Structure-activity relationship and in vitro and in vivo evaluation of the potent cytotoxic anti-microtubule agent N-(4-methoxyphenyl)-N,2,6-trimethyl-6,7- dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride and its analogues as antitumor agents

Article

pubs.acs.org/jmc

Structure−Activity Relationship and in Vitro and in Vivo Evaluation

of the Potent Cytotoxic Anti-microtubule Agent N‑(4-

Methoxyphenyl)‑N,2,6-trimethyl-6,7-

dihydro‑5H‑cyclopenta[d]pyrimidin-4-aminium Chloride and Its

Analogues As Antitumor Agents

Aleem Gangjee,*^,†,∥Ying Zhao,^†Sudhir Raghavan,^†Cristina C. Rohena,^‡Susan L. Mooberry,*^,§,∥

and Ernest Hamel^⊥

^†Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh,

Pennsylvania 15282, United States

^‡University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, United States

^§Experimental and Developmental Therapeutics Program, Cancer Therapy & Research Center, University of Texas Health Science

Center at San Antonio, 7979 Wurzbach Road, San Antonio, Texas 78229, United States

^⊥Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer

Research, National Institutes of Health, 1050 Boyles Street, Frederick, Maryland 21702, United States

S

* Supporting Information

ABSTRACT: A series of 21 substituted cyclopenta[d]pyrimidines were synthesized as an extension of our

discovery of the parent compound ( )-1·HCl as an anti-microtubule agent. The structure−activity

relationship indicates that the N-methyl and a 4N-methoxy groups appear important for potent activity. In

addition, the 6-substituent in the parent analogue is not necessary for activity. The most potent compound 30·

HCl was a one to two digit nanomolar inhibitor of most tumor cell proliferations and was up to 7-fold more

potent than the parent compound ( )-1·HCl. In addition, 30·HCl inhibited cancer cell proliferation

regardless of Pgp or βIII-tubulin status, both of which are known to cause clinical resistance to several anti-

tubulin agents. In vivo eﬃcacy of 30·HCl was demonstrated against a triple negative breast cancer xenograft mouse model.

Compound 30·HCl is water-soluble and easily synthesized and serves as a lead compound for further preclinical evaluation as an

antitumor agent.

due to its high toxicity. However, it is approved for use in the

treatment of gout and familial Mediterranean fever. While there

are no clinically approved anticancer agents that bind to the

colchicine site on tubulin, a number of combretastatins or

closely related analogues are currently being tested in clinical

trials.⁸In preclinical studies combretastatin A-4 (CA-4) was

shown to have properties superior to other colchicine binding

agents and an impressive vascular disrupting activity leading to

its clinical development.⁹⁻¹¹

While the taxanes and the vinca alkaloids have had

unprecedented success in cancer chemotherapy, innate and

acquired resistance is a major limitation to their clinical use.

Overexpression of the P-glycoprotein (Pgp) drug transporter

has been reported following treatment with microtubule

targeting agents, and Pgp overexpression correlates with poor

prognosis.¹²⁻¹⁶The clinical failure of Pgp inhibitors highlights

the need for the development of agents that are not substrates

for Pgp. The taxanes, paclitaxel and docetaxel, and the vinca

alkaloids are well-known substrates for Pgp, and a recent report

INTRODUCTION

■

Microtubule targeting agents have proven to be some of the

most valuable drugs used in the treatment of both solid tumors

and hematological malignancies.¹⁻⁴Microtubule targeting

agents are classiﬁed into two groups: microtubule stabilizers,

which promote tubulin polymerization, typiﬁed by the taxanes

and the epothilones, and inhibitors of microtubule assembly,

which include the vinca alkaloids and colchicine site agents. At

low antiproliferative concentrations, both types of agents

suppress microtubule dynamics without great eﬀects on net

microtubule mass, leading to mitotic arrest and subsequent cell

death.^2,4

Inhibitors of tubulin assembly are known to bind to a variety

of sites on tubulin, two of which have been extensively studied.

The ﬁrst is the vinca site to which vincristine, vinblastine, and

vinorelbine bind. These vinca alkaloids are important in the

treatment of hematological malignancies, non-small-cell lung

cancer, and testicular cancer.^1,5,6The second site can be

occupied by colchicine and a diverse collection of small

molecules known as colchicine site agents. This binding site is

found primarily on the β-tubulin subunit, at its interface with α-

tubulin.⁷Colchicine itself is not used in the treatment of cancer

Received: April 30, 2013

A

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Figure 1. Structure of ( )-1·HCl and A.

noted that the newer microtubule stabilizer, ixabepilone, is also

a substrate for Pgp.¹⁷A second mechanism of drug resistance

involves the overexpression of speciﬁc subtypes of β-tubulin,

particularly the overexpression of the βIII isotype, which has

been shown to be associated with resistance to paclitaxel,

docetaxel, and vinorelbine.¹⁸⁻²¹Colchicine site agents are

hypothesized to have advantages over currently available

microtubule targeting drugs because of their ability to

circumvent βIII-mediated resistance. Thus, the clinical develop-

ment of new colchicine site agents may provide new

opportunities for patients whose tumors express this tubulin

isotype.²²Another major limitation of some microtubule

targeting drugs is their poor aqueous solubility.²This property

of the taxanes and ixabepilone require formulation in

Cremophor, which is linked with hypersensitivity reactions.⁶

Thus, the discovery and development of potent, water-soluble

colchicine site agents may provide signiﬁcant advances in

cancer chemotherapy.

( )-1·HCl is replaced with a 2-NH₂moiety to determine the

importance of potential hydrogen bonding ability at this

position. The N4−H isomer (9) was also synthesized to

conﬁrm that the N4-alkyl (methyl) group is necessary for

activity as in ( )-1·HCl. Compound 30·HCl (Table 1) lacks

the 6-methyl moiety of ( )-1·HCl and was synthesized to

determine the importance of the 6-CH₃group for biological

activity.

The ﬁrst three compounds synthesized were 3·HCl to 5·HCl

(Scheme 1), which replace the 4-OCH₃with a 4-SCH₃moiety

(3·HCl) or introduce an electron-withdrawing group at the

para- (4·HCl) or meta-position (5·HCl) in the aniline ring. The

free bases of 3−5 were synthesized from 2¹⁴using the

appropriate N-methyl anilines in i-PrOH in the presence of 2−

3 drops of conc. HCl. Compounds 3·HCl to 5·HCl precipitated

out as white solids when anhydrous HCl gas was bubbled

through a solution of the corresponding free base in anhydrous

ether.

We^23,24recently reported the discovery of a potent anti-

tubulin compound (R,S)-N-(4-methoxyphenyl)-N,2,6-trimeth-

yl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-4-aminium chlor-

ide, ( )-1·HCl (Figure 1), and identiﬁed the contribution of

the two enantiomers [(S)-1·HCl and (R)-1·HCl] to the anti-

tubulin and cytotoxic properties of the racemate (Figure 1).

Compound ( )-1·HCl is a water-soluble colchicine site agent

that inhibits microtubule assembly and the growth of cancer

cells with a GI₅₀in the nanomolar range. In addition,

compound ( )-1·HCl overcomes drug resistance associated

with overexpression of Pgp and βIII-tubulin. Herein we report

the synthesis of a variety of analogues of ( )-1·HCl with

variations at R₁, R₂, R₃, and R₄in structure A (Figure 1) to

determine the structure−activity relationship (SAR) of the

cyclopenta[d]pyrimidine scaﬀold for anti-microtubule and

antitumor activity. These small molecule derivatives are readily

synthesized, are potent, and show better eﬃcacy against both

sensitive and multidrug-resistant cancer cell lines.

The next structural modiﬁcation involved the replacement of

the 2-methyl group with a 2-amino group. We have reported

molecular modeling of our lead compounds ( )-1·HCl and the

(R)- and (S)-enantiomers.^23,24Using similar methodology, we

determined that the 2-NH₂could function as both a potential

hydrogen donor and a potential hydrogen acceptor and could

aﬀord additional H-bonds with the amino acids in the

colchicine binding pocket of tubulin, such as Lys254 and

Asn258.²⁴Thus, it was of interest to synthesize 9 and 10.

Compounds 9 and 10 (Scheme 2) were obtained starting from

the commercially available 3-methyladipic acid 6 following a

modiﬁed synthetic route reported for 1.²⁴Reaction of 6 with

conc. H₂SO₄at reﬂux in ethanol and a Dieckmann

condensation in the presence of elemental sodium in toluene

followed by treatment with guanidine carbonate aﬀorded 7

(34%). Chlorination of 7 to 8 (34%) was accomplished by

heating with POCl₃for 3 h. Nucleophilic substitution of 8 with

appropriate anilines in i-PrOH gave 9 (55%) and 10 (26%),

respectively, as white solids. Compound 10·HCl was obtained

(65%) as a white precipitate when anhydrous HCl gas was

bubbled through a solution of 10 in anhydrous ether.

A logical extension of the 6-CH₃group of 1 was

homologation of the methyl group to determine spatial

requirements for biological activity at the 6-position. Target

compounds 19 and 20 were synthesized as shown in Scheme 3.

Ring-opening of 4-vinyl-1-cyclohexene 1,2-epoxide 11 in dilute

HCl aﬀorded the diol 12 (85%).^25,26Compound 12 was treated

with tris(cetylpyridinium) 12-tungstophosphate (CWP), which

was prepared independently from 12-tungstophosphoric acid

and 3 equiv of cetylpyridinium chloride as catalyst in t-BuOH in

the presence of hydrogen peroxide to give dioic acid 13.²⁷

Compound 13 was treated with thionyl chloride in methanol to

aﬀord the dimethyl ester 14 (20% yield over two steps).

Condensation of 14 with guanidine carbonate and acetamidine

hydrochloride aﬀorded bicyclic 15 and 16, respectively.

RATIONALE AND SYNTHESIS

■

The modiﬁcations of ( )-1·HCl were focused on variations of

R₁, R₂, R₃, and R₄, as shown in structure A (Figure 1). We²⁴

previously reported that the 4′-OCH₃moiety is important for

activity and that the preferable location of the methoxy group is

the para-position rather than the meta- or ortho-positions. In

addition, we established that an 4-N-methyl was preferred over

an unsubstituted NH. It was now of interest to determine the

biological activity of other variations on the aniline ring, as well

as the cyclopenta[d]pyrimidine scaﬀold, in an attempt to

optimize activity.

Compounds 3−5 (Table 1) were designed to isosterically

replace the oxygen of ( )-1·HCl with a sulfur atom at the para-

position of the phenyl ring (3), and 4−5 provide an electron

withdrawing group at the para- and meta-positions, respectively,

of the phenyl ring. In compound 10 (Table 1), the 2-CH₃of

B

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Table 1. IC₅₀Values for Growth Inhibition for Compounds Tested in MDA-MB-435 Cells and EC₅₀for Cellular Microtubule

Depolymerization

Chlorination of 15,16 to 17,18 was performed at reﬂux in

POCl₃for 3 h. Nucleophilic substitution of 17,18 with 4-

methoxy-N-methylaniline in i-PrOH aﬀorded compounds,

respectively. Compound 20 was a liquid and was converted

to the corresponding HCl salt by bubbling anhydrous HCl gas

through a solution of 20 in anhydrous ether.

and to decrease its ﬂexibility. Thus the regio 5-methyl isomer of

1 was expected to provide information on the importance of

the methyl at the 6-position in addition to the conformational

ﬂexibility of the N-methylaniline. The 5-methyl analogue

( )-26 was synthesized from commercially available methyl

3-oxoheptanoate 21 (Scheme 4). Compound 21 was treated

with p-acetamidobenzene sulfonylazide in the presence of

triethylamine via diazo-transfer to aﬀord diazo 22 (63%).²⁸

Moving the 6-methyl group to the 5-position was anticipated

to inﬂuence the conformation of the N-methylaniline moiety

C

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a

salts of 35−38 were obtained as solids when anhydrous HCl

gas was bubbled through anhydrous ether solutions of the free

bases.

