DOI:10.1021/jm901530b

Synthesis and Structure-Activity Relationships of Azamacrocyclic C-X-C Chemokine Receptor4 Antagonists:Analogues Containing a Single Azamacrocyclic Ring are Potent Inhibitors of T-Cell Tropic(X4)HIV-1Replication

Gary J.Bridger,*,†Renato T.Skerlj,†,)Pedro E.Hernandez-Abad,‡David E.Bogucki,†Zhongren Wang,†Yuanxi Zhou,†Susan Nan,†Eva M.Boehringer,†Trevor Wilson,†Jason Crawford,†Markus Metz,†,)Sigrid Hatse,§Katrien Princen,§Erik De Clercq,§and Dominique Schols§

†AnorMED Inc.now Genzyme Corporation,500Kendall Street,Cambridge,Massachusetts02142,‡Johnson Matthey Pharmaceutical Research,1401King Road,West Chester,Pennsylvania19380,and§Rega Institute for Medical Research,Katholieke Universiteit Leuven, Minderbroedersstraat10,B-3000Leuven,Belgium.)Genzyme Corp.,153Second Avenue,Waltham,Massachusetts02451.

Received October15,2009

Bis-tetraazamacrocycles such as the bicyclam AMD3100(1)are a class of potent and selective anti-HIV-1

agents that inhibit virus replication by binding to the chemokine receptor CXCR4,the coreceptor for entry

of X4viruses.By sequential replacement and/or deletion of the amino groups within the azamacrocyclic ring

systems,we have determined the minimum structural features required for potent antiviral activity in this

class of compounds.All eight amino groups are not required for activity,the critical amino groups on a per

ring basis are nonidentical,and the overall charge at physiological pH can be reduced without compromising

potency.This approach led to the identification of several single ring azamacrocyclic analogues such as

AMD3465(3d),36,and40,which exhibit EC50’s against the cytopathic effects of HIV-1of9.0,1.0,and

4.0nM,respectively,antiviral potencies that are comparable to1(EC50against HIV-1of4.0nM).More

importantly,however,the key structural elements of1required for antiviral activity may facilitate the design

of nonmacrocyclic CXCR4antagonists suitable for HIV treatment via oral administration.

Introduction

The development of antiviral agents that inhibit alternative targets in the HIV a-replicative cycle remains an important goal in order to alleviate the side effects of currently approved agents or to overcome the problem of drug resistance.In this regard,we have focused on the development of compounds that inhibit CXCR4,the coreceptor used by T-tropic(T-cell tropic)viruses for fusion and entry of HIV into target cells of the immune system.The corresponding chemokine receptor CCR5is used by M-tropic(macrophage tropic)viruses and has been associated with the early stages of infection and replication in HIV-positive patients.1,2The transition from M-tropic to T-tropic(or dual/mixed-tropic)virus during the course of HIV infection in approximately50%of patients is associated with a faster CD4þT-cell decline and a more rapid disease progression.3-5

Recently,we reported the results of clinical trials with our prototype CXCR4antagonist AMD31006-8(1)and an orally bioavailable CXCR4antagonist,(S)-N0-(1H-benzimidazol-2-ylmethyl)-N0-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-dia-mine(AMD070).9-11When administered to HIV positive patients whose virus was confirmed to use CXCR4for viral entry,both agents were able to suppress the replication of CXCR4and dual-tropic strains of HIV.Similarly,the CCR5 antagonist Maraviroc suppresses replication of HIV-1that exclusively uses CCR5for entry12and was recently approved by the FDA for combined antiretroviral therapy in treatment-experienced patients.13A combination of CCR5and CXCR4 antagonists for treatment of dual/mixed-tropic HIV infection is therefore highly desirable.

Beyond its use as a coreceptor for HIV,the CXCR4 chemokine receptor has a more fundamental role in the trafficking of white blood cells,which broadly express CXCR4.14,15A member of the superfamily of G-protein coupled receptors,the interaction of CXCR4and its ligand, stromal cell-derived factor-1(SDF-1),plays a central role in the homing and retention of cells within the bone marrow microenvironment.16Consistent with these observations,ad-ministration of1to healthy volunteers caused a dose-depen-dent leukocytosis6,7that in subsequent studies was shown to include the mobilization of CD34þstem and progenitor cells suitable for hematopoietic stem cell transplantation.17-20The ability of analogues of1to mobilize progenitors correlated with their in vitro capacity to inhibit SDF-1binding to CXCR4.21Because of the need for parenteral administration, 1was developed in combination with granulocyte colony-stimulating factor(G-CSF)to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin’s lymphoma(NHL)and multiple myeloma(MM).22-25Plerix-afor(1)was approved by the FDA in December2008.

We have previously reported the structure-activity rela-tionships of anti-HIV bis-azamacrocycles and their transition

*To whom correspondence should be addressed.Phone:617-429-

7994.Fax:617-768-9809.E-mail:gary.bridger@genzyme.com.Ad-

dress:Gary J.Bridger,Genzyme Corporation,55Cambridge Parkway,

Cambridge MA02142.

a Abbreviations:HIV,Human Immunodeficiency Virus;CXCR4,

C-X-C chemokine receptor4;CCR5,C-C-R chemokine receptor5.

