2244
`
`J . Med. Chem. 1995,38, 2244-2250
`
`New Analogs of Burimamide as Potent and Selective Histamine HS Receptor
`Antagonists: The Effect of Chain Length Variation of the Alkyl Spacer and
`Modifications of the N-Thiourea Substituent
`
`Roeland C. Vollinga,* Wiro M. P. B. Menge, Rob Leurs, and Hendrik Timmerman
`Leiden I Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry, Vrzje
`Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
`
`Received December 27, 1994@
`
`Burimamide was one of the first compounds reported to antagonize the activation of the
`histamine H3 receptor by histamine. We have prepared a large series of burimamide analogs
`by variation of the alkyl spacer length of burimamide from two methylene groups to six
`methylene groups and also by replacement of the N-methyl group with other alkyl and aryl
`groups. All analogs are reversible, competitive H3 antagonists as determined on the guinea
`pig intestine. Elongation of the alkyl chain from an ethylene chain to a hexylene chain results
`in an increase of the H3 antagonistic activity. The H3 selective pentylene and hexylene analogs
`of burimamide are about 10 times more potent than burimamide. The N-thiourea substituents,
`however, have no beneficial influence on the affinity.
`
`Introduction
`
`The existence of a third histamine receptor subtype,
`inhibiting the synthesis and release of histamine,
`located presynaptically in histaminergic nerve endings
`in rat cerebral cortex, was suggested in 1983 by h a n g
`et a1.l Confirmation of the existence of this new
`histamine receptor subtype was provided by the devel-
`opment of the H3 selective agonist (R)-a-methylhista-
`mine and the H3 selective antagonist thioperamide.2 The
`H3 receptor has since been shown to play an important
`regulatory role in the release of other neurotransmitters
`
`in the central nervous ~ y s t e m ~ - ~ and the periphery.’-12
`A few years before the identification of the H3 recep-
`tor, the antagonistic effect of the H2 antagonist burim-
`amide on the inhibitory action of histamine on electri-
`cally evoked contractions of guinea pig intestine
`preparations was described.13 This inhibitory effect of
`histamine was reversible and not mediated by adren-
`ergic nor HI re~ept0rs.l~ The histamine H2 antagonist
`burimamide was able to block this inhibitory effect of
`histamine, but insensitivity of the evoked contractions
`to H2 agonists made it doubtful that this effect was
`mediated by the Hz receptor. Further evidence for the
`distinct difference between the “classical” HZ receptors
`in the heart and these histamine-stimulated, contrac-
`tion-inhibiting receptors on the guinea pig ileum was
`given by Fjalland et a l l 5 The antagonistic effect of
`burimamide on the inhibitory guinea pig ileum receptors
`was described t o be about 25 times higher than that of
`another H2 antagonist, cimetidine, whereas on the HZ
`receptor in the heart, cimetidine was described to be at
`least 10 times more potent as an H2 antagonist than
`burimamide.
`After the discovery of the histamine H3 receptor and
`the description of the H3 antagonistic effect of burim-
`amide, the inhibitory histamine receptors on the guinea
`pig intestine were suggested to be of the H3 subtype as
`we11.16 Burimamide was therefore one of the first
`compounds discovered to antagonize the H3 receptor and
`
`@
`
`Abstract published in Advance ACS Abstracts, May 15, 1995.
`002 2-262319511838-2244$09.00/0
`
`played a major role in its elucidation. The compounds’
`lack of selectivity, however, makes it less attractive as
`a pharmacological tool for this receptor.
`The first potent and selective antagonist for the
`histamine H3 receptor was thioperamide, as derived
`from a series of rigid analogs of histamine.2 This
`compound possesses several distinct structural features,
`which are also present in the structure of burimamide:
`an N-alkyl-substituted thiourea group and an alkyl
`spacer on the 4(5)-position of an imidazole ring. The
`cyclohexyl group in the structure of thioperamide has
`been reported to be optimal for high affinity on the H3
`receptor. l7
`Thioperamide can be seen as a rigid analog of burim-
`amide but is more potent and selective as an H3
`antagonist. Two important differences in the structure
`of burimamide and thioperamide are the length of the
`alkyl spacer between the imidazole and the thiourea
`group (a butylene chain in the structure of burimamide
`and a propylene chain in the structure of thioperamide)
`and the N-alkyl substituent on the thiourea group (a
`methyl group for burimamide and a cyclohexyl group
`for thioperamide).
`This raises the question of whether burimamide has
`the optimal structure for its H3 antagonistic properties
`and whether the antagonistic activity and its selectivity
`for the H3 receptor can be increased with some struc-
`tural modifications. Not many structural variations of
`burimamide and their activity on the histamine H3
`receptor are known. A strong influence of the chain
`length of the alkyl spacer of burimamide on the H3
`activity has been demonstrated, since a burimamide
`analog with a propylene chain (norburimamide) is only
`a weak antagonist, with a pA2 value of 6.1 for the H3
`receptor, compared to a pA2 value of 7.2 of burimamide
`(both on rat cortex).l8
`We wanted to study the influence of the chain length
`of the alkyl spacer in the structure of burimamide
`derivatives on the H3 activity. We additionally wished
`to evaluate the influence of the N-thiourea substituents
`on the activity of this receptor. Therefore we prepared
`a large series of analogs of burimamide and determined
`0 1995 American Chemical Society
`
`SAWAI EX. 1020
`Page 1 of 7
`
`

`

`New Analogs of Burimamide
`
`Scheme 1. Synthesis of Burimamide Analogs 2-6 from
`4(5)-(w-Aminoalkyl)-lH-imidazoles 1
`
`w
`
`[-R
`
`dC
`
`H2)r
`
`H2
`+ R-NCS
`
`/"r[
`
`EtOH
`
`HN+N
`
`2a-h ; n d
`3a-h ; n=3
`4a-h ; n=4
`5a-i ; n=5
`6a,t ; n=6
`
`7a-i
`
`H N v N
`
`1
`
`l a ; n=2
`l b ; n=3
`IC; n=4
`I d ; n=5
`l e ; n=6
`
`R = methyl, ethyl, n-propyl, isc-propyl, cyclohexyl,
`phenyl, benzyl, phenylethyl, 4-chlorobenzyl
`
`the H3 activity of these compounds functionally on an
`in vitro test system using guinea pig jejunum prepara-
`tions.ll In this series we varied the length of the alkyl
`spacer of burimamide from two to six methylene groups
`and additionally replaced the methyl group by other
`alkyl and aryl groups. We investigated the selectivity
`of the most potent analogs as well, by determining their
`affinity for the H1 and H2 receptors.
