binds
and holds oxygen, whereas a shift toward the T-state
produces the converse: a low-affinity Hb that more
readily releases oxygen. We have had a long-standing
interest in designing agents that produce low-affinity
Hb’s. Such agents have several potential clinical ap-
plications including radiosensitization of tumors, pro-
longation of stored blood half-life, and treatment of
ischemic diseases, i.e., stroke, trauma, angina, cardio-
pulmonary bypass surgery, etc.1,2
Several synthetic agents have been reported to lower
F igu r e 1. Structures of allosteric modifiers that decrease
oxygen affinity.
the oxygen affinity of Hb (Figure 1). During the search
for an antisickling agent, Abraham and co-workers3,4
found that bezafibrate (BZF, 2), another antilipidemic
discovered that the antilipidemic drug clofibrate (1)
agent, more potently right-shifted the OBC. Lalezari et
al.6-8 developed several urea analogues of BZF with a
urea linkage replacing the four-atom bridge between the
two benzene rings in BZF. These urea analogues did
not have clinical utility due to their strong affinity for
serum proteins (albumin, etc.) at physiological concen-
trations. Structure-based drug design by Abraham et
lowered the oxygen affinity of Hb. Perutz and Poyart5
* To whom correspondence should be addressed. Phone: +1 804 828
8183. Fax: +1 804 827 3664. E-mail: donald.abraham@vcu.edu.
† VCU.
§ Allos Therapeutics.
‡ INSERM.
10.1021/jm010358l CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/13/2002

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1185
Sch em e 1a
Syn th esis
The desired compounds were synthesized from the
precursor molecule 4-[[(3,5-dimethylanilino)carbonyl]-
methyl]phenol (5) as shown in Scheme 1. The synthesis
of compound 5 has been reported previously.16 Two
different procedures were used to obtain compound 4
(J P7) from amidophenol 5. Scheme 1 involved the
reaction of amidophenol 5 with cyclopentanone in the
presence of sodium hydroxide and chloroform. This
reaction is assumed to involve the preliminary forma-
tion of a dichloroepoxide17,18 which, in turn, reacts with
the phenolate ion to give 4. An alternate synthetic route
(Scheme 2) was devised for large-scale synthesis. As
shown in Scheme 2, bromination of ethyl cyclopentane-
carboxylate with N-bromosuccinimide in refluxing car-
bon tetrachloride under an IR lamp for 6 h afforded the
corresponding bromoester. Convergence of the ami-
dophenol 5 with ethyl 2-bromocyclopentanecarboxylate
in the presence of potassium carbonate and a catalytic
amount of potassium iodide (Williamson ether synthe-
sis) produced 4. The final steps in the synthesis of 4
involved the hydrolysis of the ester under basic condi-
tions using potassium hydroxide, followed by protona-
tion to produce the subsequent free acid. Compound 4
was then coupled with several D- and L-amino acid
methyl ester hydrochlorides in the presence of 1-hy-
droxybenzotriazole hydrate, 1-(3-dimethylaminopropyl)3-
ethylcarbodiimide hydrochloride (DEC), and N-methyl-
morpholine. The corresponding amino acid methyl
esters obtained were hydrolyzed in aqueous ethanol
using lithium hydroxide. The residue was dissolved in
water and acidified to obtain the corresponding amino
acids (Scheme 3). In the case of L-glutamate and
L-aspartate-tert-butoxycarbonyl ester, the hydrolysis
was accomplished using trifluoroacetic acid (Scheme 4).
a
Reagents and conditions: (i) CHCl3, NaOH.
al.9,11 produced a series of fibrate analogues that re-
placed the urea link with an amide methylene bridge.
RSR13 {2-[4-(((3,5-dimethylanilino)carbonyl)methyl)-
phenoxy]-2-methylpropionic acid} (3) showed the high-
est activity in whole blood as well as in Hb solution
assays. RSR13 was also found to shift the oxygen
equilibrium curve to the right when administered in
vivo in rats, mice, and cats.12-14 Preclinical evaluations
of RSR13 demonstrated that the effector enhances the
effectiveness for radiation treatment of hypoxic tumors
by increasing tumor oxygenation and oxygen radical
formation.15 RSR13 is currently in phase III clinical
trials as a radiosensitizing agent for metastatic brain
cancer and for the treatment of glioblastoma multiforme
brain tumors.
This paper describes a new series of potent allosteric
effectors that represent potential candidates for in vivo
animal studies. Compound 4 (J P7), a potent allosteric
effector,16 was used as a template for modification by
conjugation with D- and L-amino acids. To determine the
effect of the amino acid stereocenter on allosteric
activity, several amino acid conjugates of J P7 were
prepared. The D-, L-J P7 conjugates were evaluated with
Hb solutions as well as with whole blood in vitro. X-ray
crystallographic studies on deoxy Hb-allosteric effector
complexes were initiated to correlate enantiomeric
allosteric effector potencies with differences in ste-
reospecific binding.
In the synthesis of Nꢀ-benzyloxycarbonyl-L- and D-
lysine, the corresponding lysines were obtained by
catalytic hydrogenation using 10% palladium on carbon.
The mixture was hydrogenated in a Parr-Shaker ap-
paratus until absorption of the hydrogen gas was
stopped. The pure product was obtained upon recrys-
tallization (Scheme 5).
Sch em e 2a
a
Reagents and conditions: (i) EtOH, H+; (ii) N-bromosuccinimide (NBS), CCl4 reflux under IR; (iii) K2CO3, EtOH; (iv) KOH.

1186 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Youssef et al.
Sch em e 3a
Sch em e 5a
a
Reagents and conditions: (i) H2, Pd, C.
oxygen pressure in mmHg at which Hb is 50% saturated
with oxygen in the presence (P50 compound) or absence
(P50 control) of an effector. The Hb OEC’s were recorded
using purified-stripped human adult Hb (Hb A) with a
Hemox analyzer (TCS, Southampton, PA) under the
following conditions: pH 7.2, 100 mM NaCl, 50-60 µM
heme, and 50 mM bis-Tris buffer at 25 °C.19,20 The
results of OEC studies in the presence of 0.5 mM of
compounds are described in Table 2 and the Discussion
section. Preparation and purification of Hb A was
performed as described earlier.10 It involved separation
of RBC’s from plasma by centrifugation followed by
lysing cells with NaCl to obtain a mixture of hemoglobin
solution. The Hb mixture was then purified by column
chromatography over DEAE-Sephacel ion-exchange resin
using pH 8. 6 Tris buffer. The purity of Hb A fractions
was checked by electrophoresis, and pure fractions were
concentrated using Schleicher and Schuell collodoin bag
and used for OEC studies as desired.
a
Reagents and conditions: (i) DEC, HOBt, NMM, DMF; (ii)
LiOH, EtOH.
Sch em e 4a
Biological evaluation for potential clinical applications
of the new allosteric effectors were run in whole blood
to monitor any loss of effector activity due to poor
transport into red cells and/or serum protein binding.
The whole blood OEC’s were obtained using multi-point
tonometry (Instrumentation Laboratories, Inc., Lexing-
ton, MA). Compounds with the highest degree of allos-
teric activity produce the largest right shift in P50
relative to control P50. The slope of the log of the oxygen
binding curves is known as the Hill coefficient (n50). The
Hill coefficient measures the degree of cooperativity in
ligand binding to an allosteric protein; the normal range
for hemoglobin in human blood being 2.7-3.2. High
cooperativity is important to oxygen binding for efficient
oxygen delivery at normal capillary oxygen pressures
(pO2 ∼ 40 mmHg) since the step portion of the sigmoi-
dal-binding curve occurs near this pO2. The whole blood
results are summarized in Table 1 and discussed below.
a
Reagents and conditions: (i) DEC, HOBt, NMM, DMF; (ii)
TFA, CH2Cl2.
Biologica l Eva lu a tion
Resu lts a n d Discu ssion
Biological evaluation for structure-activity relation-
ships was conducted with hemoglobin solutions to
exclude serum protein binding and transport differ-
ences. The new analogues were tested for their ability
to right-shift the oxygen equilibrium curves (OEC’s) and
quantified by their ability to increase in P50 (partial
pressure of oxygen at 50% Hb saturation). P50 is the
Hb Solu tion Stu d ies. The D-isomer conjugates that
showed enantiomeric differences were found to be more
potent than L-isomer conjugates in Hb solutions. Com-
pounds 21 and 22 exhibited a strong shift in the Hb A
OEC’s (P50 compound/P50 control (absence of compound) ) 7), retain-
ing high cooperativity (n50 ) 2.3 and 2.1, respectively).

