Third-generation immucillins: Syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase

1126

J. Med. Chem. 2009, 52, 1126–1143

Third-Generation Immucillins: Syntheses and Bioactivities of Acyclic Immucillin Inhibitors of

Human Purine Nucleoside Phosphorylase

Keith Clinch,^†Gary B. Evans,*^,†Richard F. G. Fro¨hlich,^†Richard H. Furneaux,^†Peter M. Kelly,^†Laurent Legentil,^†

Andrew S. Murkin,^‡Lei Li,^‡Vern L. Schramm,^‡Peter C. Tyler,^†and Anthony D. Woolhouse^†

Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31310, Lower Hutt 5040, New Zealand, Department of Biochemistry,

Albert Einstein College of Medicine of YeshiVa UniVersity, 1300 Morris Park AVenue, Bronx, New York 10461

ReceiVed NoVember 10, 2008

ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP),

developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases.

Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin

analogues (8-38) in order to identify simpliﬁed alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-

ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated

that the ﬂexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within

the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity.

SerMe-ImmH (10, K_d) 5.2 pM) was shown to be orally available in mice with a long biological residence

time on blood PNP.

Introduction

ation in the selection of a putative drug candidate over and above

1 and 2 is the “cost of goods” of that candidate. The practicalities

of synthesizing deazaguanine immucillins vs deazahypoxanthine

immucillins suggested that the latter were preferred on cost

grounds and ease of synthesis. To extend the range of cost-

effective alternatives to 1 and 2, we have prepared the achiral

azetidine derivative 7,²¹but despite being simpler to make than

1 or 2, compound 7 was not deemed a potent enough PNP

inhibitor to pursue as a drug candidate. Other workers have also

reported their approaches to ﬁnding a suitable drug contender

by synthesizing some carbocyclic²²and acyclic²³analogues of

3.

Since the discovery of the antiviral drug 9-[(2-hydroxy-

ethoxy)methyl]guanine (acyclovir) some 30 years ago,^24,25there

has been considerable interest and research into acyclic nucleo-

side analogues and their efﬁcacy as antiviral and anticancer

drugs.^26-28We recently communicated the biological activities

of several acyclic derivatives^1,29of which DATMe-ImmH (11)

was found to be a surprisingly potent inhibitor. Prompted by

this exciting discovery, we elected to explore the SAR of acyclic

immucillin derivatives in more detail. To provide a systematic

basis for identifying the target acyclic analogues required for

synthesis, we used the structures of our two clinical candidates,

1 and 2, as starting points, concentrating on alterations to the

pyrrolidine rings.³⁰Central in the selection of targets arising

out of these analyses was the incorporation of a secondary or

tertiary nitrogen atom, which after protonation, could mimic

an oxacarbenium ion, postulated to be an important transition

state feature in our original enzyme inhibitor models.^31-33

Attachment to these nitrogen atoms of a variety of branched

and linear alkyl chains, both chiral and achiral, bearing pendant

hydroxyl groups, completed the simple target structures (Figure

1). A good number of these target compounds were readily

revealed through disconnecting individual carbon-carbon bonds

of the pyrrolidine ring component in 1 or 2, although the ﬁnal

list of targets was not restricted to this approach.

The application of transition state theory and use of KIEs^a

in the rational design of inhibitors of PNPs has been recently

validated.^1-4This has resulted in the design and synthesis of a

series of putative drug candidates, two of which, D-immucillin-H

(ImmH, 1)^5-7(Figure 1) and D-DADMe-immucillin-H (DADMe-

ImmH, 2),^8-10are currently in human clinical trials for the

treatment of T- and B-cell cancers and a variety of autoimmune

diseases.^11-15Compound 1, a ﬁrst-generation immucillin and

2, a second-generation immucillin, exert their effects on human

T- and B-cells by inhibiting the human form of PNP, an enzyme

involved in recycling deoxyguanosine.¹⁶The interest of me-

dicinal chemists in developing inhibitors of PNP was piqued

by the observation that a genetic deﬁciency of PNP in some

humans caused a speciﬁc T-cell immune deﬁciency syndrome

as its primary phenotype.¹⁷Despite the considerable efforts of

several pharmaceutical companies to ﬁnd suitable small mol-

ecule PNP inhibitors to mimic this phenotype, to date, only the

work of our group has afforded inhibitors with dissociation

constants low enough to observe clinical effects in vivo in

humans.¹¹

The selective binding preference of PNP for 1 and 2 was

borneoutbytheobservationthatthecorrespondingL-enantiomers^18,19

5 and 6, respectively, were much less active inhibitors. On the

other hand, substitution of the deazahypoxanthine moiety present

in 1 and 2 with deazaguanine led to analogues 3⁵and 4,²⁰

respectively, together with a corresponding increase in the

potency of enzyme inhibition. After taking into account the

mandatory requirements of efﬁcacy and appropriate ADME as

well as a favorable PK proﬁle, the only other major consider-

* To whom correspondence should be addressed. Phone: +64-4-9313040.

Fax: +64-4-9313055. E-mail: g.evans@irl.cri.nz.

†

Carbohydrate Chemistry Team, Industrial Research Limited.

Department of Biochemistry, Albert Einstein College of Medicine of

‡

Yeshiva University.

^aAbbreviations: DADMe-ImmH, 4′-deaza-1′-aza-2′-deoxy-1′-(9-meth-

ylene)-ImmH; DATMe-ImmH, 2′-deoxy-2′-amino-tetritol-N-(9-methylene)-

ImmH; ImmH, Immucillin-H; SerMe-ImmH, serinol-N-(9-methylene)-

ImmH; KIE, kinetic isotope effect; PNP, purine nucleoside phosphorylase;

ADME, absorption, distribution, metabolism, and excretion; PK, pharma-

cokinetic; TRIS, tris(hydroxymethyl)aminomethane.

In this paper, we present full details on the syntheses and

human PNP inhibitory activity of compounds 8-38. Among

the 31 acyclic inhibitors described herein, several putative drug

10.1021/jm801421q CCC: $40.75

2009 American Chemical Society

Published on Web 01/26/2009

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1127

Figure 1. Dissociation constants for human PNP with acyclic immucillins. Compounds marked with an asterisk exhibited slow-onset inhibition

kinetics, whereby a slow protein conformational change following initial binding of the inhibitor resulted in a tighter complex. All values indicated

were ﬁnal, equilibrium dissociation constants in pM following formation of the tight complex if applicable. Relative errors were typically ( 15%

or less. Source of K_ivalue: (a) Miles et al.,⁵(b) Taylor et al.,¹(c) Rinaldo-Matthis et al.,²(d) Evans et al.,²¹(e) this manuscript, (f) Lewandowicz

et al.²⁹Abbreviations; dG, 9-deazaguanine; dHx, 9-deazahypoxanthine.

candidates, readily available on-scale from commercially avail-

able starting materials, were revealed. In particular, SerMe-

immucillin-G (SerMe-ImmG, 8) exhibited the lowest dissocia-

tion constant so far reported against human PNP. Readily

synthesized from achiral, commercially available starting ma-

terials, 8 is also the simplest structure to display such potent

enzyme inhibitory activity. Importantly, SerMe-ImmH (10) (K_d

) 5.2 pM), the 9-deazahypoxanthinyl analogue of 8, was shown

to be orally available in mice with a long biological residence

time on blood PNP.

low yields, respectively. Global deprotection of compounds

42 and 43 afforded 8 and the TRIS derivative 13, respectively

(Scheme 1). Similarly, 39 and 40 were treated under the same

conditions with aldehyde 44⁸to yield products 45 and 47 in

good to low yields, respectively. Compound 45 was readily

deprotected with reﬂuxing concentrated aqueous hydrochloric

acid to afford the acyclic immucillin, 10, in good yield, and

this was readily converted to 15 by reductive alkylation with

the masked hydroxy aldehyde, 1,4-dioxane-2,5-diol, using

sodium cyanoborohydride. Likewise, reductive alkylation of

45 with paraformaldehyde afforded the N-methyl derivative

46, which was deprotected under acidic conditions to afford

compound 14. On the other hand, the TRIS derivative,

compound 47 (vide supra), was deprotected using boron

tribromide in dichloromethane to afford the target compound

16 in a low yield.

Results and Discussion

Commercially available serinol (39) and TRIS (40) were

both treated with protected 9-deazaguanine derivative 41²³

under reductive amination/alkylation conditions with sodium

cyanoborohydride to yield compounds 42 and 43 in good to

1128 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

Scheme 1^a

^aReagents: (a) NaCNBH₃, AcCl, MeOH, room temperature. (b) (i) 1M NaOH, MeOH, room temperature; (ii) HCl, room temperature. (c) NaCNBH₃,

MeOH, room temperature. (d) cHCl, reﬂux. (e) Paraformaldehyde, NaCNBH₃, AcCl, MeOH, room temperature. (f) 1,4-Dioxane-2,5-diol, NaCNBH₃, MeOH,

room temperature. (g) BBr₃, CH₂Cl₂, room temperature.

Attempts were also made to synthesize compounds 10 and

16 by applying the more elegant Mannich reaction of 9-dea-

zahypoxanthine (75) with 39 and 40, respectively, under

conditions previously employed for the syntheses of our second-

generation immucillins.¹⁰No products were observed under

these conditions, possibly due to the formation of unreactive

1H-oxazolo[3,4-c]oxazoles arising from the reaction of the

pendant hydroxyls and amino groups with formaldehyde.³⁴

Intriguingly, when a racemic mixture consisting of 11 and

its enantiomer 19 was screened for enzyme inhibition activity,

it exhibited a relatively low inhibition constant against human

PNP. We therefore synthesized all four possible stereoisomers

11, 19, 24, and 25 (Scheme 2). The starting points were the

previously described^35-37chiral diethyl 2-amino-3-hydroxysuc-

cinates 48-51, readily available from D- and L-diethyl tartrates,

which were each reductively alkylated with aldehyde 44⁸in

either methanol or 1,2-dichloroethane using sodium cyanoboro-

hydride or sodium triacetoxyborohydride, respectively, to afford

coupled products 52-55 in good yields. Compounds 52-55

were then reduced using the reagent combination of lithium

borohydride-methanol³⁸to afford the triols 56-59 in moderate

to good yields. Global deprotection of 56-59 by treatment with

either boron tribromide at room temperature or concentrated

hydrochloric acid at reﬂux gave the acyclic immucillins 11, 19,

24, and 25.

globally deprotected to afford 9. Compound 9 was 2-3 times

more active an inhibitor of human PNP than its 9-deazahypox-

anthine analogue 11, a result consistent with our previous

observations to date when comparing the 9-deazaguanine

immucillin series with the corresponding 9-deazahypoxanthine

series.²⁶

Another expedient route to an acyclic immucillin was

accessed from the ﬁrst-generation immucillin, 1·HCl (Scheme

3). Protection of the pyrrolidine nitrogen of 1 followed by

periodate oxidation of the contiguous secondary alcohols and

then reduction of the resulting dialdehyde intermediate afforded

the triol 61 in excellent yield for the three steps. Removal of

the tert-butoxycarbonyl protecting group by acid-catalyzed

hydrolysis gave compound 12·HCl in good yield.

The synthesis of an analogue of 2 bearing an extra hydroxyl

at C-2′ was considered of interest, but as it was thought that

the hemiaminal structure so created would be hydrolytically

unstable, hence the acyclic analogue (()-17 became of interest.

cis-But-2-ene-1,4-diol (62) was reacted with N-benzylhydroxy-

lamine and formaldehyde to afford the racemic isoxazolidine

(()-64 in quantitative yield (Scheme 4). Reductive cleavage of

the N-O bond gave amino alcohol (()-66, in moderate yield,

which was reductively alkylated with aldehyde 44⁸under

standard conditions to afford (()-68, again in moderate yield.

Deprotection of (()-68 via acid-catalyzed hydrolysis followed

by hydrogenolysis gave the acyclic immucillin (()-17 in an

unoptimized yield for the two steps. trans-But-2-ene-1,4-diol

(63) underwent a similar cycloaddition reaction, although in a

much poorer yield, to afford isoxazolidine (()-65. This isox-

azolidine was then converted, as in the previous case, through

(()-67 and (()-69 to the target compound (()-38 in moderate

overall yield.

Compound 11 proved to be a highly potent inhibitor of human

PNP and its inhibition activity has been reported previously by

us.¹Reductive alkylation of 11 using paraformaldehyde and

sodium cyanoborohydride in methanol afforded the N-methyl

derivative 26 in good yield.

Compound 9, the 9-deazaguanine analogue of 11 in this

series, was also synthesized. To this end, aldehyde 41²³was

reductively aminated with (5S,6R)-6-amino-2,2-dimethyl-1,3-

dioxepan-5-ol³⁹using sodium triacetoxyborohydride in 1,2-

dichloroethane to afford the protected immucillin 60, which was

To survey a nonexhaustive series of simple achiral acyclic

immucillins, we investigated the conversion of three primary

amino-alcohols, ethanolamine (70), 3-amino-propan-1-ol, and

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1129

Scheme 2^a

^aReagents: (a) Compound 44,⁸AcOH, NaCNBH₃, MeOH, room temperature. (b) Compound 44,⁸Na(OAc)₃BH, 1,2-dichloroethane, room temperature.

(c) LiBH₄, MeOH, Et₂O, room temperature. (d) BBr₃, CH₂Cl₂, room temperature. (e) cHCl, reﬂux. (f) paraformaldehyde, NaCNBH₃, MeOH, 50 °C f room

temperature. (g) (i) AcCl, MeOH, room temperature; (ii) cHCl, reﬂux.

Scheme 3^a

^aReagents: (a) (i) Boc₂O, Et₃N, H₂O, MeOH, room temperature; (ii) NaIO₄, room temperature; (iii) NaBH₄, room temperature. (b) cHCl, MeOH, room

temperature.

4-amino-butan-1-ol, and two secondary amino-alcohols dietha-

nolamine (73) and 3-(2-hydroxyethylamino)propan-1-ol (74) to

their corresponding immucillins (Scheme 5). Compounds 70,

73, and 74 underwent the Mannich reaction with 75¹⁰and

formaldehyde to afford compounds 20, 21, and 33, respectively,

where 33 represents the acyclic analogue of 2 with the

pyrrolidine C3′-C4′ bond lysed.

Both 3-amino-propan-1-ol and 4-amino-butan-1-ol required

N-benzylation to their secondary amines 71 and 72, respectively,

before they would undergo the Mannich reaction with 75¹⁰to

afford compounds 76 and 77, respectively. Subsequent hydro-

genolysis of the N-benzyl groups in compounds 76 and 77 with

hydrogen and a palladium catalyst afforded the target com-

pounds 18 and 29, respectively, in good overall yields. It is

interesting to note that amines 71-74 underwent the Mannich

reaction despite their potential to form, through reaction with

formaldehyde, unreactive tertiary amine species in the form of

N-alkylated oxazolidines, 1,3-oxazinanes, or 1,3-oxazepanes.

This contrasts with 39 and 40 mentioned earlier, which failed

to undergo similar Mannich reactions with 75.¹⁰

from the commercially available 2-(hydroxymethyl)propane-

1,3-diol (Scheme 6).⁴⁰Displacement of the mesylate with either

methylamine or benzylamine afforded compounds 79 and 81,

respectively, in moderate to good yields. The Mannich reactions

of 79 and 81 with 75¹⁰proceeded smoothly, although in poor

yield for 81, to provide compounds 80 and 82, respectively.

Deprotection of 80 was achieved in a single step through acid

treatment at room temperature to afford 22 in good yield,

whereas deprotection of 82 was achieved in two steps via acid-

catalyzed removal of the acetonide followed by hydrogenolysis

of the benzyl group to afford 27 in unoptimized yield for the

two steps.

Naturally occurring carbohydrates are an important and

convenient source by which to obtain functionalized polyol

derivatives. Those derived from trioses, tetroses, and pentoses

were considered to be the most important in our investigations.

We therefore investigated a series of acyclic amino alcohols

containing only a single chiral center and available from the

commercial chiral acetonides 83 and 84 as examples in the triose

series (Scheme 7). Mesylation of 83 and 84 afforded the known

corresponding mesylates 85 and 86, which were treated with

benzylamine in reﬂuxing acetonitrile to provide amines 87 and

88, respectively, in good yields.^41,42

Disconnecting the N-C2′ bond or the C2′-C3′/N-C2′ bonds

in the pyrrolidine ring of 2 provided immucillin targets 22 and

27, respectively. The protected mesylate 78 was readily prepared

1130 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

Scheme 4^a

^aReagents: (a) BnNHOH ·HCl, NaOAc, 37% aq HCHO, EtOH, reﬂux. (b) Zn, AcOH, room temperature f 67 °C. (c) Compound 44,⁸AcCl, NaCNBH₃,

MeOH, room temperature. (d) (i) cHCl, reﬂux; (ii) H₂, Pd/C, MeOH.

Scheme 5^a

^aReagents: (a) 37% aq HCHO, H₂O, 85 °C. (b) 37% aq HCHO, NaOAc, H₂O, 85 °C. (c) Pd/C, H₂, iPrOH, 50 °C.

Scheme 6^a

^aReagents: (a) Aq methylamine, DMSO, 75 °C. (b) 9-Deazahypoxanthine (75),¹⁰37% aq HCHO, AcOH, 95 °C. (c) cHCl, MeOH, room temperature. (d)

Benzylamine, 80 °C. (e) (i) cHCl, MeOH, room temperature; (ii) 10% Pd/C, H₂, H₂O, room temperature.

At this point, two different routes were pursued utilizing

compounds 87 and 88. First, we investigated the direct reductive

amination of aldehyde 44⁸with 87 and 88, followed by acid-

catalyzed hydrolysis of the resulting adducts with reﬂuxing

concentrated hydrochloric acid to provide coupled products 89

and 37 in moderate to good yields, respectively. Compound 37

was screened as an inhibitor in order to observe the effect on

the inhibition constant of a large hydrophobic group. Hydro-

genolysis of 89 in water under a hydrogen atmosphere using

10% Pd/C as a catalyst afforded the immucillin 23 in moderate

yield. Similarly, hydrogenolysis of 37 under the same conditions

as 89 gave the immucillin 28 in near quantitative yield. Second,

the treatment of amines 87 and 88 with 2-bromoethanol in

reﬂuxing acetonitrile, with potassium carbonate as base, afforded

the corresponding tertiary ethanolamines, which after hydro-

genolysis were reductively alkylated with aldehyde 44⁸in the

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1131

Scheme 7^a

^aReagents: (a) Benzylamine, CH₃CN, reﬂux. (b) Compound 44,⁸Na(OAc)₃BH, 1,2-dichloroethane, MgSO₄, room temperature. (c) cHCl, reﬂux. (d) 10%

Pd/C, H₂, H₂O, room temperature. (e) 2-Bromoethanol, K₂CO₃, CH₃CN, reﬂux. (f) 10% Pd/C, H₂, MeOH, room temperature.

Scheme 8^a

By way of an example of a pentose derived immucillin the

D-ribonamide (99),⁴³the 1-C homologue of D-erythronamide

(92), was reduced with LAH in THF to afford the amine 100

in moderate yield and then reductively alkylated with 44⁸again

under standard conditions to afford the protected immucillin

101 in poor yield (Scheme 10). Deprotection of 101 in reﬂuxing

concentrated hydrochloric acid afforded immucillin 35.