Scheme 1

Finally, the 2-desmethyl-6-desmethyl compounds 43−47

(Scheme 7) were also synthesized, to determine the importance

to biological activity of the 2-substituent. Treatment of

compound 27 with ammonium formate in methanol gave 39

(91%). Compound 39 reacted with formic acid in acetic

anhydride to aﬀord 40 (77%), which was condensed with

ammonium formate in formamide to aﬀord bicyclic 41 (83%).

Chlorination of 41 with POCl₃at reﬂux for 3 h gave 42 (60%).

Nucleophilic aromatic substitution of 42 with appropriate N-

methylanilines in i-PrOH aﬀorded the free bases 43−47. The

liquid compounds 43 and 44 were converted to their HCl salts

on treatment with anhydrous HCl gas as described above.

a

Conditions: (a) (1) i-PrOH, 2−3 drops of HCl, substituted N-

methyl-aniline; (2) anhydrous HCl gas, anhydrous ether.

Compound 22 was converted to cyclopentane ( )-23 (43%)

with a rhodium(II) acetate mediated intramolecular C−H

insertion.^28,29Condensation of 23 with acetamidine hydro-

chloride in the presence of t-BuOK aﬀorded bicyclic ( )-24

(79%). Chlorination of ( )-24 to ( )-25 was performed at

reﬂux with POCl₃for 3 h (32%). Nucleophilic aromatic

substitution of ( )-25 with 4-methoxy-N-methylaniline in i-

PrOH aﬀorded the free base ( )-26, which was converted to

the HCl salt ( )-26 as described above.

It was also of interest to ascertain the importance of the 6-

CH₃group of ( )-1·HCl to biological activity. In addition, the

4N-ethoxy analogue was synthesized as a simple homologue of

( )-1·HCl to determine size requirements at the 4N-position.

The synthesis of the 2-methyl-6-desmethyl compounds 30−32

is shown in Scheme 5. Condensation of methyl-2-oxocyclo-

pentane carboxylate 27 with acetamidine hydrochloride in the

presence of t-BuOK aﬀorded bicyclic 28 (81%). Chlorination of

28 with POCl₃at reﬂux for 3 h aﬀorded 29 (69%). Compound

29 was unstable and easily reverts to 28. Thus, immediate

reaction of 29 with the appropriate N-methylanilines in i-PrOH

in the presence of 2−3 drops of concentrated HCl was

necessary to aﬀord 30−32. The liquids 30 and 31 were

converted to their HCl salts 30·HCl and 31·HCl following the

method described above.

A preliminary evaluation of the antiproliferative eﬀect of

compound 30·HCl (Table 1) showed it to be more potent than

the lead ( )-1·HCl. Thus, the 2-amino analogues of 30·HCl,

with variations in the aniline ring substitutions, were also

synthesized. Target compounds 35−38 were prepared as

shown in Scheme 6. Condensation of 27 with guanidine

carbonate in the presence of t-BuOK aﬀorded bicyclic 33

(83%). Chlorination of 33 with POCl₃at reﬂux for 3 h aﬀorded

34 (56%). Compound 34 reacted with the appropriate N-

methylanilines in i-PrOH in the presence of 2−3 drops of

concentrated HCl to aﬀord the free bases 35−38. The HCl

BIOLOGICAL EVALUATIONS AND SAR

■

Compounds 3−5·HCl, 9, 10·HCl, 19, 20·HCl, 26·HCl, 30−

31·HCl, 32, 35−38·HCl, 43−44·HCl, 43, and 45−47 were

tested for antiproliferative activity against the drug-sensitive

MDA-MB-435 cancer cell line using the sulforhodamine B

assay (SRB assay), and IC₅₀values (concentration required to

cause 50% inhibition of proliferation) were calculated.^30,31The

ability of the compounds to cause microtubule depolymeriza-

tion in cells was also evaluated, and an EC₅₀, concentration

required to cause approximately 50% microtubule loss, was

determined. For substitution on the aniline ring, the data

(Table 1) showed that an electron-withdrawing moiety was

detrimental to both antiproliferative activity and cellular

microtubule loss: the 4-chloro compound 4·HCl and the 3-

bromo compound 5·HCl were, respectively, about 130- and 90-

fold less potent in antiproliferative activity and >700- and 260-

fold less potent against microtubule depolymerization than

( )-1·HCl. When the 2-methyl group was replaced with a 2-

amino, compound 10·HCl retained the potent antiproliferative

eﬀect as it was only about 1.7-fold less active than ( )-1·HCl.

This indicated that a potential hydrogen bond donor or

acceptor at the 2-position is not necessary for potent activity.

For the 2-methyl series, an analogue with a one carbon

extension at the 6-position, compound ( ) 20·HCl, with a 6-

vinyl moiety, had a reduced antiproliferative eﬀect, with an IC₅₀

of 73 nM (about 4-fold less potent than ( )-1·HCl). When the

6-methyl group was moved to the 5-position, the resulting

compound ( )-26·HCl had activity comparable (IC₅₀of 19

nM) to that of ( )-1·HCl, and the 6-desmethyl compound 30·

HCl had an IC₅₀of 7 nM (2.4-fold more potent than ( )-1·

HCl). Thus the 6-methyl or vinyl group on the cyclopentane

ring does not play a major role in the inhibitory activity and can

be removed to aﬀord a more potent analogue, one that has no

a

Scheme 2

a

Conditions: (a) (1) Ethanol, conc. sulfuric acid, reﬂux, 8 h; (2) Na, toluene, reﬂux, 3 h; (3) guanidine carbonate, t-BuOH, t-BuOK; (b) POCl₃,

reﬂux, 3 h; (c) N-methyl-4-methoxyaniline, i-PrOH, 2−3 drops HCl; (d) anhydrous HCl gas, ether.

D

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a

Scheme 3

a

Reagents and conditions: (a) H₂O, HCl; (b) t-BuOH, H₂O₂, CWP; (c) SOCl₂, CH₃OH; (d) (1) Na, toluene, reﬂux; (2) guanidine carbonate or

acetamidine hydrochloride, t-BuOH, t-BuOK; (e) POCl₃, reﬂux; (f) i-PrOH, conc. HCl, 4-methoxy-N-methylaniline; (g) HCl gas, anhydrous ether.

a

Scheme 4

a

Reagents and conditions: (a) (1) p-acetamidobenzene sulfonylazide, CH₃CN, Et₃N, rt, 8 h; (b) Rh₂(OAc)₄, CH₂Cl₂; (c) acetamidine

hydrochloride, t-BuOK, t-BuOH; (d) POCl₃, reﬂux, 3 h; (e) 4-methoxy-N-methylaniline, i-PrOH, 2 drops of conc. HCl, reﬂux, 8 h; (f) HCl gas,

anhydrous ether.

a

Scheme 5

a

Reagents and conditions: (a) acetamidine hydrochloride, t-BuOH, t-BuOK; (b) POCl₃, reﬂux; (c) i-PrOH, conc. HCl, substituted N-methylaniline;

(d) HCl gas, anhydrous ether.

chiral carbons. Deletion of the 2-methyl group from the

heterocyclic ring (43·HCl) aﬀorded a compound with lower

activity (IC₅₀of 43 nM), 6-fold less potent than 30·HCl.

However, compound 44 with a 4′-ethoxy moiety at the 4′-

position on the aniline ring, has comparable potency (IC₅₀of

49 nM) with the 4′-methoxy analogue 43·HCl. This is

consistent with the trend shown in both the 2-methyl and

the 2-amino series when comparing 31·HCl with 30·HCl and

38·HCl with 35·HCl. When the 4′-methoxy group was replaced

with a 4′-methylthio group, the resulting analogue 47 showed

similar potency (IC₅₀of 43 nM) as 43·HCl, as is also the case

in comparing 3·HCl with ( )-1·HCl. However, when a methyl

group was introduced at the 4′-position on the aniline ring

(compound 45), the cellular microtubule depolymerization

E

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a

Scheme 6

a

Reagents and conditions: (a) guanidine carbonate, t-BuOH, t-BuOK; (b) POCl₃, reﬂux; (c) (1) i-PrOH, conc. HCl, substituted-N-methylaniline;

(2) HCl gas, anhydrous ether.

a

Scheme 7

a

Reagents and conditions: (a) ammonium formate, CH₃OH; (b) formic acid and acetic anhydride; (c) formamide, ammonium formate; (d) POCl₃,

reﬂux; (e) i-PrOH, conc. HCl, 4-methoxy-N-methylaniline; (f) HCl gas, anhydrous ether.

activity was lost (compare with compound 44·HCl). Analogue

46 with an electron-withdrawing group at the 4′-position on

the aniline ring had no activity. These results indicate that the

substituent at the 4′-position on the aniline ring is crucial for

activity, and thus far a substituent with an electron-donating

group along with a hydrogen bond acceptor atom, such as

OCH₃or SCH₃, is advantageous.

For analogues with a 2-amino scaﬀold, the potency trend is

similar to the 2-methyl scaﬀold: the aniline 4′-methoxy moiety

is important for potent activity, and the optimum location for

the methoxy group is at the 4′-position, since both 36·HCl,

which has a 3′-methoxy, and 37·HCl, which has a 2′-methoxy,

were inactive. With a vinyl group at the 6-position of the

cyclopentane ring, compound 19 was about 7-fold less potent

than 10·HCl. When the 6-methyl group was removed from the

scaﬀold, compound 35·HCl was more potent than 10·HCl, but

35·HCl was less potent than the 2-methyl 30·HCl. Comparing

30·HCl with 35·HCl and 43·HCl, the greatest antiproliferative

activity occurred with a 2-methyl moiety. When the methyl

moiety on the aniline N was removed to yield compound 9,

antiproliferative activity was lost (IC₅₀>10 μM). This

conﬁrmed that the methylation of the aniline N is crucial for

potency as observed in the 2-methyl series analogues and is in

agreement with our previous reports.^23,24

Further Mechanistic Studies of 30·HCl. Of the target

compounds listed in Table 1, compound 30·HCl was the most

potent analogue, over twice as active as ( )-1·HCl, and it was

therefore selected for further mechanistic studies.

Evaluation of Anti-microtubule and Antiproliferative

Eﬀects. The eﬀects of ( )-1·HCl and 30·HCl on interphase

microtubules (Figure 2) were evaluated in a cell-based

phenotypic screen. Compounds ( )-1·HCl and 30·HCl led

to drastic microtubule depolymerization, similar to the eﬀects

of colchicine and CA-4. The EC₅₀was determined to be 7 nM

for CA-4, 56 nM for ( )-1, and 26 nM for 30·HCl. Like other

microtubule targeting agents, ( )-1·HCl and 30·HCl cause the

formation of aberrant mitotic spindles with concurrent mitotic

Figure 2. The eﬀects of CA-4 and 30·HCl on interphase microtubules.