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1187
Ta ble 1. Results of in Vitro Whole Blood Studiesa
compound
configuration
R
P50cb
P50ec
∆P50 ( SDd
51.5 ( 1.8
n50ee
3
29.8
82.6
80.0
74.7
67.7
52.6
50.0
46.2
42.8
52.1
50.3
71.3
70.6
42.6
41.9
54.8
53.9
57.3
56.6
67.5
64.1
49.8
48.1
48.8
47.2
49.9
47.5
56.8
50.7
45.5
44.8
44.7
38.7
34.0
31.1
45.0
39.5
28.2
27.6
40.6
38.9
49.0
47.0
42.5
41.1
33.3
30.6
41.1
39.7
55.3
50.2
51.4
50.1
29.0
27.2
28.6
26.7
1.5
4
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
28.9
26.2
26.8
29.4
29.4
27.1
25.7
26.7
27.4
28.7
27.9
28.7
27.1
24.5
26.2
27.1
28.3
27.5
26.5
27.2
24.8
24.8
27.5
26.2
28.3
28.3
28.3
42.3 ( 4.9
25.1 ( 1.8
17.6 ( 2.5
21.9 ( 1.3
41.5 ( 0.5
15.2 ( 0.6
28.6 ( 0.4
30.2 ( 0.5
38.4 ( 2.4
20.3 ( 1.2
20.2 ( 1.1
20.0 ( 1.6
26.6 ( 4.3
20.6 (0.5
15.5 ( 4.2
5.4 ( 2.0
1.7
2.1
2.1
1.7
1.8
2.3
2.0
1.9
1.8
1.9
2.0
2.1
2.3
2.1
2.3
2.1
2.1
2.5
2.3
2.0
2.3
2.3
2.2
1.9
2.0
2.7
2.7
Gly
H
D-ALa
L-Ala
D-Leu
L-Leu
D-Val
L-Val
D-Phe
L-Phe
D-Trp
L-Trp
D-Met
L-Met
D-Ser
L-Ser
CH3
CH3
CH2CH(CH3)2
CH2CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2C6H5
CH2C6H5
CH2indole
CH2indole
(CH2)2SCH3
(CH2)2SCH3
CH2OH
CH2OH
Nꢀ-benzyloxycarbonyl-D-lys
(CH2)4NHCOOCH2C6H5
(CH2)4NHCOOCH2C6H5
CH(CH3)C2H5
CH2C6H4OH
(CH2)3
14.0 ( 3.9
0.4 ( 0.5
Nꢀ-benzyloxycarbonyl-L-lys
L-Ile
13.2 ( 1.2
20.8 ( 1.4
17.0 ( 1.0
7.2 ( 1.9
L-Tyr
L-Pro
S-benzyl-L-cys
L-Thr
CH2SCH2C6H5
CH(OH)CH3
CH2COOH
12.9 ( 1.0
26.6 ( 3.6
22.5 ( 0.9
-0.2 ( 1.3
-0.6 ( 1.3
L-Asp
L-Glu
CH2CH2COOH
(CH2)4NH2
D-Lys
L-Lys
(CH2)4NH2
a
b
Whole blood analyses were carried out at a final effector concentration of 5 mM. All stock solutions were prepared in DMSO. P50
control value in mmHg. P50 value in the presence of the effector in mmHg. ∆P50 ) (P50 effector - P50 control) in mmHg. e The Hill
coefficient at 50% saturation (n50) in the presence of effector (average n50 control value ) 2.7, n ) 8).
c
d
Compounds 30 and 32 exhibited an even greater shift
in the OEC, the P50 ratio (drug to control) increasing
by a factor around 10 (P50 48.6 and 43 mmHg, respec-
tively). The average control P50 of pure Hb solution in
the absence of an allosteric effector was consistent, near
4.7 mmHg. Both 30 and 32 also retained good cooper-
ativity (see Hill coefficient, Table 2). In summary, these
effectors appear to be strong candidates for further
toxicological and pharmacological studies to measure
their in vivo efficacy in animals. RSR13, currently in
clinical trials, and J P7, on the other hand, only in-
creased the P50 ratio (effector/control) by a factor of 5.
Wh ole Blood Stu d ies. In general, the enantiomers
that showed equivalent activity in hemoglobin solutions

1188 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Ta ble 2. Results of Hemoglobin Solution Studiesa
Youssef et al.
d
compound
configuration
R
P50eb
P50e/P50cc
n50
3
4
23.4
25.7
12.0
10.5
13.8
21.3
9.4
13.0
13.9
12.4
8.2
4.7
5.1
2.4
2.1
2.8
4.3
1.9
2.6
2.8
2.6
1.7
7.4
7.4
3.6
1.9
3.4
2.3
5.9
4.5
2.5
9.7
2.7
8.6
2.9
3.2
3.4
1.2
1.6
2.3
2.4
2.6
2.7
2.6
2.7
2.6
2.7
2.6
2.7
2.6
2.3
2.1
2.6
2.8
2.6
2.6
2.4
2.5
2.6
2.3
2.7
2.1
2.6
2.6
2.6
2.3
2.4
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Gly
H
CH3
CH3
CH2CH(CH3)2
CH2CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2C6H5
CH2C6H5
CH2indole
D-ALa
L-Ala
D-Leu
L-Leu
D-Val
L-Val
D-Phe
L-Phe
D-Trp
L-Trp
D-Met
L-Met
D-Ser
L-Ser
37.0
37.1
18.1
9.5
CH2indole
(CH2)2SCH3
(CH2)2SCH3
CH2OH
17.1
11.2
29.5
22.4
12.3
48.6
13.5
43.0
14.3
16.1
16.8
5.8
CH2OH
Nꢀ-benzyloxycarbonyl-D-lys
(CH2)4NHCOOCH2C6H5
(CH2)4NHCOOCH2C6H5
CH(CH3)C2H5
CH2C6H4OH
(CH2)3
CH2SCH2C6H5
CH(OH)CH3
CH2COOH
Nꢀ-benzyloxycarbonyl-L-lys
L-Ile
L-Tyr
L-Pro
S-benzyl-L-cys
L-Thr
L-Asp
L-Glu
D-Lys
L-Lys
CH2CH2COOH
(CH2)4NH2
(CH2)4NH2
8.1
a
All studies were carried out at 50-60 µM heme concentration and in the presence of 0.5 mM effector. All compound solutions were
b
prepared in 100 mM bis-Tris buffer, pH 7.2, except compounds 19 and 20 that were prepared in 30% DMSO. 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/P50c (P50 control value with no effector
d
present, 5.0 mmHg, in case of DMSO is 4.7 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).
also showed equivalent activity in the whole blood
studies. Effectors with differences in enantiomeric activ-
ity in Hb solutions also exhibited the same trends in
the whole blood studies, the D-isomer conjugates being
more potent than the L-isomer conjugates. All of the new
D- and L-analogues, with the exception of 28 (L), 36 (D),
and 37 (L), increased whole blood P50 values by varying
degrees (Table 1).
One disappointing observation occurred when it was
found that enantiomers 21/22 and compounds 30 and
32 did not show a significant rise in the P50 in the whole
blood studies probably due to the propensity of the
tryptophan, tyrosine, and benzyl-cys moieties to bind
to human serum albumin and plasma protein or due to
their poor red cell membrane permeability. To explore
whether such factors are responsible for the poor
allosteric activities of enantiomers 21/22 and compounds
30 and 32, we have initiated plasma protein binding
and red cell binding partitioning studies with these
compounds.
whole blood by 42 and 38 mmHg, respectively. Com-
pounds 15 and 19 are equipotent in whole blood with
J P7. Further pharmacokinetic evaluation of active
analogues is underway to determine if any differences
in plasma protein binding capability can be attributed
to allosteric activity variations.
Cr ysta llogr a p h ic Bin d in g Stu d ies. Hb crystal-
lographic binding studies were conducted on two pairs
of allosterically active enantiomers, constituting 17 and
18 as one pair and enantiomers 19 and 20 as the second
pair. We asked the question, does the stereochemistry
of effector side chains play a crucial role in binding the
effectors to the Hb water cavity? Therefore, we selected
enantiomeric pairs (17/18 and 19/20) for crystallo-
graphic studies. Enantiomers (17 and 18), possessing
small alkyl side chains, are equipotent in their allosteric
activities and should show no major differences in
binding, while enantiomers 19 and 20 with bulky
aromatic side chains exhibit significant differences in
their allosteric properties and should show significant
differences in binding. In efforts to evaluate these
differences in binding interactions resulting from the
extended amino acid side chains that can be correlated
to their differences in allosteric activities, enantiomers
(17/18 and 19/20) were selected for the crystallographic
studies. The difference electron density maps show all
four compounds bind to the Hb central water cavity in
symmetry related pairs, similar to that found for J P7
It appears that the lack of allosteric effector potency
of compounds 28, 36, and 37 is probably related to their
long and polar amino acid side chain groups, which
could deter transport into red blood cells. Compounds
15 {1-[4-(((3,5-dimethylanilino)carbonyl)methyl)phenoxy]-
cyclo-pentanecarbonyl-D-leucine} and 19 {D-phenylala-
nine} were found to be the most active allosteric
effectors, right-shifting the oxygen dissociation curve in

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1189
F igu r e 2. Stereoview of the allosteric site of Hb complexes of enantiomers 19 (a) and 20 (b). The effector (yellow) interacts with
R1 and R2 subunits (magenta) and â2 subunit (blue). Atoms are shown in stick representation, with oxygen and nitrogen atoms
colored red and blue, respectively. Hydrogen bonds are shown in black dashed lines.
(Abraham et al., unpublished results) and RSR13.11
However, further analysis indicates that unlike J P7 and
RSR13 effectors, the symmetry related pair of each
enantiomer (17, 18, 19, and 20) cannot be simulta-
neously accommodated in the central water cavity due
to the steric interactions between each. As also found
previously with J P7 and RSR13, each bound effector
interacts with three different subunits (two R and one
â).
The J P7 portion of each enantiomer-Hb complex
exhibited similar interactions with Hb as observed for
the unconjugated J P7 molecule, except for Lys 99R.
With RSR-13 and J P7, the bridging amide carbonyl
oxygen of the effectors points toward the terminal side
chain ammonium of Lys 99R2 forming a hydrogen bond.