`Chemistry
`The burimamide analogs 2-6 were prepared by
`reaction of the corresponding 4(5)-(w-aminoalkyl)-l.H-
`imidazoles with a series of alkyl or aryl isothiocyanates
`(see Scheme 1). The 4(5)-(u-aminoalkyl)-lH-imidazoles
`lb-e were prepared using a method described earlier
`
`by our g r ~ ~ p . ~ ~ ~ ~ ~ All isothiocyanates (7a-i) were com-
`mercially available. Most of the compounds were iso-
`lated as oxalates because of better stability and isola-
`tion.
`Pharmacology
`The H3 activity of the compounds was determined on
`an in vitro test system, on the basis of the concentration-
`dependent inhibitory effect of histamine H3 agonists on
`the electrically evoked contractile response of isolated
`guinea pig jejunum segments.ll The affnity of the
`selected compounds for the HI receptor was determined
`by the displacement of r3H1mepyramine bound to mem-
`branes of CHO cells expressing guinea pig H1 recep-
`tors.21 The affnity of the selected compounds for the
`HZ receptor was established by displacement of [12511-
`iodoaminopotentidine bound to membranes of CHO cells
`expressing human H2 receptors.22
`Results and Discussion
`All the synthesized analogs of burimamide are re-
`versible, competitive antagonists on the histamine H3
`receptor, as determined on guinea pig jejunum, with
`Schild slopes not significantly different from unity (see
`Table 1).
`The burimamide analogs 2a-h, with an ethylene
`chain, which can be seen as derivatives of histamine,
`are only weak H3 antagonists. This means that replace-
`ment of the positively charged, protonated amino group
`(at physiological pH) of histamine, by a neutral N-
`substituted thiourea group, results in loss of intrinsic
`activity on the H3 receptor. This might be due to steric
`hindrance, since Na-methylhistamine is a potent agonist
`for the H3 receptor and the replacement of the N-methyl
`group by a propyl group results in a compound without
`H3 activity.ls Moreover the reduced affinity might be
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 12 2245
`
`Table 1. Histamine H3 Antagonistic Activity of Burimamide
`Analogs 2-6 as Determined on the in Vitro Test System on
`Guinea Pig Jejunum
`compd nameorcodd nb
`Rc
`slopee N f
`pAzd
`5.5 f 0.2 1.0 f 0.1 3
`2 methyl
`W F 4 5 7 7
`2a
`5.3 f 0.2 1.1 f 0.2 4
`2 ethyl
`2b
`W F 4578
`5.4 f 0.2 1.0 f 0.1 4
`2 n-propyl
`W F 4579
`2c
`4.8 f 0.1 0.9 f 0.1 3
`2 isopropyl
`W F 4580
`2d
`2 cyclohexyl 5.9 f 0.2 1.1 fO.l 3
`W F 4581
`2e
`5.2 f 0.2 1.0 f 0.1 3
`W F 4582
`2 phenyl
`2f
`5.8 f 0.2 1.1 f 0.2 3
`W F 4583
`2 benzyl
`2g
`2 phenylethyl 5.9 f 0.1 1.0 f 0.1 3
`2h
`W F 4584
`6.4 f 0.2 1.0 f 0.1 4
`3 methyl
`norburimamide
`3a
`7.1 f 0.2 1.0 f 0.1 4
`3 ethyl
`W F 4631
`3b
`7.0 f 0.2 1.2 f 0.1 4
`3 n-propyl
`3c
`W F 4632
`7.1 f 0.2 1.0% 0.1 4
`W F 4633
`3 isopropyl
`3d
`3 cyclohexyl 6.9 i 0.2 1.1 f 0.1 4
`W F 4634
`3e
`6.9 f 0.1 1.1 i 0.1 4
`3 phenyl
`W F 4635
`3f
`6.7 f 0.2 1.1 f 0.1 4
`3 benzyl
`W F 4636
`3g
`3 phenylethyl 6.7 f 0.2 1.1 f 0.1 4
`3h
`W F 4637
`7.0 f 0.2 1.0 f O . l 5
`4 methyl
`burimamide
`4a
`7.4 f 0.2 1.1 f 0.2 4
`4 ethyl
`4b
`W F 4681
`7.3 i 0.3 1.2 f 0.3 4
`4 n-propyl
`4c
`W F 4682
`7.5 f 0.1 1.0 f 0.3 4
`4 isopropyl
`4d
`W F 4683
`4 cyclohexyl 7.1 5 0 . 2 1.1 i 0 . 3 4
`W F 4 6 8 4
`4e
`7.6 f 0.2 1.0 f 0.3 4
`4 phenyl
`4f
`W F 4685
`7.1 i 0.3 1.2 f 0.3 4
`W F 4686
`4 benzyl
`4g
`4 phenylethyl 7.0 f 0.2 1.3 i 0.1 3
`W F 4687
`4h
`5 methyl
`8.0 f O . l 1.0 fO.l 3
`W F 4613
`5a
`8.0 f 0.1 1.0 f 0.1 4
`5 ethyl
`W F 4614
`5b
`7.7 f 0.1 1.2 f 0.1 4