pubs.acs.org/jmc Published on Web12/31/2009r2009American Chemical SocietyArticle Journal of Medicinal Chemistry,2010,Vol.53,No.31251 metal complexes in detail.26-28Because of the common

structural features between a doubly protonated cyclam

(1,4,8,11-tetraazacyclotetradecane)ring present in1(at phy-

siological pH)and a kinetically labile transition metal com-

plex of cyclam with an overall charge ofþ2,we proposed that

both structural motifs may bind to the CXCR4receptor

through interactions with amino acid residues containing

carboxylate groups.29We have subsequently shown via direc-

ted mutagenesis of the aspartate and glutamic acid residues in

CXCR4that binding of1and related analogues to the seven

transmembrane,G-protein coupled receptor is highly depen-

dent upon the amino acids Asp171and Asp262,located in

transmembrane region(TM)-IV and TM-VI at each end of

the main ligand binding crevice of the receptor.30-35Mutation

of either aspartic acid to aspargine significantly reduced the

ability of1to inhibit binding of radiolabeled stromal cell

derived factor-1R(125I-Met-SDF-1R).More importantly,

however,U87cells stably transfected with CD4and the

mutant coreceptors CXCR4[D171N]and CXCR4[D262N]

were less effective at supporting infection of the CXCR4-using

HIV-1strain NL4.3compared to the wild-type receptor and

the double mutant CXCR4[D171N,D262N]completely failed

as a coreceptor for HIV infection.31Correspondingly,the

ability of1to inhibit HIV-1infection via CXCR4[D171N]and

CXCR4[D262N]was also diminished,thereby confirming

that1binds in a region of the receptor that is critical for X4

HIV-1coreceptor function.

We have also reported that binding of the bis-Zn,Ni,and

Cu complexes of1were also dependent upon D171and D262

of the receptor.36In a similar manner to1,the transition

metal complexes were found to be less effective inhibitors of

125I-Met-SDF-1R binding to the mutant receptors CXCR4-

[D171N]and CXCR4[D262N]compared to the wild-type

receptor.Incorporation of Zn,Ni,or Cu into the cyclam rings

of1increased the affinity to the wild-type CXCR4receptor,

but the enhancement was selectively eliminated by substitu-

tion of Asp262.Supporting physiochemical evidence for the

interaction of acetate(carboxylates)with metal complexes of

azamacrocycles,including1,has been recently reported.37,38

In the current study,we determine the minimum struc-

tural features of1required for potent antiviral activity, leading to the identification of the single azamacrocyclic ring analogue AMD346532,33,39,40(3d)and ultimately the design of nonmacrocyclic,orally biovailable CXCR4an-tagonists.11,41,42Given the growing body of evidence that the CXCR4/SDF-1interaction is involved in regulating several human malignancies,43-45CXCR4antagonists may have additional therapeutic applications in addition to HIV treatment.

Chemistry

Analogues containing a single1,4,8,11-tetraazacyclotetra-decane(cyclam)ring were prepared by modifications to previously published routes26,29as shown in Scheme1.Reac-tion of the selectively protected tris-diethylphosphoramidate (Dep)cyclam ring(2a)with R,R-dibromo-p-xylene in aceto-nitrile containing potassium carbonate gave the desired bro-momethyl intermediate(2b).Reaction of the bromide with an excess of the requisite amine,followed by deprotection of the Dep-groups with a saturated solution of hydrogen bromide in acetic acid at room temperature.gave analogues3a-i as the corresponding hydrobromide salts.

To prepare analogues of3d in which the cyclam ring was replaced by a series of14-membered azamacrocyclic rings,we prepared a series of selectively protected macrocyclic ring systems containing a single(unprotected)secondary amine. This approach ensures the regiochemical outcome of the reaction with a benzylic halide during final construction (as shown in Scheme6).The syntheses of appropriate pre-cursors are shown in Schemes2-5.To incorporate fluorine groups at the desired position in the macrocyclic ring,suitably fluorinated bis-electrophiles were prepared,starting from 4-oxo-heptanedioic acid diethyl ester(4)and heptane-1,4,7-triol(8)as depicted in Scheme2.Reaction of the ketone(4) with neat(diethylamino)-sulfur trifluoride46,47(DAST)at room temperature for12days gave the corresponding di-fluoro-intermediate(5)in43%yield.Reduction of the ester groups with LAH(to give the diol6),followed by derivatiza-tion with toluenesulfonyl chloride,gave the bis-electrophile (7)required for the impending macrocyclization reaction.The corresponding monofluorinated intermediate was prepared in a similar manner.Protection of the primary alcohols in8as the acetyl group using acetic anhydride gave the secondary alcohol9,which was rapidly(and virtually quantitatively) converted to the fluorinated intermediate(10)with DAST (2.0equiv)in dichloromethane.Removal of the acetyl pro-tecting groups with saturated ammonia in methanol,followed by reaction of the diol(11)with p-toluenesulfonyl chloride, Scheme1

a

a Reagents:(a)R,R0-dibromo-p-xylene,K

2

CO3,CH3CN,reflux;

(b)amine,K2CO3,CH3CN,reflux;(c)HBr,acetic acid,room temp. Scheme2

a

a Reagents:(a)Et

2

NSF3(neat),room temp;(b)LAH,Et2O;

(c)Ts-Cl,Et3N,CH2Cl2;(d)acetic anhydride,pyridine;(e)Et2NSF3, CH2Cl2,-78°C,then room temp;(f)NH3/MeOH,room temp;

(g)Ts-Cl,Et3N,CH2Cl2.

1252Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.

gave the desired bis-electrophile 12containing a single fluorine group.

