N-(3-Acyloxy-2-benzylpropyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea Analogues:Novel Potent and High Affinity Antagonists and Partial Antagonists of the Vanilloid Receptor
Jeewoo Lee,*,?Jiyoun Lee,?Myungshim Kang,?Myoungyoup Shin,?Ji-Min Kim,?Sang-Uk Kang,?Ju-Ok Lim,?Hyun-Kyung Choi,?Young-Ger Suh,?Hyeung-Geun Park,?Uhtaek Oh,?Hee-Doo Kim,?Young-Ho Park,§
Hee-Jin Ha,|Young-Ho Kim,|Attila Toth,⊥Yun Wang,⊥Richard Tran,⊥Larry V.Pearce,⊥
Daniel J.Lundberg,⊥and Peter M.Blumberg⊥
Research Institute of Pharmaceutical Sciences,College of Pharmacy,Seoul National University,Shinlim-Dong,Kwanak-Ku, Seoul151-742,Korea,College of Pharmacy,Sookmyung Women’s University,Seoul140-742,Korea,AmorePacific R&D Center, Yongin-Si,Kyounggi-Do449-900,Korea,Digital Biotech,Ansan,Kyounggi-Do425-839,Korea,and Laboratory of Cellular Carcinogenesis and Tumor Promotion,Center for Cancer Research,National Cancer Institute,NIH,Bethesda,Maryland20892 Received February24,2003
Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor(VR1)agonists
with the alkylsulfonamido group provides a series of compounds which are effective antagonists
to the action of the capsaicin on rat VR1heterologously expressed in Chinese hamster ovary
(CHO)cells.In particular,compound61,N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N′-
[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin,
displayed a K i value of7.8nM(compared to520nM for capsazepine and4nM for5-iodoRTX),
and showed excellent analgesic activity in mice.Structure-activity analysis of the influence
of modifications in the A-and C-regions of4-methylsulfonamide ligands on VR1agonism/
antagonism indicated that3-fluoro substitution in the A-region and a4-tert-butylbenzyl moiety
in the C-region favored antagonism,whereas a3-methoxy group in the A-region and3-acyloxy-
2-benzylpropyl moieties in the C-region favored agonism.
Introduction
The vanilloid receptor VR11is a cation permeable ion channel present on polymodal nociceptors that is acti-vated by protons,heat,2and ligands such as capsaicin (CAP),3the pungent ingredient in chilli peppers,and resiniferatoxin(RTX),4a natural diterpene isolated from the cactuslike succulent Euphobia resinifera.VR1has been cloned from rat dorsal root ganglia(DRG)5and more recently from the human.6It is a member of the Trp family of channel proteins,7oligomerizing as a tetramer8and functioning as a relatively nonselective cation channel with some preference for calcium ions.9 The vanilloid receptor is expressed predominantly on unmyelinated pain-sensing nerve fibers(C-fibers)and smallΑδfibers in the dorsal root,trigeminal,and nodose ganglia.The activation of the vanilloid receptor by pungent agonists triggers cation influx resulting in excitation of primary sensory neurons,action potentials, and ultimately the central perception of pain.Following excitation,the C-fiber sensory neurons may become desensitized,and this desensitization forms a basis for the therapeutic use of vanilloid receptor agonists.1,10 Potential therapeutic applications include chronic pain, such as that associated with diabetic neuropathy,post-herpetic neuralgia,arthritis and cluster headache,urologic problems such as detrusor hyperreflexia and bladder hypersensitivity,and pruritus.1
A strategy complementary to desensitization of C-fiber sensory neurons by VR1agonists is the blocking of pain-producing endogenous agonists by VR1antago-nists.Proof of principle for this approach has been pro-vided with capsazepine and with some N-alkylglycine trimers.11,12Because of the long duration of desensitiza-tion following treatment with VR1agonists,antagonists may be of particular utility where short-term blockade of VR1is desired.Whereas several agonists are cur-rently marketed(e.g.,capsaicin)or undergoing clinical trial(e.g.,resiniferatoxin,13DA-5018,14SDZ-24966515), only a few VR1antagonists have been described so far. Capsazepine,16the most extensively characterized,has only modest potency and is somewhat nonspecific,also antagonizing voltage-sensitive calcium channels and the nicotinic cholinergic receptor.17,18The N-alkylglycine trimers appear to function as noncompetitive antago-nists,presumably as channel blockers,with potencies around1μM.12Iodo-RTX,prepared semisynthetically from RTX by iodination,was also reported to display potent antagonism in rat and human VR1.19
The identification of RTX as a novel vanilloid with a binding potency approximately4orders of magnitude greater than that of capsaicin(RTX:K i)0.13nM, CAP:K i)1,700nM in CHO/VR1)20suggested a more complex pharmacophore for vanilloids than that ap-preciated previously.In the basis of part on previously published SAR studies on RTX,21we have proposed a hypothetical pharmacophore model for interaction with the capsaicin binding site of VR1in which four func-
*To whom correspondence should be addressed.Phone:82-2-
880-7846.Fax:82-2-888-0649.E-mail:jeewoo@snu.ac.kr.
?Seoul National University.
?Sookmyung Women’s University.
§AmorePacific R&D Center.
|Digital Biotech.
⊥National Cancer Institute.
3116J.Med.Chem.2003,46,3116-3126
10.1021/jm030089u CCC:$25.00?2003American Chemical Society
Published on Web05/30/2003
tional groups,4-hydroxy-3-methoxyphenyl,C 20-ester,C 3-keto,and orthophenyl,represent principal pharma-cophores.On the basis of this model,we have syn-thesized and recently described the ultrapotent VR1agonists 1and 2,with K i values of 19nM and 11nM,respectively,in a [3H]RTX binding assay on DRG neurons;the compounds are thus approximately 280-and 480-fold more potent than capsaicin,respectively,for receptor binding.22The Novartis group 3,24has des-ignated the structural regions of vanilloids as regions A -C shown in Figure 1,and our current work involves replacement of the isodecenoyl moiety of the C-region with a 3-acyloxy-2-benzylpropyl moiety,which we de-signed based on our RTX-derived pharmacophore model and which represents a motif conferring significantly enhanced binding affinity in RTX competition for VR1compared to capsaicin.
The series of compounds described here arise out of our ongoing program to find potent VR1agonists and antagonists.This series is based on the structural modification on the A-region of the potent agonists,N -(3-acyloxy-2-benzylpropyl)-N ′-(4-hydroxy-3-methoxybenzyl)-thioureas,which we had previously shown to have high affinities for rat VR1.22Extensive structure activity studies on the capsaicinoids have indicated that the 4-phenolic hydroxyl group represents a crucial pharma-cophore although it also contributes to the metabolic lability in vivo.23,24We reasoned that isosteric replace-ment of the pungent phenolic OH group in our potent agonists might maintain their strong affinities,change their metabolic lability,and modify the biological char-acteristic of the ligands.The alkylsulfonylamido group has been utilized as a bioisostere of the phenolic OH because the acid dissociation constant of the NH group in alkylsulfonamides is close to that of the phenolic OH group (both are approximately p K a )9).Its effectiveness as a bioisostere implies that the NH portion of the alkylsulfonamide hydrogen bonds with a receptor site by aligning itself in a manner closely approximating the phenolic OH group with respect to both bond distances and bond angles.25Consequently,the isosteric replace-ment of the phenolic OH group in our potent agonists
with the alkylsulfonamido group led to a series of potent antagonists with high binding affinities.We have previ-ously reported a different strategy for design of antago-nists in which we incorporated our potent agonist templates with bicycles such as tetrahydrobenzazepine or tetrahydroisoquinoline as an A-region derived from capsazepine.16This approach afforded several novel antagonists with potencies similar to or modestly better than capsazepine;however,further modification of the structures was problematic because their agonistic/antagonistic character was very sensitive to small modifications.26
In the present study,we have synthesized and characterized in detail,using CHO cells heterologously expressing rat VR1,a series of the potent and selective VR1antagonists having a N -4-alkylsulfonylamino-benzyl thiourea template.We have also analyzed SAR on the effect of A and C-regions in 4-alkylsulfonylamino ligands and describe significant substitution aspects on agonism/antagonism.
Chemistry
The syntheses of target thioureas were achieved by the general coupling methods between the correspond-ing isothiocyanates and amines.22The syntheses of benzylamines with a different A-region were described in Schemes1-3.4-Alkylsulfonylamino and 4-acylamino-benzylamines (13-20)were prepared from 4-amino-benzylamine (3)in three steps in good yields as shown in Scheme 1.3-Methylsulfonylamino (24)and 4-(methyl-sulfonylamino)methylbenzylamines (28)were also pre-pared from the corresponding aminobenzyl alcohols (21,25)in a conventional manner as shown in Scheme 2.For the synthesis of 3-methoxy-4-(methylsulfonylamino)-benzylamine as outlined in Scheme 3,3-methoxy-4-nitrobenzyl alcohol (29)was converted to the corre-sponding N -Boc amine (31)in three steps.The nitro group of 31was reduced and then mesylated to furnish 33whose protecting group was hydrolyzed to afford amine 34.The synthesis of the 3-fluoro-4-(methyl-sulfonylamino)benzylamine (35)was described in a previous report.27For the synthesis of sulfonamide thiourea analogues,2-(3,4-dimethylbenzyl)-3-pivaloyl-oxypropyl isothiocyanate (39)was coupled with the amines (13-20,24,28)to afford thioureas (40-49)as shown in Scheme 5.For the synthesis of different A/C region analogues as outlined in Scheme 6,3-acyloxy-2-benzylpropyl azides (50-53)26were reduced under the Lindler’s catalyzed hydrogenation and then coupled with appropriate isothiocyanates (36-38),which were prepared from the corresponding amines (13,34,35)by the reaction of 1,1′-thiocarbonyldiimidazole as shown in Scheme 4to afford the desired thioureas (54-61).Biological Results
The agonistic and antagonistic activities of the syn-thesized VR1ligands were assessed in vitro by a 45Ca 2+uptake assay,which was carried out using rat VR1heterologously expressed in Chinese hamster ovary cells (CHO/VR1cells)as previously described.20,27,28The in vitro antagonistic potencies of the compounds were evaluated by measuring antagonism of the 45Ca 2+uptake induced by 50nM capsaicin and expressed as the K i (SEM,respectively,correcting for
competition
Figure 1.
Antagonists of the Vanilloid Receptor Journal of Medicinal Chemistry,2003,Vol.46,No.143117
by capsaicin.All compounds were also evaluated as agonists.Potencies as agonists were expressed as EC 50(SEM,and absolute levels of 45Ca 2+uptake were compared with that induced by a maximally effective concentration of capsaicin in this system,namely 300nM.Receptor binding affinities were assessed in terms of the ability of the compounds to compete for specific binding of [3H]RTX in the CHO/VR1system and were expressed as the K i (SEM.All values represent the mean of at least three experiments.The results are summarized in Table 1.
Replacement of the 4-phenolic OH group in our previously reported high affinity and potent agonist 122with a 4-methylsulfonamido group provided a potent antagonist 40which inhibited the 45Ca 2+uptake in-duced by capsaicin with a K i of 67nM.The antagonistic potency of 40was thus approximately 10-fold better than that of capsazepine (K i )520nM).Evaluated as
an agonist,compound 40proved not to be a true,full antagonist but rather retained weak efficacy as an agonist,yielding about 7%of the 45Ca 2+uptake of a maximally effective dose of capsaicin (Table 1).The extent of the partial agonism depended on assay condi-tions.The detailed characterization of this weak partial agonism and its modulation by VR1coregulators is presented elsewhere.28The receptor binding affinity of 40yielded a K i value of 29.3nM,which was significantly improved compared to capsazepine (K i )1300nM)and was almost unchanged compared to that of the corre-sponding agonist,https://www.doczj.com/doc/9d10969278.html,pound 40established that replacement of the 4-hydroxyl with a 4-methylsulfon-amido group in a vanilloid can change the biological behavior of the ligand from agonist to antagonist while preserving binding affinity.
To determine the optimal size of the alkyl group of the 4-alkylsulfonamide,derivatives with alkyl groups of ethyl,propyl,isopropyl,and phenyl (41-44)were prepared.Their binding affinities and antagonistic potencies gradually decreased with increasing size although they showed partial agonism as did 40.Indeed,for the 4-phenylsulfonylamino analogue (44),the an-tagonistic activity was completely abolished and the binding affinity was found to be weak.These results
Scheme
1
Scheme
2
Scheme
3
Scheme
4
3118Journal of Medicinal Chemistry,2003,Vol.46,No.14Lee et al.
suggest that the capsaicin binding site of the receptor probably possesses a small pocket for the phenolic OH group and is subject to steric interference by bulky
groups.The 4-trifluoromethylsulfonylamino analogue (45)exhibited little binding affinity to the receptor.In this case,the size of the alkyl group was almost the same as that for the methylsulfonylamino group,and the dramatic loss in receptor potency may be attributed to the markedly reduced p K a value of the NH group in NHSO 2CF 3(calculated p K a )9.47for NHSO 2CH 3;p K a )4.83for NHSO 2CF 3in a substituted benzene)due to a strong withdrawing effect of CF 3.As another isostere of the phenolic OH,the amide analogues (46,47)of the potent agonist,1,were also characterized.However,they were devoid of any antagonism in blocking the entry of calcium induced by capsaicin and proved to be pure agonists with moderate binding affinities.To dis-cern the positional importance of the 4-methylsulfonyl-amino in 40for antagonism,the corresponding 3-methylsulfonylamino analogue (48)and one-carbon elongated analogue (49)were examined;they proved to be full agonists with binding affinities similar to cap-saicin.These results indicate that the binding and antagonistic potency of 40is attenuated by the above structural and positional modifications on the 4-methylsulfonylamino group.
Starting with the lead antagonist 40,we explored structural modifications in the C region and elsewhere in the A region.Substitution of the 3,4-dimethylphenyl on 40with the 4-tert -butylphenyl to provide 54(K i )86nM)resulted in a little decrease in the antagonistic potencies.Likewise,the binding affinity of 54with a K i value of 64nM was reduced by 2-fold relative to 40.This
Scheme
5
Scheme
6
Table 1.Potencies of Vanilloid Ligands for Binding to Rat VR1and for Inducing Calcium Influx in CHO/VR1Cells
K i (nM)binding affinity
EC 50(nM)agonism K i (nM)antagonism capsaicin 1800((270)44.8((3.8)
NE capsazepine
1300((150)NE
520((12)117.4((4.1) 1.97((0.56)NE a 2 6.35((0.48) 2.83((0.55)
NE 4029.3((7.6)WE b 67((25)41108((11)WE b 309((91)42313((52)WE b
940((130)
43227((40)790((210)c
WE 442500((660)VWE NE 4515.5%at 10μM VWE NE 461700((500)450((140)NE 472600((780)1140((260)NE 48355((61)990((79)NE 493000((980)1700((150)
NE 5464((21)WE b
86((17)55410((110)1730((450)c NE 56239((69)2790((860)b NE 5749((15)22((10)c NE 5825.2((6.9)40((11)NE 59148((19)166((15)NE 6077.2((7.5)252((13)NE 61
54((28)
NE
7.8((3.0)
a
NE:not effective,VWE:very weakly effective,WE:weakly effective at 30μM.b Only fractional calcium uptake compared to 300nM capsaicin (38,7%;39,14%;40,15%;52,30%).c Only fractional calcium uptake compared to 300nM capsaicin (23-59%).
