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  • Bioorganic & Medicinal Chemistry 25 (2017) 31713181

    Contents lists available at ScienceDirect

    Bioorganic & Medicinal Chemistry

    journal homepage: www.elsevier .com/locate /bmc

    Synthesis, biochemical evaluation, and molecular modeling studies ofaryl and arylalkyl di-n-butyl phosphates, effective butyrylcholinesteraseinhibitors

    http://dx.doi.org/10.1016/j.bmc.2017.04.0020968-0896/ 2017 Elsevier Ltd. All rights reserved.

    Corresponding authors.E-mail addresses: Kensaku.nakayama@csulb.edu (K. Nakayama), Jason.schwans

    @csulb.edu (J.P. Schwans), Eric.sorin@csulb.edu (E.J. Sorin).

    Kensaku Nakayama a,, Jason P. Schwans a,, Eric J. Sorin a,, Trina Tran a, Jeannette Gonzalez a, Elvis Arteaga a,Sean McCoy a, Walter Alvarado b

    aDepartment of Chemistry and Biochemistry, California State University, Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840, USAbDepartment of Physics, California State University, Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840, USA

    a r t i c l e i n f o a b s t r a c t

    Article history:Received 4 January 2017Revised 29 March 2017Accepted 3 April 2017Available online 5 April 2017

    Keywords:CholinesteraseAlzheimers diseaseOrganophosphatesEnzyme inhibitionComputational docking

    A series of dialkyl aryl phosphates and dialkyl arylalkyl phosphates were synthesized. Their inhibitoryactivities were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Thedi-n-butyl phosphate series consistently displayed selective inhibition of BChE over AChE. The mostpotent inhibitors of butyrylcholinesterase were di-n-butyl-3,5-dimethylphenyl phosphate (4b)[KI = 1.0 0.4 lM] and di-n-butyl 2-naphthyl phosphate (5b) [KI = 1.9 0.4 lM]. Molecular modelingwas used to uncover three subsites within the active site gorge that accommodate the three substituentsattached to the phosphate group. Phosphates 4b and 5b were found to bind to these three subsites inanalogous fashion with the aromatic groups in both analogs being accommodated by the lower region,while the lone pairs on the P@O oxygen atoms were oriented towards the oxyanion hole. In contrast, di-n-butyl-3,4-dimethylphenyl phosphate (4a) [KI = 9 1 lM], an isomer of 4b, was found to orient its aro-matic group in the upper left region subsite as placement of this group in the lower region resultedin significant steric hindrance by a ridge-like region in this subsite. Future studies will be designed toexploit these features in an effort to develop inhibitors of higher inhibitory strength againstbutyrylcholinesterase.

    2017 Elsevier Ltd. All rights reserved.

    1. Introduction

    Cholinesterase inhibitors are currently the major drug type inuse to manage the progression of Alzheimers disease (AD), themost common type of adult-onset dementia.1 AD-associated cogni-tive impairment is correlated with acetylcholine (ACh) level reduc-tion in the brain. This cognitive loss takes place in conjunction withchanges in cholinesterase activity where acetylcholinesterase(AChE) activity decreases and butyrylcholinesterase (BChE) activityincreases.2 BChE activity in the AD brain is known to be elevated4065% above normal while AChE activity decreases to about65% of the normal level.2

    Since both AChE and BChE hydrolyze ACh, the use of cholines-terase inhibitors as treatment for AD is based on the hypothesisthat the inhibition of these enzymes will increase the concentra-tion of acetylcholine in the brain.3 Thus, current treatment for

    AD involves the administration of reversible dual cholinesteraseinhibitors, such as galantamine and tacrine, to suppress the activityof both AChE and BChE.4 Recently, increased efforts are being madeto develop cholinesterase inhibitors that target BChE in order totreat AD at more advanced stages.5,6

    For example, cymserine analogs that selectively inhibited BChEwere shown to raise ACh levels, improve cognition and lower b-amyloid peptide in rodent.7 In addition, N1-phenethylnorcymser-ine, a BChE-selective inhibitor, suppressed cognitive dysfunctionin mice challenged by amyloid-b peptide.8 These results suggestthat abnormal BChE activity increases the severity of cognitive dys-function associated with AD. Therefore, it is possible that suitableBChE-inhibitors may have a significant role to play in the treat-ment of cognitive loss associated with AD.