The selectively protected azamacrocyclic rings were pre-pared via directed combinatorial macrocyclization of bis-2-nitrobenzenesulfonamides 48(Ns)(15a -c ,16a -c ,18)with bis-electrophiles (7,12,17)using previously optimized condi-tions 28(Scheme 3).To incorporate a phenyl or heterocyclic ring into the macrocycle,the corresponding bis-2-nitrobenze-nesulfonamide (15a -c )was prepared from the bis-aminoethyl intermediates 28(13a -c )by reaction with nosyl chloride (Et 3N,CH 2Cl 2).Similarly,16a ,b were obtained by reac-tion of commercially available intermediates 14a ,b with nosyl chloride or in the case of 16c (X=S)by reduction of 3,30-thiodipropionitrile with BH 33Me 2S and reaction of the intermediate diamine (14c )with nosyl chloride to give 16c .Macrocyclization was accomplished by dropwise addition of a DMF solution of the bis-electrophile to a DMF solution of the bis-2-nitrobenzenesulfonamide containing Cs 2CO 3maintained at a temperature of 80°C.Standard workup,followed by purification of the crude product by column chromatography on silica gel,gave the desired macrocycles 19a -c ,20a -c ,and 21a ,b in yields of 19-55%.Reaction of the

intermediates from above with HBr/acetic acid at room temperature gave 22a -c ,23a -c ,and 24a ,b ,respectively.Because of synthetic convenience,we also prepared the selectively protected “isomers”of 22a ,b and 23a in which the alternative secondary amine was available for the alkylation reaction.We reasoned that reaction of 19a ,b and 20a with approximately 1equiv of thiophenol 49(our reagent of choice for nosyl deprotections)may allow pseudoselective deprotec-tion of a single nosyl group,leaving the Dep group intact.After some optimization,we found that reaction of 19a ,b and 20a with 0.8equiv of thiophenol and potassium carbonate in DMF (or acetonitrile)gave the precursors 25and 26a ,b in manageable,albeit modest yields (20-50%)following col-umn purification on silica gel (Scheme 4).Finally,the inter-mediates 27a ,b and 28(Scheme 5)were synthesized as recently described by palladium(0)catalyzed coupling of organozinc iodide reagents with bromopyridines.50

Having completed the series of selectively protected aza-macrocycles,we proceeded to completion of the desired analogues by straightforward installation of the right-hand portion containing the aminomethyl pyridine moiety.As shown in Scheme 6,this was accomplished in all cases by direct alkylation of the available secondary amine of the macrocycle with the benzylic chlorides 34a ,b .Intermediate 34a was prepared in four steps from 4-bromomethyl benzoic acid methyl ester (29)and 2-aminomethylpyridine (31):con-version of 31to the 2-nitrobenzenesulfonamide 32,followed by alkylation with the benzyl bromide 30(obtained by reduc-tion of 29with DIBAL-H)gave the desired alcohol 33.As previously reported,28reaction of benzylic alcohols such as 33with methanesulfonyl chloride gave the chloride 34a rather than the corresponding mesylate,presumably via in situ nucleophilic substitution of the initially formed mesylate with chloride.Intermediate 34b (Scheme 6)containing a Dep-protecting group was prepared by an alternative synthesis

Scheme 3

a

a

Reagents:(a)Ns-Cl,Et 3N,CH 2Cl 2;(b)Cs 2CO 3,DMF,80°C;(c)HBr(g),AcOH,room temp.

Scheme

4

Scheme

5

Article Journal of Medicinal Chemistry,2010,Vol.53,No.31253

(procedures in Supporting Information).Alkylation of the available secondary amine of the macrocycles with 34a (or 34b )in CH 3CN in the presence of K 2CO 3gave the penultimate intermediates 35a -n .Deprotection of the nosyl groups with thiophenol and K 2CO 3in DMF gave the free base of the desired analogues,which in the vast majority of cases were converted to the corresponding hydrobromide salts.For analogues derived from the macrocyclic precursors 25and 26a ,b ,the intermediates isolated prior to the deprotection also contained a residual Dep group in addition to nosyl groups.For compound 45,we found that conversion to the hydro-bromide salt using a saturated solution of HBr in acetic acid resulted in concomitant deprotection of the remaining Dep group to obtain compound 45.For compounds 44and 46,the residual Dep group was removed prior to nosyl deprotection and salt formation.

The thioether analogue 41a was also used to prepare the corresponding sulfoxide and sulfone analogues for antiviral evaluation as shown in Scheme 7.Initially,we globally protected the amino groups of 41a with Boc and subjected this intermediate to oxidation with oxone in MeOH 51at -10°C to give a mixture of the sulfoxide and sulfone that were separated by column chromatography on silica gel.However,while deprotection of the Boc groups with simulta-neous conversion to the hydrobromide salt proceeded without incident for the sulfone (to give 41c ),we found that deprotec-tion of the corresponding sulfoxide led to substantial reduc-tion and hence recovery of the starting analogue 41a .To overcome this problem,the sulfoxide was synthesized by direct oxidation of 41a with 1equiv of oxone in MeOH to give 41b in a 21%isolated yield and was subsequently tested as the free base in antiviral assays.

Finally,we prepared a short series of analogues containing a carbon atom in place of a tertiary nitrogen group at the ring junction.To economize on the number of synthetic steps,we

elected to synthesize the dimesylate 54(Scheme 8),an inter-mediate that could be commonly used for the synthesis of multiple analogues via macrocylization with the bis-2-nitro-benzenesulfonamide precursors already in our possession (namely 15a ,16a ,b from Scheme 3).Intermediate 54was prepared from the commercially available starting material bromo-p -tolunitrile via a double one-carbon homologation of the malonate 51,followed by derivatization to gave the requisite bis-methanesulfonate 54.Macrocyclizations of 54with bis-sulfonamides 15a and 16a ,b were performed as described above.Deprotection of the nosyl groups followed by conversion to the corresponding hydrobromide salts gave analogues 56and 58a ,b .Discussion

Having previously established the optimum ring size and distance between the amines of both aliphatic and

Scheme 6

a a

Reagents:(a)DIBAL-H,CH 2Cl 2;(b)Ns-Cl,Et 3N,CH 2Cl 2;(c)K 2CO 3,CH 3CN,60°C;(d)Ms-Cl,Et 3N,CH 2Cl 2;(e)K 2CO 3,CH 3CN,80°C;(f)R =Ns:thiophenol,K 2CO 3,DMF,or R =Dep:HBr(g),AcOH,room temp.