Antagonists of the Vanilloid Receptor Journal of Medicinal Chemistry,2003,Vol.46,No.143119
finding is somewhat different from our previous obser-vation 22that a 4-tert -butylphenyl-substituted agonist,2,exhibited a higher affinity than the corresponding 3,4-dimethylphenyl-substituted derivative,1,as shown in Table 1.Like compound 40,compound 54showed partial agonism,30%of the level of calcium uptake induced by 300nM capsaicin.This difference of partial agonism is consistent with effects of C-region modifica-tions on the pattern of antagonism for this template.Incorporation of a 3-methoxy group on compounds 40and 54provided 57and 58,which were found to be agonists,without antagonistic activity,and with good potency.The EC 50values were 22nM and 40nM,respectively.The binding affinities of 57and 58,with K i values of 49nM and 25.2nM,were 2-fold weaker and 3-fold stronger than those of 40and 54,respectively.A structural comparison between 40and 57(or 54and 58)reveals that the 3-methoxy group on the A-region plays an important role in eliciting VR1agonistic activity.It is noteworthy that in the structure -activity studies of a series of capsaicinoids,a 3-methoxy group on the A-region proved to be a crucial pharmacophore in terms of agonism in the calcium uptake assay.For example,the 3-demethoxy analogue of N -octylhomo-vanillic amide was ca.20-fold less potent than that of the parent compound.3Replacement of 3-pivaloyl groups of 40,54,57,and 58with 3-benzoyl groups,compounds 55,56,59and 60,respectively,led to a decrease in their binding affinities and,for the first two,a shift toward greater agonism.It should be noted that several of the compounds in this series,namely 55,56,and 57,induced a lower level of calcium uptake than did 300nM capsaicin.This did not represent partial agonism,in that no corresponding activity was observed in assays for antagonism.The mechanistic basis for this behavior remains under investigation.
When,as a replacement of the 3-methoxy group,a 3-fluoro group was introduced to produce compound 61,there was little change in its affinity as measured in the binding assay (K i of 54nM compared to 49nM for 57).On the other hand,consistent with the important role of the 3-methoxy group for agonism,its replacement with a 3-fluoro group generated a potent antagonist,with a K i of 7.8nM.61thus represents a significant improvement in antagonist potency compared to com-
pound 40.Furthermore,unlike 40,it did not show residual weak efficacy as an agonist.We conclude that the 3-fluoro-4-methylsulfonylaminophenyl group is a promising antagonistic pharmacophore in the A-region.The analgesic activities of potent antagonists (40and 61)were evaluated in the acetic acid (AA)-induced writhing model in mice,following the previously re-ported procedure,29and were compared to that of the clinically used analgesic ketorolac (Figure 2).Whereas compound 40showed a comparable potency (ED 50)2,620(2380μg/kg)relative to ketorolac (ED 50)2800(1400μg/kg),compound 61exhibited dramatically enhanced analgesic potency,with an ED 50of 7.43(6.4μg/kg that was approximately 350-fold more potent than that of compound 40.Discussion
Recently,we have demonstrated detailed biochemical characterization of N -(4-tert -butylbenzyl)-N ′-[4-(methyl-sulfonylamino)benzyl]thiourea,its 3-fluoro and 3-meth-oxy analogues as VR1antagonists or partial ago-nists.27,28Structurally,they have the same A and B-regions relative to the VR1ligands described above;however,their C-region contains a 4-tert -butylbenzyl group different from the 3-acyloxy-2-benzylpropyl moi-ety in above compounds.To investigate the effect on VR1agonism/antagonism of 3-substituents in the A re-gion and their modulation by the C-regions in 4-methyl-sulfonamide ligands,we compared the structure -activ-ity relationships of two series of N -4-methylsulfonyl-amino thioureas.Their structures and biochemical properties as VR1ligands are described in Figure 3.Their pattern of activity was consistent with our emerg-ing understanding of vanilloid structure -activity rela-tionships.Our findings can be summarized as
follows:
Figure https://www.doczj.com/doc/9d10969278.html,parison of the analgesic activities of compound 40and 61with ketorolac in the acetic acid-induced writhing model in
mice.
Figure 3.The SAR diagram of N -[4-(methylsulfonylamino)-benzyl]thiourea analogues.
3120Journal of Medicinal Chemistry,2003,Vol.46,No.14Lee et al.
(1)we have demonstrated that substitution of the 4-phenolic hydroxyl of the A-region in agonists with an 4-methylsulfonamido group shifted the ligands from agonism toward antagonism,and a3-fluoro substitution further favored antagonism,whereas a3-methoxy group favored agonism.(2)Although the A-region makes a major contribution to the extent of agonism/antagonism, it is clear that the C-region also contributes.Thus,the N-(4-tert-butylbenzyl)moiety(top series in Figure3) tended to give more extensive antagonism than did the N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]moiety (bottom series in Figure3).
In the4-tert-butylbenzyl series(top series),the re-placement of4-hydroxy-3-methoxyphenyl in a potent and full agonist(65)previously reported by Wriggles-worth et al.23with4-methylsulfonamide produced a full antagonist(63).Interestingly,compound63was a full antagonist of capsaicin stimulation but was only a partial antagonist for stimulation by temperature or pH.27The addition of a3-fluoro substituent to the A-region in63further enhanced antagonism,as evi-denced by compound62being a full antagonist for stimulation not only by capsaicin but also by tempera-ture(44°C)or pH6.0.27It is noted that the3-fluoro-4-hydroxyphenylacetamide analogue,generated by the replacement of3-methoxy in65with3-fluoro,was shown to have10-fold less agonistic potency in calcium influx than the corresponding3-methoxy analogue but was a weak partial agonist with ca.40%efficacy.3 Conversely,the incorporation of a3-methoxy substitu-ent to the A-region in63enhanced agonism,indicating that compound64proved to be a partial agonist like 40.28Recently,we have reported in detail the biochemi-cal characterization of40and compound64as partial agonists,including the demonstration that their frac-tional efficacy reflects their functioning as partial ago-nists with a corresponding degree of partial antago-nism.28
In a similar manner,in the2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl series(bottom series),in which40 showed a low level of partial agonism,the corresponding derivative with a4-hydroxy-3-methoxyphenyl group in the A-region was a full and potent agonist(compound 1)as reported previously.22Conversely,the addition of a3-fluoro group rendered it a full antagonist(61).The incorporation of a3-methoxy substituent to40shifted its partial agonism to the full agonist57.
In conclusion,we investigated the isosteric replace-ment of the phenolic hydroxyl group in potent VR1 agonists with alkylsulfonamido groups and their struc-ture-activity relationships as agonists and antagonists. We obtained compounds which are antagonists of cap-saicin in CHO cells heterologously expressing VR1. Compound61with a3-fluoro-4-methylsulfonylamino pattern of substitution in the A-region represents a potent and full VR1antagonist.In vivo,it produced an excellent analgesic effect in the acetic-acid induced writhing test compared to ketorolac and40with partial agonism.
Experimental Section
General Method.All chemical reagents were commercially available.Melting points were determined on a Melting Point Bu¨chi B-540apparatus and are uncorrected.Silica gel column chromatography was performed on silica gel60,230-400mesh,Merck.Proton NMR spectra were recorded on a JEOL JNM-LA300at300MHz.Chemical shifts are reported in ppm units with Me4Si as a reference standard.Mass spectra were recorded on a VG https://www.doczj.com/doc/9d10969278.html,bustion analyses were performed on an EA1110Automatic Elemental Analyzer,CE Instruments,and were within0.4%of the calculated values unless otherwise noted.
tert-Butyl N-(4-Aminobenzyl)carbamate(4).A mixture of4-aminobenzylamine(3)(10g,82mmol)and di-tert-butyl dicarbonate(20g,90mmol)in THF(100mL)was stirred for 2h at room temperature and concentrated in vacuo.The residue was purified by flash column chromatography over silica gel with hexane/ethyl acetate(3:2)as eluant to afford4 as a yellow solid(17.5g,96%);mp)74-75°C;1H NMR (CDCl3)δ7.06(d,2H,J)8.3Hz,H-2,6),6.63(dt,2H,J) 2.7,8.3Hz,H-3,5),4.73(bs,1H,NHBoc),4.18(d,2H,J) 5.6Hz,CH2NH),3.60(bs,2H,NH2),1.45(s,9H,C(CH3)3).
General Procedure for the Synthesis of5-12.A cooled solution of4(5mmol)at0°C in pyridine(10mL)was treated with the corresponding acyl chloride(6mmol)and stirred for 16h at room temperature.The reacton mixture was cooled in an ice-bath,carefully neutralized with1M hydrochloric acid solution,diluted with water,and extracted with dichloro-methane several times.The combined organic layers were washed with water and brine,dried over magnesium sulfate, and filtered.The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:1)as eluant to afford5-12.
tert-Butyl N-[(4-methylsulfonylamino)benzyl]carbam-ate(5):92%yield,1H NMR(CDCl3)δ7.28(d,2H,J)8.3 Hz,H-2,6),7.18(dd,2H,J)2.2,6.3Hz,H-3,5),6.76(s,1H, NHSO2),4.88(bs,1H,NHBoc),4.28(d,2H,J)5.6Hz, CH2NH),2.99(s,3H,SO2CH3),1.46(s,9H,C(CH3)3).
tert-Butyl N-[(4-ethylsulfonylamino)benzyl]carbamate (6):73%yield,1H NMR(CDCl3)δ7.25(d,2H,J)8.5Hz, H-2,6),7.17(d,2H,J)8.5Hz,H-3,5),6.85(s,1H,NHSO2), 4.89(bs,1H,NHBoc),4.28(d,2H,J)5.6Hz,CH2NH),3.09 (q,2H,J)7.2Hz,SO2C H2CH3),1.46(s,9H,C(CH3)3),1.34 (t,3H,J)7.2Hz,SO2CH2C H3).
tert-Butyl N-[(4-propylsulfonylamino)benzyl]carbamate (7):99%yield,1H NMR(CDCl3)δ7.25(d,2H,J)8.5Hz, H-2,6),7.17(d,2H,J)8.5Hz,H-3,5),6.85(s,1H,NHSO2), 4.89(bs,1H,NHBoc),4.28(d,2H,J)5.6Hz,CH2NH),3.05 (t,2H,J)7.8Hz,SO2C H2CH2),1.85(m,2H,SO2CH2C H2), 1.46(s,9H,C(CH3)3),1.01(t,3H,J)7.6Hz,CH3).
tert-Butyl N-[(4-isopropylsulfonylamino)benzyl]car-bamate(8):78%yield,1H NMR(CDCl3)δ7.25(d,2H,J) 8.3Hz,H-2,6),7.17(d,2H,J)8.3Hz,H-3,5),6.85(s,1H, NHSO2),4.87(bs,1H,NHBoc),4.27(d,2H,J)5.6Hz, CH2NH),3.28(m,1H,SO2CH),1.46(s,9H,C(CH3)3),1.38 (d,6H,J)6.8Hz,CH(C H3)3).
tert-Butyl N-[(4-phenylsulfonylamino)benzyl]carbam-ate(9):78%yield,1H NMR(CDCl3)δ7.75(d,2H,J)8.5 Hz,Ph),7.54(t,1H,J)7.3Hz,Ph),7.44(t,2H,J)7.8Hz, Ph),7.15(d,2H,J)8.5Hz,H-2,6),7.02(d,2H,J)8.5Hz, H-3,5),6.68(s,1H,NHSO2),4.80(bt,1H,NHBoc),4.23(d,2 H,J)5.8Hz,CH2NH),1.44(s,9H,C(CH3)3).
tert-Butyl N-[(4-trifluoromethylsulfonylamino)benzyl]-carbamate(10):15%yield,1H NMR(CDCl3)δ7.46(d,2H, J)8.3Hz,H-2,6),7.28(d,2H,J)8.3Hz,H-3,5),4.98(bs, 1H,NHBoc),4.30(d,2H,J)5.8Hz,CH2NH),1.47(s,9H, C(CH3)3).
tert-Butyl N-[(4-acetylamino)benzyl]carbamate(11): 96%yield,1H NMR(CDCl3)δ7.45(d,2H,J)8.3Hz,H-2,6), 7.23(d,2H,J)8.3Hz,H-3,5),4.81(bs,1H,NHBoc),4.27 (d,2H,J)5.1Hz,CH2NH),2.18(s,3H,COCH3),1.46(s,9 H,C(CH3)3).
tert-Butyl N-[(4-isobutylamino)benzyl]carbamate(12): 99%yield,1H NMR(CDCl3)δ7.48(d,2H,J)8.3Hz,H-2,6), 7.23(d,2H,J)8.3Hz,H-3,5),7.11(s,1H,NHSO2),4.79 (bs,1H,NHBoc),4.26(d,2H,J)5.1Hz,CH2NH),2.50(m, 1H,COCH),1.46(s,9H,C(CH3)3),1.25(d,6H,J)6.8Hz, CH(C H3)2).
Antagonists of the Vanilloid Receptor Journal of Medicinal Chemistry,2003,Vol.46,No.143121
General procedure for the synthesis of13-20.A cooled solution of5-12(4mmol)in dichloromethane(20mL)in an ice-bath was slowly treated with trifluoroacetic acid(5mL) and stirred for 1.5h at0°C.The mixture was carefully concentrated in vacuo to give13-20in a quantitative yield. The amine salts were washed with ethyl ether and used for the next step without further purification.
N-[4-(Aminomethyl)phenyl]methanesulfonamide tri-fluoroacetate(13):1H NMR(DMSO-d6)δ9.87(s,1H, NHSO2),8.14(bs,3H,NH3),7.40(d,2H,J)8.5Hz,H-2,6), 7.22(d,2H,J)8.5Hz,H-3,5),3.97(s,2H,CH2),2.99(s,3 H,SO2CH3).
N-[4-(Aminomethyl)phenyl]ethanesulfonamide tri-fluoroacetate(14):1H NMR(DMSO-d6)δ9.92(s,1H, NHSO2),8.21(bs,3H,NH3),7.39(d,2H,J)8.5Hz,H-2,6), 7.22(d,2H,J)8.5Hz,H-3,5),3.96(s,2H,CH2),3.08(q,2 H,J)7.3Hz,SO2C H2CH3), 1.17(t,3H,J)7.3Hz, SO2CH2C H3).
N-[4-(Aminomethyl)phenyl]-1-propanesulfonamide tri-fluoroacetate(15):1H NMR(DMSO-d6)δ9.92(s,1H, NHSO2),8.21(bs,3H,NH3),7.39(d,2H,J)8.5Hz,H-2,6), 7.21(d,2H,J)8.5Hz,H-3,5),3.96(s,2H,CH2),3.06(t,2 H,J)7.5Hz,SO2C H2CH2),1.66(m,2H,SO2CH2C H2),0.91 (t,3H,J)7.3Hz,SO2CH2C H3).
