CHAPITRE 2 : DU DÉVELOPPEMENT DE PETITES MOLÉCULES HÉTÉROCYCLIQUES ANTAGONISTES DU
VII. CONCLUSION
Suite à la découverte du composé sr 4321, par criblage in silico de la banque de composé du CERMN, nous avons souhaité développer de nouveaux inhibiteurs de l’interaction VEGF/VEGFR1.
Pour cela une nouvelle voie de synthèse a été mise au point. Bien que présentant de nombreux avantages par rapport à celle développé au CERMN, une limitation de notre voie de synthèse est vite apparue et a fait l’objet d’une étude méthodologique.
Afin d’initier une étude de Relation Structure-Activité, le composé sr 4321 a été re-synthétisé selon notre schéma réactionnel. Cependant, contrairement au composé du CERMN, notre molécule s’est avérée inactive sur le test de déplacement VEGFR1-ECD. Suite à une analyse comparative de ces deux lots de composés, nous avons mis en évidence que la molécule du CERMN était moins pure que la notre. Bien que nous n’ayons pu déterminer la nature exacte des impuretés, nous avons cependant mis en évidence que l’activité observée sur notre test de déplacement n’était pas due à proprement parler aux impuretés. Nous avons montré que ces impuretés n’interagissent à priori pas directement avec le récepteur, mais que par contre elles ont un effet sur la molécule sr 4321. En effet, alors que pur, le composé ne peut se lier au récepteur, en présence d’impureté(s) il est capable de se fixer au VEGFR1 et de perturber l’interaction VEGF/VEGFR1.
En parallèle de cette étude sur le composé sr 4321, le développement de nouveaux analogues de cette molécule a été entrepris. Par réaction de chimie click catalysée au cuivre nous avons obtenu des composés semblant bloquer la fixation du btVEGF sur le récepteur. Cependant, l’analyse approfondie de ces composés nous a conduits à découvrir que la capacité de ces molécules à bloquer in vitro l’interaction VEGF/VEGFR1 était en réalité due à la présence de cuivre résiduelle provenant de notre voie de synthèse.
Au cours de cette première approche, les principales limitations rencontrées ont donc été des problèmes de reproductibilité et de faux positifs. Ceci met donc l’accent sur l’importance de bien
115 connaitre les molécules testées ainsi que les voies de synthèses utilisées afin de pouvoir réaliser les contrôles adéquats lors de l’évaluation in vitro de l’activité et ainsi distinguer une réelle d’inhibition d’artefacts de liaison.
Suite aux résultats obtenus sur le composé sr 4321 ainsi qu’à la constatation que nous n’avions finalement pas obtenu de composé organique actif, le développement de petites molécules anti- angiogéniques a donc été suspendu. Cependant, bien que ce projet n’ai pas abouti, nous avons découvert que l’ion Cu2+ possède un fort pouvoir inhibiteur de l’interaction VEGF/VEGFR1 (CI
50 = 0,97
µM). Sans que son mécanisme d’action ne soit complètement connu, il est décrit que le cuivre a un effet pro-angiogénique. Notre découverte pouvant présenter un début d’explication, nous avons alors poursuivi notre étude afin d’essayer d’élucider son mécanisme d’action et son implication dans le contrôle de l’angiogenèse. Cette étude est présentée dans le chapitre suivant.