Scheme 7

a

a

Reagents:(a)oxone,MeOH,-10°C;(b)(Boc)2O,THF;(c)HBr(g),AcOH,room temp.

Scheme 8

a

a

Reagents:(a)NaH,R -bromo-tolunitrile,THF;(b)LiAlH 4,THF;(c)Ns-Cl,Et 3N,CH 2Cl 2;(d)2-picolyl chloride,Et 3N,K 2CO 3,KBr,CH 3CN,reflux;(e)Ms-Cl,Et 3N,CH 2Cl 2;(f)cetyltrimethyammonium bromide,NaCN,benzene,H 2O,reflux;(g)conc HCl/AcOH (4:1),reflux;(h)BH 3.Me 2S,THF;(i)Ms-Cl,Et 3N,CH 2Cl 2;(j)Cs 2CO 3,DMF,80°C;(k)thiophenol,K 2CO 3,CH 3CN (or DMF),40°C.

1254Journal of Medicinal Chemistry,2010,Vol.53,No.3

Bridger et al.

pyridine-fused bis-tetraazamacrocycles required for potent X4anti-HIV activity,we designed a series of compounds to address the question of structural redundancy.The prototype bis-macrocycle 1has a center of symmetry and contains eight amino groups,of which four are positively charged at phy-siological pH.In the current study,we aimed to answer two specific questions:(1)Are all four positive charges required for potent anti-HIV activity?(2)On a per ring basis,what are the minimum structural requirements for activity?

Assuming that the structural requirements are not iden-tical for both rings of 1,we reasoned that the simplest replacement for a single tetraaza-macrocyclic ring would be a pseudo diamine-segment,representing the first two amino groups of the macrocyclic ring from the point of attachment at the benzylic position.A judicious choice of “diamine”would also reduce the overall charge to þ1.Having previously established that the optimum distance between the first two amino groups was a two-carbon unit,we prepared a series of aminomethyl-substituted analogues in which the second amino group was a substituent upon an aromatic ring or part of a heterocyclic ring.In either case,the second p K a would be sufficiently low to prevent a second protonation at physiological pH.The compounds were tested for their ability to inhibit replication of HIV-1III B in MT-4cells,a strain of HIV-1that uses exclusively CXCR4for fusion and viral entry into target cells.The results are shown in Table 1.

Compared to 1,the introduction of a benzylamine group (3a )in place of the azamacrocyclic ring substantially reduced anti-HIV potency,although the compound remained active at submicromolar concentrations.The concentration of 3a re-quired to inhibit HIV-1replication by 50%(the EC 50)was 0.49μM,which was approximately 100-fold higher than the 50%inhibitory concentration of 1.Aromatic amino groups at the 2-position (3b )or 4-position (3c )did not affect antiviral potency.Both 3b ,c exhibited comparable EC 50’s to the un-substituted benzyl group (3a ).However,we observed a sub-stantial increase in anti-HIV potency when the benzyl group was replaced by a pyridyl group (3d ).Compound 3d exhibited a 50%inhibitory concentration of 0.009μM,which was only ca.2-fold higher than the EC 50of 1.Furthermore,the 50%cytotoxic concentration (CC 50)of compound 3d in MT-4cells was greater than 112μM.Thus 3d exhibits a selectivity index of greater than 12000.

The positional specificity of the pyridine-N in 3d was also examined.Replacement of the 2-pyridyl group with the 3-pyridyl (3e )or 4-pyridyl (3f )group had a detrimental effect on anti-HIV potency.For example,the EC 50’s of analogues 3e ,f were approximately 3orders of magnitude higher than the concentration of 3d required to inhibit HIV-1replication by 50%(the EC 50’s of 3e and 3f were 8.470and 9.977μM,respectively).Methylation of the amine in 3d (to give 3g )or extension of the connectivity to an aminoethyl pyridine group (to give 3h )also adversely affected the anti-HIV potency.Finally,we replaced the pyridine moiety with a comparable heterocycle of lower p K a than pyridine,namely the pyrazine group (3i ).Perhaps not surprisingly,the antiviral potency of analogue 3i was approximately comparable to the benzyl analogue 3a ,which did not contain a vicinal heterocycle nitrogen atom.

With the optimized “right-hand”replacement for the aza-macrocycle ring of 1fixed as the 2-aminomethyl pyridine group,we then turned our attention to the “left-hand”ring.Needless to say,the mandatory synthesis of the symmetrical analogue in which both rings were replaced by a 2-amino-methyl pyridine group turned out to be a predictably fruitless exercise (EC 50was >250μM,data not shown).We therefore focused on systematically replacing individual amine groups of the left ring.As shown in Table 2,we first prepared an analogue in which the [14]aneN 4(cyclam)ring had been replaced by the optimized and equally suitable,py[iso -14]-aneN 4ring (to give compound 36).Consistent with the structure -activity relationship of py[iso -14]aneN 4bis-azama-crocycles,compound 36proved to be a potent inhibitor of HIV-1replication,exhibiting an EC 50of 0.001μM,that is,around 9-fold and 4-fold lower,respectively,than the con-centration of 3d or 1required to inhibit viral replication by 50%.Although the pyridine-N of the macrocyclic ring in 36was previously found to be critical for high antiviral potency,we reasoned that a precise determination of the pyridine-N contribution to potency could help redesign a less basic mimic.Compounds 37and 38were then prepared to answer this question.Both analogues 37,containing a phenyl replacement and 38,containing an “exocyclic”pyridine fused group,retained reasonable anti-HIV potency (the EC 50’s of 37and 38were 0.040and 0.104μM,respectively)but were at least 40-to 100-fold less potent than analogue 36.So what role does the pyridine group play?