N-[4-(Aminomethyl)phenyl]-2-propanesulfonamide tri-fluoroacetate(16):1H NMR(DMSO-d6)δ9.88(s,1H, NHSO2),8.17(bs,3H,NH3),7.38(d,2H,J)8.4Hz,H-2,6), 7.24(d,2H,J)8.4Hz,H-3,5),3.95(s,2H,CH2),3.20(m,1 H,SO2CH),1.22(d,6H,J)6.6Hz,CH(C H3)2).
N-[4-(Aminomethyl)phenyl]benzenesulfonamide tri-fluoroacetate(17):1H NMR(DMSO-d6)δ8.11(bs,3H,NH3), 7.78(dd,2H,J)1.5,6.8Hz,Ph),7.5-7.65(m,3H,Ph),7.28 (d,2H,J)8.4Hz,H-2,6),7.11(d,2H,J)8.4Hz,H-3,5), 3.89(s,2H,CH2).
N-[4-(Aminomethyl)phenyl]trifluoromethanesulfon-amide trifluoroacetate(18):1H NMR(DMSO-d6)δ8.12(bs, 3H,NH3),7.28(d,2H,J)8.4Hz,H-2,6),7.11(d,2H,J) 8.4Hz,H-3,5),3.98(s,2H,CH2).
N-[4-(Aminomethyl)phenyl]acetamide trifluoroace-tate(19):1H NMR(D2O)δ7.50(d,2H,J)8.5Hz,H-2,6), 7.46(d,2H,J)8.5Hz,H-3,5),4.18(s,2H,CH2),2.70(m,1 H,SO2CH),1.21(d,6H,J)7.1Hz,CH(C H3)2).
N-[4-(Aminomethyl)phenyl]-2-methylpropanamide tri-fluoroacetate(20):1H NMR(D2O)δ7.52(d,2H,J)8.5 Hz,H-2,6),7.46(d,2H,J)8.5Hz,H-3,5),4.18(s,2H,CH2), 2.19(s,3H,COCH3).
N-[3-(Chloromethyl)phenyl]methanesulfonamide(22).
A cooled suspension of3-aminobenzyl alcohol(21)(0.2g,1.6 mmol),lithium chloride(0.136g,3.2mmol),and2,6-lutidine (0.410g,3.52mmol)in DMF(5mL)at0°C was treated dropwise with methanesulfonyl chloride(0.272g,3.52mmol) and stirred for10min.After being stirred for5h at room temperature,the reaction mixture was diluted with H2O and extracted with ether several times.The combined organic layer was washed successively with H2O and brine,dried over MgSO4,and concentrated in vacuo.The residue was purified by flash column chromatography over silica gel with EtOAc/ hexanes(3:1)as eluent to afford22(0.27g,76%)as a white solid:mp)99.5°C;1H NMR(CDCl3)δ7.15-7.4(m,4H, Ar),6.38(bs,1H,NH),4.57(s,2H,CH2Cl),3.04(s,3H, SO2CH3);IR(KBr)3258(NH),1326,1149(SO2N)cm-1 N-[3-(Azidomethyl)phenyl]methanesulfonamide(23).
A mixture of22(0.279g,1.27mmol)and sodium azide(0.247 g,3.81mmol)in DMF(5mL)was refluxed for7h.The reaction mixture was cooled to room temperature,diluted with H2O and extracted with ethyl acetate several times.The combined organic layer was washed successively with H2O and brine, dried over MgSO4,and concentrated in vacuo.The residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(3:1)as eluent to afford23(0.273g,95%)as an oil.;1H NMR(CDCl3)δ7.15-7.4(m,4H,Ar),6.46(bs,1H, NH),4.36(s,2H,CH2N3),3.04(s,3H,SO2CH3);IR(neat) 3265(NH),2101(N3),1326,1150(SO2N)cm-1
N-[3-(Aminomethyl)phenyl]methanesulfonamide(24).
A suspension of23(100mg,0.442mmol)and palladium on carbon(20mg)in MeOH(5mL)was hydrogenated under a balloon of hydrogen for2h.The reaction mixture was filtered, and the filtrate was concentrated in vacuo to give the corre-sponding amine(88mg,99%)as a white solid which was pure enough to be used for the next step.
[4-(Aminomethyl)phenyl]methanol(25).To a suspen-sion of lithium aluminum hydride(0.58g,15.25mmol)in THF (40mL)was added dropwise a solution of4-cyanobenzaldehyde (0.5g, 3.8mmol)in THF(10mL),and the mixture was refluxed for5h.The reaction mixture was cooled in an ice bath,quenched by successive addition of H2O(15mL),15% aqueous NaOH(3mL),and H2O(20mL),and stirred for30 min.The resulting suspension was filtered,and the residue was washed with CH2Cl2.The combined filtrate and washings were washed successively with H2O and brine,dried over MgSO4,and concentrated in vacuo to give25as a solid(0.5g, 96%),which was used for the next step without further purification:1H NMR(CDCl3)δ7.33(dd,4H,Ar),4.69(s,2 H,CH2OH),3.88(s,2H,CH2NH2).
N-[4-(Chloromethyl)benzyl]methanesulfonamide(26). The compound was prepared from25by following the proce-dure described for the synthesis of22as a white solid in52% yield:mp)75°C;1H NMR(CDCl3)δ7.35(dd,4H,Ar),4.58 (s,2H,CH2Cl),4.34(s,2H,CH2NH2SO2),2.91(s,3H, NHSO2CH3);IR(KBr)3235(NH),1302,1134(NHSO2) N-[4-(Azidomethyl)benzyl]methanesulfonamide(27). The compound was prepared from26by following the proce-dure described for the synthesis of23as an oil in78%yield: 1H NMR(CDCl3)δ7.35(dd,4H,Ar),4.35(m,4H,CH2N3 and CH2NH2SO2),2.90(s,3H,NHSO2CH3);IR(KBr)3266 (NH),2101(N3),1305,1424(NHSO2)
N-[4-(Aminomethyl)benzyl]methanesulfonamide(28). The compound was prepared from27by following the proce-dure described for the synthesis of24as a solid in a quantita-tive yield and was directly used for the next step.
3-Methoxy-4-nitrobenzyl Azide(30).A cooled solution of 3-methoxy-4-nitrobenzyl alcohol(29,1g, 5.46mmol)and triphenylphosphine(2.146g,8.19mmol)in DMF(30mL)at0°C was treated with carbon tetrabromide(2.716g,8.19mmol) and stirred for10min at0°C.The reaction mixture was treated with sodium azide(1.065g,16.38mmol)and stirred for24h at room temperature.The mixture was diluted with water and extracted with ethyl acetate several times.The combined organic layers were washed with water and brine, dried over magnesium sulfate,and filtered.The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes (1:2)as eluant to afford30as a yellow solid(1.055g,93%): mp)35-37°C,1H NMR(CDCl3)δ7.87(d,1H,J)8.3Hz, H-5),7.04(s,1H,H-2),6.97(dd,1H,J)1.7,8.3Hz,H-6), 4.45(s,2H,CH2N3),3.99(s,3H,OCH3).
tert-Butyl N-(3-Methoxy-4-nitrobenzyl)carbamate(31).
A mixture of30(0.833g,4mmol),triphenylphosphine(1.573 g,6mmol),and water(0.144g,8mmol)in tetrahydrofuran (30mL)was stirred for24h at room temperature and concentrated in vacuo.The residue was dissolved in ethanol (20mL)and treated with di-tert-butyl dicarbonate(1.746g,8 mmol).After being stirred for2h at room temperature,the mixture was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:2)as eluant to afford31as a yellow oil (0.971g,86%):1H NMR(CDCl3)δ7.84(d,1H,J)8.3Hz, H-5),7.02(s,1H,H-2),6.93(dd,1H,J)8.3Hz,H-6),4.99 (bs,1H,NHBoc),4.35(d,2H,J)6.1Hz,CH2NH),3.96(s, 3H,OCH3),1.47(s,9H,C(CH3)3).
tert-Butyl N-(4-Amino-3-methoxybenzyl)carbamate(32).
A suspension of31(0.847g,3mmol)and10%palladium on carbon(0.085g)in methanol(20mL)was hydrogenated under a balloon of hydrogen for1h at room temperature.The mixture was filtered and concentrated in vacuo.The residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:1)as eluant to afford32as a white solid
3122Journal of Medicinal Chemistry,2003,Vol.46,No.14Lee et al.
(0.684g,90%):mp)76-77.5°C;1H NMR(CDCl3)δ6.6-6.75(m,3H,Ar),4.20(d,2H,J)5.6Hz,CH2NH),3.84(s, 3H,OCH3),1.46(s,9H,C(CH3)3).
tert-Butyl N-[3-Methoxy-4-(methylsulfonylamino)ben-zyl]carbamate(33).A cooled solution of32(0.634g,2.5 mmol)in pyridine(5mL)at0°C was treated with methane-sulfonyl chloride(0.23mL,3mmol)and stirred for16h at room temperature.The reaction mixture was cooled in an ice-bath,carefully neutralized with1M hydrochloric acid solution, diluted with water,and extracted with dichloromethane several times.The combined organic layers were washed with water and brine,dried over magnesium sulfate,and filtered. The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:1)as eluant to afford33as a white solid (0.595g,72%):mp)125.5-127.5°C;1H NMR(CDCl3))δ7.46 (d,1H,J)8.5Hz,H-5),6.86(m,2H,H-2,6),6.74(s,1H, NHSO2),4.88(bs,1H,NHBoc),4.28(d,2H,J)5.8Hz, CH2NH),3.88(s,3H,OCH3),2.94(s,3H,SO2CH3),1.47(s,9 H,C(CH3)3).
3-Methoxy-4-(methylsulfonylamino)benzylamine Tri-fluoroacetate(34).A cooled solution of33(1mmol)in dichloromethane(4mL)in an ice-bath was slowly treated with trifluoroacetic acid(1mL)and stirred for1.5h at0°C.The mixture was carefully concentrated in vacuo to give34as a white solid in a quantitative yield.The amine salts were washed with ethyl ether and used for the next step without further purification,respectively:mp)161-163.5°C;1H NMR(DMSO-d6)δ8.16(bs,3H,NH3),7.28(d,1H,J)8.0 Hz,H-5),7.20(s,1H,H-2),6.99(d,1H,J)8.0Hz,H-6), 4.00(s,2H,CH2NH3),3.82(s,3H,OCH3),2.95(s,3H, SO2CH3).
3-Fluoro-4-(methylsulfonylamino)benzylamine(35). This compound was prepared from2-fluoro-4-iodoaniline in three steps by our previously reported procedure.27 General Procedure for the Synthesis of Isothiocyan-ate.Method A.A solution of amine salt(10mmol)in DMF (5mL)was treated with triethylamine(1.4mL,10mmol), stirred for1h at room temperature,and added with1,1′-thiocarbonyldi-2(1H)-pyridone(2.326g,10mmol).After being stirred for24h at room temperature,the mixture was diluted with water and extracted with ethyl acetate.The combined organic layers were washed with water and brine,dried over magnesium sulfate,and filtered.The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography on silica gel with EtOAc/hexanes(1:1)as eluant to afford isothiocyanate.
Method B.To a solution of1,1′-thiocarbonyl diimidazole (12mmol)dissolved completely in DMF(20mL)at50°C was added dropwise a preprepared solution of amine salt(10mmol) and triethylamine(10mmol)in DMF(3mL)for5min.After the mixture was stirred for20min at room temperature,the usual workup and purification described above afforded isothio-cyanate.
4-(Methylsulfonylamino)benzyl isothiocyanate(36): 63%yield,white solid,mp)122-124°C;1H NMR(CDCl3)δ7.32(d,2H,J)8.5Hz)7.24(d,2H,J)8.5Hz),6.62(s,1 H,NHSO2),4.70(s,2H,CH2)3.04(s,3H,SO2CH3).
3-Methoxy-4-(methylsulfonylamino)benzyl isothio-cyanate(37):59%yield,white solid,mp)100-103°C;1H NMR(CDCl3)δ7.46(d,1H,J)8.5Hz,H-5),6.86(m,2H, H-2,6),6.74(s,1H,NHSO2),4.68(s,2H,CH2),3.88(s,3H, OCH3),2.98(s,3H,SO2CH3).
3-Fluoro-4-(methylsulfonylamino)benzyl isothiocyan-ate(38):62%yield,white solid,mp)93-96°C;1H NMR (CDCl3)δ7.61(t,1H,J)8.3Hz,H-5),7.1-7.2(m,2H, H-2,6),6.58(bs,1H,NHSO2),4.71(s,2H,CH2),3.05(s,3H, SO2CH3).
