Synthesis and Reactivity of Laquinimod, a Quinoline-3-carboxamide: Intramolecular Transfer of the Enol Proton to a Nitrogen Atom as a Plausible Mechanism for Ketene Formation Karl Jansson,* Tomas Fristedt, Arne Olsson, Bo Svensson, and Stig Jo¨nsson R&D Laboratories, ActiVe Biotech Research AB, Box 724, SE-220 07 Lund, Sweden
[email protected] ReceiVed NoVember 16, 2005
5-Chloro-N-ethyl-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-N-phenyl-3-quinolinecarboxamide (laquinimod, 2) is an oral drug in clinical trials for the treatment of multiple sclerosis. The final step in the synthesis of 2 is a high-yielding aminolysis reaction of ester 1 with N-ethylaniline. An equilibrium exists between 1 and 2, and removal of formed methanol during the reaction is a prerequisite for obtaining high yields of 2 from 1. The reactivity of 1 and 2 is explained by a mechanistic model that involves a transfer of the enol proton to the exocyclic carbonyl substituent with concomitant formation of ketene 3. This proton transfer is especially facilitated for 2 because the intramolecular hydrogen bond to the carbonyl oxygen is weakened due to steric interactions. Both 1 and 2 undergo solvolosis reactions that obey first-order reaction kinetics, further supporting the theory that these two molecules are able to decompose unimolecularly into ketene 3. The solvent-dependent spectroscopic features of 2 indicate that the molecule mainly resides in two conformations. One conformation is favored in nonpolar solvents and is likely the result of intramolecular hydrogen bonding. The other conformation is favored in polar solvents and probably exhibits less intramolecular hydrogen bonding.
Introduction
SCHEME 1
Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system (CNS). The etiology of the disease is unknown, but the pathophysiological mechanism is similar to other autoimmune diseases where there is an infiltration of mononuclear cells into the target tissue. 5-Chloro-N-ethyl-1,2dihydro-4-hydroxy-1-methyl-2-oxo-N-phenyl-3-quinolinecarboxamide (laquinimod, 2, Scheme 1) was developed by Active Biotech Research AB with roquinimex as a lead compound1,2 and has successfully undergone a clinical phase II trial as MS medication.3 The potency of laquinimod is very high, and proof (1) Noseworthy, J. H.; Wolinsky, J. S.; Lublin, F. D. Neurology 2000, 54, 1726. (2) (a) Jo¨nsson, S.; Andersson, G.; Fex, T.; Fristedt, T.; Hedlund, G.; Jansson, K.; Abramo, L.; Fritzson, I.; Pekarski, O.; Runstro¨m, A.; Sandin, H.; Thuvesson, I.; Bjo¨rk, A. J. Med. Chem. 2004, 47, 2075. (b) Bjo¨rk, A.; Jo¨nsson, S.; Fex, T.; Hedlund, G. US Patent 2000, 6,077,851. (c) Sih, J. C. US Patent 1999, 5,912,349. (3) Polman, C.; Barkhof, F.; Sandberg-Wollheim, M.; Linde, A.; Nordle, O ¨ .; Nederman, T. Neurology 2005, 64, 987.
of concept was established using doses as low as 0.3 mg/person/ day. The exact mechanism of action by which laquinimod exerts its effect on MS is still under investigation. The large-scale production of laquinimod utilizes the synthetic route outlined in Scheme 2. However, in this paper we will not discuss the 10.1021/jo052368q CCC: $33.50 © 2006 American Chemical Society
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J. Org. Chem. 2006, 71, 1658-1667
Published on Web 01/21/2006
Synthesis and ReactiVity of Laquinimod SCHEME 2.
Synthetic Route for Large-Scale Production of the Sodium Salt of 2
SCHEME 3
full details of large-scale synthesis such as process development and critical parameters. Instead, we will focus on the final pivotal step where ester 1 reacts with N-ethylaniline in heptane to afford compound 2 in almost quantitative yield. We will present evidence that this reaction occurs via the ketene intermediate 3, rather than via a tetrahedral intermediate. Conformational analysis and solvent dependent spectroscopic properties of 2 are also discussed. Results and Discussion A number of synthetic routes were evaluated for the largescale synthesis of laquinimod 2.2,4 The route depicted in Scheme 2 was chosen because it was reliable, easy to perform, and gave a good overall yield of 2 without the need of complicated purification methods. The nonhygroscopic sodium salt of 2 (2Na) was chosen as the final drug form due to its excellent stability during storage and the fact that some minor impurities in 2 were removed in the salt-forming step. More details of the large-scale synthesis will be published later. The laboratoryscale synthesis of 2-Na essentially follows the same route and is described in the Experimental Section. The first three steps of the synthetic route are straightforward. However, the final aminolysis step in which amide 2 is formed in almost quantitative yield from ester 1 deserves further comments. In general, amides are considered to be more stable than their corresponding esters, and a bimolecular reaction of an ester with an amine (4) Jansson, K. US Patent 2005, 6, 875, 869 B2.