A-10 cells were treated with vehicle or compounds as indicated for 18

h and ﬁxed, and microtubules were visualized by indirect

immunoﬂuorescence techniques.

accumulation when measured by ﬂow cytometry (Figure 3).

These data show that compound 30·HCl is a potent

antimicrotubule agent.

The ability to circumvent Pgp-mediated drug resistance was

evaluated using an SK-OV-3 isogenic cell line pair (Table 2).

Similar to ( )-1·HCl, 30·HCl was 1.5-times more resistant to

cells overexpressing the Pgp drug transporter. On the other

hand, paclitaxel, a known Pgp substrate, was 868-times more

resistant. These data suggest that 30·HCl is a poor substrate for

Pgp and therefore might have advantages over some clinically

useful tubulin-targeting drugs.

A second clinically relevant mechanism of drug resistance to

these microtubule agents is the expression of the βIII isotype of

tubulin. An isogenic HeLa cell line pair was used to test the

F

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Figure 3. Cell cycle distribution. MDA-MD-435 cells were treated with vehicle (A), 12.5 nM paclitaxel (B), or 25 nM 30·HCl (C) for 24 h, the cells

were harvested, stained, and evaluated by ﬂow cytometry.

Table 2. ( )-1 and 30·HCl Circumvent Clinically Relevant Models of Drug Resistance

a

b

c

eﬀect of Pgp on drug sensitivity

IC₅₀(nM)

parental and βIII-tubulin transfected HeLa cells

IC₅₀(nM)

c

drug

SK-OV-3

SK-OV-3 MDR-M6-6

Rr

864

1.5

HeLa

WT βIII

Rr

paclitaxel

CA-4

3.0 0.06

4.5 0.2

2,600 270

6.6 1.3

1.6 0.2

4.7 0.2

37.3 4.1

9.3 0.3

7.7 0.2

5.7 0.4

23.9 1.7

8.2 0.8

4.7

1.2

0.6

0.8

( )-1·HCl

30·HCl

38.6 3.1

11.3 0.2

44.4 3.2

16.4 0.4

1.2

1.5

a

Antiproliferative eﬀects of ( )-1·HCl and 30 HCl in parental and PgP overexpressing cell lines in comparison with other microtubule disrupting

agents (n = 3 SD). Eﬀects of the expression of βIII-tubulin on the sensitivity of cell lines to microtubule-targeting agents. The IC₅₀values were

determined using SRB assay (n = 3 SD). Relative resistance. The Rr was calculated by dividing the IC₅₀of the transduced cell line by the IC₅₀of

b

c

the parental cell line.

Table 3. Comparison of Cancer Cell Growth Inhibitory Activity (NCI) GI₅₀(nM) of 30·HCl with ( )-1·HCl

GI₅₀(nM)

panel/cell

line

panel/cell

line

( )-1·HCl 30·HCl

( )-1·HCl 30·HCl panel/cell line ( )-1·HCl 30·HCl

panel/cell line

renal cancer

18.4 786 - 0

( )-1·HCl 30·HCl

leukemia

CCRF-CEM

HL-60(TB)

K-562

colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

melanoma

16.3

<10

31.8

15.7

<10

18.3

24.0

15.1

<10

LOX IMVI

22.7

10.2

34.8

<10

14.6

17.8

<10

16.4 M14

<10

A498

<10

MDA-MB-435

SK-MEL-2

<10

33.1

<10

ACHN

<10

MOLT-4

RPMI-8226

SR

27.8

14.3

<10

11.8

<10

11.9 CAKI-1

SK-MEL-28

SK-MEL-5

<10

RXF 393

SN12C

TK10

<10

31.8

292

KM12

<10

33.0

NSCLC

SW-620

CNS cancer

SF-268

UACC-62

42.7

46.1

A549/ATCC

EKVX

24.4

19.6

19.4

55.4

31.5

16.4

59.0

15.0

34.8

ovarian cancer

UO-31

13.4

15.2

<10

11.4

36.9

<10

12.5

13.9 IGROVI

11.0

<10

26.8

12.8 prostate cancer

HOP-62

<10

SF-295

<10

OVCAR-3

OVCAR-4

<10

PC-3

14.6

21.4

<10

19.6

HOP-92

16.0

34.7

22.3

48.8

SF-539

22.9 DU-145

36.4 breast cancer

21.8 MCF7

NCI-H226

NCI-H23

NCI-H322M

SNB-19

SNB-75

U251

17.0 OVCAR-5

38.5

32.8

<10

OVCAR-8

<10

24.6

<10

25.9

NCI/ADR-

RES

<10

MDA-MB-231/

ATCC

NCI-H460

NCI-H522

23.4

10.3

SK-OV-3

27.5

14.6 HS 578T

<10

21.9

<10

21.8

<10

BT-549

MDA-MB-468

ability of these compounds to overcome the overexpression of

βIII. Compound 30·HCl has a Rr value of 0.8 (Table 2) in this

cell line pair suggesting that it can overcome drug resistance

mediated by βIII tubulin compared with paclitaxel with a Rr of

4.7.

NCI Cytotoxicity Studies. The improved potency of

compound 30·HCl prompted its selection and evaluation

against the NCI 60 tumor cell line panel, and the results (Table

3) are compared with those for ( )-1·HCl. The data (Table 3)

showed that 30·HCl was a potent inhibitor (GI₅₀of one to two

digit nanomolar) of almost all of the cancer cell lines and in

G

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Journal of Medicinal Chemistry

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most instances was more potent than ( )-1·HCl, displaying up

to 7-fold greater potency than ( )-1·HCl, further establishing

30·HCl as the lead analogue for further preclinical develop-

ment.

In Vivo Activity of 30·HCl. Compound 30·HCl was further

evaluated for potential antitumor eﬀects in the MDA-MB-231

triple negative breast cancer murine xenograft model. Total

doses ranging from 50.8 to 75.0 mg/kg were well tolerated,

resulting in no greater than 9% weight loss, even when dosing

was done twice a day for a total of 12 doses of 50 mg/kg. At

these concentrations, 30·HCl was eﬃcacious at preventing

tumor growth, causing a 27−44% tumor growth delay (T-C/C)

with net log kills of 1.1−1.5 (Table 4 and Figure 4). There were

μM concentrations, 30·HCl inhibited the binding of [³H]-

colchicine, again somewhat more potently than ( )-1·HCl and

somewhat less potently than CA-4. It is therefore likely that 30·

HCl binds in the colchicine site.

Molecular Modeling and Computational Studies. In an

attempt to determine the structural basis of the inhibition of

tubulin assembly at the colchicine site, it was of interest to

determine the possible conformation(s) and binding poses of

the most potent analogue, 30 HCl, in the known colchicine

binding site as deﬁned by the crystal structure at 3.58 Å

resolution (PDB ID 1SAO).⁷This crystal structure is

composed of two αβ-tubulin dimers complexed with N-

deacetyl-N-(2-mercaptoacetyl) colchicine (DAMA-colchicine),

a close structural analogue of colchicine (Figure 6). The crystal

structure indicates a rather large binding site. A variety of small

molecules have been docked into this binding site, and

pharmacophores of the various small molecule colchicine site

compounds have been proposed.³²⁻³⁴

Docking of DAMA-Colchicine. To validate the utility of

MOE 2007.09³⁵for docking ligands into the binding site,

DAMA-colchicine, the ligand in the crystal structure (PDB

1SA0) was built using the molecule builder, energy minimized,

and redocked into the binding site. The best-docked pose of

DAMA-colchicine displayed an rmsd of 0.95 Å compared with

the crystal structure pose of DAMA-colchicine. MOE 2007.09

was thus validated for our docking studies.

Binding of DAMA-Colchicine to Tubulin. A description

of the binding of colchicine to the tubulin dimer has been

published.^33,36,37The colchicine binding site is located

primarily in β-tubulin at its interface with α-tubulin. The

large colchicine binding site (∼10 Å × ∼10 Å × ∼4−5 Å) is

composed of the T7 loop, S8 and S9 strands, and H7 and H8

helices, where the β-subunit joins loop T5 of the α-subunit.^33,36

Thrα179 and Valα181 from α-tubulin have been shown to form

crucial interactions with DAMA-colchicine and other colchicine

site inhibitors.^33,37Additionally, the oxygen atom of the 3-

methoxy in the A-ring of colchicine forms hydrogen bonds with

Cysβ241. Side chain atoms of Valα181 and Metβ259 provide

additional stabilization for DAMA-colchicine binding.^33,36The

C-ring carbonyl group of DAMA-colchicine hydrogen bonds

with Lysβ352 (residue numbers follow the convention in ref 7).

A variety of low-energy conformations (within 1 kcal/mol of

the best pose) were obtained on docking the most potent

analogue 30·HCl, as well as other analogues. Figure 7 shows

one of the docked conformations of 30·HCl. This pose was

ranked fourth in the results and had a score (−6.9850 kcal/

mol) within 1 kcal/mol of the best-scored pose. In this pose,

the 4-methoxy of 30·HCl approximates the 3-methoxy group in

the A-ring of DAMA-colchicine. A hydrogen bond between

Cysβ241 and the oxygen atom of the 4′-methoxy of 30·HCl, as

seen with the 3-methoxy group of DAMA-colchicine, is formed

with this conformation of 30·HCl. The phenyl ring of 30·HCl

mimics the A-ring of colchicine and could form hydrophobic

interactions with Leuβ248, Alaβ250, Leuβ255, and Alaβ316

from the β-tubulin subunit. The 4′-methoxy methyl moiety of

30·HCl could interact with the side chain of Valβ318 or with

the side chain of Ileβ378. The N-CH₃group of 30·HCl is

oriented toward the C5- and C6-bridging carbon atoms of the

B-ring of colchicine and interacts with the side chain carbon

atoms of Lysβ254 and Alaβ250. Additionally, the N-CH₃

moiety of 30·HCl could form hydrophobic interactions with

the side chain carbon atom of Leuβ248 aided by the ﬂexible

nature of the protein. These interactions could help stabilize

Table 4. In Vivo Activity of 30·HCl in the MDA-MB-231

a

Breast Cancer Murine Xenograft Model

max %

tumor

free on

day 41

relative

weight

loss

optimal

% T/C

(day)

growth

delay

% T-C/C cell kill

net log

vehicle control, 10%

DMSO in saline/

Tween 80

0

6.7

30·HCL, 50 mg/kg,

QD × 5, day 14(27)

30·HCL, 75 mg/kg,

QD × 5, day 14(27)

30·HCL, 75 mg/kg,

Q2D × 5, day

14(27)

0

7.7

7.9

9.0

20 (31)

17 (31)

19 (31)

27

40

44

−1.40

−1.10

−1.50

30·HCL, 50 mg/kg,

BID × 6, day

14(16,18,27,29,31)

0

7.8

25 (31)

32

−1.30

a

T/C is the median tumor mass of a speciﬁc treatment group (T)

divided by the median tumor mass of the control group (C), expressed

as percent. T-C/C is the median number of days between the time the

treatment groups (T) and control group (C) take to reach a

predetermined size. This number is normalized to the control group

(C) and expressed as a percent. QD refers to daily administration of

drug dose, Q2D refers to administration of dose every other day, and

BID refers to twice daily dosing. Animals were dosed according to

their schedule for 5 or 6 days starting on day 14 and allowed to

recover, and dosing started again on day 27. Study consisted of 8

animals per experimental treatment group and 16 animals in the

control group.

no drug related deaths, and the maximum tolerated dose was

never reached. A total dose of 75 mg/kg seemed to be the most

eﬃcacious, as it resulted in the smallest tumors 17% and 19%

(T/C) at day 21. These initial studies suggest 30·HCl has

antitumor eﬃcacy in this murine model of triple negative breast

cancer and should be evaluated further in additional preclinical

models.