Finally, we investigated the synthesis from commercially

available (()-1,2,4-butanetriol (102), of (()-36, the N-Me, 3′-

deoxy variant of (()-30 and (()-31. Acetalization of (()-102

with benzaldehyde gave benzylidene acetal (()-103, and this

compound in turn was sulfonated with methane sulfonyl chloride

and the resulting mesylate treated with aqueous methylamine

to afford methylamino acetal (()-104 in good yield for the three

steps (Scheme 11). Compound (()-104 underwent a Mannich

reaction with 75¹⁰under standard conditions and in good yield

^aReagents: (a) LAH, THF, room temperature. (b) Compound 44,⁸

Na(OAc)₃BH, 1,2-dichloroethane, MgSO₄, room temperature. (c) cHCl,

reﬂux.

to afford adduct (()-105, which was readily deprotected to

provide immucillin (()-36.

Inhibition Studies. The inhibition of the phosphorolysis of

inosine catalyzed by human PNP was tested with third-

generation compounds 8-38 and compared to the ﬁrst-genera-

tion inhibitors 1 and 3 and the second-generation inhibitors 2

and 4 (Figure 1). Among the acyclic analogues evaluated were

those that could be conceptually formed by a carbon-carbon

bond cleavage in the pyrrolidine rings of the parent compounds.

Although weaker than the 56 pM dissociation constant for 1,

the ImmH-based analogues 24 and 12 maintained moderate to

strong inhibition at 4300 and 210 pM, respectively. The

DADMe-ImmH-based analogues (()-17, 22, 33, and (()-36

yielded widely varying inhibition, becoming progressively

weaker as the cleavage site was moved around the pyrrolidine

ring, ranging from 780 pM with (()-17 to 227000 pM with

(()-36. Comparing the two series of seco-immucillins, it

therefore seems that human PNP tolerates a loss in binding to

the 2′-position much better than at other positions in the ring.

Intriguingly, inversion of conﬁguration of the 3′-hydroxyl

group found in 24 to that present in 11 yielded a K_i* of 8.6

pM, corresponding to a 500-fold improvement in inhibition.¹It

is possible that the more ﬂexible nature of the third-generation

inhibitors enables 11 to adopt a conformation resembling 1 and

2 within the active site of PNP. Differential binding afﬁnity

due to increased rotational freedom has also been observed with

the L-enantiomers of 1 and 2, where protein structures showed

the more ﬂexible L-DADMe-ImmH (6; K_i) 380 pM) was bound

in an orientation similar to its D-enantiomer, but L-ImmH (5;

K_i* ) 12000 pM) was not.¹⁹

presence of sodium triacetoxyborohydride to afford compounds

90 and 91 in poor to moderate yields, respectively, for the three

steps. Acid-catalyzed hydrolysis of 90 and 91 afforded the

immucillins 32 and 34 in good yields.

Following this, we investigated a tetrose derived series of

immucillins for screening. The D-erythronamide 92^43,44was

readily reduced using LAH in THF at room temperature to

afford the amino alcohol 93 in excellent yield (Scheme 8).

Compound 93 was then reductively alkylated with aldehyde 44⁸

and sodium triacetoxyborohydride in 1,2-dichloroethane to

afford the protected immucillin 94 in moderate yield. Standard

acid-catalyzed deprotection of 94 gave immucillin 30 in

unoptimized yield.

Immucillin (()-31, as a racemic mixture of stereoisomers,

was synthesized as shown (Scheme 9). The D,L-threo-acetonide

(()-95 was prepared in good yield from (()-diethyl tartrate

using dimethoxypropane and TsOH in reﬂuxing benzene,

conditions which led to a partial transesteriﬁcation. Treatment

of (()-95 with 1 mol equiv of 7 M methanolic ammonia solution

at room temperature provided amido ester (()-96 in moderate

yield. Reduction of (()-96 with LAH gave the amino alcohol

(()-97 in excellent yield, and this product was reductively

alkylated with aldehyde 44⁸under standard conditions to afford

the protected immucillin (()-98 in good yield. Deprotection of

the racemic mixture (()-98 with reﬂuxing hydrochloric acid

afforded immucillin (()-31 in unoptimized yield.

1132 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

Scheme 9^a

^aReagents: (a) DMP, TsOH, benzene, reﬂux. (b) 7M NH₃in MeOH, room temperature. (c) LAH, THF, reﬂux. (d) Compound 44,⁸Na(OAc)₃BH, 1,2-

dichloroethane, MgSO₄, room temperature. (e) cHCl, reﬂux.

Scheme 10^a

Figure 2. Bioavailability of SerMe-ImmH (10) in mice. Enzyme

activity was assayed following administration of 50 nmol by oral

treatment (4) or intraperitoneal injection (×). Activities were normal-

ized for protein concentration and are reported relative to uninhibited

samples assayed prior to dose administration. The t_1/2for onset is the

time following treatment required to achieve a relative activity of 0.5.

The t_1/2for offset is the time following treatment required to achieve

^aReagents: (a) LAH, THF, reﬂux. (b) Compound 44,⁸NaBH(OAc)₃,

a relative activity recovery of 0.5. Oral treatment lags only marginally

1,2-dichloroethane, room temperature. (c) cHCl, reﬂux.

behind ip injection for onset of inhibition but remains nearly as effective

with a similarly long t_1/2offset of 25 days.

Scheme 11^a

in 18 to that in 29 resulted in poorer binding (K_i) 25000 pM).

Compound 27, which contains a methylene group inserted into

10, making it a closer mimic of 2, gave similarly weaker afﬁnity

(K_i) 14100 pM). Thus, it is apparent that the positioning of

the two hydroxyl groups is critical to picomolar inhibition.

Reintroduction of hydroxymethyl groups into 10, although

signiﬁcantly less potent, resulted in good (compounds 12-16;

K_i) 210-620 pM) to moderate (compound 24; K_i) 4300 pM)

levels of inhibition. Like 10, four of these compounds, 13-16,

are achiral, making them particularly attractive for synthetic

development as inhibitors of PNP.

^aReagents: (a) PhCHO, TsOH, toluene, reﬂux. (b) (i) iPr₂NEt, MsCl,

Bioavailability. SerMe-ImmH (10) was administrated to mice

CH₂Cl₂, 0 °C f room temperature; (ii) 40% aq MeNH₂, DMSO, 80 °C.

(c) Compound 75,¹⁰AcOH, 37% aq HCHO, 1,4-dioxane, 80 °C. (d) cHCl,

orally or by intraperitoneal (ip) injection with a single dosage

of 50 nmol. Complete inhibition of PNP catalytic activity was

observed in the ip injection group within 20 min, yielding a t_1/2

of 10 min for the onset of inhibition (Figure 2). For the orally

treated group, the t_1/2of onset was 18 min. Thus, SerMe-ImmH

(10) is orally available and only modestly less effective than

direct ip injection. Both ip and oral groups regained 50% of

the initial PNP catalytic activity after 25 days, yielding a t_1/2of

offset greater than the lifetime of mouse erythrocytes (25 days).

Therefore, it can be concluded that after the inhibited erythrocyte

MeOH, room temperature.

The potent inhibition observed with 11 prompted further

exploration of acyclic analogues possessing variations in the

amino alcohol moiety. The most potent human PNP inhibitor

in the 9-deazahypoxanthine series with a K_i* of only 5.2 pM,

was found to be 10, which differs from 11 by the removal of a

hydroxymethyl moiety. Further simpliﬁcation was evaluated

with 20, which with a K_iof 1100 pM bound weaker to human

PNP by a factor of 500. Lengthening of the alkyl chain found

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1133

silica gel (∼1 g) and puriﬁed by two chromatography columns,

eluting the ﬁrst one with EtOAc/MeOH/28% aq NH₄OH ) 80:

20:1 and the second one with CHCl₃/MeOH/28% aq NH₄OH )

80:20:1 to afford 43 (41 mg, 49%) as an amorphous white solid.

¹H NMR (500 MHz, CD₃OD): δ 8.55 (s, 1H), 7.24 (s, 1H), 6.31

(s, 2H), 3.88 (s, 2H), 3.66 (s, 6H), 3.18 (s, 3H), 3.06 (s, 3H), 1.15

(s, 9H). ¹³C NMR (125.7 MHz, CD₃OD): δ 179.0, 158.7, 156.4,

155.6, 144.5, 128.4, 115.5, 115.3, 67.1, 62.5, 62.5, 62.5, 61.9, 41.1,

39.8, 36.4, 35.1, 27.4. ESI-HRMS for C₂₀H₃₃N₆O₆[MH⁺] calcd,

473.2462; found, 473.2458.

is degraded, some of the released inhibitor becomes recycled

by newer cells, prolonging the period of inhibition.

Conclusions

Expanded chemical scaffolds were designed to simplify

chemical approaches to transition state analogue inhibitors of

human PNP. Inhibitors have been obtained with dissociation

constants as low as 2.1 pM. Compounds 8 (K_d) 2.1 pM) and

10 (K_d) 5.2 pM) are achiral molecules and thereby simplify

synthesis and lower “the cost of goods” relative to compounds

1 and 2, both currently in clinical trials. Remarkably, compound

10 showed equivalent bioavailability to mouse blood PNP by

oral or intraperitoneal administration. A single oral dose

inhibited >50% of blood PNP catalytic activity for a period of

25 days. Closely related compounds were weaker inhibitors.

Thus, a new family of powerful PNP inhibitors is described

with favorable pharmacokinetic properties in mice.

2-Amino-7-({[1,3-dihydroxy-2-(hydroxymethyl)propan-2-

yl]amino}methyl)-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one Hy-

drochloride Salt (13 ·HCl). Compound 43 (38 mg, 84 µmol) was

dissolved in THF (2 mL) and aqueous sodium hydroxide (1 mL, 1

mmol, 1.0 M) was added, and the resulting mixture was stirred

overnight. After 28 h, water (5 mL) was added and the resulting

solution concentrated in vacuo. Puriﬁcation of the residue by

chromatography (iPrOH/28% aq NH₄OH ) 4:1) afforded an

N-CHO derivative of 13, which was treated with 5% aqueous HCl

1

to afford 13·HCl (22 mg, 92%) as a white foam. H NMR (500

Experimental Section

MHz, D₂O, internal 1,4-dioxane at δ 3.75): δ 7.64 (s, 1H), 4.44 (s,

2H), 3.88 (s, 6H). ¹³C NMR (125.7 MHz, D₂O, internal 1,4-dioxane

at 67.19): δ 154.2, 151.1, 132.8, 132.3, 112.3, 102.0, 66.8, 58.7,

58.7, 58.7, 35.8. ESI-HRMS for C₁₁H₁₈N₅O₄[MH⁺] calcd, 284.1359;

found, 284.1365. Anal. (C₁₁H₁₇N₅O₄·3HCl) C, H, N.

Anhydrous solvents were obtained commercially. Air sensitive

reactions were carried out under argon. Organic solutions were dried

over MgSO₄. TLC was performed on glass or aluminum sheets

coated with 60 F254 silica gel. Organic compounds were visualized

under UV light or by use of a dip of cerium(IV) sulfate (0.2%,

w/v) and ammonium molybdate (5%) in sulfuric acid (2M), or one

of I₂(0.2%) and KI (7%) in H₂SO₄(1M) or one of p-(N,N-

dimethylamino)benzaldehyde in cHCl-MeOH (Ehrlich′s reagent).

Chromatography (ﬂash column) was performed on Scharlau or

Merck silica gel 60 (40-60 µm). Melting points are uncorrected.

Optical rotations were recorded with a path length of 1 dm and are

in units of 10^-1deg cm²g^-1; concentrations are in g/100 mL. NMR

spectra were recorded on a Bruker AC-300 instrument at 300 MHz

(¹H) or 75.5 MHz (¹³C) unless otherwise stated. Analytical and

preparative HPLC were carried out on Phenomenex Synergi Polar

RP 80A columns eluting with 3-50% MeOH in 0.1% aqueous TFA

with detection of products at 230 nm. Microanalyses were carried

out by the Campbell Microanalytical Laboratory, University of

Otago, New Zealand. Positive electrospray high resolution mass

spectra (ESI-HRMS) were recorded on a Waters Q-TOF Premier

tandem mass spectrometer.

5-(Benzyloxymethyl)-7-{[(1,3-dihydroxypropan-2-yl)amino]m-

ethyl}-4-methoxy-5H-pyrrolo[3,2-d]pyrimidine (45). Acetyl chloride

(0.117 mL, 1.65 mmol) was added to a stirred solution of serinol

(39) (300 mg, 3.29 mmol) and aldehyde 44⁸(196 mg, 0.66 mmol)

in MeOH (5 mL). Sodium cyanoborohydride (62 mg, 0.99 mmol)

was added, and the resulting reaction mixture was stirred at room

temperature for 16 h. The mixture was then concentrated in vacuo

and the residue puriﬁed by chromatography (CH₂Cl₂/MeOH/28%

aq NH₄OH ) 95:5:0.5 f 9:1:0.05) to afford 45 (188 mg, 77%) as

a colorless solid. ¹H NMR (CD₃OD): δ 8.42, (s, 1H), 7.65 (s, 1H),

7.25-7.16 (m, 5H), 5.75 (s, 2H), 4.50 (s, 2H), 4.10 (s, 3H), 4.03

(s, 2H), 3.68 (dd, J ) 11.2, 5.4 Hz, 2H), 3.58 (dd, J ) 11.2, 5.9

Hz, 2H), 2.81 (pentet, J ) 5.6 Hz, 1H). ¹³C NMR (CD₃OD, center

line at δ 49.0): δ 157.9, 150.8, 150.6, 138.7, 134.1, 129.3, 128.8,

128.6, 117.0, 116.2, 78.5, 71.5, 62.5, 62.5, 61.3, 54.3, 41.4. ESI-

HRMS for C₁₉H₂₅N₄O₄[MH⁺] calcd, 373.1876; found, 373.1865.

7-{[(1,3-Dihydroxypropan-2-yl)amino]methyl]}-3,5-dihydro-4H-

pyrrolo[3,2-d]pyrimidin-4-one (SerMe-ImmH, 10) and Its Hydro-

chloride Salt (10 ·HCl). Compound 45 (180 mg, 0.48 mmol) was

heated under reﬂux in concentrated aqueous HCl (2 mL) for 1.5 h.

The reaction mixture was concentrated in vacuo and the residue

dissolved in a 1:1 mixture of MeOH:H₂O, neutralized with

Amberlyst A-21 resin, ﬁltered, and the ﬁltrate concentrated in vacuo.

The residue was puriﬁed by chromatography (iPrOH/H₂O/28% aq

NH₄OH ) 92:4:4) to afford 10, which was treated with 5% aqueous

HCl to afford 10·HCl (85 mg, 64%) as a colorless hygroscopic

solid. An analytical sample was recrystallized from H₂O/EtOH; mp

217-219 °C. ¹H NMR (500 MHz, D₂O + drop DCl, internal

CH₃CN at δ 2.06): δ 9.07 (s, 1H), 7.92 (s, 1H), 4.60 (s, 2H), 3.97

(dd, J ) 12.6, 4.4 Hz, 2H), 3.89 (dd, J ) 12.6, 5.9 Hz, 2H), 3.54

(m, 1H). ¹³C NMR (125.7 MHz, D₂O + drop DCl, internal CH₃CN

at δ 1.47): δ 152.8, 145.3, 133.4, 132.0, 118.7, 103.5, 60.4, 58.3,

58.3, 39.2. ESI-HRMS for C₁₀H₁₄N₄O₃Na [MNa⁺] calcd, 261.0964;

found, 261.0964. Anal. (C₁₀H₁₅ClN₄O₃0.5H₂O) C, H, N.

Chemistry. 2-Amino-7-{[(1,3-dihydroxypropan-2-yl)amino]m-

ethyl}-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (SerMe-Im-

mG, 8) and Its Hydrochloride Salt (8 ·HCl). Acetyl chloride (0.072

mL, 1.01 mmol) was added to stirred MeOH (5 mL), followed by

serinol 39 (180 mg, 2.02 mmol), aldehyde 41²³(140 mg, 0.403

mmol), and sodium cyanoborohydride (38 mg, 0.61 mmol). After

4 h, the solvent was evaporated and the residue chromatographed

on silica gel (CHCl₃/7 M NH₃-MeOH, 95:5 f 9:1) to give 42 as

a yellow solid (65 mg). Compound 42 was stirred in a 1:1 mixture

of MeOH:1 M NaOH (4 mL) at room temperature for 48 h. The

mixture was acidiﬁed with 5% aqueous HCl and concentrated in

vacuo. Chromatography (iPrOH/H₂O/28% aq NH₄OH ) 80:15:0.5)

of the resulting residue gave 8 as a colorless solid, which was

triturated with 7 M NH₃-MeOH and the colorless solid ﬁltered off,

dried, and converted to its HCl salt with 5% aqueous HCl to afford

1

8·HCl (14 mg 11%) as a white solid. H NMR (D₂O, internal

CH₃CN at δ 2.06): δ 7.63 (s, 1H), 4.45 (s, 2H), 3.95 (dd, J ) 12.6,

4.5 Hz, 2H), 3.86 (dd, J ) 12.6, 5.9 Hz, 2H), 3.48 (m, 1H). ¹³C

NMR (D₂O, internal CH₃CN at δ 1.47): δ 154.3, 151.1, 133.1,

132.2, 112.5, 101.6, 60.2, 58.4, 58.4, 39.3. ESI-HRMS for

C₁₀H₁₆N₅O₃[MH⁺] calcd, 254.1253; found, 254.1253.

5-(Benzyloxymethyl)-7-{[(1,3-dihydroxypropan-2-yl)(methyl)ami-

no]methyl}-4-methoxy-5H-pyrrolo[3,2-d]pyrimidine (46). Acetyl

chloride (8.91 µL, 0.125 mmol) was added to MeOH (5 mL) with

stirring and then 45 (155 mg, 0.42 mmol), paraformaldehyde (62

mg, 2.08 mmol), and sodium cyanoborohydride (39 mg, 0.62 mmol)

were added. After 16 h, the reaction mixture was concentrated in

vacuo and the residue puriﬁed by chromatography (CHCl₃/MeOH/

28% aq NH₄OH ) 95:5:0.5) to afford 46 (115 mg, 72%) as a

colorless gum. ¹H NMR (500 MHz, CD₃OD): δ 8.40 (s, 1H), 7.61

(s, 1H), 7.26-7.18 (m, 5H), 5.74 (s, 2H), 4.50 (s, 2H), 4.10 (s,

3H), 3.95 (s, 2H), 3.77 (dd, J ) 11.4, 7.3 Hz, 2H), 3.65 (dd, J )

11.4, 5.6 Hz, 2H), 2.93 (m, 1H), 2.37 (s, 3H). ¹³C NMR (125.7

{7-[(1,3-Dihydroxypropan-2-ylamino)methyl]-2-[(dimethylami-

no)methyleneamino]-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimi-

din-3-yl}methyl 2,2-Dimethylpropanoate (43). To a mixture of TRIS

(40) (128 mg, 1.06 mmol) and 41²³(64 mg, 0.18 mmol) in MeOH

(5 mL) was added sodium cyanoborohydride (20 mg, 0.30 mmol)

and acetyl chloride (32 µL, 0.45 µmol), and the resulting bright-

yellow heterogeneous mixture was stirred overnight at room

temperature. The reaction mixture was concentrated in vacuo onto

1134 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

MHz, CD₃OD, center line at δ 49.0): δ 157.9, 151.1, 150.6, 138.8,

134.6, 129.3, 128.7, 128.6, 117.2, 115.8, 78.5, 71.5, 66.3, 60.3,

60.3, 54.3, 48.1, 38.2. ESI-HRMS for C₂₀H₂₇N₄O₄[MH ⁺] calcd,

387.2032; found, 387.2034.