`W F 4615
`5 n-propyl
`5c
`7.7 f 0.1 1.2 f 0.1 4
`W F 4616
`5 isopropyl
`5d
`7.5 f 0.1 1.0 f 0.1 4
`5 cyclohexyl
`W F 4617
`5e
`7.6 f 0.2 1.0 f 0.2 3
`5 phenyl
`W F 4618
`5f
`7.7 f 0.2 1.0 f 0.1 3
`5 benzyl
`W F 4619
`5g
`5 phenylethyl 7.5 f 0.2 1.1 f 0.2 3
`5h
`W F 4620
`Si
`5 4-C1-benzyl 8.1 f 0.2 0.9 f 0.1 3
`W F 4742
`7.9 f 0.1 1.0 f 0.1 5
`6 methyl
`W F 4740
`6a
`8.0 f 0.2 0.9 f 0.2 3
`W F 4741
`6 phenyl
`6f
`
`-
`-
`a Compound code number. Alkyl chain length of 2-6 (number
`of methylene units). Substituent of 2-6. Antagonistic param-
`eter as determined on the described in vitro H3 assay representing
`the negative logarithm of the abscissa1 intercept from the Schild
`plot f SD. e Slope of Schild plot f SD, not significantly different
`from unity. f Number of different animal preparations.
`
`s
`
`n
`
`
`
`Histamine; R'=R2=H
`(R)-a-Methylhistamine; R'=Me; R'=H
`Na-Methylhistamine; R'=H; R2=Me
`
`Thioperamide
`
`Burimamide
`
`lmmepip
`
`lmetit
`; n=2, R=H
`; n=2, R=Me
`VUF 8621
`VUF 8328
`; n=3, R=H
`Clobenpropit ; n=3, R4chlorobenzyl
`Figure 1. Several discussed structures.
`
`the result of the changed electronic properties. Most
`of the described potent H3 agonists so far are compounds
`
`SAWAI EX. 1020
`Page 2 of 7
`
`

`

`2246 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 12
`
`Vollinga et al.
`
`t (R)a-Methylhistamine
`-e-
`+3.106Mcomp.5a
`--t + 1.1 0.' M comp. 5 a
`d- + 3 1 0 ' M comp.5a
`10dMcomp.5a
`+ + 1
`I
`.4
`
`-7
`
`.9
`
`-8
`
`-5
`-6
`log [(R)-a-Methylhistamine]
`Figure 2. Concentration-response curves of (R)-a-methyl-
`histamine, with a rightward parallel shift upon addition of
`compound 5a ( W F 4613) (corrected to 100%). The Schild plot
`of these results is shown in the inset.
`
`/y
`
`A
`
`- n = 2
`
`6.04
`
`I +---4-4
`5.0 1
`
`R-group
`Figure 3. Influence of the alkyl chain length ( n ) and the
`N-thiourea substituent (R) of burimamide analogs 2-6 on the
`p A 2 value on the histamine H3 receptor. Lines have been drawn
`for easy recognition of these influences.
`
`Table 2. Selectivity of the Pentylene Analogs of Burimamide
`5a-i, Compared to That of Burimamide Itself (4a), for the
`Histamine H3 Receptor
`
`PK*
`
`results in an increase of the H3 antagonistic activity.
`pAz H3e
`Hzd
`HiC
`Rb
`compd
`na
`The pentylene chain seems to be optimal in length for
`3.5 f 0 . 9 5.4 f 0.1 7.0 f 0.2
`4
`methyl
`4a
`H3 antagonistic activity for these analogs. Replacement
`4.7 f 0.1 4.7 f 0.1 8.0 f 0.1
`5
`methyl
`Sa
`of the pentylene chain of 5a, for instance, by a hexylene
`4.8 i: 0.1 5.0 f 0.1 8.0 f 0.1
`ethyl
`5b
`5
`chain, does not lead to increased H3 activity (see 6a).
`5.5 f 0.1 5.3 f 0.1 7.7 f 0.1
`5
`n-propyl
`5c
`4.9 f 0.1 5.0 f 0.1 7.7 f 0.1
`5
`isopropyl
`5d
`The aflinity for the HI and H2 receptors is determined
`5.1 f 0.1 5.4 f 0.1 7.5 f 0.1
`cyclohexyl
`5e
`5
`for these potent pentylene analogs (5a-i) (see Table 2).
`5.6 f 0.1 4.9 f 0.1 7.6 f 0.2
`phenyl
`5f
`5
`Clearly these compounds are selective for the H3 recep-
`5.4 f 0.1 5.8 i: 0.2 7.7 f 0.2
`benzyl
`5
`5g
`5.5 f 0.1 5.5 f 0.3 7.5 f 0.2
`tor, although the N-methyl-substituted pentylene analog
`phenylethyl
`5h
`5
`5.8 f 0.1 5.8 f 0.2 8.1 i: 0.2
`5
`5a is more selective than the more lipophilic N44-
`4-C1-benzyl
`5i
`chlorobenzy1)-substituted analog 5i. This pentylene
`a Alkyl chain length of 5 (number of methylene units). Sub-
`homolog of burimamide Sa is 10 times more potent and
`stituent of 5. log value of the binding affinity for the histamine
`Hi receptor f SEM.