BIOCHEMICAL EVALUATIONS

■

The eﬀects of ( )-1·HCl and 30·HCl on the polymerization of

puriﬁed tubulin were evaluated. This allows for the study of the

direct interaction of the compounds with their intracellular

target. The data (Figure 5) show that this compound is an

eﬀective and potent inhibitor of tubulin assembly. Further

quantitative studies were conducted and conﬁrmed that 30·HCl

inhibits tubulin assembly with somewhat greater potency than

( )-1·HCl and somewhat less potency than CA-4 (Table 5).

The ability of 30·HCl to bind to the colchicine site on tubulin

was evaluated by measuring inhibition of [³H]colchicine

binding to tubulin. From Table 5, it is clear that at 1 and 5

H

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Figure 4. In vivo evaluation of 30·HCl.

Figure 6. Colchicine and DAMA-colchicine.

analogues (not shown) in this series and explains, in part, the

loss of activity of the NH analogue 9, which would lack these

additional N-CH₃interactions with tubulin. Docked poses of

compounds with the N-CH₃group consistently scored higher

(by ∼1 kcal/mol) than those of compounds that lack the N-

CH₃group.

Figure 5. Eﬀects of select compounds on puriﬁed tubulin polymer-

ization. Colchicine was used as a positive control. The assays were

conducted using porcine tubulin in the presence of 10% glycerol, 2

mg/mL puriﬁed tubulin, and GTP.

The C-ring of DAMA-colchicine partly overlaps the

cyclopenta[2,3-d]pyrimidine ring of 30·HCl and interacts

with side chain carbon atoms of Lysβ252, Leuβ255, and

Asnβ258. The C7 of 30·HCl overlaps the C9-carbonyl carbon

atom of DAMA-colchicine. The C2-methyl group of 30·HCl

could be involved in a potential hydrophobic interaction with

Alaα180.

In summary, 21 analogues of the lead compound ( )-1·HCl

were synthesized and evaluated, and SAR for substitutions in

the 2-, 4-, 4N-anilinyl, and 6-positions were studied. Compound

30·HCl, the 6-desmethyl analogue of the lead compound

( )-1·HCl, was 2.4-fold more potent than the parent

compound as an antiproliferative agent, with single digit

nanomolar potency. This analogue also showed promising

Table 5. Inhibition of Tubulin Assembly and Colchicine

Binding by CA-4, ( )-1·HCl and 30·HCl

inhibition of

colchicine binding, %

inhibition SD

inhibition of tubulin assembly, IC₅₀

compound

(μM) SD

1 μM

5 μM

CA-4

1.0 0.09

1.9 0.01

1.6 0.1

88

2

99 0.2

( )-1·HCl

30·HCl

60

70

84

3

92 0.7

the docked conformation of 30·HCl. The N-CH₃moiety was

similarly oriented in the docked conformations of other

I

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 7. Docking mode of 30·HCl (white) overlaid with DAMA-colchicine (red).

was diluted with chloroform, neutralized with NH₄OH, and washed

with water (2 × 30 mL). After drying with anhydrous Na₂SO₄, the

organic solvent with 1 g silica gel was evaporated under reduced

pressure to give a dry plug. This plug was placed on the top of a silica

gel column and eluted with chloroform. Fractions containing the

product were pooled and evaporated to aﬀord pure compound.

General Method To Make HCl Salt from Base. HCl gas was

bubbled through the solution of free base in anhydrous ether until no

more solid was precipitated. The solid was collected by ﬁltration and

dried to give the HCl salt.

N,2,6-Trimethyl-N-(4-(methylthio)phenyl)-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-aminium Chloride (3·HCl). Compound 3·

HCl was synthesized from 2 (0.06 g, 0.39 mmol) and N-methyl-4-

methylmercaptoaniline (0.049 g, 0.46 mmol) following the general

procedure for nucleophilic displacement from the chlorinated

compound and the general method to make a HCl salt from the

base to aﬀord, after puriﬁcation 0.052 g (40% over two steps) as a gray

solid: TLC R_f0.33 (chloroform/methanol, 10:1); mp 214.4−215.5 °C;

¹H NMR (DMSO-d₆) δ 0.89 (d, J = 6.8 Hz, 3H), 1.44−1.53 (m, 1H),

antitumor activity in vivo and serves as the analogue for further

preclinical development.

EXPERIMENTAL SECTION

■

Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY

drying apparatus over P₂O₅at 50 °C. Melting points were determined

on a digital MEL-TEMP II melting point apparatus with FLUKE 51

K/J electronic thermometer and are uncorrected. Nuclear magnetic

resonance spectra for protons (¹H NMR) were recorded on a Bruker

Avance II 400 (400 MHz) or on a 500 (500 MHz) NMR systems. The

chemical shift values are expressed in ppm (parts per million) relative

to tetramethylsilane as an internal standard: s, singlet; d, doublet; t,

triplet; q, quartet; m, multiplet; br, broad singlet. Thin-layer

chromatography (TLC) was performed on Whatman Sil G/UV254

silica gel plates with a ﬂuorescent indicator, and the spots were

visualized under 254 and 366 nm illumination. Proportions of solvents

used for TLC are by volume. Column chromatography was performed

on a 230−400 mesh silica gel (Fisher Scientiﬁc) column. Elemental

analyses were performed by Atlantic Microlab, Inc., Norcross, GA.

Elemental compositions are within 0.4% of the calculated values and

indicate >95% purity of the compounds. Fractional moles of water or

organic solvents found in some analytical samples could not be

prevented despite 24−48 h of drying in vacuo and were conﬁrmed

1.97−2.06 (m, 1H), 2.30−2.39 (m, 1H), 2.53 (s, 3H), 2.63 (s, 3H),

3.03−3.08 (m, 2H), 3.54 (s, 3H), 7.37 (s, 4H), 15.02 (bs, 1H); Anal.

calcd for (C₁₇H₂₂ClN₃S·0.1H₂O) C, H, N, Cl, S.

N-(4-Chlorophenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-aminium Chloride (4·HCl). Compound 4·HCl was

synthesized from 2 (0.25 g, 1.37 mmol) and 4-chloro-N-methylaniline

(0.19 g, 1.37 mmol) following the general procedure for nucleophilic

displacement from the chlorinated compound and the general method

to make a HCl salt from the base to aﬀord after puriﬁcation 0.228 g

(58% over two steps) as a white solid: TLC R_f0.50 (CHCl₃/CH₃OH,

1

where possible by their presence in the H NMR spectra. All solvents

and chemicals were purchased from Sigma-Aldrich Co. or Fisher

Scientiﬁc Inc. and were used as received.

General Procedure for Chlorination. A mixture of the

appropriate 4-oxo compound and POCl₃(10 mL) was heated at

reﬂux for 3 h. The reaction mixture was cooled and evaporated at

reduced pressure. The residue was diluted with chloroform (50 mL)

and neutralized slowly in an ice bath with NH₄OH. The organic

portion was washed with water (3 × 30 mL) and dried with anhydrous

Na₂SO₄. Concentration of the organic solvent with 1 g of silica gel

aﬀorded a dry plug. This plug was placed on the top of a silica gel

column and eluted with chloroform. Fractions containing the product

were pooled and evaporated to aﬀord the chlorinated compound.

General Procedure for Nucleophilic Displacement from

Chlorinated Compound. Chlorinated compound and the appro-

priate substituted N-methyl anilines or aniline were dissolved in

isopropyl alcohol (5 mL). To this solution was added 37% HCl (2−3

drops). The mixture was heated at reﬂux for 3−6 h. Then the reaction

mixture was cooled and evaporated at reduced pressure. The residue

1

10:1); mp 114.4−116.0 °C; H NMR (DMSO-d₆) δ H NMR (400

MHz, DMSO) δ 0.89 (d, J = 6.8 Hz, 3H), 1.48 (dd, J = 16.3, 6.1 Hz,

1H), 1.99 (dd, J = 16.9, 5.9 Hz, 1H), 2.27−2.41 (m, 1H), 2.62 (s, 3H),

3.05 (dd, J = 17.6, 7.9 Hz, 1H), 3.54 (s, 3H), 7.48 (d, J = 8.7 Hz, 2H),

7.59 (d, J = 8.7 Hz, 2H)., 15.04 (bs, 1H); Anal. calcd. for

(C₁₆H₁₈ClN₃) C, H, N, Cl.

N-(3-Bromophenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-aminium Chloride (5·HCl). Compound 5·HCl was

synthesized from 2 (0.085 g, 0.55 mmol) and 3-bromo-N-methyl

aniline (0.06 mL) following the general procedure for nucleophilic

displacement from chlorinated compound and the general method to

make a HCl salt from base to aﬀord after puriﬁcation 0.090 g (52%

over two steps) as a white solid: TLC R_f0.40 (CHCl₃/CH₃OH, 10:1);

mp 200.5−202.0 °C; ¹H NMR (DMSO-d₆) δ 0.86 (d, J = 6.8 Hz, 3H),

J

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.37−1.47, 1.89−1.99, 2.43−2.44, 2.98−3.05 (m, 5H), 2.60 (s, 3H),

3.51 (s, 3H), 3.80 (s, 3H), 7.01−7.03, 7.33−7.35 (m, 4H), 14.88 (br,

1H); Anal. calcd. for (C₁₆H₁₉BrN₃Cl·0.2H₂O) C, H, N, Cl, Br.

2-Amino-6-methyl-3,5,6,7-tetrahydro-4H-cyclopenta[d]-

pyrimidin-4-one (7). 3-Methyladipic acid (1.60 g, 10 mmol) was

heated under reﬂux in an ethanol/conc. H₂SO₄solution (35 mL, v/v =

2.5/1) for 8 h. The solution was neutralized with NH₄OH to pH = 7,

then diluted with ethyl acetate (100 mL), and washed with water. The

organic phase was dried with anhydrous Na₂SO₄and evaporated to

aﬀord a light yellow liquid used in the next step. The liquid was diluted

with anhydrous toluene (100 mL), and sodium (0.23 g) was added to

the solution. The mixture was heated under reﬂux for 3 h and cooled,

neutralized with a 1 N HCl solution, and washed with water. After

drying with anhydrous Na₂SO₄, the organic phase was evaporated to

aﬀord a light brown liquid used in the next step. The liquid was diluted

with t-BuOH. Guanidine carbonate (2.70 g, 15 mmol) and potassium

tert-butoxide (1.68 g, 15 mmol) were added, and the mixture was

heated under reﬂux overnight. The reaction mixture was cooled, and a

precipitate was removed by ﬁltration. The precipitate was washed

twice with warm methanol (30 mL × 1, 15 mL × 1). The ﬁltrate and

washings were combined and evaporated under reduced pressure, and

the residue was puriﬁed by column chromatography using chloroform/

methanol (100/1) as eluent to aﬀord 7 as a white solid (0.23 g, 19%

yield for 3 steps): TLC R_f0.36 (CHCl₃/CH₃OH, 10:1); mp 319−321

room temperature overnight. The reaction mixture was diluted with

distilled water (30 mL) and extracted with ethyl acetate (20 mL × 3).