134.2, 133.0, 119.4, 104.7, 67.6, 59.3, 59.3, 59.3, 36.4. ESI-HRMS

for C₁₁H₁₇N₄O₄[MH⁺] calcd, 269.1250; found, 269.1263.

5-Benzyloxymethyl-4-methoxy-7-({[(2R,3S)-1,3,4-trihydroxybu-

tan-2-yl]amino}methyl)-5H-pyrrolo[3,2-d]pyrimidine (56). A mix-

ture of diethyl (2S,3S)-2-amino-3-hydroxysuccinate (48)³⁶(0.87 g,

4.24 mmol, prepared from diethyl-D-tartrate according to literature

methods^35,36), sodium cyanoborohydride (0.44 g, 7.07 mmol) and

44⁸(1.05 g, 3.54 mmol) were evaporated from MeOH (3×). The

residue was dissolved in MeOH (20 mL) and acetic acid added

(10 drops). The reaction mixture was stirred at room temperature

for 18 h and then silica gel added and the mixture concentrated in

vacuo. Puriﬁcation of the resulting residue by chromatography

(Et₃N/MeOH/CH₂Cl₂) 1:3:99) afforded crude 52 (2.58 g, 150%)

as a colorless oil. To a stirred solution of crude 52 (1.73 g, 2.33

mmol, 66%) in diethyl ether (30 mL) was added MeOH (1.43 mL,

35.3 mmol) and then lithium borohydride³⁸(8.83 mL, 17.7 mmol,

2.0 M in THF). After 1 h, MeOH (1.43 mL, 35.3mmol) was added

to the reaction mixture and stirring continued. After 1 h further,

the reaction mixture was diluted with MeOH and then concentrated

in vacuo and the residue dissolved in MeOH (20 mL), diluted with

HCl (20 mL, 1M), and concentrated in vacuo. The resulting residue

was puriﬁed by chromatography (7 M NH₃-MeOH/CH₂Cl₂) 15:

85) to afford 56 (0.940 g, 98% based on crude 52) as a white solid.

¹H NMR (CD₃OD): δ 8.43 (s, 1H), 7.69 (s, 1H), 7.28-7.15 (m,

5H), 5.77 (s, 2H), 4.52 (s, 2H), 4.12 (m, 2H), 4.11 (s, 3H), 3.80

(dd, J ) 11.7, 5.3 Hz, 1H), 3.80-3.60 (m, 3H), 3.59 (dd, J )

11.0, 4.9 Hz, 1H), 2.90 (q, J ) 4.9 Hz, 1H). ¹³C NMR (CD₃OD):

δ 158.4, 151.4, 151.0, 139.1, 135.0, 129.7, 129.2, 129.1, 117.5,

115.3, 79.0, 72.5, 72.0, 65.6, 62.0, 61.6, 54.8, 42.1.

7-{[(1,3-Dihydroxypropan-2-yl)(methyl)amino]methyl]}-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (14) and its Hydrochlo-

ride salt (14 ·HCl). Compound 46 (110 mg, 0.285 mmol) was heated

under reﬂux in concentrated aqueous HCl (4 mL) for 1.5 h. The

solution was concentrated in vacuo and the residue dissolved in

MeOH/H₂O (1:1, 3 mL) and neutralized with Amberlyst A-21 resin.

The resin was removed by ﬁltration and the ﬁltrate concentrated in

vacuo and the resulting residue puriﬁed by chromatography

(CHCl₃/7 M NH₃-MeOH ) 9:1 f 85:15) to afford 14 as a colorless

solid. Conversion of 14 to the HCl salt with 5% aqueous HCl

1

afforded 14·HCl (54 mg, 66%). H NMR (D₂O, pH ∼ 1, internal

CH₃CN at δ 2.06): δ 8.73 (s, 1H), 7.87 (s, 1H), 4.64, 4.59 (2

partially coalescing br s, 2H), 4.10-3.86 (m, 4H), 3.65 (pentet, J

1

) 5.9 Hz, 1H), 2.96 (s, 3H). H NMR (D₂O + NaOD pH ∼ 10,

internal CH₃CN at δ 2.06) 7.99 (s, 1H), 7.48 (s, 1H), 3.88 (s, 2H),

3.82 (dd, J ) 11.8, 6.1 Hz, 2H), 3.72 (dd, J ) 11.8, 5.7 Hz,2 H),

2.89 (pentet, J ) 5.9 Hz, 1H), 2.33 (s, 3H). ¹³C NMR (D₂O, pH

∼1, internal CH₃CN at δ 1.47) 153.9, 145.0, 136.8, 133.6, 118.8,

103.4, 65.7, 57.3, 57.1, 49.1, 37.0. ESI-HRMS for C₁₁H₁₇N₄O₃

[MH⁺] calcd, 253.1301; found, 253.1292.

7-{[(1,3-Dihydroxypropan-2-yl)(2-hydroxyethyl)amino]methyl}-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (15) and its Hydro-

chloride salt (15 ·HCl). Sodium cyanoborohydride (34 mg, 0.54

mmol) was added to a solution of compound 10 (105 mg, 0.38

mmol) and 1,4-dioxane-2,5-diol (69 mg, 0.57 mmol) in MeOH (5

mL) and the mixture stirred at room temperature for 16 h. The

solution was concentrated in vacuo and the residue puriﬁed by

chromatography (CHCl₃/MeOH/28% aq NH₄OH ) 85:15:0.1) to

afford 15 as a colorless solid. Conversion of the free base to the

HCl salt using 5% aqueous HCl afforded 15·HCl (43 mg, 35%) as

7-({[(2R,3S)-1,3,4-Trihydroxybutan-2-yl]amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (DATMe-ImmH, 11) and

Its Triﬂuoroacetate Salt (11 ·TFA) and Hydrochloride Salt (11 ·HCl).

To a stirred solution of compound 56 (0.940 g, 2.34 mmol) in

CH₂Cl₂(30 mL) at -78 °C was added boron tribromide (23.4 mL,

23.4 mmol, 1.0 M in CH₂Cl₂). After 15 min, the reaction mixture

was warmed to room temperature and coevaporated with MeOH

(3×). The residue was stirred in 7M NH₃MeOH solution for 10

min, after which time silica gel was added and the resulting mixture

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography (CHCl₃/MeOH/28% aq NH₄OH ) 10:9:1) to afford

11 and then further puriﬁed by preparative HPLC to afford 11·TFA

(0.234 g, 26%) as a crystalline white solid. A small portion of the

product was evaporated from 5% aqueous HCl to afford 11·HCl.

¹H NMR (D₂O + drop DCl, internal CH₃CN at δ 2.06): δ 8.95 (s,

1H), 7.89 (s, 1H), 4.66 (d, J ) 14.3 Hz, 1H), 4.57 (d, J ) 14.3 Hz,

1H), 4.05-3.94 (m, 2H), 3.91 (dd, J ) 13.0, 5.4 Hz, 1H), 3.78

(dd, J ) 12.4, 3.3 Hz, 1H), 3.66 (dd, J ) 12.4, 4.4 Hz, 1H), 3.47

(m, 1H). ¹³CNMR (D₂O + drop DCl, internal CH₃CN at δ 1.47)

153.4, 145.2, 133.7, 133.3, 118.9, 104.0, 68.4, 63.4, 60.6, 57.7,

39.6. ESI-HRMS for C₁₁H₁₆N₄O₄Na [MNa⁺] calcd, 291.1069;

found, 291.1071.

1

a colorless solid. H NMR (D₂O, internal CH₃CN at δ 2.06): δ

8.56 (s, 1H), 7.85 (s, 1H), 4.79 (s, partly coalesced with HOD,

2H), 4.00-3.89 (m, 6H), 3.79 (pentet, J ) 6.2 Hz, 1H), 3.59 (br s,

1H), 3.50 (m, 1H). ¹³C NMR (D₂O, internal CH₃CN at δ 1.47): δ

154.3, 144.7, 138.6, 133.1, 118.8, 104.0, 64.5, 56.9, 55.9, 55.6,

52.8, 47.2. ESI-HRMS for C₁₂H₁₉N₄O₄[MH⁺] calcd, 283.1406;

found, 283.1400.

5-Benzyloxymethyl-7-({[1,3-dihydroxy-2-(hydroxymethyl)propan-

2-yl]amino}methyl)-4-methoxy-5H-pyrrolo[3,2-d]pyrimidine (47).

Sodium cyanoborohydride (18 mg, 0.28 mmol) was added to a

suspension of compound 44⁸(50 mg, 0.17 mmol) and TRIS (40)

(20 mg, 0.17 mmol) in MeOH (5 mL) and the resulting reaction

mixture stirred for 16 h at room temperature. The crude reaction

mixture was absorbed onto silica gel and concentrated in vacuo

and then the residue puriﬁed by chromatography (MeOH/EtOAc

) 1:4) to afford 47 (24 mg, 36%) as a syrup. ¹H NMR (CD₃OD):

δ 8.42 (s, 1H), 7.65 (s, 1H), 7.23 (m, 5H), 5.74 (s, 2H), 4.51 (s,

2H), 4.11 (s, 3H), 4.04 (s, 2H), 3.68 (s, 6H). ¹³C NMR (CD₃OD):

δ 158.4, 151.2, 150.9, 139.1, 134.4, 129.7, 129.2, 129.0, 117.5,

116.4, 78.9, 71.9, 62.9, 62.9, 62.9, 62.6, 54.8, 36.6. ESI-HRMS

for C₂₀H₂₇N₄O₅[MH⁺] calcd, 403.1981; found, 403.1985.

7-({Methyl[(2R,3S)-1,3,4-trihydroxybutan-2-yl]amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (26) and Its Hy-

drochloride Salt (26 ·HCl). To a suspension of compound 11 (30

mg, 0.11 mmol) in MeOH (1 mL) was added paraformaldehyde

(3.4 mg, 0.11 mmol). The mixture was stirred at 50 °C for 30 min,

and then sodium cyanoborohydride (7.4 mg, 0.12 mmol) was added

and the resulting reaction mixture stirred at room temperature for

16 h. The mixture was concentrated in vacuo, and the resulting

residue puriﬁed by chromatography (CH₂Cl₂/MeOH/28% aq

NH₄OH ) 8:2:0.1) to afford 26 (22 mg, 70%) as a white solid; mp

193 °C. [R]²_D¹-5.3 (c 1, H₂O). ¹H NMR (D₂O + 0.1% DCl, internal

CH₃CN at 2.06): δ 8.04 (s, 1H), 7.68 (s, 1H), 4.61 (d, J ) 13.7

Hz, 1H), 4.53 (d, J ) 13.7 Hz, 1H), 4.06-3.99 (m, 2H), 3.94 (dd,

J ) 13.5, 6.9 Hz, 1H), 3.75 (d, J ) 12.3 Hz, 1H), 3.57 (dd, J )

12.3, 4.2 Hz, 1H), 3.50-3.43 (m, 1H), 2.94 (s, 3H). ¹³C NMR (D₂O

+ 0.1% DCl, internal CH₃CN at 1.47): δ 155.7, 144.6, 143.6, 132.0,

118.4, 106.5, 67.6, 64.8, 63.3, 56.6, 49.8, 37.8. ESI-HRMS for

C₁₂H₁₉N₄O₄[MH⁺] calcd, 283.1406; found, 283.1404.

7-({[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (16) and Its Hy-

drochloride Salt (16 ·HCl). Boron tribromide (1 mL, 1.0 mmol, 1.0

M in CH₂Cl₂) was added dropwise to a solution of compound 47

(30 mg, 74.5 µmol) in CH₂Cl₂(5 mL) and stirred at room

temperature. A white solid precipitated from the reaction after 1 h,

and the reaction mixture was then quenched with MeOH, concen-

trated in vacuo, and codistilled with MeOH (3 × 25 mL) to afford

crude product. The residue was dissolved in MeOH, concentrated

in vacuo onto silica gel, and puriﬁed by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 5:4.5:0.5) to afford 16 (7 mg, 35%) as

a white solid and converted with 5% aqueous HCl to 16·HCl; mp

223-224 °C (plates from EtOH). ¹H NMR (D₂O, internal acetone

at 2.225 ppm): δ 9.06, (s, 1H), 7.92 (s, 1H), 4.59 (s, 2H), 3.91 (s,

6H). ¹³C NMR (D₂O, internal acetone at δ 31.5): δ 153.7, 146.0,

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1135

5-Benzyloxymethyl-4-methoxy-7-({[(2S,3R)-1,3,4-trihydroxybu-

tan-2-yl]amino}methyl)-5H-pyrrolo[3,2-d]pyrimidine (57). A mix-

ture of diethyl (2R,3R)-2-amino-3-hydroxysuccinate (49)³⁶(83 mg,

0.40 mmol, prepared from diethyl-L-tartrate by a known literature

method³⁶), sodium cyanoborohydride (42 mg, 0.67 mmol), and 44⁸

(100 mg, 0.34 mmol) were evaporated from MeOH (3×) and then

dissolved in MeOH (10 mL) and acetic acid added (5 drops). After

stirring at room temperature for 16 h, silica gel was added and the

mixture concentrated in vacuo and the resulting residue puriﬁed

by chromatography (Et₃N/EtOAc/hexanes ) 1:66:33) to afford

3.83-3.68 (m, 3H), 3.63 (d, J ) 5.5 Hz, 2H), 3.31 (pentet, J )

1.6 Hz, 1H). ¹³C NMR (CD₃OD, center line δ 49.0): δ 158.0, 150.8,

150.7, 138.8, 134.2, 129.3, 128.8, 128.6, 117.0, 116.1, 78.5, 72.1,

71.5, 65.6, 62.3, 61.0, 54.3, 41.6. ESI-HRMS for C₂₀H₂₇N₄O₅

[MH⁺] calcd, 403.1981; found, 403.1980.

7-({[(2R,3R)-1,3,4-Trihydroxybutan-2-yl]amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (24) and Its Hydrochlo-

ride Salt (24 ·HCl). Compound 58 (100 mg, 0.25 mmol) was heated

under reﬂux in concentrated HCl (4 mL) for 2 h. After cooling,

the solution was concentrated in vacuo and the residue dissolved

in MeOH and neutralized with Amberlyst A-21 resin. The mixture

was ﬁltered to remove the resin and the ﬁltrate concentrated in

vacuo. Puriﬁcation of the resulting residue by chromatography

(CH₂Cl₂/MeOH/28% aq NH₄OH ) 7:3:0.3 f 5:4.5:0.5) gave 24,

which was then converted with 5% aqueous HCl to 24·HCl (55

mg, 73% yield) as a colorless solid. A small portion was further

puriﬁed by preparative HPLC and the product evaporated from 5%

aqueous HCl to afford 24·HCl. ¹H NMR (D₂O + drop DCl, internal

CH₃CN at δ 2.06): δ 9.03 (s, 1H), 7.90 (s, 1H), 4.61 (s, 2H), 4.16

(m, 1H), 4.02-3.89 (m, 2H), 3.69 (d, J ) 5.5 Hz, 2H), 3.55 (m,

1H). ¹³CNMR (D₂O + drop DCl, internal CH₃CN at δ 1.47): δ

153.1, 145.4, 133.5, 132.6, 118.9, 103.5, 68.8, 62.7, 60.9, 57.4,

39.7. ESI-HRMS for C₁₁H₁₆N₄O₄Na [MNa⁺] calcd, 291.1069;

found, 291.1067.

1

crude 53 (114 mg, 70%) as a colorless oil. H NMR revealed the

product was slightly contaminated with (copolar) starting amine.

To a stirred solution of crude 53 (114 mg, 0.23mmol) in diethyl

ether (10 mL) was added MeOH (0.10 mL, 2.34mmol) and then

lithium borohydride³⁸(0.59 mL, 1.17 mmol, 2.0 M in THF). After

30 min, the reaction mixture was diluted with MeOH, silica gel

was added, and the mixture concentrated in vacuo. The resulting

residue was puriﬁed by chromatography (28% aq NH₄OH/MeOH/

CH₂Cl₂) 0.5:5:95 f 0.5:15:85) to afford 57 (56 mg, 59%) as a

1

colorless gum. The H and ¹³C NMRs were identical to those of

the enantiomer, 56.

7-({[(2S,3R)-1,3,4-Trihydroxybutan-2-yl]amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (19) and Its Hydrochlo-

ride Salt (19 ·HCl). Boron tribromide (1.39 mL, 1.39 mmol, 1.0 M

in CH₂Cl₂) was added dropwise to a stirred solution of compound

57 (56 mg, 0.14 mmol) in CH₂Cl₂(7 mL) at -78 °C. After 15

min, the reaction mixture was warmed to room temperature and

coevaporated with MeOH (3×). The residue was stirred in 7 M

NH₃-MeOH for 10 min and then silica gel was added and the

resulting mixture concentrated in vacuo. The residue was puriﬁed

by chromatography (CHCl₃/MeOH/28% aq NH₄OH ) 10:9:1) to

afford 19 (17 mg, 46%) as a white solid. A small portion was

puriﬁed by preparative HPLC and the product evaporated from 5%

aqueous HCl to afford 19·HCl. The ¹H and ¹³C NMR data were as

for 11·HCl. The minor differences in chemical shift between

11·HCl and 19·HCl were likely due to concentration effects. ESI-

HRMS for C₁₁H₁₆N₄O₄Na [MNa⁺] calcd, 291.1069; found, 291.1065.

Diethyl (2S,3R)-2-Hydroxy-3-({[5-benzyloxymethyl-4-methoxy-

5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)butanedioate (54).

Sodium triacetoxyborohydride (0.545 g, 2.57 mmol) was added to

a solution of diethyl (2S,3R)-2-amino-3-hydroxysuccinate (50)³⁶

(0.53 g, 2.57 mmol, prepared from diethyl-L-tartrate by known

literature methods^36,37) and compound 44⁸(0.588 g, 1.98 mmol)

in 1,2-dichloroethane (30 mL) and the resulting reaction mixture

stirred at room temperature for 4 h. The mixture was then diluted

with CH₂Cl₂and washed with aqueous saturated NaHCO₃, dried,

and concentrated in vacuo. The residue was puriﬁed by chroma-

tography (EtOAc/hexanes ) 9:1 f EtOAc) to afford 54 (0.67 g,

Diethyl (2R,3S)-2-Hydroxy-3-({[5-benzyloxymethyl-4-methoxy-

5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)butanedioate (55).

