Synthesis of ethylene-bridged (N(δ) to N(ω)) analogues of arginine

Full text of DOI:10.1021/jo990551b

J . Org. Chem. 1999, 64, 9265-9267
9265
Syn th esis of Eth ylen e-Br id ged (Nδ to Nω)
An a logu es of Ar gin in e
J oseph T. Lundquist, IV, Kevin S. Orwig, and
Thomas A. Dix*
Department of Pharmaceutical Sciences, Medical University
of South Carolina, 280 Calhoun Street, P.O. Box 250140,
Charleston, South Carolina 29425
F igu r e 1. Arginine (1), Nω-methylarginine (2), and ethylene-
bridged analogues 3a -c.
Received March 29, 1999
guanylation of ornithine with the requisite methyl-
isothiourea salt.2a Analogues 3a -c are not accessible by
this route since the δ-nitrogen of ornithine must undergo
both guanylation and alkylation to form the ethylene
bridge. We first envisioned that alkylation of the δ-ni-
trogen of ornithine with an N-alkylated 1-bromo-2-
aminoethane derivative or, less directly, with dibromo-
ethane followed by substitution with ammonia or the
requisite alkylamine would provide the ethylenediamino
side chain. Diamine cyclization with cyanogen bromide
(CNBr) as described by Ishikawa et al.7 would then lead
to facile production of the cylic guanidinium group.
However, reaction of the δ-amino functionality of L-N-R-
Boc-ornithine with Cbz-1-amino-2-bromoethane or dibro-
moethane under a variety of conditions failed to produce
significant product yields.
In tr od u ction
Based on insights from earlier work,1 we have designed
and synthesized a series of nonnatural analogues of the
cationic, ion-pairing amino acids L-arginine (1, Figure 1)
and L-lysine, which feature incorporation of alkyl groups
into the endogenous cationic side chains to increase
overall lipophilicity and modulate electrostatic character.2
The analogues may improve several important param-
eters relevant to the in vivo activity of bioactive peptides
such as binding potency and selectivity, lipophilicity (and
thus bloodstream and blood-brain barrier access), and
stability profiles.3 Additional interest in structural vari-
ants of L-arginine arises from the ability of related
compounds, such as Nω-methyl-L-arginine (2, Figure 1),4
to act as nitric oxide (NO) synthase (NOS) isoform
inhibitors. These enzymes produce NO during conversion
of L-arginine to L-citrulline, and L-arginine analogues
typically serve as competitive inhibitors. While 2 and
related compounds are potent in vitro inhibitors of
different NOS isoforms, their use in vivo is confounded
by deguanylation by the enzyme arginase to L-ornithine,
which can be enzymatically converted back to L-arginine,
the substrate for NOS.5 We envisioned that Nδ-alkylated
analogues of L-arginine (e.g., 3a -c) would hinder deguan-
ylation by arginase and result in enhanced in vivo
inhibitory activity toward NOS. Further, identification
of stable compounds that selectively inhibit different
isoforms of NOS is of significant clinical interest, and
substitution of larger alkyl groups for the Nω-methyl of
2 improves the selectivity of this group of inhibitors.6 The
synthesis and characterization of arginine analogues 3a -
c, designed to improve peptide bioactivity and yield
improved NOS inhibitors, are described in this paper.
Reversing the alkylation procedure, with some elabo-
ration, proved to be a more feasible route (Scheme 1).
Previously, we synthesized (2S)-2-azido-5-bromovaleric
acid (4) using the Evans chiral auxiliary8 to stereoselec-
tively introduce the R-azido group on the appropriate
ω-bromo acid.9 This strategy fixes the chiral center early,
allowing the aqueous nucleophilic substitution reactions
required for construction of the guanidino functionalities
to be performed later in the syntheses. The R-amino
group is masked as the azido functionality, a protective
strategy necessary for the diamine cyclization reactions.
We first treated 4 with ethylenediamine and noted the
formation of the desired Nω-substituted product 8 with
a major side product that proved to be the six-membered
lactam10 9, which was previously shown to be easily
formed with analogous compounds in another labora-
tory.11
Resu lts a n d Discu ssion
We have produced various Nω mono- and dialkylated
analogues of arginine including 2 (Figure 1) through
* To whom correspondence should be addressed. Tel: (843)-953-
6614. Fax: (843)-953-6615. E-mail: dixta@musc.edu.
(1) (a) Beeson, C. C.; Dix T. A. J . Org. Chem. 1992, 57, 4386-4394.