The organic layer was dried over Na₂SO₄. Concentration of the

organic solvent with 6 g of silica gel aﬀorded a dry plug. This plug was

placed on the top of a silica gel column and eluted with chloroform/n-

hexane (1:1). Fractions containing the product were pooled and

evaporated to aﬀord 12 as a colorless liquid (2.81 g, 85%): TLC R_f

1

0.17 (chloroform/methanol, 10:1); H NMR (400 MHz, CDCl3) δ

1.31−1.72 (m, 6 H), 2.27−2.35 (m,1 H), 3.41 (p, J = 4.4, 3.6; 1 H),

3.51 (p, J = 4.4, 4.0; 1 H), 4.47 (d, J = 3.6; 1 H, exch), 4.53 (d, J = 4.0;

1 H, exch), 4.87−4.93 (m, 2H), 5.71−5.79 (m, 1 H).

3-Vinylhexanedioic Acid (13). A mixture of 12 (2.0 g, 14 mmol),

35% H₂O₂(4.2 mL), and CWP (1.069 g, 5.86 mmol) in t-BuOH (50

mL) was heated at reﬂux for 1 d. Then 10% sodium bisulfate was

added to decompose the excess H₂O₂. The mixture was extracted with

ethyl acetate (40 mL × 3) and dried over Na₂SO₄. Concentration of

the organic solvent with 5 g of silica gel aﬀorded a dry plug. This plug

was placed on the top of a silica gel column and eluted with

chloroform/methanol (50:1). Fractions containing the product were

pooled and evaporated to aﬀord 13 as a colorless liquid (0.68 g, 28%):

TLC R_f0.11 (chloroform/methanol, 10:1); ¹H NMR (500 MHz,

DMSO-d₆) δ 1.42−1.68 (m, 2 H), 2.13−2.31 (m, 4 H), 2.34−2.45 (m,

1 H), 4.97−5.01 (m, 2 H,), 5.56−5.65 (m, 1 H), 12.05 (bs, 2 H).

Dimethyl 3-Vinylhexanedioate (14). Thionyl chloride (1.43 mL,

19.6 mmol) was added dropwise to a solution of compound 13 (0.68

g, 3.95 mmol) in methanol (6 mL) in an ice-bath. Then the mixture

was stirred at room temperature overnight. Concentration of the

organic solvent with 6 g of silica gel aﬀorded a dry plug. This plug was

placed on the top of a silica gel column and eluted with hexane/ethyl

acetate (50:1). Fractions containing the product were pooled and

evaporated to aﬀord 14 as a colorless liquid (0.38 g, 48%): TLC R_f

1

°C; H NMR (DMSO-d₆) δ 1.11 (d, J = 6.8 Hz, 3H), δ 1.35−1.46,

1.99−2.20, 2.38−2.72, 2.92−2.98 (m, 5H), 6.32 (bs, 2H), 10.47 (bs,

1H); Anal. calcd. for (C₈H₁₁N₃O·0.1CH₃OH) C, H, N.

4-Chloro-6-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-

amine (8). Compound 8 was synthesized from compound 7 (0.297 g,

1.8 mmol) following the general procedure for chlorination described

above to aﬀord after puriﬁcation 0.112 g (34%) as an oﬀ-white solid:

TLC R_f0.48 (CHCl₃/CH₃OH, 10:1); mp 181.5−182.9 °C; ¹H NMR

(DMSO-d₆) δ 1.17 (d, J = 6.8 Hz, 3H), 1.55−1.65, 2.15−2.38, 2.49−

2.62, 2.83−2.92 (m, 5H), 6.83 (bs, 2H); Anal. calcd. for

(C₈H₁₀ClN₃₃OH) C, H, N, Cl.

1

0.43 (hexane/ethyl acetate, 3:1); H NMR (500 MHz, DMSO-d₆) δ

1.47−1.70 (m, 2 H), 2.23−2.43 (m, 5 H), 3.55 (s, 3 H), 3.56 (s, 3 H),

4.96−5.00 (m, 2 H), 5.54−5.64 (m, 1 H).

2-Amino-6-vinyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-

one (15). To a solution of 14 (1.10 g, 5.5 mmol) in anhydrous

toluene, elemental sodium (0.19 g, 8.2 mmol) was added in portions.

The mixture was heated at reﬂux for 3 h. The reaction mixture was

neutralized with 1 N HCl and extracted with ethyl acetate (20 mL ×

3). The organic layer was dried over Na₂SO₄and concentrated. The

resulting yellow liquid was diluted with tert-butanol (15 mL). To this

solution were added potassium tert-butoxide (1.85 g, 16.4 mmol) and

guanidine carbonate (1.49 g, 8.2 mmol). The mixture was heated at

reﬂux overnight. The solid was ﬁltered and washed with hot methanol.

Silica gel (3 g) was added to the ﬁltrate, and the solvent was

evaporated. The resulting silica gel plug was loaded onto a silica gel

column and eluted with chloroform/methanol (50:1). The fractions

containing the product (TLC) were pooled, and the solvent was

evaporated to give a sticky solid. The solid was washed with anhydrous

ether and ﬁltered to aﬀord pure 15 as a white solid (0.21 g, 21% for

two steps): TLC R_f0.17 (chloroform/methanol, 10:1); mp >260 °C;

¹H NMR (500 MHz, DMSO-d₆) δ 1.62−2.97 (m, 5 H), 4.85−5.06

N⁴-(4-Methoxyphenyl)-6-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine-2,4-diamine (9). Compound 9 was synthesized from 8

(0.1 g, 0.54 mmol) and 4-methoxylaniline (0.074 g, 0.65 mmol)

following the general procedure for nucleophilic displacement from

chlorinated compound described above to aﬀord after puriﬁcation

0.045 g (55%) as a light pink solid: TLC R_f0.28 (CHCl₃/CH₃OH,

1

10:1); mp 187.4−189.5 °C; H NMR (DMSO-d₆) δ 1.09 (d, J = 6.8

Hz, 3H), 2.16−2.25 (m, 2H), 2.38−2.47 (m, 1H), 2.74 (dd, J = 16.3,

8.3 Hz, 1H), 2.83 (dd, J = 15.3, 8.2 Hz, 1H), 3.71 (s, 3H), 5.84(bs,

2H), 6.83 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 8.05 (bs, 1H);

Anal. calcd. for (C₁₅H₁₈N₄O·0.1CHCl₃) C, H, N.

N⁴-(4-Methoxyphenyl)-N⁴,6-dimethyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidine-2,4-diamine (10). Compound 10 was

synthesized from 8 (0.094 g, 0.51 mmol) and N-methyl-4-

methoxylaniline (0.084 g, 0.61 mmol) following the general procedure

for nucleophilic displacement from chlorinated compound described

above to aﬀord after puriﬁcation 0.08 g (55%) as a white solid: TLC R_f

0.26 (CHCl₃/CH₃OH, 10:1); mp 146.2−147.5 °C; ¹H NMR

(m, 2 H), 5.82−5.92 (m, 1 H), 6.36 (bs, 2 H), 10.13 (bs, 1 H); Anal.

calcd. for (C₉H₁₁N₃O·0.15C₄H₁₀O) C, H, N.

1

(DMSO-d₆) δ H NMR (400 MHz, DMSO) δ 0.81 (d, J = 6.1 Hz,

3H), 1.26−1.40 (m, 1H), 1.82 (dd, J = 15.2, 7.3 Hz, 2H), 2.00−2.14

(m, 1H), 2.54−2.66 (m, 1H), 3.25 (s, 3H), 3.75 (s, 3H), 5.90 (bs, 2H,

NH₂, exch), 6.92 (d, J = 8.4 Hz, 5H), 7.10 (d, J = 8.6 Hz, 5H); Anal.

calcd. for (C₁₆H₂₀N₄O) C, H, N.

2-Methyl-6-vinyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-

one (16). To a solution of 14 (1.00 g, 5 mmol) in anhydrous toluene,

sodium metal (0.23 g, 10 mmol) was added in small portions. The

mixture was heated at reﬂux for 3 h. The reaction mixture was

neutralized with 1 N HCl and extracted with ethyl acetate (20 mL ×

3). The organic layer was dried over Na₂SO₄and concentrated. The

resulting yellow liquid was diluted with t-BuOH (15 mL). To this

solution were added potassium tert-butoxide (1.69 g, 15 mmol) and

acetamidine hydrochloride (7.5 mmol). The mixture was heated at

reﬂux overnight. The solid obtained on cooling was ﬁltered, and the

ﬁltered material was washed with hot methanol. To the ﬁltrate was

added silica gel (3 g), and the solvent was removed by evaporation.

The resulting silica gel plug was loaded onto a silica gel column and

eluted with chloroform/methanol (50:1). The fractions containing the

product (TLC) were pooled, and the solvent was evaporated to aﬀord

16 as an oﬀ-white solid (0.17 g, 19% over two steps): TLC R_f0.19

2-Amino-N-(4-methoxyphenyl)-N,6-dimethyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-aminium Chloride (10·HCl). The 10·HCl

was prepared from 10 (0.15 g) following the general method to make

a HCl salt from base described above to yield 0.11 g (65%) as a white

1

solid: mp 232.8−233.4 °C; H NMR (DMSO-d₆) δ 0.84 (d, J = 6.8

Hz, 3H), 1.24−1.36 (m, 1H), 1.74−1.88 (m, 1H), 2.17−2.37 (m, 2H),

2.87 (dd, J = 17.4, 8.2 Hz, 1H), 3.41 (s, 3H), 3.80 (s, 3H), 7.01 (d, J =

8.7 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.70 (bs, 2H), 12.93 (bs, 1H);

Anal. calcd. for (C₁₆H₂₁N₄OCl·0.6CH₃OH) C, H, N, Cl.

4-Vinylcyclohexane-1,2-diol (12). The suspension of 4-vinyl-1-

cyclohexene 1,2-epoxide 11 (3 mL) and conc. H₂SO₄(3 mL) in

distilled water (10 mL) in a 50 mL round-bottom ﬂask was stirred at

K

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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1

(chloroform/methanol, 10:1); mp 189.0−190.9 °C; H NMR (500

MHz, DMSO-d₆) δ 1.62−2.97 (m, 5 H), 2.24 (s, 3 H), 4.90−5.08 (m,

2 H), 5.83−5.98 (m, 1 H), 12.20 (bs, 1 H); Anal. calcd. for

(C₁₀H₁₂N₂O) C, H, N.

silica gel plug obtained was loaded onto a silica gel column and eluted

with chloroform/methanol (100:1). Fractions containing the product

(TLC) were pooled, and the solvent was evaporated to aﬀord 24 as a

white solid (0.083 g, 79%): TLC R_f0.28 (chloroform/methanol,

1

4-Chloro-6-vinyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-

amine (17). Compound 17 was synthesized from 15 (0.3 g, 1.69

mmol) following the general chlorination procedure described above

to aﬀord after puriﬁcation 0.152 g (46%) as an oﬀ white solid: TLC R_f

10:1); H NMR (500 MHz, DMSO-d₆) δ 1.16 (d, J = 6.9 Hz, 3H),

1.44−1.52 (m, 1H), 1.75 (s, 1H), 2.12−2.20 (m, 1H), 2.24 (s, 3H),

2.54−2.62 (m, 1H), 2.68−2.76 (m, 1H), 3.03−3.08 (m, 1H), 12.14

(bs, 1H); Anal. calcd. for (C₉H₁₂N₂O·0.2H₂O) C, H, N.

