Synthesis and biological characterization of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide, a prodrug of a selective iNOS inhibitor

Article abstract of DOI:10.1021/jm010420e

The 5-tetrazole amide of L-N⁶-(1-iminoethyl)lysine (L-NIL), L-N⁶-(1-iminoethyl)lysine 5-tetrazole amide (1), has been prepared and evaluated. In contrast to L-NIL, 1 is a stable, nonhygroscopic, crystalline solid. Unlike L-NIL, 1 has

Full text of DOI:10.1021/jm010420e

1686
J . Med. Chem. 2002, 45, 1686-1689
Syn th esis a n d Biologica l Ch a r a cter iza tion of L-N6-(1-Im in oeth yl)lysin e
5-Tetr a zole-a m id e, a P r od r u g of a Selective iNOS In h ibitor
E. Ann Hallinan,* Sofya Tsymbalov, Clifford R. Dorn, Barnett S. Pitzele, and Donald W. Hansen, J r.
Department of Medicinal Chemistry, Pharmacia Corporation, 4901 Searle Parkway, Skokie, Illinois 60077, and
800 North Lindbergh Boulevard, St. Louis, Missouri 63167
William M. Moore, Gina M. J erome, J ane R. Connor, Linda F. Branson, Deborah L. Widomski, Yan Zhang,
Mark G. Currie, and Pamela T. Manning
Department of Arthritis and Inflammation Pharmacology, Pharmacia Corporation, 4901 Searle Parkway,
Skokie, Illinois 60077, and 800 N. Lindbergh Boulevard, St. Louis, Missouri 63167
Received September 5, 2001
The 5-tetrazole amide of L-N6-(1-iminoethyl)lysine (L-NIL), L-N6-(1-iminoethyl)lysine 5-tetrazole
amide (1), has been prepared and evaluated. In contrast to L-NIL, 1 is a stable, nonhygroscopic,
crystalline solid. Unlike L-NIL, 1 has minimal inhibitory activity in vitro on human inducible
nitric oxide synthase (iNOS). However, it is rapidly converted in vivo to L-NIL and produces
dose-dependent inhibition of iNOS in acute and chronic models of inflammation in the rodent
with efficacy comparable to L-NIL. In addition, both 1 and L-NIL exhibit significant and
comparable in vivo selectivity for the inhibition of iNOS vs endothelial NOS. Doses ap-
proximately 80-fold greater than those that inhibited inflammation do not elevate systemic
blood pressure. In summary, both the physical properties and the pharmacological profile of 1
make it an ideal molecule for preclinical and clinical studies on the role of selective iNOS
inhibitors in mediating inflammatory disease processes.
In tr od u ction
duction of local prostaglandins may subsequently aug-
ment the inflammation and tissue damage elicited by
the cytotoxic actions of NO. This observation is of
particular importance with regard to iNOS and COX-
2, since the expression of these two inducible proteins
appears to be regulated by the same factors produced
during chronic inflammation.5 The activation of COX-2
by NO generated from iNOS may increase and/or
sustain inflammation during both acute and chronic
inflammatory conditions.
Selective iNOS inhibitors, including L-NIL,6 have
been shown to suppress the increase in plasma nitrites
and/or paw swelling associated with the overproduction
of NO in animal models of acute and chronic inflam-
mation including nonlethal endotoxemia,6b carrageenan-
induced paw edema,7,8 and adjuvant-induced arthri-
tis.9,10 Importantly, L-NIL was found to be both orally
active and produce marked efficacy at doses that did
not produce an elevation in systemic blood pressure,
demonstrating in vivo selectivity.8,9 This suggests that
selective iNOS inhibitors may have therapeutic poten-
tial for treatment of diseases mediated by the overpro-
duction of NO.
Nitric oxide (NO) is involved in the regulation of many
physiological processes, as well as in the pathophysiol-
ogy of a number of diseases. It is synthesized enzymati-
cally from L-arginine via a five electron transfer process
in numerous tissues and cell types by three distinct
isoforms of the enzyme, nitric oxide synthase (NOS).