(b) Beeson, C. C.; Dix T. A. J . Am. Chem. Soc. 1993, 115, 10275-10281.
(2) (a) Kennedy, K. J .; Simandan T. L.; Dix T. A. Synth. Commun.
1997, 28, 741-746. (b) Kennedy, K. J .; Lundquist, J . T., IV; Simandan
T. L.; Beeson, C. C.; Dix T. A. Bioorg. Med. Chem. Lett. 1997, 7, 1937-
1940.
(3) Lundquist, J . T., IV; Dix T. A. J . Med. Chem., in press.
(4) Fukuto, J . M.; Mayer, B. In Methods in Nitric Oxide Research;
Feelisch, M., Stampler, J . S., Eds.; J . Wiley & Sons: New York, 1996;
pp 147-160.
(5) Hecker, M.; Billiar, T. R. In Methods in Nitric Oxide Research;
Feelisch, M., Stampler, J . S., Eds.; J . Wiley & Sons: New York, 1996;
pp 262-264.
(6) Zhang, H. Q.; Fast, W.; Marletta, M. A.; Martasek, P.; Silverman,
R. B. J . Med. Chem. 1997, 40, 3869-70.
(7) Ishikawa, F.; Kosasayama, A.; Nakamura, S.; Konno, T. Chem.
Pharm. Bull. 1978, 26, 3658-3665.
(8) Evans, D. A.; Britton, T. C.; Ellman, J . C.; Dorow, R. L. J . Am.
Chem. Soc. 1990, 112, 4011-4030.
(9) Lundquist, J . T., IV; Dix, T. A. Tetrahedron Lett. 1997, 39, 775-
778.
(10) Evidence supporting lactamization was displayed in the test
reaction of 4 with 30 equiv dimethylamine. The 1H NMR spectra of
the completed reaction mixture for the formation of (2S)-2-azido-Nδ-
dimethylvaleric acid shows only one product since there is no ex-
changeable hydrogen on the δ-nitrogen available for cyclic condensa-
tion. This reaction follows the general nucleophilic substitution scheme
used to form 7a in the Experimental Section.
10.1021/jo990551b CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/20/1999

9266 J . Org. Chem., Vol. 64, No. 25, 1999
Notes
Sch em e 1
We were unable to separate 8 and 9 by Dowex ion
exchange and believe that further acid-catalyzed lactam-
ization could occur during purification. This problem was
circumvented by protecting the carboxylic acid of 4 as
the benzyl amide 5 using the mixed anhydride method.12
Next, 5 was treated with excess ethylenediamine or
N-ethylethylenediamine in aqueous ethanol at room
temperature overnight.13 Products 6a and 6c, respec-
tively, were extracted into butanol and treated im-
mediately with CNBr to give crude cyclized products 7a
and 7c. The benzyl amide enhances lipophilicity for the
anhydrous cyclization and provides a means for facile RP-
HPLC purification of the products. Reduction of the azido
group and acid-catalyzed cleavage of the amide bond
gives the cyclic arginine derivatives 3a 14 and 3c. Reaction
of N-methylethylenediamine with 5, however, resulted
in significantly lower yields due to the production of a
mixture of two isomers 6b and 10.
easily solvated exocyclic 2-imino group.17 All of these
compounds show more nonpolar character in octanol-
water partitioning and HPLC experiments. Each of the
analogues has been NR-Fmoc-protected18 and incorpo-
rated into short (<10 residue) peptides of potential
biological interest.3 It was not necessary to utilize the
side-chain protecting groups usually required for solid-
phase peptide synthesis of guanidino-containing amino
acids. Currently, we are attempting to protect the side-
chain functionalities so that these analogues will be
better suited for lengthy solid-phase syntheses. Potency
and selectivity of these compounds as NOS isoform
inhibitors will be reported shortly.