appears to be a potentially useful method for the synthesis of amides. However, relatively few examples of this kind of amide synthesis are reported in the literature, in comparison to the more common method of reacting an acyl chloride with amines or the coupling of carboxylic acids with amines using coupling reagents such as DCC. The reason for this is probably that in the bimolecular reaction of an ester with an amine, high temperature is needed to form the transition state preceding the tetrahedral intermediate. In many cases, the amide is isolated in low yield because the high reaction temperature causes unwanted side reactions. Aliphatic amines are often more reactive than aromatic amines, and very few examples of secondary aromatic amines reacting with esters are reported in the literature. During our work with the synthesis of laquinimod 2, we realized that this amide is less stable than the corresponding ester 1 and that equilibrium exists between 1 and 2 in the aminolysis reaction. The equilibrium is probably maintained via a stabilized ketene intermediate 3 rather than via the tetrahedral intermediate. Laboratory Trials. Synthesis of 2 Using Toluene as Solvent. Compound 2 was isolated in 90% yield when a mixture of 1 and 2.0 equiv of N-ethylaniline in toluene was heated to the boiling point, and most of the volatiles were distilled off during approximately 6 h. However, the purity profile of the resulting 2 was not considered to be satisfactory, as substantial amounts of unreacted ester 1 and quinoline 7 (Scheme 3) were found in the isolated crude product (see the Experimental Section and Table 1). An interesting observation is that ester 1 and amide J. Org. Chem, Vol. 71, No. 4, 2006 1659
Jansson et al. TABLE 1. Yield and Purity Profile of the Isolated Product 2
TABLE 2. Reaction of Esters with N-Ethylanilinea
impurities in 2 (w/w, %)a solvent
yield (%)
toluene heptane
90 98
a
contenta 94.0 99.4
(%)
1
7
4.55 0.02
0.54 0.03
Determined by HPLC.
2 rapidly reached equilibrium at relatively low temperatures. When 1 reacted at 140 °C with 1.75 equiv of N-ethylaniline in sealed tubes (no volatiles can leave the reaction vessel), 2 was formed in 21% and 22% molar yield after 35 min and after 70 min, respectively (79% and 78% of 1 remained in the mixtures, see the Experimental Section for more details). The fact that the yields were almost identical indicates that equilibrium was reached. In another experiment where 2 was heated for 1 or 3 h at 100 °C with 3.0 equiv of methanol in sealed tubes, rapid methanolysis occurred and gave ester 1 in 98% molar yield in both cases (2% of 2 remained in the mixtures). Together, these experiments demonstrate the high reactivity of 1 and 2 and, unexpectedly, that ester 1 is favored in the equilibrium between 1 and 2. They also indicate that a prerequisite for obtaining a high yield of 2 from 1 is that methanol is efficiently removed from the reaction mixture by distillation of the volatiles. Laboratory Trials. Synthesis of 2 Using n-Heptane as Solvent. Apart from the finding that it was necessary to efficiently remove the formed methanol, another factor that dramatically increased the yield and purity of 2 was found. When using n-heptane (bp 98 °C) as solvent instead of toluene, 2 was isolated in 98% yield as white to off-white crystals with no single impurity exceeding 0.1% (Table 1). Only 1 and 7 were found in low amounts in the isolated product. This improvement in yield and purity was achieved by simply using a nontraditional solvent for synthesis such as n-heptane and resulted in a process patent covering the synthesis of compound 2 as well as similar structures.4 Both reaction rate and yield are higher in heptane than in toluene, despite the lower boiling point of heptane, and this can probably be attributed to the low solubility of 2 in heptane at the reaction temperature. The solubility of 1 and 2 in heptane at room temperature was determined by means of HPLC to be 0.15 and 0.03 mg/mL respectively. The solubility at higher temperatures was not determined, but still it is expected that the solubility of 2 is lower than that of 1. In heptane, precipitation of 2 occurs as small white needles during the reaction. In contrast, when using toluene as solvent, both 1 and 2 are fully soluble at the reaction temperature. Thus, the low solubility of 2 in heptane probably accounts for the superior yield, reaction rate, and purity in comparison to when toluene was used as a solvent. Our method was very promising and was later successfully implemented in the large-scale production of 2. The byproduct 7 is formed in a reaction between 2 and small amounts of water present in the reaction mixture. Water may be present from the beginning of the synthesis (in solvents, etc.) or may enter the reaction vessel during the reaction. In fact, quinoline 7 was isolated in quantitative yield when 2 was heated at 140 °C with 2.0 equiv of water in toluene for 4 h (see the Experimental Section). The same reaction was also studied at 100 °C for 1 h and resulted in an almost quantitative yield of carboxylic acid 6 (contaminated with a small amount of 7). Thus, the primary hydrolysis product of 2 is carboxylic acid 6, which decarboxylates into 7 at elevated temperatures. 1660 J. Org. Chem., Vol. 71, No. 4, 2006
ester
product
yield (%)
1 14 10 12 8
2 15 11 13 9
90 67 0 0 0
a The esters and 2.0 equiv of N-ethylaniline were heated in toluene, and most of the volatiles were distilled off during 6 h.