Diethyl (2R,3S)-2-amino-3-hydroxysuccinate (51)³⁶(109 mg, 0.53

mmol, prepared from diethyl-D-tartrate by known literature

methods^36,37), sodium cyanoborohydride (55 mg, 0.88 mmol), and

compound 44⁸(131 mg, 0.44 mmol) were evaporated from MeOH

(3×) and then dissolved in MeOH (10 mL) and acetic acid added

(5 drops). The reaction mixture was stirred at room temperature

for 2 h, and then silica gel was added and concentrated in vacuo.

The resulting residue was puriﬁed by chromatography (EtOAc/

hexanes ) 2:1 f EtOAc/Et₃N ) 1:99) to afford 55 (166 mg, 77%)

1

as a colorless oil. The H NMR was identical to its enantiomer,

54.

7-({[(2S,3S)-1,3,4-Trihydroxybutan-2-yl]amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (25) and Its TFA Salt

(25 ·TFA) and Hydrochloride Salt (25 ·HCl). To a stirred solution

of compound 55 (166 mg, 0.34mmol) in diethyl ether (10 mL) was

added MeOH (0.14 mL, 3.41 mmol) and then lithium borohydride³⁸

(0.85 mL, 1.71 mmol, 2.0 M in THF). After 30 min, the reaction

mixture was diluted with MeOH and concentrated in vacuo. The

residue was dissolved in MeOH, diluted with concentrated aqueous

ammonia (1 mL), and concentrated in vacuo onto silica gel.

Puriﬁcation of the resulting residue by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 85:15:2 f 70:30:2 f 50:50:4) afforded

crude 59 (103 mg, 0.26 mmol, 75%) as a colorless oil, which was

dissolved in CH₂Cl₂(10 mL), cooled to -78 °C, and boron

tribromide (2.56 mL, 2.56 mmol, 1.0 M in CH₂Cl₂) added. After

15 min, the reaction mixture was warmed to room temperature and

coevaporated with MeOH (3×). The residue was stirred in

methanolic ammonia solution (7 M) for 10 min, silica gel was

added, and the mixture concentrated in vacuo. The resulting residue

was puriﬁed by chromatography (CHCl₃/MeOH/28% aq NH₄OH

) 10:9:1) to give 25 and then further puriﬁed by preparative HPLC

to afford 25·TFA (18 mg, 26%) as a white solid. A small portion

1

69%) as a pale-yellow gum. [R]²_D¹-5.9 (c 0.54, EtOH). H NMR

(CDCl₃): δ, 8.55 (s, 1H), 7.34 (s, 1H), 7.33-7.23 (m, 5H), 5.70 (s,

2H), 4.62 (d, J ) 3.4 Hz, 1H), 4.48 (s, 2H), 4.23-4.15 (m, 5H),

4.10 (s, 3H), 3.99 (d, J ) 13.8 Hz, 1H), 3.85 (d, J ) 3.3 Hz, 1H),

2.25 (br s, exchanged to D₂O, 2H), 1.26 (t, J ) 7.1 Hz, 3H), 1.25

(t, J ) 7.1 Hz, 3H). ¹³C NMR (CDCl₃, center line at δ 77.0): δ

171.9, 171.1, 156.2, 150.0, 149.8, 136.9, 130.9, 128.4, 127.9, 127.6,

116.1, 77.0, 72.0, 70.2, 63.8, 61.4, 61.3, 53.5, 42.6, 14.1, 14.1. ESI-

HRMS for C₂₄H₃₁N₄O₇[MH⁺] calcd, 487.2193; found, 487.2174.

5-Benzyloxymethyl-4-methoxy-7-({[(2R,3R)-1,3,4-trihydroxybu-

tan-2-yl]amino}methyl)-5H-pyrrolo[3,2-d]pyrimidine (58). Solid

lithium borohydride³⁸(268 mg, 12.3 mmol) was added portionwise

to a reﬂuxing solution of compound 54 (600 mg, 1.23 mmol) in

THF (10 mL) and MeOH (1.0 mL, 24.6 mmol) over 2 h. After

cooling, the solution was concentrated in vacuo and the residue

puriﬁed by chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH ) 95:

5:0.5 f 85:15:0.5) to afford 58 (219 mg, 44%) as a colorless gum,

which crystallized on standing. The product was of sufﬁcient purity

to proceed to the next step, but a small quantity was recrystallized

for characterization purposes from ethyl acetate; mp 108-109 °C.

1

was evaporated from 5% aqueous HCl to afford 25·HCl. The H

and ¹³C NMR data were the same as for compound 24·HCl, its

enantiomer. The minor differences in chemical shift between 24

and 25 were likely due to concentration effects. ESI-HRMS for

C₁₁H₁₆N₄O₄Na [MNa⁺] calcd, 291.1069; found, 291.1066.

(2-[(Dimethylamino)methyleneamino]-7-{[(5R,6S)-6-hydroxy-

2,2-dimethyl-1,3-dioxepan-5-ylamino]methyl}-4-oxo-4,5-dihydro-

3H-pyrrolo[3,2-d]pyrimidin-3-yl)methyl Pivalate (60). To a mixture

of compound 41²³(66 mg, 0.19 µmol) and (5S,6R)-6-amino-2,2-

dimethyl-1,3-dioxepan-5-ol³⁹(44 mg, 0.27 mmol) in 1,2-dichlo-

roethane (6.5 mL) was added MgSO₄(400 mg, 3.3 mmol) and

sodium triacetoxyborohydride (61 mg, 0.29 mmol). The resulting

mixture was stirred overnight at room temperature and then ﬁltered,

1

[R]¹_D⁸-6.1 (c 0.59, MeOH). H NMR (CD₃OD): δ 8.42 (s, 1H),

7.64 (s, 1H), 7.28-7.16 (m, 5H), 5.75 (s, 2H), 4.50 (s, 2H), 4.10

(s, 3H), 4.07 (d, J ) 13.5 Hz, 1H), 4.00 (d, J ) 13.5 Hz, 1H),

1136 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

and the solids were rinsed with CH₂Cl₂(20 mL). The ﬁltrate was

washed with saturated aqueous NaHCO₃and brine (20 mL each),

dried, ﬁltered, and then silica gel (1 g) was added and the mixture

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography (CHCl₃/MeOH/28% aq NH₄OH ) 90:10:1) to

mmol) and sodium acetate (9.31 g, 114 mmol) were stirred together

in ethanol (75 mL) at room temperature for 15 min. Aqueous 37%

formaldehyde solution (12.68 mL, 170 mmol) was added and

stirring continued for 30 min and then cis-but-2-ene-1,4-diol (62)

(4.67 mL, 56.8 mmol) added and the mixture heated under reﬂux

for 16 h. The solvent was evaporated and the residue dissolved in

CHCl₃and washed with aqueous saturated NaHCO₃, dried, ﬁltered,

and then concentrated in vacuo to afford (()-64 (12.5 g, 98%) as

a brown syrup suitable for use without further puriﬁcation. An

aliquot of (()-64 was puriﬁed by chromatography (EtOAc f

EtOAc/MeOH ) 95:5). ¹³C NMR (CDCl₃): δ 136.5, 129.0, 128.4,

127.6, 78.5, 62.4, 61.3, 60.4, 56.8, 45.8.

(2R/S,3R/S)-3-[(Benzylamino)methyl]butane-1,2,4-triol (66). Zinc

dust (11.1 g, 170 mmol) was added to a solution of (()-64 (12.5

g, 56.1 mmol) in acetic acid (150 mL) (exotherm to 67 °C) and

the mixture stirred at room temperature for 1 h. The mixture was

ﬁltered, the ﬁltrate concentrated in vacuo, and the resulting residue

puriﬁed by chromatography (CH₂Cl₂/7 M NH₃in MeOH ) 9:1

f8:2) to afford (()-66 (5.8 g, 45%) as a colorless syrup. ¹H NMR

(CDCl₃): δ 7.33-7.23 (m, 5H), 4.11 (br s, 4H), 3.76-3.66 (m,

5H), 3.61-3.51 (m, 2H), 2.81-2.70 (m, 2H), 1.82 (sextet, J ) 5.5

Hz, 1H). ¹³C NMR (CDCl₃): δ 138.8, 128.5, 128.2, 127.3, 73.3,

64.5, 63.1, 54.0, 49.7, 43.3.

1

afford 60 (86 mg, 92%) as an oil. [R]²_D⁰+2.4 (c 2.37, CHCl₃). H

NMR (500 MHz, CDCl₃): δ 10.60 (br s, D₂O-exchangeable, 1H),

8.56 (s, 1H), 7.09 (s, 1H), 6.35 (s, 2H), 4.02 (d, J ) 13.4 Hz, 1H),

3.89 (d, J ) 13.4 Hz, 1H), 3.78 (dd, J ) 12.4, 2.8 Hz, 1H), 3.75

(dd, J ) 11.3, 1.8 Hz, 1H), 3.65-3.57 (m, 2H), 3.56 (dd, J )

12.4, 7.6 Hz, 1H), 3.15 (s, 3H), 3.04 (s, 3H), 2.71 (ddd, J ) 7.3,

6.8, 2.6 Hz, 1H), 2.32 (br s, 1H, D₂O exchangeable), 1.33 (s, 3H),

1.32 (s, 3H), 1.15 (s, 9H). ¹³C NMR (125.7 MHz, CDCl₃): δ 178.0,

157.3, 155.7, 154.0, 143.5, 127.0, 115.2, 115.0, 101.5, 73.0, 66.3,

63.1, 62.7, 60.5, 41.7, 41.2, 39.2, 35.3, 27.4, 25.1, 25.0. ESI-HRMS

for C₂₃H₃₇N₆O₆[MH⁺] calcd, 493.2764; found, 493.2775.

2-Amino-7-({[(2R,3S)-1,3,4-trihydroxybutan-2-yl]amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (DATMe-ImmG, 9).

A mixture of acetyl chloride (70 µL, 0.98 mmol) in MeOH (5 mL)

was added to 60 (49 mg, 99 µmol) and stirred at room temperature

for 16 h. The resulting solution was concentrated in vacuo, and

the residue was treated with water (2 mL) and concentrated HCl

(2 mL) at 100 °C for 75 min. The solution was again concentrated

in vacuo, dissolved in MeOH, and concentrated in vacuo onto silica

gel (∼1 g). Puriﬁcation of the residue by chromatography (CHCl₃/

MeOH/28% aq NH₄OH ) 6:4:1) afforded 9 (20 mg, 71%) as an

amorphous white solid. [R]²_D⁰-15 (c 0.21, MeOH). ¹H NMR (500

MHz, D₂O, internal 1,4-dioxane at δ 3.75): δ 7.30 (s, 1H), 3.95

(d, J ) 13.7 Hz, 1H), 3.85 (d, J ) 13.7 Hz, 1H), 3.83-3.77 (m,

2H), 3.73-3.66 (m, 2H), 3.58 (dd, J ) 12.0, 6.2 Hz, 1H), 2.93-2.87

(m, 1H). ¹³C NMR (125.7 MHz, D₂O, internal 1,4-dioxane at 67.2)

δ 158.0, 152.6, 143.9, 129.5, 113.0, 109.6, 71.1, 63.7, 59.8, 59.8,

40.5. ESI-HRMS for C₁₁H₁₈N₅O [MH⁺] calcd, 284.1359; found,

284.1352.

5-Benzyloxymethyl-7-({benzyl[(2R/S,3R/S)-3,4-dihydroxy-2-

(hydroxymethyl)butyl]amino}methyl)-4-methoxy-5H-pyrrolo[3,2-

d]pyrimidine (68). Acetyl chloride (21 µL, 0.30 mmol), compound

44⁸(179 mg, 0.60 mmol), compound (()-66 (136 mg, 0.60 mmol),

and sodium cyanoborohydride (57 mg, 0.90 mmol) were succes-

sively added to MeOH (6 mL) and the resulting reaction mixture

stirred at room temperature for 64 h. The mixture was concentrated

in vacuo and the residue puriﬁed by chromatography (CH₂Cl₂/7 M

NH₃in MeOH ) 96:4) to afford (()-68 (159 mg, 52%) as a

1

colorless gum. H NMR (CD₃OD): δ 8.43 (s, 1H), 7.58 (s, 1H),

7.32-7.12 (m, 10H), 5.75 (s, 2H), 4.50 (s, 2H), 4.10 (s, 3H), 3.85

(s, 2H), 3.71 (dd, J ) 10.9, 4.8 Hz, 1H), 3.65-3.51 (m, 4H),

3.48-3.37 (m, 2H), 2.69-2.57 (m, 2H), 2.19 (m, 1H). ¹³C NMR

(CD₃OD, center line at δ 49.0): δ 157.9, 151.3, 150.9, 139.8, 138.7,

135.1, 130.4, 129.3, 128.7, 128.6, 128.2, 116.8, 114.2, 78.6, 74.1,

71.6, 65.4, 63.7, 59.9, 55.5, 54.3, 48.5, 42.1. ESI-HRMS for

C₂₈H₃₅N₄O₅[MH⁺] calcd, 507.2607; found, 507.2604.

tert-Butyl (1,3-Dihydroxypropan-2-yl)[(1S)-2-hydroxy-1-(4-hy-

droxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)ethyl]carbamate (61). Di-

tert-butyl dicarbonate (91 mg, 0.42 mmol) was added to a solution

of ImmH (1·HCl)⁷(70 mg, 0.23 mmol) and Et₃N (65 µL, 0.46

mmol) in a mixture of water (1 mL) and MeOH (3 mL). The

solution was stirred for 30 min and then concentrated in vacuo to

afford 110 mg of a colorless solid, which consisted, as estimated

7-({[(2R/S,3R/S)-3,4-Dihydroxy-2-(hydroxymethyl)butyl]am-

ino}methyl)-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (17)

and Its Hydrochloride Salt (17 ·HCl). Compound (()-68 (150

mg, 0.30 mmol) was heated under reﬂux in concentrated HCl (4

mL) for 1.5 h. The solution was concentrated in vacuo to a cream-

colored foam, which was dissolved in a 1:1 mixture of MeOH/

water (10 mL, v/v) and the solution neutralized with Amberlyst

A-21 resin. The resin was ﬁltered off and 10% Pd-C (50 mg) added

to the ﬁltrate and the mixture stirred under an atmosphere of

hydrogen for 1 h. The suspension was ﬁltered through celite and

concentrated in vacuo and the resulting residue puriﬁed by

chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH ) 5:4.5:0.5) to

afford (()-17 as a colorless solid, which was further puriﬁed by

prep HPLC and the product evaporated from 5% aqueous HCl to

afford (()-17 ·HCl (26 mg, 28%). ¹H NMR (D₂O, internal CH₃CN

at δ 2.06): δ 8.60 (s, 1H), 7.81 (s, 1H), 4.45 (s, 2H), 3.84-3.78

(m, 2H), 3.69-3.53 (m, 3H), 3.35-3.23 (m, 2H), 2.22 (m, 1H).

¹³C NMR (D₂O, internal CH₃CN at δ 1.5): δ 154.3, 144.6, 137.9,

132.6, 118.7, 104.8, 71.4, 63.4, 60.6, 47.8, 41.6, 41.0. ESI-HRMS

for C₁₂H₁₉N₄O₄[MH⁺] calcd, 283.1406; found, 283.1406.

(4R/S,5S/R)-(2-Benzylisoxazolidine-4,5-diyl)dimethanol (65). A

mixture of N-benzylhydroxylamine (4.4 g, 27.7 mmol) and anhy-

drous NaOAc (3.0 g, 36.7 mmol) in EtOH (35 mL) was stirred at

room temperature for 15 min, after which time aqueous 37%

formaldehyde (4.1 mL, 55.1 mmol) was added and the mixture

stirred for another 30 min. A solution of trans-but-2-ene-1,4-diol

(63) (1.63 g, 18.5 mmol) in ethanol (20 mL) was then introduced

to the mixture in a single portion and the resulting solution reﬂuxed

for 18 h. After cooling, the mixture was concentrated in vacuo and

the residue dissolved in CH₂Cl₂and the solution washed with

aqueous saturated NaHCO₃solution, dried, ﬁltered, and the ﬁltrate

1

by H NMR, of about 78 mg, 0.21 mmol of (2S,3S,4R,5R)-tert-

butyl 3,4-dihydroxy-2-(4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-

yl)-5-(hydroxymethyl)pyrrolidine-1-carboxylate with the rest being

triethylamine hydrochloride. This mixture was dissolved in MeOH

(4 mL) and water (3 mL) and sodium periodate (55 mg, 0.26 mmol)

added. After stirring for 15 min, a precipitate formed and sodium

borohydride (24 mg, 0.64 mmol) was added and the mixture stirred

for an additional 15 min, ﬁltered through celite, and the mixture

concentrated in vacuo. The residue was puriﬁed by chromatography

(CH₂Cl₂/MeOH ) 85:15) to afford 61 (72 mg, 92%) as a colorless

1

solid. [R]²_D⁰+35.8 (c 0.505, MeOH). H NMR (CD₃OD): δ 7.89

(s, 1H), 7.50 (br s 1H), 5.32 (br s, 0.5 H), 5.08 (br s, 0.5H), 4.28

(br s 1H), 4.17-3.50 (br m, 6H), 1.39 (br d, 9H). ESI-HRMS for

C₁₆H₂₅N₄O₆[MH⁺] calcd, 369.1774; found, 369.1760.

7-{(1S)-1-[(1,3-Dihydroxypropan-2-yl)amino]-2-hydroxyethyl}-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one Hydrochloride (12).

Compound 61 (68 mg, 0.19 mmol) was dissolved in a mixture of

MeOH (2 mL) and concentrated HCl (0.2 mL). After a few minutes,

the solution was concentrated in vacuo to give a yellow foam, which

was crystallized from EtOH to afford 12 (42 mg, 75%) as a colorless

solid; mp >300 °C. [R]²_D⁰+24.1 (c 0.435, H₂O + 1 drop

1

concentrated HCl). H NMR (D₂O + DCl, internal acetone at δ

2.225): δ 8.95 (s, 1H), 7.96 (s, 1H), 5.09 (t, J ) 4.7 Hz, 1H), 4.24

(dd, J ) 12.2, 4.3 Hz, 1H), 4.11 (dd, J ) 12.2, 5.1 Hz, 1H),

3.97-3.77 (m, 4H), 3.46 (pentet, J ) 5.3 Hz, 1H). ¹³C NMR (D₂O

+ DCl, internal acetone at δ 31.5): δ 154.0, 146.0, 133.4, 132.5,

119.7, 106.6, 62.1, 60.1, 59.3, 59.1, 54.7. ESI-HRMS for

C₁₁H₁₇N₄O₄[MH⁺] calcd, 269.1250; found, 269.1239.

(4R/S,5R/S)-(2-Benzylisoxazolidine-4,5-diyl)dimethanol (64). A

mixture of N-benzylhydroxylamine hydrochloride (13.59 g, 85.15

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1137

then concentrated in vacuo. The crude product was puriﬁed by

chromatography (EtOAc) to afford (()-65 (1.46 g, 35%) as an

immobile syrup. ¹H NMR (CDCl₃): δ 7.41-7.22 (m, 5H),

4.10-3.82 (m, 2H), 3.80-3.60 (m, 4H), 3.5-2.3 (m, 5H). ¹³C NMR

(CDCl₃): δ 137.0, 129.3, 128.8, 128.0, 81.6, 65.0, 64.1, 63.0, 58.7,

47.7.