`log value of the binding affinity for the
`about 50 times more selective than burimamide itself.
`histamine Hz receptor f SEM. e Antagonistic parameter as de-
`The large influence of the length of the alkyl spacer
`termined on the described in vitro H3 assay representing the
`(up to five methylene units) on the H3 activity of the
`negative logarithm of the abscissa1 intercept from the Schild plot
`f SD. f Apparent -log Kb as determined by Black et aLZ9 on a
`burimamide analogs is clearly visible in Figure 3. From
`conventional in vitro assay on guinea pig ileum, using histamine
`this figure, the lack of influence of the N-thiourea
`as agonist; however, since the Schild slope was significantly
`substituent on the H3 activity, however, is also appar-
`different from unity, it is doubtful that this is an HI antagonistic
`ent. If we consider the analogs Sa-i with a pentylene
`effect.
`chain (n = 51, there is not a great difference in the p A 2
`with an imidazole ring and an amino group (e.g., (R)-
`value between the compounds containing a small alkyl
`group, a large alkyl group, or an aromatic substituent.
`a-methylhistamine and immepip), separated by an alkyl
`This suggests that the receptor binding of this part of
`spacer.
`The imidazole ring seems to be essential for activa-
`the burimamide analogs is not through a hydrophobic
`interaction nor through an electrostatic ?t-n interaction
`tion, since replacement of the imidazole ring by other
`between aromatic systems. These results are rather
`heterocyclic rings resulted in less active compounds or
`surprising, since it has been proposed that an H3
`
`compounds deprived of any agonistic a ~ t i v i t y . ~ ~ > ~ ~ The
`antagonist should consist of an N-containing heterocycle
`amino group of histamine, however, which is protonated ,
`linked to a polar group by an alkyl chain with a
`at physiological pH, has been replaced with other basic
`lipophilic residue attached to the polar group for en-
`groups, like an isothiourea group, resulting in potent
`hancement of the affinity.24 A clear example of the
`H3 agonists (e.g., imetit25-28). The pKa of the isothiourea
`affinity-enhancing effect of lipophilic residues can be
`group (pKa = 9-10) has been described to be similar t o
`observed in the series of analogs of imetit, as described
`that of aliphatic amines (pKa = 9-11).27 Monomethyl-
`by Van der Goot et a1.25 In this series, derivatization
`ation of the isothiouronium moiety in imetit does not
`of the potent H3 antagonist W F 8328 (pA2 value of 8.0
`drastically affect the agonistic activity on the H3 recep-
`on guinea pig ileum) leads to compounds with even
`tor (pD2 value of W F 8621 is 7.3, compared to a pD2
`higher affinity for the H3 receptor. The introduction of
`value of 8.1 for imetit on the guinea pig
`ap-chlorobenzyl group on the isothiourea group of W F
`whereas the ethylene homolog of burimamide 2a is a
`8328 resulted in the most potent H3 antagonist de-
`weak H3 antagonist. Because the thiourea group of 2a
`scribed so far (clobenpropit), with a p A 2 value of 9.9 on
`is uncharged at physiological pH, it seems that a specific
`the guinea pig ileum. The introduction of lipophilic
`ionic binding site at the H3 receptor for cationic groups
`residues on the thiourea group of the burimamide
`of Hs agonists, probably a carboxylate (e.g., an aspartate
`analogs, however, does not enhance the H3 antagonistic
`residue), exists.
`activity. This seems to rule out a possible interaction
`Elongation of the alkyl chain of the burimamide
`of this series of antagonists in the same manner as the
`analogs from a propylene chain to a pentylene chain
`
`SAWAI EX. 1020
`Page 3 of 7
`
`

`

`New Analogs of Burimamide
`Thioperamide
`isothiourea derivatives of Van der
`also binds in a distinct manner to the H3 receptor, other
`than the burimamide analogs, since 3e is about 100
`times less potent as an H3 antagonist than thioperam-
`ide, which can be seen as its rigid analog. Since there
`is no large influence of the N-thiourea substituents of
`the burimamide analogs on the p A 2 value, only an
`interaction of the thiourea group with the receptor via
`hydrogen bonding seems likely.
`It can be concluded that the intrinsic activity of
`histamine on the H3 receptor is lost when the amino
`group is replaced by an N-substituted thiourea group.
`Elongation of the alkyl spacer up to five methylene units
`leads to an increase of affinity. Replacement of the
`pentylene chain O f 5a by a hexylene chain does not lead
`to increased H3 activity (see 6a) indicating an additional
`binding site for the pentylene and higher analogs of
`burimamide. The chain length of the alkyl spacer has
`a large influence on the H3 antagonistic activity, with
`5a being 10 times more potent than burimamide. The
`N-thiourea substituents, however, have no great influ-
`ence on the affinity. The results indicate a binding
`behavior for the burimamide analogs in a nonlipophilic
`environment different from other H3 antagonists like
`thioperamide and clobenpropit. Although burimamide
`was originally described as an Hz antagonist, the
`pentylene analogs of burimamide are more potent and
`selective for the histamine H3 receptor.
`Experimental Section
`Chemistry. 'H NMR spectra were recorded on a Bruker
`AC-200 (200 MHz) spectrometer with tetramethylsilane or
`sodium 3-(trimethylsily1)propionate as an internal standard.
