Synthesis and enzymatic evaluation of 2- and 4-aminothiazole-based inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.3762/bjoc.5.28

Highly potent and selective inhibitors of neuronal nitric oxide synthase (nNOS) possessing a 2-aminopyridine group were recently designed and synthesized in our laboratory and were shown to have significant in vivo efficacy. In this work, analogs of our lead compound possessing 2- and 4-aminothiazole rings in place of the aminopyridine were synthesized. The less basic aminothiazole rings will be less protonated at physiological pH than the aminopyridine ring, and so the molecule will carry a lower net charge. This could lead to an increased ability to cross the blood-brain barrier thereby increasing the in vivo potency of these compounds. The 2-aminothiazole-based compound was less potent than the 2-aminopyridine-based analogue. 4-Aminothiazoles were unstable in water, undergoing tautomerization and hydrolysis to give inactive thiazolones.

Full text of DOI:10.3762/bjoc.5.28

Synthesis and enzymatic evaluation of 2- and
4-aminothiazole-based inhibitors of neuronal nitric
oxide synthase
Graham R. Lawton1, Haitao Ji1, Pavel Martásek2,3, Linda J. Roman2
and Richard B. Silverman*,1
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2009, 5, No. 28.
1Department of Chemistry, Center for Molecular Innovation and Drug
Discovery, and Chemistry of Life Processes Institute, Northwestern
University, Evanston, Illinois 60208-3113 (USA), 2Department of
Biochemistry, University of Texas Health Science Center, San
Antonio, Texas (USA) and 3Department of Pediatrics and Center for
Applied Genomics, 1st School of Medicine, Charles University,
Prague, Czech Republic
doi:10.3762/bjoc.5.28
Received: 27 March 2009
Accepted: 14 May 2009
Published: 04 June 2009
Associate Editor: S. Flitsch
Email:
© 2009 Lawton et al; licensee Beilstein-Institut.
License and terms: see end of document.
Richard B. Silverman* - r-silverman@northwestern.edu
* Corresponding author
Keywords:
2-aminothiazole; 4-aminothiazole; nitric oxide synthase inhibitor;
nNOS
Abstract
Highly potent and selective inhibitors of neuronal nitric oxide synthase (nNOS) possessing a 2-aminopyridine group were recently
designed and synthesized in our laboratory and were shown to have significant in vivo efficacy. In this work, analogs of our lead
compound possessing 2- and 4-aminothiazole rings in place of the aminopyridine were synthesized. The less basic aminothiazole
rings will be less protonated at physiological pH than the aminopyridine ring, and so the molecule will carry a lower net charge.
This could lead to an increased ability to cross the blood-brain barrier thereby increasing the in vivo potency of these compounds.
The 2-aminothiazole-based compound was less potent than the 2-aminopyridine-based analogue. 4-Aminothiazoles were unstable
in water, undergoing tautomerization and hydrolysis to give inactive thiazolones.
Introduction
Neuronal nitric oxide synthase (nNOS) is the constitutive must be inhibited selectively without inhibition of the other
isoform of nitric oxide synthase (NOS) found in the CNS. It is isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS);
believed to play a role in many neurological diseases, including inhibition of eNOS could lead to side effects such as hyperten-
Parkinson’s disease [1], Alzheimer’s disease [2], damage due to sion [5].
stroke [3], and cerebral palsy caused by pre-natal hypoxia [4].
To fully explore the role nNOS plays in these and other Designing therapeutically useful nNOS-selective inhibitors is a
diseases, and to design inhibitors with therapeutic value, nNOS difficult task as the substrate for all three isoforms is L-arginine,
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Beilstein Journal of Organic Chemistry 2009, 5, No. 28.
and so they have similar active sites. In recent years, we have greater in vivo efficacy. The conjugate acid of the ring nitrogen
developed several potent and highly selective inhibitors of of the aminopyridine has a pKa ≈ 7 [11], therefore, the ring will
nNOS that have solved this problem by exploiting subtle differ- be partially protonated and only partially charged in biological
ences among the isoforms [6]. In addition, the active site of systems. Crystal structures of 2 bound to the active site of
nNOS is polar with multiple acidic groups, and so most inhib- nNOS show that the aminopyridine group interacts with a
itors that bind with high potency are polar with multiple basic glutamate residue (Glu592, rat nNOS), presumably via
groups. In general, highly charged, hydrophilic molecules do hydrogen bonding and electrostatic interactions [8,12]. The
not diffuse passively across the blood-brain barrier (BBB), thus aminopyridine ring nitrogen must be protonated for this interac-
limiting the concentration of inhibitor in the CNS [7].