3-(Benzyl((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl)am-

ino)propanol (76) and Its Hydrochloride Salt (76 ·HCl). A mixture

of 3-(benzylamine)propanol (71) (159 mg, 0.96 mmol), 9-deaza-

hypoxanthine¹⁰(75) (100 mg, 0.74 mmol), and 37% aqueous

formaldehyde (72 µL, 0.96 mmol) in water (5 mL) was stirred and

heated at 85 °C in a stoppered ﬂask for 16 h. After cooling, the

solution was concentrated in vacuo and the residue puriﬁed by

chromatography (CH₂Cl₂/MeOH ) 9:1 f 8:2) to afford 76 (37

mg, 16%) as a white solid; mp 200-205 °C. ¹H NMR (CD₃OD +

DCl): δ 9.09 (s, 1H), 7.99 (s, 1H), 7.64-7.59 (m, 2H), 7.45-7.42

(m, 3H), 4.80 (d, J ) 14.5 Hz, 1H), 4.70 (d, J ) 14.5 Hz, 1H),

4.65 (d, J ) 13.5 Hz, 1H), 4.49 (d, J ) 13.5 Hz, 1H), 3.55 (t, J )

5.6 Hz, 2H), 3.32-3.26 (m, 2H), 2.02-1.95 (m, 2H). ¹³C NMR

(CD₃OD + DCl): δ 153.0, 147.1, 134.6, 133.4, 132.7, 131.4, 131.2,

130.7, 120.4, 103.7, 60.65, 58.6, 51.9, 27.8. ESI-HRMS for

C₁₇H₂₁N₄O₂[MH⁺] calcd, 313.1665; found, 313.1661.

(2S/R,3R/S)-[(Benzylamino)methyl]butane-1,2,4-triol (67). Zinc

dust (470 mg, 7.2 mmol) was added to a solution of compound

(()-65 (320 mg, 1.43 mmol) in acetic acid (6 mL) and the mixture

stirred vigorously for 1.5 h. The suspension was ﬁltered through

celite and the ﬁltrate concentrated in vacuo and the resulting residue

dissolved in CH₂Cl₂, silica gel added, and the resulting mixture

concentrated in vacuo. The residue was puriﬁed by chromatography

(CH₂Cl₂/7 M NH₃-MeOH ) 4:1) to afford (()-67 (260 mg, 81%)

1

as an immobile syrup. H NMR (CDCl₃): δ 7.41-7.21 (m, 5H),

3.90 (q, J ) 4.0 Hz, 1H), 3.80 (s, 2H), 3.75 (d, J ) 5.1 Hz, 2H),

3.66 (dq, J ) 11.7, 3.9 Hz, 2H), 3.18 (br s, 4H), 2.94 (d, J ) 6.9

Hz, 2H), 1.78 (m, 1H). ¹³C NMR (CDCl₃): δ 138.9, 129.1, 128.7,

127.9, 73.7, 65.4, 65.3, 54.4, 48.9, 43.9.

3-((4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)-

propanol (18) and Its Hydrochloride Salt (18 ·HCl). To a solution

of 76 (35 mg, 0.11 mmol) in iPrOH (2 mL) was added 10% Pd-C

(20 mg). The mixture was stirred at 50 °C under an atmosphere of

hydrogen for 16 h. The resulting suspension was ﬁltered through

celite and the ﬁltrate concentrated in vacuo and the residue puriﬁed

by chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH ) 5:5:0.1) to

5-Benzyloxymethyl-7-({benzyl[(2R/S,3S/R)-3,4-dihydroxy-2-

(hydroxymethyl)butyl]amino}methyl)-4-methoxy-5H-pyrrolo[3,2-

d]pyrimidine (69). To a solution of (()-67 (250 mg, 1.11 mmol)

and compound 44⁸(330 mg, 1.11 mmol) in MeOH (11 mL) was

added acetyl chloride (40 µL, 0.56 mmol), and this was followed

by the addition of sodium cyanoborohydride (105 mg, 1.67 mmol).

The mixture was stirred at room temperature for 72 h and then

diluted with CH₂Cl₂, washed with aqueous saturated NaHCO₃

solution, dried, ﬁltered, and the ﬁltrate concentrated in vacuo. The

resulting residue was puriﬁed by chromatography (7 M NH₃in

MeOH/CH₂Cl₂) 4:96) to afford (()-69 (310 mg, 55%) as an

immobile syrup. ¹H NMR (CDCl₃): δ 8.56 (s, 1H), 7.46-7.17 (m,

11H), 5.74 (s, 2H), 5.33 (s, 2H), 4.14 (s, 3H), 4.05 (d, J ) 14.2

Hz, 1H), 3.92 (d, J ) 14.2 Hz, 1H), 3.80-3.41 (m, 7H), 2.90 (dd,

J ) 13.2, 9.0 Hz, 1H), 2.72 (dd, J ) 12.9, 5.1 Hz, 1H), 2.38 (m,

1H). ¹³C NMR (CDCl₃): δ 156.9, 150.9, 150.7, 138.0, 137.2, 133.5,

129.7, 129.0, 128.9, 128.4, 128.0, 127.9, 112.3, 77.2, 74.3, 70.7,

65.1, 64.5, 59.4, 54.3, 54.1, 46.0, 40.9. ESI-HRMS for C₂₈H₃₅N₄O₅

[MH⁺] calcd, 507.2607; found, 507.2592.

7-({[(2R/S,3S/R)-3,4-Dihydroxy-2-(hydroxymethyl)butyl]am-

ino}methyl)-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (38)

and Its Hydrochloride Salt (38 ·HCl). A solution of compound

(()-69 (310 mg, 0.61 mmol) was reﬂuxed in concentrated HCl (8

mL) for 2.5 h and the solution concentrated in vacuo. The crude

residue was dissolved in MeOH (10 mL) and water (2 mL) and

neutralized with Amberlyst A-21 resin. The resin was ﬁltered off

and the ﬁltrate concentrated in vacuo. The residue was dissolved

in MeOH (6 mL) and water (3 mL) and then 10% Pd/C (0.1 g)

added and the mixture stirred under a hydrogen atmosphere for

2 h. The suspension was ﬁltered through celite and the ﬁltrate

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH ) 5:4.5:0.5) to

afford (()-38 (60 mg, 35%) as a colorless solid. ¹H NMR (D₂O +

DCl): δ 8.64 (s, 1H), 7.76 (s, 1H), 4.40 (s, 2H), 3.75-3.48 (m,

5H), 3.27-3.20 (m, 2H), 2.07 (m, 1H). ¹³C NMR (D₂O + DCl): δ

153.7, 144.6, 136.3, 132.5, 118.5, 104.3, 71.2, 63.9, 61.4, 47.1,

41.2, 40.2. ESI-HRMS for C₁₂H₁₉N₄O₄[MH⁺] calcd, 283.1406;

found, 283.1413.

1

afford 18 (18 mg, 74%) as a white solid; mp 189 °C. H NMR

(D₂O + 0.1% DCl, internal acetonitrile at δ 2.06): δ 8.76 (s, 1H),

7.82 (s, 1H), 4.43 (s, 2H), 3.69 (t, J ) 6 Hz, 2H), 3.21 (t, J ) 7.5

Hz, 2H), 1.97-1.91 (m, 2H). ¹³C NMR (D₂O + 0.1% DCl, internal

acetonitrile at δ 1.47): δ 153.9, 144.9, 136.0, 132.7, 118.7, 104.6,

59.5, 45.3, 41.0, 28.5. ESI-HRMS for C₁₀H₁₅N₄O₂[MH⁺] calcd,

223.1195; found, 223.1194.

7-{[Benzyl(4-hydroxybutyl)amino]methyl}-3,5-dihydro-4H-pyr-

rolo[3,2-d]pyrimidin-4-one (77) and Its Hydrochloride Salt (77 ·HCl).

A mixture of 4-(benzylamino)butan-1-ol (72) (172 mg, 0.96 mmol),

9-deazahypoxanthine (75)¹⁰(100 mg, 0.74 mmol), and 37% aqueous

formaldehyde (72 µL, 0.96 mmol) in water (5 mL) was stirred and

heated in stoppered ﬂask at 85 °C for 16 h. After cooling, the

solution was concentrated in vacuo and the residue puriﬁed by

chromatography (CH₂Cl₂/MeOH ) 9:1 f 8:2) to afford 77 (130

1

mg, 55%) as a white solid; mp >250 °C. H NMR (CD₃OD +

DCl): δ 9.10 (s, 1H), 8.00 (s, 1H), 7.63 (m, 2H), 7.45 (m, 3H),

4.79 (d, J ) 14.1 Hz, 1H), 4.72 (d, J ) 14.1 Hz, 1H), 4.62 (d, J )

13 Hz, 1H), 4.50 (d, J ) 13 Hz, 1H), 3.55 (t, J ) 6 Hz, 2H), 3.20

(m, 2H), 1.93 (m, 2H), 1.50 (m, 2H). ¹³C NMR (CD₃OD + DCl):

δ 153.0, 147.1, 134.5, 133.5, 132.7, 131.4, 130.7, 120.4, 103.7,

100.2, 62.3, 58.2, 53.6, 50.0, 30.8, 22.2. ESI-HRMS for C₁₈H₂₃N₄O₂

[MH⁺] calcd, 327.1821; found, 327.1815.

7-{[(4-Hydroxybutyl)amino]methyl}-3,5-dihydro-4H-pyrrolo[3,2-

d]pyrimidin-4-one (29) and Its Hydrochloride Salt (29 ·HCl). To

compound 77 (230 mg, 0.70 mmol) in iPrOH (3 mL) was added

10% Pd-C (50 mg) and the mixture stirred at 50 °C under an

atmosphere of hydrogen for 16 h. While still warm, the solution

was ﬁltered through celite and the ﬁltrate concentrated in vacuo.

The resulting residue was puriﬁed by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 8:2:0.1) to afford 29 (133 mg, 80%) as

1

a white solid; mp >250 °C. H NMR (D₂O + 0.1% DCl, internal

acetonitrile at δ 2.06): δ 8.32 (s, 1H), 7.73 (s, 1H), 4.39 (s, 2H),

3.60 (t, J ) 6.3 Hz, 2H), 3.12 (t, J ) 7.7 Hz, 2H), 1.75 (pentet, J

) 7.7 Hz, 2H), 1.59 (pentet, J ) 6.3 Hz, 2H). ¹³C NMR (D₂O +

0.1% DCl, internal acetonitrile at δ 1.47): δ 155.1, 144.0, 141.2,

131.9, 118.5, 106.0, 61.4, 47.2, 40.9, 29.0, 23.0. ESI-HRMS for

C₁₁H₁₇N₄O₂[MH⁺] calcd, 237.1352; found, 237.1349.

7-{[(2-Hydroxyethyl)amino]methyl}-3,5-dihydro-4H-pyrrolo[3,2-

d]pyrimidin-4-one (20) and Its Hydrochloride Salt (20 ·HCl). A

mixture of ethanolamine (70) (78 µL, 0.96 mmol), 9-deazahypox-

anthine¹⁰(75) (100 mg, 0.74 mmol), and 37% aqueous formalde-

hyde (72 µL, 0.96 mmol) in water (5 mL) was stirred and heated

in a stoppered ﬂask at 85 °C for 16 h. After cooling, the solution

was concentrated in vacuo and the residue was puriﬁed by

chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH ) 6:3.5:0.5) to

7-{[Bis(2-hydroxyethyl)amino]methyl}-3,5-dihydro-4H-pyrrolo[3,2-

d]pyrimidin-4-one (21). Aqueous 37% formaldehyde (160 µL, 2.0

mmol) was added to a solution of diethanolamine hydrochloride

(73) (283 mg, 2.0 mmol) and sodium acetate (160 mg, 2.0 mmol)

in water (2 mL) followed by the addition of 9-deazahypoxanthine

(75)¹⁰(300 mg, 2.2 mmol). The mixture was stirred at 95 °C (bath

temperature) for 12 h, and after cooling, silica gel (1.0 g) was added

and the suspension concentrated in vacuo. Puriﬁcation of the

resulting residue by chromatography (CH₂Cl₂/MeOH/28% aq

NH₄OH ) 5:4.8:0.2) afforded 21 (244 mg, 44%) as a syrup, which

1

afford 20 (100 mg, 65%) as a pale-brown solid; mp 214 °C. H

NMR (D₂O + 0.1% DCl): δ 8.75 (s, 1H), 7.83 (s, 1H), 4.47 (s,

2H), 3.86 (t, J ) 5.1 Hz, 2H), 3.26 (t, J ) 5.1 Hz, 2H). ¹³C NMR

(D₂O + 0.1% DCl): δ 154.0, 144.9, 136.3, 132.8, 118.7, 104.5,

57.2, 49.0, 40.9. ESI-HRMS for C₉H₁₃N₄O₂[MH⁺] calcd, 209.1039;

found, 209.1031.

1138 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

1

solidiﬁed on standing; mp 178-180 °C. H NMR (D₂O): δ 8.21

7-({[3-Hydroxy-2-(hydroxymethyl)propyl]amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (27). Acetic acid (0.122

mL, 2.13 mmol) was added dropwise to a solution of compound

81 (100 mg, 0.43 mmol) in 1,4-dioxane (2 mL) followed by

9-deazahypoxanthine (75)¹⁰(115 mg, 0.85 mmol) and the resulting

suspension heated at 95 °C (bath temperature) for 16 h. After

cooling to room temperature silica gel (1.0 g) was added and the

mixture concentrated in vacuo. The resulting residue was puriﬁed

by chromatography (7 M NH₃MeOH/CH₂Cl₂) 1:19) to afford,

presumably, compound 82 (56 mg, 35%), which was committed

to the next step without characterization. Concentrated HCl (1 mL)

was added to a stirred solution of compound 82 (50 mg, 0.13 mmol)

in MeOH (2 mL). After 0.5 h, the solution was concentrated in

vacuo and the residue dissolved in MeOH to which silica gel was

added and then concentrated in vacuo. The residue was puriﬁed

by chromatography (7 M NH₃in MeOH/CH₂Cl₂) 1:4) to afford

9-{[(2,2-dimethyl-1,3-dioxan-5-yl)methylamino]methyl}-9-deaza-

hypoxanthine (19 mg, 0.56 mmol, 42%) as a white solid, which

was dissolved in H₂O (2 mL), 10% Pd/C (56 mg) was added, and

the mixture was stirred under an atmosphere of hydrogen for 72 h.

After ﬁltering through celite, the ﬁltrate was concentrated in vacuo

and the residue puriﬁed by chromatography (CH₂Cl₂/MeOH/28%

aq NH₄OH ) 5:4.5:0.5) to afford 27 (4 mg, 28%) as a white solid.

¹H NMR (D₂O): δ 8.03 (s, 1H), 7.31 (s, 1H), 4.71 (s, 2H), 3.79 (s,

2H), 3.54 (d, J ) 6.0 Hz, 2H), 2.59 (d, J ) 6.6 Hz, 2H), 1.83

(septet, J ) 6.0 Hz, 1H).¹³C NMR (D₂O): δ 163.5, 151.6, 144.8,

126.4, 118.9, 112.0, 61.2, 61.2, 47.0, 42.2. ESI-HRMS for

C₁₁H₁₇N₄O₃[MH⁺] calcd, 253.1301; found, 253.1292.

(s, 1H), 7.77 (s, 1H), 4.63 (s, 2H), 3.94 (d, J ) 3.0 Hz, 4H), 3.37

(d, J ) 3.0 Hz, 4H). ¹³C NMR (D₂O): δ 154.8, 143.5, 142.5, 132.4,

118.1, 103.4, 55.4, 55.4, 54.1, 54.1, 47.9. ESI-HRMS for

C₁₁H₁₇N₄O₃[MH⁺] calcd, 253.1301; found, 253.1301.

7-{[(2-Hydroxyethyl)(3-hydroxypropyl)amino]methyl}-3,5-dihy-

dro-4H-pyrrolo[3,2-d]pyrimidin-4-one (33). 3-(Hydroxyethylamino)-

propanol hydrochloride (74) (280 mg, 1.8 mmol) and sodium acetate

(150 mg, 1.8 mmol) were dissolved in water (2 mL) and added to

the solution were aqueous formaldehyde (150 µL, 1.8 mmol) and

9-deazahypoxanthine (75)¹⁰(250 mg, 1.85 mmol). The reaction

mixture was stirred at 95 °C (bath temperature) for 12 h and then

silica gel (1.0 g) was added and the mixture concentrated in vacuo.

Puriﬁcation of the resulting residue by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 5:4.8:0.2) afforded 33 (331 mg, 68%)

as a syrup, which solidiﬁed on standing; mp 179-180 °C. ¹H NMR

(D₂O): δ 7.94 (s, 1H), 7.62 (s, 1H), 4.37 (s, 2H), 3.86 (t, J ) 5.4

Hz, 2H), 3.61 (t, J ) 6.0 Hz, 2H), 3.18 (m, 4H), 1.95 (m, 2H). ¹³

NMR (D₂O): δ 155.3, 144.6, 143.3, 131.9, 118.0, 105.2, 59.6, 56.2,

54.4, 51.2, 47.4, 26.5. ESI-HRMS for C₁₂H₁₉N₄O₃[MH⁺] calcd,

267.1457; found, 267.1454.

C

(2,2-Dimethyl-1,3-dioxan-5-yl)-N-methylmethanamine (79). An

aqueous methylamine solution (3 mL, 34.8 mmol) was added to a

solution of (2,2-dimethyl-1,3-dioxan-5-yl)methyl methanesulfonate

(78)⁴⁰(900 mg, 4.0 mmol) in DMSO (7 mL) and stirred at 75 °C

for 16 h. After cooling, the reaction mixture was diluted with CHCl₃

and washed with water (×2), dried, ﬁltered, and the ﬁltrate

concentrated in vacuo. The crude material was puriﬁed by chro-

matography (CH₂Cl₂f MeOH/ CH₂Cl₂) 1:4 f 7 M NH₃in

(R)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl Methanesulfonate

(85). The title compound was prepared from (S)-(2,2-dimethyl-1,3-

dioxolan-4-yl)methanol (83) (Sigma-Aldrich, 99% ee) by known

literature procedures.^41,42[R]_D²¹-3.1 (c 0.83, CHCl₃). [R]²_J¹-3.3

1

MeOH/CH₂Cl₂) 1:4) to afford 79 (330 mg, 52%) as an oil. H

NMR (CD₃OD): δ 3.94 (dd, J ) 12.0, 4.4 Hz, 2H), 3.66 (dd, J )

12.0, 7.6 Hz, 2H), 2.53 (d, J ) 6.9 Hz, 2H), 2.36, (s, 3H), 1.89 (m,

1H), 1.38 (s, 3H), 1.37 (s, 3H). ¹³C NMR (CD₃OD): δ 99.7, 64.4,

64.4, 52.3, 36.9, 36.0, 26.0, 23.5.

(c 0.83, CHCl₃). Lit.⁴¹[R]_J²⁵-3 (c 0.028, CHCl₃). The ¹H and ¹³

C

NMRs were in agreement with those reported in the literature.^41,42

(S)-N-Benzyl-1-(2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (87).