General Procedure for the Synthesis of Thiourea(40-49).A solution of amine salt(13-20,0.5mmol)in dimethyl-formamide(1mL)was treated with triethylamine(0.5mmol) (not needed for free amine24and28)and stirred for30min at room temperature.To the mixture was added{[2-(3,4-dimethylbenzyl)-3-pivaloyloxy]propyl}isothiocyanate(39)(0.5mmol).After being stirred for24h at room temperature,the mixture was diluted with water and extracted with ethyl acetate several times.The combined organic layers were washed with water and brine,dried over magnesium sulfate, and filtered.The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:1)as eluant to afford the thiourea in85-95%yields.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(methylsulfonylamino)benzyl]thiourea(40):white solid, mp)47°C;1H NMR(CDCl3)δ7.29(d,2H,J)8.3Hz),7.18 (d,2H,J)8.3Hz),6.85-7.05(m,3H),6.68(s,1H,NHSO2), 6.34(m,1H,NHCS),6.09(bs,1H,NHCS),4.51(bs,2H, CSNHCH2Ar),4.17(m,1H,CH2OCO),3.7-3.85(m,2H, CH2OCO and CHC H2NHCS),3.22(m,1H,CHC H2NHCS), 2.99(s,3H,SO2CH3),2.5-2.7(m,2H,CH2Ar),2.2-2.4(m,7 H,2×CH3and CH),1.23(d,9H,J)3.9Hz,C(CH3)3);MS (FAB)m/z520(MH+);Anal.(C26H37N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(ethylsulfonylamino)benzyl]thiourea(41):white solid, mp)48°C;1H NMR(CDCl3)δ7.25(d,2H,J)8.6Hz),7.16 (d,2H,J)8.6Hz),6.85-7.1(m,3H),6.40(m,1H,NH), 6.21(bs,1H,NH),4.50(bs,2H,CSNHCH2Ar),4.15(m,1H, CH2OCO),3.65-3.85(m,2H,CH2OCO and CHC H2NHCS), 3.24(m,1H,CHC H2NHCS), 3.09(q,3H,J)7.2Hz, SO2C H2CH3),2.5-2.7(m,2H,CH2Ar),2.15-2.4(m,7H,2×CH3and CH),1.34(t,3H,J)7.2Hz,SO2CH2C H3),1.24(d, 9H,J)3.9Hz,C(CH3)3);MS(FAB)m/z534(MH+);Anal. (C27H39N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(propylsulfonylamino)benzyl]thiourea(42):white solid, mp)54°C;1H NMR(CDCl3)δ7.25(d,2H,J)8.6Hz),7.15 (d,2H,J)8.6Hz),6.85-7.1(m,3H),6.45(bs,1H,NH), 6.30(bs,1H,NH),4.51(bs,2H,CSNHCH2Ar),4.15(m,1H, CH2OCO),3.7-3.85(m,2H,CH2OCO and CHC H2NHCS), 3.24(m,1H,CHC H2NHCS),3.04(t,3H,SO2C H2CH2CH3), 2.5-2.7(m,2H,CH2Ar),2.15-2.4(m,7H,2×CH3and CH), 1.82(m,2H,SO2CH2C H2CH3),1.24(d,9H,J)3.7Hz, C(CH3)3),1.00(t,3H,SO2CH2CH2C H3);MS(FAB)m/z548 (MH+);Anal.(C28H41N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(isopropylsulfonylamino)benzyl]thiourea(43):white solid,mp)48°C;1H NMR(CDCl3)δ7.34(s,1H,NHSO2), 7.22(d,2H,J)8.5Hz),7.16(d,2H,J)8.5Hz),6.8-7.05 (m,3H),6.48(bs,1H,NH),6.40(bs,1H,NH),4.50(bs,2H, CSNHCH2Ar),4.13(m,1H,CH2OCO),3.65-3.85(m,2H, CH2OCO and CHC H2NHCS),3.2-3.35(m,2H,CHC H2NHCS and SO2C H(CH3)2),2.5-2.7(m,2H,CH2Ar),2.15-2.4(m,7 H,2×CH3and CH),1.36(s,3H,SO2CH(C H3)2),1.34(s,3H, SO2CH(C H3)2),1.23(d,9H,J)3.6Hz,C(CH3)3);MS(FAB) m/z548(MH+);Anal.(C28H41N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(phenylsulfonylamino)benzyl]thiourea(44):white solid, mp)66°C;1H NMR(CDCl3)δ7.76(d,2H,J)7.3Hz),7.51 (t,1H),7.41(t,2H),7.14(d,2H,J)8.3Hz),6.8-7.1(m,5 H),6.40(bs,1H,NH),6.19(bs,1H,NH),4.43(bs,2H, CSNHCH2Ar),4.13(m,1H,CH2OCO),3.65-3.85(m,2H, CH2OCO and CHC H2NHCS),3.20(m,1H,CHC H2NHCS), 2.5-2.7(m,2H,CH2Ar),2.1-2.4(m,7H,2×CH3and CH), 1.23(d,9H,J)3.8Hz,C(CH3)3);MS(FAB)m/z582(MH+); Anal.(C31H39N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(trifluoromethylsulfonylamino)benzyl]thiourea(45): white solid,mp)51-52°C;1H NMR(CDCl3)δ7.28(d,2H, J)8.7Hz),7.22(d,2H,J)8.7Hz),6.85-7.05(m,3H), 6.36(bs,1H,NH), 6.16(bs,1H,NH), 4.51(bs,2H, CSNHCH2Ar),4.08(dd,1H,CH2OCO),3.65-3.85(m,2H, CH2OCO and CHC H2NHCS),3.20(m,1H,CHC H2NHCS), 2.5-2.7(m,2H,CH2Ar),2.15-2.3(m,7H,2×CH3and CH), 1.22(d,9H,J)3.8Hz,C(CH3)3);MS(FAB)m/z574(MH+); Anal.(C26H34F3N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(acetylamino)benzyl]thiourea(46):white solid,mp) 74°C;1H NMR(CDCl3)δ7.40(d,2H,J)8.3Hz),7.23(d,2
Antagonists of the Vanilloid Receptor Journal of Medicinal Chemistry,2003,Vol.46,No.143123
H,J)8.3Hz),6.8-7.05(m,3H),6.28(m,1H,NH),6.08(bs, 1H,NH),4.44(bs,2H,CSNHCH2Ar),4.17(m,1H,CH2OCO), 3.7-3.85(m,2H,CH2OCO and CHC H2NHCS),3.27(m,1H, CHC H2NHCS),2.5-2.7(m,2H,CH2Ar),2.1-2.4(m,10H,2×CH3,COCH3and CH),1.23(d,9H,J)3.9Hz,C(CH3)3) MS(FAB)m/z484(MH+);Anal.(C27H37N3O3S)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[4-(isobutylcarbonylamino)benzyl]thiourea(47):white solid,mp)65°C;1H NMR(CDCl3)δ7.50(d,2H,J)8.3 Hz),7.24(d,2H,J)8.3Hz),6.8-7.05(m,3H),6.25(m,1H, NH),6.00(bs,1H,NH),4.42(bs,2H,CSNHCH2Ar),4.12(m, 1H,CH2OCO),3.7-3.85(m,2H,CH2OCO and CHC H2-NHCS),3.25(m,1H,CHC H2NHCS),2.5-2.7(m,3H,CH2Ar and CHMe2),2.15-2.3(m,7H,2×CH3and CH),1.25(m,15 H,C(CH3)3and CH(C H3)2);MS(FAB)m/z512(MH+);Anal. (C29H41N3O3S)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[3-(methylsulfonylamino)benzyl]thiourea(48):white solid, mp)51-54°C;1H NMR(CDCl3)δ6.85-7.35(m,7H,Ar), 6.3-6.4(bs,3H,NH),4.57(bs,2H,NHCH2Ar),4.12(m,1H, O d COCH2),3.85(m,1H,O d COCH2),3.71(m,1H,CHC H2-NHC d S),3.29(m,1H,CHC H2NHC d S),2.99(s,3H,SO2CH3),
2.55-2.64(m,2H,CH2Ph),2.2-2.3(m,7H,2×CH3and
C H CH2NHC d S),1.24(s,9H,(CH3)3CO2);IR(KBr)3364(NH), 3250(NH),1715(C d O),1326(SO2N),1286(N-CS-N),1150 (SO2N)cm-1;MS(FAB)m/z520(MH+);Anal.(C26H37N3O4S2) C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-{[4-(methylsulfonylamino)methyl]benzyl}thiourea(49): white solid,mp)39°C;1H NMR(CDCl3)δ7.25-7.35(m,4 H),6.85-7.05(m,3H,aromatic),6.31(bs,1H,NH),6.22(bs, 1H,NH),5.04(bs,1H,NH),4.49(bs,2H,NHCH2Ar),4.35 (d,2H,J)5.7Hz,CH2NHSO2CH3),3.92(m,1H,O d COCH2), 3.72(m,1H,O d COCH2),3.48(m,1H,CHC H2NHC d S),3.22 (m,1H,CHC H2NHC d S),2.89(s,3H,SO2CH3),2.5-2.64(m, 2H,CH2Ph),2.2-2.3(m,7H,2×CH3and C H CH2NHC d S), 1.22(s,9H,(CH3)3CO2);IR(KBr)3355(NH),1716(C d O), 1318(SO2N),1286(N-CS-N),1150(SO2N)cm-1;MS(EI)m/z 532(M+-1);Anal.(C27H39N3O4S2)C,H,N,S.
General Procedure for the Synthesis of Thioureas (54-61).A suspension of azide(50-53)(0.5mmol)and Lindlar catalyst(50mg)in ethanol(5mL)was hydrogenated under a balloon of hydrogen for2h.The mixture was filtered,and the filtrate was concentrated in vacuo.The residue was dissolved in dichloromethane(5mL)and added with isocyanate(36-38)(0.5mmol).After being stirred for24h at room temper-ature,the mixture was concentrated in vacuo,and the residue was purified by flash column chromatography over silica gel with EtOAc/hexanes(1:1)as eluant to afford thiourea.
N-[2-(4-tert-Butylbenzyl)-3-pivaloyloxypropyl]-N′-[4-(methylsulfonylamino)benzyl]thiourea(54):white solid, mp)61°C;1H NMR(CDCl3)δ7.30(d,4H,J)8.5Hz),7.18 (d,2H,J)8.6Hz),7.11(d,2H,J)8.6Hz),6.51(s,1H, NHSO2),6.33(m,1H,NH),6.02(bs,1H,NH),4.53(bs,2H, CSNHCH2Ar),4.16(dd,1H,CH2OCO),3.7-3.85(m,2H, CH2OCO and CHC H2NHCS),3.22(m,1H,CHC H2NHCS), 2.99(s,3H,SO2CH3),2.6-2.7(m,2H,CH2Ar),2.32(m,1H, CH),1.29(s,9H,C(CH3)3),1.23(s,9H,C(CH3)3);MS(FAB) m/z548(MH+);Anal.(C28H41N3O4S2)C,H,N,S.
N-[3-Benzoyloxy-2-(3,4-dimethylbenzyl)propyl]-N′-[4-(methylsulfonylamino)benzyl]thiourea(55):white solid, mp)46°C;1H NMR(CDCl3)δ8.0(m,2H),7.59(m,1H), 7.45(m,2H),7.29(bs,1H,NHSO2),7.24(d,2H,J)8.3Hz), 7.15(d,2H,J)8.3Hz),6.85-7.05(m,3H),6.40(m,1H, NH),6.09(bs,1H,NH),4.52(bs,2H,CSNHCH2Ar),4.37(m, 1H,CH2OCO),4.10(m,1H,CH2OCO),3.80(m,1H,CHC H2-NHCS),3.41(m,1H,CHC H2NHCS),2.94(s,3H,SO2CH3), 2.6-2.8(m,2H,CH2Ar),2.44(m,1H,CH),2.2-2.4(m,6H, 2×CH3);MS(FAB)m/z540(MH+);Anal.(C28H33N3O4S2)C, H,N,S.
N-[3-Benzoyloxy-2-(4-tert-butylbenzyl)propyl]-N′-[4-(methylsulfonylamino)benzyl]thiourea(56):white solid, mp)68°C;1H NMR(CDCl3)δ8.02(d,2H),7.60(t,1H) 7.47(t,2H),7.25-7.35(m,4H),7.1-7.2(m,4H),6.54(s,1H,NHSO2),6.39(m,1H,NH),6.12(bs,1H,NH),4.53(bs,2 H,CSNHCH2Ar), 4.40(m,1H,CH2OCO), 4.08(m,1H, CH2OCO),3.80(m,1H,CHC H2NHCS),3.38(m,1H,CHC H2-NHCS),2.97(s,3H,SO2CH3),2.6-2.75(m,2H,CH2Ar),2.48 (m,1H,CH),1.29(s,9H,C(CH3)3);MS(FAB)m/z568(MH+); Anal.(C30H37N3O4S2)C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea(57): white solid,mp)49°C;1H NMR(CDCl3)δ7.48(d,1H,J) 8.0Hz),6.85-7.1(m,5H),6.77(s,1H,NHSO2),6.31(m,1 H,NH),5.98(bs,1H,NH),4.49(bs,2H,CSNHCH2Ar),4.18 (m,1H,CH2OCO),3.7-3.85(m,2H,CH2OCO and CHC H2-NHCS),3.88(s,3H,OCH3),3.22(m,1H,CHC H2NHCS),2.93 (s,3H,SO2CH3),2.5-2.7(m,2H,CH2Ar),2.2-2.4(m,7H,2×CH3and CH),1.23(d,9H,J)4.1Hz,C(CH3)3);MS(FAB) m/z550(MH+);Anal.(C27H39N3O5S2)C,H,N,S.
N-[2-(4-tert-Butylbenzyl)-3-pivaloyloxypropyl]-N′-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea(58): white solid,mp)62°C;1H NMR(CDCl3)δ7.43(d,1H,J) 8.1Hz),7.30(d,2H,J)8.3Hz),7.10(d,2H,J)8.3Hz), 6.93(s,1H),6.88(d,1H,J)8.1Hz),6.81(s,1H,NHSO2), 6.39(t,1H,NH), 6.24(bs,1H,NH), 4.52(bs,2H, CSNHCH2Ar),4.15(dd,1H,J)3.6,11.2Hz,CH2OCO),3.7-3.9(m,2H,CH2OCO and CHC H2NHCS),3.87(s,3H,OCH3), 3.25(m,1H,CHC H2NHCS),2.92(s,3H,SO2CH3),2.5-2.7 (m,2H,CH2Ar),2.30(m,1H,CH),1.29(s,9H,C(CH3)3), 1.23(s,9H,C(CH3)3);MS(FAB)m/z578(MH+);Anal. (C29H43N3O5S2)C,H,N,S.
N-[3-Benzoyloxy-2-(3,4-dimethylbenzyl)propyl]-N′-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea(59): white solid,mp)62°C;1H NMR(CDCl3)δ8.02(m,2H), 7.60(m,1H),7.4-7.5(m,3H),6.8-7.1(m,5H),6.77(s,1H, NHSO2),6.38(m,1H,NH),6.10(bs,1H,NH),4.50(bs,2H, CSNHCH2Ar),4.42(m,1H,CH2OCO),4.05(m,1H,CH2OCO), 3.82(m,1H,CHC H2NHCS),3.85(s,3H,OCH3),3.37(m,1 H,CHC H2NHCS),2.92(s,3H,SO2CH3),2.6-2.8(m,2H, CH2Ar),2.45(m,1H,CH),2.2-2.3(m,6H,2×CH3);MS (FAB)m/z570(MH+);Anal.(C29H35N3O5S2)C,H,N,S.
N-[3-Benzoyloxy-2-(4-tert-butylbenzyl)propyl]-N′-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea(60): white solid,mp)57°C;1H NMR(CDCl3)δ8.02(dd,2H,J) 7.1,1.5Hz),7.60(m,1H),7.47(m,3H),7.32(d,2H,J)8.3 Hz),7.15(d,2H,J)8.3Hz),6.85-6.95(m,2H),6.77(s,1 H,NHSO2),6.38(t,1H,NH),6.08(bs,1H,NH),4.51(bs,2 H,CSNHCH2Ar),4.42(dd,1H,J)3.9,11.7Hz,CH2OCO), 4.05(m,1H,CH2OCO),3.82(m,1H,CHC H2NHCS),3.86(s, 3H,OCH3), 3.37(m,1H,CHC H2NHCS), 2.92(s,3H, SO2CH3),2.5-2.7(m,2H,CH2Ar),2.45(m,1H,CH),1.29(s, 9H,C(CH3)3);MS(FAB)m/z598(MH+);Anal.(C31H39N3O5S2) C,H,N,S.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea(61): white solid,mp)56°C;1H NMR(CDCl3)δ7.45(d,2H,J) 8.3Hz),6.85-7.15(m,5H),6.53(bs,2H,NHCS),4.58(bs,2 H,CSNHCH2Ar),4.14(dd,1H,J)3.9,11.7Hz,CH2OCO), 3.6-3.9(m,2H,CH2OCO and CHC H2NHCS),3.25(m,1H, CHC H2NHCS),2.99(s,3H,SO2CH3),2.5-2.7(m,2H,CH2Ar), 2.2-2.4(m,7H,2×CH3and CH),1.23(d,9H,J)3.9Hz, C(CH3)3);MS(FAB)m/z538(MH+);Anal.(C26H36FN3O4S2)C, H,N,S.