tion to occur. Replacement of the aminopyridine with less basic
aminothiazoles (3 and 4), whose ring nitrogens have pKa values
Recently, a new strategy for fragment-based de novo design, ≈ 6, should reduce the overall charge on the molecule at
called fragment hopping, was described and utilized to design 1 physiological pH for more efficient bioavailability. In the acidic
(Figure 1), a new nNOS-selective inhibitor [8], which showed environment of the nNOS active site, however, protonation
nanomolar nNOS inhibitory potency and more than 1000-fold should occur to allow the aminothiazole to interact with the
selectivity over eNOS. Lead compound 1 was then evolved into active site glutamate and maintain tight binding. The methyl
highly potent and selective inhibitor 2 with better drug-like group at the 4 position on the aminopyridine ring contributes to
properties [9]. Intravenous administration of 2 resulted in signi- binding through an interaction with a hydrophobic pocket in the
ficant protection against neuronal damage in a cerebral palsy nNOS active site. The R-groups at the 5-position of 4 should
rabbit model [10].
allow us to probe the hydrophobic binding pocket defined by
P565, A566, V567, and F584 in the substrate binding site and
Replacement of basic functional groups of our lead molecule optimize this interaction. Figure 2 shows the docking mode for
with less basic groups will reduce the overall charge on the 3 and 4a in rat nNOS.
molecule and should increase BBB penetration, leading to
Figure 1: Lead compounds 1 and 2; 2- and 4-aminothiazole analogs 3 and 4a-c.
Figure 2: A: The docking conformation of 3 in the active site of rat nNOS; B: The docking conformation of 4b in the active site of rat nNOS. Cofactors
heme and H4B are shown in orange and purple, respectively.
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Beilstein Journal of Organic Chemistry 2009, 5, No. 28.
Scheme 1: Attempts to open epoxide 5 with deprotonated aminothiazoles. i) n-BuLi, 2 equiv, THF, −78 °C; ii) 5, THF, −78 °C to rt.
Initially, attempts were made to construct 3 and 4 using chem- Ethyl glycinate was alkylated with p-chlorobenzyl chloride to
istry analogous to that used to construct 2 (Scheme 1). Briefly, give secondary amine 18, which was protected with a Boc
Boc protected 2-amino-4-methylthiazoles, and 4-amino-2- group. The ester was hydrolyzed and converted to the Weinreb
methylthiazoles were treated with n-butyllithium, and then amide. Reduction with LAH gave aldehyde 22 (Scheme 3).
epoxide 5 was added in an attempt to form trans alcohols 6 and
7. However, this method failed to give any of the desired Compounds 17 and 22 were condensed to form an imine prior
products.
to the addition of NaHB(OAc)3, giving secondary amine 23.
Removal of the Boc groups with acid gave 3 as a tetrahydro-
A successful route to 2-aminothiazoles is outlined in Scheme 2. chloride salt.
Epoxide 5 was opened with allylmagnesium bromide, and the
resulting alcohol was protected as a TBS ether. The double The 4-aminothiazoles were constructed as shown in Scheme 4.
bond was converted to an epoxide, which was then opened with Treatment of acetonitrile with LDA followed by addition of
bromide under acidic conditions [13]. A mixture of diastereo- epoxide 5 gave trans-alcohol 24 [14]. The nitrile group was
mers was formed, but both were oxidized to α-bromoketone 12. converted to a thioamide using ammonium sulfide [15]. The
Condensation with thiourea gave the 2-aminothiazole (13). thioamide was condensed with either ethyl bromopyruvate or an
Diprotection of the amine with Boc groups and deprotection of epoxide (30) [16]. Condensation produces an equivalent of acid,
the TBS ether gave trans-alcohol 15. A Mitsunobu reaction was which was sufficient to cleave the Boc group. Buffering the
used to install a nitrogen atom in the form of a phthalimide reaction resulted in incomplete conversion to the thiazole
group with the desired cis-stereochemistry. The amine was because acidic conditions are necessary to catalyze the final
deprotected, but that resulted in the loss of one of the Boc dehydration step in the reaction [17]. However, the problem
groups to give 17.
was solved by simply neutralizing the mixture on completion of
the reaction and reprotecting the amine. The resulting esters
Scheme 2: Assembly of 2-aminothiazole fragment. i) AllylMgBr, ether, 0 °C, 15 min.; ii) TBSCl, imidazole, DMF, 35 °C, 16 h; iii) m-CPBA, rt, 48 h; iv)
LiBr, AcOH, THF, rt, 16h; v) (COCl)2, DMSO, TEA, CH2Cl2, −78 °C, 1 h; vi) thiourea, EtOH, reflux, 5 h; vii) Boc2O (2.5 equiv), DMAP, THF, rt, 16 h;
viii) TBAF, THF, rt 16 h; ix) PPh3, DIAD, phthalimide, THF, rt, 16 h; x) H2NNH2 (aq), MeOH, rt, 16 h, then 2N HCl, rt, 30 min.
