Synthesis and Spectroscopic Study of Plant Growth Regulators Phenylpyridylureas An "Agrorganic" Undergraduate Laboratory Experiment Alexandre Hocquet and Jacques Tohier ENS Cachan, Depatiement de chimie, F-94235 Cachan Cedex, France Josette Fournier CREPA, 8, rue Becquerel, F-49040 Beaucouze, France
I n agropharmaceutical synthesis laboratories, new molecules are synthesized everyday, hut scarce are the ones t h a t pass successfully the numerous toxicological tests. One synthesis strategy consists in focusing on a particular family that possesses the desired biological activity (pyridylphenylureas have cytokinin activity (1, 21, that is to say they regulate the growth of plants as does cytokinin, a plant growth hormone (3)),and then make a few substituent changes, in order to increase the possibilities of obtaining a n authorized active substance (4). I n this context, we will prepare and study the 'H NMR spectra of three molecules: N-phenyl-N'-2-pyridylurea(11, N-4-chlorophenyl-N'-2-pyridylurea(21, and N-4-chlorophenyl-N'-3pyridylurea (31, shown in Figure 1. These three molecules belong to the pyridylphenylurea family. The variation consists in the presence or lack of a C1 substituent on the phenyl group, and the position of the N in the pyridyl group. Differences and similarities will be checked during synthesis and analysis of 'HNMR spectra. We can illustrate
..
ing oven too. I n order not to waste a graduated pipet, a pencil mark can be done on a simple one, corresponding to 1.5 mL. Phenslisocyanates must be manipulated with care, due to thk possibility of irritations caused by vapors. Nevertheless. these substrates are common in ortranic svnthesis, especially in pesticide chemistry wherLthey are used to synthesize a lot of ureas and carbamates (5,6). The three ureas are synthesized following the same path. The reagents corresponding to each urea are listed in Table 2. Table 1. Physical Properties of Substrates TI("
phenyiisocyanate 4-chiorophenyiisocyanate 2-aminopyridine 3-aminoovridine
c)
MM (g morl)
30 29-31
119.12 153.57
55-58 6C43
94.12 94.12
(qualitatively)the influence ofthe C1 suhstituent upon the synthesis reaction. the influence of the N position upon the internal structure of the molecule.
As a whole. this could represent an undereraduate comparative synthesis and anaiysis of biologicaliy active molecules, suitable for a n introductory lab session. Synthesis Disubstituted ureas are traditionally synthesized by adding an amine to a n isocyanate (Fig. 21, in a one-step condensation reaction. The apparatus and reagents used are conventional ones. The manipulation is accessible to freshmen. Solvents and chemicals used are l,4-dioxane and absolute ethanol of Normapur grade, purchased from Prolabo; phenylisocyanate, 4-chlorophenylisocyanate, 2-aminopyridine, and 3-aminopyridine of synthesis grade, obtained from Merck. Characteristics of substrates are mentioned in Table 1. 3 At ambient temperature, phenylisocyanates are crystalline. For ~ r a c t i c areasons l (volume measurements, diluFigure 1. Studied phenyipyridyiureas. tion rate), the isocyanate is manipulated above melting point, so that it is in a liquid form. The isocyanate flask is set in a water bath or drying oven, a t 50 "C. Caution: The stopper m u s t be held @* co sealed because of the irritating vapors of 0 the isocyanate, which hydrolyzes quickly. Also, to prevent crystallization of the isocyanate, the pipet should be placed in the dry- Figure 2. Synthesisof phenylpyridylurea from aminopyridine and phenyiisocyanate. 1092
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After a period of time varying from 1to 50 s, depending on the urea to be synthesized, a white precipitate appears, and the reaction can be regarded as completed with a satisfactory yield after 1h. If the mixture is too bulky, a few milliliters of solvent can be added. The mixture is then filtered on a glass frit funnel. The recrystallization solvent is ethanol. A suficient amount of solvent is poured into a 250-mL flask. Areflux condenser is adapted to the apparatus. The crude product is then poured in boiling solvent. In Table 3, times of precipitation during synthesis, sufficient volumes for recrystallization and yields obtained in our sessions are listed. Also, these three molecules can be characterized by their melting point.
One gram of nminopyridine (10 mmol) is poured into a 50-mL conical flask containing 10 mL of dioxane and the resulting mixture is stirred with a magnetic stirrer until the amine is completely dissolved. Then, 10 mmol of the isocyanate (which correspond roughly to 1.5 mL) are added to the mixture, as quickly as possible, under a fume hood to prevent the inhalation of irritating vapors. A reflux condenser is adapted after the mixing of the reagents. The dilution of the isocyanate and the reaction both are exothermic, and the slight rise of temperature induced could evaporate part of the solvent. Table 2. Corresponding Substrates and Products
am~ne phenyltsocyanate 2-aminopyridme chlorophenyl~socyanate 2-aminopyridine chlorophenyl~socyanate 3-aminopyndine isocyanate
1 2
Mechanisms
The only difference between 1and 2 is the chlorine located on the phenyl group, in a para position, on 2. In order to compare the reactions rates, precipitation 3 times are observed. The faster precipitation occurs, the sooner the urea is synthesized. However, this correlation devends on the solubilities of the Table 3. Svnthesis Characteristics products in the reaction solvent. Compounds 1 precipitation recrystallization yield (%) Tt (" C ) MM(g inor') and 2 have similar solubilities in dioxane; thus, time (s) solvent volume (mL) precipitation rate and reaction kinetics can be 1 50 150 52% 194 213.24 correlated. Comparison of times of precipitation (Table 100 55% 214 247.69 2 instantaneous 3) ofboth 1and 2 shows that the inductive effect 30 21% 224 247.69 3 10 of the C1 substituent is of primary significance. The reaction mechanism corresponds to a nucleophilic addition of an aminopyridine to the phenylisocyanate (Fig. 3). The carbon of the carbonvl . erouo ., . is suooosed . . to be more electroohilic when a CI substituent is present, due to its electron-withdrawing inductive effect (71.As a matter of fact, the reaction evolves faster for 2.