Two of these isoforms are expressed in a constitutive
manner, predominantly in the vascular endothelium
(eNOS, type III NOS) and in the nervous system (nNOS,
type I NOS). Under normal physiological conditions,
these constitutive forms of NOS generate low, transient
levels of NO (picomolar to nanomolar concentrations)
in response to increases in intracellular calcium con-
centrations. These low levels of NO act to regulate blood
pressure, platelet adhesion, gastrointestinal motility,
bronchomotor tone, and neurotransmission.1 The ex-
pression of the third isoform (iNOS, type II NOS) is
induced by endotoxin and/or cytokines and generates
high, sustained levels of NO (up to micromolar concen-
trations). These elevated levels of NO and resulting NO-
derived metabolites cause cellular cytotoxicity and
tissue damage and are thought to contribute to the
pathophysiology of a number of human diseases.2
NO, or more likely peroxynitrite, a product of the
reaction of NO and superoxide, has also been found to
directly activate both the constitutive and inducible
cyclooxygenase enzymes (COX-1 and COX-2, respec-
tively) by serving as a substrate for the peroxidase
activities of these enzymes3,4 resulting in an increase
in local prostaglandin production. This enhanced pro-
The pharmaceutical properties of L-NIL are sub-
optimal for carrying out chronic studies, particularly
preclinical studies in large animals as well as clinical
studies. Therefore, an attempt was made to identify a
prodrug form of L-NIL that possessed the necessary
physical properties required for more in depth charac-
terization. Compound 1, a tetrazole-amide of L-NIL, was
synthesized and found to be a stable crystalline solid
that exhibited comparable biological activity to L-NIL.
The synthesis and the preliminary biological charac-
terization of this prodrug (1) are described.
* To whom correspondence should be addressed. Tel.: 847-982-7753.
Fax: 847-982-7103. E-mail: ann.hallinan@pharmacia.com.
10.1021/jm010420e CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/13/2002

Prodrug of a Selective iNOS Inhibitor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1687
Sch em e 1a
a
Key: (a) BOP, NMM, DMF, room temperature, 20 h, 31%. (b) H2, 5% Pd/C, EtOH, HOAc, 5 psi, room temperature, 9 h, quantitative.
(c) Methyl acetimidate hydrochloride, TEA, DMF or ethyl acetimidate HCl, EtOH, NaOH, o.n., room temperature. (d) 4 N HCl/dioxane,
HOAc, 2 h, 91% from step 2.
Ta ble 1. Comparison of IC50 Values for Inhibition of Human
Syn th esis
NOS Isoforms (IC50 - µM)a
As shown in Scheme 1, the synthetic sequence re-
compd
iNOS
5.9
>1850
eNOS
nNOS
quired acylation of 5-amino-tetrazole with NR-Boc-Nꢀ-
Z-L-Lys-OH. Coupling of 5-aminotetrazole to protected
lysine was more difficult than expected. The mixed
anhydride coupling method using iso-butyl chlorofor-
mate or a carbodiimide-mediated acylation with 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride and N-hydroxybenzotriazole resulted in 2 in poor
yields. Benzotriazole-1-yl-oxy-tris(dimethylamino)phos-
phonium hexafluorophosphate (BOP) proved to be the
most effective coupling agent, with yields of approxi-
mately 60% on a small scale and 30% on a large scale.
The poor nucleophilicity of 5-aminotetrazole probably
accounted for the poor coupling yield. The product of
the coupling was subsequently digested in dichlo-
romethane to give pure, crystalline product with a
melting point of 196.6 °C. Alternatively, the reaction
mixture could be poured onto cold water. With stirring,
a solid formed that could be filtered and washed with
H2O to provide 2. Removal of the carbobenzoxy group
using Pd/C was quantitative. Recrystallization from
ethyl acetate gave a crystalline acetate salt 3 with a
melting point of 205.5 °C. Amidine formation was
uneventful, but immediate protonation of the amidine
was necessary to avoid decomposition to the ꢀ amide.
The Boc group was removed under anhydrous acid
conditions using HCl in dioxane. The final product 1
was purified by washing with HOAc and Et2O followed
by recrystallization from methanol and acetone yielding
a white solid with a melting point of 168.2 °C. The
tetrazole amide was stable under normal reaction
conditions.
L-NIL
1
138
2420
35
>1850
a
IC50 values were determined with recombinant human induc-
ible, endothelial, and neuronal NOS isoforms as described in the
Experimental Section.
F igu r e 1. Efficacy of L-N6-(1-iminoethyl)lysine 5-tetrazole
amide (1) and L-NIL in the nonlethal rat endotoxin model.
Values are means ( SEM for n ) 6 animals/dose and represent
the percent inhibition in plasma nitrite levels of animals
treated with endotoxin alone as compared to saline-treated
controls. Data shown is from a representative experiment.