The title compound was prepared according to the literature

method⁴¹from compound 85. [R]_D²¹+4.0 (c 0.69, CHCl₃). Lit.⁴⁵

[R]²_D⁰+5.8 (c 1.04, CHCl₃). [R]_J²¹+4.3 (c 0.69, CHCl₃). Lit.⁴¹[R]²_J⁵

+5.5 (c 0.054, CHCl₃). The ¹H and ¹³C NMRs were in agreement

with those reported in the literature.^41,42,45

7-({[3-Hydroxy-2-(hydroxymethyl)propyl](methyl)amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (22). Acetic acid

(0.180 mL, 3.10 mmol) was added dropwise to a solution of

compound 79 (100 mg, 0.63 mmol) in 1,4-dioxane (2 mL) followed

by 9-deazahypoxanthine (75)¹⁰(170 mg, 1.3 mmol) and the

resulting suspension heated at 95 °C (bath temperature) for 16 h.

After cooling, silica gel (1 g) was added to the solution and

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography (7 M NH₃in MeOH/CH₂Cl₂) 1:9) to afford crude

80 (120 mg, 62%). Concentrated HCl (1 mL) was added to a

solution of crude 80 (100 mg, 0.33 mmol) in MeOH (2 mL) and

the resulting reaction mixture left to stand for 30 min. After

concentration in vacuo, the resulting residue was dissolved in

MeOH, silica gel (1 g) was added and the mixture again

concentrated in vacuo. The residue was puriﬁed by chromatography

(7 M NH₃in MeOH/CH₂Cl₂) 1:4) to afford 22 (80 mg, 92%) as

a solid. ¹H NMR (D₂O): δ 7.92 (s, 1H), 7.64 (s, 1H), 4.39 (s, 2H),

3.62 (dd, J ) 11.2, 5.0 Hz, 2H), 3.47 (dd, J ) 11.2, 7.0 Hz, 2H),

3.19 (d, J ) 6.5 Hz, 2H), 2.83 (s, 3H), 2.28 (septet, J ) 6.3 Hz,

1H). ¹³C NMR (D₂O): δ 155.1, 144.4, 143.3, 132.1, 118.0, 104.4,

61.3, 61.3, 56.7, 50.5, 40.9, 38.3. ESI-HRMS for C₁₂H₁₉N₄O₃

[MH⁺] calcd, 267.1457; found, 267.1449.

N-Benzyl(2,2-dimethyl-1,3-dioxan-5-yl)methanamine (81). A so-

lution of (2,2-dimethyl-1,3-dioxan-5-yl)methyl methanesulfonate

(78)⁴⁰(1.70 g, 7.58 mmol) in neat benzylamine (10 mL, 92 mmol)

was stirred at 80 °C for 2 h. After cooling to room temperature,

the solvent was evaporated, the residue dissolved in toluene

(containing a small amount of EtOAc) and washed with water,

dried, ﬁltered, and the ﬁltrate concentrated in vacuo. The resulting

residue was puriﬁed by chromatography (EtOAc) to afford 81 (1.51

g, 85%) as a yellow oil. ¹H NMR (CD₃OD): δ 7.32-7.18 (m, 5H),

3.93 (dd, J ) 12.0, 6.0 Hz, 2H), 3.72 (s, 2H), 3.64 (dd, J ) 12.0,

6.0 Hz, 2H), 2.54 (d, J ) 6.0 Hz, 2H), 1.89 (m, 1H), 1.31 (s, 6H).

¹³C NMR (CD₃OD): δ 140.8, 129.4, 129.4, 128.1, 99.3, 64.0, 64.0,

54.7, 33.7, 25.6, 23.0.

7-({Benzyl[(2S)-2,3-dihydroxypropyl]amino}methyl)-3,5-dihydro-

4H-pyrrolo[3,2-d]pyrimidin-4-one (89). To a solution of compound

87 (0.67 g, 3.03 mmol) and compound 44⁸(0.9 g, 3.03 mmol) in

1,2-dichloroethane (30 mL) was added sodium triacetoxyborohy-

dride (0.834 g, 3.94 mmol) and anhydrous MgSO₄(2 g) and the

mixture stirred for 5 h. The reaction mixture was then diluted with

CH₂Cl₂, washed with aqueous saturated NaHCO₃, brine, dried,

ﬁltered, and the ﬁltrate concentrated in vacuo. The residue was

puriﬁed by chromatography (EtOAc/hexanes ) 1:1) to afford

7 - [ ( b e n z y l { [ ( 4 S ) - 2 , 2 - d i m e t h y l - 1 , 3 - d i o x o l a n - 4 -

yl]methyl}amino)methyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-

4-one (1.18 g, 78%) as a pale-yellow gum. [R]²_D¹+21.6 (c 0.92,

MeOH). ¹H NMR (CDCl₃): δ 8.55 (s, 1H), 7.41 (s, 1H), 7.37-7.18

(m, 10H), 5.73 (s, 2H), 4.46 (s, 2H), 4.38-4.29 (m, 1H), 4.10, (s,

3H), 4.05-3.98 (m, 2H), 3.92 (d, J ) 14.4 Hz, 1H), 3.79 (d, J )

13.9 Hz, 1H), 3.63 (d, J ) 13.9 Hz, 1H), 3.53, (t, J ) 7.9 Hz, 1H),

2.73 (dd, J ) 13.3, 5.9 Hz, 1H), 2.64 (dd, J ) 13.3, 5.9 Hz, 1H),

1.32 (s, 6H). ¹³C NMR (CDCl₃, center line at δ 77.0): δ 156.2,

150.7, 150.0, 139.6, 136.9, 132.2, 128.8, 128.4, 128.2, 128.0, 127.7,

126.9, 115.7, 114.7, 109.0, 77.0, 74.7, 70.1, 68.4, 59.2, 56.2, 53.5,

47.8, 26.8, 25.6. ESI-HRMS for C₂₉H₃₅N₄O₄[MH⁺] calcd, 503.2658;

found, 503.2635. 7-[(Benzyl{[(4S)-2,2-dimethyl-1,3-dioxolan-4-

yl]methyl}amino)methyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-

4-one (1.1 g, 2.189 mmol) was heated to 100 °C in concentrated

HCl (15 mL) for 3 h. After cooling to room temperature, the

solution was evaporated in vacuo and the residue dissolved in

MeOH, neutralized with Amberlyst A-21 resin, ﬁltered, and the

ﬁltrate concentrated in vacuo. The residue was puriﬁed by chro-

matography (CH₂Cl₂/7 M NH₃MeOH ) 9:1 f 85:15) to afford

89 (0.427 g, 59%) as a colorless solid. [R]²_D⁰-12.7 (c 0.715,

MeOH). ¹H NMR (CD₃OD): δ 7.90 (s, 1H), 7.37 (s, 1H), 7.31-7.16

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1139

(m, 5H), 3.97-3.78 (m, 3H), 3.69 (d, J ) 13.8 Hz, 1H), 3.63 (d,

J ) 13.8 Hz, 1H), 3.50 (dd, J ) 11.2, 4.8 Hz, 1H), 3.42 (dd, J )

11.2, 5.7 Hz, 1H), 2.60 (d, J ) 6.4 Hz, 2H). ¹³C NMR (CD₃OD,

center line at δ 49.0): δ 156.1, 145.5, 142.5, 140.2, 130.2, 129.4,

129.2, 128.1, 119.3, 115.0, 70.6, 66.3, 60.0, 57.6, 49.0. ESI-HRMS

for C₁₇H₂₁N₄O₃[MH⁺] calcd, 329.1614; found, 329.1618.

ethanol (96 µL, 1.36 mmol) in CH₃CN (4 mL) and the mixture

heated under reﬂux for 64 h. After cooling to room temperature,

the mixture was diluted with EtOAc and washed with saturated

NaHCO₃, dried, ﬁltered, and the ﬁltrate concentrated in vacuo. The

resulting residue was puriﬁed by chromatography (EtOAc/hexanes

) 1:1 f 7:3) to afford (S)-2-[benzyl(2,2-dimethyl-1,3-dioxolan-

4-yl)methylamino]ethanol (172 mg, 72%), which was dissolved in

MeOH (5 mL), 10% Pd-C (50 mg) was added, and the mixture

stirred under an atmosphere of hydrogen for 1 h. The mixture was

then ﬁltered through celite and the ﬁltrate concentrated in vacuo.

The resulting residue was puriﬁed by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 97:3:0.5 f 9:1:0.1) and the product

obtained distilled on a Kugelrohr apparatus at 120 °C/0.05 mmHg

to afford (S)-2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methylamino]et-

hanol (70 mg, 62%) as a colorless gum. [R]²_D⁰-9.6 (c 0.555,

MeOH). ¹H NMR (CD₃OD): δ 4.24 (pentet, J ) 6.2 Hz, 1H), 4.06

(dd, J ) 8.2, 6.3 Hz, 1H), 3.71-3.59 (m, 3H), 2.76-2.66 (m, 4H),

1.39 (s, 3H), 1.33 (s, 3H). ¹³C NMR (CD₃OD, center line at δ 49.0):

δ 110.4, 76.4, 68.6, 61.6, 53.4, 52.5, 27.3, 25.7. ESI-HRMS for

C₈H₁₈NO₃[MH⁺] calcd, 176.1287; found, 176.1274. (S)-2-[(2,2-

Dimethyl-1,3-dioxolan-4-yl)methylamino]ethanol (48 mg, 0.27

mmol), MgSO₄(500 mg), aldehyde 44⁸(81 mg, 0.27 mmol), and

sodium triacetoxyborohydride (75 mg, 0.36 mmol) were stirred

together at room temperature in 1,2-dichloroethane (3 mL) for 16 h.

The mixture was then diluted with CH₂Cl₂and washed with

saturated aqueous NaHCO₃solution, dried, ﬁltered, and the ﬁltrate

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography (EtOAc f EtOAc/MeOH/28% aq NH₄OH ) 97:

3:0.01) to afford 90 (100 mg, 80%) as a colorless gum, which turned

pale-yellow on standing. ¹H NMR (CDCl₃): δ 8.53 (s, 1H),

7.36-7.23 (s, 6H), 5.71 (s, 2H), 4.60 (br s, exchanged to D₂O,

1H), 4.46 (s, 2H), 4.20 (pentet, J ) 6.3 Hz, 1H), 4.10 (s, 3H), 4.02

(d, J ) 14.1 Hz, 1H), 3.88-3.67 (m, 4H), 3.26 (dd, J ) 8.0, 6.9

Hz, 1H), 2.86-2.70 (m, 3H), 2.61 (dd, J ) 13.2, 6.7 Hz, 1H),

1.32 (s, 3H), 1.28 (s, 3H). ¹³C NMR (CDCl₃, center line at δ 77.0):

δ 156.3, 150.2, 149.9, 136.8, 131.5, 128.5, 128.0, 127.7, 116.2,

115.1, 108.9, 76.9, 74.2, 70.1, 68.4, 59.9, 57.2, 56.5, 53.6, 47.8,

26.8, 25.4. ESI-HRMS for C₂₄H₃₃N₄O₅[MH⁺] calcd, 457.2451;

found, 457.2469.

7-({[(2S)-2,3-Dihydroxypropyl](2-hydroxyethyl)amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (32). Compound 90

(100 mg, 0.22 mmol) was dissolved in concentrated HCl (4 mL)

and heated at 100 °C for 1.5 h and then concentrated in vacuo.

The residue was dissolved in MeOH and a little water and the

solution neutralized with Amberlyst A-21 resin. The mixture was

ﬁltered, the ﬁltrate concentrated in vacuo, and the residue puriﬁed

by chromatography (iPrOH/H₂O/28% aq NH₄OH ) 9:0.5:0.5 f

8:1.5:0.5) to afford 32 (42 mg, 68%) as a colorless solid. [R]_D²⁰

-17.9 (c 0.545, H₂O). ¹H NMR (D₂O + NaOD, internal acetonitrile

at δ 2.06): δ 8.07 (s, 1H), 7.35 (s, 1H), 3.94-3.65 (m, 3H), 3.68

(t, J ) 6.1 Hz, 2H), 3.50 (dd, J ) 11.7, 4.2 Hz, 1H), 3.40 (dd, J

) 11.6, 6.3 Hz, 1H), 2.75-2.48 (m, 4H). ¹³C NMR (D₂O, internal

acetone at δ 31.5) δ 156.3, 145.4, 143.7, 132.0, 118.6, 109.2, 68.9,

65.1, 58.5, 56.7, 56.3, 48.8. ESI-HRMS for C₁₂H₁₉N₄O₄[MH⁺]

calcd, 283.1406; found, 283.1404.

7-({[(2S)-2,3-Dihydroxypropyl]amino}methyl)-3,5-dihydro-4H-

pyrrolo[3,2-d]pyrimidin-4-one (23) and Its Hydrochloride Salt

(23 ·HCl). Compound 89 (100 mg, 0.305 mmol) was dissolved in

MeOH (10 mL), diluted with water (10 mL), and 10% Pd-C (50

mg) added and the resulting mixture stirred under an atmosphere

of hydrogen at room temperature for 45 min. The reaction mixture

was ﬁltered through celite and the ﬁltrate concentrated in vacuo.

The crude residue was puriﬁed by chromatography (CH₂Cl₂/MeOH/

28% aq NH₄OH ) 5:4.5:0.5) to give 23 as a colorless solid, which

was converted with 5% aqueous HCl to 23 ·HCl as a colorless foam

and crystallized from MeOH (35 mg, 42%) as a white solid; mp

241-242 °C. [R]²_D⁰-12.9 (c 0.535, H₂O). ¹H NMR (D₂O + DCl):

δ 9.07 (s, 1H), 7.91 (s, 1H), 4.53 (s, 2H), 4.06 (m, 1H), 3.69-3.57

(m, 2H), 3.33 (dd, J ) 12.9, 2.9 Hz, 1H), 3.17 (dd, J ) 12.9, 9.8

Hz, 1H). ¹³C NMR (D₂O + DCl): δ 153.8, 146.1, 134.1, 133.1,

119.6, 104.1, 68.5, 64.5, 50.2, 41.8. ESI-HRMS for C₁₀H₁₅N₄O₃

[MH⁺] calcd, 239.1144; found, 239.1136.

(S)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl Methanesulfonate (86).

Compound 86 was prepared from (R)-(2,2-dimethyl-1,3-dioxolan-

4-yl)methanol (84) (Sigma-Aldrich, 99% ee) as described for its

enantiomer, 85. [R]_D²⁵+3.1 (c 0.72, CHCl₃). The ¹H and ¹³C NMRs

were identical to those of the enantiomer, compound 85.

(R)-N-Benzyl-1-(2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (88).

A solution of compound 86 (3.0 g, 14.27 mmol) and benzylamine

(6.23 mL, 57.1 mmol) were reﬂuxed together in CH₃CN (38 mL)

for 48 h. After cooling to room temperature, the solvent was

evaporated and the residue dissolved in EtOAc and washed with

aqueous saturated NaHCO₃, dried, ﬁltered, and the ﬁltrate concen-

trated in vacuo. The resulting residue was puriﬁed by chromatog-

raphy (EtOAc/hexanes ) 6:4 f 8:2) to afford 88 (2.56 g, 81%) as

1

a yellow oil. [R]²_D¹-3.7 (c 0.885, CHCl₃). The H and ¹³C NMRs

were identical to those of the enantiomer, compound 87.

7-({Benzyl[(2R)-2,3-dihydroxypropyl]amino}methyl)-3,5-dihydro-

4H-pyrrolo[3,2-d]pyrimidin-4-one (37). Compound 88 (372 mg,

1.68 mmol) was converted into 7-[(benzyl{[(4R)-2,2-dimethyl-1,3-

dioxolan-4-yl]methyl}amino)methyl]-3,5-dihydro-4H-pyrrolo[3,2-

d]pyrimidin-4-one (631 mg, 75%) as a colorless gum as described

for its enantiomer in the preparation of 89. [R]²_D¹-21.9 (c 0.905,

1

MeOH). The H and ¹³C NMRs were identical to those of the S

enantiomer. ESI-HRMS for C₂₉H₃₅N₄O [MH⁺] calcd, 503.2658;

found, 503.2643. 7-[(Benzyl{[(4R)-2,2-dimethyl-1,3-dioxolan-4-

yl]methyl}amino)methyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-

4-one (600 mg, 1.19 mmol) was converted, as described for the

S-enantiomer in the preparation of 89, into 37 (360 mg, 92%) as a

1

colorless solid. [R]_D²²+13.0 (c 0.715, MeOH). The H and ¹³C

NMRs were identical to those of the enantiomer, compound 89.

ESI-HRMS for C₁₇H₂₁N₄O₃[MH⁺] calcd, 329.1614; found, 329.1600.

7-({[(2R)-2,3-Dihydroxypropyl]amino}methyl)-3,5-dihydro-4H-

pyrrolo[3,2-d]pyrimidin-4-one (28) and Its Hydrochloride Salt

(28 ·HCl). Compound 37 (100 mg, 0.31 mmol) was dissolved in

hot water (20 mL), cooled to room temperature, and 10% Pd-C

(50 mg) added and the resulting mixture stirred under an atmosphere

of hydrogen at room temperature for 4 h. The hydrogen was

replaced with Ar and the mixture heated to 80 °C to ensure product

was in solution and then the mixture was ﬁltered through celite

and the ﬁltrate concentrated in vacuo to afford 28 (72 mg, 99%) as

a colorless solid. Compound 28 was treated with 5% aqueous HCl

5-(Benzyloxymethyl)-7-({[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl](2-

hydroxyethyl)amino}methyl)-4-methoxy-5H-pyrrolo[3,2-d]pyrimi-

dine (91). In the same way as that described for the preparation of

90, compound 88 (200 mg, 0.94 mmol) was converted ﬁrst into

intermediate (R)-2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methylamino-

]ethanol (103 mg, 78%) [R]²_D⁰+9.5 (c, 0.525, MeOH). The ¹H and

¹³C NMRs were identical to those of the enantiomer. ESI-HRMS

for C₈H₁₈NO₃[MH⁺] calcd, 176.1287; found, 176.1278. Reductive

alkylation of this intermediate then afforded 91 (180 mg, 86%) as

1

to afford 28·HCl. [R]_D¹⁸+12.6 (c 0.565, H₂O). The H and ¹³C

1

a colorless gum. H and ¹³C NMRs were identical to those of 90,

NMRs were identical to those of the enantiomer, compound 23.

ESI-HRMS for C₁₀H₁₄N₄O₃Na [MNa⁺] calcd, 261.0964; found,

261.0952.

its enantiomer. ESI-HRMS for C₂₄H₃₃N₄O₅[MH⁺] calcd, 457.2451;

found, 457.2467.