VR1Binding Assays.Cell Culture.The pUHG102VR1 plasmid was transfected into CHO cells containing the pTet Off regulatory plasmid(Clontech).In these cells,expression of the VR1is repressed in the presence of tetracycline but is induced upon removal of the antibiotic.Stable clones were isolated in culture medium containing puromycin(10μg/mL) and maintained in HAM F12medium supplemented with tetracycline(1μg/mL),5μg/mL geniticin,25mM HEPES,10% FBS.Cells utilized for assays were grown in culture medium without antibiotic for48h before use.Cells were seeded in T75cell culture flasks in media without antibiotics and grown to approximately90%confluence.The flasks were then washed with PBS and harvested in0.25%trypsin,1mM EDTA.The
3124Journal of Medicinal Chemistry,2003,Vol.46,No.14Lee et al.
cells were pelleted by gentle centrifugation and stored at-20
°C until assay.
Competition Binding Assay.Binding studies with
[3H]resiniferatoxin(RTX)were carried out as described previ-
ously with minor modifications(Szallasi et al.,1992).Binding
assay mixtures were set up on ice and contained50-100pM
[3H]RTX,various concentrations of competing ligands,0.25mg/
mL BSA(Cohn fraction V),and about5×105VR1-transfected
cells.The final volume was adjusted to350μL with DPBS with
Ca2+and Mg2+and0.25mg/mL bovine serum albumin.
Nonspecific binding was determined in the presence of100nM
nonradioactive RTX.The binding reaction was initiated by
transferring the assay mixtures to a37°C water bath and was
terminated after a60min incubation period by cooling the
tubes on ice.To reduce nonspecific binding,200μg/mL R-glyco-
protein was added.Membrane-bound RTX was then separated
from the free by pelleting the membranes in a Beckman12
benchtop centrifuge(15min,maximal velocity),the tips of the
tubes containing the pellets were cut off,and the radioactivity
was determined by scintillation counting.Equilibrium binding
parameters(K i and cooperativity)were determined by fitting
the Hill equation to the measured values with the aid of the
program MicroCal Origin6.0.
Compound Preparation.Initial stocks were dissolved in
DMSO.For the binding assays,compounds were diluted in
with DPBS with Ca2+and Mg2+and0.25mg/mL bovine serum
albumin.For the calcium uptake assays,compounds were
diluted in DMEM with0.25mg/mL bovine serum albumin.
Functional Characterization for Agonist/Antagonist
Activity.45Ca2+Uptake.Molecules were characterized to
determine whether they were full agonists,partial agonists,
or antagonists.For studies of45Ca2+uptake by CHO/VR1cells
(Tet-off cells),the cells were plated in24-well plates to yield a
cell density20-40%of that required to produce confluence.
The next day the medium was changed to remove the
tetracycline and induce VR1expression.Experiments were
performed approximately36-40h after induction.For45Ca2+
uptake assay,cells were incubated for5min at37°C in a total
volume of400μL of serum free DMEM(containing1.8mM
CaCl2)in the presence of0.25mg/mL BSA(Sigma),1μCi/mL 45Ca2+(5-30Ci/g from ICN,CA),and increasing concentra-tions of the compound to be tested.Immediately after the
incubation,extracellular45Ca2+was removed by washing the
cells three times with cold DPBS(containing1.8mM CaCl2).
Then400μL of RIPA buffer(50mM Tris pH7.4;150mM
NaCl;1%Triton X-100;0.1%SDS;1%sodium deoxycholate)
was added to each well in order to lyse the cells.Plates were
shaken slowly for20min;then300μL of cell lysate was
transferred from each well into a scintillation vial and
radioactivity was determined by scintillation counting.For
each data point in each experiment,four wells were assayed.
Data from these experiments were analyzed by computer fit
to the Hill equation.At least three separate experiments were
carried out for each compound.To determine antagonist
activity,studies were performed in exactly the same fashion
with the exception that50nM capsaicin was added to the
assay mixture to stimulate45Ca2+uptake.
Writhing Test.Experimental protocols involving animals
in this study were reviewed by the Animal Care and Use
Committee of the College of Pharmacy,Seoul National Uni-
versity,according to the NIH guidelines(NIH publication
number85-23,revised1985)of“Principles of Laboratory
Animal Care”.Male ICR mice(Bio Genomics,Korea),weighing ～25g,were maintained on a12h light-dark cycle(light on between6:00p.m.and6:00a.m.)and allowed free access to
food and water.The temperature and humidity of the animal
room were maintained at22(2°C and50(5%,respectively.
Mice were allowed to habituate for～30min in the testing room
on the day of experimentation.Animals then received an
intraperitoneal injection of0.3mL of an acetic acid solution
(1.2%,diluted in0.9%saline)and were placed in a transparent
acrylic cage.Five minutes later the number of writhing
movements(abnormal stretching)was counted for a20min
period.Animals(10animals/dose)were pretreated with test compounds or vehicle(0.2mL,i.p.)30min before the injection of acetic acid.Test compounds were dissolved in either ethanol/ Tween-80/saline(10/10/80)mixture or Cremophor EL/DMSO/ distilled water(10/10/80)mixture.The effect of each compound was tested at4-7different doses.A reduction in the number of writhing movements compared to the vehicle-treatment group(the mean number of writhing movements in this group was35)was considered to be indicative of an antinociceptive effect of a compound.The percentage antinociceptive efficiency (eff)was calculated as follows:%eff)100-[(#of writhing movements/#of writhing movement control)×100].
Data are expressed as ED50values that indicate the concentration at which a given compound reduces the number of writhing by50%compared to that of a vehicle-treatment group.ED50values were obtained based on dose-response curves using mean data and fitted to by nonlinear regression analysis(Winnonlin version3.1,Pharsight Corp.,Mountain-view,CA)on a PC.
Acknowledgment.This work was supported by a Fund2002grant(ES0024)from the Korea Research Foundation.
References
(1)Szallasi,A.;Blumberg,P.M.Vanilloid(Capsaicin)Receptors and
Mechanisms.Pharmacol.Rev.1999,51,159-211.
(2)Kress,M.;Zeihofer,H.U.Capsaicin,Protons and Heat:New
Excitement about Nociceptors.TIPS1999,20,112-118.
(3)Walpole C.S.J.;Wrigglesworth,R.Capsaicin in the Study of
Pain;Academic Press:San Diego,CA,1993;pp63-82.
(4)Appendino,G.;Szallasi,A.Euphorbium:Modern Research on
Its Active Principle,Resiniferatoxin,Revives an Ancient Medi-cine.Life Sci.1997,60,681-696.
(5)Caterina,M.J.;Schumacher,M.A.;Tominaga,M.;Rosen,T.
A.;Levine,J.D.;Julius,D.The Capsaicin Receptor:a Heat-
activated Ion Channel in the Pain Pathway.Nature1997,389, 816-824.
(6)Hayes,P.;Meadows,H.J.;Gunthorpe,M.J.;Harries,M.H.;
Duckworth,D.M.;Cairns,W.;Harrision,D.C.;Clarke,C.E.;
Ellington,K.;Prinjha,R.K.;Barton,A.J.L.;Medhurst,A.D.;
Smith,G.D.;Topp,S.;Murdock,P.;Sanger,G.J.;Terrett,J.;
Jenkins,O.;Benham,C.D.;Randall,A.D.;Gloger,I.S.;Davis, J.B.Cloning and Functional Expression of a Human Orthologue of Rat Vanilloid Receptor-1.Pain2000,88,205-215.
(7)Gunthorpe,M.J.;Benham,C.D.;Randall,A.;Davis,J.B.The
Diversity of the Vanilloid Receptor Family of Ion Channels.
Trends in Pharmacol.Sci.2002,23,183-191.
(8)Kedei,N.;Szabo,T.;Lile,J.D.;Treanor,J.J.;Olah,Z.;Iadarola,
M.J.;and Blumberg,P.M.Analysis of the Native Quaternary Structure of the Vanilloid Receptor1.J.Biol.Chem.2001,276, 28613-28619.
(9)Oh,U.;Hwang,S.W.;Kim,D.Capsaicin Activates a Nonselec-
tive Cation Channel in Cultured Neonatal Rat Dorsal Root Ganglion Neurons.J.Neurosci.1996,16,1659-1667.
(10)Szallasi,A.;Blumberg,P.M.;Mechanisms and Therapeutic
Potential of Vanilloids(Capsaicin-like Molecules).Adv.Phar-macol.1993,24,123-155.
(11)Kwak,J.;Jung,J.Y.;Hwang,S.W.;Lee,W.T.;and Oh,U.A
Capsaicin-Receptor Antagonist,Capsazepine,Reduces Inflam-mation-induced Hyperalgesic Responses in the Rat:Evidence for an Endogenous Capsaicin-like Substance.Neuroscience1998, 86,619-626.
(12)Garcia-Martinez,C.;Humet,M.;Planells-Cases,R.;Gomis,A.;
Caprini,M.;Viana, F.;De le Pena, E.;Sanchez-Baeza, F.;
Carbonell,T.;De Felipe, C.;Perez-Paya, E.;Belmonte, C.;
Messeguer,A.;and Ferrer-Montiel,A.Attenuation of Thermal Nociception and Hyperalgesia by VR1Blockers.Proc.Natl.Acad.
Sci.U.S.A.2002,99,2374-2379.
(13)Lazzeri,M.;Beneforti,P.;Spinelli,M.;Zanollo,A.;Barbagli,G.;
Turini,D.Intravesical Resiniferatoxin for the Treatment of Hypersensitive Disorder:a Randomized Placebo Controlled Study.J.Urol.2000,164,676-679.
(14)Park,N.S.;Seong,C.M.;Jung,Y.S.;Kim,W.B.;Kim,S.H.
DA-5018(Capsavanil,KR-25018).Drugs Future2000,25,1131-1137.
(15)Urban,L.;Campbell,E.A.;Panesar,M.;Patel,S.;Chaudhry,
N.;Kane,S.;Buchheit,K.-H.;Sandells,B.;James,I.F.In Vivo Pharmacology of SDZ249-665,a Novel,Nonpungent Capsaicin Analogue.Pain2000,89,65-74.
Antagonists of the Vanilloid Receptor Journal of Medicinal Chemistry,2003,Vol.46,No.143125
(16)Walpole,C.S.J.;Bevan,S.;Bovermann,G.;Boelsterli,J.J.;
Breckenridge,R.;Davies,J.W.;Hughes,G.A.;James,I.;Oberer, L.;Winter,J.;Wrigglesworth,R.The Discovery of Capsazepine, the First Competitive Antagonist of the Sensory Neuron Exci-tants Capsaicin and Resiniferatoxin.J.Med.Chem.1994,37, 1942-1954.
(17)Docherty,R.J.;Yeats,J.C.;Piper A.S.Capsazepine Block of
Voltage-gated Calcium Channels in Adult Rat Dorsal Root Ganglion Neurons in Culture.Br.J.Pharmacol.1997,121, 1461-1467.
(18)Liu,L.;Simon,S.A.Capsazepine,a Vanilloid Receptor Antago-
nist,Inhibits Nicotinic Acetylcholine-Receptors in Rat Tri-geminal Ganglia.Neurosci.Lett.1997,228,29-32.
(19)(a)Wahl,P.;Foged,C.;Tullin,S.;Thomsen,C.Iodo-Resinifera-
toxin,a New Potent Vanilloid Receptor Antagonist.Mol.Phar-macol.2001,59,9-15.(b)Seabrook,G.R.;Sutton,K.G.;
Jarolimek,W.;Hollingworth,G.J.;Teague,S.;Webb,J.;Clark, N.;Boyce,S.;Kerby,J.;Ali,Z.;Chou,M.;Middleton,R.;
Kaczorowski,G.;Jones,A.B.Functional Properties of the High-Affinity TRPV1(VR1)Vanilloid Receptor Antagonist(4-Hydroxy-5-iodo-3-methoxyphenylacetate ester)Iodo-Resiniferatoxin.J.
Pharmacol.Exp.Ther.2002,303,1052-1060.Other Iodo-RTX:
(c)McDonnell,M.E.;Zhang,S.-P.;Dubin,A.E.;Dax,S.L.
Synthesis and In Vitro Evaluation of a Novel Iodinated Resin-iferatoxin Derivatives that is an Agonist at the Human Vanilloid VR1Receptor.Bioorg.Med.Chem.Lett.2002,12,1189-1192.
(20)Szallasi,A.;Blumberg,P.M.;Annicelli,L.L.;Krause,J.E.;
Cortright,D.N.The Cloned Rat Vanilloid Receptor VR1Medi-ates Both R-type Binding and C-type Calcium Response in Dorsal Root Ganglion Neurons.Mol.Pharmacol.1999,56,581-587.
(21)Walpole,C.S.J.;Bevan,S.;Bloomfield,G.;Breckenridge,R.;
James,I.F.;Ritchie,T.;Szallasi,A.;Winter,J.;Wrigglesworth, R.Similarities and Differences in the Structure-Activity Rela-tionships of Capsaicin and Resiniferatoxin Analogues.J.Med.
Chem.1996,39,2939-2952.
(22)Lee,J.;Lee,J.;Kim,J.;Kim,S.Y.;Chun,M.W.;Cho,H.;
Hwang,S.W.;Oh,U.;Park,Y.H.;Marquez,V.E.;Beheshti, M.;Szabo,T.;Blumberg,P.M.N-(3-Acyloxy-2-benzylpropyl)-
N′-(4-hydroxy-3-methoxybenzyl)thiourea Derivatives as Potent Vanilloid Receptor Agonists and Analgesics.Bioorg.Med.Chem.
2001,9,19-32.
(23)Wriggleworth,R.;Walpole,C.S.J.;Bevan,S.;Campell,E.A.;
Dray,A.;Hughes,G.A.;James,I.;Masdin,K.J.;Winter,J.
Analogues of Capsaicin with Agonist Activity as Novel Analgesic Agents:Structure-Activity Studies.4.Potent,Orally Active Analgesics.J.Med.Chem.1996,39,4942-4951.
(24)Walpole,C.S.J.;Wrigglesworth,R.;Bevan,S.;Campbell,E.
A.;Dray,A.;James,I.F.;Perkins,M.N.;Reid,D.J.;Winter,J.
Analogues of Capsaicin with Agonist Activity as Novel Analgesic Agents;Structure-Activity Studies.1.The Aromatic“A-Region”.
J.Med.Chem.1993,36,2362-2372.
(25)Larsen,A.A.;Lish,P.M.A New Bio-isostere:Alkylsulphonamido-
phenethanolamines.Nature1964,203,1283-1284.
(26)Lee,J.;Lee,J.;Szabo,T.;Gonzalez, A. F.;Welter,J. D.;
Blumberg,P.M.N-(3-Acyloxy-2-benzylpropyl)-N-Dihydroxy Tetra-hydrobenzazepine and Tetrahydroisoquinoline Thiourea Ana-logues as Vanilloid Receptor Ligands.Bioorg.Med.Chem.2001, 9,1713-1720.
(27)Wang,Y.;Szabo,T.;Welter,J.D.;Toth,A.;Tran,R.;Lee,J.;
Kang,S.U.;Lee,Y.-S.;Min,K.H.;Suh,Y.-G.;Park,M.-K.;Park,
H.-G.;Park,Y.-H.;Kim,H.-D.;Oh,U.;Blumberg,P.M.;Lee,J.