At physiological pH,the overall charge of the py[iso -14]-aneN 4ring in 36is also þ2(in a similar manner to cyclam 52)and the likely protonation sequence is indicated in Figure 1A,based on the sequence reported by Delgado et al.53for similar 14-membered tetraazamacrocyclic rings contain-ing pyridine.Presumably,the secondary amino groups are predominantly protonated and the overall structure is stabi-lized by intramolecular hydrogen bond interactions from the adjacent hydrogen-bond acceptors,the pyridine and tertiary benzylic amine groups (while minimizing the elec-trostatic repulsion of two positive charges in a confined macrocyclic ring).This is confirmed by a conformational analysis of 36on B3LYP/6-31G*level followed by single point energy calculations.In the energetically most stable ring conformation (LMP2/6-311þG*þZPE),the pyridine nitro-gen forms two six-membered intramolecular hydrogen bond interactions with the two adjacent protonated nitrogens as shown in Figure 2.Potential five-membered intramolecular hydrogen bond interactions are formed with the tertiary amine.

Table 1.Antiviral Activity of Single Ring

Azamacrocycles

n

R 1R 2

HIV-1(III B )EC 50(μM)MT-4cells CC 50(μM)3a 1H Ph

0.4911603b 1H 2-amino-Ph 1.825243c 1H 4-amino-Ph 0.7172273d 1H 2-pyridine 0.009>1123e 1H 3-pyridine 8.470373f 1H 4-pyridine 9.977>2793g 1Me 2-pyridine 0.416383h 2H 2-pyridine 49.135>1103I 1

H

5-Me-pyrazine

1.5781

0.004

>421

Article

Journal of Medicinal Chemistry,2010,Vol.53,No.31255

The stabilization provided by this “shared”protonated structure could account for the high basicity of azamacrocyc-lic rings,as suggested by Kimura et al.54It did not seem unreasonable,therefore,that a potential role of the pyridine group is the contribution of a single intramolecular hydrogen-bond,which locks the conformation of the protonated aza-macrocyclic ring in manner that is beneficial to antiviral potency.To test this hypothesis,we prepared a series of analogues (depicted in Figure 1B,data in Table 2)in which the fused aromatic group had been removed and replaced by an aliphatic group,in some cases containing a hydrogen-bond

acceptor at the key position “x,”the position occupied by the pyridine nitrogen in compound 36.

Consistent with the hydrogen-bonding hypothesis,the alkyl analogue 39exhibited an anti-HIV potency that was compar-able to the phenyl and exocyclic pyridine analogues 37and 38(the EC 50’s of 37and 39,were 0.040and 0.043μM,re-spectively).This result categorically rules out the possibility that the conformational restrictions imposed by the fused aromatic groups in compounds 37,38were even partially responsible for the high potency of 36.However,incorpora-tion of a hydrogen-bond acceptor at position x (Figure 1B)in some cases restored activity comparable to 36.For example,the oxygen analogue 40exhibited an EC 50that was only 4-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%(the EC 50of 40was 0.004μM).The corresponding thioether analogue 41a exhibited an EC 50of 0.013μM,which is approximately 3-fold higher than com-pound 40.Although the antiviral potency of the thioether analogue 41a compared to the ether analogue 41is greater than one would predict from the strength of the hydrogen-bond acceptor acceptor capabilities (thioether groups are considerably weaker H-bond acceptors than the oxygen in

Table 2.Antiviral Activity of Single Ring

Azamacrocycles

Figure 1.Proposed hydrogen-bond structure of protonated aza-macrocycles.

40),this result can be reconciled by considering the nature of the H-bond required;a six-membered intramolecular H-bond constrained by the macrocyclic ring (Figure 2).

With the thioether compound 41a in hand,we also pre-pared the sulfoxide (41b )and sulfone (41c )analogues by direct oxidation of 41a .We reasoned that the oxygen atoms of the sulfoxide and sulfone are stronger H-bond acceptors than the sulfur atom of 41a and may consequently improve the anti-HIV potency.However,both 41b and 41c were considerably weaker antiviral agents,exhibiting 50%effective concentra-tions for inhibition of HIV-1replication that were at least 79-fold higher than the EC 50of 41a (the EC 50’s of 41b and 41c were 0.485and 11.878μM,respectively).The precise reason for the poor antiviral activity exhibited by analogues 41b ,c was unclear;although the sulfoxide and sulfone are more sterically demanding than the thioether and could induce a ring conformation that is detrimental to antiviral activity,we could not rule out the possibility that the H-bond acceptor oxygen is now “one-bond”outside of the ring,and the intramolecular H-bond itself induces an unfavorable confor-mation (a seven-membered ring H-bond in 41b ,c (Figure 2)compared to a six-membered in 41a ).To complete this series of compounds therefore,we decided to introduce the fluoro and difluoro substituents at position x (Figure 1B).Several reports have demonstrated that the fluoro group can partici-pate as an acceptor for intramolecular H-bonds,particularly within highly constrained ring structures.55-57This is also confirmed by our calculations,as shown in Figure 2.The fluoro (43)and difluoro (42)analogues were also attractive substituents for two other reasons:(1)the substituents would be situated at the fourth carbon from the adjacent amine group,thereby minimizing the affect on p K a ;(2)in a similar manner to the sulfoxide and sulfone,the H-bond acceptor would be one-bond outside of the macrocyclic ring.However in this case,because the fluorine atom in C -F groups is isostructural with hydrogen,a negative effect of the fluoro substituents on antiviral activity can only be attributed to an inappropriately positioned H-bond rather than steric requirements (that is,in the absence of an H-bond,we would expect the fluoro or difluoro analogues to exhibit an EC 50comparable to the methylene analogue 39).In antiviral test-ing,the fluoro (43)and difluoro (42)analogues displayed EC 50’s that were greater than 20-fold higher than the methy-lene analogue 39(the EC 50’s of 39,42,and 43were 0.043,0.920,and 1.239μM,respectively),confirming the negative consequences of an incorrectly positioned hydrogen-bond (Figure 2).