.
,,
Spectral Analysis
II CI1
.
\01
Figure 3. Mechanism of the condensation of aminopyridine and phenylisocyanate.
The three molecules are characterized by their 'H NMR spectra, measured on a Bruker AC200 soectrometer. in deuterated DMSO as the solvent. NMR spectra of the three molecules show different peaks corresponding to both aromatic moups . . around 7.5 ppm .. (8). Signals corresponding to protons bonded to the nitrogen atoms of the urea function yield two separate peaks (with a difference of one or more than one ppm) for 1 (9.4 and 10.4 ppm)
,
@I Hq 'g
u,
f-
2
S
;i I
Yl,Yty$iy,, ,
100
90
Flgure 4 NMR spectrum of 1
,
80
100
90
80
70
70
F~gure5 NMR Spectrum of 2 Volume 71
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Comparison ofthe solubilities of2 and 3 in ethanol supports this hypothe~is.Owing to entropic contribution, as it &uallv oc&rs (12). this intramolecdar hvdroeen bond is assumkd to be stronger than usual inte&ole&lar hydrogen bonds such a s the ones formed between the urea and ;he solvent. Thus, 2 is less tied to the solvent and is, therefore. less soluble in ethanol than 3 (see volumes for recrvstallization). Like ethanol. dioxane is a hvdrogen bond acceotor solvent. Thus, 3 ismore soluble in dioxane too. ~ h i s ' f a c act counts for the poor yield of the synthesis of 3 compared to
- -
2 -.
This intramolecular hydrogen bond also is responsible for differences of retention by soil. Compound 2 is less available for hydrogen bonding with the argilo-humic complex of soil. I t is, thus, less adsorbed by soil than 3. Two molecules of the same family, with similar biological activities (1,2)show different behaviors in ecotoxicological studies (13). Literature Cited 1. Vassilev,G.; Izanush,P.;Yonova, P;Dimcheva,Z.Dokl. Bolg Akod Nauk. I987,40, 1W-112. 2. Yamsguehi, K.; Shudo, K J Agr Fond C k m . 1991.39,793-796. 3. Koshimizu, K; Iwamura, H. CkomistryofPIonl Hormones; Mahashi. N. Ed., CRC Press: Boca Raton. FL. 1986, pp 153-199. 4. Plummer, E. Pesticide Chemistry; Frehae, H.Ed., VCH: Weinheim, Germany. 1991, ""Sl-fi" r- -- --.
5. Geissbuhler, H.; Martin, H.;Voss, GHarbiCrdes; Keamey, P andKaufman, D., Ed&, Marcel Dekker: NewYork, 1976, Vol. 1, Chapter% pp20S291. Cultures ef Techniques: Nantes, France, 1988. 6. Fournier, J. ChimiCdPspestieid~s; 7. Issaer, N. Phrsccal O w n & Chemistry; Wiley: New York, 1989, pp 131-143. 8. C1erc.T.: Pretsch, E.; Seibl. J.; Simon, W. SpdrolDofofor SIruclurpMf~nninafion ofOrganic Compounds, 2nd ed.; Sp"nge~Ver1ag: New York. 1989. 9. Joe8ten.M. J Chem Educ 1982,59.362366 10. Sudha,L. ;Sathyanarayana, N. J. Mol. Strub. 1985,131.141-146. 11. Ouahab, L.: LeMagueres, P: Hoquet, A : Fournier. J . submitted for publication in Ada Cryst C. : I andZshow Z, E mnfipation around the two partial double N-C bonds of the urea moiety. auowing intramolemlar hydrogen bonding; whereas, 3 oresents 2. Z confieuration IRz. 51.
Figure 6. NMR Spectrum of 3.
(Fig. 4) and 2 (9.4 and 10.6 ppm) (Fig. 51, whereas they appear very close for 3 (8.8 and 8.9 ppm) (Fig. 6). These downfield N-H peaks are not surprising for aromatic substituted ureas (8).This is due to both the effects of vicinitv of electrone~ativeatoms (urea group) a n d of t h e ring current of the aromatic H-N'n) moups. However, two distinctly separate signals for similar protons is much less common. This can be at\ tributed to intramolecular hydrogen bonding (9). 0 H This hvdroeen bond involving- a hvdrogen " " - of the urea group and the nitrogen of the pyridyl group forms a 3 2 six-membered ring (Fig. 7). I t cannot exist in 3 because the pyridyl nitrogen is too far from the urea Z,E configuration Z,Zconfiguration hydrogen (10). This structure has been confirmed for Figure 7. Intramolecular hydrogen bond for 2 but not for 3. 2 and 3 by X-ray analysis (11).
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