High Affinity Antagonists of the Vanilloid Receptor.Mol.Phar-macol.2002,62,947-956.(Published erratum appears in Mol.
Pharmacol.2003,63,958.)
(28)The biochemical behavior of38and compound III will be
described in Wang,Y,Toth,A,Tran,R.;Szabo,T.;Welter,J.
D.;Blumberg,P.M.;Lee,J.;Kang,S.-U.;Lim,J.-O.;Lee,J.High
Affinity Partial Agonists of Vanilloid Receptor,2003,Mol.
Pharm.In press.
(29)Lee,J.;Lee,J.;Kang,M.-S.;Kim,K.-P.;Chung,S.-J.;Blumberg,
P.M.;Yi,J.-B.;Park,Y.H.Phenolic Modification as an Approach to Improve the Pharmacology of the3-Acyloxy-2-benzylpropyl Homovanillic Amides and Thioureas,a Promising Class of Vanilloid Receptor Agonist and Analgesics.Bioorg.Med.Chem.
2002,10,1171-1179.
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化学通报
CHEMISTRY 1999年 第3期 No.3 1999
1，3-二氨基硫脲的合成研究
孙晓红 关键词 二氨基硫脲 合成 催化 刘源发
1，3-二氨基硫脲(简称TCH)是一种重要的有机合成中间体，在一些杂环类医药、 农药的合成中有广泛的用途，同时它的一些金属衍生物也具有较大的应用价值。关于 其合成方法文献已有报道［1］，且一直受到研究工作者的重视。在几种不同的合成方 法中，通常采用的是以二硫化碳和水合肼为原料，经两步反应制得TCH，以反应式表示 如下：
从原料来源及工艺条件来看，这是一条合理的工艺路线，二硫化碳与水合肼在较 低温度下反应，先生成二硫代肼基甲酸钅 井(简称HDTC)，后者经加热分解，放出硫 化氢，冷却后过滤，即可得到TCH。但此种方法早期文献报道收率一般低于70%［2,3］, 如加热温度控制不当，反应剧烈，难以控制，TCH的收率会更低，且不安全，目前国内 有关生产厂家仍采用此工艺路线。后有一些文献报道了有关此方法的改进研究，发现 过量的水合肼存在可提高收率［4］，加入水及一些低烷基醇有利于反应进行，但并不 增加反应收率；一些胺或强碱如四亚甲基二胺、氢氧化钠存在下可增加TCH的收率 ［5］；在巯基乙醇存在下，二硫化碳与过量水合肼反应不仅可提高收率，同时可减少 副产物生成，可使水合肼循环套用次数增加，TCH的平均收率～90%［6］。但是以上方 法存在反应时间过长，一般需20h左右及催化剂巯基乙醇价格贵，来源困难的问题。 我们在文献［6］的基础上，对此方法进行了改进研究，研究成功以氯乙醇等卤代 醇代替巯基乙醇，并适当提高脱硫化氢的反应温度，使反应时间大为缩短，在10h以内 即可完成反应，过量的水合肼可循环套用的工艺条件，TCH的收率一般均在90%以上。
1 实验部分
1.1 主要原料及规格 二硫化碳，化学纯； 80%水合肼，化学纯； 2-氯乙醇，分析纯； 1，3-氯-2丙醇，自制； 巯基乙醇，化学纯。 1.2 实验步骤 1.2.1 操作方法 在装有搅拌器、温度计、滴液漏斗及冷凝器(上口连有尾气导出管) 的四口烧瓶中加入80%水合肼3mol及适量水，2-氯乙醇12g，冰水浴冷却至15℃左右， 搅拌下滴加二硫化碳1mol，约1h加完，然后在室温下搅拌30min，此时有黄色结晶HDTC 析出。加入6g氢氧化钠，加热升温并控制反应温度在75～85℃之间反应10h，所放出的 硫化氢气体经导气管用稀氢氧化钠吸收。冷却至室温，过滤析出的白色颗粒状TCH。用 150mL甲醇洗涤，干燥，得产物重97.5g,收率92%。 将过滤所得母液及甲醇洗涤液合并，加入反应瓶，搅拌下于15℃左右，30min之 内，滴加0.52mol二硫化碳，继续在此温度下反应1h。冷却至0℃，30min后，过滤析出 https://www.doczj.com/doc/9d10969278.html,/web/chemistry/2000/https://www.doczj.com/doc/9d10969278.html,/col/1999/hxtb/hxtb9903/... 2011-10-27
文件编号：RHD-QB-K5166 （管理制度范本系列） 编辑：XXXXXX 查核：XXXXXX 时间：XXXXXX 硫脲合成工序工艺操作规程及安全规定标准版 本
硫脲合成工序工艺操作规程及安全 规定标准版本 操作指导：该管理制度文件为日常单位或公司为保证的工作、生产能够安全稳定地有效运转而制定的，并由相关人员在办理业务或操作时必须遵循的程序或步骤。，其中条款可根据自己现实基础上调整，请仔细浏览后进行编辑与保存。 一、工艺操作规程式 1、开车前准备 （1）接碳化工序准备开车通知后（一般提前半小时通知），班长通知合成工、投料工做好开车前准备，检查各自使用的设备处于良好状态。 （2）检查硫化氢总管阀门、合成罐、气体进气阀、母液进液阀、夹套进水、进气、退水、退气阀、合成罐放液阀、石灰氮投料孔盖均处于关闭状态。 （3）投料工根据班长要求准备投小成石灰氮原料，并检查提升机是否工作正常，等待合成工进一步
投料通知。 （4）合成工检查母液池母液是否达规定量及一次渣水是否抽入并准备好。 2、开车操作 （1）合成罐打二次母液：合成工先将二次吸收液地槽泵出口管头放入合成罐人孔口内，然后将二次吸收罐底阀打开，放液至二次吸收液地槽，同时开启地槽杆式移动泵，将二次吸收液全部批入合成罐内。关闭二次吸收罐底阀，关闭地槽杆式移动打液泵，将打液胶管从合成罐人孔口取出。二次吸收液打液操作结束。 （2）合成罐打循环液：打开合成罐上循环母液进液阀，开启循环母液打液泵（潜水泵），补充循环母液，使合成罐内混合液量达8.5M3。然后关闭循环母液泵，关闭合成罐上循环母液进液阀。
（3）小成投料 合成工操作：合成工将提升机二层石灰氮出料口移动出料口接好，并对准合成罐石灰氮进料口，确认牢靠后，开启合成罐搅拌器，确定小成投料数量，并通知投料工准备投石灰氮。 投料工操作：开启提升机，待运转正常后，将规定量石灰氮投入提升机进料口内。 投料完毕后，投料工通知合成工，合成工将合成罐上投料孔盖盖好，并把移动出料口收回原来位置。 （4）二次吸收罐投料：在投小成过程中，合成工可打开二次吸收罐上循环母液进液阀，然后开启循环母液泵，往二次吸收罐内打母液2.5—3M3。然后合成工将二次吸收罐上投料孔盖盖好。 （5）依次打开硫化氢管进口管总阀，投小成后
本科毕业论文 学 院 化学化工学院 专 业 化 学 年 级 2009 级 姓 名 罗红辰 论文（设计）题目 1,3,4-噻二唑类化合物的合成 指导教师 张玉霞 职称 教授 2013 年 5月 16日 学号：
信阳师范学院本科学生毕业论文（设计）开题报告
信阳师范学院本科学生毕业论文（设计）中期检查表
目录 摘要 (1) Abstract (1) 前言 (3) 1试验部分 (3) 1.1 主要仪器和实验试剂 (3) 1.2 1,3,4-噻二唑类化合物的合成 (3) 1.3 产物的结构与性能分析 (4) 2结果和讨论 (4) 2.1溶解性及熔点 (4) 2.2红外光谱 (5) 2.3 紫外光谱 (7) 2.4荧光光谱 (9) 3结语 (10) 参考文献 (11)
1,3,4-噻二唑类化合物的合成 学生姓名：罗红辰学号：20095051109 化学化工学院化学专业 指导教师：张玉霞职称：教授 摘要：乙酸在浓盐酸的催化下与氨基硫脲反应生成脂肪族类2,5-二取代-1,3,4-噻二唑，取代苯甲醛与氨基硫脲在六水合氯化铁的催化下关环生成芳香族类2,5-二取代-1,3,4-噻二唑类化合物，并对其进行了结构表征和荧光分析。 关键词：噻二唑；取代苯甲醛；氨基硫脲；合成 Abstract:Under the catalysis of concentrated hydrochloric, acetic acid react with thiosemicarbazide and generate an aliphatic 2,5 - disubstituted -1,3,4 – thiadiazole．under the catalysis of ferric chloride hexahydrate，the product of substituted benzaldehyde reacting with thiosemicarbazide synthesize Aromatic 2,5- disubstituted-1,3,4- thiadiazole compounds．And，Their structural characterization and fluorescence analysis were done after synthesis． Keywords:thiadiazole；substituted benzaldehyde；thiosemicarbazide；synthesize 前言 20世纪末以来，化学工作者发现l,3,4-噻二唑在许多领域都有重要应用。在工业方面，1,3,4-噻二唑类化合物主要被用作润滑油脂抗磨极压剂，也用作钼、石墨等矿石的浮选剂[2]。在农业方面，1,3,4-噻二唑类化合物主要用作除莠剂、灭草剂、杀菌、抑菌剂、植物生长调节剂等，用来防治水稻百叶枯病、柑橘溃疡病、蕃茄青枯病等[3]。在医药方面，l,3,4-噻二唑是一类具有较高生物活性的杂环化合物，常作为药物中间体主要用来合成具有抗菌，抗焦虑，抗癌活性的药物[4-12]。噻二唑化合物的“碳氮硫”结构作为活性中心已引起广泛关[13-17]，含有3个杂原子的1,3,4-噻二唑衍生物是一类重要的杂环化合物，因该类化合物具N-C-S毒性基而具有广谱生物活性，其应用广泛，发展前景广阔。 以下是脂肪族1,3,4-噻二唑类化合物和芳香族1,3,4-噻二唑类化合物的合成路线：化合物（Ⅰ）的合成路线：
尿素制硫脲 目前，我国硫}}1:生产主要是利用各种含硫化氢的尾气，生产规模都在5000t/a以下。现有硫眠生产厂家约30多家，年产量约为50000吨，其中生产规模较大的厂家有: 江苏昆山化工厂、 湖南衡南县化工农药厂、 山东临体县化工厂、 山西运城鸿运化工集团公司、 天津跃进化工厂、 宁夏大荣实业集团有限公司、 湖南衡阳宏湘化工有限公司等 传统工艺过程为:生石灰和水经消化反应生成的石灰乳进入吸收釜，在吸收釜中石灰乳吸收硫化氢气体生成硫氢化钙溶液。该溶液与固体粉末状石灰氮反应生成硫}}1:溶液。此溶液过滤后，将清液进行减压蒸发浓缩，浓缩液经冷却结晶、
离心分离、烘干即得成品硫眠。其工艺流程示意图如图1-1所示 目前改进的工艺方法是直接把工业生产过程中产生的废气硫化氢与石灰氮水溶液一步法直接反应，减少了石灰的消耗，工业废渣量也减少了。 具体的流程为:将石灰氮与水(回流母液或洗液)在合成反应釜中混合均匀，边搅拌边通入硫化氢气体进行合成反应生成硫}}1:溶液。此溶液过滤后，进入冷却结晶器进行冷冻结晶，结晶液离心分离，将晶体烘干即得成品。其工艺流程示意图如图1-2所示 该工艺是由石灰氮溶液直接吸收硫化氢气体生成硫}}1:，硫}}1:溶液中产品的析出则由原来的蒸发浓缩、冷却结晶改为冷冻结品。该工艺减少一种原料生石灰，省去石灰乳的配制、硫氢化钙溶液的制备、硫}}1:溶液的蒸发浓缩三个工序。该工艺与老工艺比较主要有以下优缺点。一是少用一种原料生石灰，同时，硫化氢和氰氨化钙的消耗定额都有所降低，降低了原料成本。二是传统工艺需用蒸汽加热蒸发硫}}1:溶液中的部分水份，蒸汽耗量很大;该工艺需要冷冻盐水进行冷冻结晶，电的消耗量有所增加。但从整个水、电、汽的消耗定额来看，该工艺比传统工艺每吨产品的费用降低了37.6。三是该工艺由于工艺流程缩短、操作人员减少、设备投资降低，因此，整个硫眠生产成本下降。 氰胺一硫化氢合成法是目前国外生产硫}}1:的主要工艺方法。该法是将氰胺溶于乙酸乙酷制得氰胺的乙酸乙酷溶液，加入浓氨水溶液中，同时通入过量硫化氢气体并充分搅拌，即得到大量硫}}1:晶体，过滤晶体并用无水乙醇洗涤，再经重结晶得到产品，纯度可达99%以上。上层滤液可作为母液循环利用，硫眠总
摘要 现代生活中，癌症对人类生命和健康的威胁与日俱增，研发有效的抗癌药物一直备受各界关注。吡咯以各种形式的衍生物广泛地存在于自然界, 在生物体的发育、生长、能量储存和转换，生物体的各种信息传递等生命过程中起着重要的作用。氨基硫脲作为一种含硫酰肼，是合成抗结核药氨硫脲和磺胺药物的原料，是一个重要的有机合成中间体。因此，本论文以2,4-二甲基-5-甲酰基-吡咯-3-甲酸和硫代氨基脲、4-甲基氨基硫脲、4-乙基氨基硫脲、4-异丙基氨基硫脲、4-苯基氨基硫脲为原料，合成了一系列含羧酸的吡咯缩氨基硫脲化合物。此外，我们对目标产物进行溶解性的测试，并通过红外光谱、核磁共振等光谱手段对产物进行了表征，希望能从中筛选治疗癌症的候选药物。 关键词：缩氨基硫脲、吡咯、羧基、溶解性