117
ARTICLE 2
Thienopyrimidinedione Formation Versus Ester Hydrolysis from Ureido
Carboxylic Acid Methyl Ester
Marie Reille-Seroussi, Raphaël Labruère, Nicolas Inguimbert, Sylvain Broussy,
Nathalie Gagey-Eilstein, Wang-Quing Liu, Michel Vidal, Florent Huguenot
PAPER ▌479
paper
Thienopyrimidinedione Formation Versus Ester Hydrolysis from Ureido
Carboxylic Acid Methyl Ester
Studies on the Hydrolysis of Ureidothienyl Carboxylates
Marie Reille-Seroussi,a Raphaël Labruère,a Nicolas Inguimbert,b Sylvain Broussy,a Nathalie Gagey-Eilstein,a Wang-Qing Liu,a Michel Vidal,*a,c Florent Huguenot*a,d
a Université Paris Descartes, Sorbonne Paris Cité, CNRS UMR 8638, UFR Faculté des Sciences Pharmaceutiques et Biologiques,
4 avenue de l’Observatoire, 75270 Paris cedex 06, France Fax +33(1)43291403; E-mail: florent.huguenot@parisdescartes.fr
b Université de Perpignan Via Domitia, Laboratoire de Chimie des Biomolécules, 52 avenue Paul Alduy, 66860 Perpignan, France c UF de Pharmacocinétique et Pharmacochimie, AP-HP, Hôpital Cochin, 75014 Paris, France
E-mail: michel.vidal@parisdescartes.fr
d Université Paris Descartes, Sorbonne Paris Cité, UFR Biomédicale des Saints-Pères, 45 rue des Saints-Pères, 75270 Paris cedex 06, France Received: 10.10.2012; Accepted after revision: 20.12.2012
Abstract: The basic hydrolysis of ureidothienyl carboxylic esters
was found to depend on the substitution pattern of the ureido moi- ety. While various hindered substituents led to carboxylic acid for- mation, unhindered substituents preferentially resulted in a cyclization step, yielding thienopyrimidinedione derivatives.
Key words: carboxylic acids, esters, hydrolysis, heterocycles,
steric hindrance
Among the broad range of available templates, heterocy- clic scaffolds represent the most promising lead structures for the discovery of novel synthetic drugs. Our current work, focused on antitumor and anti-angiogenic thera- pies, has recently led to the identification of some thio- phene derivatives as ‘hits’ for the Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) binding interface (VEGFR1 plays a key role in angiogenesis, one of the hallmarks of cancer) through an in silico screening pro- cess (Figure 1).1 These candidates could be divided into two chemical families: the (3-carboxy-2-ureido) thio- phene family and the (2-carboxy-3-ureido) thiophene family.2 We chose to study the ureido thiophene carbox- ylic acids because of their properties: indeed, these mole- cules exert various biological effects depending on their substitution pattern.3 As the compounds selected were sufficiently different from previously reported series, we hypothesized that this would preclude binding to targets already described. Furthermore, their selectivity for VEGFR could be adjusted by varying substituents in the ureido moiety.
Figure 1 Selected ‘hit’ molecules
Keeping this in mind, we synthesized 2-ureido-3-carbox- ylic acid thiophene derivatives, which have also been de- scribed in the formation of thienopyrimidines and other analogues.4 Thienopyrimidinediones result from an un- wanted cyclization process that occurs during the synthe- sis of 2-ureido-3-carboxylic acid thiophenes in the presence of an organic or inorganic base. However, sub- stituted thienopyrimidinediones and derivatives, present in the core of many physiologically active agents, display interesting therapeutic properties. Such compounds have been shown to inhibit several enzymes as well as to mod- ulate the activity of many receptors, and could be attrac- tive scaffold molecules for anti-angiogenic therapy.5 Therefore, ester cleavage under acidic conditions is re- quired to avoid cyclization when starting from tert-butyl carboxylic acid ester,6 but the thiophene derivative was not commercially available, and the transesterification re- action or tert-butyl ester formation starting from the acid is sensitive to decarboxylation reaction.7 Here, we de- scribe factors that control the steric and electron with- drawal/donation properties of the thienopyrimidinedione cyclization step. We were interested in exploring this re- action and determining the rules that predict the cycliza- tion, in particular the nature of the R substituent.