Exp er im en ta l Section
1H and 13C NMR spectra were acquired on a 300 or 400 MHz
spectrometer. All chemical reagents and reagent-grade solvents
were obtained from commercial suppliers. Flash chromatography
was performed using Whatman silica gel 60 (230-400 mesh)
and monitored using Whatman UV254 thin-layer chromatography
plates. Semipreparative HPLC was performed on a Waters dual
pump HPLC system in combination with a Kontrosorb 10C18
column using the following mobile phase system: 0.1% trifluo-
roacetic acid in water (solvent A) and 0.084% trifluoroacetic acid
in acetonitrile (solvent B). Optical rotations were measured in
1 dm cells of 1 or 10 mL capacity. Optical purity was assessed
at >95% by the use of a modified Mosher’s method.19
Cyclization was conducted on this mixture since the
two products could not be resolved by RP-HPLC. Appar-
ently, while the N-ethyl group provided enough steric
hindrance to afford predominantly the desired ω-alkyl
diamine isomer 6c, this was not the case with 6b. The
overall yields of analogues 3a -c were thus 61%, 25%,
and 49%, respectively, from 4.
Synthesis of analogues 3a -c provides compounds with
interesting differences in properties when compared to
arginine. We have measured pKa’s of approximately 11
for 3a -c by potentiometric titration,15 which is about two
units lower than arginine. The lowered basicity can be
attributed to decreased resonance for the alkylated
guanidinium groups16 and the lipophilicity imparted by
alkyl substituents that favors protonation on the more
(2S)-2-Azid o-N-ben zyl-5-br om ova ler a m id e (5). Following
the method of Anderson et al.,12 (2S)-2-azido-5-bromovaleric acid
(4) (0.96 g, 4.32 mmol) was dissolved in dry THF (27.9 mL) and
equilibrated to -15 °C, at which time N-methylmorpholine (0.48
mL, 4.32 mmol) was added followed by isobutyl chloroformate
(0.59 mL, 4.54 mmol). The temperature was maintained at -15
°C for 4 min when benzylamine (0.50 mL, 4.54 mmol) was added.
The reaction was stirred mechanically at this temperature for
1 min before being allowed to warm to room temperature over
60 min. The reaction mixture was concentrated in vacuo and
then partitioned between 0.1 M pH 7.5 phosphate buffer and
dichloromethane. The organic fractions were pooled, dried over
anhydrous MgSO4, filtered, and evaporated under reduced
pressure. The crude product was purified by flash chromatog-
raphy on silica gel (ethyl acetate/hexanes 1:1, Rf ) 0.32), giving
1.21 g of the clear oil (5) in 90% yield: [R]25 ) +10.4 (c ) 1.0
D
in MeOH); 1H NMR (300 MHz, CDCl3) δ 7.42-7.21 (m, 5H), 4.47
(11) Tsuji, S.; Kusumoto, S.; Shiba, T. Chem. Lett. 1975, 1281-1284.
(12) Anderson, G. W.; Zimmerman, J . E.; Callahan, F. M. J . Am.
Chem. Soc. 1967, 89, 5012-5017.
(13) March, J . Advanced Organic Chemistry. Reactions, Mechanism,
and Structure; 3rd ed.; J . Wiley & Sons: New York, 1985; pp 364-
365.
(14) The unsymmetrical guanidinium group of 3a exhibits a defini-
tive 1H NMR spectrum for the constrained ethylene protons which are
nearly identical to the analogous protons in 2-imino-1-imidazolidine-
acetic acid obtained from Aldrich (see the Supporting Information).
(15) Schmidt, C. A.; Kirk, P. L.; Appleman, W. K. J . Biol. Chem.
1930, 88, 285-293.
(d, J ) 5.8 Hz, 2H), 4.10 (t, J ) 5.7 Hz, 1H), 3.44 (t, J ) 6.1 Hz,
(16) Angyal, S. J .; Warburton, W. K. J . Chem. Soc. 1951, 2492-
2494.
(17) Stein, W. D. The Movement of Molecules across Cell Membranes;
Academic Press: New York, 1967; pp 75-76.
(18) Ten Kortenaar, P. B.; Van Dijk, B. G.; Peeters, J . M.; Raaben,
B. J .; Adams, P. J .; Tesser, G. I. Int. J . Peptide Protein Res. 1986, 27,
398-400.
(19) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95, 512-
519.

Notes
J . Org. Chem., Vol. 64, No. 25, 1999 9267
2H), 2.25-1.90 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 168.6,
137.4, 128.8, 127.8, 127.8, 63.6, 43.6, 32.5, 30.9, 28.3.
13C NMR (100 MHz, CD3OD) δ 171.8, 158.5, 139.7, 129.6, 128.7,
128.4, 64.0, 46.7, 46.2, 45.7, 44.2, 41.0, 29.6, 23.9, 12.0.