On the other hand, the conjugated amino acid carboxy-
late of the new conjugates makes a hydrogen bond with
Lys 99R2. For all complexes, 3,5-dimethyl phenyl ring
is nestled in a pocket surrounded by the following
hydrophobic residues Phe 36R2, Lys 99R2, Leu 100R2,
and His 103R2 (Figures 2 and 3). As previously indi-
cated, the hydrophobic interaction between the 3,5-
dimethyl group and the residues Lys 99R2, Leu 100R2,
and His 103R2 constrains the T-state of deoxy Hb by
preventing subunit rotation during the allosteric transi-
tion. Similar to J P7 and RSR13 complexes, only one of
the two 3,5-dimethyl groups on the terminal phenyl ring
was oriented toward these hydrophobic residues. The
other methyl was directed into the water cavity. The
amide group of Asn 108â2 forms an interaction with the
π electron cloud of the 3,5-dimethyl phenyl group via a
Lewis acid-base like interaction. Perutz et al.21 pro-
posed that the π cloud of the benzene ring of the
structurally similar BZF interacts with the electron-
deficient side chain of Asn 108â2. This novel type of
aromatic/polar hydrogen bond has about one-half the
strength of a normal hydrogen bond and may make a
significant contribution to protein stability.22 Hydro-
phobic interactions were also observed between the
phenoxy ring and Trp 37â2. The cyclopentane ring made
hydrophobic interactions with Tyr 140R1, Pro 95R1, and
Thr 137R1. By engaging in interdimer interactions with
three of the four protein subunits, the effector stabilizes
the T-state deoxy Hb and thus is able to shift the
allosteric equilibrium to the right.
Unlike the enantiomeric pair 17 and 18, the extended
amino acid side chains of the enantiomer-Hb complexes
of 19 and 20 are oriented differently from each other
resulting in additional but different interactions. In the
case of 19 (Figure 2a), the phenyl group of the amino
acid side chain of the effector makes hydrophobic
interactions with Pro 95R1, Phe 98R1, Lys 99R1, and Ser

1190 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Youssef et al.
F igu r e 3. Stereoview of the allosteric site of Hb complexes of enantiomers 17 (a) and 18 (b). The effector (yellow) interacts with
R1 and R2 subunits (magenta) and â2 subunit (blue). Atoms are shown in stick representation, with oxygen and nitrogen atoms
colored red and blue, respectively. Hydrogen bonds are shown in black dashed lines.
133R1. On the other hand, enantiomer 20, which is a
weaker acting effector than 19, exhibited hydrophobic
interactions with only Ser 133R2 (Figure 2b). We believe
this accounts for the lower activity observed for 20.
The binding sites of enantiomers 17 and 18 with Hb
are shown in parts a and b of Figure 3, respectively.
The best fit to the difference electron density maps for
each showed that the amino acid conjugate in both
complexes does not make strong interactions with the
protein. The lack of differential contact would account
for the similar activity observed for both enantiomers.
The crystallographic studies therefore suggest that the
amino acid conjugate derivatives of J P7 reported here
play a crucial role in contributing to the allosteric
activity.
mines the observed allosteric activity. SARs demon-
strated that, in general, D-isomers are more potent than
the corresponding L-isomers. D-Orientation enables
greater polar hydrophobic side chain interactions be-
tween the effectors and binding site residues. Within
the Hb solution data, the amino acid conjugates con-
taining a heteroatom or polar group such as hydroxyl
are more active than effectors with branched chain alkyl
and arylalkyl susbtituents. However, in whole blood
studies, the activity profile is reversed where effectors
with branched chain alkyl and arylalkyl susbtituents
are more active than amino acid conjugates containing
a heteroatom or polar group. The loss of activity by the
polar effector conjugates is probably due to serum
protein binding and lack of transport into erythrocytes.
Studies are underway to test this hypothesis. The X-ray
studies indicate that the orientation and interactions
of the chiral center with the protein plays an important
role in determining the observed allosteric activity. In
general, all effectors function by preventing the subunit
rotation during the allosteric transition. The effectors
Su m m a r y a n d Con clu sion s
The chiral allosteric amino acid conjugates of J P7
synthesized in this study decreased the oxygen affinity
of hemoglobin and exhibit a wide range of allosteric
activities. The orientation of the chiral center deter-

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1191
by water. It was then dried over anhydrous magnesium
sulfate. The removal of the solvent afforded a pale yellow
residue, ethyl cyclopentanecarboxylate. The analytical pure
sample was obtained by flash chromatography using hexane
stabilize the T-state to varying degrees, and this is
reflected in the observed allosteric activities.
1
Exp er im en ta l Section
as eluent; yield 17.6, 94%. H NMR (CDCl3): δ 1.22 (t, 3H, J
) 7 Hz), 1.54-1.89 (m, 8H), 2.69 (m, 1H), 4.09 (q, 2H, J ) 7
Ch em istr y. All reagents and starting materials used in the
synthesis were purchased from Aldrich Chemical Co., Sigma,
Fluka, and Advanced-Chem-Tech and were used directly
without further purification. Specifically, glycine methyl ester,
D- and L-alanine, leucine, valine, phenylalanine, tryptophan,
methionine, and serine methyl ester hydrochlorides in 99%
purity were purchased from Aldrich. Nꢀ-benzyloxycarbonyl-D-
and L-lysine, L-tyrosine, L-isoleucine, L-proline, S-benzyl-L-
cysteine, L-threonine methyl ester hydrochlorides, L-aspartyl-
γ-tert-butyl, L-glutamate-γ-tert-butyl ester hydrochlorides in
98% purity were purchased from Advanced-Chem-Tech. All
solvents were purchased from Fischer Scientific Co. and
Aldrich Chemical Co. Silica gel coated plates (0.25 mm
thickness) were purchased from Analtech Inc. and used for
thin-layer chromatography (TLC). The separations were spot-
ted by visualizing under UV light (λ ) 254 nm) or iodine
chamber. Flash chromatographic separations were done using
Merck, grade 9385, 230-400 mesh, 60 Å silica gel. The proton
nuclear magnetic magnetic resonance (1H NMR) spectra were
recorded on a Varian Gemini 300 MHz spectrophotometer and
are reported in parts per million (δ ) ppm) using tetrameth-
ylsilane (TMS) as an internal standard. Elemental analyses
were performed by Atlantic Microlab, Inc. (Norcross, GA), and
results are within (0.4% of the theoretical value. All inter-
mediate compounds were analyzed by TLC and 1H NMR but
are not reported. Melting points (mp) were determined on
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Optical rotations were determined using a Digital
Polarimeter (DIP 1000) Ver. 1.31.00 (J asco Corp.).
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r boxylic Acid (4, J P 7). Sch em e 1. To a
tetrahydrofuran (25 mL) solution of 4-[[(3,5-dimethylanilino)-
carbonyl]methyl]phenol (2.0 g, 7.84 mmol) stirred at 0 °C were
added pulverized sodium hydroxide (3.14 g, 78.4 mmol) and
cyclopentanone (6.59 g, 78.4 mmol). To this mixture was added
3.74 g (31.4 mmol) of chloroform dropwise over a 20 min period.
The reaction mixture was stirred and maintained below 15
°C for the first hour and then stirred at room temperature
overnight. Tetrahydrofuran was evaporated under reduced
pressure to give a solid residue that was dissolved in water
and extracted with ethyl acetate, the aqueous layer was
acidified with 37% hydrochloric acid, and the organic materials
re-extracted with ethyl acetate.
Hz).
(2) A mixture of ethyl cyclopentanecarboxylate (15.0 g, 105.6
mmol), N-bromosuccinimide (19.7 g, 110.7 mmol), and carbon
tetrachloride (120 mL) was heated to reflux under the infrared
lamp for 6 h. The yellow reaction mixture turned red after
initiation and then slowly became almost colorless. Separation
of white solid, succinimide, was observed during this time. The
reaction progress was monitored by TLC (hexane:ethyl acetate,
9:1). The product can be detected only by the difference in color
after complexation with iodine. After completion of the reac-
tion, the reaction mixture was filtered while hot. Evaporation
of solvent furnished a yellow liquid residue, which was cooled
to 5 °C and kept refrigerated for an additional 16 h. It was
then filtered, and the organic layer was evaporated under
reduced pressure to get a pale yellow liquid, ethyl-2-bromocy-
clopentanecarboxylate. The analytically pure sample was
obtained by flash chromatography using hexane as eluent;
yield 21.3 g, 91.4%. 1H NMR (CDCl3): δ 1.33 (t, 3H, J ) 7
Hz), 1.8 (m, 2H), 1.98 (m, 2H), 2.28 (t, 4H, J ) 6 Hz), 4.24 (q,
2H, J ) 7 Hz).
(3) A mixture of N-(3,5-dimethylphenyl)-4-hydroxypheny-
lacetamide (5.0 g, 19.6 mmol), ethyl-2-bromocyclopentanecar-
boxylate (15.2 g, 68.8 mmol), 325 mesh potassium carbonate
(8.1 g, 58.7 mmol), potassium iodide (0.01 g, 0.06 mmol), and
ethanol (50 mL) was heated to reflux for 25 h. The reaction
mixture was filtered, and the residue was washed with ethanol
(30 × 50 mL). The combined filtrate was evaporated under
reduced pressure to get a dark liquid, which solidified on
standing. The pure ester, ethyl 1-[4-(((3,5-dimethylanilino)-
carbonyl)methyl)phenoxy]cyclopentanecarboxylate was ob-
tained by flash chromatography using hexane-chloroform
mixture in 5% increments up to 50% chloroform maximum as
eluent; yield 7.1 g, 92%. Mp: 80-81 °C. 1H NMR (CDCl3) δ
1.2 (t, 3H, J ) 7 Hz), 1.81 (m, 4H), 2.2-2.4 (m, 10H), 3.6 (s,
2H), 4.15 (q, 2H, J ) 7 Hz), 6.6 (s, 1H), 6.78 (d, 2H, J ) 8 Hz),
7.03 (s, 2H), 7.18 (d, 2H, J ) 8 Hz).