1

0.36 (chloroform/methanol, 10:1); mp 142.8−144.6 °C; H NMR

4-Chloro-2,5-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine

(25). Compound 25 was synthesized from 24 (0.3 g, 1.83 mmol)

following the general procedure for chlorination described above to

aﬀord after puriﬁcation 0.105 g (32%) as a colorless oil: TLC R_f0.29

(500 MHz, DMSO-d₆) δ 1.54−3.74 (m, 5 H), 4.94−5.11 (m, 2 H),

5.79−5.90 (m, 1 H), 6.85 (bs, 2 H); Anal. calcd. for (C₉H₁₀ClN₃) C,

H, N, Cl.

1

4-Chloro-2-methyl-6-vinyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine (18). Compound 18 was synthesized from 16 (0.460 g,

2.61 mmol) following the general procedure for chlorination described

above to aﬀord after puriﬁcation 0.414 g (81%) as a colorless liquid:

TLC R_f0.45 (chloroform/methanol, 10:1); ¹H NMR (500 MHz,

DMSO-d₆) δ 1.17−3.93 (m, 5 H), 2.56 (s, 3 H), 5.00−5.14 (m, 2 H),

5.84−6.01 (m, 1 H).

(hexane/ethyl acetate, 3:1); H NMR (CDCl₃) δ 1.28 (d, J = 7.0 Hz,

3H), 1.76 (ddd, J = 17.2, 10.7, 6.4 Hz, 1H), 2.26−2.36 (m, 1H), 2.64

(s, 3H), 2.84−2.95 (m, 1H), 3.07 (dt, J = 17.5, 8.6 Hz, 1H), 3.31−3.41

(m, 1H).

N-(4-Methoxyphenyl)-N,2,5-trimethyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-aminium Chloride (26·HCl). Compound

26 was synthesized from 25 (0.1 g, 0.55 mmol) and 4-methoxy-N-

methylaniline (0.083 g, 0.60 mmol) following the general procedure

for nucleophilic displacement from a chlorinated compound described

above to aﬀord after puriﬁcation a colorless oil. Following the general

method to make a HCl salt from a base, the oil was converted to 26·

HCl, 0.108 g (62% over two steps) as a white solid: TLC R_f0.31

N⁴-(4-Methoxyphenyl)-N⁴-methyl-6-vinyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidine-2,4-diamine (19). Compound 19 was

synthesized from 17 (0.1 g, 0.51 mmol) and N-methyl-4-methoxyani-

line (0.077 g, 0.56 mmol) following the general procedure for

nucleophilic displacement from a chlorinated compound described

above to aﬀord after puriﬁcation 0.084 g (56%) as a white solid: TLC

1

(chloroform/methanol, 10:1); mp 178.3−180.7 °C; H NMR (500

1

R_f0.30 (chloroform/methanol, 10:1); mp 144.8−147.0 °C; H NMR

MHz, DMSO-d₆) δ 0.65 (d, J = 6.6 Hz, 3H), 1.48−1.57 (m, 1H),

1.88−2.02 (m, 2H), 2.62 (s, 3H), 2.84 (dd, J = 18.2, 9.4 Hz, 1H), 3.01

(dt, J = 18.1, 9.1 Hz, 1H), 3.54 (s, 3H), 3.81 (s, 3H), 7.04−7.05 (m,

2H), 7.40 (bs, 2H), 15.10 (s, 1H); Anal. calcd. for (C₁₇H₂₂ClN₃O) C,

H, N, Cl.

(400 MHz, DMSO-d₆) δ 1.58 (m, 1H), 1.81 (m, 1H), 2.30−2.40 (m,

1H), 2.54−2.70 (m, 2H), 3.27 (s, 3H), 3.76 (s, 3H), 4.84 (dd, J = 20.1,

15.0 Hz, 2H), 5.63−5.77 (m, 1H), 5.92 (bs, 2H), 6.93 (d, J = 8.9 Hz,

2H), 7.13 (d, J = 8.8 Hz, 2H); Anal. calcd. for (C₁₇H₂₀N₄O) C, H, N.

N-(4-Methoxyphenyl)-N,2-dimethyl-6-vinyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-amine Hydrochloride (20·HCl). Com-

pound 20·HCl was synthesized from 18 (0.4 g, 2.05 mmol) and N-

methyl-4-methoxyaniline (0.310 g, 2.26 mmol) following the general

procedure for nucleophilic displacement from a chlorinated compound

described above to aﬀord after puriﬁcation a light yellow liquid: TLC

R_f0.28 (chloroform/methanol, 10:1). Using the general procedure to

make a HCl salt described above, the yellow liquid was converted to

20·HCl, 0.3 g (44% over two steps) as a white solid: mp 191−193.5

°C; ¹H NMR (400 MHz, DMSO-d₆) δ 1.64−1.75 (m, 1H), 1.88−1.97

(m, 1H), 2.62 (s, 3H), 2.72−2.76 (m, 1H), 2.88−2.92 (m, 1H), 3.04−

3.10 (m, 1H), 3.53 (s, 3H), 3.82 (s, 3H), 4.90−4.95 (m, 2H), 5.70−

5.77 (m, 1H), 7.04 (d, J = 9.1 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 15. 06

(bs, 1H); Anal. calcd. for (C₁₈H₂₂ClN₃O) C, H, N, Cl.

2-Methyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (28).

A mixture of compound 27 (1.25 mL, 9.9 mmol), acetamidine

hydrochloride (1.42 g, 15.6 mmol), and t-BuOK (3.1 g, 27.7 mmol) in

t-BuOH (50 mL) was heated at reﬂux overnight. The reaction mixture

was cooled, and the precipitate was removed by ﬁltration and washed

with warm methanol twice (30 mL × 1, 15 mL × 1). The ﬁltrate and

washings were combined and evaporated under reduced pressure. The

residue was puriﬁed by column chromatography using chloroform/

methanol (100/1) as eluent to aﬀord 28 as a white solid (1.2 g, 81%):

TLC R_f0.38 (CHCl₃/CH₃OH, 10:1); mp 234.4−236.6 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 1.86−2.00 (m, 2H), 2.24 (s, 3H), 2.58 (t, J =

7.4 Hz, 2H), 2.69 (t, J = 7.7 Hz, 2H), 12.17 (s, 1H); Anal. calcd. for

(C₈H₁₀N₂O·0.2H₂O) C, H, N.

4-Chloro-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (29).

Compound 29 was synthesized from 28 (0.55 g, 3.66 mmol) following

the general procedure for chlorination described above to aﬀord after

puriﬁcation 0.35 g (56%) as a yellow liquid: TLC R_f0.67 (CHCl₃/

Methyl 2-Diazo-3-oxoheptanoate (22). A mixture of methyl 3-

oxoheptanoate 21 (0.2 mL, 1.26 mmol), 4-acetamidobenzene

sulfonylazide (0.319 g, 1.33 mmol), and trimethylamine (0.35 mL,

2.51 mmol) in acetonitrile (3 mL) was stirred at room temperature for

8 h. The reaction mixture was diluted with 1 N NaOH and extracted

with ethyl acetate. After drying with anhydrous Na₂SO₄, the organic

solvent with 1 g of silica gel was evaporated under reduced pressure to

give a dry plug. This plug was placed on the top of a silica gel column

and eluted with 1% ethyl acetate in hexane. Fractions containing the

product were pooled and evaporated to aﬀord 22 as a light yellow oil

1

CH₃OH, 10:1); H NMR (500 MHz, DMSO-d₆) δ 2.03−2.12 (m,

2H), 2.55 (s, 3H), 2.90 (t, J = 7.5 Hz, 2H), 2.96 (t, J = 7.8 Hz, 2H).

N-(4-Methoxyphenyl)-N,2-dimethyl-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-aminium Chloride (30·HCl). Compound 30 was

synthesized from 29 (0.35 g, 2.1 mmol) and N-methyl-4-methoxylani-

line (0.31 g, 2.3 mmol) using the general procedure for nucleophilic

displacement from chlorinated compound described above to aﬀord

after puriﬁcation a light yellow liquid. Using the general procedure to

make a HCl salt from a base, the light yellow liquid was converted to

30·HCl, 0.362 g (57% for two steps) as a white solid: TLC R_f0.19

1

(0.145 g, 63%): TLC R_f0.52 (hexane/ethyl acetate, 5:1); H NMR

(500 MHz, DMSO-d₆) δ 0.87 (t, J = 7.4 Hz, 3H), 1.29 (dq, J = 14.7,

7.4 Hz, 2H), 1.51 (dt, J = 15.1, 7.5 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H),

3.76 (s, 3H).

1

(CHCl₃/CH₃OH, 10:1); mp 233.7−234.4 °C; H NMR (400 MHz,

2,5-Dimethyl-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one

(24). Rh₂(OAc)₄(0.036 g, 0.08 mmol) was suspended in dry

dichloromethane (10 mL). A solution of 22 (0.3 g, 1.63 mmol) in

dichloromethane was then added dropwise to a stirred slurry of

Rh₂(OAc)₄over 3 h. The reaction mixture was then ﬁltered through a

fritted funnel to recover the catalyst (20 mg). The solvent was

evaporated to give 23 (0.110 g, 43%) as a light green oil. A mixture of

23 (0.1 g, 0.64 mmol), t-BuOK (0.215 g, 1.92 mmol), and acetamidine

hydrochloride (0.091 g, 0.96 mmol) in t-BuOH (20 mL) was heated at

reﬂux overnight. The reaction mixture was then ﬁltered and washed

with methanol. To the ﬁltration was added silica gel (300 mg), and the

solvent was evaporated under reduced pressure to aﬀord a plug. The

DMSO-d₆) δ 1.71−1.87 (m, 4H), 2.62 (s, 3H), 2.81−2.92 (m, 2H),

3.53 (s, 3H), 3.81 (s, 3H), 7.03 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.9

Hz, 2H), 15.18 (s, 1H); Anal. calcd. for (C₁₅H₁₈N₄O·0.7HCl·1.3H₂O)

C, H, N, Cl.