We wanted to synthesize the target molecules in large quantities for in vivo tests and animal assays, and also to assess the efficacy of the corresponding esters. The solu- bility of carboxyureido thiophenes being very limited, even in organic solvents such as DMSO or DMF, a small library of ureido carboxylic acid methyl esters were pre- pared from the corresponding amino esters 1, available (Scheme 1) using a phenyl oxy carbonyl (POC) strategy, instead of using thiaisatoic anhydride ring opening.8 In ac- cordance with the synthetic strategy shown in Scheme 2, carbamate 2 was prepared in large amounts by the con- densation of phenyl chloroformate with methyl 2-amino- 3-thiophenecarboxylic ester without the addition of a base, to avoid the release of the phenolate from the carba- mate. Compound 2 was then converted into ureas 3a–n by the substitution of the phenolate moiety by alkylamines, with moderate to good yields. Next, hydrolysis under
S CO2H NH O H N S CO2H NH O H N OMe SYNTHESIS 2013, 45, 0479–0490
Advanced online publication: 17.01.20130039-78811437-210X
DOI: 10.1055/s-0032-1318100; Art ID: SS-2012-T0791-OP © Georg Thieme Verlag Stuttgart · New York
480 M. Reille-Seroussi et al. PAPER
Synthesis 2013, 45, 479–490 © Georg Thieme Verlag Stuttgart · New York
basic conditions was performed to yield the targeted car- boxylic acids.
Scheme 1 Retrosynthetic strategy for 2-ureido-3-carboxylic acid
thiophenes
Scheme 2 Strategy for the synthesis of ureido thiophenecarboxylic
acids. Reagents and conditions: (a) PhCO2Cl, THF, r.t., 24 h, 82%;
(b) RNH2, Et3N, CHCl3, reflux, 24 h, 60–91%; (c) (i) KOH (1 M),
MeOH–H2O (1:1), Δ, 2 h, (ii) concd HCl, 0–86%.
By using a refluxing solution of KOH in methanol, com- pound 3a, in which R is a tert-butyl moiety, was entirely converted to the carboxylic acid 4a in good yield, without a trace of the thienopyrimidinedione (Table 1, entry 1). Notably, refluxing the aqueous KOH (1 M) solution was necessary to hydrolyze the methyl carboxylic ester to the corresponding acid, the use of 1 M LiOH, NaOH, or KOH at room temperature for three days being ineffective. At the outset, various linear aliphatic chains, branched chains, cyclic substituents, and aromatic rings were used. All compounds were prepared according to the procedure described for 4 and 5 (Table 1). For methyl-2-ureido-3- carboxylic acid esters 3a–n, the steric size of the R sub- stituent determined the ratio of pyrimidinedione to car- boxylic acid: for ‘linear’ groups (e.g., in which CH2 was bounded to NH), no traces of carboxylic acid was detected (Table 1, entries 9–14). Carboxylic acids were formed with more hindered substituents (Table 1, entries 1–6). A comparison of the cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups (Table 1, entries 7, 6, 5, and 4, re- spectively) showed that acid formation was clearly corre- lated with ring size. Indeed, larger substituents yielded a higher percentage of carboxylic acids. Unexpectedly,
when a phenyl substituent was linked to nitrogen, thieno- pyrimidinedione was the only product obtained in good yield (80%) (Table 1, entry 8), although the size of this substituent is comparable to that of the cyclohexyl group. As the first step of the cyclization process is the formation of an amide anion stabilized by the phenyl ring, this would favor the formation of the pyrimidinedione ring by attack- ing the ester moiety. Thus, ring formation is favored by unhindered or electron-withdrawing substituents at the ureido site.
Subsequently, the limitations of this steric effect were tested using methyl ureido arylcarboxylic esters contain- ing different substituents (Table 2), in accordance with the strategy for the synthesis shown in Scheme 3. The thieno- pyrimidinedione/carboxylic acid ratio was affected by the size of the substituent for the all four series tested. In all cases, a tert-butyl substituent afforded the carboxylic acid derivative, whereas thienopyrimidinediones were obtained with the 4-nitrobenzyl [CH2(4-O2NC6H4)] substituent.
CO2H NH O H N R O H N O O CO2t-Bu NH O H N R O N O R H N S S S S S CO2Me NH2 S CO2Me NH O NH a S CO2Me NH O OPh b S CO2H NH O NH c 1 2 3a–n 4a–n R R
Table 1 Hydrolysis of Methyl 2-Ureido-3-carboxylate Thiophenes
Entry R Ratioa 4/5 Yield (%)b
1 3a t-Bu 100:0 78 2 3b CHEt2 100:0 79 3 3c s-Bu 100:0 82 4 3d c-C6H11 100:0 86 5 3e c-C5H9 85:15 70 6 3f c-C4H7 45:55 82 7 3g c-C3H5 0:100 72 8 3h Ph 0:100 80 9 3i CH2(4-O2NC6H4) 0:100 67 10 3j CH2C≡CH 0:100 66 11 3k CH2CH2OH 0:100 71 12 3l CH2CH2CN 0:100 78 13 3m CH2CH2(4-O2NC6H4) 0:100 88 14 3n CH2CH2(4-MeOC6H4) 0:100 84 a Determined by 1H NMR analysis of the crude mixture. b Sum of 4 and 5 as isolated product.