(4) The ethyl ester (6.0 g, 15.2 mmol) was then dissolved in
ethanol (60 mL). To this solution were added potassium
hydroxide (2.13 g, 38.0 mmol) and water (3 mL). The mixture
was heated to reflux for 50 h. The solvent was evaporated
under reduced pressure, and the solid residue obtained was
dissolved in water (70 mL). The resultant solution was filtered,
and the filtrate was acidified using concentrated hydrochloric
acid, filtered, and washed with water.
The pure product 4, J P7, was obtained upon recrystalliza-
tion from ethyl acetate and hexane; yield 5.1 g, 91%. Mp and
1H NMR data were the same as the compound prepared in
Scheme 1. Anal. (C22H25NO4) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n e Ca r bon yl Glycin e (12). Sch em e 3. To a
stirring solution of 1-[4-(((3,5-dimethylanilino)carbonyl)meth-
yl)phenoxy]cyclopentanecarboxylic acid (2.21 g, 6.0 mmol),
glycine methyl ester hydrochloride (0.75 g, 6.0 mmol), and
1-hydroxybenzotriazole hydrate (0.88 g, 6.5 mmol) in dimeth-
ylformamide (30 mL) were added N-methylmorpholine (0.9 g,
8.9 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (1.36 mg, 7.1 mmol) under nitrogen at room
temperature. After being stirred for another 24 h, the reaction
mixture was diluted with ethyl acetate (100 mL) and washed
with water (40 mL). The ethyl acetate solution was further
washed with 10% potassium hydrogen sulfate solution (2 ×
50 mL), brine (50 mL), saturated sodium bicarbonate solution
(2 × 50 mL), and brine (50 mL). The organic phase was dried
over anhydrous magnesium sulfate, filtered, and evaporated
under reduced pressure. The pure ester product was obtained
by flash chromatography using hexane:ethyl acetate (1:1) as
eluent; yield 2.32 g, 88.5%. Mp: 142-143 °C. 1H NMR
(CDCl3): δ 1.71-1.8 (m, 4H), 2.1-2.34 (m, 10H), 3.63 (s, 2H),
The title compound was extracted into aqueous sodium
bicarbonate, acidified, and re-extracted with ethyl acetate. The
organic layer was dried over anhydrous magnesium sulfate,
filtered, and evaporated under reduced pressure. The analyti-
cally pure product was obtained upon recrystallization from
ethyl acetate and hexane; yield 1.88 g, 65%. Mp: 170-171 °C.
1H NMR (CDCl3): δ 1.75-1.81 (m, 4H), 2.17-2.4 (m, 10H),
3.60 (s, 2H), 6.75 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H),
7.18 (d, 2H, J ) 8 Hz). Anal. (C22H25NO4) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r boxylic Acid (4, J P 7). Sch em e 2. The title
compound was synthesized in four steps:
(1) A mixture of cyclopentanecarboxylic acid (15.0 g, 131.6
mmol), ethanol (70 mL, 200% proof), and concentrated sulfuric
acid (1 mL, 98%) was refluxed for 5 h using a water condenser.
The reaction can be monitored by TLC (hexane:ethyl acetate,
9:1, developed in iodine chamber) because the starting com-
pound as well as the product are not visible under UV light.
After completion of the reaction, ethanol was evaporated from
the reaction mixture under reduced pressure. The pale yellow
liquid obtained was cooled to room temperature and then
slowly poured over crushed ice under stirring. The product
separated as an oil and was isolated using a separatory funnel,
and the aqueous portion was extracted with chloroform (2 ×
50 mL). The combined organic layer was washed with water,
100 mL of saturated solution of sodium bicarbonate, followed

1192 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Youssef et al.
3.68 (s, 3H), 4.04 (d, 2H, J ) 5.8 Hz), 6.7 (s, 1H), 6.84 (d, 2H,
J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J ) 8 Hz).
The final acid was prepared similarly to the previous
reaction subjecting the corresponding l-leucine methyl ester
for hydrolysis (1.14 g, 2.3 mmol); yield 0.99 g, 89.2%. Mp: 89-
90 °C. [R]D -37.7° (c ) 1.03, methanol). Anal. (C28H36N2O5) C,
H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-va lin e (17). This compound was
synthesized similarly to the previous reaction using D-valine
methyl ester hydrochloride (1.0 g, 6.0 mmol). The crude ester
obtained after workup was purified by flash chromatography
using hexane:ethyl acetate (2:1) as eluent. The product was
recrystallized using ether and hexane; yield 2.44 g, 85%. Mp:
104-105 °C. 1H NMR (CDCl3): δ 0.73 (d, 3H, J ) 6.8 Hz),
0.81 (d, 3H, J ) 6.8 Hz), 1.71-1.81 (m, 4H), 2.04-2.18 (m,
2H), 2.21-2.4 (m, 9H), 3.6 (s, 2H), 3.65 (s, 3H), 4.5 (dd, 1H, J
) 5,10), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d,
2H, J ) 8 Hz).
Compound 17 was synthesized similarly to the previous
reaction using the corresponding D-valine methyl ester (1.1 g,
2.3 mmol) for hydrolysis; yield 1.0 g, 93.5%. Mp: 81-82 °C.
[R]D +14.5° (c ) 1, methanol). 1H NMR (CDCl3): δ 0.73 (d,
3H, J ) 6.8), 0.81 (d, 3H, J ) 6.8 Hz), 1.71-1.81 (m, 4H), 2.04-
2.18 (m, 2H), 2.21-2.4 (m, 9H), 3.6 (s, 2H), 4.5 (dd, 1H, J )
5,10 Hz), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18
(d, 2H, J ) 8 Hz). Anal. (C27H34N2O5‚0.25H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-va lin e (18). l-Valine methyl ester
hydrochloride (1.0 g, 6.0 mmol) was used to prepare compound
19. The crude ester obtained after workup was purified by
flash chromatography using hexane:ethyl acetate (2:1) as
eluent; yield 2.64 g, 92%. Mp: 104-106 °C.
The corresponding glycine methyl ester (1.0 g, 2.3 mmol) in
ethanol (30 mL) and lithium hydroxide (0.11 g, 4.6 mmol)
dissolved in water (10 mL) were stirred at room temperature
overnight. The solvent was evaporated on a rotavap at room
temperature. The residual product was dissolved in water (100
mL) and extracted with ethyl acetate (2 × 50 mL). The aqueous
phase was acidified with hydrochloric acid and extracted with
ethyl acetate (4 × 40 mL). The organic phase was washed with
brine (2 × 50 mL), dried over anhydrous magnesium sulfate,
filtered, and evaporated to give a pure product; yield 0.8 g,
82.5%. Mp: 160-161 °C. 1H NMR (CDCl3): δ 1.71-1.8 (m,
4H), 2.1-2.34 (m, 10H), 3.63 (s, 2H), 4.04 (d, 2H, J ) 5.8 Hz),
6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J
) 8 Hz). Anal. (C24H28N2O5) C, H, N.
Compounds 13-33 were prepared using the same procedure
as described above for compound 12.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-a la n in e (13). J P7 (2.21 g, 6.0
mmol) was reacted with D-alanine methyl ester hydrochloride
(0.84 g, 6.0 mmol), 1-hydroxybenzotriazole hydrate (0.88 g, 6.5
mmol), N-methylmorpholine (0.9 g, 8.9 mmol), and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.36
mg, 7.1 mmol). The crude product obtained after workup was
purified by flash chromatography using hexane:ethyl acetate
1
(1:1); yield 2.35 g, 86.7%. Mp: 117-118 °C. H NMR (CDCl3):
δ 1.35 (d, 3H, J ) 7.2 Hz), 1.68-1.76 (m, 4H), 2.04-2.14 (m,
2H), 2.2-2.4 (m, 8H), 3.6 (s, 2H), 3.65 (s, 3H), 4.59 (m, 1H),
6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J
) 8 Hz).
The final compound was prepared using the D-alanine
methyl ester (1.04 g, 2.3 mmol). The product was obtained
upon recrystallization from ether and hexane; yield 0.9 g,
The L-valine methyl ester (1.1 g, 2.3 mmol) was then
hydrolyzed as before to yield 0.92 g, 86% of compound 19.
Mp: 81-82 °C. [R]D -18.2° (c ) 0.5, methanol). Anal.
(C27H34N2O5‚0.5H2O) C, H, N.
1
89.1%. Mp: 168-169 °C. [R]D +23.8° (c ) 0.5, methanol). H
NMR (CDCl3): δ 1.35 (d, 3H, J ) 7.2 Hz), 1.68-1.76 (m, 4H),
2.04-2.14 (m, 2H), 2.2-2.4 (m, 8H), 3.6 (s, 2H), 4.59 (m, 1H),
6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J
) 8 Hz). Anal. (C25H30N2O5) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-a la n in e (14). This compound was
synthesized similarly to the previous reaction using L-alanine
methyl ester hydrochloride (0.84 g, 6 mmol). The crude product
was purified by flash chromatography using hexane:ethyl
acetate (1:1) as eluent; yield 2.0 g, 73.8%. Mp: 117-118 °C.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-p h en yla la n in e (19). This com-
pound was synthesized similarly to the previous reaction using
D-phenylalanine methyl ester hydrochloride (1.29 g, 6.0 mmol).
The crude ester obtained after workup was recrystallized using
ether-hexane mixture; yield 2.88 g, 91.1%. Mp: 106-108 °C.
1H NMR (CDCl3): δ 1.7-1.81 (m, 4H), 1.9-2.35 (m, 10H), 2.97
(dd, 1H, J ) 7.6,14.2 Hz), 3.11 (dd, 1H, J ) 5.3,14.2 Hz), 3.6
(s, 2H), 3.65 (s, 3H), 4.85 (m, 1H), 6.65 (s, 1H), 6.71 (d, 2H, J
) 8 Hz), 6.95-7.18 (m, 9H).