N-(4-Ethoxyphenyl)-N,2-dimethyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-aminium Chloride (31·HCl). Compound 31 was

synthesized from 29 (0.080 g, 0.48 mmol) and 4-ethoxy-N-

methylaniline (0.079 g, 0.52 mmol) following the general procedure

described for 30·HCl to aﬀord after puriﬁcation 0.074 g (79%) as a

light brown solid: TLC R_f0.36 (chloroform/methanol, 10:1); mp

1

209.5−211.3 °C; H NMR (500 MHz, DMSO-d₆) δ 1.35 (t, J = 6.9

Hz, 3H), 1.79 (bs, 4H), 2.62 (s, 3H), 2.86 (bs, 2H), 3.52 (s, 3H), 4.07

L

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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(q, J = 6.9 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H),

15.11 (s, 1H); Anal. calcd. for (C₁₇H₂₂ClN₃O·0.3CH₃OH) C, H, N,

Cl.

puriﬁcation 0.091 g (48%) as a light brown solid: TLC R_f0.31

(CHCl₃/CH₃OH, 10:1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.33 (t, J

= 6.9 Hz, 3H), 1.58 (dt, J = 14.1, 7.2 Hz, 2H), 1.68 (t, J = 7.0 Hz, 2H),

2.44 (t, J = 7.7 Hz, 2H), 3.27 (s, 3H), 4.02 (q, J = 6.9 Hz, 2H), 5.89

(bs, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H); Anal.

calcd. for (C₁₆H₂₀N₄O·0.3H₂O) C, H, N.

Methyl 2-Aminocyclopent-1-enecarboxylate (39). A mixture of

methyl-2-oxocyclopentane carboxylate 27 (2 mL, 15.5 mmol),

ammonium formate (5.4 g, 77.4 mmol), and methanol (30 mL) was

heated at reﬂux for 24 h. To the solution was added silica gel (6 g),

and the mixture was evaporated under reduced pressure to aﬀord a

plug. This plug was loaded on a dry silica gel column and ﬂashed

chromatographed with hexane/ethyl acetate (10:1). Fractions showing

the major spot on TLC were pooled and evaporated to give 39, 2 g

(91%) as a white solid: TLC R_f0.26 (hexane/ethyl acetate, 3:1); mp

94.3−95.8 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 1.67−1.73 (m, 2H),

2.34−2.44 (m, 4H), 3.53 (s, 3H), 6.74 (bs, 2H); Anal. calcd. for

(C₇H₁₁NO₂) C, H, N.

N,2-Dimethyl-N-p-tolyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-

4-amine (32). Compound 32 was synthesized from 29 (0.140 g, 0.83

mmol) and N-methyl-p-tulidine (0.101 g, 0.83 mmol) following the

general procedure for nucleophilic displacement from a chlorinated

compound described above to aﬀord after puriﬁcation 0.102 g (48%)

as a light brown solid: TLC R_f0.30 (chloroform/methanol, 10:1); mp

97.5−99.3 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 1.65 (p, J = 7.6 Hz,

2H), 1.78 (t, J = 7.3 Hz, 2H), 2.32 (s, 3H), 2.41 (s, 3H), 2.60 (t, J =

7.7 Hz, 2H), 3.36 (s, 3H), 7.10 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.1 Hz,

2H); Anal. calcd. for (C₁₆H₁₉N₃·0.1H₂O) C, H, N.

2-Amino-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (33).

A mixture of 27 (1.25 mL), guanidine carbonate (2.71 g, 15 mmol),

and t-BuOK (3.1 g, 27.7 mmol) in t-BuOH (50 mL) was heated at

reﬂux overnight. The reaction mixture was cooled, and a precipitate

was removed by ﬁltration. The ﬁltered material was washed twice with

warm methanol (30 mL × 1, 15 mL × 1). The ﬁltrate and washings

were combined and evaporated under reduced pressure, and the

residue was puriﬁed by column chromatography using chloroform/

methanol (5/1) as eluent to aﬀord 33 (1.53 g, 83%) as a white solid:

Methyl 2-Formamidocyclopent-1-enecarboxylate (40). Formic

acid (4.3 mL) was added to acetic anhydride (6.5 mL) while cooling in

an ice bath. Compound 37 (1 g, 7.08 mmol) was added to the cold

solution in small portions. The cooling bath was removed, and the

resulting solution was stirred at room temperature for 4 h. The

reaction mixture was extracted with ethyl acetate (30 mL × 3). The

organic layer was dried over anhydrous Na₂SO₄and then ﬁltered. To

the ﬁltrate was added silica gel (5 g), and the mixture was evaporated

under reduced pressure to aﬀord a plug. This plug was loaded on a dry

silica gel column and ﬂashed chromatographed with hexane/ethyl

acetate (10:1). Fractions showing the major spot on TLC were pooled

and evaporated to give 40, 0.927 g (77%) as a white solid: TLC R_f0.26

1

TLC R_f0.17 (CHCl₃/CH₃OH, 10:1); H NMR (DMSO-d₆) δ 1.88−

1.95 (m, 2H), 2.54−2.58 (m, 2H), 2.67 (t, J = 7.8 Hz, 2H), 12.16 (bs,

1H); Anal. calcd. for (C₈H₁₀N₂O·0.2H₂O) C, H, N.

4-Chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-amine (34).

Compound 34 was synthesized from 33 (0.55 g, 3.66 mmol)

following the general procedure for chlorination described above to

aﬀord after puriﬁcation 0.35 g (56%) as a yellow liquid: TLC R_f0.67

1

(CHCl₃/CH₃OH, 10:1); H NMR (400 MHz, DMSO-d₆) δ 1.96−

1

(hexane/ethyl acetate, 3:1); mp 76.7−78.0 °C; H NMR (DMSO-d₆)

2.06 (m, 2H), 2.69−2.77 (m, 2H), 2.82 (t, J = 7.8 Hz, 2H), 8.01 (bs,

2H).

δ 1.75−1.95 (m, 2 H), 2.82−2.94 (m, 1H), 2.98−3.10 (m, 1H), 3.69

(s, 3H), 8.28 (bs, 0.5H), 8.57 (bs, 0.5H), 9.71 (bs, 0.5H), 10.28 (bs,

0.5H); Anal. calcd. for (C₈H₁₁NO₃) C, H, N.

N⁴-(4-Methoxyphenyl)-N⁴-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine-2,4-diamine (35). Compound 35 was synthesized from 34

(0.120 g, 0.71 mmol) and 4-methoxy-N-methylaniline (0.116 g, 0.85

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.136 g (71%) as a white solid: TLC R_f0.41 (CHCl₃/

CH₃OH, 10:1); mp 187.7−189.5 °C; ¹H NMR (400 MHz, DMSO-d₆)

δ 1.52−1.64 (m, 2H), 1.64−1.73 (t, J = 7.2 Hz, 2H), 2.44 (t, J = 7.7

Hz, 2H), 3.28 (s, 3H), 3.76 (s, 3H), 5.90 (bs, 2H), 6.93 (d, J = 8.8 Hz,

2H), 7.12 (d, J = 8.8 Hz, 2H); Anal. calcd. for (C₁₅H₁₈N₄O·0.3H₂O)

C, H, N.

6,7-Dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (41). To a

solution of ammonium formate (1 g, 16 mmol) in formamide (2

mL) at 150 °C was added compound 40 (0.5 mg, 2.96 mmol). The

resulting solution was heated at 150 °C for 4 h and then allowed to

stand at room temperature for 12 h. This mixture was loaded on a wet

silica gel column and ﬂashed chromatographed with chloroform/

methanol (50:1). Fractions showing the major spot on TLC were

pooled and evaporated to give 41, 0.335 g (83%) as a white solid: TLC

1

R_f0.34 (chloroform/methanol, 10:1); mp 245.2−246.9 °C; H NMR

N⁴-(3-Methoxyphenyl)-N⁴-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine-2,4-diamine (36). Compound 36 was synthesized from 34

(0.060 g, 0.35 mmol) and 3-methoxy-N-methylaniline (0.048 g, 0.35

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.054 g (56%) as a white solid: TLC R_f0.36 (CHCl₃/

CH₃OH, 10:1); mp 153.0−154.1 °C; ¹H NMR (500 MHz, DMSO-d₆)

δ 1.58−1.67 (m, 2H), 1.77 (t, J = 7.1 Hz, 2H), 2.47 (d, J = 7.7 Hz,

2H), 3.34 (s, 3H), 3.74 (s, 3H), 5.94 (bs, 2H), 6.69−6.77 (m, 2H),

6.80 (d, J = 8.9 Hz, 1H), 7.27 (t, J = 8.4 Hz, 1H); Anal. calcd. for

(C₁₅H₁₈N₄O·0.1C₄H₁₀O) C, H, N.

(DMSO-d₆) δ 1.90−1.99 (m, 2H), 2.62 (t, J = 7.4 Hz, 2H), 2.75 (t, J =

7.6 Hz, 2H), 8.01 (s, 1H), 12.26 (s, 1H); Anal. calcd. for (C₇H₈N₂O)

C, H, N.

4-Chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidine (42). Com-

pound 42 was synthesized from 41 (0.330 g) following the general

procedure for chlorination described above to aﬀord after puriﬁcation

0.223 g (60%) as a light yellow solid: TLC R_f0.55 (chloroform/

1

methanol, 10:1); mp 41.5−43.0 °C; H NMR (DMSO-d₆) δ 2.05−

2.14 (m, 2H), 2.96 (t, J = 7.6 Hz, 2H), 3.03 (t, J = 7.8 Hz, 2H), 8.78

(s, 1H); Anal. calcd. for (C₇H₇ClN₂) C, H, N, Cl.

N-(4-Methoxyphenyl)-N-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-amine (43). Compound 43 was synthesized from 42

(0.16 g, 1.04 mmol) and 4-methoxy-N-methylaniline (0.157 g, 1.14

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.231 g (87%) as a light yellow solid: TLC R_f0.33

N⁴-(2-Methoxyphenyl)-N⁴-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine-2,4-diamine (37). Compound 37 was synthesized from 34

(0.085 g, 0.50 mmol) and 2-methoxy-N-methylaniline (0.067 g, 0.50

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.065 g (48%) as a light yellow solid: TLC R_f0.21

1

(CHCl₃/CH₃OH, 10:1); mp 95.7−96.5 °C; H NMR (DMSO-d₆) δ

1

(CHCl₃/CH₃OH, 10:1); mp 170.3−172.0 °C; H NMR (500 MHz,

1.62−1.71 (m, 2H), 1.83 (t, J = 7.3 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H),

3.35 (s, 3H), 3.78 (s, 3H), 6.96 (d, J = 8.9 Hz, 2H), 7.19 (d, J = 8.8

Hz, 2H), 8.42 (s, 1H); Anal. calcd. for (C₁₅H₁₇N₃O) C, H, N.

N-Methyl-N-p-tolyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-

amine (45). Compound 45 was synthesized from 42 (0.1 g, 0.65

mmol) and N-methyl-p-toluidine (0.087 g, 0.71 mmol) following the

general procedure for nucleophilic displacement from a chlorinated

compound described above to aﬀord after puriﬁcation 0.09 g (58%) as

an oﬀ-white solid: TLC R_f0.67 (CHCl₃/CH₃OH, 5:1); mp 79.1−80.5

°C; ¹H NMR (500 MHz, DMSO-d₆) δ 1.67 (p, J = 7.6 Hz, 2H), 1.83

DMSO-d₆) δ 1.51−1.63 (m, 4H), 2.42 (t, J = 7.6 Hz, 2H), 3.21 (s,

3H), 3.76 (s, 3H), 5.84 (bs, 2H), 6.94 (t, J = 7.5 Hz, 1H), 7.08 (d, J =

8.3 Hz, 1H), 7.16 (d, J = 9.3 Hz, 1H), 7.28−7.34 (m, 1H); Anal. calcd.

for (C₁₅H₁₈N₄O·0.3CH₃OH) C, H, N.