Gen er a l P r oced u r e for Dia m in e Su bstitu tion a n d Su b-
sequ en t Cycliza tion . (2S)-2-Azid o-N-ben zyl-5-(2-im in oim -
id a zolid in e)va ler a m id e (7a ). Compound 5 (342 mg, 1.10
mmol) was dissolved in EtOH/H2O (90:10, 6.5 mL). Neat ethyl-
enediamine (991 mg, 16.50 mmol) was added to the stirring
solution and allowed to react overnight in a closed container at
ambient temperature. Next, the EtOH was evaporated and the
crude product extracted from 1.5 N NH4OH into saturated
butanol. The butanol fractions were combined, washed with NH4-
OH, and concentrated under reduced pressure with mild heating
at 50 °C. This procedure removes most of the aqueous soluble
ethylenediamine while ensuring the newly formed diamine side
chain of 6a is in the un-ionized form for subsequent cyclization.
The product 6a (ca. 95% purity) was used without further
purification in the guanidinium-forming cyclization reaction
following the procedure of Ishikawa et al.7 Briefly, CNBr (112
mg, 1.10 mmol) was dissolved in benzene (3.0 mL) and stirred
at room temperature. Next, 6a , dissolved in benzene (1.0 mL),
was added dropwise over 5 min to the CNBr solution. The
formation of 7a was noted by precipitation of the insoluble HBr
salt. The reaction was stirred for a minimum of 3 h, upon which
time the benzene was removed under reduced pressure and the
product purified by RP-HPLC. Purification of about 400 mg of
crude product was effected by elution at a flow rate of 6 mL/
min using a linear gradient from 3 to 35% B over 60 min and
monitoring the effluent by UV absorbance at 254 nm. The yield
Gen er al P r ocedu r e for Fin al Depr otection . (2S)-2-Am in o-
5-(2-im in oim id a zolid in e)va ler ic Acid (3a ). Compound 7a
(362 mg, 0.84 mmol) was dissolved in MeOH (30 mL) to which
palladium, 10 wt % on activated carbon (Pd-C 10%) (25 mg),
was added under nitrogen. The hydrogenation flask was placed
on a Parr reactor at 30 psi and shaken overnight. Next, the
Pd-C 10% was removed by filtration and the MeOH removed
in vacuo. The resulting amine was then refluxed in HCl 37%
overnight, at which time the reaction was cooled and evaporated
to dryness under reduced pressure. The crude material was
dissolved in 1.5 N NH4OH and washed with dichloromethane
to remove residual benzylamine. The final product was purified
on a Dowex-50 ion-exchange column eluting with 1.5 N NH4OH
while monitoring by TLC (phenol/H2O 3:1) with ninhydrin
detection. The yield of the white solid 3a from 7a was 88%: [R]25
D
) +16.9 (c ) 1.0 in 6 N HCl); mp 275-300 °C dec; 1H NMR
(300 MHz, D2O) δ 4.15 (t, J ) 6.2 Hz, 1H), 3.78-3.58 (m, 4H),
3.37 (t, J ) 6.8 Hz, 2H) 2.12-1.90 (m, 2H), 1.90-1.66 (m, 2H);
13C NMR (100 MHz, D2O) δ 178.3, 165.5, 59.1, 54.2, 50.3, 47.3,
33.4, 28.7; ESI MS calcd for C8H16N4O2 (MH+) 201.3, found
201.2.
(2S)-2-Am in o-5-(2-im in o-3-m et h ylim id a zolid in e)va ler -
ic Acid (3b). Compound 7b was used in the general procedure
above to produce arginine analogue 3b, a white solid, in 93%
yield: [R]25 ) -4.4 (c ) 1.0 in 6 N HCl); mp 275-300 °C dec;
D
1H NMR (300 MHz, D2O) δ 3.78 (t, J ) 5.8 Hz, 1H), 3.64 (s,
4H), 3.34 (t, J ) 6.9 Hz, 2H), 2.93 (s, 3H), 1.96-1.82 (m, 2H),
1.82-1.59 (m, 2H); 13C NMR (100 MHz, D2O) δ 178.4, 160.6,
57.4, 50.8, 48.6, 47.4, 34.5, 30.8, 25.1; ESI MS calcd for
C9H18N4O2 (MH+) 215.3, found 215.2.
of 7a , a yellowish oil, from 5 was 77%: [R]25 ) -1.8 (c ) 1.0 in
D
MeOH); 1H NMR (300 MHz, CD3OD) δ 7.28-7.12 (m, 5H), 4.33
(s, 2H), 3.90 (t, J ) 6.3 Hz, 1H), 3.53 (s, 4H), 3.23 (t, J ) 7.0 Hz,
2H), 1.85-1.65 (m, 2H), 1.65-1.49 (m, 2H); 13C NMR (100 MHz,
CD3OD) δ 171.8, 160.4, 139.7, 129.6, 128.7, 128.4, 64.1, 48.8,
45.0, 44.2, 41.9, 29.7, 24.0.