The title compound was obtained on hydrolysis of the
corresponding L-alanine methyl ester (1.04 g, 2.3 mmol). A
pure final product was obtained upon recrystallization from
ether and hexane; yield 0.92 g, 91.1%. Mp: 169-171 °C. [R]D
-25.0° (c ) 0.4, methanol). Anal. (C25H30N2O5) C, H, N.
The final acid was obtained using the corresponding D-
phenylalanine methyl ester (1.21 g, 2.3 mmol) for hydrolysis;
yield 1.1 g, 93.2%. Mp: 87-88 °C. [R]D +8.2° (c ) 1.05,
methanol). 1H NMR (CDCl3) δ 1.7-1.81 (m, 4H), 1.9-2.35 (m,
10H), 2.97 (dd, 1H, J ) 7.6,14.2 Hz), 3.11 (dd, 1H, J ) 5.3,-
14.2 Hz), 3.6 (s, 2H), 4.85 (m, 1H), 6.65 (s, 1H), 6.71 (d, 2H, J
) 8 Hz), 6.95-7.18 (m, 9H). Anal. (C31H34N2O5‚0.25H2O) C,
H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-p h en yla la n in e (20). This com-
pound was prepared using L-phenylalanine methyl ester
hydrochloride (1.29 g, 6.0 mmol). The crude ester obtained
after workup was purified by flash chromatography using
hexane:ethyl acetate (2:1) as eluent; yield 2.52 g, 79.7%. Mp:
107-109 °C.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-leu cin e (15). This compound was
synthesized similarly to the previous reaction using D-leucine
methyl ester hydrochloride (1.1 g, 6.0 mmol). The crude ester
was recrystallized using ether-hexane mixture; yield 2.22 g,
1
75%. Mp: 119-120 °C. H NMR (CDCl3): δ 0.82 (2d, 6H, J )
5.3 Hz), 1.42-1.6(m, 3H), 1.74-1.8 (m, 4H), 2.04-2.12 (m, 2H),
2.21-2.34 (m, 8H), 3.6 (s, 2H), 3.65 (s, 3H), 4.6 (m, 1H), 6.7
(s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J ) 8
Hz).
The end product was produced by hydrolysis of the corre-
sponding D-leucine methyl ester (1.14 g, 2.3 mmol); yield 0.94
g, 84.7%. Mp: 87-88 °C. [R]D +37.3° (c ) 1.05, methanol). H
The hydrolysis of L-phenylalanine methyl ester (1.21 g, 2.3
mmol) afforded 20; yield 1.0 g, 84.7%. Mp: 87-88 °C. [R]D
-9.9° (c ) 0.88, methanol). Anal. (C31H34N2O5‚0.25H2O) C, H, N.
1
NMR (CDCl3): δ 0.82 (2d, 6H, J ) 5.3 Hz),1.42-1.6 (m, 3H),
1.74-1.8 (m, 4H), 2.04-2.12 (m, 2H), 2.21-2.34 (m, 8H), 3.6
(s, 2H), 4.6 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s,
2H), 7.18 (d, 2H, J ) 8 Hz). Anal. (C28H36N2O5) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-leu cin e (16). This compound was
synthesized similarly to the previous reaction using L-leucine
methyl ester hydrochloride (1.1 g, 6.0 mmol). The crude ester
was purified by flash chromatography using hexane:ethyl
acetate (2:1) as eluent; yield 2.31 g, 78%. Mp: 120-122 °C.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-tr yp top h a n (21). This compound
was synthesized as above using D-tryptophan methyl ester
hydrochloride (1.53 g, 6.0 mmol). The crude ester obtained
after workup was purified by flash chromatography using
hexane:ethyl acetate (2:1) as eluent; yield 2.72 g, 80%. Mp:
1
83-84 °C. H NMR (DMSO-d6): δ 1.6-1.8 (m, 4H), 2.16-2.2
(m, 2H), 2.21-2.4 (s, 8H), 3.08 (dd, 1H, J ) 8.7,15 Hz), 3.3
(dd, 1H, J ) 4, 15 Hz), 3.5 (s, 2H), 3.55 (s, 3H), 4.76 (m, 1H),
6.6 (s, 1H), 6.7 (d, 2H, J ) 8 Hz), 6.79 (s, 1H), 6.9 (t, 1H, J )

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1193
7.5 Hz), 7.01 (t, 1H, J ) 7.5 Hz) 7.1 (s, 2H), 7.2 (d, 2H, J ) 8
Hz), 7.25 (d, 1H, J ) 7.5 Hz), 7.46 (d, 1H, J ) 7.5 Hz).
The corresponding ester (1.08 g, 2.3 mmol) was then
hydrolyzed to furnish 26, yield 0.93 g, 88.6%. Mp: 73-74 °C.
[R]D -20.3° (c ) 0.4, methanol). Anal. (C25H30N2O6‚0.25H2O)
C, H, N.
The hydrolysis of D-tryptophan methyl ester (1.3 g, 2.3
mmol) produced 22; yield 1.2 g, 94.5%. Mp: 107-108 °C. [R]D
+62.8° (c ) 0.5, methanol). 1H NMR (DMSO-d6): δ 1.6-1.8
(m, 4H), 2.16-2.2 (m, 2H), 2.21-2.4 (s, 8H), 3.08 (dd, 1H, J )
8.7,15 Hz), 3.3 (dd, 1H, J ) 4, 15 Hz), 3.5 (s, 2H), 4.76 (m,
1H), 6.6 (s, 1H), 6.7 (d, 2H, J ) 8 Hz), 6.79 (s, 1H), 6.9 (t, 1H,
J ) 7.5 Hz), 7.01 (t, 1H, J ) 7.5 Hz) 7.1 (s, 2H), 7.2 (d, 2H, J
) 8 Hz), 7.25 (d, 1H, J ) 7.5 Hz), 7.46 (d, 1H, J ) 7.5 Hz).
Anal. (C33H35N3O5) C, H, N.
Nr-1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en -
oxy]cyclopen tan e-car bon yl-NE-ben zyloxycar bon yl-D-lysin e
(27). This lysine derivative was synthesized using Nꢀ-benzy-
loxycarbonyl-D-lysine methyl ester hydrochloride (1.99 g, 6.0
mmol). The crude ester was purified by flash chromatography
using hexane:ethyl acetate (1:1) as eluent; yield 3.6 g, 93.5%.
1
Mp: 53-55 °C. H NMR (CDCl3): δ 1.1-1.35 (m, 4H), 1.76-
1.85 (m, 4H), 2.02-2.4 (m, 10H), 2.95-3.05 (m, 4H), 3.6 (s,
2H), 3.65 (s, 3H), 4.55 (m, 1H), 5.15 (s, 2H), 6.7 (s, 1H), 6.84
(d, 2H, J ) 8 Hz), 7.1-7.35 (m, 9H).
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-tr yp top h a n (22). This compound
was obtained from L-tryptophan methyl ester hydrochloride
(1.53 g, 6.0 mmol). The crude ester obtained after workup was
purified by flash chromatography using hexane:ethyl acetate
(2:1) as eluent; yield 2.95 g, 86.8%. Mp: 86-87 °C.
The ester (1.3 g, 2.3 mmol) was then hydrolyzed as before
to furnish 23; yield 1.2 g, 94.5%. Mp: 107-108 °C. [R]D -63.1°
(c ) 0.5, methanol). Anal. (C33H35N3O5‚0.75H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-m eth ion in e (23). This compound
was synthesized by using D-methionine methyl ester hydro-
chloride (1.2 g, 6.0 mmol). The crude ester obtained after
workup was purified by flash chromatography using hexane:
ethyl acetate (2:1) as eluent; yield 2.8 g, 96%. Mp: 54-55 °C.
1H NMR (CDCl3): δ 1.76-1.81 (m, 4H), 1.91-2.0 (m, 4H), 2.1-
2.4 (m, 13H), 3.6 (s, 2H), 3.7 (s, 3H), 4.66 (m, 1H), 6.7 (s, 1H),
6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J ) 8 Hz).
The final acid was obtained from the corresponding D-
methionine methyl ester (1.64 g, 2.3 mmol) on hydrolysis; yield
1.28 g, 80%. Mp: 62-63 °C. [R]D +24.9° (c ) 0.3, methanol).
1H NMR (CDCl3): δ 1.76-1.81 (m, 4H), 1.91-2.0 (m, 4H), 2.1-
2.4 (m, 13H), 3.6 (s, 2H), 4.66 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H,
The title compound was obtained after base hydrolysis of
the corresponding Nꢀ-benzyloxycarbonyl-D-lysine methyl ester
(1.48 g, 2.3 mmol); yield 1.34 g, 92.4%. Mp: 61-62 °C. [R]D
+18.5° (c ) 3, methanol). 1H NMR (CDCl3): δ 1.1-1.35 (m,
4H), 1.76-1.85 (m, 4H), 2.02-2.4 (m, 10H), 2.95-3.05 (m, 4H),
3.6 (s, 2H), 4.55 (m, 1H), 5.15 (s, 2H), 6.7 (s, 1H), 6.84 (d, 2H,
J ) 8 Hz), 7.1-7.35 (m, 9H). Anal. (C36H43N3O7) C, H, N.
N r-1-[4-(((3,5-D im e t h y la n ilin o )c a r b o n y l)m e t h y l)-
p h en oxy]cyclop en ta n e-ca r bon yl-NE-ben zyloxyca r bon yl-
L-lysin e (28). The corresponding L-isomer was obtained by
reacting Nꢀ-benzyloxycarbonyl-L-lysine methyl ester hydro-
chloride (0.93 g, 6.0 mmol). The crude ester obtained after
workup was purified by flash chromatography using hexane:
ethyl acetate (1:1) as eluent; yield 3.28 g, 85.2%. Mp: 53-55
°C.