TABLE 3. Stability of Esters and Amides under Neutral or Basic Conditions compd
conditionsa
16 16
MeOH/100 °C/6 min 0.5 M NaOMe/MeOH/ 100 °C/6 min 1 M NaOH/rt/1 h 1 M NaOH/100 °C/1 h MeOH/100 °C/6 min 0.5 M NaOMe/MeOH/ 100 °C/2 min MeOH/100 °C/6 min 0.5 M NaOMe/MeOH/100 °C/6 min 1 M NaOH/100 °C/1 h MeOH/100 °C/6 min MeOH/140 °C/10 min
16 16 10 10 2 2 2 13 17
product 1 1
% conversion to productb 24 9
no reaction 7 no reaction methyl ester
0 40 0 100
1 no reaction no reaction no reaction no reaction
100 0 0 0 0
a Reactions in methanol were heated in sealed tubes in a microwave oven. Analysis was performed by 1H NMR. The remaining part was starting material.
b
Further Examination of the Reaction Characteristics of Esters and Amides. A study was undertaken to investigate how various esters (Scheme 3) behave in their reactions with N-ethylaniline (Table 2). The esters and 2.0 equiv of Nethylaniline were heated in toluene, and most of the volatiles were distilled off at atmospheric pressure during 6 h. Esters 1 and 145 gave their corresponding N-ethylanilides 2 and 15 in good yields. However, ethyl trans-cinnamate 10, ethyl benzoate 12, and the 4-O-methyl analogue 8 yielded no trace of their corresponding products 11, 13, and 9. The activation energies for formation of 11, 13, and 9 are apparently much higher than can be provided by the temperature in toluene. Even when the reaction temperature was increased by using xylene (bp 138142 °C) instead of toluene as solvent, the products 11, 13, and 9 could not be detected in their respective reaction mixtures. These experiments indicate that β-ketoesters such as 1 and 14, which have enolizable protons in their structures, are reactive and that this structural feature is important for the reaction with the aniline. Therefore, the importance of pH in the methanolysis of some esters and amides under neutral or basic conditions were studied (Table 3). Thus, heating ethyl ester 16 (Scheme 3) at 100 °C in a microwave reactor for 6 min in MeOH led to formation of methyl ester 1 in 24% yield. The same experiment using 0.5 M NaOMe in MeOH yielded only 9% of 1. The opposite result was obtained for ethyl trans-cinnamate 10, which was inert in methanol at 100 °C but was completely transformed to the corresponding methyl ester under basic conditions. Ester 16 is soluble and stable in 1 M NaOH when stirred for 1 h at room temperature, in contrast to most other soluble carboxylic esters, which are hydrolyzed under such conditions. An increase of the temperature to 100 °C for 1 h gave 40% yield of compound 7. Amide 2 is easily solvolyzed into 1 in methanol in quantitative yield but is inert at 100 °C in 0.5 M NaOMe in MeOH or when heated at 100 °C in 1 M NaOH for 1 h. The
Synthesis and ReactiVity of Laquinimod
amide 13 was not solvolyzed to methyl benzoate when heated in MeOH at 100 °C. These experiments further underline that the enol proton, found in compounds 16 and 2, is fundamental for the reaction with methanol and that amide 2 is more reactive than ester 16. Apparently, the exocyclic amide bond in 2 is weak and reactive when the enol proton is present in the molecule whereas it is very stable when the molecule is in its enolate form. Compound 2 is rather acidic (pKa ) 4.2), and the stable enolate form dominates under physiological conditions. In vivo pharmacokinetic studies on compound 2 have not revealed any reactions related to those discussed here, and 2 is metabolized mainly by aryl hydroxylation and oxidative N-dealkylation6 and the half-life elimination in man is 3 days. Compound 17 is similar in structure to 2 but is extremely stable when heated in MeOH at 140 °C and shows no sign of the methanolysis product or any other byproducts. Unlike compound 2, which is readily soluble in water at pH 7.5 (100 mg/mL), compound 17 is virtually insoluble (