`Mass spectra were recorded on a Finnigan MAT-90 spectrom-
`eter. Melting points were measured on a Mettler FP-5 + FP-
`52 apparatus and are uncorrected. Elemental analyses was
`performed by MHW Laboratories, Phoenix, AZ. Histamine
`dihydrochloride (la) was purchased from Janssen Chimica. 4-
`(5)-(3-Aminopropyl)-lH-imidazole dihydrobromide (lb), 4(5)-
`(4-aminobutyl)-lH-imidazole dihydrobromide (IC), and 4(5)-
`(5-aminopentyl)-lH-imidazole dihydrobromide (Id) were
`prepared as described earlier by our
`4(5)-(6-Amino-
`hexyl)-lH-imidazole (le) was prepared using the same method.20
`Methyl (7a) and ethyl (7b) isothiocyanate were purchased from
`Aldrich; n-propyl (7~1, isopropyl (7d), benzyl (7g), and phen-
`ylethyl(7h) isothiocyanate were from Maybridge Chemical Co.
`(MCC); cyclohexyl (7e) and phenyl (70 isothiocyanate were
`from Janssen Chimica, and chlorobenzyl isothiocyanate (7i)
`was purchased from Lancaster. The isothiocyanates were used
`without purification. The purity of the products was checked
`on thin layer chromatography (Merck silica gel 60, F254,0.25
`mm). The free bases of all compounds gave one spot using
`either ethyl acetate (Rf = 0-0.11, methanol (Rf 0.9-LO), or
`CHC13 (Rf = 0.5). The yields of the purified salts are given.
`General Procedure. The required 4(5)-(w-aminoalkyl)-
`1H-imidazole 1, either as dihydrochloride or as dihydrobro-
`mide, was added to 2 equiv of sodium ethanolate in absolute
`ethanol. This solution was refluxed for 30 min and cooled to
`room temperature. The formed precipitate was removed by
`filtration, and 3 equiv of the needed isothiocyanate 7 was
`added to the filtrate. The ethanol was removed under reduced
`pressure, after 2 h of refluxing. The residue was purified by
`column chromatography, by washing with ethyl acetate as
`eluent (isothiocyanate eluted Rf = 1.0). The product was
`subsequently eluted with methanol as eluent (unreacted amine
`remained on column). After removal of the methanol under
`reduced pressure, the free base was converted into a hydro-
`bromide or an oxalate.
`The hydrobromides were prepared by the solvation of the
`free base in 10% HBr solution. After stirring at room tem-
`perature for 15 min, the acidic solution was concentrated in
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 12 2247
`
`vacuo, triturated three times with absolute ethanol, and
`recrystallized from ethanollethyl acetate.
`The oxalates were prepared by solvation of the free base in
`ethyl acetate and the addition of an excess of a saturated
`solution of oxalic acid in ethyl acetate (slowly). The formed
`precipitate was collected by centrifugation, washed with ethyl
`acetate (three times), and recrystallized from absolute ethanol.
`N-Methyl-N'-[2-(4(5)-imidazolyl)ethyllthiourea hydro-
`bromide (2a): mp 99.9-100.8 "C; yield 49%.
`'H NMR
`(D2O): 6 2.87 (s, 3H, CH3), 3.03 (t, 2H, J = 7 Hz, imidazole-
`CHz), 3.78 (t, 2H, J = 7 Hz, CH2NH), 7.30 (5, l H , imidazole-
`5(4)H), 8.62 (s, l H , imidazole-2H). MS (EI, re1 intensity): m l z
`184 (M+, 57), 153 (M+ - CH3NH2,47), 150 (M+ - HzS, 54),95
`([ImC2H4]+, loo), 81 ([ImCHz]+, 84). HRMS: m l z 184.0782;
`
`calcd for C7H12N4S, 184.0783. Anal. ( C ~ H ~ Z N ~ S . ~ H B ~ ) C, H,
`N.
`N-Ethyl-N'-[2-(4(5)-imidazolyl)ethyllthiourea hydro-
`bromide (2b): mp 164.5-165.0 "C; yield 74%. 'H NMR
`(D2O): 6 1.12 (t, 3H, J = 7 Hz, CH3), 3.03 (t, 2H, J = 7 Hz,
`imidazole-CHz), 3.32 (9, 2H, J = 7 Hz, CH2CH31, 3.78 (t, 2H,
`J = 7 Hz, CHzNH), 7.39 (s, l H , imidazole-5(4)H), 8.62 (s, l H ,
`imidazole-2H). MS (EI, re1 intensity): m l z 198 (M+, 501, 164
`(M+ - H2S, 321,153 (M+ - CZH5NH2, 18),95 ([ImC2H41+, 1001,
`81 ([ImCHz]+, 51). HRMS: m l z 198.0940; calcd for CSHMN~S,
`198.0939. Anal. (CsH14N4S.1.96HBr) C, H, N.
`N-n-Propyl-N'-[2-(4(5)-imidazolyl)ethyllthiourea hy-
`drobromide (2c): mp 172.6-173.1 "C; yield 36%. 'H NMR
`(D2O): 6 0.88 (t, 3H, J = 7 Hz, CH3), 1.53 (m, 2H, CHZCH~),
`3.04 (t, 2H, J = 7 Hz, imidazole-CHz), 3.10-3.45 (m, 2H, CHZ-
`CHzCHs), 3.70-3.92 (m, 2H, CHzNH), 7.30 (s, l H , imidazole-
`5(4)H), 8.64 (s, l H , imidazole-2H). MS (EI, re1 intensity): mlz
`212 (M+, 62), 178 (M+ - HzS, 51,153 (M+ - C ~ H ~ N H Z ,
`13),95
`([ImCzH4]+, loo), 81 ([ImCH21+, 35). HRMS: m l z 212.1100;
`calcd for C9H1&J4S, 212.1096. Anal. (CSHXN~S-HB~) C, H, N.