Hydrolysis of Nꢀ-benzyloxycarbonyl-L-lysine methyl ester
(1.48 g, 2.3 mmol) produced 28; yield 1.3 g, 87.9%. Mp: 63-
65 °C. [R]D -19.9° (c ) 2.4, methanol). Anal. (C36H43N3O7‚
0.5H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-isoleu cin e (29). This compound
was prepared by using L-isoleucine methyl ester hydrochloride
(1.1 g, 6.0 mmol). The crude ester obtained after workup was
purified by flash chromatography using hexane:ethyl acetate
J
) 8 Hz), 7.1 (s, 2H), 7.18 (d, 2H, J ) 8 Hz). Anal.
(C27H34N2O5S) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-m eth ion in e (24). This compound
was prepared using L-methionine methyl ester hydrochloride
(1.2 g, 6.0 mmol). As usual, the crude ester obtained after
workup was purified by flash chromatography using hexane:
ethyl acetate (2:1) as eluent; yield 2.74 g, 93.8%. Mp: 55-57
°C.
1
(2:1) as eluent; yield 2.31 g, 78%. Mp: 127-128 °C. H NMR
(CDCl3): δ 0.82 (t, 3H, J ) 7.3 Hz), 0.9 (d, 3H, J ) 6.8 Hz),
0.98-1.4 (m, 2H), 1.69-1.81 (m, 4H), 2.02-2.18 (m, 2H), 2.2-
2.45 (m, 9H), 3.6 (s, 2H), 3.65 (s, 3H), 4.48 (dd, 1H, J ) 6, 8.4
Hz), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.2 (d, 2H,
J ) 8 Hz).
The ester L-isoleucine methyl ester (1.14 g, 2.3 mmol) was
further hydrolyzed to give 29; yield 0.97 g, 87.4%. Mp: 72-
73 °C. [R]D -11.6° (c ) 0.5, methanol). 1H NMR (CDCl3): δ
0.82 (t, 3H, J ) 7.3 Hz), 0.9 (d, 3H, J ) 6.8 Hz), 0.98-1.4 (m,
2H), 1.69-1.81 (m, 4H), 2.02-2.18 (m, 2H), 2.2-2.45 (m, 9H),
3.6 (s, 2H), 4.48 (dd, 1H, J ) 6, 8.4 Hz), 6.7 (s, 1H), 6.84 (d,
2H, J ) 8 Hz), 7.1 (s, 2H), 7.2 (d, 2H, J ) 8 Hz). Anal.
(C28H36N2O5‚0.5H2O) C, H, N.
The final compound was obtained by hydrolysis of the
corresponding L-methionine methyl ester (1.64 g, 2.3 mmol);
yield 1.33 g, 83.1%. Mp: 64-65 °C. [R]D -25.5° (c ) 0.25,
methanol). Anal. (C27H34N2O5S) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-ser in e (25). This compound was
synthesized using D-serine methyl ester hydrochloride (0.93
g, 6.0 mmol). The crude ester obtained after workup was
purified by flash chromatography using ethyl acetate as eluent;
yield 2.52 g, 94.7%. Mp: 58-59 °C. 1H NMR (DMSO-d6): δ
1.64-1.81 (m, 4H), 2.16-2.2 (m, 2H), 2.21-2.4 (m, 8H), 3.5
(s, 2H), 3.55 (s, 3H), 3.6 (dd, 1H, J ) 3, 11 Hz), 3.72 (dd, 1H,
J ) 4, 11 Hz), 4.24 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz),
7.1 (s, 2H), 7.2 (d, 2H, J ) 8 Hz).
The ester D-serine methyl ester (1.08 g, 2.3 mmol) was then
hydrolyzed to afford 25; yield 0.94 g, 89.5%. Mp: 73-75 °C.
[R]D +19.2° (c ) 0.5, methanol). 1H NMR (DMSO-d6): δ 1.64-
1.81 (m, 4H), 2.16-2.2 (m, 2H), 2.21-2.4 (m, 8H), 3.5 (s, 2H),
3.6 (dd, 1H, J ) 3, 11 Hz), 3.72 (dd, 1H, J ) 4, 11 Hz), 4.24
(m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.2 (d,
2H, J ) 8 Hz). Anal. (C25H30N2O6‚0.5H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-ser in e (26). This compound was
synthesized using corresponding L-isomer of D-serine methyl
ester hydrochloride (0.93 g, 6.0 mmol). The crude ester
obtained after workup was purified by flash chromatography
using ethyl acetate as eluent; yield 2.51 g, 94.4%. Mp: 60-62
°C.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-tyr osin e (30). This derivative was
synthesized using L-tyrosine methyl ester hydrochloride (1.39
g, 6.0 mmol). The crude ester obtained after workup was
purified by flash chromatography using hexane:ethyl acetate
1
(2:1) as eluent; yield 2.84 g, 87.1%. Mp: 74-75 °C. H NMR
(CDCl3): δ 1.7-1.81 (m, 4H), 2.03-2.17 (m, 2H), 2.2-2.34 (m,
8H), 2.75 (dd, 1H, J ) 9.5, 13.5 Hz), 3.15 (dd, 1H, J ) 4.3,
13.5 Hz), 3.6 (s, 2H), 3.65 (s, 3H), 4.6 (m, 1H), 6.4 (s, 1H), 6.45
(d, 2H, J ) 8 Hz), 6.65 (d, 2H, J ) 8.5 Hz), 6.7 (d, 2H, J ) 8.5
Hz), 6.8(s, 2H), 7.0 (d, 2H, J ) 8 Hz).
L-Tyrosine methyl ester (1.25 g, 2.3 mmol) was then
hydrolyzed to give 31; yield 1.1 g, 90.2%. Mp: 97-98 °C. [R]D
-26.2° (c ) 0.52, methanol). 1H NMR (CDCl3): δ 1.7-1.81 (m,
4H), 2.03-2.17 (m, 2H), 2.2-2.34 (m, 8H), 2.75 (dd, 1H, J )
9.5, 13.5 Hz), 3.15 (dd, 1H, J ) 4.3, 13.5 Hz), 3.6 (s, 2H), 4.6
(m, 1H), 6.4 (s, 1H), 6.45 (d, 2H, J ) 8 Hz), 6.65 (d, 2H, J )
8.5 Hz), 6.7 (d, 2H, J ) 8.5 Hz), 6.8 (s, 2H), 7.0 (d, 2H, J ) 8
Hz). Anal. (C31H34N2O6‚0.25H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-p r olin e (31). The proline deriva-
tive was synthesized using the corresponding L-proline methyl

1194 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Youssef et al.
ester hydrochloride (0.99 g, 6.0 mmol). The crude ester
obtained after workup was purified by flash chromatography
using hexane:ethyl acetate (2:1) as eluent; yield 2.3 g, 80.1%.
Mp: 71-72 °C. 1H NMR (CDCl3): δ 1.69-1.81 (m, 4H), 1.89-
2.43 (m, 14H), 3.6 (s, 2H), 3.64-3.7 (m, 5H), 4.5 (dd, 1H, J )
4.3, 7.3 Hz), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.2
(d, 2H, J ) 8 Hz).
The pure product was obtained by flash chromatography
using ethyl acetate as eluent; yield 1.0 g, 87.7%. Mp: 63-65
°C. [R]D -11.2° (c ) 0.65, methanol). 1H NMR (CDCl3) δ 1.75-
1.81 (m, 4H), 2.02-2.19 (m, 2H), 2.21-2.34 (m, 8H), 2.82 (dd,
2H, J ) 4.5, 7.8 Hz), 3.61 (s, 2H), 3.68 (s, 3H), 4.6 (m, 1H), 6.7
(s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s, 2H), 7.03 (d, 2H, J ) 8
Hz). Anal. (C27H32N2O7) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en t a n eca r b on yl-L-glu t a m a t e (35). This compound
was synthesized by following a previous reaction but using
l-glutamate-γ-tert-butyl ester hydrochloride (1.52 g, 6.0 mmol).
The crude ester obtained after workup was purified by flash
chromatography using hexane:ethyl acetate (2:1) as eluent;
yield 2.38 g, 70%. Mp: 50-51 °C. 1H NMR (CDCl3): δ 1.41 (s,
9H), 1.7-1.9 (m, 4H), 2.02-2.4 (m, 14H), 3.6 (s, 2H), 3.7 (s,
3H), 4.55 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s,
2H), 7.03 (d, 2H, J ) 8 Hz).
The ester L-glutamate-γ-tert-butyl ester (1.27 g, 2.3 mmol)
was hydrolyzed as before to afford 35. The crude product was
purified by flash chromatography using ethyl acetate as eluent;
yield 0.94 g, 80.3%. Mp: 60-61 °C. [R]D -14.2° (c ) 0.46,
methanol). 1H NMR (CDCl3): δ 1.7-1.9 (m, 4H), 2.02-2.4 (m,
14H), 3.6 (s, 2H), 3.7 (s, 3H), 4.55 (m, 1H), 6.7 (s, 1H), 6.84 (d,
2H, J ) 8 Hz), 6.97(s, 2H), 7.03 (d, 2H, J ) 8 Hz). Anal.
(C28H34N2O7) C, H, N.
The final acid was obtained by hydrolysis of the correspond-
ing L-proline methyl ester (1.1 g, 2.3 mmol); yield 0.94 g, 93.1%.