S CO2Me NH O H N R S CO2H NH O H N R S N H N O O R 4a–n 5a–n 3a–n + i) KOH (1 M) MeOH, Δ ii) concd HCl
PAPER Studies on the Hydrolysis of Ureidothienyl Carboxylates 481
© Georg Thieme Verlag Stuttgart · New York Synthesis 2013, 45, 479–490
Scheme 3 Strategy for the synthesis of ureido thiophenecarboxylic
acids. Reagents and conditions: (a) PhCO2Cl, THF, r.t., 74–100%; (b)
RNH2, Et3N, CHCl3, reflux, 24 h, 24–91%.
For each thiophene series, the ratios of the resulting com- pounds were affected by the steric size of the substituent. The most bulky substituents yielded a high percentage of carboxylic acids. In general, linear alkyl substituents trig- gered cyclization to thienopyrimidinediones. When a phe- nyl core was involved, we never observed a mixture of thienopyrimidinediones and carboxylic acids. Except for very bulky groups, thienopyrimidinediones were prefer- entially formed (Table 2, entries 13–16). Interestingly, the relative position of the sulfur atom in the thiophene moi- ety of methyl ureidocarboxylate has a strong influence on
the cyclization reaction. For example, three different ra- tios were obtained with the cyclobutyl substituent. Only carboxylic acid 4q was obtained when starting from 3q (Table 2, entry 7), whereas a mixture of thienopyrimidin- ediones and carboxylic acids were obtained from 3f and 3u (Table 2, entries 3 and 11).
In all cases, refluxing was required to obtain the conver- sion of esters, indicating that the hydroxide anion was not sufficiently nucleophilic to be added directly to the car- bonyl of the ester, at room temperature. The reaction me- dium displayed a yellow color only with the deprotonation of nitrogen of the urea group, this species being stabilized by the conjugation of the electron pair with the aromatic ring and the carbonyl of the urea. Taking into account the addition of the amide carbonyl group of the ester, the first factor affecting carboxylic acid formation is the steric hin- drance asserted by the R substituent (Scheme 4). It is pos- sible that the carboxylic acid is formed by the ring opening of the thienoxazinone (benzoxazinone, respec- tively to the benzene core) by the hydroxide anion. An ad- ditional effect that promotes cyclization can be seen with the 3-ureido-2-carboxythiophene core and is due to the electron-withdrawing and inductive effect of sulfur, which increases the electrophilicity of the carbonyl group of the carboxylic ester. In these structures, the propagation of the inductive effect occurs when the sulfur atom is clos- est to the carbonyl group.
CO2Me NH2 CO2Me NH O OPh a 2, 6, 7, 8 Ar Ar CO2Me NH O H N b 3a–z Ar R CO2Me NH2 Ar where Ar : S S S
Table 2 Hydrolysis of Methyl Aryl-2-ureido-3-carboxylates
Entry Ar R Ratioa 4/5 Yield (%)b
1 2 3 4 3a 3d 3f 3i t-Bu c-C6H11 c-C4H7 CH2(4-O2NC6H4) 4a/5a 4d/5d 4f/5f 4i/5i 100:0 100:0 45:55 0:100 78 86 82 67 5 6 7 8 3o 3p 3q 3r t-Bu c-C6H11 c-C4H7 CH2(4-O2NC6H4) 4o/5o 4p/5p 4q/5q 4r/5r 100:0 100:0 100:0 0:100 75 79 64 81 9 10 11 12 3s 3t 3u 3v t-Bu c-C6H11 c-C4H7 CH2(4-O2NC6H4) 4s/5s 4t/5t 4u/5u 4v/5v 100:0 67:33 28:72 0:100 80 81 76 72 13 14 15 16 3w 3x 3y 3z t-Bu c-C6H11 c-C4H7 CH2(4-O2NC6H4) 4w/5w 4x/5x 4y/5y 4z/5z 100:0 10:90 0:100 0:100 71 96 63 90
a Determined by 1H NMR analysis of the crude mixture. b Sum of 4 and 5 as isolated product.