Next,we focused on the sequence of aliphatic amine groups in the macrocyclic ring required for potent antiviral activity.By straightforward synthetic manipulation of our collection of ring systems,we prepared the structural isomers of analo-gues 36,37,and 39in which the side-chain (R,in Table 2)was connected to the alternative secondary amine group to give compounds 44,45,and 46.In antiviral testing,analogue 44was substantially less potent than its corresponding regioi-somer 39:the EC 50of 44was 11.131μM,which was approxi-mately 260-fold higher than the EC 50of 39.A similar loss of antiviral potency was observed with the phenyl analogue 46and its isomer 37(the EC 50’s of 46and 37were 14.106and 0.040μM,respectively).Interestingly,the loss of antiviral potency with the pyridine-fused isomer 45compared to 36was significant but not as substantial;the EC 50of 45was 0.063μM,around 60-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%.There was a possibility,therefore,that while the “tri-aza”ring configura-tion required for potent antiviral activity is clearly

represented

Figure 2.Lowest energy conformations of compounds 36,40,41c ,and 42.View from top on a plane defined by three nitrogens and X (see Figure 1).Dashed lines indicate hydrogen bond interactions:the hydrogen bond acceptors in 36and 40are in one plane with the three nitrogens.This is not the case for 41c and 42.Bond angles:36:—(N 333H -N þ)=140.5°,122.4°,102.1°,108.4°.40:—(O 333H -N þ)=135.1°,141.5°;—(N 333H -N þ)=104.6°,102.8°.41c :—(O 333H -N þ)=112.8°,112.8°;—(N 333H -N þ)=108.2°,108.0°.42:—(F 333H -N þ)=142.2°,142.2°;—(N 333H -N þ)=114.7°,114.7°.

(isomeric14-membered triaza rings)inhibited replication of HIV-1but were approximately70-fold and20-fold less po-tent,respectively,than analogue45.Consistent with the ring configuration of45(a three carbon unit connecting the tertiary amine and pyridine-N groups),the optimum config-uration was a4,7,17-triazabicyclo system(48,structurally related to45)rather than a3,6,17-triazabicyclo ring(ana-logue47,structurally related to39).Compound48inhibited HIV-1replication with an EC50that was3-fold lower than47. Because of synthetic convenience,an analogue of48contain-ing the nonbasic amide group(49)was also completed for antiviral testing.As expected,removing the positively charged secondary amine group was highly detrimental to antiviral potency(the EC50of49was126.4μM).

Finally,we prepared a short series of analogues in which the tertiary amine group in analogue36(and analogues39,40 in Table1)connecting the side-chain R to the macrocyclic ring,has been replaced by a carbon(CH)group.Using56as an example,one would predict that the loss of a hydrogen-bond acceptor provided by(in this case)the tertiary amine group(H-bond no.1in Figure1A)would lead to a similar reduction in antiviral potency compared to the replacement of the pyridine group in36with a phenyl group(to give37). Consistent with this analogy,the antiviral activity of56was comparable to37(the EC50’s were0.217and0.040μM, respectively)and both compounds were at least40-fold less potent inhibitors of HIV-1replication than36(EC50= 0.001μM).Interestingly,replacement of the tertiary amine group in39or40(to give58a and58b)led to a substantially greater reduction in antiviral potency:the EC50’s of58a and58b were ca.40-to50-fold higher than the concentration of39or40required to inhibit viral replication by50%. Significantly,however,the simple diaza-macrocycle58b re-mained active,exhibiting,albeit,modest antiviral potency (EC50=2.185μM).These combined results clearly supported our original pharmacophore hypothesis that(1)the minimum macrocyclic requirements for potent activity are the proto-nated secondary amine groups in a14-membered ring and(2) the activity is improved by hydrogen-bond acceptors which presumably lock the ring in a favorable conformation for antiviral activity.

To complete the study and our intitial goals,several analogues were selected for p K a determinations in an attempt to confirm the overall charge.The results are shown in Table3.As expected,the compounds in general exhibit two high p K a’s,consistent with the azamacrocyclic literature and therefore most likely due to double protonation of the aza-macrocyclic ring.The third p K a is closer to physiological pH, precluding the absolute assignment of protonation status on the aminomethylpyridine moiety during the HIV inhibitory step.Nevertheless,we can estimate the overal charge of these analogues to be in the rangeþ2toþ3,which compares favorably with1(þ4).

In summary,we have determined the key structural features of1required for potent antiviral activity and,in the process, identified several single azamacrocyclic ring structures with comparable or improved antiviral inhibitory potency.As shown in Figure3,there is considerable structural redundency in1:all eight amino groups are not required for activity,the critical amino groups on a per ring basis are nonidentical,and the overall charge at physiological pH can be reduced without compromising potency.These features have been used to design nonmacrocyclic analogues that will be reported in a subsequent manuscript.41

Experimental Section

Compound1(Mozobil(plerixafor))is1,10-[1,4-phenylenebis-(methylene]-bis-1,4,8,11-tetraazacyclotetradecane octahydrochlo-ride,dihydrate(formula weight=830.51).