Mp: 90-91 °C. [R]D -57.9° (c ) 1, methanol). 1H NMR
(CDCl3): δ 1.69-1.81 (m, 4H), 1.89-2.43 (m, 14H), 3.6 (s, 2H),
3.65 (m, 2H), 4.5 (dd, 1H, J ) 4.3, 7.3 Hz), 6.7 (s, 1H), 6.84 (d,
2H, J ) 8 Hz), 7.1 (s, 2H), 7.2 (d, 2H, J ) 8 Hz). Anal.
(C27H33N2O5‚0.25H2O) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-S-ben zyl-L-cystein e (32). This com-
pound was synthesized using S-benzyl-L-cysteine methyl ester
hydrochloride (1.57 g, 6.0 mmol). The crude ester was sepa-
rated as oil and purified by flash chromatography using
hexane:ethyl acetate (1:2) as eluent; yield 3.12 g, 90.7%. 1H
NMR (CDCl3): δ 1.78-1.81 (m, 4H), 2.08-2.18 (m, 2H), 2.21-
2.4 (m, 8H), 2.74 (dd, 1H, J ) 7, 14 Hz), 2.84 (dd, 1H, J ) 5,
14 Hz), 3.6 (s, 2H), 3.65 (s, 3H), 3.85(s, 2H), 4.72 (m, 1H), 6.7
(s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s, 2H), 7.03 (d, 2H, J ) 8
Hz), 7.14-7.27 (m, 5H).
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-D-lysin e (36). Sch em e 5. To the
corresponding Nꢀ-benzyloxycarbonyl-D-lysine (18) (0.5 g, 0.79
mmol) in ethanol (10 mL) was added 10% palladium on carbon.
The mixture was hydrogenated in the Parr-Shaker until
absorption of the hydrogen gas was stopped. The catalyst was
filtered and washed with ethanol (2 × 25 mL), and the
combined filtrate was evaporated under reduced pressure. The
pure product was obtained upon recrystallization from chlo-
roform; yield 0.33 g, 84.6%. Mp: 124-125 °C. [R]D +30.0° (c
) 0.5, methanol). 1H NMR (CDCl3): δ 1.1-1.35 (m, 4H), 1.76-
1.85 (m, 4H), 2.02-2.4 (m, 10H), 2.95-3.05 (m, 4H), 3.6 (s,
2H), 4.55 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s,
2H), 7.03 (d, 2H, J ) 8 Hz). Anal. (C28H37N3O5.0.5C2H5OH) C,
H, N.
The ester S-benzyl-L-cysteine methyl (1.32 g, 2.3 mmol) was
further hydrolyzed to produce 32; yield 1.23 g, 95.3%. Mp:
130-132 °C. [R]D -40.1° (c ) 0.79, methanol). 1H NMR
(CDCl3): δ 1.78-1.81 (m, 4H), 2.08-2.18 (m, 2H), 2.21-2.4
(m, 8H), 2.74 (dd, 1H, J ) 7, 14 Hz), 2.84 (dd, 1H, J ) 5, 14
Hz), 3.6 (s, 2H), 3.85(s, 2H), 4.72 (m, 1H), 6.7 (s, 1H), 6.84 (d,
2H, J ) 8 Hz), 6.97(s, 2H), 7.03 (d, 2H, J ) 8 Hz), 7.14-7.27
(m, 5H). Anal. (C32H36N2O5S) C, H, N.
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-th r eon in e (33). This compound
was synthesized similar to the previous reaction using L-
threonine methyl ester hydrochloride (1.02 g, 6.0 mmol). The
crude ester obtained after workup was purified by flash
chromatography using hexane:ethyl acetate (2:1) as eluent;
yield 2.7 g, 93.4%. Mp: 64-66 °C. 1H NMR (CDCl3): δ 1.02(d,
3H, J ) 6.5 Hz), 1.76-1.81 (m, 4H), 2.07-2.18 (m, 2H), 2.21-
2.4 (m, 8H), 3.6 (s, 2H), 3.65 (s, 3H), 4.32 (m, 1H), 4.47 (dd,
1H, J ) 2.4, 8.5 Hz), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s,
2H), 7.2 (d, 2H, J ) 8 Hz).
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-lysin e (37). This compound was
synthesized as described above using the corresponding Nꢀ-
benzyloxycarbonyl-L-lysine (19) (0.5 g, 0.79 mmol). The final
product was obtained upon recrystallization from chloroform;
yield 0.35 g, 89.7%. Mp: 124-125 °C. [R]D -29.6° (c ) 0.5,
The L-threonine methyl ester (1.11 g, 2.3 mmol) was
hydrolyzed to give 33; yield 0.84 g, 83.2%. Mp: 82-83 °C. [R]D
1
methanol). H NMR (CDCl3): δ 1.1-1.35 (m, 4H), 1.76-1.85
1
-12.3° (c ) 0.5, methanol). H NMR (CDCl3) δ 1.02(d, 3H, J
(m, 4H), 2.02-2.4 (m, 10H), 2.95-3.05 (m, 4H), 3.6 (s, 2H),
4.55 (m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s, 2H),
7.03 (d, 2H, J ) 8 Hz). Anal. (C28H37N3O5.0.5 C2H5OH) C, H,
N.
) 6.5 Hz), 1.76-1.81 (m, 4H), 2.07-2.18 (m, 2H), 2.21-2.4
(m, 8H), 3.6 (s, 2H), 4.32 (m, 1H), 4.47 (dd, 1H, J ) 2.4, 8.5
Hz), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 7.1 (s, 2H), 7.2 (d, 2H,
J ) 8 Hz). Anal. (C26H32N2O6) C, H, N.
Oxygen Equ ilibr iu m Stu d ies. 1. Wh ole Blood An a lysis.
A multi-point tonometry was used to measure the OEC of
whole blood. This technique measures the Hb saturation using
a co-oximeter on individual blood samples after equilibrium
of each sample with a gas of known oxygen concentrations.
The whole blood samples were collected in heparinzed tubes
from healthy volunteers and stored over ice. A 200 mM stock
solution of the compound was prepared in DMSO. J ust before
tonometry, 50 µL of the compound stock solution was mixed
with 1950 µL of whole blood to achieve a final concentration
of 5 mM. OEC study controls included the addition of 50 µL of
DMSO (the same amount of solvent used to dissolve the
effectors). Each sample was run in duplicate. The samples were
incubated in tonometers (IL 237 Instrumentation Laboratories,
Inc., Lexington, MA) for approximately 10 min at 37 °C against
gas mixtures containing 2.95%, 5.85%, and 8.75% concentra-
tions of O2, a fixed concentration of 5.8% CO2, and a balance
of N2. The samples were allowed to equilibrate at three
separate concentrations of O2 mixtures 20%, 40%, and 60%,
respectively. After equilibration at each concentration of O2,
a sample was removed via syringe and aspirated into the blood
gas analyzer (IL 1420 Instrumentation Laboratories, Inc.,
Lexington, MA) and co-oximeter (IL 482 and IL 682 Instru-
1-[4-(((3,5-Dim eth yla n ilin o)ca r bon yl)m eth yl)p h en oxy]-
cyclop en ta n eca r bon yl-L-a sp a r ta te (34). Sch em e 4. Fol-
lowing the procedure as described above, J P7 (2.21 g, 6.0
mmol) was reacted with L-aspartate-γ-tert-butyl ester hydro-
chloride (1.44 g, 6.0 mmol), 1-hydroxybenzotriazole hydrate
(0.88 g, 6.5 mmol), N-methylmorpholine (0.9 g, 8.9 mmol), and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(1.36 mg, 7.1 mmol). The crude product was purified by flash
chromatography using hexane:ethyl acetate (2:1) as eluent;
1
yield 2.31 g, 70%. Mp: 47-48 °C. H NMR (CDCl3): δ 1.4 (s,
9H), 1.75-1.81 (m, 4H), 2.02-2.19 (m, 2H), 2.21-2.34 (m, 8H),
2.82 (dd, 2H, J ) 4.5, 7.8 Hz), 3.61 (s, 2H), 3.68 (s, 3H), 4.6
(m, 1H), 6.7 (s, 1H), 6.84 (d, 2H, J ) 8 Hz), 6.97(s, 2H), 7.03
(d, 2H, J ) 8 Hz).
Trifluoroacetic acid (2 mL) was then added to the corre-
sponding tertiarybutoxycarbonyl ester (1.27 g, 2.3 mmol) in
dry dichloromethane (30 mL) at 0 °C. The mixture was stirred
at room temperature overnight. After completion of the reac-
tion, the mixture was diluted with dichloromethane (40 mL)
and washed with water (3 × 30 mL) and brine (30 mL). The
organic phase was dried over anhydrous magnesium sulfate,
filtered, and evaporated under reduced pressure.

Synthesis of Chiral Allosteric Modifiers of Hemoglobin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1195
(3) Abraham, D. J .; Perutz, M. F.; Phillips, S. E. V. Physiological
and X-ray Studies of Potential Antisickling Agents. Proc. Natl.
Acad. Sci. U.S.A. 1983, 80, 324-328.
(4) Abraham, D. J .; Kennedy, P. E.; Mehanna, D. C.; Patwa, D. C.;
Williams, F. L. Design, Synthesis, and Testing of Potential
Antisickling Agents. 4. Structure-Activity Relationships of
Benzyloxy and Phenoxy Acids. J . Med. Chem. 1984, 27, 967-
978.
mentation Laboratories, Inc., Lexington, MA) to determine the
pH, pCO2, and pO2 and to obtain the oxygen saturation values
(sO2), respectively. The measured values for pO2 and sO2 at
each oxygen saturation point was then subjected to the
nonlinear regression analyses to calculate P50 and n50 using
the program Scientist (Micromath, Salt Lake City, UT). The
control was prepared by adding 1950 µL of whole blood to 50
µL of DMSO.
(5) Perutz, M. F.; Poyart, C. Bezafibrate Lowers Oxygen Affinity of
Haemoglobin. Lancet 1983, 15, 881-882.