Abstract In our modern society, the threat of cancer disease to human life and health grows with time elapsing. More attentions have been paid to develop effective anti-cancer drugs. Various forms of pyrrole derivatives are widely existed in nature and play an important role in an organism's development, growth, energy storage and conversion and biometric information transmission of various life processes. Thiosemicarbazide, a sulfur-containing hydrazide, is the raw material of synthesized anti-TB thiourea and sulfa drugs and an important intermediate for organic synthesis. Therefore, in this paper, the series of pyrrole thiosemicarbazones compounds containing Carboxyl group were successfully synthesized by reacting 2,4-dimethyl-5-formyl-pyrrole-3 -carboxylic acid with thiosemicarbazide, 4-methyl thiosemicarbazide, 4-ethyl thiosemicarbazide, 4-iso propyl thiosemicarbazide, 4-phenyl thiosemicarbazide. Solubility of the target product was tested and then confirmed by IR and 1H NMR spectroscopy, we hope to search drug candidates for cancer treatment. Key word: thiosemicarbazone, pyrrole, carboxyl, solubility
硫脲合成工序工艺操作规程及安全规 一、工艺操作规程式 1、开车前准备 （1）接碳化工序准备开车通知后（一般提前半小时通知），班长通知合成工、投料工做好开车前准备，检查各自使用的设备处于良好状态。 （2）检查硫化氢总管阀门、合成罐、气体进气阀、母液进液阀、夹套进水、进气、退水、退气阀、合成罐放液阀、石灰氮投料孔盖均处于关闭状态。 （3）投料工根据班长要求准备投小成石灰氮原料，并检查提升机是否工作正常，等待合成工进一步投料通知。 4）合成工检查母液池母液是否达规定量及一次渣水是否抽入并准备好。 2、开车操作 （1）合成罐打二次母液：合成工先将二次吸收液地槽泵出口管头放入合成罐人孔口内，然后将二次吸收罐底阀打开，放液至二次吸收液地槽，同时开启地槽杆式移动泵，将二次吸收液全部批入合成罐内。关闭二次吸收罐底阀，关闭地槽杆式移动打液泵，将打液胶管从合成罐人孔口取出。二次吸收液打液操作结束。 （2）合成罐打循环液：打开合成罐上循环母液进液阀，开启循环母液打液泵（潜水泵），补充循环母液，使合成罐内混合液量达8.5M3 。然后关闭循环母液泵，关闭合成罐上循环母液进液阀。 3）小成投料
合成工操作：合成工将提升机二层石灰氮出料口移动出料口接好，并对准合成罐石灰氮进料口，确认牢靠后，开启合成罐搅拌器，确定小成投料数量，并通知投料工准备投石灰氮。 投料工操作：开启提升机，待运转正常后，将规定量石灰氮投入提升机进料口内 投料完毕后，投料工通知合成工，合成工将合成罐上投料孔盖盖好，并把移动出料口收回原来位置。 （4）二次吸收罐投料：在投小成过程中，合成工可打开二次吸收罐上循环母液进液阀，然后开启循环母液泵，往二次吸收罐内打母液2.5 —3M3 。然后合成工将二次吸收罐上投料孔盖盖好。 （5）依次打开硫化氢管进口管总阀，投小成后合成罐上的硫化氢进气阀，出气阀及对应二次吸收硫化氢尾气进气阀及该罐的排气阀，检查无误后可通知上工序碳化工开碳。 （6）开碳后开启进气管路上气体冷却水上水阀，使冷却器处于正常工作状态，同时观察二次吸收罐上排气管口是否有气体排出，同时检查合成罐上人孔、投料孔是否盖严、密封，若出现漏气现象，应及时停车，重新盖盖。均正常后，即可进入巡检工作状态。 （7）正常小成吸收硫化氢气体总量约4—5 塔（每塔钡水 6.5M3 硫化钡浓度约为110—120g/l ）硫化氢气体 680—740kg。当吸收进入最后一塔时，为控制小成吸收温度，这时可根据气温，入塔母液温度情况，确定合成釜冷却水的通冷时间，确保吸收结束时，吸收液温度在60―― 70C之间。通冷却水操作，可由合成工打开 合成釜夹套冷却水进口阀及出口阀完成。 （8）在一个合成釜吸收至最后一塔时，与碳化工联系下一个合成能否正常连续吸收，如接通知可进行。此时即可按上述开车操作（1）—（4）条进行操作，使第二个合成釜处于吸收等待阶段。 （9）当第一个合成釜吸收结束时，即可先打开第二个合成釜硫化氢进气阀、排气阀，对应二次吸收的进气阀、 排气阀。然后依次关闭第一个合成釜的进气阀、排气阀及对应的二次吸收的进气阀、排气阀。观察第二个合成的二次吸收罐排气阀是 否正常。若正 常后，第二个吸收过程即可进入巡检操作阶段。
硫脲https://www.doczj.com/doc/9d10969278.html,/309/ Keyword：硫脲硫脲价格硫脲作用硫脲性质硫脲类药物 中文名：硫脲 中文别名：硫代尿素 英文名称：Thiourea 英文别名：2-Thiourea; Isothiourea; sulfocarbamide; sulfouren; sulourea; Thiocarbamide; Thiurea; THU; thi o arbamid; sulphourea CAS No.：62-56-6 EINECS号：200-543-5 分子式：CH4N2S 分子量：76.12 熔点：171-175℃ 沸点：186.8 °C at 760 mmHg 折射率： 1.637 (30 C) 闪光点：66.8 °C Inchi：InChI=1/CH4N2S/c2-1(3)4/h(H4,2,3,4) 密度： 1.405 水溶性：13.6 g/100 mL (20℃) 危险类别码：R22,R40,R51/53,R63, 危险品运输编号：UN 2877/2811 安全说明：S36/37,S61, 海关编码：29309070 包装等级：III
危险类别： 6.1 储存温度：Store at RT. 急性毒性：口服-大鼠LD50: 125 毫克/公斤; 腹腔-小鼠LD50: 100 毫克/公斤 灭火剂：水、二氧化碳、砂土、泡沫 火警危险：Slightly flammable. 毒性分级：高毒 刺激数据：眼睛-兔子14% 中度 储运特性：库房通风低温干燥; 与食品原料分开储运 可燃性危险特性：受热放出有毒氧化硫和氧化氮气体 健康危害：Poisonous inhaled or swallowed. Irritating to skin; may cause allergic skin eruptions. 危险品标志： Xn:Harmful 分子结构式： 上游原料：硫化钡硫化氢硫酸氰氨化钙盐酸重晶石 下游产品：硫尿嘧啶苄硫醇反丁烯二酸巯基丙酸二氧化硫脲2-氨基噻唑2-氨基-5-甲基噻唑氨噻肟酸2-肟基-2-(2-氨基噻唑)-4-乙酸乙酯乙亚氨基-3-(α-羟基苯乙烯)噻唑啉盐酸盐2-氨基-5-硝基噻唑噻嗪酮灭多威拌种灵二硫氰基甲烷 硫脲https://www.doczj.com/doc/9d10969278.html,/309/ 物化性质
1总论 1.1项目建设的意义 近年来随着新生抗生素的广泛应用，头孢类抗生素的品种日益增多，需求也以每年20%的速度增长，目前仅临床应用的头孢类抗生素就有30多种，而头孢曲松用量列头孢类抗生素第一位。头孢曲松属第三代抗生素，它具有疗效高、抗菌谱广、抗菌性强、副作用小的优越疗效而被广泛应用于临床。该品种已被列为国家基本药物和基本保险用药。 生产头孢曲松的重要原料为三嗪酸，甲基肼是一种重要的医药中间体，广泛用于新生抗生素头孢曲松原料三嗪酸的合成。国内三嗪酸生产厂家均从青海购运低含量甲基肼合成甲基氨基硫脲来生产三嗪酸，由于运费较高，致使三嗪酸的生产成本居高不下。因此，石家庄市美斯特化工有限责任公司决定在赞皇县建设年产300吨甲基肼生产项目，来支持我国抗生素的发展，从而为头孢曲松的生产降低成本打下基础。 随着城乡医疗应用普及，头孢曲松的市场需求越来越大。随着头孢曲松药物生产的发展，甲基肼作为头孢曲松药物生产的源头原料也将出现旺盛市场。根据市场调查，国内外三嗪酸生产厂家均大量需求甲基肼，并且石家庄市美斯特化工有限责任公司已经和抚顺美强化工有限公司、河北金通医药化工有限责任公司两大三嗪酸生产厂达成协议，为这两家公司提供甲基肼。因此，该项目建成后，产品市场前景非常看好。 1.2编制依据 (1)《中华人民共和国环境影响评价法》，2003.9.1； (2) 《中华人民共和国水污染防治法》，1984.5.11； (3)《中华人民共和国大气污染防治法》，2000.4.29； (4)《中华人民共和国环境噪声污染防治法》，1996.10.29； (5)《中华人民共和国固体废物污染环境防治法》，1995.10.30； (6)《中华人民共和国清洁生产促进法》， 2002.6.9； (7)中华人民共和国国务院令第253号《建设项目环境保护管理条例》，1998.11.29； (8)河北省第八届人民代表大会常务委员会公告第80号《河北省建设项目环境保护管理条例》，1996.12.17； (9)中华人民共和国国务院国发(1996)31号《国务院关于环境保护若干问题的决
书山有路勤为径，学海无涯苦作舟 硫脲法提进金及其溶金原理 硫脲又名硫化尿素，白色有光泽的菱形面晶体，味苦，易溶于水，水溶 液呈中性。硫脲能够用来浸金，是由于在氧化剂存在的条件下，金可溶解于含 有硫脲的酸性溶液中： Au+2CS（NH2）2==== Au（SCN2H4）2++e 提金使用氰化物，由于其为剧毒品，不仅对人体有害，而且会污染环境， 因此人们都在寻求无毒的代用品，硫脲法便是在这种情况下应运而生的浸金工 艺方法。由于氰化法污染环境，多年来在寻找无毒微毒的浸金溶剂方面做了大 量的研究工作，各种非氰化法应运而生，值得注意的是硫脲法和氯化法，其中 认为最有前途的是硫脲法。在有氧化剂存在的条件下，使作酸性硫脲溶液直接 溶解金的方法称为硫脲法提金。其优点是硫脲毒性低，贵液易处理，硫脲可 再生重用；金矿石中的杂质不易被溶解；浸出速度快。缺点是硫脲价格高，耗 量大，因而成本高；消耗硫酸，且对设备腐蚀严重要在酸性溶液中浸出，不适 于处理碱性矿石。作业操作不稳定，而且从硫脲液中回收金的工艺还存在技术 上有待解决的问题。其溶金原理是：在含有高价铁离子的酸性稀硫脲溶液中，金被氧化并与硫脲络合生成阳离子络合物进入溶液。金被氧化和络合和反 应式为： 2Au+4CS（NH2）2+Fe2（SO4）3→{Au[CS（NH2）2]2}2SO4+2FeSO4 同时硫脲将继续被氧化，形成一些其他产物，其第一个氧化产品是甲脒化二硫。 2CS（NH2）2 ←→NH2（NH）CSSC（NH）NH2+2H++2e- 甲脒化二硫是活性很高的氧化剂，人们认为，它对于实际的金的溶解是必要的。甲脒化二硫又生 成硫脲和亚磺酸化合物，最后分解为氨基氰和元素硫。这些反应会引起硫脲的 损失。硫脲溶金时的浸出率主要取决于介质PH 值、氧化剂类型与用量、硫脲
硫脲 白色而有光泽的晶体。味苦。密度1.405。熔点180～182℃。更热时分解。溶于水，加热时能溶于乙醇，极微溶于乙醚。熔融时部分地起异构化作用而形成硫氰比铵。用于制造药物、染料、树脂、压塑粉等的原料，也用作橡胶的硫化促进剂、金属矿物的浮选剂等。由硫化氢与石灰浆作用成硫氢化钙，再与氰氨（基）化钙作用而成。也可将硫氰化铵熔融制取，或将氨基氰与硫化氢作用制得。 中文名硫脲 外文名thiourea 别名硫代尿素 分子式CN2H4S 相对分子质量76.12 化学品类别有机物--硫化物 管制类型不管制 储存密封保存 基本信息 中文名称：硫脲 中文别名：硫代尿素 英文名称：Thiourea 英文别名：2-Thiourea; Isothiourea; sulfocarbamide; sulfouren; sulourea; Thiocarbamide; Thiurea; THU; thio arbamid; sulphourea; carbamoylsulfamic acid CAS号：62-56-6 EINECS号：200-543-5[1] 1、理化性质 物理性质
外观与性状：白色光亮苦味晶体。 熔点(℃)：176～178 相对密度（水=1）：1.41 沸点(℃)：分解 分子式：CH4N2S 分子量：76.12 辛醇/水分配系数的对数值：2.5 溶解性：溶于冷水、乙醇，微溶于乙醚。[2] 化学性质 遇明火、高热可燃。受热分解，放出氮、硫的氧化物等毒性气体。与氧化剂能发生强烈反应。[2] 在空气中易潮解。在150℃时转变成硫氰酸铵。在真空下150-160℃时升华,180℃时分解。具有还原性,能使游离态碘还原成碘离子。本品富于反应性,用以制备各种化合物。能与多种氧化剂反应生成脲、硫酸及其他有机化合物,也能与无机化合物制成易溶解的加成化合物。 本品一次作用时毒性小,反复作用时能经皮肤吸收,抑制甲状腺和造血器官的机能,引起中枢神经麻痹及呼吸和心脏功能降低等症状。生产本品1～15年的工人,会出现头痛、嗜眠、全身无力、皮肤干燥、口臭、口苦、腹上部疼痛、便秘、尿频等症状。典型症状为面色苍白、面部虚肿、腹胀、基础代谢降低,血压降低,脉搏变慢,心电图有变化。还会出现皮肤病等症状。本品对蛙的LD50为10G/KG,对鼠皮下注射的D50为4g/kg。对人的致死量,文献记载为10g/kg。生产本品的工人应戴防毒口罩,穿防护服,注意安全。生产设备应密闭,无跑、冒、滴、漏现象。[3] 2、作用与用途 用以合成磺胺噻唑、蛋氨酸和肥猪片等药物的原料。用作染料及染色助剂、树脂及压塑
编号：SY-AQ-01054 ( 安全管理) 单位：_____________________ 审批：_____________________ 日期：_____________________ WORD文档/ A4打印/ 可编辑 硫脲合成工序工艺操作规程及 安全规定 Operation procedures and safety regulations of thiourea synthesis process
硫脲合成工序工艺操作规程及安全 规定 导语：进行安全管理的目的是预防、消灭事故，防止或消除事故伤害，保护劳动者的安全与健康。在安全管 理的四项主要内容中，虽然都是为了达到安全管理的目的，但是对生产因素状态的控制，与安全管理目的关 系更直接，显得更为突出。 一、工艺操作规程式 1、开车前准备 （1）接碳化工序准备开车通知后（一般提前半小时通知），班长通知合成工、投料工做好开车前准备，检查各自使用的设备处于良好状态。 （2）检查硫化氢总管阀门、合成罐、气体进气阀、母液进液阀、夹套进水、进气、退水、退气阀、合成罐放液阀、石灰氮投料孔盖均处于关闭状态。 （3）投料工根据班长要求准备投小成石灰氮原料，并检查提升机是否工作正常，等待合成工进一步投料通知。 （4）合成工检查母液池母液是否达规定量及一次渣水是否抽入