General experimental procedures are provided in refs 26-28.1H and13C NMR spectra were recorded at300and 75MHz,respectively,on a Bruker Avance300spectrometer. Electrospray mass spectral analysis was performed on a Bruker Esquire spectrometer.Fast atom bombardment mass spectral analysis was carried out by M-Scan(West Chester, PA).Microanalyses for C,H,N,and halogen were per-formed by Atlantic Microlabs(Norcross,GA)and were within(0.4%of theoretical values.Purity was determined by reversed phase HPLC and was g95%for all compounds tested.

Preparation of Cyclam Analogues.To a stirred solution of4, 8,11-tris(diethoxyphosphoryl)-1,4,8,11-tetra-azacyclotetradeca-ne(2a26,27)(6.1g,0.01mol)and K2CO3(1.g,0.013mol)in CH3CN(150mL)was added R,R0-dibromo-p-xylene(13.2g, 0.05mol)and the reaction mixture stirred at70°C for1h.The solution was cooled to room temperature and the solvent removed under reduced pressure.The residue was partitioned between brine(50mL)and CH2Cl2(100mL).The organic phase was separated,dried(Na2SO4),and concentrated to a minimum volume.The solid was filtered off and the solvent evaporated under reduced pressure to give the crude product.Purification by column chromatography on silica gel(25:1CH2Cl2/CH3OH) gave1-[1-methylene-4-(bromo-methylene)phenylene]-4,8,11-tris-(diethoxyphosphoryl-1,4,8,11-tetraazacyclotetra-decane(4.7g, 59%)(2b)as a pale-yellow oil.1H NMR(CDCl3)δ1.21-1.37 (m,18H),1.66-1.74(m,2H),1.82-1.91(m,2H),2.30-2.35(m, 2H),2.58-2.63(m,2H),2.99-3.16(m,12H),3.48(s,2H), 3.95-4.07(m,12H),4.48(s,2H),7.21-7.35(4H).

To a solution of the appropriate amine(5.0equiv)in dry CH3CN(5mL)containing a suspension of K2CO3(1.5equiv) at80°C was added dropwise with stirring a solution of1-[1-methylene-4-(bromomethylene)phenylene]-4,8,11-tris(diethoxy-phosphoryl-1,4,8,11-tetraazacyclotetradecane(2b)(0.6mmol)in CH3CN(10mL)over15-20min.After stirring for a further1h at80°C,the solution was concentrated to dryness and the residue was partitioned between CH2Cl2and water.The organic layer was separated and washed with water(3Â)and then dried (MgSO4)and evaporated.The crude residue was purified by

Table3.Protonation Constants of Selected Azamacrocycles

compd p K a1p K a2p K a3 378.(0.027.73(0.02 6.90(0.02 399.66(0.028.60(0.027.53(0.02 41a9.59(0.028.15(0.027.34(

0.02

Figure3.Key nitrogen atoms(bold)per ring of1,required for

potent antiviral activity.column chromatography on silica gel eluting with5-15% MeOH/CH2Cl2to afford a viscous oil.

To a stirred solution of the protected cyclam derivative from above(0.1-0.5mmol)in acetic acid(3mL)was added a saturated solution of HBr(g)in acetic acid(5mL)and the solution was stirred at room temperature for14h.The resulting precipitate was collected by filtration and washed with acetic acid then Et2O.The solid was then dissolved in H2O(3mL)and treated with charcoal(100mg)and the mixture was heated to 80°C for30min.The hot solution was filtered through celite and the filtrate was concentrated to approximately1mL,after which acetic acid was added,resulting in the immediate formation of a white precipitate.The white solid was collected by filtration and dried in vacuo.

Compounds3a-i were prepared by these methods.

N-[1,4,8,11-Tetraazacyclotetradecanyl-1,4-phenylenebis(methy-lene)]-2-(amino-methyl)pyridine hexahydrobromide(3d).White solid:mp200-205°C(dec).1H NMR(D2O)δ2.04(m,4H),

3.20-3.40(m,8H),3.40-3.60(m,8H),

4.34(s,2H),4.38(s,2H),

4.51(s,2H),7.50(m,4H),7.75(t,1H,J=6.6Hz),7.82(d,1H, J=7.9Hz),8.26(t,1H,J=7.9Hz),8.63(d,1H,J=

5.3Hz).13C NMR(D2O)δ18.30,18.96,37.04,37.28,37.40,40.92,41.13, 41.49,44.26,47.61,48.01,51.29,58.88,127.46,127.75,130.40, 131.05,131.23,131.47,132.10,132.44,144.95,145.81,14

6.01. FAB MS m/z493(MþH81Br,7),491(MþH79Br,7),411(MþH,100).Anal.(C24H38N636HBr)C,H,N,Br.

General Procedure A:Macrocyclization.To a stirred solution of the requisite bis-nitrobenzenesulfonamide and anhydrous Cs2CO3(2.5equiv)in DMF(50mL of DMF per mmol of bis-nitrobenzenesulfonamide)maintained at80°C under N2was added a solution of the bis-electrophile(1.0-1.5equiv)in DMF (5mL of DMF per mmol of bis-electrophile),dropwise over 10h.The reaction mixture was allowed to stir at80°C for a further30h and then cooled to room temperature and concen-trated in vacuo.The residue was partitioned between EtOAc and water,and the organic layer was separated,washed with satd NaHCO3and then brine and dried over MgSO4or Na2SO4. Evaporation of the solvent and purification of the residue by column chromatography on silica gel(conditions indicated) gave the desired Dep-protected macrocycle.