Mean values from multiple experiments gave an ED50 of 0.6
mg/kg for 1 and 0.5 mg/kg for L-NIL.
statistically significant) between the two dose-response
curves observed in Figure 1 could be attributed to the
higher molecular weight of 1 due to the aminotetrazole
moiety. The mean ED50 for 1 is 0.6 vs 0.5 mg/kg for
L-NIL. Compound 1 was also assessed in an acute model
of inflammation, the rat carrageenan-induced paw
edema model, and was found to reduce paw swelling in
a dose-dependent manner (ED50 between 10 and 30 mg/
kg, Figure 2). This is in good agreement with data
reported for L-NIL (50% reduction in paw swelling at
approximately 30 mg/kg) found 3 h after administration
of compound.7 For further characterization, the efficacy
of 1 was assessed in a chronic model of inflammation,
rat adjuvant-induced arthritis. As shown in Figure 3, 1
gave dose-dependent inhibition of paw swelling/joint
inflammation, producing complete inhibition between
Biologica l Assa ys
As shown in Table 1, 1 in comparison to L-NIL
produced essentially no inhibition of the human NOS
isoforms. The IC50 values for human recombinant iNOS,
eNOS, and nNOS were greater than 1850 µM, indicat-
ing that the prodrug exhibited little to no iNOS inhibi-
tory activity. However, when evaluated in vivo for
efficacy following oral administration in a nonlethal
endotoxemia model in the rat, 1 gave dose-dependent
inhibition of elevated plasma nitrites, similar to that
which occurred with L-NIL6a (Figure 1). The shift (not

1688 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Hallinan et al.
toxicity has been associated with the aminotetrazole
portion of the prodrug (data not shown), in agreement
with data in the literature.11
Con clu sion s
Compound 1 was synthesized and shown to be a
stable, nonhygroscopic, highly water soluble crystalline
solid, clearly distinct from L-NIL, which rapidly deli-
quesces on exposure to air. Importantly, these physical
properties of 1 are preferable for use in chronic pre-
clinical studies, particularly in larger animals such as
the dog and primate, as well as for use in clinical
studies.
While 1 is a poor inhibitor of the human recombinant
NOS isoforms in enzyme assays in comparison to L-NIL
(Table 1), it is readily converted in vivo to generate
L-NIL and aminotetrazole. The pharmacological proper-
ties of 1 have been assessed and compared to L-NIL in
vivo in several models of acute and chronic inflamma-
tion. Both compounds are potent inhibitors of iNOS in
a rodent nonlethal endotoxin model, exhibiting dose-
dependent inhibition of nitric oxide production following
the systemic induction of iNOS. Treatment with either
1 or L-NIL7,8 reduced paw inflammation following
carageenan administration in a dose-dependent fashion
and with similar efficacy. In a chronic inflammatory
model, rat adjuvant-induced arthritis, iNOS expression
is elevated in the inflamed, swollen joint tissue. In this
model, the prophylactic treatment with either 1 or
L-NIL9,10 reduced the paw swelling with similar efficacy
as in the acute models.
F igu r e 2. Efficacy of L-N6-(1-iminoethyl)lysine 5-tetrazole
amide (1) in the carrageenan-induced model of acute paw
inflammation. Compound was administered by gavage 3 h
after carrageenan injection. Edema was measured by the
change in paw volume. Values are means ( SEM for n ) 5
animals/dose.
In addition to characterizing their pharmacological
activity, these compounds were also examined for effects
on blood pressure (regulated by eNOS) in order to
provide an indication of their functional selectivity in
vivo. Neither compound produced an increase in blood
pressure at doses up to approximately 80-fold of the
ED50 (∼0.5 mg/kg) in the rat nonlethal endotoxin model,
demonstrating in vivo selectivity for the inhibition of
iNOS vs eNOS. In summary, we have found that 1
possesses significantly improved physical properties as
compared to L-NIL, while maintaining its favorable
pharmacological properties. Finally, we are further
exploring the potential of the prodrug, 1, in both
preclinical and clinical settings.
F igu r e 3. Efficacy of L-N6-(1-iminoethyl)lysine 5-tetrazole
amide (1) in the adjuvant-induced arthritis model on changes
in hind paw volume 21 days following adjuvant administration.
Doses expressed are milligrams per kilogram per day. Com-
pound 1 was administered at doses of 0.3, 1, 3, or 10 mg/kg/
dose twice daily throughout the course of the study starting
at the time of adjuvant administration. Values are means (
SEM for n ) 7-10 animals/dose.
6 and 20 mg/kg/day. This value agrees well with the
literature value reported for L-NIL (maximal inhibition
at 20 mg/kg/day).10 In addition, similar efficacy was
observed with 1 and L-NIL when evaluating plasma or
paw exudate nitrite/nitrate levels (data not shown).