并准备好。 2、开车操作 （1）合成罐打二次母液：合成工先将二次吸收液地槽泵出口管头放入合成罐人孔口内，然后将二次吸收罐底阀打开，放液至二次吸收液地槽，同时开启地槽杆式移动泵，将二次吸收液全部批入合成罐内。关闭二次吸收罐底阀，关闭地槽杆式移动打液泵，将打液胶管从合成罐人孔口取出。二次吸收液打液操作结束。 （2）合成罐打循环液：打开合成罐上循环母液进液阀，开启循环母液打液泵（潜水泵），补充循环母液，使合成罐内混合液量达8.5M3。然后关闭循环母液泵，关闭合成罐上循环母液进液阀。 （3）小成投料 合成工操作：合成工将提升机二层石灰氮出料口移动出料口接好，并对准合成罐石灰氮进料口，确认牢靠后，开启合成罐搅拌器，确定小成投料数量，并通知投料工准备投石灰氮。 投料工操作：开启提升机，待运转正常后，将规定量石灰氮投入提升机进料口内。
硫脲为有机络合剂，可与许多金属离子形成络合物。 为白色有光泽的菱形六面结晶体，味苦，微毒，无腐蚀作用。密度为 1.405g/cm3，熔点为180～182℃，温度更高时分解，易溶于水，20℃时在水中溶解度为9%-10%（142g/L），溶解热为2 2.57kJ/d，其水溶液呈中性。 近40年来，由于物理技术的迅速发展，硫脲分子的化学结构已被进一步认定为以下的共振式： 即它是通过分子中的N十和s原子所具有的自由电子对，吸附于金粒表面而使金的氧化还原电位大大降低，使金易于氧化而溶解进人溶液中。 硫脲在碱性溶液中不稳定，易分解为硫化物和氨基氰： SC（NH 2） 2 ＋2NaOH====Na 2 S＋CNNH 2 ＋2H 2 O 分解生成的氨基氰可转变为尿素： CNNH 2＋H 2 O=====CO（NH 2 ） 2 从而造成硫脲的消耗。在碱性介质中，硫脲分解生成的S2-还可与溶液中的Au+、Ag+及Cu2+等各种金属阳离子生成硫化物沉淀。 硫脲在酸性（pH1-6）溶液中具有还原性质，可被氧化而生成多种产物。在 室温下比较容易氧化为二硫甲脒（SCN 2H 3 ） 2 ： 硫脲的稳定性主要取决于介质的pH,硫脲浓度和温度。在适宜的温度下，当硫脲浓度一定时，随着介质pH的下降，硫脲趋向于更稳定；反之，当介质的pH 一定时，随着硫脲浓度的增大，硫脲越易被氧化。为保持硫脲在溶金过程中的稳定性，提金作业宜采用低pH的硫脲溶液。也只有降低溶液的pH,才能适当地提高溶液中的硫脲浓度。298K硫脲的主要热力学数据如表1。
温度的提高虽能加快硫脲溶金的初始速度，但它会严重影响硫脲的稳定性，使得溶金才度随时间的延长而不断下降，甚至无效。在多数文献中，选定的硫脲溶金介质温度不高了25℃，虽然它不一定是最佳的选择，但试验证明，随着介质（不论是酸性、中性或碱性）温度的升高硫脲的氧化速度会加快。当硫脲在酸性或碱性溶液中，加热至60℃时，均会发生水解而生成氨、二氧化碳和液态H 2 S： SC（NH 2） 2 ＋2H 2 O====CO 2 ＋2NH 3 ＋H 2 S H 2 S还可进一步分解成S。当煮沸硫脲液时，硫脲便因快速水解而生成S2-、S、 HSO 4-和SO 4 2-等而失效。 硫脲的重要特性是在水溶液中与过渡金属离子生成稳定的络阳离子，反应通 式如下： Me n++x（TU）====[Me（TU） x ]n+ 其中：Tu-硫脲；n-化合价；x—配位数。 几种金属硫脲络合物的累计生成常数β x 列于表2中。 2 硫脲作为一种强配位体和金属离子键结合可以通过氮原子的非键电子对，或 硫原子与金属离子选择络合。Au（I）-硫脲络离子Au（TU） 2 +的阳离子性质与对 应的氰络阴离子Au（CN） 2 -完全不同，尽管前者稳定性比后者稍差，但从上表2可见，除汞的硫脲络离子较金稳定外，其他金属的硫脲络合物都不如金稳定，故硫脲对金还是有一定选择性的。当然，Cu2+、Bi3+等也可与硫脲形成较稳定的络阳离子。当原料中含有这些组分时，将增加硫脲的消耗并降低其溶金效率。
1M 硫脲+10mM EDTA+1M 尿素可否用于解决DNA-蛋白质交联？？？？ 同时含有高浓度的解聚剂尿素和硫脲，两者联合发挥协同作用，增强溶解疏水蛋白质效应. 一同使用的时候通常尿素的浓度是5-7M，而硫脲是2M。含有尿素的溶液在操作时一定要注意控制温度，温度过高（>37℃）会造成分解，而温度过低可能造成析出。 裂解液［7M 尿素 / 2M 硫脲 / 1%(w/v) ASB14（amidosulfobetaine 14） / 0.5%(v/v) Triton X-100 / 30mM DTT / 40mM Tris碱 / 0.5% Biolytes pH3-10 7M尿素＋２Ｍ硫脲＋４％ＣＨＡＰＳ构成的变性环境已经足以抑制大部分蛋白酶的活性，２Ｍ硫脲＋４％ＣＨＡＰＳ对于抽提膜蛋白有很大的帮助，不过如果您专职做膜蛋白建议采用分级抽提法。 ＥＤＴＡ用于灭活金属蛋白酶（主要是其鏊合作用） 蛋白酶K是一种枯草蛋白酶类的高活性蛋白酶，从林伯氏白色念球菌（Tritirachium album Limber）中纯化得到。该酶有两个Ca2+结合位点，它们离酶的活性中心有一定距离，与催化机理并无直接关系。然而，如果从该酶中除去Ca2+，由于出现远程的结构变化，催化活性将丧失80%左右，但其剩余活性通常已足以降解在一般情况下污染酸制品的蛋白质。所以，蛋白酶K消化过程中通常加入EDTA（以抑制依赖于Mg2+的核酸酶的作用）。但是，如果要消化对蛋白酶K具有较强耐性的蛋白，如角蛋白一类，则可能需要使用含有1mmol/l Ca2+而不含EDTA的缓冲液。在消化完毕后、纯化核酸前要加入EGT（pH8.0）至终浓度为2mmol/L,以鳌合Ca2+。 蛋白酶K，是一种切割活性较广的丝氨酸蛋白酶。它切割脂族氨基酸和芳香族氨基酸的羧基端肽键。此酶经纯化去除了RNA酶和DNA酶活性。由于蛋白酶K在尿素和SDS中稳定，还具有降解天然蛋白质的能力，因而它应用很广泛，包括制备脉冲电泳的染色体DNA，蛋白质印迹以及去除DNA和RNA制备中的核酸酶。蛋白酶K的一般工作浓度是50—100μg/ml。在较广的pH范围内（pH 4-12.5）均有 。 活性。推荐反应缓冲液：50mM Tris-HCl (pH7.5),10mM CaCl 2 值得注意的是，蛋白酶K在原位杂交技术中通常用于杂交前的处理,它具有消化包围靶DNA蛋白质的作用,以增加探针与靶核酸结合的机会,提高杂交信号.但蛋白酶K的浓度过高、消化时间过长或孵育温度过高时,都会对细胞的结构有一定的破坏,导致组织切片的脱落,细胞核的消失,从而影响杂交结果.EDTA缓冲液可
硫脲强化还原漂白高岭土工艺 CN 101774595 B 摘要 本发明涉及一种硫脲强化还原漂白高岭土工艺，以连二亚硫酸钠为主要还原剂和硫脲为辅助还原剂，在常温、pH值为3.0～6.5的氧化铝催化条件下，对高岭土进行联合强化漂白，同时进行屏蔽反应和络合反应，解决了高岭土漂白后泛黄问题，具有成本低、漂白效果好、环境污染小的特点，高岭土粉体产品自然白度大于85％，煅烧白度达到90％以上，具有高的亮度、稳定性和分散性，可广泛用于高档精细陶瓷、涂料和造纸等行业。 权利要求(2) 1. 一种硫脲强化还原漂白高岭土工艺，包括将高岭土矿经水力分级后，进行分散沉降提纯、漂白反应，然后进行洗涤、压滤、烘干，获得成品，其特征在于所述的漂白反应是在常温、PH值为3. 0?6. 5的氧化铝催化条件下，以连二亚硫酸钠为主要还原剂和硫脲为辅助还原剂对高岭土进行联合强化漂白，其中连二亚硫酸钠加入量为高岭土重量的0. 2%?0. 7 %，硫脲加入量为高岭土重量的0. 05%?0. 5% ；在还原漂白反应后，再引入铝盐作为屏蔽剂进行屏蔽反应，以阻止铁离子与粘土的再结合，其中屏蔽剂加入量为高岭土重量的0.2% _1.5%，然后加入络合剂络合被还原的铁离子，其加入量为高岭土重量的0.3%?0.6% ；
所述的络合剂为腐殖酸、草酸、柠檬酸和EDTA其中的一种或任意两种的组合。 2.根据权利要求1所述硫脲强化还原漂白高岭土工艺，其特征在于所述铝盐为氯化铝。 说明 硫脲强化还原漂白高岭土工艺 技术领域 [0001] 本发明涉及一种对高岭土进行漂白的生产工艺，具体为一种硫脲强化还原漂白高岭土工艺，广泛适用于对粘土类矿物的漂白。 背景技术 [0002] 高岭土是一种无机非金属矿物原料，主要由高岭石、云母、残余长石和石英组成，易分散悬浮于水中，具有良好的可塑性，高的粘结性和离子交换能力等独特性能，因此广泛用于陶瓷、造纸、化工和医药等重要的工业领域。纯净的高岭土呈白色，但自然界的高岭土中往往伴生一些暗色矿物如赤铁矿、磁铁矿针铁矿和含铁的云母等；另外高岭石本身还吸附一些着色物质，部分参与到高岭石矿物晶格当中形成结构铁（取代三八面体中的铝），而使高岭土呈现灰色和黄色等颜色，从而难以直接作为工业原料使用，一般都必须采用物理和化学的方法除去高岭土的杂
文件编号：TP-AR-L5166 There Are Certain Management Mechanisms And Methods In The Management Of Organizations, And The Provisions Are Binding On The Personnel Within The Jurisdiction, Which Should Be Observed By Each Party. （示范文本） 编制：_______________ 审核：_______________ 单位：_______________ 硫脲合成工序工艺操作规程及安全规定正式样本
硫脲合成工序工艺操作规程及安全 规定正式样本 使用注意：该管理制度资料可用在组织/机构/单位管理上，形成一定的管理机制和管理原则、管理方法以及管理机构设置的规范，条款对管辖范围内人员具有约束力需各自遵守。材料内容可根据实际情况作相应修改，请在使用时认真阅读。 一、工艺操作规程式 1、开车前准备 （1）接碳化工序准备开车通知后（一般提前半 小时通知），班长通知合成工、投料工做好开车前准 备，检查各自使用的设备处于良好状态。 （2）检查硫化氢总管阀门、合成罐、气体进气 阀、母液进液阀、夹套进水、进气、退水、退气阀、 合成罐放液阀、石灰氮投料孔盖均处于关闭状态。 （3）投料工根据班长要求准备投小成石灰氮原 料，并检查提升机是否工作正常，等待合成工进一步
投料通知。 （4）合成工检查母液池母液是否达规定量及一次渣水是否抽入并准备好。 2、开车操作 （1）合成罐打二次母液：合成工先将二次吸收液地槽泵出口管头放入合成罐人孔口内，然后将二次吸收罐底阀打开，放液至二次吸收液地槽，同时开启地槽杆式移动泵，将二次吸收液全部批入合成罐内。关闭二次吸收罐底阀，关闭地槽杆式移动打液泵，将打液胶管从合成罐人孔口取出。二次吸收液打液操作结束。 （2）合成罐打循环液：打开合成罐上循环母液进液阀，开启循环母液打液泵（潜水泵），补充循环母液，使合成罐内混合液量达8.5M3。然后关闭循环母液泵，关闭合成罐上循环母液进液阀。
书山有路勤为径，学海无涯苦作舟 硫脲的性质 硫脲又称硫化尿素，是一种白色而有光泽的菱形六面结晶体，味苦，微毒，无腐蚀作用。分子式为SC（NH2）2，相对分子质量76.12，密度1.405g／cm3，熔点180～182℃。它易溶于水，在20℃水中的溶解度为9%～10%，25 ℃为14%，能满足硫脲浸出金对浓度的任何要求。硫脲的水溶液呈中性，无腐蚀作用。 硫脲在碱性溶液中不稳定，易分解成硫离子和氨基氰而消耗，氨基氰又可水 解为尿素： SC（NH2）2＋2NaOH Na2S＋CNNH2＋2H2O CNNH2＋H2O CO（NH2）2 且在碱性介质中，硫脲分解生成的S2－还可与溶液中的Au＋、Ag＋及Cu2 ＋等各种金属阳离子生成硫化物沉淀。 在酸性（pH1～6）溶液中硫脲具有还原性质，但它的配制液经较长时间存 放，自身也能氧化生成多种产物，故通常应现配现用。如在室温下的酸性介质中，硫脲放置时间过长就能自行氧化为二硫甲脒或者二聚硫脲： 或者2SC（NH2）2 （SCN2H3）2＋2H＋＋2e（1） 2SC（NH2）2 （SCN2H4）22＋＋2e （2） 而（SCN2H3）2∕SC（NH2）2 电对的标准电位为0.42V，比25℃时硫脲溶金过程Au（SCN2H4）2＋∕Au电对的标准电位0.38V 高，故氧化生成的二硫甲脒在适当pH 值的溶金过程中，实际上又成为活性氧化剂，并使自身还原成硫脲。在室温的酸性介质中，硫脲还会氧化生成S0 和HSO4－、SO42－等氧化态较高的产物，但它们的反应速度很慢。N.P.芬克尔斯坦（Finkelstein）还证实，即使在pH＜4 的溶液中，硫脲也少量发生酸分解而生成H2S：
硫脲合成工序工艺操作规程及 安全规定 Through the process agreement to achieve a unified action policy for different people, so as to coordinate action, reduce blindness, and make the work orderly. 编制：___________________ 日期：___________________
硫脲合成工序工艺操作规程及安全规定 温馨提示：该文件为本公司员工进行生产和各项管理工作共同的技术依据，通过对具体的工作环节进行规范、约束，以确保生产、管理活动的正常、有序、优质进行。 本文档可根据实际情况进行修改和使用。 一、工艺操作规程式 1、开车前准备 （1）接碳化工序准备开车通知后（一般提前半小时通知）, 班长通知合成工、投料工做好开车前准备, 检查各自使用的设备处于良好状态。 （2）检查硫化氢总管阀门、合成罐、气体进气阀、母液进液阀、夹套进水、进气、退水、退气阀、合成罐放液阀、石灰氮投料孔盖均处于关闭状态。 （3）投料工根据班长要求准备投小成石灰氮原料, 并检查提升机是否工作正常, 等待合成工进一步投料通知。 （4）合成工检查母液池母液是否达规定量及一次渣水是否抽入并准备好。 2、开车操作 （1）合成罐打二次母液：合成工先将二次吸收液地槽泵出口管头放入合成罐人孔口内, 然后将二次吸收罐底阀打开, 放液至二次吸收液地槽, 同时开启地槽杆式移动泵, 将二次吸收液全部批入合成罐内。关闭二次吸收罐底阀, 关闭地槽杆式移动打液泵, 将打液胶管从合成罐人孔口取出。二次吸收液打液操作结束。 （2）合成罐打循环液：打开合成罐上循环母液进液阀, 开启循环母液打液