General Procedure B:Deprotection of the Diethoxyphosphoryl (Dep)group.To a stirred solution of the Dep-protected macro-cycle in acetic acid(ca.2.5mL of acetic acid per mmol of Dep-macrocycle)was added a freshly prepared solution of saturated HBr(g)in acetic acid(10mL per mmol of Dep-macrocycle),and the resulting homogeneous solution was stirred at room tem-perature for a further22h.Addition of diethyl ether(125mL per mmol of macrocycle)to the reaction mixture gave a precipitate that was allowed to settle to the bottom of the flask, and the supernatant solution was decanted.The precipitate was washed with ether by decantation(repeated3Â),and the residue was then partitioned between CH2Cl2and1N aq NaOH.The separated aqueous layer was extracted with CH2Cl2(2Â),and the combined organic extracts were washed with brine and then dried(MgSO4or Na2SO4)and concen-trated in vacuo.The macrocycle was purified by column chromatography on silica gel or used directly without further purification in the next step.

General Procedure C:Alkylation of the Macrocycle with N-[1-Methylene-4-(chloromethylene)phenylene]-N-(2-nitrobenzenesul-fonyl)-2-(aminomethyl)pyridine.To a stirred solution of the macrocycle and anhydrous K2CO3(5.0equiv)in anhydrous CH3CN(10-15mL per mmol of macrocycle)under N2was added N-[1-methylene-4-(chloromethylene)phenylene]-N-(2-nitrobenzenesulfonyl)-2-(aminomethyl)pyridine(34a)(1.0-3.0 equiv),and the reaction mixture was allowed to stir at80°C for 18h and then concentrated in vacuo.The residue was parti-tioned between EtOAc and water,and the organic layer was separated,washed with satd NaHCO3and then brine and dried over MgSO4or Na2SO4.Evaporation of the solvent and pur-ification of the residue by column chromatography on silica gel gave the fully Ns-protected product.

General Procedure D:Deprotection of the2-Nitrobenzenesul-fonyl(Ns)Groups.To a stirred solution of the intermediate from the above procedure and anhydrous K2CO3(3.0-4.0equiv per Ns group)in anhydrous DMF(12mL per mmol of inter-mediate)under N2was added dropwise,thiophenol(1.0-2.5 equiv per Ns group).The reaction mixture was allowed to stir at room temperature for a further4h and then concentrated in vacuo.The residue was partitioned between EtOAc and water, and the organic layer was separated,washed with satd NaHCO3 and then brine and dried over MgSO4or Na2SO4.Evaporation of the solvent and purification of the residue by column chro-matography on silica gel or alumina gave the desired product as the free base.

General Procedure E:Conversion to the Hydrobromide Salt. The free base was dissolved in MeOH(15mL per mmol of free base),and a freshly prepared solution of saturated HBr(g)in MeOH(35mL per mmol of free base)was added giving a precipitate.The mixture was stirred for5min,and diethyl ether was added(50mL per mmol of free base).The solid was allowed to settle to the bottom of the flask,and the supernatant solution decanted.The solid was washed by decantation with MeOH (5Â)and then ether(10Â),and the last traces of ether were removed by evaporation in vacuo followed by drying in vacuo at 40-50°C overnight to give the desired product as the hydro-bromide salt.

Anti-HIV Activity Assays.Inhibition of HIV-1(III B)replica-tion assays were performed as previously described.26-28Anti-HIV activity and cytotoxicity measurements were carried out in parallel.They were based on the viability of MT-4cells that had been infected with HIV in the presence of various concentrations of the test compounds.After the MT-4cells were allowed to proliferate for5days,the number of viable cells was quantified by a tetrazolium-based colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium bromide(MTT)procedure in96-well microtrays.In all of these assays,viral input(viral multi-plicity of infection,MOI)was0.01,or100times the50%cell culture infective dose(CCID50).The EC50was defined as the concentration required to protect50%of the virus-infected cells against viral cytopathicity.The50%cytotoxic concentration (CC50)was defined as the compound concentration required to reduce the viability of mock-infected cells by50%.The greater than symbol(>)is used to indicate the highest concentrations at which the compounds were tested and still found to be non-cytotoxic.Average EC50and CC50values for several separate experiments are presented as defined above.As a rule,the individual values did not deviate by more than2-fold up or down from the EC50and CC50values indicated in Tables1and2.

Potentiometric Titrations.Aza-macrocylic p K a determina-tions were obtained by potentiometric titration in aqueous solution(I=0.16,NaCl)under an argon atmosphere at25°C in the pH range2.5-11.0.Error limits in Table3were estimated from multiple independent titrations.

Computational Details.Three-dimensional conformations for all compounds were obtained with Macromodel9.7within Maestro9.0using the OPLS2005force field.58Standard options have been used for all other parameters of the conformational search panel.These geometries were further optimized on B3LYP/6-31G*level of theory.All conformations are local energy minima with0imaginary frequencies as identified by B3LYP/6-31G*frequency calculations.The energies of con-formations were determined on LMP2/6-311þG*level includ-ing zero-point correction energies(ZPE)from frequency calculations.Elimination of redundant low energy conforma-tions provided mostly conformations displaying intramolecular hydrogen bonds resembling the respective lowest energy con-formation.59Therefore we limited our discussion to this energy conformation.Three-dimensional representations have been generated with Vida4.0.0.60Supporting Information Available:Experimental procedures and characterization data for the synthesis of intermediate34b and compounds30through58b.Characterization data for compounds3a-i.This material is available free of charge via the Internet at http://pubs.acs.org.

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