Finally, the in vivo selectivity for iNOS vs eNOS was
determined by assessing the effect on systemic blood
pressure following oral administration of both com-
pounds in the rat. No increase in blood pressure
occurred following the oral administration of 40 mg/kg
of either 1 or L-NIL, while a modest increase occurred
(∼10%) following the administration of 50 mg/kg of
either compound, demonstrating substantial in vivo
selectivity in the rat. Importantly, ex vivo studies
demonstrate that 1 is rapidly metabolized in whole
blood of rat, dog, and monkey to generate L-NIL and
aminotetrazole (data not shown). In vivo studies in the
rat indicate that 1 is rapidly metabolized to L-NIL
following iv administration and L-NIL is rapidly avail-
able in the systemic circulation after oral administration
of 1 (data not shown). In our initial in vivo studies, no
Exp er im en ta l Section
All solvents and reagents were used without further puri-
fication unless otherwise noted. Thin layer chromatograms
were run on 0.25 mm EM precoated plates of silica gel 60 F254.
Visualization was achieved by exposure to Ca(ClO)2 followed
by spraying with a 0.5% KI 0.5% potato starch solution. High-
performance liquid chromatograms (HPLC) were obtained
from YMC AQ C-18 reverse phase columns. 1H nuclear
magnetic resonance (NMR) spectra were obtained from either
General Electric QE-300 or Bruker-400 MHz Ultrashield
spectrometers with tetramethylsilane or 3-(trimethylsilyl)-
propionic-2,2,3,3-d4 acid, sodium salt as an internal standard.
13C NMR were obtained from a Bruker-400 MHz Ultrashield
spectrometer at 125.8 MHz with tetramethylsilane or 3-(tri-
methylsilyl)propionic-2,2,3,3-d4 acid, sodium salt as an internal
standard. Melting points are obtained by differential scanning
calorimetry.
P h en ylm eth yl (5S)-5-[[(1,1-Dim eth yleth oxy)ca r bon yl]-
a m in o]-6-oxo-6-(1H -t et r a zol-5-yla m in o)h exyl]ca r b a m -
a te (2). A solution of NR-Boc-Nꢀ-Z-L-Lys-OH (100 g, 0.263 mol),
5-aminotetrazole hydrate (27.1 g, 0.263 mol), 4-methylmor-
pholine (NMM, 78.9 g, 0.780 mol), and BOP (116.4 g, 0.263
mol) in dimethyl formamide (DMF, 400 mL) was stirred at

Prodrug of a Selective iNOS Inhibitor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1689
room temperature under nitrogen for 20 h. About 350 mL of
DMF was removed under high vacuum. The resulting syrup
was added slowly to 2 L of mechanically stirred ice-water and
stirred for 90 min. A tan solid was filtered, washed with cold
H2O, and dried at 60 °C yielding 100 g of 2. The solid was
recrystallized from MeOH:EtOAc (1:1) yielding 36.0 g of a
white solid (31% of theory). 1H NMR (DMSO-d6): δ 7.21-7.40
(m, 5H), 5.00 (s, 2H), 3.95-4.18 (m, 1H), 2.90-3.08 (m, 2H),
1.58-1.70 (m, 2H), 1.40 (s, 9H), 1.20-1.32 (m, 4H). Anal.
(C20H29N7O5) C, H, N.
1,1-Dim eth yleth yl [(1S)-5-Am in o-1-[(1H-tetr azol-5-ylam i-
n o)ca r bon yl]p en tyl]ca r ba m a te Mon oa ceta te (3). The ben-
zyloxycarbonyl group of amide 2 (37 g, 0.0827 mol) was
removed in the presence of 4% Pd/C in EtOH:HOAc (9:1) at 5
psi and room temperature for 9 h. The thick orange oil was
digested with EtOAc. When solidification was complete, a
white solid was filtered, washed with EtOAc, and dried in a
vacuum oven at 35 °C to give 31.9 g of 3 (quantitative).