`N-Isopropyl)-N'-[2-(4(5)-imidazolyl)ethyll thiourea
`oxalate (2d): mp 123.1 "C; yield 53%. 'H NMR (DzO): 6 1.01
`(d, 6H, J = 7 Hz, 2*CH3), 2.90 (t, 2H, J = 7 Hz, imidazole-
`CH2), 3.58-3.75 (m, 2H, CH2NH), 3.75-4.10 (b s, l H , CHI,
`7.16 (s, l H , imidazole-5(4)H), 8.49 (s, l H , imidazole-2H). MS
`(EI, re1 intensity): m l z 212 (M+, 591,178 (M+ - HzS, 12), 153
`(M+ - C3H7NHz, 191, 95 ([ImC2H41+, loo), 81 ([ImCHzI+, 52).
`HRMS: m l z 212.1090; calcd for CgH1&S, 212.1096. Anal.
`
`( C S H I ~ N ~ S C ~ H ~ O ~ ) C, €3, N.
`N-Cyclohexyl-N'-[2-(4(5)-imidazolyl)ethyl] thiourea
`oxalate (2e): mp 161.7 "C; yield 92%. 'H NMR (DzO): 6
`0.99-1.85 (m, 10H, cyclohexyl-CHz's), 2.97 (t, 2H, J = 7 Hz,
`imidazole-CHz), 3.50-3.90 (m, 3H, CH + CHzNH), 7.22 (s, l H ,
`imidazole-5(4)H), 8.53 (s, l H , imidazole-2H). MS (EI, re1
`intensity): m l z 252 (M+, 57), 218 (M+ - HzS, 121, 153 (M+ -
`C6H11NH2, 30), 95 ([ImCzH41+, 1001, 81 ([ImCHzl+, 72).
`
`HRMS: m l z 252.1401; calcd for C ~ ~ H Z O N ~ S , 252.1409. Anal.
`(CizHzoN4S.0.5CzHz04) C, H, N.
`N-Phenyl-N'-[2-(4(5)-imidazolyl)ethyl] thiourea hydro-
`bromide (20: mp 148.6-148.9 "C; yield 74%. 'H NMR
`(DzO): 6 2.94-3.03 (m, 2H, imidazole-CHz), 3.75-3.97 (m, 2H,
`CH2NH), 7.11-7.57 (m, 6H, phenyl-H + imidazole-5(4)H), 8.61
`(s, l H , imidazole-2H). MS (EI, re1 intensity): m l z 246 (M+,
`3), 212 (M+ - HzS, 7), 153 (M+ - C ~ H ~ N H Z , 411, 135 ([CsH5-
`NCS]+, loo), 93 ([C~&NHZ]+, 621, 95 ([ImCzH41+, 121, 81
`([ImCHz]+, 72),77 ([CF&,]+, 51). HRMS: m l z 246.0931; calcd
`
`for C12H14N4S, 246.0939. Anal. ( C I ~ H M N ~ S * H B ~ ) C, H, N.
`N-Benzyl-N'-[2-(4(5)-imidazolyl)ethyl] thiourea oxalate
`(2g): mp 153.7-155.0 "C; yield 18%. 'H NMR (DMSO-&): 6
`2.89 (t, 2H, J = 7 Hz, imidazole-CHz), 3.59-3.83 (m, 2H, CH2-
`NH), 4.53-4.77
`(m, 2H, CH2-phenyl), 7.18-7.38
`(m, 6H,
`phenyl-H + imidazole-4(5)H), 7.85-8.00 (m, 1H, NH), 8.72 (t,
`l H , J = 6 Hz, NH), 8.72 (s, l H , imidazole-2H), 11.15-11.85
`(m, NH + oxalate). MS (EI, re1 intensity): m l z 260 (M+, 281,
`226 (M+ - HzS, a), 153 (M+ - C7H7NHz, 201, 95 ([ImCzH41+,
`44), 91 ([C7H7]+, 1001, 81 ([ImCHzI+, 38). HRMS: m l z
`260.1101; calcd for C&1&S,
`260.1096. Anal. (CI~HI&J~S*
`CzH204) C, H, N.
`N-(2-Phenylethyl)-N'-[2-(4(5)-imidazolyl)ethyll thio-
`urea oxalate (2h): mp 145.1-145.5 "C; yield 18%. 'H NMR
`(DzO): 6 2.66-2.91 (m, 4H, imidazole-CHz + CH2-phenyl),
`3.32-3.80 (m, 4H, 2*CHzNH), 7.15 (s, l H , imidazole-5(4)H),
`
`SAWAI EX. 1020
`Page 4 of 7
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`

`2248 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 12
`
`Vollinga et al.
`
`CH2), 2.82 (t, 2H, J = 7 Hz, CH2-phenyl), 3.10-3.44 (m, 2H,
`7.10-7.34 (m, 5H, phenyl-H), 8.47 (s, lH, imidazole-2H). MS
`CHzNH), 3.44-3.79 (m, 2H, CHzCH2-phenyl), 7.12 (s, l H ,
`(EI, re1 intensity): m l z 274 (M+, 39), 220 (M+ - H2S, 21, 153
`imidazole-5(4)H), 7.16-7.36 (m, 5H, phenyl-H), 8.49 (s, lH,
`(M+ - C8HgNH2, 22), 105 ([C8H91f, 42), 95 ([ImC2H41f, 1001,
`imidazole-2H). MS (EI, re1 intensity): m l z 288 (M+, 0.21, 95
`91 ([C,H7]+, 641, 81 ([ImCH21+, 43). HRMS: m l z 274.1253;
`([ImCzH4]+, 81, 91 ([C7H71+, 471, 82 ([ImCH31+, 181, 45 ([C2H5-
`
`calcd for C14H18N4S1 274.1252. Anal. ( C M H I B N ~ S G H ~ O ~ ) C,
`NH2]+, 100). HRMS: m l z 288.1414; calcd for C I ~ H ~ O N ~ S ,
`H, N.