CO2Me NH O H N R CO2H NH O H N R N H N O O R i) KOH (1 M) MeOH, Δ 3a,d,f,i,o–z + Ar Ar Ar 4a,d,f,i,o–z 5a,d,f,i,o–z ii) concd HCl S S S
482 M. Reille-Seroussi et al. PAPER
Synthesis 2013, 45, 479–490 © Georg Thieme Verlag Stuttgart · New York
Scheme 4 Plausible mechanism of thienopyrimidinedione 5 forma-
tion
An additional factor influencing the process is an intramo- lecular nonbonded [1,4]-type S···O interaction, pinpointed in several thiophene and thiazole derivatives.9 This inter- action would promote the addition of nitrogen by increas- ing the electrophilicity of the carbonyl group and the rotation of the ester group to more easily attain the Bürgi– Dunitz angle. The nonbonded intramolecular [1,5]-type S···O interaction in the 2-ureidothiophene structure could explain the difference in reactivity of 3f when compared to 3q. After deprotonation, the excess negative charge would deter nonbonded interaction and promote the rota- tion of the urea group to allow the cyclization process. In the absence of this nonbonded interaction (for 3o–q), the energy required for the rotation would favor the hydroly- sis of the ester to the corresponding carboxylic acid. To overcome the cyclization process, the possibility using an enzymatic reaction was checked. For the most chemi- cally sensitive compound 3j, which is also the most attrac- tive from the point of view of an easy access to structural diversity via click chemistry, a rapid screening of proteo- lytic enzymes was carried out (Table 3). We used en- zymes frequently described for their hydrolytic activity towards esters. Subtilisin, lipase A and horse pancreatic
lipase are serine proteases/esterases and act via an acyla-
tion-deacylation mechanism. Moreover, these enzymes have already permitted the hydrolysis of some methyl es- ters.10 Pig liver esterase (PLE) was the most effective en- zyme for the full conversion of the ester to the carboxylic acid 4j (Table 3, entry 7): no trace of thienopyrimidinedi- one formation was observed by 1H NMR analysis of the crude reaction mixture. It is also worth noting that the oth- er enzymes were incapable of converting 3j to 4j, al- though this was theoretically feasible based on results with benzoxazinones.11 The first problem that we faced was finding a suitable solvent in which ester 3j was solu- ble and that would not undermine the activity of the en- zyme. The best compromise was the use of a phosphate buffered saline (PBS) supplemented with 10% DMSO. At higher concentrations of DMSO, PLE was found to be de- natured. When the reaction was performed on a larger scale with the same amount of enzyme, the yield dropped rapidly (Table 3, entry 8); the conversion was complete because of the formation of compound 5j due to the basic- ity of the buffer.
In summary, we have studied the basic hydrolysis of aro- matic ureido carboxylate derivatives. The nature of the ar- omatic ring affected the cyclization reaction leading to the production of thienopyrimidinediones. For the same aro- matic ring, the nature of the substituted urea moiety deter-
mined the relative proportion of carboxylic acid and thienopyrimidinedione produced. Bulky substituents yielded higher percentages of carboxylic acids, while less hindered substituents or electron-withdrawing substitu- ents promoted thienopyrimidinedione formation. We are continuing our study on the hydrolysis catalyzed by en- zymes, in particular regarding the identification of a given compound as a substrate for an enzyme.
Reagent grade solvents (SDS-Carlo Erba), alkyne reagents (Alfa Aesar), NMR solvents (Eurisotop), and all other reagents (Aldrich, Fluka, Acros) were purchased ready to use. Organic solutions were concentrated under vacuum using a Büchi rotary evaporator. Chro- matographic purification of products was carried out using 32–64 mesh silica gel. TLC was performed on 0.25 mm silica gel 60-F plates (EM reagents). The developed chromatogram was visualized by fluorescence. IR spectra were recorded on a Nicolet 510FT-IR.