(2S)-2-Am in o-5-(3-et h yl-2-im in oim id a zolid in e)va ler ic
Acid (3c). Compound 7c was used in the general procedure
above to produce arginine analogue 3c, a white solid, in 90%
(2S)-2-Azido-N-ben zyl-5-(2-im in o-3-m eth ylim idazolidin e)-
va ler a m id e (7b). The synthesis of 7b follows the general
procedure described above except that N-methylethylenediamine
was used in place of ethylenediamine. The mixture of isomers
6b and 10, which could not be separated by chromatography,
was treated with CNBr to give crude 7b. Approximately 350 mg
of crude product was purified by RP-HPLC as above using a
linear gradient from 8 to 35% B over 60 min to give pure 7b as
yield: [R]25 ) -6.6 (c ) 1.0 in 6 N HCl); mp 275-300 °C dec;
D
1H NMR (300 MHz, D2O) δ 4.16 (t, J ) 5.5 Hz, 1H), 3.68 (s,
4H), 3.45-3.27 (m, 4H), 2.14-1.91 (m, 2H), 1.91-1.68 (m, 2H),
1.19 (t, J ) 7.1 Hz, 3H); 13C NMR (100 MHz, D2O) δ 178.4, 163.4,
59.2, 52.2, 51.6, 50.8, 46.4, 33.3, 28.7, 17.6; ESI MS calcd for
C10H20N4O2 (MH+) 229.3, found 229.2.
a yellowish oil in 30% yield from 5: [R]25 ) +8.0 (c ) 1.0 in
Ack n ow led gm en t. J .T.L. is an American Founda-
tion for Pharmaceutical Education predoctoral scholar.
The authors thank Professor Kevin Schey of the MUSC
Mass Spectometry Facility and Dr. J ack Pelletier (Wy-
eth-Ayerst) for support of this work. The MUSC NMR
Facility also was used in support of the work.
D
MeOH); 1H NMR (300 MHz, CD3OD) δ 7.28-7.10 (m, 5H), 4.31
(s, 2H), 3.88 (t, J ) 6.3 Hz, 1H), 3.55-3.38 (m, 4H), 3.21 (t, J )
7.0 Hz, 2H), 2.86 (s, 3H), 1.83-1.63 (m, 2H), 1.63-1.46 (m, 2H);
13C NMR (100 MHz, CD3OD) δ 171.8, 159.4, 139.7, 129.6, 128.7,
128.4, 64.0, 49.2, 46.7, 45.8, 44.2, 32.4, 29.6, 24.0.
(2S)-2-Azid o-N-ben zyl-5-(3-eth yl-2-im in oim id a zolid in e)-
va ler a m id e (7c). The synthesis of 7c follows the general
procedure described above except that N-ethylethylenediamine
was used in place of ethylenediamine. Approximately 375 mg
of crude product was purified by RP-HPLC as above using a
linear gradient from 8 to 30% B over 60 min. The yield was 60%
for the yellowish oil 7c from 5: [R]25D ) +15.8 (c ) 1.0 in MeOH);
1H NMR (300 MHz, CD3OD) δ 7.30-7.13 (m, 5H), 4.32 (s, 2H),
3.90 (t, J ) 6.3 Hz, 1H), 3.61-3.42 (m, 4H), 3.33-3.16 (m, 4H),
1.87-1.65 (m, 2H), 1.65-1.48 (m, 2H), 1.12 (t, J ) 7.2 Hz, 3H);
1
Su p p or tin g In for m a tion Ava ila ble: 300 MHz H NMR
spectra for 5, 6a , the mixture of 6b and 10, 6c, 7a -c, 2-imino-
1-imidazolidineacetic acid, and 3a -c, 400 mHz 1H NMR
spectrum of Mosher’s derivatized 3a , 100 MHz 13C NMR
spectra for 5, 7a -c, and 3a -c, and ESI MS for 3a -c. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O990551B