`
`288.1409. Anal. ( C ~ ~ H ~ O N ~ S . ~ . ~ C ~ H Z O ~ ) C, H, N.
`N-Methyl-N'-[3-(4(5)-imidazolyl)propyl] thiourea oxa-
`N-Methyl-N'-[4-(4(5)-imidazolyl)bu~ll thiourea oxalate
`late (3a): mp 126.1-128.9 "C; yield 49%. 'H NMR (D2O): 6
`(4a): mp 120.1-122.6 "C; yield 18%. 'H NMR (D2O): 6 1.61
`1.96 (m, 2H, CHZCH~NH), 2.77 (t, J = 7 Hz, 2H, imidazole-
`(m, 4H, central CHz's), 2.72 (t, 2H, J = 7 Hz, imidazole-CHd,
`CHz), 2.86 (b s, 3H, CH3), 3.30-3.67 (m, 2H, CHzNH), 7.23 (s,
`2.82 (m, 3H, CH31, 3.22-3.62 (m, 2H, CH2NH), 7.17 (s, 1H,
`1H, imidazole-5(4)H), 8.57 (s, l H , imidazole-2H). MS (EI, re1
`imidazole-5(4)H), 8.52 (s, l H , imidazole-2H). MS (EI, re1
`intensity): m l z 198 (M+, 201,167 (M+ - CH3NH2,6), 164 (M+
`intensity): m l z 212 (M+, 791, 181 (M+ - CH3NH2, 201, 179
`- H2S, 5), 109 ([Im&Hs]+, 12),95 ([ImC2&1+, 941, 82 ([ImCHd+,
`(M+ - HS, 91, 123 ([ImC4H81+, 421, 109 ([ImC3Hsl+, 431, 95
`100). HRMS: m l z 198.0929; calcd for C B H ~ ~ N ~ S ,
`198.0939.
`([ImC2H41+, 1001, 81 ([ImCHz]+, 69). HRMS: m l z 212.1091;
`Anal. (C8H14N4S.O.84C2H204) C, H, N.
`calcd for CgH1&S, 212.1096. Anal. (CgH16N4S@.8C2H204) C,
`N-Ethyl-N'-[3-(4(5)-imidazolyl)propyllthiourea oxalate
`H, N.
`(3b): mp 116.1 "C; yield 44%. 'H NMR (DzO): 6 1.12 (t, 3H,
`N-Ethyl-N'-[4-(4(5)-imidazolyl)butyllthiourea oxalate
`J = 7 Hz, CH3), 1.95 (m, 2H, CH2CH2NH), 2.77 (t, 2H, J = 8
`(4b): mp 120.3 "C; yield 29%. 'H NMR (DzO): 6 1.08 (t, 3H,
`Hz, imidazole-CHz), 3.15-3.62 (m, 4H, 2*CHzNH), 7.23 (s, lH,
`J = 7Hz, CH3), 1.61 (m, 4H, central CHz's), 2.72 (t, 2H, J = 7
`imidazole-5(4)H), 8.57 (s, l H , imidazole-2H). MS (EI, re1
`Hz, imidazole-CH2), 3.22-3.51 (m, 4H, 2*CHzNH), 7.17 (s, l H ,
`intensity): m l z 212 (M+, 531, 178 (M+ - HzS, 101, 167 (M+ -
`imidazole-5(4)H), 8.52 (s, l H , imidazole-2H). MS (EI, re1
`C2H5NH2, 41, 109 ([ImC3Hsl+, 311, 95 ([ImC2H43+, 851, 82
`intensity): m l z 226 (M+, 81), 193 (M+ - HS, 71, 181 (M+ -
`([ImCH31+, 100). HRMS: m l z 212.1092; calcd for C ~ H M N ~ S ,
`C2H5NH2, 251, 123 ([ImC4H81f, 47), 109 ([ImC3Hd+, 401, 95
`
`212.1096. Anal. ( C ~ H I ~ N ~ S C ~ H ~ O ~ ) C, H, N.
`([ImC2H41+, 1001, 81 ([ImCH21+, 75). HRMS: m l z 226.1250;
`N.n.Propyl-N'-[3-(4(5)-imidazolyl)propyllthiourea
`calcd for C ~ O H ~ ~ N ~ S ,
`226.1252. Anal. ( C I O H ~ ~ N ~ S . ~ . ~ C ~ H ~ O ~ )
`oxalate (3c): mp 123.2-125.2 "C; yield 24%.
`'H NMR
`C, H, N.
`(DzO): 6 0.87 (t, 3H, J = 7 Hz, CH31, 1.53 (m, 2H, CH2CHd,
`N-n-Propyl-N'-[4-(4(5)-imidazolyl)butyl]thiourea oxa-
`1.97 (m, 2H, CH2CH2NH), 2.77 (t, 2H, J = 7 Hz, imidazole-
`late (412): mp 146.9 "C; yield 55%. 'H NMR (D2O): 6 0.84 (t,
`3H, J = 7 Hz, CH3), 1.42-1.78 (m, 6H, central CHis + CH2-
`CHz), 3.10-3.65 (m, 4H, 2*CHzNH), 7.23 (s, l H , imidazole-
`5(4)H), 8.56 (s, lH, imidazole-2H). MS (EI, re1 intensity): mlz
`CH3), 2.73 (t, 2H, J = 7 Hz, imidazole-CHz), 3.10-3.62 (m,
`226 (M+, 91, 192 (M+ - H2S, 41, 167 (M+ - C ~ H ~ N H Z , 41, 109
`
`4H, 2*CHzNH), 7.18 (s, lH, imidazole-5(4)H), 8.53 (s, lH,
`([ImC3Hs]+, 91, 95 ([ImCzHd+, 1001, 82 ([ImCH31+, 33).