    The inhibition of the cholinesterases by organophosphorusinsecticides and nerve toxins is widely appreciated.911 Investiga-tions into the selective inhibition of BChE over AChE byorganophosphorus compounds is also receiving increased atten-tion recently. For example, the exploration of organophosphatesas AD therapeutics that are BChE-selective have been recently

    http://crossmark.crossref.org/dialog/?doi=10.1016/j.bmc.2017.04.002&domain=pdfhttp://dx.doi.org/10.1016/j.bmc.2017.04.002mailto:Kensaku.nakayama@csulb.edumailto:Jason.schwans @csulb.edumailto:Jason.schwans @csulb.edumailto:Eric.sorin@csulb.eduhttp://dx.doi.org/10.1016/j.bmc.2017.04.002http://www.sciencedirect.com/science/journal/09680896http://www.elsevier.com/locate/bmc

  • 3172 K. Nakayama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 31713181

    reported by Richardson and co-workers.12 Meanwhile, Sultatos andco-workers have reported the higher reactivity of the organophos-phate chlorpyrifos towards BChE over AChE.13 More recently,Kaboudin and Emadi found that phosphorothioates were moder-ately more selective inhibitors of BChE over AChE.14 A recent studyby Vinsova and co-workers has established that salicyanilidediethyl phosphates are excellent inhibitors of both AChE and BChE,with a preference for the latter enzyme, surpassing in some casesthe inhibitory activity of the currently approved AD medicationsrivastigmine and galantamine.15

    Some time ago, we systematically studied the inhibitory prop-erties of a small library of dialkyl phenyl phosphates (DAPPs;Fig. 1) against AChE and BChE,16 a family of compounds that pos-sess some structural similarities to metrifonate, a dual cholinester-ase inhibitor that raised acetylcholine levels and cognitive abilityin AD patients during clinical trials.17 We expected that the DAPPsshould act as reversible inhibitors since they lack a good leavinggroup. We found that phosphate 1a displayed the weakest inhibi-tion against BChE among the series, while it was shown to be themost efficient inhibitor of AChE. Meanwhile, compound 1d wasthe best inhibitor of BChE, while displaying no inhibition againstAChE. Encouraged by these findings, we decided to explore theeffects on inhibition against the cholinesterases, with particularfocus on BChE, when systematic changes were introduced intothe aromatic portion of the overall scaffold. The results of thesestudies are described herein.

    2. Results and discussion

    2.1. Chemistry

    We studied a library of phosphates comprised of aryl di-n-butylphosphates (3a3c, 4ac, 5a, 5b), benzyl di-n-butyl phosphate(6a), di-n-butyl methylphenylmethyl phosphates (6a6d), and di-n-butyl phenylethyl phosphate (7) (Fig. 2). As mentioned earlier,the library of compounds examined in our previous work16 didnot probe the effects of changes in the phenyl portion of the inhi-bitor scaffold on enzyme inhibition. In this study, one main goalwas to explore how the methyl group location on the aromatic ringaffects inhibition. We also changed the size of the phenyl ring bysubstituting it with a naphthyl group. In addition, we incorporateda methylene group as well as an ethyl chain between the phenylring and the phosphate oxygen atom to study the effects of thesechanges on enzyme inhibition.

    In addition to the library of dibutyl phosphates listed in Fig. 2,we also prepared two aryl diethyl phosphates 8 and 9 (Fig. 3)which allowed us to study the effects of shortened alkyl chainson inhibition. These phosphates were prepared in acceptable yieldsand purity (>95% by GC/MS) by allowing either diethyl chlorophos-phate 10 or di-n-butyl chlorophosphate 11 to react with an excessof the appropriate alcohol, ROH, in the presence of pyridine(Scheme 1). Typical reaction times were approximately 12 h,except for the case when 1-naphthol was employed as the nucle-ophile, which required three days. All compounds were smoothly

    O PO

    OROR

    O PO

    OR'OR

    1a: R = CH31b: R = CH2CH31c: R = CH2CH2CH31d: R = CH2(CH2)2CH31e: R = CH2(CH2)3CH31f: R = c-C6H11

    2: R = CH2CH2; R' = CH2(CH2)2CH3

    Fig. 1. Library of dialkyl phenyl phosphates employed as inhibitors.

    purified via column chromatography to afford colorless or near col-orless oils that gave satisfactory 1H NMR, 13C NMR and mass spec-tral data.

    2.2. Enzyme inhibition studies

    2.2.1. Evaluating the effect of adding substituents on the phenyl groupof alkyl phenyl phosphates

    As described above, Law et al. previously measured inhibitionconstants for a series of alkyl phenyl phosphates for BChE andAChE (Fig. 1).16 Increasing the length of the alkyl groups fromone to five carbons led to more potent inhibitors. Although com-parison of the reported KI values suggests the dipentyl phenylphosphate was the most potent inhibitor relative to the shorterchain analogs (KI = 6 lM (1e) versus KI = 4580 lM (1c, 1d), werecently observed that limited solubility affects the determinationof the inhibition behavior of the dipentyl phenyl analog (unpub-lished results). The di-n-butyl phenyl analog, however, was solublein the range of concentrations used to measure inhibition con-stants. We therefore investigated the effect of adding phenyl sub-stituents in the di-n-butyl phenyl scaffold.

    We first investigated the inhibitory behavior of introducing amethyl group on the phenyl ring at the 2-, 3-, or 4-position. Thesolubility of each analog in 2% methanol was evaluated usingabsorbance spectra. The absorbance increased linea

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