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Beilstein Journal of Organic Chemistry 2009, 5, No. 28.
Scheme 3: Synthesis of compound 3. i) 4-chlorobenzylchloride, EtOH, reflux, 4 h; ii) Boc2O, TEA, MeOH, 3 h; iii) 1 N NaOH, MeOH, rt, 4 h; iv) EDC,
HOBt, TEA, HN(OMe)Me·HCl, CH2Cl2, 16 h; v) LAH, THF, 0 °C, 1 h; vi) (±)-17, CH2Cl2, NaHB(OAc)3, 1 h; vii) 4N HCl, dioxanes, rt, 16 h.
Scheme 4: Assembly of the 4-aminothiazole fragments. i) LiCH2CN, THF, 0 °C, 4 h; ii) (NH4)2S (aq), MeOH, 16 h; iii) ethyl brompyruvate (for R = H)
or 30, MeOH, reflux, 5 h, then DIEA, Boc2O, rt, 16 h; iv) 1N NaOH (aq), MeOH, rt, 16 h; v) DPPA, TEA, 3 Å mol. sieves, t-BuOH, reflux, 16 h; vi)
PPh3, DIAD, phthalimide, THF, rt, 16 h; vii) H2NNH2 (aq), MeOH, rt, 16 h, then 2N HCl, rt, 30 min.
(26a, R = H; 26b, R= Me; 26c, R = i-Pr) were hydrolyzed, and presumably because the thiazole nitrogen is less nucleophilic.
a Curtius rearrangement performed in tert-butanol gave the Cleavage of the phthalimide group gave amines 29a-c.
protected 4-aminothiazoles (7a-c) [18]. Unlike the case of the
aminopyridine analogues [19], the aminothiazole does not need The syntheses of 4a-c were completed by reductive amination
to be diprotected to allow the Mitsunobu reaction with followed by removal of the Boc groups (Scheme 5). Although
phthalimide as the nucleophile to proceed (28a-c). This is the final deprotection did give the desired product, as evi-
Scheme 5: Synthesis of inhibitor 4a-c. The 4-aminothiazoles were not stable in water undergoing tautomerization and hydrolysis. i) MeOH, 30 min,
then NaHB(OAc)3, rt, 1 h; ii) 4N HCl, dioxanes, rt, 16 h.
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Beilstein Journal of Organic Chemistry 2009, 5, No. 28.
denced by mass spectrometry, on addition of water, the product
decomposed. The 4-aminothiazole tautomerized to the
thiazoline, which was then hydrolyzed [20]. Test reactions on
model 4-aminothiazoles prepared by an analogous route showed
that none of the desired product remains in aqueous solution.
Further degradation occurred following hydrolysis, and the
products could not be identified.
Supporting Information
Supporting Information features full experimental details
for all synthetic steps, characterization of intermediates (1H
NMR, 13C NMR, ESMS), details of Autodock analysis,
details of in vitro enzyme assay, and HPLC chromatograms
for determining purity of compound 3.
Supporting Information File 1
Experimental and analytical data
Results and Discussion
Compound 3 was tested for in vitro activity against rat nNOS
[21], bovine eNOS [22], and murine iNOS [23] using a hemo-
globin capture assay [24], giving Ki values of 10 μM, 1 mM and
50 μM, respectively. This corresponds to a significant loss in
potency toward nNOS relative to lead compound 2 [Ki(nNOS)
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-5-28-S1]
= 0.085 μM, Ki(eNOS) = 85 μM, Ki(iNOS] = 9 μM), suggesting Acknowledgments
that the aminopyridine ring is critical for high affinity binding. The authors are grateful to the National Institutes of Health
It has been shown that the addition of electron-withdrawing (GM49725 to R.B.S. and GM52419 to Professor Bettie Sue
groups to a 2-aminopyridine significantly decreases NOS Siler Masters, with whose laboratory P.M. and L.J.R. are asso-
affinity [25]. This is possibly the result of a lowering of the pKa ciated) for financial support of this research. B.S.S.M. also is
such that the ring nitrogen is insufficiently protonated to grateful to the Welch Foundation for a Robert A. Welch Found-
interact with the glutamate. We had hoped that the high acidity ation Distinguished Professorship in Chemistry (AQ0012). P.M.
of the active site would protonate the aminothiazole so that it is supported by grants 0021620806 and 1M0520 from MSMT
would interact well with the glutamate residue. This may not be of the Czech Republic. We thank Marc Sala for preliminary
the case. An alternative explanation for the loss of potency is studies on the synthesis of 6.
that the 2-aminothiazole ring is much smaller than 2-amino-4-
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