5-(Benzyloxymethyl)-7-({[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl](2-

hydroxyethyl)amino}methyl)-4-methoxy-5H-pyrrolo[3,2-d]pyrimi-

dine (90). Potassium carbonate (125 mg, 0.904 mmol) was added

to a solution of compound 87 (200 mg, 0.90 mmol) and 2-bromo-

7-({[(2R)-2,3-Dihydroxypropyl](2-hydroxyethyl)amino}methyl)-

3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (34). Compound 91

(165 mg, 0.36 mmol) was converted into 34 (74 mg, 73%) as a

colorless solid as described for the preparation of 32. [R]²_D⁰+18.4

1140 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

1

(c, 0.56, H₂O). The H and ¹³C NMR data were identical to those

Methyl (2R/S,3R/S)-5-carbamoyl-2,2-dimethyl-1,3-dioxolane-4-

carboxylate (96). Compound (()-95 (1.78 g, 7.66 mmol) was

dissolved in MeOH (10 mL) and stirred at room temperature as

methanolic ammonia (1.09 mL, 7.66 mmol, 7M) was added. The

solution was stirred for two days at room temperature before silica

gel (10 g) was added the mixture concentrated in vacuo. The

resulting residue was puriﬁed by chromatography (EtOAc/hexanes

) 1:1) to afford (()-96 (0.67 g, 43%) as an oil that solidiﬁed on

of 32 its enantiomer. ESI-HRMS for C₁₂H₁₉N₄O₄[MH⁺] calcd,

283.1406; found, 283.1398.

(2S,3R)-O-Isopropylidene-4-hydroxybutylamine (93). A solution

of 2,3-O-isopropylidene-D-erythronamide (92)⁴³(2.80 g, 16.0 mmol)

in anhydrous THF (45 mL) was added dropwise over 30 min to a

stirred suspension of LAH (2.43 g, 64.0 mmol) in THF (40 mL) at

such a rate as to maintain the reaction at room temperature. The

resulting mixture was then heated under reﬂux for 16 h and then

cooled to room temperature and worked up by the cautious addition

of water (2.5 mL), 15% aqueous NaOH (2.5 mL), and more water

(7.5 mL). The resulting suspension was ﬁltered through celite and

the ﬁltrate concentrated in vacuo to afford 92 (2.17 g, 84%) as a

mobile syrup. ¹H NMR (CDCl₃): δ 4.31 (td, J ) 6.7, 4.2 Hz, 1H),

4.23 (td, J ) 6.7, 3.9 Hz, 1H), 3.76 (dd, J ) 12.0, 7.0 Hz, 1H),

3.69 (dd, J ) 12.0 Hz, 4.1 Hz, 1H), 3.06 (dd, J ) 12.6, 7.1 Hz,

1H), 2.95 (dd, J ) 12.6, 3.9 Hz, 1H), 2.72 (brs, 3H), 1.45 (s, 3H),

1.36 (s, 3H). ¹³C NMR (CDCl₃); δ 108.3, 77.6, 77.5, 60.8, 41.2,

27.8, 25.3.

1

standing. H NMR (CDCl₃): δ 6.52 (br s, 1H), 6.34 (br s, 1H),

4.73 (s, 2H), 3.84 (s, 3H), 1.52 (s, 3H), 1.48 (s, 3H). ¹³C NMR

(CDCl₃): δ 173.0, 170.9, 113.9, 77.8, 77.4, 53.2, 27.0, 26.5. ESI-

HRMS for C₈H₁₃NO₅Na [MNa⁺] calcd, 226.0691; found 226.0696.

5-Benzyloxymethyl-7-[({[(4R/S,5R/S)-5-(hydroxymethyl)-2,2-di-

methyl-1,3-dioxolan-4-yl]methyl}amino)methyl]-4-methoxy-5H-

pyrrolo[3,2-d]pyrimidine (98). Compound (()-96 (830 mg, 4.08

mmol) was dissolved in anhydrous THF (10 mL) and added

dropwise to a suspension of LAH (608 mg, 16.0 mmol) in THF

(60 mL) at room temperature. The resulting suspension was heated

under reﬂux for 4 h and after cooling to 0 °C quenched cautiously

with water (0.7 mL), 15% aqueous NaOH (0.7 mL), and water (2

mL) again. The resulting suspension was ﬁltered through celite and

the ﬁltrate concentrated in vacuo to afford (()-97 (600 mg, 91%)

as a colorless syrup that was used in the next step without

puriﬁcation. To a mixture of compound 44⁸(590 mg, 1.98 mmol)

and compound (()-97 (410 mg, 2.54 mmol) in 1,2-dichloroethane

(10 mL) containing anhydrous MgSO₄(1.0 g) was added, in a single

portion, sodium triacetoxyborohydride (700 mg, 3.31 mmol) and

the resulting mixture stirred for 16 h at room temperature. The

mixture was then diluted with CH₂Cl₂and washed with saturated

aqueous NaHCO₃and then dried, ﬁltered, and the ﬁltrate concen-

trated in vacuo. The crude residue was puriﬁed by chromatography

(MeOH/EtOAc ) 2:98 f 1:19) to afford (()-98 (650 mg, 58%)

as an immobile, colorless syrup. ¹H NMR (CDCl₃): δ 8.45 (s, 1H),

7.28 (s, 1H), 7.27-7.13 (m, 5H), 5.63 (s, 2H), 4.41 (s, 2H), 4.03

(s, 3H), 3.95 (s, 2H), 3.80-3.69 (m, 3H), 3.49 (m, 1H), 3.21 (br s,

2H), 3.07 (dd J ) 12.1, 3.0 Hz, 1H), 2.63 (m, 1H), 1.32 (s, 3H),

1.31 (s, 3H). ¹³C (CDCl₃): δ 156.7, 150.5, 150.3, 137.3, 131.5,

128.8, 128.4, 128.1, 116.3, 115.2, 108.9, 82.2, 80.1, 77.4, 70.6,

62.8, 54.0, 50.6, 43.7, 27.3, 27.1. ESI-HRMS for C₂₃H₃₀N₄O₅Na

[MNa⁺] calcd, 465.2114; found, 465.2144.

5-Benzyloxymethyl-7-[({[(4S,5R)-5-(hydroxymethyl)-2,2-dimeth-

yl-1,3-dioxolan-4-yl]methyl}amino)methyl]-4-methoxy-5H-pyrro-

lo[3,2-d]pyrimidine (94). To a mixture of compound 44⁸(390 mg,

1.30 mmol) and compound 93 (210 mg, 1.30 mmol) in 1,2-

dichloroethane (8 mL) containing anhydrous MgSO₄(500 mg) was

added, in a single portion, sodium triacetoxyborohydride (360 mg,

1.70 mmol) and the resulting reaction stirred overnight at room

temperature. The mixture was diluted with CH₂Cl₂and washed with

saturated aqueous NaHCO₃before being dried, ﬁltered, and

concentrated to dryness. The resulting residue was puriﬁed by

chromatography (MeOH/EtOAc ) 2:98 f 1:19) to afford 94 (230

1

mg, 40%) as a syrup. H (CDCl₃): δ 8.45 (s, 1H), 7.28 (s, 1H),

7.26-7.13 (m, 5H), 5.62 (s, 2H), 4.41 (s, 2H), 4.30-4.21 (m, 2H),

4.03 (s, 3H), 3.95 (s, 2H), 3.88 (br s, 2H), 3.67 (dd, J ) 12.0, 7.4

Hz, 1H), 3.59 (dd, J ) 12.0, 3.7 Hz, 1H), 2.93 (dd, J ) 12.2, 7.6

Hz, 1H), 2.83 (dd, J ) 12.2, 3.1 Hz, 1H), 1.34 (s, 3H), 1.26 (s,

3H). ¹³C NMR (CDCl₃): δ 156.7, 150.4, 150.3, 137.3, 131.5, 128.8,

128.3, 128.0, 116.3, 115.1, 108.3, 77.9, 77.4, 76.2, 70.6, 60.8, 53.9,

48.4, 43.5, 27.8, 25.3. ESI-HRMS for C₂₃H₃₁N₄O₅[MH⁺] calcd,

443.2294; found, 443.2327.

7-({[(2S,3R)-2,3,4-Trihydroxybutyl]amino}methyl)-3,5-dihydro-

4H-pyrrolo[3,2-d]pyrimidin-4-one (30) and Its Hydrochloride

(30 ·HCl). Concentrated HCl (10 mL) was added to compound 94

(480 mg, 1.08 mmol) and the resulting solution heated at reﬂux

for 2 h. After cooling to room temperature, the solvent was

concentrated in vacuo and the residue dissolved in MeOH (10 mL)

and water (2 mL). The solution was neutralized with Amberlyst

A-21 resin and then ﬁltered and concentrated in vacuo onto silica

(1 g). The residue was puriﬁed by chromatography (CH₂Cl₂/MeOH/

28% aq NH₄OH ) 5:4:1) to afford 30 (61 mg, 21%) as a white

solid. ¹H NMR (500 MHz, D₂O/DCl): δ 8.87 (s, 1H), 7.71 (s, 1H),

4.32 (s, 2H), 3.77 (m, 1H), 3.54 (dd, J ) 11.6, 3.4 Hz, 1H), 3.49

(m, 1H), 3.42 (dd, J ) 11.6, 5.9 Hz, 1H), 3.24 (dd, J ) 13.0, 3.1

Hz, 1H), 3.01 (dd, J ) 13.0, 9.6 Hz, 1H). ¹³C NMR (D₂O/DCl): δ

152.5, 144.9, 132.9, 131.8, 118.3, 102.8, 73.0, 67.1, 62.1, 49.0,

40.6. ESI-HRMS for C₁₁H₁₇N₄O₄[MH⁺] calcd, 269.1250; found,

269.1242. Anal. (C₁₁H₁₆N₄O₄) C, H, N.

7-({[(2R/S,3R/S)-2,3,4-Trihydroxybutyl]amino}methyl)-3,5-dihy-

dro-4H-pyrrolo[3,2-d]pyrimidin-4-one (31) and Its Triﬂuoroac-

etate Salt (31·TFA). A solution of compound (()-98 (480 mg, 1.09

mmol) in concentrated HCl (10 mL) was heated at reﬂux for 3 h.

After cooling to room temperature, the solution was evaporated

and the crude residue dissolved in MeOH (10 mL) containing water

(3 mL) and neutralized with Amberlyst A-21 resin. The resin was

removed by ﬁltration and the ﬁltrate concentrated in vacuo to afford

(()-31 (110 mg, 38%) as an immobile syrup, a portion of which

was puriﬁed by preparative HPLC to afford (()-31·TFA. ¹H NMR

(CD₃OD): δ 8.08 (s, 1H), 7.55 (s, 1H), 4.32 (s, 2H), 3.93 (septet,

J ) 2.8 Hz, 1H), 3.59-3.44 (m, 1H), 3.53 (s, 2H), 3.14 (d, J )

5.9 Hz, 2H). ¹³C NMR (CD₃OD): δ 161.5 (q, J ) 38 Hz), 161.2,

160.7, 155.6, 144.8, 143.3, 131.6, 120.1, 117.6 (q, J ) 283 Hz),

107.7, 74.3, 71.1, 63.9, 51.0, 42.3. ESI-HRMS for C₁₁H₁₇N₄O₄

[MH⁺] calcd, 269.1250; found, 269.1241.

(1R)-1-[(4R,5S)-5-(Aminomethyl)-2,2-dimethyl-1,3-dioxolan-4-

yl]ethane-1,2-diol (100). To a stirred suspension of LAH (7.1 g,

0.187 mol) in anhydrous THF (80 mL) was added, dropwise at

room temperature, a solution of 2,3-O-isopropylidene D-ribonamide

(99)⁴³(3.3 g, 16.1 mmol) in THF (90 mL). After the addition was

complete, the mixture was heated under reﬂux and maintained as

such for 20 h. After cooling to 0 °C, the reaction was quenched

cautiously with water (7 mL), 15% aqueous NaOH (7 mL), and

water (21 mL) again. The resulting suspension was ﬁltered through

celite and the inorganics washed successively with small volumes

of warm ethyl acetate. The combined ﬁltrates were concentrated

Ethyl Methyl (2R/S,3R/S)-2,2-dimethyl-1,3-dioxolane-4,5-dicar-

boxylate (95). To a solution containing (()-diethyl tartrate (2.0 g,

9.7 mmol) and dimethoxypropane (2.0 g, 19.4 mmol) in benzene

(30 mL) was added TsOH (0.1 g) and the mixture reﬂuxed for 3 h.

After cooling to room temperature, the solution was diluted with

EtOAc (200 mL) and washed with a saturated brine/sodium

bicarbonate solution and then dried, ﬁltered, and the ﬁltrate

concentrated in vacuo to afford (()-95 (1.78 g, 79%) as a mobile

liquid, a sample of which was puriﬁed by Kugelrohr distillation

(120-140 °C/18 mmHg). ¹H NMR (CDCl₃): δ 4.79-4.74 (m, 2H),

4.26 (q, J ) 7.1 Hz, 2H), 3.81 (s, 3H) 1.48 (s, 6H), 1.32 (t, J ) 7.1

Hz, 3H). ¹³C NMR (CDCl₃): δ 170.4, 170.0, 114.1, 77.6, 77.4, 62.3,

53.1, 26.7, 14.4.

1

in vacuo to afford 100 (1.45 g, 47%) as an immobile syrup. H

NMR (CDCl₃): δ 4.17 (m, 2H), 3.83 (m, 3H), 3.77 (m, 1H), 3.24

(br d, 3H), 3.02 (m, 2H), 1.40 (s, 3H), 1.33 (s, 3H). ¹³C NMR

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1141

(CDCl₃): δ 108.8, 78.2, 78.0, 69.6, 65.0, 41.7, 28.3, 25.7. ESI-

HRMS for C₈H₁₈NO₄[MH⁺] calcd, 192.1236; found, 192.1237.

7-({[(2S,3S,4R)-2,3,4,5-Tetrahydroxypentyl]amino}methyl)-3,5-

dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (35) and Its Hydro-

chloride Salt (35 ·HCl). To a mixture containing 100 (210 mg,

1.08 mmol), 44⁸(320 mg, 1.08 mmol), and anhydrous MgSO₄

(600 mg) in 1,2-dichloroethane (8 mL) was added, in a single

portion, sodium triacetoxyborohydride (310 mg, 1.40 mmol), and

the resulting reaction mixture was then stirred for 16 h at room

temperature. After dilution with CH₂Cl₂(50 mL) and a small

volume of water, the solution was washed with saturated aqueous

NaHCO₃solution and then dried, ﬁltered, and the ﬁltrate

concentrated in vacuo. The residue was puriﬁed by chromatog-

raphy (MeOH/EtOAc ) 7:93) to afford crude 101 (160 mg, 32%)

as an immobile syrup. A solution of 101 (160 mg, 0.30 mmol)

in concentrated HCl was heated under reﬂux for 2 h. After

cooling to room temperature, the solvent was evaporated and

the residue dissolved in MeOH (2 mL) and H₂O (10 mL) and

then neutralized with Amberlyst A-21 resin. The resin was

removed by ﬁltration and silica added to the ﬁltrate and the

resulting mixture concentrated in vacuo. The resulting residue

was puriﬁed by chromatography (CH₂Cl₂/MeOH/28% aq NH₄OH

inseparable mixture. Further chromatography (CHCl₃/EtOAc/

MeOH ) 5:2:1 f 4:2:3 f 7 M NH₃in MeOH/CH₂Cl₂) 1:4)

of this material afforded (()-105 (406 mg, 65%) as a white solid.

¹H NMR (CDCl₃/CD₃OD): δ 7.84 (s, 1H), 7.48-7.45 (m, 2H),

7.39 (s, 1H), 7.37-7.28 (m, 3H), 5.56 (s, 1H), 4.30-4.11 (m,

2H), 4.02 (dt, J ) 11.9, 2.5 Hz, 1H), 3.91 (d, J ) 13.8 Hz, 1H),

3.83 (d, J ) 13.8 Hz, 1H), 2.73 (dd, J ) 13.5, 7.5 Hz, 1H),

2.57 (dd, J ) 13.5, 3.4 Hz, 1H), 2.39 (s, 3H), 1.74 (dd, J )

12.5, 4.9 Hz, 1H), 1.53 (d, J ) 12.4 Hz, 1H). ¹³C NMR (CDCl₃/

CD₃OD): δ 156.8, 146.2, 143.2, 140.6, 130.6, 130.5, 129.8,

128.0, 119.7, 113.9, 103.0, 77.4, 68.7, 62.5, 52.0, 44.3, 31.5.

7-({[(2R/S)-2,4-Dihydroxybutyl](methyl)amino}methyl)-3,5-di-

hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (36). A solution of (()-

105 (70 mg, 0.20 mmol) in MeOH (2 mL) and concentrated

aqueous HCl (2 mL) was allowed to stand for 2 h at room

temperature. The solution was diluted with water, extracted with

CHCl₃(×2), and the aqueous phase concentrated in vacuo. The

resulting residue was puriﬁed by chromatography (CH₂Cl₂/

MeOH/28% aq NH₄OH ) 5:4:1) to afford (()-36 (40 mg, 76%)

1

as a white solid. H NMR (D₂O): δ 8.00 (s, 1H), 7.71 (s, 1H),

4.51 (d, J ) 14.0 Hz, 1H), 4.44 (d, J ) 14.0 Hz, 1H), 4.19 (br

s, 1H), 3.68 (t, J ) 6.4 Hz, 2H), 3.17 (br s, 2H), 2.86 (s, 3H),

1.71-1.61 (m, 2H). ¹³C NMR (D₂O): δ 155.3, 144.6, 143.4,

132.4, 118.2, 104.2, 63.1, 60.0, 58.0, 50.0, 40.2, 36.8. ESI-HRMS

for C₁₂H₁₉N₄O₃[MH⁺] calcd, 267.1457; found, 267.1449.

Inhibition Assays. Human PNP was expressed as an N-

terminal histidine-tagged fusion protein, as previously de-

scribed.³²PNP activity assays in the presence of inhibitors were

monitored by a xanthine-oxidase coupled assay, as previously

described.⁵Inosine (K_m) 40 µM), inhibitor, and protein

concentrations were determined spectrophotometrically using an

1

) 5:4:1) to afford 35 (12 mg, 12%) as a colorless powder. H

1

NMR (500 MHz, D₂O/DCl): H NMR (500 MHz, D₂O/DCl): δ

8.90 (s, 1H), 7.81 (s, 1H), 4.44 (s, 2H), 4.08 (m, 1H), 3.72-3.59

(m, 3H), 3.55 (dd, J ) 11.5, 6.1 Hz, 1H), 3.32 (dd, J ) 12.7,

1.8 Hz, 1H), 3.18 (dd, J ) 12.7, 9.6 Hz, 1H). ¹³C NMR (125.7

MHz, D₂O/DCl): δ 155.4, 147.3, 135.6, 135.3, 120.9, 105.7,

75.4, 74.4, 69.8, 65.1, 50.7, 43.1. ESI-HRMS for C₁₂H₁₉N₄O₅

[MH⁺] calcd, 299.1355; found, 299.1347.

(2R/S,4R/S)-(2-Phenyl-1,3-dioxan-4-yl)methanol (103). A mixture

of (()-1,2,4-butanetriol (102) (3.0 g, 28.3 mmol) and benzal-

dehyde (11.48 mL, 113 mmol) in dry toluene (50 mL) with TsOH

(0.269 g, 1.413 mmol) was heated under reﬂux in a Dean-Stark

apparatus. After 1 h, the solution was washed with saturated

aqueous NaHCO₃, and then dried, ﬁltered, and the ﬁltrate

concentrated in vacuo. The resulting residue was puriﬁed by

chromatography to afford (()-103 (2.88 g, 53%) as a syrup.