1H and 13C NMR spectra were recorded on a Bruker spectrometer
(250, 300, 400, 500 MHz and 63, 75, 100, 125 MHz, respectively), and were internally referenced to residual protonated solvent sig- nals. Data for 1H NMR are reported as follows: chemical shift (δ
ppm), multiplicity (standard abbreviations were used to indicate the spin multiplicities), coupling constant (Hz), integration. Data for
13C NMR are reported in terms of the chemical shift. Assignments
shown as C4 refer to quaternary carbons. Melting points were deter-
mined with a Köfler apparatus or were measured on a Stuart SMP3 melting point apparatus and are uncorrected. Mass spectrometry spectra were recorded on a Waters ZQ 2000 spectrometer. High-
R N N O OH R OMe N N OH O N H N O O R Ar Ar Ar Δ
Table 3 Enzymatic Hydrolysis of 3j
Entry Enzyme Conditions Yield (%) Conv 1 Subtilisin cyclohexane–MeOH, PBS (0.1 M) – 0 2 Subtilisin PBS–DMSO (9:1) – 0 3 PLAPa PBS–DMSO (9:1) – 0 4 PLAP PBS–DMSO (3:1) – 0 5 CalAb PBS–DMSO (9:1) – 0 6 HPLc PBS–DMSO (9:1) – 0 7 PLEd PBS–DMSO (9:1) 92e 100% 8 PLE PBS–DMSO (9:1) 52f 100% 8 PLE PBS–CHCl3 (3:2) – 0 9 PLE PBS–toluene (2:1) – 0
a Pig liver acetone powder.
b Lipase A from the yeast Candida antarctica. c Horse pancreatic lipase.
d Pig liver esterase.
e Reaction performed with 30 mg of 3j. f Reaction performed with 500 mg of 3j.
S CO2Me NH O H N S CO2H NH O H N 4j 3j enzyme buffer solvent
PAPER Studies on the Hydrolysis of Ureidothienyl Carboxylates 483
© Georg Thieme Verlag Stuttgart · New York Synthesis 2013, 45, 479–490
resolution mass spectra were recorded on a Bruker MicrO-Tof-Q 2 spectrometer at the CRMPO (Rennes, France).
Carbamates 2, 6, 7, 8; General Procedure
To a stirred solution of the appropriate aromatic amine (2.0 mmol) in THF (10 mL) was added phenyl chloroformate (251 μL, 2.0 mmol) in one portion. The reaction mixture was stirred at r.t. until complete consumption of starting materials as monitored by TLC (eluent: cyclohexane–EtOAc 5:1), then diluted with H2O (20 mL)
and EtOAc (10 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), and evaporated to dryness un-
der reduce pressure. The products were purified by column chroma- tography on silica gel using cyclohexane–EtOAc (5:1) as eluent.
Methyl 2-[(Phenoxycarbonyl)amino]thiophene-3-carboxylate (2)
Yield: 600 mg (quant); white solid; mp 126–127 °C.
IR (neat): 3286, 2953, 2923, 1740, 1680, 1554, 1491, 1441, 1253, 1208, 1191 cm–1. 1H NMR (500 MHz, CDCl 3): δ = 3.93 (s, 3 H, CH3), 6.76 (d, 3JH,H= 5.5 Hz, 1 H, CHAr), 7.2–7.3 (m, 4 H, CHAr), 7.43 (m, 2 H, CHAr), 10.47 (br, 1 H, NH). 13C NMR (125 MHz, CDCl 3): δ = 51.7 (CH3), 112.4 (C4), 115.6 (CH), 121.3 (2 × CH), 124.3 (CH), 126.1 (CH), 129.5 (2 × CH), 150.1 (C4), 150.4 (C4), 151.4 (C4), 165.7 (C4). MS (ESI+): m/z = 300 [M + Na]+. Methyl 4-[(Phenoxycarbonyl)amino]thiophene-3-carboxylate (6)
Yield: 570 mg (95%); white solid; mp 93–94 °C.