2. Hb Solu tion Stu d ies. All of the effectors (except
compound 19 and 20) were prepared as 10 mM stock solutions
in 100 mM NaCl bis-Tris buffer, pH 7.2. After the addition of
an excess of NaHCO3, the solution was warmed to 60 °C and
stirred for several hours and back-titrated carefully to pH 7.7
at 25 °C prior to use. Compounds 19 and 20 were prepared in
30% DMSO because of their poor solubility in NaCl bis-Tris
buffer. Oxygen equilibrium measurements were performed
with the HEMOX analyzer (TCS Medical Products, Southamp-
ton, PA) using purified stripped human adult hemoglobin as
described previously.19,20 A total of 4 mL of buffer (100 mM
NaCl, 50 mM bis-Tris at pH 7.2 at 25 °C) is added to a cuvette
in the HEMOX, followed by 200 µL of the 10 mM effector stock
solution to achieve 0.5 mM of the effectors. Hb is then added
to have a final Hb concentration of 50-60 µM in heme basis.
Catalase (20 µg/mL) and 50 mM EDTA were added to limit
oxidation of the hemes. The solution was then fully oxygen
saturated. The oxygen pressure was gradually decreased to
record the curve continuously from the right to the left. The
saturation of Hb was determined spectrophotometrically with
a dual wavelength spectrophotometer (577 and 586.2 nm). The
(6) Lalezari, I.; Rahbar, S.; Lalezari, P.; Fermi, G.; Perutz, M. F.
LR16, A Compound with Potent Effects on the Oxygen Affinity
of Hemoglobin, on Blood Cholesterol, and on Low-Density
Lipoprotein. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6117-6121.
(7) Lalezari, I.; Lalezari, P. Synthesis and Investigation of Effects
of 2-[4-[[(Arylamino)carbonyl]amino]phenoxy]-2-methylpropionic
Acids on the Affinity of Hemoglobin for Oxygen: Structure-
Activity Relationships. J . Med. Chem. 1989, 32, 2352-2357.
(8) Lalezari, I.; Lalezari, P.; Poyart, C.; Marden, M.; Kister, J .; Bohn,
B.; Fermi, G.; Perutz, M. F. New Effectors of Human Hemoglo-
bin: Structure and Function. Biochemistry 1990, 29, 1515-1523.
(9) Abraham, D. J .; Wireko, F. C.; Randad, R. S.; Poyart, C.; Kister,
J .; Bohn, B.; Liard, J .; Kunert, M. P. Allosteric Modifiers of
Hemoglobin: 2-[4-[[(3,5-disubstitutedanilino)carbonyl] amino]-
phenoxy]-2-methylpropionic Acid Derivatives that Lower the
Oxygen Affinity of Hemoglobin in Red Cell Suspension, in Whole
Blood and in Vivo in Rats. Biochemistry 1992, 31, 9141-9149.
(10) Randad, R. S.; Mahran, M. A.; Mehanna, A. S.; Abraham, D. J .
Allosteric Modifiers of Hemoglobin. 1. Design, Synthesis, Testing
and Structure-Allosteric Activity Relationship of Novel Hemo-
globin Oxygen Affinity Decreasing Agents. J . Med. Chem. 1991,
34, 752-757.
(11) Abraham, D. J .; Kister, J .; J oshi, G. S.; Wireko, F. C.; Safo, M.
K.; Marden, M. C.; Poyart, C. Structure/Function of Allosteric
Effectors of Hemoglobin. Med. Chem. Res. 1998, 8, 478-486.
(12) Kunert, M. P.; Liard, J . F.; Abraham, D. J . RSR-13, An Allosteric
Effector of Hemoglobin, Increases Systemic and Iliac Vascular
Resistance in Rats. Am. J . Physiol. 1996, 271, H602-H613.
(13) Khandelwal, S. R.; Randad, R. S.; Lin, P.-S.; Meng, H.; Pittman,
R. N.; Kontos, H. A.; Choi, S. C.; Abraham, D. J .; Schmidt-
Ullrich, R. Enchanced Oxygenation in Vivo by Allosteric Inhibi-
tors of Hemoglobin Saturation. Am. J . Physiol. 1993, 265,
H1450-H1453.
(14) Wei, E. P.; Randad, R. S.; Levasseur, J . E.; Abraham, D. J .;
Kontos, H. A. Effect of Local Change in Oxygen Saturation of
Hemoglobin on Cerebral Vasodilation from Hypoxia and Hy-
potension. Am. J . Physiol. 1993, 265, H1439-H1443.
(15) Teicher, B. A.; Ara, G.; Emi, Y.; Kakeji, Y.; Ikebe, M.; Maehara,
Y.; Buxton, D. RSR13: Effects on Tumor Oxygenation and
Response to Therapy. Drug Dev. Res. 1996, 38, 1-11.
(16) Abraham, D. J .; J oshi, G. S.; Randad, R. S.; Panikker, J .
Allosteric Modifiers of Hemoglobin Useful for Decreasing Oxygen
Affinity and Preserving Oxygen Carrying Capability of Stored
Blood. U.S. Patent 5,731,454 (March 24, 1990).
(17) Weizman, C.; Sulzbacher, M.; Bergman, E. The Synthesis of
R-Alkoxyisobutyric Acids and Alkyl Methacrylates from Ac-
etonechloroform. J . Am. Chem. Soc. 1948, 70, 1153-1158.
(18) Reeve, W. Reactions of Aryl Trichloromethyl Carbinols with
Nucleophiles. Synthesis 1971, 131-138.
(19) Poyart, C.; Marden, M. C.; Kister, J . Bezafibrate Derivatives as
Potent Allosteric Effectors of Hemoglobin. Methods Enzymol.
1994, 232, 496-513.
(20) Kister, J .; Poyart, C.; Edelstein, S. J . An Expanded Two-state
Allosteric Model for Interactions of Human Hemoglobin A with
Nonsaturating Concentrations of 2,3-Diphosphoglycerate. J .
Biol. Chem. 1987, 262, 12085-12091.
(21) Perutz, M. F.; Fermi, G.; Abraham, D. J .; Poyart, C.; Bursaux,
E. Hemoglobin as a Receptor of Drugs and Peptides: X-ray
Studies of the Stereochemistry of Binding. J . Am. Chem. Soc.
1986, 108, 1064-1078.
(22) Burley, S. K.; Petsko, G. A. Amino-Aromatic Interactions in
Proteins. FEBS 1986, 203, 139-143.
(23) Safo, M. K.; Moure, C. M.; Burnett, J . C.; J oshi, G. S.; Abraham,
D. J . High-Resolution Crystal Structure of Deoxy Hemoglobin
Complexed with a Potent Allosteric Effector. Protein Science
2001, 10, 951-957.
(24) Fermi, G.; Perutz, M. F.; Shannon, B.; Fourme, R. The Crystal
Structure of Human Deoxyhemoglobin at 1.7 Å Resolution. J .
Mol. Biol. 1984, 175, 159-174.
P
50 and n50 values were calculated by linear regression analysis
from data points comprised between 40% and 60% oxygen
saturation.
X-r a y Da ta An a lysis. Crystals of deoxyHb complexed with
the effectors 17, 18, 19, and 20 were obtained under similar
conditions as previously described for the RSR13-Hb com-
plex.23 As expected, all Hb complexes yielded crystals that are
isomorphous to that of the native Hb crystal.24 The crystals
belong to the space group P21 and have one tetramer in their
asymmetric unit. X-ray diffraction data were collected using
R-axis II image plate detector equipped with a Rigaku RU-
200 generator operating at 50 kV and 180 mA. All data sets
were processed using the Molecular Structure Corporation
(MSC) Biotex software and the CCP4 program suite.25
Binding of the effectors to deoxyHb were ascertained from
difference Fourier electron density maps, computed from [from
F
obs(complex) - Fobs(native)] amplitude at 2.0 Å resolutions,
where Fobs(complex) is the observed structure factors of the
effector-Hb complexes and Fobs(native) is the observed struc-
ture factors from the previously determined structure of native
T-state Hb.24 For all complexes, a model of the effector
molecule was built with the program Sybyl26 and fitted to the
observed effector electron density.
Ack n ow led gm en t. This work was supported in part
by NIH Grant RO1 HL 32793 (D.J .A.) and a grant from
Allos Therapeutics, Inc. We also gratefully acknowledge
the Cultural and Educational Bureau-Embassy of Arab
Republic of Egypt for a fellowship to A.M.Y.
Su p p or tin g In for m a tion Ava ila ble: Atomic coordinates
for four unrefined hemoglobin-allosteric effector complexes,
deposited as D-valine-17.pdb, L-valine-18.pdb, D-phenylala-
nine-19.pdb, and L-phenylalanine-20.pdb. This material is
available free of charge via the Internet at http://pubs.acs.org.
[The coordinates are also available from the corresponding
author (dabraham@hsc.vcu.edu).]
Refer en ces
(25) The CCP4 Suite: Programs for Protein Crystallography. Acta
Crystallogr. 1994, D50, 760-763.
(26) Sybyl Molecular Modeling System (version 6.6), Tripos, Inc., 1699
Hanley Road, St. Louis, MO 63144.
(1) Hirst, D. G.; Wood, P. J .; Schwartz, H. C. The Modification of
Hemoglobin Affinity for Oxygen and Tumor Radiosensitivity by
Antilipedimic Drugs. Radiat. Res. 1987, 112, 164-172.
(2) Siemann, D. W.; Macler, L. M. Tumor Radiosensitization
Through Reductions in Hemoglobin Affinity. Int. J . Radiat.
Oncol. Biol. Phys. 1986, 12, 1295-1297.
J M010358L