1,1-Dim eth yleth yl [(1S)-5-[(1-Im in oeth yl)am in o]-1-[(1H-
t et r a zol-5-yla m in o)ca r b on yl]p en t yl]ca r b a m a t e Mon o-
a ceta te (4). To a stirring suspension of 3 (31.8 g, 0.0827 mol)
and triethylamine (TEA, 25.8 g, 0.255 mol) in DMF (300 mL)
was added solid methyl acetimidate hydrochloride (6.57 g,
0.060 mol) in one portion. Addition of methyl acetimidate
hydrochloride was repeated two times at 1 h intervals. After
the mixture was stirred at room temperature for 16 h, the
NEt3‚HC1 was removed by filtration and washed with a small
amount of DMF. The filtrate was acidified with glacial HOAc
(20 mL) and concentrated under high vacuum to an orange
oil. The oil was dissolved in 50% aqueous HOAc (200 mL) and
concentrated under high vacuum to give 65 g of an oil. The
crude product was used in the next step without further
purification.
(2S)-2-Am in o-6-[(1-im in oeth yl)a m in o]-N-(1H-tetr a zol-
5-yl) Hexa n a m id e, Hyd r a te, Dih yd r och lor id e (1). Crude
amidine (94 g, estimated as 0.107 mol) was dissolved in glacial
HOAc (800 mL) with magnetic stirring, and the solution was
treated with 4 N HC1 in dioxane (80 mL, 0.32 mol). The
mixture was stirred at room temperature for 2 h as an oil
formed on the sides of the flask. The acetic acid was decanted
from product, and the oil was triturated with HOAc (300 mL)
followed by anhydrous Et2O (500 mL). The oil was then
triturated in MeOH to obtain a crystalline solid, which was
then diluted with acetone. The solid was filtered, washed with
acetone, and dried in a vacuum oven at 35 °C to give 29.5 g of
tan powder.
The MeOH/acetone filtrate from above was concentrated and
diluted with acetone to give a second crop of 6.7 g. On the basis
of the starting material in step 2, the yield is 34.12 g (91%).
The product was washed with HOAc, followed by Et2O, and
then recrystallized from MeOH:acetone (15:85). 1H NMR
(D2O): δ 4.38 (t, 1H, J ) 6.0 Hz), 3.29 (t, 2H, J ) 8.0 Hz),
2.21 (s, 3H), 2.06-2.17 (m, 2H), 1.70-1.78 (m, 2H), 1.50-1.59
(m, 2H). 13C NMR (D2O): δ 171.40, 167.62, 152.86, 56.38,
44.51, 32.96, 29.20, 24.15, 21.26. Anal. (C9H18N8O‚2HCl‚
1.25H2O) C, H, N, Cl.
administered orally 3 h following the intraplantar administra-
tion of carrageenan at which time near maximal edema had
developed. Edema was measured by the increase in paw
volume determined using a plethysmometer. Paws were
measured at the start of the experiment and served as their
own baseline controls.
Ad ju va n t-In d u ced Ar th r itis. Adjuvant-induced arthritis
was induced by the intradermal injection of Mycobacterium
butyricum in Lewis rats as previously reported.9 Compound 1
was administered orally by gavage, twice daily, beginning on
the day of adjuvant administration. Twenty-one days following
adjuvant administration, each hindpaw was assessed for
swelling using a plethysmometer.
Deter m in a tion of Mea n Ar ter ia l P r essu r e in th e Ra t.
Male Wistar rats (Harlan Sprague Dawley, Inc., Indianapolis
IN) weighing 250-350 g were anesthetized, and the femoral
artery was cannulated. The rats were placed in restraining
cages, and the cannula was connected to a Cobe disposable
pressure transducer (Cobe Laboratories, Inc., Lakewood CO).
Following recovery from anesthesia, hemodynamic parameters
were measured using a Gould Transducer Signal Conditioner
and TA Recording System (Gould Instrument Systems Inc.,
Valley View, OH). Baseline data were collected for approxi-
mately 30 min prior to the administration of a single dose of
either vehicle (water) or compound dissolved in water. Com-
pounds were administered orally by gavage. Mean arterial
pressure and heart rate were recorded for 4 h following
compound administration (n ) 3-4 rats for compound-treated
groups, 2-3 rats for vehicle-treated groups).
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as previously described.6b ADP-Sepharose affinity purified
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determine IC50 values by testing each compound at eight
concentrations in the presence of a final arginine concentration
of 30 µM.
Non leth a l En d otoxin Mod el. The in vivo efficacy of 1 and
L-NIL was determined following oral administration in the
rat following the systemic induction of iNOS by endotoxin
administration, as previously described.6b Compounds were
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plasma nitrite/nitrate levels were measured 5 h following
endotoxin administration.
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Ca r r a geen a n -In d u ced P a w Ed em a . The efficacy of 1 in
the carrageenan-induced rat model of acute paw inflammation
was determined as previously described.7 Compound 1 was
J M010420E