The ¹H and ¹³C NMR spectra were the same as those previously

reported.⁴⁶

ε₂₄₈of 12.3 mM^-1cm^-1(pH 6),⁴⁷an ε₂₆₁of 9.54 mM^-1cm^-1

49

(pH 7),⁴⁸and an ε₂₈₀of 31.65 mM^-1cm^-1

,

respectively. In

most cases, the inhibitor concentration was at least 10-fold

greater than the enzyme concentration, as required for simple

analysis of slow-onset tight-binding inhibition. In the few cases

of particularly tight binding where the inhibitor-enzyme ratio

was less than 10, the concentration of free inhibitor, [I], was

corrected by eq 1:

[I] ) [I]_total- (1 - V_i⁄ V_o) × [E]_total

(1)

(2R/S,4R/S)-N-Methyl-1-(2-phenyl-1,3-dioxan-4-yl)metha-

namine (104). To a solution of (()-103 (0.80 g, 4.12 mmol) in

CH₂Cl₂(20 mL) was added N,N-diisopropylethylamine (1.702

mL, 10.30 mmol), and the solution was cooled in an ice bath.

Methanesulfonyl chloride (0.414 mL, 5.35 mmol) was added,

and the solution was allowed to warm to room temperature. After

1 h, the solution was washed with 2 M aqueous HCl, saturated

aqueous NaHCO₃, dried, ﬁltered, and the ﬁltrate concentrated

in vacuo to a syrup (1.15 g). A solution of this syrup (0.9 g,

3.30 mmol) in DMSO (8 mL) containing 40% aqueous methy-

lamine (2.85 mL, 33.0 mmol) was stoppered and heated at

∼75-80 °C for 24 h. After cooling, the solution to room

temperature CHCl₃was added and the mixture washed twice

with water, dried, ﬁltered, and the ﬁltrate concentrated in vacuo.

The resulting residue was puriﬁed by chromatography to afford

(()-104 (0.465 g, 68%) as a syrup. ¹H NMR (CDCl₃): δ

7.50-7.47 (m, 2H), 7.39-7.30 (m, 3H), 5.52 (s, 1H), 4.78 (m,

1H), 4.09-3.93 (m, 2H), 2.80 (dd, J ) 12.3, 8.0 Hz, 1H), 2.67

(dd, J ) 12.3, 3.4 Hz, 1H), 2.45 (s, 3H), 1.96-1.83 (m, 2H),

1.49 (dd, J ) 13.2, 1.2 Hz, 1H). ¹³C NMR (CDCl₃): δ 139.0,

129.2, 128.6, 126.5, 101.6, 76.8, 67.2, 57.0, 36.8, 29.5.

(2R/S,4R/S)-7-({Methyl[(2-phenyl-1,3-dioxan-4-yl)methyl]am-

ino}methyl)-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (105).

A mixture of (()-104 (368 mg, 1.78 mmol), 9-deazahypoxan-

thine (75)¹⁰(288 mg, 2.13 mmol), acetic acid (0.508 mL, 8.88

mmol), and 37% aqueous formaldehyde (0.396 mL, 5.33 mmol)

in 1,4-dioxane (10 mL) was stirred and heated in a stoppered

ﬂask at 80 °C for 24 h. After cooling to room temperature, the

solution was concentrated in vacuo and the resulting residue

puriﬁed by chromatography (7 M NH₃in MeOH/CH₂Cl₂) 1:9)

to afford the product and 9-deazahypoxanthine (75)¹⁰as an

where [I]_totaland [E]_totalare the total concentrations of inhibitor and

enzyme, respectively, and V_iand V_oare the reaction rates in the

presence and absence of inhibitor, respectively. Data were ﬁtted to

eq 2 for competitive inhibition

V_i) V_o[S] ⁄ ([S] + K_m(1 + [I] ⁄ K_i))

(2)

where [S] is the concentration of substrate (usually ﬁxed at 1 mM).

Bioavailability of SerMe-ImmH.¹⁰Male Balb/c mice (∼25

g) were purchased from the National Cancer Institute (NCI).

The mice were fasted overnight before drug administration. A

single dose of 50 nmol of SerMe-ImmH (10) was administrated

to the mice orally or by intraperitoneal (ip) injection (three mice

in each treatment). Blood samples (5 µL) were taken at different

times, mixed with the same volume of 1% heparin and 0.3%

Triton X-100 in PBS, and stored at 4 °C before assays. The

erythrocyte PNP catalytic activity was monitored spectropho-

tometrically by adding 1 µL of the blood sample to the reaction

mixture containing 50 mM potassium phosphate (pH 7.4), 1 mM

inosine, and 60 mU of xanthine oxidase. The PNP catalytic

activity of each sample was normalized to protein concentration.

The normalized rate at each time point was divided by the

normalized rate at time zero and then plotted against time. The

time (t_1/2) required for 50% of the PNP catalytic activity to be

lost and the time (t_1/2) required for a 50% regain of the PNP

catalytic activity were interpolated from the plot.

Acknowledgment. This work was supported by research

grant GM41916 from the NIH and by a contract from the

New Zealand Foundation for Research, Science & Technol-

1142 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4

Clinch et al.

Inhibitors Bound to Human Purine Nucleoside Phosphorylase. J. Am.

Chem. Soc. 2008, 130, 842–844.

(20) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.;

Tyler, P. C. Synthesis of second-generation transition state analogues

of human purine nucleoside phosphorylase. J. Med. Chem. 2003, 46,

5271–5276.

(21) Evans, G. B.; Furneaux, R. H.; Greatrex, B.; Murkin, A. S.; Schramm,

V. L.; Tyler, P. C. Azetidine Based Transition State Analogue

Inhibitors of N-Ribosyl Hydrolases and Phosphorylases. J. Med. Chem.

2008, 51, 948–956.

ogy. We are grateful to Jennifer Mason for analytical HPLC

analysis and Dr. Herbert Wong for NMR analysis.

Supporting Information Available: Analytical data, HPLC

traces, and NMR spectra of compounds 8-38. This material is

available free of charge via the Internet at http://pubs.acs.org.

References

(22) Chun, B. K.; Song, G. Y.; Chu, C. K. Stereocontrolled syntheses of

carbocyclic C-nucleosides and related compounds. J. Org. Chem. 2001,

66, 4852–4858.

(1) Taylor, E. A.; Clinch, K.; Kelly, P. M.; Li, L.; Evans, G. B.; Tyler,

P. C.; Schramm, V. L. Acyclic ribooxacarbenium ion mimics as

transition state analogues of human and malarial purine nucleoside

phosphorylases. J. Am. Chem. Soc. 2007, 129, 6984–6985.

(2) Rinaldo-Matthis, A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Gupta,

A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Almo, S. C.; Wang,

C. C.; Schramm, V. L. Inhibition and structure of Trichomonas

Vaginalis purine nucleoside phosphorylase with picomolar transition

state analogues. Biochemistry 2007, 46, 659–668.

(3) Murkin, A. S.; Birck, M. R.; Rinaldo-Matthis, A.; Shi, W.; Taylor,

E. A.; Almo, S. C.; Schramm, V. L. Neighboring group participation

in the transition state of human purine nucleoside phosphorylase.

Biochemistry 2007, 46, 5038–5049.

(4) Taylor Ringia, E. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.;

Murkin, A. S.; Schramm, V. L. Transition state analogue discrimination

by related purine nucleoside phosphorylases. J. Am. Chem. Soc. 2006,

128, 7126–7127.

(5) Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.;

Schramm, V. L. One-third-the-sites transition-state inhibitors for purine

nucleoside phosphorylase. Biochemistry. 1998, 37, 8615–8621.

(6) Kicska, G. A.; Long, L.; Horig, H.; Fairchild, C.; Tyler, P. C.;

Furneaux, R. H.; Schramm, V. L.; Kaufman, H. L. Immucillin H, a

powerful transition-state analog inhibitor of purine nucleoside phos-

phorylase, selectively inhibits human T lymphocytes. Proc. Natl. Acad.

Sci. U.S.A. 2001, 98, 4593–4598.

(7) Evans, G. B.; Furneaux, R. H.; Gainsford, G. J.; Schramm, V. L.;

Tyler, P. C. Synthesis of Transition State Analogue Inhibitors for

Purine Nucleoside Phosphorylase and N-Riboside Hydrolases. Tetra-

hedron 2000, 56, 3053–3062.

(8) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.;

Tyler, P. C. Exploring structure-activity relationships of transition

state analogues of human purine nucleoside phosphorylase. J. Med.

Chem. 2003, 46, 3412–3423.

(9) Lewandowicz, A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.;

Schramm, V. L. Achieving the ultimate physiological goal in transition

state analogue inhibitors for purine nucleoside phosphorylase. J. Biol.

Chem. 2003, 278, 31465–31468.

(10) Evans, G. B.; Furneaux, R. H.; Schramm, V. L.; Tyler, P. C. Synthesis

of a transition state analogue inhibitor of purine nucleoside phospho-

rylase via the Mannich reaction. Org. Lett. 2003, 5, 3639–3640.

(11) Galmarini, C. M. Drug evaluation: forodesinesPNP inhibitor for the

treatment of leukemia, lymphoma and solid tumor. IDrugs 2006, 9,

712–722.

(23) Semeraro, T.; Lossani, A.; Botta, M.; Ghiron, C.; Alvarez, R.; Manetti,

F.; Mugnaini, C.; Valensin, S.; Focher, F.; Corelli, F. Simpliﬁed

analogues of immucillin-G retain potent human purine nucleoside

phosphorylase inhibitory activity. J. Med. Chem. 2006, 49, 6037–6045.

(24) Elion, G. B.; Furman, P. A.; Fyfe, J. A.; de Miranda, P.; Beauchamp,

L.; Schaeffer, J. Selectivity of action of an antiherpetic agent, 9-(2-

hydroxyethoxymethyl)guanine. Proc. Natl. Acad. Sci. U.S.A. 1977,

74, 5716–5720.

(25) Elion, G. B. Acyclovir: Discovery, Mechanism of Action and

Selectivity. J. Virol. Med. Supp. 1993, 1, 2–6.

(26) De Clercq, E. The acyclic nucleoside phosphonates from inception to

clinical use: historical perspective. AntiViral Res. 2007, 75, 1–13.

(27) Khandazhinskaya, A.; Yasko, M.; Shirokova, E. The synthesis and

antiviral properties of acyclic nucleoside analogues with a phospho-

nomethoxy fragment in the side chain. Curr. Med. Chem. 2006, 13,

2953–2980.

(28) Horton, D.; Thomas, S.; Gallucci, J. A synthetic route to 9-(polyhy-

droxyalkyl)purines. Carbohydr. Res. 2006, 341, 2211–2218.

(29) Lewandowicz, A.; Ringia, E. A.; Ting, L. M.; Kim, K.; Tyler, P. C.;

Evans, G. B.; Zubkova, O. V.; Mee, S.; Painter, G. F.; Lenz, D. H.;

Furneaux, R. H.; Schramm, V. L. Energetic mapping of transition state

analogue interactions with human and Plasmodium falciparum purine

nucleoside phosphorylases. J. Biol. Chem. 2005, 280, 30320–30328.

(30) Schramm, V. L.; Tyler, P. C. Imino-sugar-based nucleosides. Curr.

Top. Med. Chem. 2003, 3, 525–540.

(31) Schramm, V. L. Enzymatic transition state theory and transition state

analogue design. J. Biol. Chem. 2007, 282, 28297–28300.

(32) Lewandowicz, A.; Schramm, V. L. Transition state analysis for human

and Plasmodium falciparum purine nucleoside phosphorylases. Bio-

chemistry 2004, 43, 1458–1468.

(33) Schramm, V. L. Enzymatic transition states: thermodynamics, dynam-

ics and analogue design. Arch. Biochem. Biophys. 2005, 433, 13–26.

(34) Banphavichit, V.; Bhanthumnavin, W.; Vilaivan, T. Synthesis of new

tridentate chiral aminoalcohols by a multicomponent reaction and their

evaluation as ligands for catalytic asymmetric Strecker reaction.

Tetrahedron 2007, 63, 8727–8734.

(35) Chehade, K. A. H.; Baruch, A.; Verhelst, S. H. L.; Bogyo, M. An

improved preparation of the activity-based probe JPM-OET and in

situ applications. Synthesis 2005, 240–244.

(36) Tang, Z.; Yang, Z.-H.; Chen, X.-H.; Cun, L.-F.; Mi, A.-Q.; Jiang,

Y.-Z.; Gong, L.-Z. A highly efﬁcient organocatalyst for direct aldol

reactions of ketones with aldehydes. J. Am. Chem. Soc. 2005, 127,

9285–9289.

(37) Breuning, A.; Vicik, R.; Schirmeister, T. An improved synthesis of

aziridine-2,3-dicarboxylates via azido alcohols-epimerisation studies.

Tetrahedron: Asymmetry 2003, 14, 3301–3312.

(38) Soai, K.; Ookawa, A. Mixed Solvents Containing Methanol as Useful

Reaction Media for Unique Chemoselective Reductions with Lithium

Borohydride. J. Org. Chem. 1986, 51, 4000–4005.

(39) Inaba, T.; Birchler, A. G.; Yamada, Y.; Sagawa, S.; Yokata, K.; Ando,

K.; Uchida, I. A Practical Synthesis of Nelﬁnavir, an HIV-Protease

Inhibitor, Using a Novel Chiral C4 Building Block: (5R,6S)-2,2-

Dimethyl-5-hydroxy-1,3-dioxepan-6-ylammonium Acetate. J. Org.

Chem. 1998, 63, 7582–7583.

(12) Duvic, M. Systemic monotherapy vs combination therapy for CTCL:

rationale and future strategies. Oncology 2007, 21, 33–40.

(13) Robak, T.; Lech-Maranda, E.; Koerycka, A.; Robak, E. Purine

nucleoside analogs as immunosuppressive and antineoplastic agents:

mechanism of action and clinical activity. Curr. Med. Chem. 2006,

13, 3165–3189.

(14) Ravandi, F.; Gandhi, V. Novel purine nucleoside analogues for T-cell-

lineage acute lymphoblastic leukaemia and lymphoma. Expert Opin.

InVestig. Drugs 2006, 15, 1601–1613.

(15) Balakrishnan, K.; Nimmanpalli, R.; Ravandi, F.; Keating, M. J.;

Gandhi, V. Forodesine, an inhibitor of purine nucleoside phosphory-

lase, induces apoptosis in chronic lymphocytic leukemia cells. Blood

2006, 108, 2392–2398.

(16) Taylor Ringia, E. A.; Schramm, V. L. Transition states and inhibitors

of the purine nucleoside phosphorylase family. Curr. Top. Med. Chem.

2005, 5, 1237–1258.

(17) Giblett, E. R.; Ammann, A. J.; Wara, D. W.; Sandman, R.; Diamond,

L. K. Nucleoside-phosphorylase deﬁciency in a child with severely

defective T-cell immunity and normal B-cell immunity. Lancet 1975,

1, 1010–1013.

(40) Harnden, M. R.; Parkin, A.; Parratt, M. J.; Perkins, R. M. Novel

Acyclonucleotides: Synthesis and Antiviral Activity of Alkenylphos-

phonic Acid Derivatives of Purines and a Pyrimidine. J. Med. Chem.

1993, 36, 1343–1355.

(41) Lemaire, M.; Posada, F.; Gourcy, J.-G.; Jeminet, G. Formal Synthesis

of a 2- and 8-Functionalized 1,4,7-Trioxa-10-azaspiro[5.5]undecane.

Synthesis 1995, 627–629.

(18) Clinch, K.; Evans, G. B.; Fleet, G. W.; Furneaux, R. H.; Johnson,

S. W.; Lenz, D. H.; Mee, S. P.; Rands, P. R.; Schramm, V. L.; Taylor

Ringia, E. A.; Tyler, P. C. Syntheses and bio-activities of the

L-enantiomers of two potent transition state analogue inhibitors of

purine nucleoside phosphorylases. Org. Biomol. Chem. 2006, 4, 1131–

1139.

(42) Kim, H. S.; Barak, D.; Harden, T. K.; Boyer, J. L.; Jacobson, K. A.

Acyclic and Cyclopropyl Analogues of Adenosine Bisphosphate

Antagonists of the P2Y₁Receptor: Structure-Activity Relationships

and Receptor Docking. J. Med. Chem. 2001, 44, 3092–3108.

(43) Mitchell, D. L. Degradation of ^D-ribono-1f4-lactone to D-erythro-

1f4-lactone. Can. J. Chem. 1963, 41, 214–221.

(19) Rinaldo-Matthis, A.; Murkin, A. S.; Ramagopal, U. A.; Clinch, K.;

Mee, S. P. H.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Almo,

S. C.; Schramm, V. L. ^L-Enantiomers of Transition State Analogue

(44) Xu, B; Stephens, A.; Kirschenheuter, G; Greslin, A. F.; Cheng, X.;

Sennelo, J.; Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S.-A.; Kim,

H. S.; Bischofberger, N.; Cook, G.; Jacobson, K. A. Acyclic Analogues

Third-Generation Immucillins

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1143

of Adenosine Bisphosphates as P2Y Receptor Antagonists: Phosphate

Substitution Leads to Multiple Pathways of Inhibition of Platelet

Aggregation. J. Med. Chem. 2002, 45, 5694–5709.

(49) The protein extinction coefﬁcient was calculated from the primary

sequence using the Protparam tool (Gasteiger, E. Hoogland, C.

Gattiker, A. Duvaud, S. Wilkins, M. R. Appel, R. D. Bairoch, A.

Protein Identiﬁcation and Analysis Tools on the ExPASy Server. In

The Proteomics Protocols Handbook; Humana Press: Totowa, NJ,

2005; 571-607. ). The value differs from that previously reported

(28.83 mM^-1cm^-1) due to a recent modiﬁcation in the calculation

(45) Janqueta, F.; Mercha´n, F. L.; Merino, P.; Tejero, T. A Straightforward

Synthesis of L-isoserinal. Tetrahedron 1996, 52, 7045–7052.

(46) Sa´nchez-Sancho, F.; Valverde, S.; Herrado´n, B. Stereoselective

syntheses and reactions of chiral oxygenated R,ꢀ-unsaturated-γ- and

δ-lactones. Tetrahedron: Asymmetry 1996, 7, 3209–3246.

(47) Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for

Biochemical Research, 3rd ed.; Clarendon Press: Oxford, UK, 1986.

(48) Lim, M.-I.; Ren, Y.-Y.; Otter, B. A.; Klein, R. S. Synthesis of “9-

deazaguanosine” and other new pyrrolo[3,2-d]pyrimidine C-nucleo-

sides. J. Org. Chem. 1983, 48, 780–788.

JM801421Q

Article Doi

Third-generation immucillins: Syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase

DOI: 10.1021/jm801421q

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm801421q

Full text of DOI:10.1021/jm801421q

Products guided by the article

R&D Labs maybe for 1010701-19-5

Relevant to this article

Hot Product