N⁴-(4-Ethoxyphenyl)-N⁴-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine-2,4-diamine (38). Compound 38 was synthesized from 34

(0.085 g, 0.51 mmol) and 4-ethoxy-N-methylaniline (0.076 g, 0.51

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

M

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(t, J = 7.3 Hz, 2H), 2.33 (s, 3H), 2.66 (t, J = 7.7 Hz, 2H), 3.37 (s, 3H),

7.13 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 8.45 (s, 1H); Anal.

calcd. for (C₁₅H₁₇N₃) C, H, N.

In the polymerization assay,⁴¹10 μM (1.0 mg/mL) tubulin was

preincubated for 15 min at 30 °C with varying compound

concentrations in 0.8 M monosodium glutamate, taken from a 2.0

M stock solution adjusted to pH 6.6 with HCl. Reaction mixtures also

contained 4% (v/v) dimethyl sulfoxide (compound solvent).

Following the preincubation, reaction mixtures were placed on ice,

and 10 μL of 10 mM GTP (0.4 mM ﬁnal concentration) was added to

each mixture. Reaction volume was 0.24 mL prior to GTP addition,

and all concentrations are in terms of the ﬁnal reaction volume of 0.25

mL. Reaction mixtures were transferred to 0 °C cuvettes in Beckman

DU7400/7500 recording spectrophotometers equipped with elec-

tronic temperature controllers. Polymer formation was measured

turbidimetrically at 350 nm. After baselines were established at 0 °C,

temperature was rapidly jumped to 30 °C (less than 1 min), and the

IC₅₀was deﬁned as the compound concentration inhibiting the extent

of assembly by 50% after 20 min at 30 °C.

N-(4-Chlorophenyl)-N-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-amine (46). Compound 46 was synthesized from 42

(0.080 g, 0.52 mmol) and 4-chloro-N-methylaniline (0.081 g, 0.57

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.115 g (85%) as an oﬀ white solid: TLC R_f0.36

1

(CHCl₃/CH₃OH, 10:1); mp 106.8−108.1 °C; H NMR (500 MHz,

DMSO-d₆) δ 1.67−1.79 (m, 2H), 1.90 (t, J = 7.2 Hz, 2H), 2.70 (t, J =

7.7 Hz, 2H), 3.41 (s, 3H), 7.26 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.6 Hz,

2H), 8.49 (s, 1H); Anal. calcd. for (C₁₄H₁₄ClN₃) C, H, N, Cl.

N-Methyl-N-(4-(methylthio)phenyl)-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-amine (47). Compound 47 was synthesized from 42

(0.1 g, 0.65 mmol) and N-methyl-4-(methylthio)aniline (0.109 g, 0.71

mmol) following the general procedure for nucleophilic displacement

from a chlorinated compound described above to aﬀord after

puriﬁcation 0.135 g (77%) as a white solid: TLC R_f0.68 (CHCl₃/

CH₃OH, 5:1); mp 111.7−112.8 °C ; ¹H NMR (DMSO-d₆) δ 1.70 (p,

J = 7.6 Hz, 2H), 1.89 (t, J = 7.3 Hz, 2H), 2.50 (s, 3H), 2.67 (t, J = 7.7

Hz, 2H), 3.38 (s, 3H), 7.19 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.5 Hz,

2H), 8.46 (s, 1H); Anal. calcd. for (C₁₅H₁₇N₃S) C, H, N, S.

In the colchicine binding assay,⁴²0.1 mL reaction volumes

contained 1.0 μM tubulin, 5.0 μM [³H]colchicine (from Perkin-

Elmer), and potential inhibitors at 1.0 or 5.0 μM, together with

reaction components that strongly stabilize the colchicine binding

activity of tubulin.^42,43Reaction mixtures also contained 5% dimethyl

sulfoxide (compound solvent). Tubulin was the last component added

to the reaction mixtures, which were prepared on ice. Binding of

colchicine was initiated by transferring the reaction mixtures to a 37

°C water bath, and the mixtures were incubated for 10 min (a time

when the reaction without inhibitor is 40−60% complete). Reactions

were stopped with ice water, and the diluted reaction mixtures were

ﬁltered through a stack of two Whatman DEAE-cellulose ﬁlters

obtained from GE Healthcare Life Sciences. Radiolabel bound to the

ﬁlters was quantitated by scintillation counting.

In Vivo Evaluation. The purpose of this experiment was to

determine the eﬃcacy of 30·HCl, when administered in diﬀerent

treatment regimens, in the MDA-MB-231T solid tumor xenograft

model. A fragment of this tumor was implanted sc in female athymic

nude mice, and tumors were staged to approximately 125 mg.

Treatment to all groups commenced day 14 post-tumor implant. A

second course of treatment was administered to all groups. This was a

valid control.

Compound 30·HCl was administered as a solution in 10% DMSO

in saline/Tween 80. Groups 2 and 3 received 75 or 50 mg/kg per

injection, respectively, ip for 5 consecutive days commencing days 14

and 27. Group 4 received 75 mg/kg per injection ip every other day

for a total of ﬁve treatments commencing days 14 and 27. Group 5

received 50 mg/kg per injection ip twice a day, every other day, for a

total of six treatments commencing on days 14 and 27.

A MTD was not administered to any group because there was no

indication of toxicity as expressed by either excessive animal body

weight loss or lethality. The following are the optimum T/C values for

each group: group 2 (17), group 3 (20), group 4 (19) and group 5

(25). Eﬃcacy was obtained in every treatment regimen with no

marked diﬀerences between the regimens.

N-(4-Methoxyphenyl)-N-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-aminium Chloride (43·HCl). Compound 43·HCl was

synthesized from 43 (0.130 g, 0.51 mmol) using the general procedure

to make a HCl salt from a base described above to aﬀord 0.142 g

(95%) as a light yellow solid: mp 224.1−225.9 °C; ¹H NMR (DMSO-

d₆) δ 1.73−1.91 (m, 4H), 2.89 (t, J = 7.7 Hz, 2H), 3.54 (s, 3H), 3.81

(s, 3H), 7.04 (d, J = 8.9 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 8.86 (s,

1H), 15.19 (bs, 1H); Anal. calcd. for (C₁₅H₁₈ClN₃O) C, H, N, Cl.

N-(4-Ethoxyphenyl)-N-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-aminium Chloride (44·HCl). Compound 44·HCl was

synthesized from 42 (0.092 g, 0.60 mmol) and 4-ethoxy-N-ethylaniline

(0.090 g, 0.60 mmol) following the general procedure for nucleophilic

displacement from a chlorinated compound and the general method to

make a HCl salt described above to aﬀord after puriﬁcation 0.13 g

(89%) as an oﬀ-white solid: TLC R_f0.41 (CHCl₃/CH₃OH, 10:1); mp

1

228.0−229.2 °C ; H NMR (500 MHz, DMSO-d₆) δ 1.35 (t, J = 7.0

Hz, 3H), 1.81 (ddd, J = 21.8, 15.7, 6.3 Hz, 4H), 2.89 (t, J = 7.7 Hz,

2H), 4.08 (q, J = 6.9 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8

Hz, 2H), 8.86 (s, 1H),15.20 (bs, 1H); Anal. calcd. for (C₁₆H₂₀N₃OCl)

C, H, N, Cl.

Cellular Studies. Eﬀects on Cellular Microtubules. A-10 cells

were used to evaluate the eﬀects of the compounds on cellular

microtubules using indirect immunoﬂuorescence. EC₅₀values were

calculated as previously described and represent a minimum of three

independent experiments.³⁸

SRB Assay. The antiproliferative and cytotoxic activity of all

compounds was evaluated using the SRB assay as previously

described.³⁹The IC₅₀’s represent an average of at least three

independent experiments plus or minus the standard deviation.

Cell Cycle Analysis. MDA-MB-435 cells were plated and allowed

to adhere for 24 h. Drugs were then added, and cells were harvested 24

h later. Paclitaxel (12.5 nM) was used as a positive control. Once cells

were harvested, they were stained with Krishan’s reagent and analyzed

for DNA content using a FacsCalibur ﬂow cytometer.

ASSOCIATED CONTENT

* Supporting Information

■

S

Elemental Analysis. This material is available free of charge via

the Internet at http://pubs.acs.org.

In Vitro Tubulin Polymerization. The eﬀects of the compounds

on tubulin polymerization were initially measured using puriﬁed

porcine brain tubulin (Cytoskeleton Inc.). The reactions contained 2

mg/mL of tubulin incubated with 1 mM GTP and 10% glycerol and

drug as indicated. The polymerization of tubulin was monitored by

measuring the absorbance at 340 nm at 37 °C in a SpectraMax 96-well

plate spectrophotometer. Colchicine was used as a control.

Quantitative Tubulin Studies. Puriﬁed bovine brain tubulin⁴⁰

was used in these studies to determine IC₅₀values for tubulin

polymerization and compound inhibition of colchicine binding. The

techniques used in both assays have previously been described in

detail^41,42but are summarized below.

AUTHOR INFORMATION

Corresponding Authors

*Phone 412-396-6070, fax 412-396-5593, e-mail gangjee@duq.

■

edu.

*Phone 210-567-4788, fax 210-567-4300, e-mail mooberry@

uthscsa.edu.

Author Contributions

^∥A.G. and S.L.M. contributed equally to this manuscript.

Notes

The authors declare no competing ﬁnancial interest.

N

dx.doi.org/10.1021/jm400639z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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M. A.; Kavallaris, M. Microtubule dynamics, mitotic arrest, and

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Expression of class III beta tubulin in non-small cell lung cancer is

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ACKNOWLEDGMENTS

■

We gratefully acknowledge the support of the Developmental

Therapeutics Program (DTP), Frederick National Laboratory

for Cancer Research, National Cancer Institute, National

Institutes of Health, for performing the in vitro antitumor

evaluation in their 60 tumor preclinical screening program and

for conducting the in vivo xenograft studies. Grant support

from the National Cancer Institute, CA142868 (A.G., S.L.M.),

is acknowledged as well as support provided by an NSF

equipment grant for NMR instrument (NMR CHE 0614785),

the CTRC Cancer Center Support Grant, P30 CA054174, and

the Duquesne University Adrian Van Kaam Chair in Scholarly

Excellence (A.G.).

ABBREVIATIONS USED

■

Pgp, P-glycoprotein; DAMA-colchicine, N-deacetyl-N-(2-mer-

captoacetyl) colchicine; SAR, structure−activity relationship;

CWP, tris(cetylpyridinium) 12-tungstophosphate; SRB, sulfo-

rhodamine B; CA-4, combretastatin A-4

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L. Synthesis and biological activities of (R)- and (S)-N-(4-

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pyrimidin-4-aminium chloride as potent cytotoxic antitubulin agents. J.

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Risinger, A. L.; Hamel, E.; Mooberry, S. L. Synthesis and discovery of

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epoxides. Patent Application US 2004/0192976 A1. Sept. 30, 2004.

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Article Doi

Structure-activity relationship and in vitro and in vivo evaluation of the potent cytotoxic anti-microtubule agent N-(4-methoxyphenyl)-N,2,6-trimethyl-6,7- dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride and its analogues as antitumor agents

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