IR (neat): 3312, 2953, 1754, 1674, 1578, 1282, 1186 cm–1. 1H NMR (400 MHz, CDCl 3): δ = 3.94 (s, 3 H, CH3), 7.2–7.3 (m, 3 H, CHAr), 7.4–7.5 (m, 2 H, CHAr), 7.68 (d, J = 3.5 Hz, 1 H, CHAr), 8.08 (d, J = 3.5 Hz, 1 H, CHAr), 9.70 (s, 1 H, NH). 13C NMR (100 MHz, CDCl 3): δ = 51.8 (CH3), 108.3 (C4), 121.1 (CH), 121.4 (2 × CH), 125.5 (CH), 129.2 (2 × CH), 132.6 (CH), 136.0 (C4), 150.4 (C4), 151.6 (C4), 163.9 (C4). MS (ESI+): m/z = 300 [M + Na]+. Methyl 3-[(Phenoxycarbonyl)amino]thiophene-2-carboxylate (7)
Yield: 570 mg (95%); white solid; mp 117–118 °C. IR (neat): 3340, 2953, 1742, 1693, 1529, 1255, 1200 cm–1. 1H NMR (250 MHz, CDCl 3): δ = 3.92 (s, 3 H, CH3), 7.2–7.3 (m, 3 H, CHAr), 7.3–7.4 (m, 2 H, CHAr), 7.48 (d, J = 5.5 Hz, 1 H, CHAr), 7.90 (d, J = 5.5 Hz, 1 H, CHAr), 9.88 (br, 1 H, NH). 13C NMR (63 MHz, CDCl 3): δ = 51.9 (CH3), 109.5 (C4), 121.1 (CHAr), 121.4 (CHAr), 125.7 (CHAr), 129.3 (CHAr), 131.7 (CHAr), 144.3 (C4), 150.4 (C4), 151.2 (C4), 164.4 (C4). MS (ESI+): m/z = 316 [M + K]+. Methyl 2-[(Phenoxycarbonyl)amino]benzoate (8)
Yield: 436 mg (74%); white solid; mp 101–102 °C.
IR (neat): 3268, 2953, 1756, 1695, 1588, 1528, 1267, 1190 cm–1. 1H NMR (250 MHz, CDCl 3): δ = 3.96 (s, 3 H, CH3), 7.08 (t, J = 7.5 Hz, 1 H, CHAr), 7.2–7.3 (m, 3 H, CHAr), 7.3–7.5 (m, 2 H, CHAr), 7.56 (d, J = 7.5 Hz, 1 H, CHAr), 8.06 (d, J = 8.0 Hz, 1 H, CHAr), 8.46 (d, J = 8.0 Hz, 1 H, CHAr), 10.91 (br, 1 H, NH). 13C NMR (75 MHz, CDCl 3): δ = 52.4 (CH2), 114.9 (C4), 118.9 (CH), 121.7 (CH), 122.1 (CH), 125.6 (CH), 129.3 (CH), 130.9 (CH), 134.7 (CH), 141.3 (C4), 150.5 (C4), 151.8 (C4), 168.6 (C4). MS (ESI+): m/z = 294 [M + Na]+.
Ureas 3a–z; General Procedure
A mixture of carbamate 2 (277 mg, 1.0 mmol), Et3N (418 μL, 3.0
mmol), and the selected amine (1.3 mmol) in CHCl3 (10 mL) was
refluxed for 24 h. The cooled reaction mixture was diluted with CHCl3 (10 mL) and washed with sat. aq NaHCO3 (20 mL) and brine
(20 mL). The organic phase was dried (MgSO4) and evaporated to
dryness. Purification by flash chromatography (3:1 cyclohexane– EtOAc) gave the respective pure product.
Methyl 2-[(tert-Butylcarbamoyl)amino]thiophene-3-carboxyl- ate (3a)
Yield: 218 mg (78%); yellowish solid; mp 178–179 °C. IR (neat): 3335, 2966, 1660, 1537, 1502, 1245, 1209 cm–1.