`imidazole-2H). MS (EI, re1 intensity): m l z 240 (M+, 691, 207
`HRMS: mlz 226.1265; calcd for C ~ O H I ~ N ~ S ,
`226.1252. Anal.
`(M+ - HS, 7), 181 (M+ - C3H7NH2, 231, 123 ([ImC4Hsl+, 551,
`(CloHiaN4S.0.8C2H204) C, H, N.
`109 ([ImC3H6]+, 381, 95 ([ImC2H41+, 1001, 81 ([ImCH21+, 801,
`N-Isopropyl)-N'-[3-(4(5)-imidazolyl)propyllthiourea
`45 ([CzHsNHz]+, 71). HRMS: m l z 240.1409; calcd for
`oxalate (3d): mp 146.0 "C; yield 43%. 'H NMR (D20): 6 1.13
`
`CllH20N4S, 240.1409. Anal. ( C I I H ~ O N ~ S C ~ H ~ O ~ ) C, H, N.
`(d, 6H, J = 7 Hz, CH3), 1.94 (m, 2H, CH~CHZNH), 2.77 (t, 2H,
`N-Isopropyl-N'-[4-(4(5)-imidazolyl)butyll thiourea oxa-
`J = 7 Hz, imidazole-CH2), 3.37-3.58 (m, 2H, CHzNH), 3.89-
`late (4d): mp 151.3 "C; yield 64%. 'H NMR (DzO): 6 1.16 (d,
`4.17 (m, lH, CH), 7.23 (s, l H , imidazole-5(4)H), 8.57 (s, lH,
`6H, J = 7 Hz, 2*CH3), 1.65 (m, 4H, central CHz's), 2.76 (t, 2H,
`imidazole-2H). MS (EI, re1 intensity): m l z 226 (M', 301, 192
`J = 7 Hz, imidazole-CHz), 3.43 (m, 2H, CHzNH), 4.08 (m, lH,
`(M+ - HzS, 61, 167 (M+ - C ~ H ~ N H Z ,
`71, 109 ([ImC3Hd+, 231,
`CH), 7.21 (s, lH, imidazole-5(4)H), 8.55 (s, lH, imidazole-2H).
`95 ([ImC2H4]+, 79),82 ([ImCH#, 66). HRMS: m l z 226.1271;
`MS (EI, re1 intensity): m l z 240 (M+, 601, 207 (M+ - HS, 51,
`calcd for C10H18N4S, 226.1252. Anal. ( C ~ O H ~ ~ N ~ S . O . ~ C ~ H ~ O ~ )
`181 (M+ - C3H7NH2, 171, 123 ([ImC4Hsl+, 511, 109 ([ImC3Hd+,
`C, H, N.
`25), 95 ([ImC2H41+, 701, 81 ([ImCH21+, 521, 45 ([CzHsNH21+,
`N-Cyclohexyl-N'-[3-(4(5)-imidazolyl)propyll thiourea
`100). HRMS: m l z 240.1401; calcd for C I I H ~ ~ N ~ S ,
`240.1409.
`oxalate (3e): mp 102.2 "C; yield 50%. 'H NMR (D2O): 6
`0.93-1.97 (m, 12H, CH2CH2NH + cyclohexyl-CH2's), 2.70 (t,
`Anal. (CllH2oN4S-1.76CzHz04) C, H, N.
`2H, J = 8 Hz, imidazole-CHz), 3.23-3.90 (m, 3H, CHzNH +
`N-Cyclohexyl-N'-[4-(4(5)-imidazolyl)butyll thiourea
`oxalate (4e): mp 109.5 "C; yield 24%. 'H NMR (DMSO-&):
`6 1.00-1.95 (m, 14H, central CHz's + cyclohexyl-CHz's), 2.64
`CHNH), 7.18 (s, l H , imidazole-5(4)H), 8.52 (s, lH, imidazole-
`2H). MS (EI, re1 intensity): m l z 266 (M+, 291,232 (M+ - H2S,
`(m, 2H, imidazole-CHz), 3.37 (m, 2H, CHzNH), 3.93 (m, l H ,
`C m , 7.20 (s, lH, imidazole-5(4)H), 7.28-7.62 (m, 4H, NH +
`8), 167 (M+ - CdllNH2, 61, 109 ([ImC3Hd+, 241, 95 ([ImCzH43+,
`73), 82 ([ImCH31+, 100). HRMS: m l z 266.1572; calcd for
`C O a , 8.50 (s, l H , imidazole-2H). MS (EI, re1 intensity): m l z
`
`C ~ ~ H Z ~ N ~ S , 266.1565.
`280 (M+, 661, 247 (M+ - HS, 71, 181 (M+ - CsHiiNH2, 281,
`N-Phenyl-N'-[3-(4(5)-imidazolyl)propyllthiourea oxa-
`123 ([ImC4H81+, 621, 109 ([ImC3&]+, 321, 95 ([ImC2H41+, loo),
`late (30: mp 126.7 "C; yield 47%. lH NMR (DzO): 6 1.90 (m,
`81 ([ImCH2]+, 761, 45 ([CzHaHzl+, 65). HFMS mlz 280.1724;
`2H, CH~CHZNH), 2.70 (t, 2H, J = 7 Hz, imidazole-CHd, 3.39-
`3.65 (m, 2H, CH2NH), 7.27